Research ArticleHigh SHIP2 Expression Indicates Poor Survival inColorectal Cancer

Ju Yang1 Maoying Fu2 Yaoguang Ding3 Yajing Weng4 Weifei Fan5

Xiaolin Pu5 Zhijun Ge6 Feng Zhan7 Huihui Ni2 Wei Zhang2 Feng Jin2

Ning Xu8 Jiang Li9 Liang Qiu9 Jun Wang5 and Xuefeng Gu2

1 Department of Gastroenterology Kunshan Hospital of Traditional Chinese MedicineThe Affiliated Hospital of Nanjing University of Chinese Medicine Suzhou 215000 China

2Department of Infectious Diseases The First Peoplersquos Hospital of Kunshan Affiliated with Jiangsu University Suzhou 215000 China3Department of Oncology Kunshan Hospital of Traditional Chinese MedicineThe Affiliated Hospital of Nanjing University of Chinese Medicine Suzhou 215000 China

4Department of Geriatric Rehabilitation Kunshan Hospital of Traditional Chinese MedicineThe Affiliated Hospital of Nanjing University of Chinese Medicine Suzhou 215000 China

5 Department of Hematology and Oncology Jiangsu Province Geriatric Institute Nanjing 210000 China6Department of Anesthesiology Yixing Peoplersquos Hospital The Affiliated Yixing Hospital of Jiangsu University Yixing 214200 China7Department of Hepatobiliary and Laparoscopic Surgery Yixing Peoplersquos Hospital The Affiliated Yixing Hospital of Jiangsu UniversityYixing 214200 China

8Department of Pathology Nanjing Medical University Nanjing 210000 China9Department of Pathology Jiangsu Province Geriatric Institute Nanjing 210000 China

Correspondence should be addressed to Jun Wang wangjjs2013163com and Xuefeng Gu sani666163com

Received 3 August 2014 Revised 23 October 2014 Accepted 1 November 2014 Published 24 November 2014

Academic Editor Chao Hung Hung

Copyright copy 2014 Ju Yang et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

SH2-containing inositol 51015840-phosphatase 2 (SHIP2) which generally regulates insulin signaling cytoskeleton remodeling andreceptor endocytosis has been suggested to play a significant role in tumor development and progressionHowever the associationsbetween SHIP2 expression and the clinical features to evaluate its clinicopathologic significance in colorectal cancer (CRC)have not been determined yet In the present study one-step quantitative real-time polymerase chain reaction (qPCR) test andimmunohistochemistry (IHC) analysis with CRC tissue microarrays (TMA) were employed to evaluate the mRNA and proteinexpression of SHIP2 in CRCThe results showed that SHIP2 expression in the mRNA and protein levels was significantly higher inCRC tissues than that in corresponding noncancerous tissues (both 119875 lt 005) The expression of SHIP2 protein in CRC was relatedto lymph node metastasis (119875 = 0036) distant metastasis (119875 = 0001) and overall survival (119875 = 0009) Kaplan-Meier methodand Cox multifactor analysis suggested that high SHIP2 protein level (119875 = 0040) and positive distant metastasis (119875 = 0048) werecritically associated with the unfavorable survival of CRC patients The findings suggested that SHIP2 may be identified as a usefulprognostic marker in CRC and targeting CRC may provide novel strategy for CRC treatment

1 Introduction

Colorectal cancer (CRC) is the third most common malig-nancy in the world with an estimated incidence of morethan 12 million new cases and over 600 000 deaths globally[1ndash3] The number of patients who are suffering continuesto rise especially in most Asian countries [4] During

the last two decades several countries includingChina SouthKorea and Japan have witnessed a two- to threefold rise inincidence of CRC In China for example the total numberof CRC cases increased by 191 and 177 in Chinese malesand females from 2000 to 2005 respectively [5 6] Generallyspeaking ulcerative colitis familial adenomatous polyposisand hereditary nonpolyposis colon cancer syndrome are

Hindawi Publishing CorporationDisease MarkersVolume 2014 Article ID 218968 7 pageshttpdxdoiorg1011552014218968

2 Disease Markers

the three major risks that contribute to the CRC develop-ment [7] Despite the development of combined therapeuticmodalities for CRC treatment including surgical operationand combination of chemotherapy and adjuvant therapythe overall prognosis of CRC remains unsatisfactory andthe 5-year survival rate of CRC patients with metastasis isstill under 10 [8 9] It is valuable and critical to discovermolecular predictive markers for the prognosis which wouldoptimize the selection of therapeutic strategies and furtherimprove patientsrsquo survival for CRC [10]

SH2-containing inositol 51015840-phosphatase 2 (SHIP2) be-longs to the phosphoinositol phosphatases family which playsimportant role in modulating signaling pathways relevantto both diabetes and cancer and generally regulates insulinsignaling cytoskeleton remodeling and receptor endocytosis[11ndash13] As one of the important lipid phosphatases that actdownstream of phosphoinositide-31015840 kinase (PI3K) SHIP2converts phosphatidylinositol (345)-trisphosphate (PIP3)into phosphatidylinositol (34)-biphosphate (PIP2) and sub-sequently reduces the activation of PI3KAkt signaling cas-cade which exerts energetic function in tumor developmentand progression [14ndash16] Under this circumstance SHIP2is supposed to show anticancer effects and several studiessupport this presumption [17 18] However on the contraryseveral studies elucidate the prooncogenic function of SHIP2in certain type of cancer For instance high SHIP2 expressionenhances cancer cell proliferation while inhibition of SHIP2suppresses cancer growth in vitro and distant metastasesin vivo [12] Similarly SHIP2 performs oncogenically andhigh expression of SHIP2 indicates poor survival of breastcancer and laryngeal squamous cell carcinoma [19 20]SHIP2 also promotes cancer cell proliferation and metastasisby preventing epidermal growth factor receptor (EGFR)turnover and enhancing EGF-induced Akt activation [12 21]In light of the dual characteristics of SHIP2 the association ofSHIP2 expression and cancer development still seems vagueand little is known about the role of SHIP2 in CRC especiallythe prognostic significance

In this present paper we detected SHIP2 expression ina number of CRC samples and analyzed the associationsbetween SHIP2 expression and clinicopathologic items inCRC patients Finally the prognostic significance of SHIP2was evaluated in 102 CRC cases

2 Materials and Methods

21 Patients and Tissue Samples A total of 15 fresh CRCtissues and matched tumor-adjacent noncancerous tissueswere collected from the tissue bank of the Affiliated Hospitalof Nantong University to perform quantitative real-timepolymerase chain reaction (qPCR) test 102 formalin-fixedparaffin-embedded CRC tissues and corresponding tumor-adjacent normal tissues were also enrolled in this presentstudy from the Affiliated Hospital of Nantong Universityfrom 2002 to 2005 to execute immunohistochemistry (IHC)analysis Other pieces of clinicopathological information of102 CRC cases such as gender age tumor size tumorlocation histological type tumor differentiation serum CEA

level metastasis status TNM stage and overall survival werecollected from each patientrsquos medical records simultaneouslyClinical staging was performed according to the latest revi-sion of American Joint Committee on CancerInternationalUnion Against Cancer TNM staging system [22]

22 Detection of the SHIP2 Expression by One-Step qPCR TestTotal RNA was extracted from 15 cases of the frozen CRCtissues and thematched noncancerous tissues using theTrizolreagent (Invitrogen Carlsbad CA USA) according to themanufacturerrsquos guidelines Total RNA extraction and one-step qPCR analysis were performed as previously described[23] The primers for SHIP2 were as follows forward primer51015840-TCG TCA CCA GCG ACC ATT C-31015840 and reverse primer51015840-AGC CCT TTC TTG GAG ATG AAC TG-31015840 The glycer-aldehyde 3-phosphate dehydrogenase (GAPDH)mRNA levelwas used to standardize the measurements of SHIP2 and theprimers for GAPDHwere as follows forward primer 51015840-TGCACCACCAACTGCTTAGC-31015840 and reverse primer 31015840-GGCATG GAC TGT GGT CAT GAG-51015840

23 Detection of the SHIP2 Expression by IHC AnalysisTissue microarray (TMA) was produced by Alenabio Biotech(Xirsquoan China) Core tissue biopsies (2mm in diameter) werecollected from individual paraffin-embedded CRC sectionsand arranged in the recipient paraffin blocks IHC anal-ysis was performed as previously described [24] Tissuesections were incubated with polyclonal goat anti-SHIP2antibody (Santa Cruz Biotechnology Santa Cruz CA USA)in TBS containing 1 bovine serum albumin for 1 hourThe secondary antibody used was horseradish peroxidase-conjugated anti-goat antibody (Dako Cytomation Carpinte-ria CA USA)

SHIP2 immunostaining was scored by two independentpathologists according to intensity and percentage of positivecells simultaneously Staining intensity was scored as follows0 (negative) 1 (weakly positive) 2 (moderately positive) and3 (strongly positive) The percentage of SHIP2-positive cellswas also scored according to four categories as follows 1 wasgiven for 0 to 10 2 for 11 to 50 3 for 51 to 80and 4 for 81 to 100 The product of the intensity andpercentage scores was used as the final staining score Thedegree of SHIP2 staining was quantified using a two-levelgrading system as follows samples with a sum score lt 4 wereconsidered as low SHIP2 expression while those with a sumscore ge 4 were considered as high SHIP2 expression

24 Statistical Analysis Statistical analyses were performedby using SPSS 160 (SPSS Inc Chicago IL USA) and STATA120 (Stata Corporation College Station TX USA) Theexpression of SHIP2 mRNA in fresh frozen CRC tissuesand matched tumor-adjacent normal tissues normalized toGAPDH was analyzed with the Wilcoxon nonparametricsigned-rank test The relationship between SHIP2 proteinexpression and clinicopathological factors was evaluated byChi-square test Univariate and multivariate analyses wereperformed using Coxrsquos proportional hazards regressionmod-els Survival curves were calculated using the Kaplan-Meier

Disease Markers 3

10

8

6

4

2

0CRC Noncancerous

SHIP

2 ex

pres

sion

norm

aliz

ed b

y G

APD

Hlowast

P = 0004

Figure 1One-step quantitative real-time polymerase chain reaction(qPCR) test was employed to evaluate the expression of SHIP2mRNA in colorectal cancer (CRC) and tumor-adjacent noncancer-ous tissues When normalized to GAPDH the expression of SHIP2mRNA in CRC tissues (511 plusmn 0419) was significantly higher thanthat in matched noncancerous tissues (348 plusmn 0295) (119875 = 0004)

method For all tests the significance level for statisticalanalysis was set at 119875 lt 005

3 Results

31 Summarization of the Characteristics of 102 CRC PatientsA total of 102 CRC cases were enrolled from 63 men and 39women in this present study The mean age of all patients atthe time of diagnosis was 6308 plusmn 10852 years The tumordiameters of 44 cases were ge5 cm and of 58 cases were lt5 cmThe tumors of 87 cases were located in colon 11 cases in rec-tum and 4 cases in ileocecal junction The histological typeof tumor in 99 cases was adenocarcinoma whereas the other3 cases were identified as nonadenocarcinoma Four patientssuffered from a well-differentiated tumor 82 had moderatetumor differentiation and 7 had poor tumor differentiationThere were 11 cases with serum CEA level ge 15 ngmL and59 cases with serum CEA level lt 15 ngmL Positive lymphnode metastasis was noticed in 34 patients while distantmetastasis was observed in 19 patients According to TNMstaging system 63 patients were in stages I-II and 39 patientswere in stages III-IV Of all the 102 cases 61 patients survivedwhile 41 patients died

32 Evaluation of SHIP2 mRNA Expression by qPCR TestTotal RNA was extracted from 15 CRC tissues and matchednoncancerous tissues and subsequently subjected to one-step qPCR for the evaluation of the expression of SHIP2mRNA When normalized to GAPDH the means of SHIP2mRNA expression in CRC tissues and the correspondingnoncancerous tissues were 511 plusmn 0419 and 348 plusmn 0295respectively (119905 = 3174 119875 = 0004) SHIP2 mRNA expres-sion averaged 147-fold higher in CRC samples than that innoncancerous tissue samples (Figure 1)

A1

B1

C1

D1

Figure 2 Representative images of SHIP2 protein expression incolorectal cancer (CRC) tissues and corresponding noncanceroustissues with tissue microarray (TMA) A1 A2 and A3 highimmunohistochemical (IHC) staining of SHIP2 protein expressionin CRC tissue samples B1 B2 and B3 low IHC staining of SHIP2protein expression in CRC tissue samples C1 C2 and C3 highIHC staining of SHIP2 protein expression in noncancerous tissuesamples D1 D2 and D3 low IHC staining of SHIP2 proteinexpression in noncancerous tissue samples Original magnificationtimes40 in A1 B1 C1 and D1 times200 in A2 B2 C2 and D2 times400 in A3B3 C3 and D3

33 Evaluation of SHIP2 Protein Expression by IHC AnalysisIHC analysis was further performed to detect the SHIP2protein expression in CRC High SHIP2 expression wasdetected in 51 (500) out of 102 CRC tissues and in 24(235) out of 102 tumor-adjacent noncancerous tissues Theresults exhibited significant difference (119875 lt 005) and wereconsistent with results of SHIP2 mRNA expression in qPCRtest Positive staining was mainly localized in the cytoplasmof CRC cells Typical IHC staining patterns for SHIP2 in CRCare shown in Figure 2

34 Relationships between SHIP2 Protein Expression andClinicopathological Items The relationships between highSHIP2 protein expression and selected clinicopathologicalitems were shown in Table 1 High SHIP2 protein expressionwas significantly related to lymph node metastasis (119875 =0036) distant metastasis (119875 = 0001) and overall survival(119875 = 0009) (Table 1)

35 Survival Analysis In order to evaluate the prognosticrole of SHIP2 survival analyses were subsequently executedIn univariate analysis 4 items including high SHIP2 proteinexpression (119875 = 0017) lymph node metastasis (119875 = 0001)

4 Disease Markers

Table 1 The relationship between SHIP2 expression and clinicalattributes of 102 CRC patients

Groups Number SHIP21205942119875 value

+ Gender

Male 63 31 492 004 0839Female 39 20 513

Age (years)ge60 68 34 500 000 1000lt60 34 17 500

Tumor size (cm)ge5 44 21 477 016 0689lt5 58 30 517

Tumor locationColon 87 43 494

110 0576Rectum 11 5 455Ileocecal junction 4 3 750

Histological typeAdenocarcinoma 99 48 485 309 0079Nonadenocarcinoma 3 3 1000

Tumor differentiationWell 4 1 250

132 0518Moderately 82 44 537Poorly 7 4 571Insufficient data 9 2

Serum CEA level (ngmL)ge15 11 6 545

030 0581lt15 59 25 424Insufficient data 32 20

Lymph node metastasisPositive 34 22 647 441 0036lowastNegative 68 29 426

Distant metastasisPositive 19 17 895 1455 0001lowastNegative 83 34 410

TNM stageStages I II 63 27 429 336 0067Stages III IV 39 24 615

Overall survivalSurvival 61 24 393 689 0009lowastDeath 41 27 659

lowast119875 lt 005

distant metastasis (119875 = 0001) and TNM stage (119875 = 0001)showed a significant correlation with the overall survivalrate of 102 CRC patients Furthermore multivariate analysisconfirmed that high SHIP2 expression (119875 = 0040) anddistant metastasis (119875 = 0001) were two independentprognostic factors for CRC (Table 2) Kaplan-Meier survivalcurves demonstrated that CRC patients with high SHIP2expression and positive distant metastasis encountered asignificantly poorer survival time (Figure 3)

4 Discussion

In insulin signaling SHIP2 plays a substantial role in thenegative regulation of insulin sensitivity [14] SHIP2 hydro-lyzes the PI3K product PIP3 to PIP2 and inhibits theperformance of PI3KAkt signaling simultaneously [25]In turn SHIP2 suppresses cell growth through regulatingintracellular insulin sensitivity On the other hand SHIP2also regulates cell adhesion pathways which take great effectsin the movement of cells Considering the fact that theSHIP2 may function diversely in adhesion signaling bycomparing to its role in insulin signaling it is highly possiblethat abnormal expression of SHIP2 in some pathologicalconditions may impart its capability on cancer developmentfrom insulin regulation in normal physiological situations[12] Accordingly a review by Suwa et al states that SHIP2shows both prooncogenic effects and anticancer effects forSHIP2 function may differ based on interactive and imbal-ancedmodulation by upstream stimulators including insulinEGF and IGF-I and downstream molecules including Akt2tyrosine kinase receptor and focal adhesion adaptor proteins[26] For CRC a great number of studies pointed the crucialrelationships in the expression of adhesion proteins andcatenin pathway with CRC development [27ndash29] the exactrole of SHIP2 in CRC is barely explored

In this study the qPCR result showed that the SHIP2mRNA expressions in CRC tissues were higher than those innormal noncancerous tissues Furthermore TMA with CRCsamples was constructed and IHC analysis was performed toconfirm that higher SHIP2 protein expression was detectedin CRC samples than that in matched noncancerous samplesThe data elucidated the oncogenic role of SHIP2 in tumorige-nesis in CRC and were consistent with previous studies thatindicated high expression of SHIP2 in cancer tissues [19 20]Besides high SHIP2 expression in CRC was correlated withseveral clinical attributes including lymph node metastasisdistant metastasis and overall survival The results showingthe prooncogenic effect of SHIP2 were in accord with ourlatest study concerning SHIP2 expression in lung cancer[23] Similarly a number of mechanical speculations havebeen raised to explore the characteristics of SHIP2 in cancerdevelopment SHIP2 may regulate cell spreading and inducegeneration of local pools of PIP2 which play critical partsin the actin remodeling involved in the movement of cells[30] SHIP2 may perform as a scaffolding protein to promotespecific interaction with substantial cytoskeleton regulatorsthereby subsequently controlling actin remodeling [26 31]To summarize SHIP2 is believed to modulate the ability ofcells to migrate and attach via maintenance and remodelingof the actin cytoskeleton Hence the loss of focal adhesionleading to inhibitory spreading ability indicates defective celllocomotion which in turn regulates cancer cell migrationand invasion during metastasis Our data of the associationsbetween SHIP2 expression and clinicopathologic items inCRC in this present study were also in agreement with theabove reports concerning the prooncogenic effects of SHIP2

In addition high SHIP2 protein expression lymph nodemetastasis distant metastasis and TNM stage were revealedto be correlated with overall survival of CRC patients by

Disease Markers 5

Table 2 Univariate and multivariate analyses of prognostic factors in 102 CRC patients for overall survival

Univariate analysis Multivariate analysisHR 119875 gt |119911| 95 CI HR 119875 gt |119911| 95 CI

SHIP2 expressionHigh versus low 220 0017lowast 1151ndash4189 205 0040lowast 1031ndash3820

GenderMale versus female 144 0281 0744ndash2774

Age (years)ge60 versus lt60 083 0562 0439ndash1565

Tumor size (cm)ge5 versus lt5 129 0410 0701ndash2388

Tumor locationColon versus rectum versus ileocecal junction 068 0354 0306ndash1528

Histological typeAdenocarcinoma versus nonadenocarcinoma 105 0960 0145ndash7662

Tumor differentiationWell and moderately versus poorly 150 0413 0569ndash3943

Serum CEA level (ngmL)ge15 versus lt15 233 0057 0976ndash5573

Lymph node metastasisPositive versus negative 291 0001lowast 1573ndash5408 126 0668 0443ndash3568

Distant metastasisPositive versus negative 706 0001lowast 3687ndash13521 509 0001lowast 2034ndash12736

TNM stageStages I-II versus stages III-IV 025 0001lowast 0129ndash0466 072 0638 0188ndash2789

lowast119875 lt 005

10

08

06

04

02

00

000 10000 120004000 6000 80002000

Cum

surv

ival

SHIP2 expression

High expressionLow expression

High expression-censoredLow expression-censored

Analysis time (months)

(a) Kaplan-Meier survival estimates by SHIP2 expression

10

08

06

04

02

00

000 10000 120004000 6000 80002000

Cum

surv

ival

Analysis time (months)

Distant metastasisNegative distant metastasis

Positive distant metastasis Positive distant metastasis-

Negative distant metastasis-censored

censored(b) Kaplan-Meier survival estimates by distant metastasis

Figure 3 Survival analysis of colorectal cancer (CRC) patients by Kaplan-Meier method (a) Overall survival rate in patients with high SHIP2protein expression (green line) was significantly lower than that in patients with low SHIP2 expression (blue line) (b) Overall survival rate inpatients with positive distant metastasis (green line) was significantly lower than that in patients with negative distant metastasis (blue line)

6 Disease Markers

univariate analysis while high SHIP2 expression and distantmetastasis were further authenticated as independent predic-tors for unfavorable overall survival of CRC by multivariateanalysis Kaplan-Meier analysis finally demonstrated thatthe life span of patients with high SHIP2 expression waslonger than that of patients with negative expression Severalstudies including two researches of ourselves support ourdata announcing the prognostic role of SHIP2 in cancer[19 20 23 24] Hence it is rational to presume that SHIP2plays oncogenic role dominantly in CRC based on thispresent research Detailed studies of PI3KAkt signaling andadhesion protein interacting with SHIP2 in various cell typesor tissues are necessary to clarify the potential role of SHIP2in procarcinogenesis

In conclusion we identified for the first time that ahigher expression of SHIP2 in CRC tissues compared to thenoncancerous tissues and SHIP2 might play an essential roleas a prognostic marker of survival in patients with CRC Ourstudy is helpful in understanding the characteristics of SHIP2in CRC development

Ethical Approval

The study protocol was approved by the Ethics Committee ofNanjing Medical University and each local hospital Writteninformed consent was acquired from all of the patients thatwere enrolled in this paper

Conflict of Interests

The authors declare no conflict of interests

Authorsrsquo Contribution

Jun Wang and Xuefeng Gu conceived and designed theexperiments Yaoguang Ding Yajing Weng Huihui Ni WeiZhang Feng Jin Ning Xu Jiang Li and Liang Qiu performedthe experiments Ju Yang Maoying Fu Weifei Fan XiaolinPu Zhijun Ge and Feng Zhan collected the data Ju YangMaoying Fu Jiang Li and Liang Qiu analyzed the dataXuefeng Gu wrote the paper Jun Wang and Xuefeng Gusupervised the study Ju Yang and Maoying Fu contributedequally to this work

Acknowledgments

This work was supported by a grant from Natural ScienceFoundation of Jiangsu Province (Grant no BK20130895)and Natural Science Research Program for Colleges andUniversities in Jiangsu Province (Grant no 11KJB320006)

References

[1] C-W Huang H-L Tsai Y-T Chen et al ldquoThe prognostic val-ues of EGFR expression and KRAS mutation in patients withsynchronous or metachronous metastatic colorectal cancerrdquoBMC Cancer vol 13 article 599 2013

[2] M Yasunaga and YMatsumura ldquoRole of SLC6A6 in promotingthe survival and multidrug resistance of colorectal cancerrdquoScientific Reports vol 4 article 4852 2014

[3] T-W Ke H-L Hsu Y-H Wu W T-L Chen Y-W Chengand C-W Cheng ldquoMicroRNA-224 suppresses colorectal cancercell migration by targeting Cdc42rdquo Disease Markers vol 2014Article ID 617150 11 pages 2014

[4] F-T Duan F Qian K Fang K-Y Lin W-T Wang and Y-QChen ldquomiR-133b a muscle-specific microRNA is a novel prog-nosticmarker that participates in the progression of human col-orectal cancer via regulation of CXCR4 expressionrdquo MolecularCancer vol 12 no 1 article 164 2013

[5] S-C ZhangW JinH Liu et al ldquoRPSAgenemutants associatedwith risk of colorectal cancer among the Chinese populationrdquoAsian Pacific Journal of Cancer Prevention vol 14 no 12 pp7127ndash7131 2013

[6] MC SWong J Y L ChingHHHirai et al ldquoPerceived obsta-cles of colorectal cancer screening and their associated factorsamong 10078 Chinese participantsrdquo PLoS ONE vol 8 no 7Article ID e70209 2013

[7] X Lin Z Yi J Diao et al ldquoShaoYao decoction amelioratescolitis-associated colorectal cancer by downregulating proin-flammatory cytokines and promoting epithelial-mesenchymaltransitionrdquo Journal of Translational Medicine vol 12 no 1article 105 2014

[8] W S Atkin R Edwards I Kralj-Hans et al ldquoOnce-only flexiblesigmoidoscopy screening in prevention of colorectal cancer amulticentre randomised controlled trialrdquo The Lancet vol 375no 9726 pp 1624ndash1633 2010

[9] Y-F Yang X-Y ZhangMYang et al ldquoPrognostic role of nucle-ophosmin in colorectal carcinomasrdquo Asian Pacific Journal ofCancer Prevention vol 15 no 5 pp 2021ndash2026 2014

[10] Y Li JWei C Xu Z Zhao and T You ldquoPrognostic significanceof cyclin D1 expression in colorectal cancer a meta-analysisof observational studiesrdquo PLoS ONE vol 9 no 4 Article IDe94508 2014

[11] W-H Leung and S Bolland ldquoThe inositol 51015840-phosphataseSHIP-2 negatively regulates IgE-induced mast cell degranula-tion and cytokine productionrdquoThe Journal of Immunology vol179 no 1 pp 95ndash102 2007

[12] N K Prasad M Tandon S Badve P W Snyder and H Nak-shatri ldquoPhosphoinositol phosphatase SHIP2 promotes cancerdevelopment and metastasis coupled with alterations in EGFreceptor turnoverrdquo Carcinogenesis vol 29 no 1 pp 25ndash342008

[13] A Koch AMancini O El Bounkari and T Tamura ldquoThe SH2-domian-containing inositol 5-phosphatase (SHIP)-2 binds to c-Met directly via tyrosine residue 1356 and involves hepatocytegrowth factor (HGF)-induced lamellipodium formation cellscattering and cell spreadingrdquo Oncogene vol 24 no 21 pp3436ndash3447 2005

[14] S Sumie T Kawaguchi M Komuta et al ldquoSignificance of glu-cose intolerance and SHIP2 expression in hepatocellular carci-noma patients with HCV infectionrdquo Oncology Reports vol 18no 3 pp 545ndash552 2007

[15] X B Xu J Qin andW Y Liu ldquoCurcumin inhibits the invasionof thyroid cancer cells via down-regulation of PI3KAkt signal-ing pathwayrdquo Gene vol 546 no 2 pp 226ndash232 2014

[16] J J Wheler S L Moulder A Naing et al ldquoAnastrozole andeverolimus in advanced gynecologic and breast malignanciesactivity and molecular alterations in the PI3KAKTmTORpathwayrdquo Oncotarget vol 5 no 10 pp 3029ndash3038 2014

Disease Markers 7

[17] S Giuriato D Blero B Robaye C Bruyns B Payrastre andC Erneux ldquoSHIP2 overexpression strongly reduces the prolif-eration rate of K562 erythroleukemia cell linerdquo Biochemical andBiophysical Research Communications vol 296 no 1 pp 106ndash110 2002

[18] T Sasaoka K Kikuchi TWada et al ldquoDual role of SRC homol-ogy domain 2-containing inositol phosphatase 2 in the reg-ulation of platelet-derived growth factor and insulin-likegrowth factor I signaling in rat vascular smooth muscle cellsrdquoEndocrinology vol 144 no 9 pp 4204ndash4214 2003

[19] N K Prasad M Tandon A Handa et al ldquoHigh expressionof obesity-linked phosphatase SHIP2 in invasive breast cancercorrelates with reduced disease-free survivalrdquo Tumor Biologyvol 29 no 5 pp 330ndash341 2008

[20] X Zhou Y Liu andG Tan ldquoPrognostic value of elevated SHIP2expression in laryngeal squamous cell carcinomardquo Archives ofMedical Research vol 42 no 7 pp 589ndash595 2011

[21] N K Prasad ldquoSHIP2 phosphoinositol phosphatase positivelyregulates EGFR-Akt pathway CXCR4 expression and cellmigration in MDA-MB-231 breast cancer cellsrdquo InternationalJournal of Oncology vol 34 no 1 pp 97ndash105 2009

[22] F ObroceaM Sajin E CMarinescu andD Stoica ldquoColorectalcancer and the 7th revision of the TNM staging system reviewof changes and suggestions for uniform pathologic reportingrdquoRomanian Journal of Morphology and Embryology vol 52 no2 pp 537ndash544 2011

[23] M Fu W Fan X Pu et al ldquoElevated expression of SHIP2 cor-relates with poor prognosis in non-small cell lung cancerrdquoInternational Journal of Clinical and Experimental Pathologyvol 6 no 10 pp 2185ndash2191 2013

[24] M Fu X Gu H Ni et al ldquoHigh expression of inositol pol-yphosphate phosphatase-like 1 associates with unfavorablesurvival in hepatocellular carcinomardquo International Journal ofClinical and Experimental Pathology vol 6 no 11 pp 2515ndash2522 2013

[25] V Taylor M Wong C Brandts et al ldquo51015840 Phospholipid phos-phatase SHIP-2 causes protein kinase B inactivation and cellcycle arrest in glioblastoma cellsrdquoMolecular amp Cellular Biologyvol 20 no 18 pp 6860ndash6871 2000

[26] A Suwa T Kurama and T Shimokawa ldquoSHIP2 and its involve-ment in various diseasesrdquo Expert Opinion on Therapeutic Tar-gets vol 14 no 7 pp 727ndash737 2010

[27] B Ducarouge M Pelissier-Rota M Laine et al ldquoCRF2 sig-naling is a novel regulator of cellular adhesion and migrationin colorectal cancer cellsrdquo PLoS ONE vol 8 no 11 Article IDe79335 2013

[28] N Vassos T Rau S Merkel et al ldquoPrognostic value of 1205731integrin expression in colorectal liver metastasesrdquo InternationalJournal of Clinical and Experimental Pathology vol 7 no 1 pp288ndash300 2014

[29] J F Groulx V Giroux M Beausejour et al ldquoIntegrin alpha6Asplice variant regulates proliferation and the Wntbeta-cateninpathway in human colorectal cancer cellsrdquo Carcinogenesis vol35 no 6 pp 1217ndash1227 2014

[30] J M Haugh F Codazzi M Teruel and T Meyer ldquoSpatialsensing in fibroblasts mediated by 31015840 phosphoinositidesrdquo TheJournal of Cell Biology vol 151 no 6 pp 1269ndash1280 2000

[31] A Y Alexandrova ldquoEvolution of cell interactions with extracel-lular matrix during carcinogenesisrdquo Biochemistry vol 73 no 7pp 733ndash741 2008

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2 Disease Markers

the three major risks that contribute to the CRC develop-ment [7] Despite the development of combined therapeuticmodalities for CRC treatment including surgical operationand combination of chemotherapy and adjuvant therapythe overall prognosis of CRC remains unsatisfactory andthe 5-year survival rate of CRC patients with metastasis isstill under 10 [8 9] It is valuable and critical to discovermolecular predictive markers for the prognosis which wouldoptimize the selection of therapeutic strategies and furtherimprove patientsrsquo survival for CRC [10]

SH2-containing inositol 51015840-phosphatase 2 (SHIP2) be-longs to the phosphoinositol phosphatases family which playsimportant role in modulating signaling pathways relevantto both diabetes and cancer and generally regulates insulinsignaling cytoskeleton remodeling and receptor endocytosis[11ndash13] As one of the important lipid phosphatases that actdownstream of phosphoinositide-31015840 kinase (PI3K) SHIP2converts phosphatidylinositol (345)-trisphosphate (PIP3)into phosphatidylinositol (34)-biphosphate (PIP2) and sub-sequently reduces the activation of PI3KAkt signaling cas-cade which exerts energetic function in tumor developmentand progression [14ndash16] Under this circumstance SHIP2is supposed to show anticancer effects and several studiessupport this presumption [17 18] However on the contraryseveral studies elucidate the prooncogenic function of SHIP2in certain type of cancer For instance high SHIP2 expressionenhances cancer cell proliferation while inhibition of SHIP2suppresses cancer growth in vitro and distant metastasesin vivo [12] Similarly SHIP2 performs oncogenically andhigh expression of SHIP2 indicates poor survival of breastcancer and laryngeal squamous cell carcinoma [19 20]SHIP2 also promotes cancer cell proliferation and metastasisby preventing epidermal growth factor receptor (EGFR)turnover and enhancing EGF-induced Akt activation [12 21]In light of the dual characteristics of SHIP2 the association ofSHIP2 expression and cancer development still seems vagueand little is known about the role of SHIP2 in CRC especiallythe prognostic significance

In this present paper we detected SHIP2 expression ina number of CRC samples and analyzed the associationsbetween SHIP2 expression and clinicopathologic items inCRC patients Finally the prognostic significance of SHIP2was evaluated in 102 CRC cases

2 Materials and Methods

21 Patients and Tissue Samples A total of 15 fresh CRCtissues and matched tumor-adjacent noncancerous tissueswere collected from the tissue bank of the Affiliated Hospitalof Nantong University to perform quantitative real-timepolymerase chain reaction (qPCR) test 102 formalin-fixedparaffin-embedded CRC tissues and corresponding tumor-adjacent normal tissues were also enrolled in this presentstudy from the Affiliated Hospital of Nantong Universityfrom 2002 to 2005 to execute immunohistochemistry (IHC)analysis Other pieces of clinicopathological information of102 CRC cases such as gender age tumor size tumorlocation histological type tumor differentiation serum CEA

level metastasis status TNM stage and overall survival werecollected from each patientrsquos medical records simultaneouslyClinical staging was performed according to the latest revi-sion of American Joint Committee on CancerInternationalUnion Against Cancer TNM staging system [22]

22 Detection of the SHIP2 Expression by One-Step qPCR TestTotal RNA was extracted from 15 cases of the frozen CRCtissues and thematched noncancerous tissues using theTrizolreagent (Invitrogen Carlsbad CA USA) according to themanufacturerrsquos guidelines Total RNA extraction and one-step qPCR analysis were performed as previously described[23] The primers for SHIP2 were as follows forward primer51015840-TCG TCA CCA GCG ACC ATT C-31015840 and reverse primer51015840-AGC CCT TTC TTG GAG ATG AAC TG-31015840 The glycer-aldehyde 3-phosphate dehydrogenase (GAPDH)mRNA levelwas used to standardize the measurements of SHIP2 and theprimers for GAPDHwere as follows forward primer 51015840-TGCACCACCAACTGCTTAGC-31015840 and reverse primer 31015840-GGCATG GAC TGT GGT CAT GAG-51015840

23 Detection of the SHIP2 Expression by IHC AnalysisTissue microarray (TMA) was produced by Alenabio Biotech(Xirsquoan China) Core tissue biopsies (2mm in diameter) werecollected from individual paraffin-embedded CRC sectionsand arranged in the recipient paraffin blocks IHC anal-ysis was performed as previously described [24] Tissuesections were incubated with polyclonal goat anti-SHIP2antibody (Santa Cruz Biotechnology Santa Cruz CA USA)in TBS containing 1 bovine serum albumin for 1 hourThe secondary antibody used was horseradish peroxidase-conjugated anti-goat antibody (Dako Cytomation Carpinte-ria CA USA)

SHIP2 immunostaining was scored by two independentpathologists according to intensity and percentage of positivecells simultaneously Staining intensity was scored as follows0 (negative) 1 (weakly positive) 2 (moderately positive) and3 (strongly positive) The percentage of SHIP2-positive cellswas also scored according to four categories as follows 1 wasgiven for 0 to 10 2 for 11 to 50 3 for 51 to 80and 4 for 81 to 100 The product of the intensity andpercentage scores was used as the final staining score Thedegree of SHIP2 staining was quantified using a two-levelgrading system as follows samples with a sum score lt 4 wereconsidered as low SHIP2 expression while those with a sumscore ge 4 were considered as high SHIP2 expression

24 Statistical Analysis Statistical analyses were performedby using SPSS 160 (SPSS Inc Chicago IL USA) and STATA120 (Stata Corporation College Station TX USA) Theexpression of SHIP2 mRNA in fresh frozen CRC tissuesand matched tumor-adjacent normal tissues normalized toGAPDH was analyzed with the Wilcoxon nonparametricsigned-rank test The relationship between SHIP2 proteinexpression and clinicopathological factors was evaluated byChi-square test Univariate and multivariate analyses wereperformed using Coxrsquos proportional hazards regressionmod-els Survival curves were calculated using the Kaplan-Meier

Disease Markers 3

10

8

6

4

2

0CRC Noncancerous

SHIP

2 ex

pres

sion

norm

aliz

ed b

y G

APD

Hlowast

P = 0004

Figure 1One-step quantitative real-time polymerase chain reaction(qPCR) test was employed to evaluate the expression of SHIP2mRNA in colorectal cancer (CRC) and tumor-adjacent noncancer-ous tissues When normalized to GAPDH the expression of SHIP2mRNA in CRC tissues (511 plusmn 0419) was significantly higher thanthat in matched noncancerous tissues (348 plusmn 0295) (119875 = 0004)

method For all tests the significance level for statisticalanalysis was set at 119875 lt 005

3 Results

31 Summarization of the Characteristics of 102 CRC PatientsA total of 102 CRC cases were enrolled from 63 men and 39women in this present study The mean age of all patients atthe time of diagnosis was 6308 plusmn 10852 years The tumordiameters of 44 cases were ge5 cm and of 58 cases were lt5 cmThe tumors of 87 cases were located in colon 11 cases in rec-tum and 4 cases in ileocecal junction The histological typeof tumor in 99 cases was adenocarcinoma whereas the other3 cases were identified as nonadenocarcinoma Four patientssuffered from a well-differentiated tumor 82 had moderatetumor differentiation and 7 had poor tumor differentiationThere were 11 cases with serum CEA level ge 15 ngmL and59 cases with serum CEA level lt 15 ngmL Positive lymphnode metastasis was noticed in 34 patients while distantmetastasis was observed in 19 patients According to TNMstaging system 63 patients were in stages I-II and 39 patientswere in stages III-IV Of all the 102 cases 61 patients survivedwhile 41 patients died

32 Evaluation of SHIP2 mRNA Expression by qPCR TestTotal RNA was extracted from 15 CRC tissues and matchednoncancerous tissues and subsequently subjected to one-step qPCR for the evaluation of the expression of SHIP2mRNA When normalized to GAPDH the means of SHIP2mRNA expression in CRC tissues and the correspondingnoncancerous tissues were 511 plusmn 0419 and 348 plusmn 0295respectively (119905 = 3174 119875 = 0004) SHIP2 mRNA expres-sion averaged 147-fold higher in CRC samples than that innoncancerous tissue samples (Figure 1)

A1

B1

C1

D1

Figure 2 Representative images of SHIP2 protein expression incolorectal cancer (CRC) tissues and corresponding noncanceroustissues with tissue microarray (TMA) A1 A2 and A3 highimmunohistochemical (IHC) staining of SHIP2 protein expressionin CRC tissue samples B1 B2 and B3 low IHC staining of SHIP2protein expression in CRC tissue samples C1 C2 and C3 highIHC staining of SHIP2 protein expression in noncancerous tissuesamples D1 D2 and D3 low IHC staining of SHIP2 proteinexpression in noncancerous tissue samples Original magnificationtimes40 in A1 B1 C1 and D1 times200 in A2 B2 C2 and D2 times400 in A3B3 C3 and D3

33 Evaluation of SHIP2 Protein Expression by IHC AnalysisIHC analysis was further performed to detect the SHIP2protein expression in CRC High SHIP2 expression wasdetected in 51 (500) out of 102 CRC tissues and in 24(235) out of 102 tumor-adjacent noncancerous tissues Theresults exhibited significant difference (119875 lt 005) and wereconsistent with results of SHIP2 mRNA expression in qPCRtest Positive staining was mainly localized in the cytoplasmof CRC cells Typical IHC staining patterns for SHIP2 in CRCare shown in Figure 2

34 Relationships between SHIP2 Protein Expression andClinicopathological Items The relationships between highSHIP2 protein expression and selected clinicopathologicalitems were shown in Table 1 High SHIP2 protein expressionwas significantly related to lymph node metastasis (119875 =0036) distant metastasis (119875 = 0001) and overall survival(119875 = 0009) (Table 1)

35 Survival Analysis In order to evaluate the prognosticrole of SHIP2 survival analyses were subsequently executedIn univariate analysis 4 items including high SHIP2 proteinexpression (119875 = 0017) lymph node metastasis (119875 = 0001)

4 Disease Markers

Table 1 The relationship between SHIP2 expression and clinicalattributes of 102 CRC patients

Groups Number SHIP21205942119875 value

+ Gender

Male 63 31 492 004 0839Female 39 20 513

Age (years)ge60 68 34 500 000 1000lt60 34 17 500

Tumor size (cm)ge5 44 21 477 016 0689lt5 58 30 517

Tumor locationColon 87 43 494

110 0576Rectum 11 5 455Ileocecal junction 4 3 750

Histological typeAdenocarcinoma 99 48 485 309 0079Nonadenocarcinoma 3 3 1000

Tumor differentiationWell 4 1 250

132 0518Moderately 82 44 537Poorly 7 4 571Insufficient data 9 2

Serum CEA level (ngmL)ge15 11 6 545

030 0581lt15 59 25 424Insufficient data 32 20

Lymph node metastasisPositive 34 22 647 441 0036lowastNegative 68 29 426

Distant metastasisPositive 19 17 895 1455 0001lowastNegative 83 34 410

TNM stageStages I II 63 27 429 336 0067Stages III IV 39 24 615

Overall survivalSurvival 61 24 393 689 0009lowastDeath 41 27 659

lowast119875 lt 005

distant metastasis (119875 = 0001) and TNM stage (119875 = 0001)showed a significant correlation with the overall survivalrate of 102 CRC patients Furthermore multivariate analysisconfirmed that high SHIP2 expression (119875 = 0040) anddistant metastasis (119875 = 0001) were two independentprognostic factors for CRC (Table 2) Kaplan-Meier survivalcurves demonstrated that CRC patients with high SHIP2expression and positive distant metastasis encountered asignificantly poorer survival time (Figure 3)

4 Discussion

In insulin signaling SHIP2 plays a substantial role in thenegative regulation of insulin sensitivity [14] SHIP2 hydro-lyzes the PI3K product PIP3 to PIP2 and inhibits theperformance of PI3KAkt signaling simultaneously [25]In turn SHIP2 suppresses cell growth through regulatingintracellular insulin sensitivity On the other hand SHIP2also regulates cell adhesion pathways which take great effectsin the movement of cells Considering the fact that theSHIP2 may function diversely in adhesion signaling bycomparing to its role in insulin signaling it is highly possiblethat abnormal expression of SHIP2 in some pathologicalconditions may impart its capability on cancer developmentfrom insulin regulation in normal physiological situations[12] Accordingly a review by Suwa et al states that SHIP2shows both prooncogenic effects and anticancer effects forSHIP2 function may differ based on interactive and imbal-ancedmodulation by upstream stimulators including insulinEGF and IGF-I and downstream molecules including Akt2tyrosine kinase receptor and focal adhesion adaptor proteins[26] For CRC a great number of studies pointed the crucialrelationships in the expression of adhesion proteins andcatenin pathway with CRC development [27ndash29] the exactrole of SHIP2 in CRC is barely explored

In this study the qPCR result showed that the SHIP2mRNA expressions in CRC tissues were higher than those innormal noncancerous tissues Furthermore TMA with CRCsamples was constructed and IHC analysis was performed toconfirm that higher SHIP2 protein expression was detectedin CRC samples than that in matched noncancerous samplesThe data elucidated the oncogenic role of SHIP2 in tumorige-nesis in CRC and were consistent with previous studies thatindicated high expression of SHIP2 in cancer tissues [19 20]Besides high SHIP2 expression in CRC was correlated withseveral clinical attributes including lymph node metastasisdistant metastasis and overall survival The results showingthe prooncogenic effect of SHIP2 were in accord with ourlatest study concerning SHIP2 expression in lung cancer[23] Similarly a number of mechanical speculations havebeen raised to explore the characteristics of SHIP2 in cancerdevelopment SHIP2 may regulate cell spreading and inducegeneration of local pools of PIP2 which play critical partsin the actin remodeling involved in the movement of cells[30] SHIP2 may perform as a scaffolding protein to promotespecific interaction with substantial cytoskeleton regulatorsthereby subsequently controlling actin remodeling [26 31]To summarize SHIP2 is believed to modulate the ability ofcells to migrate and attach via maintenance and remodelingof the actin cytoskeleton Hence the loss of focal adhesionleading to inhibitory spreading ability indicates defective celllocomotion which in turn regulates cancer cell migrationand invasion during metastasis Our data of the associationsbetween SHIP2 expression and clinicopathologic items inCRC in this present study were also in agreement with theabove reports concerning the prooncogenic effects of SHIP2

In addition high SHIP2 protein expression lymph nodemetastasis distant metastasis and TNM stage were revealedto be correlated with overall survival of CRC patients by

Disease Markers 5

Table 2 Univariate and multivariate analyses of prognostic factors in 102 CRC patients for overall survival

Univariate analysis Multivariate analysisHR 119875 gt |119911| 95 CI HR 119875 gt |119911| 95 CI

SHIP2 expressionHigh versus low 220 0017lowast 1151ndash4189 205 0040lowast 1031ndash3820

GenderMale versus female 144 0281 0744ndash2774

Age (years)ge60 versus lt60 083 0562 0439ndash1565

Tumor size (cm)ge5 versus lt5 129 0410 0701ndash2388

Tumor locationColon versus rectum versus ileocecal junction 068 0354 0306ndash1528

Histological typeAdenocarcinoma versus nonadenocarcinoma 105 0960 0145ndash7662

Tumor differentiationWell and moderately versus poorly 150 0413 0569ndash3943

Serum CEA level (ngmL)ge15 versus lt15 233 0057 0976ndash5573

Lymph node metastasisPositive versus negative 291 0001lowast 1573ndash5408 126 0668 0443ndash3568

Distant metastasisPositive versus negative 706 0001lowast 3687ndash13521 509 0001lowast 2034ndash12736

TNM stageStages I-II versus stages III-IV 025 0001lowast 0129ndash0466 072 0638 0188ndash2789

lowast119875 lt 005

10

08

06

04

02

00

000 10000 120004000 6000 80002000

Cum

surv

ival

SHIP2 expression

High expressionLow expression

High expression-censoredLow expression-censored

Analysis time (months)

(a) Kaplan-Meier survival estimates by SHIP2 expression

10

08

06

04

02

00

000 10000 120004000 6000 80002000

Cum

surv

ival

Analysis time (months)

Distant metastasisNegative distant metastasis

Positive distant metastasis Positive distant metastasis-

Negative distant metastasis-censored

censored(b) Kaplan-Meier survival estimates by distant metastasis

Figure 3 Survival analysis of colorectal cancer (CRC) patients by Kaplan-Meier method (a) Overall survival rate in patients with high SHIP2protein expression (green line) was significantly lower than that in patients with low SHIP2 expression (blue line) (b) Overall survival rate inpatients with positive distant metastasis (green line) was significantly lower than that in patients with negative distant metastasis (blue line)

6 Disease Markers

univariate analysis while high SHIP2 expression and distantmetastasis were further authenticated as independent predic-tors for unfavorable overall survival of CRC by multivariateanalysis Kaplan-Meier analysis finally demonstrated thatthe life span of patients with high SHIP2 expression waslonger than that of patients with negative expression Severalstudies including two researches of ourselves support ourdata announcing the prognostic role of SHIP2 in cancer[19 20 23 24] Hence it is rational to presume that SHIP2plays oncogenic role dominantly in CRC based on thispresent research Detailed studies of PI3KAkt signaling andadhesion protein interacting with SHIP2 in various cell typesor tissues are necessary to clarify the potential role of SHIP2in procarcinogenesis

In conclusion we identified for the first time that ahigher expression of SHIP2 in CRC tissues compared to thenoncancerous tissues and SHIP2 might play an essential roleas a prognostic marker of survival in patients with CRC Ourstudy is helpful in understanding the characteristics of SHIP2in CRC development

Ethical Approval

The study protocol was approved by the Ethics Committee ofNanjing Medical University and each local hospital Writteninformed consent was acquired from all of the patients thatwere enrolled in this paper

Conflict of Interests

The authors declare no conflict of interests

Authorsrsquo Contribution

Jun Wang and Xuefeng Gu conceived and designed theexperiments Yaoguang Ding Yajing Weng Huihui Ni WeiZhang Feng Jin Ning Xu Jiang Li and Liang Qiu performedthe experiments Ju Yang Maoying Fu Weifei Fan XiaolinPu Zhijun Ge and Feng Zhan collected the data Ju YangMaoying Fu Jiang Li and Liang Qiu analyzed the dataXuefeng Gu wrote the paper Jun Wang and Xuefeng Gusupervised the study Ju Yang and Maoying Fu contributedequally to this work

Acknowledgments

This work was supported by a grant from Natural ScienceFoundation of Jiangsu Province (Grant no BK20130895)and Natural Science Research Program for Colleges andUniversities in Jiangsu Province (Grant no 11KJB320006)

References

[1] C-W Huang H-L Tsai Y-T Chen et al ldquoThe prognostic val-ues of EGFR expression and KRAS mutation in patients withsynchronous or metachronous metastatic colorectal cancerrdquoBMC Cancer vol 13 article 599 2013

[2] M Yasunaga and YMatsumura ldquoRole of SLC6A6 in promotingthe survival and multidrug resistance of colorectal cancerrdquoScientific Reports vol 4 article 4852 2014

[3] T-W Ke H-L Hsu Y-H Wu W T-L Chen Y-W Chengand C-W Cheng ldquoMicroRNA-224 suppresses colorectal cancercell migration by targeting Cdc42rdquo Disease Markers vol 2014Article ID 617150 11 pages 2014

[4] F-T Duan F Qian K Fang K-Y Lin W-T Wang and Y-QChen ldquomiR-133b a muscle-specific microRNA is a novel prog-nosticmarker that participates in the progression of human col-orectal cancer via regulation of CXCR4 expressionrdquo MolecularCancer vol 12 no 1 article 164 2013

[5] S-C ZhangW JinH Liu et al ldquoRPSAgenemutants associatedwith risk of colorectal cancer among the Chinese populationrdquoAsian Pacific Journal of Cancer Prevention vol 14 no 12 pp7127ndash7131 2013

[6] MC SWong J Y L ChingHHHirai et al ldquoPerceived obsta-cles of colorectal cancer screening and their associated factorsamong 10078 Chinese participantsrdquo PLoS ONE vol 8 no 7Article ID e70209 2013

[7] X Lin Z Yi J Diao et al ldquoShaoYao decoction amelioratescolitis-associated colorectal cancer by downregulating proin-flammatory cytokines and promoting epithelial-mesenchymaltransitionrdquo Journal of Translational Medicine vol 12 no 1article 105 2014

[8] W S Atkin R Edwards I Kralj-Hans et al ldquoOnce-only flexiblesigmoidoscopy screening in prevention of colorectal cancer amulticentre randomised controlled trialrdquo The Lancet vol 375no 9726 pp 1624ndash1633 2010

[9] Y-F Yang X-Y ZhangMYang et al ldquoPrognostic role of nucle-ophosmin in colorectal carcinomasrdquo Asian Pacific Journal ofCancer Prevention vol 15 no 5 pp 2021ndash2026 2014

[10] Y Li JWei C Xu Z Zhao and T You ldquoPrognostic significanceof cyclin D1 expression in colorectal cancer a meta-analysisof observational studiesrdquo PLoS ONE vol 9 no 4 Article IDe94508 2014

[11] W-H Leung and S Bolland ldquoThe inositol 51015840-phosphataseSHIP-2 negatively regulates IgE-induced mast cell degranula-tion and cytokine productionrdquoThe Journal of Immunology vol179 no 1 pp 95ndash102 2007

[12] N K Prasad M Tandon S Badve P W Snyder and H Nak-shatri ldquoPhosphoinositol phosphatase SHIP2 promotes cancerdevelopment and metastasis coupled with alterations in EGFreceptor turnoverrdquo Carcinogenesis vol 29 no 1 pp 25ndash342008

[13] A Koch AMancini O El Bounkari and T Tamura ldquoThe SH2-domian-containing inositol 5-phosphatase (SHIP)-2 binds to c-Met directly via tyrosine residue 1356 and involves hepatocytegrowth factor (HGF)-induced lamellipodium formation cellscattering and cell spreadingrdquo Oncogene vol 24 no 21 pp3436ndash3447 2005

[14] S Sumie T Kawaguchi M Komuta et al ldquoSignificance of glu-cose intolerance and SHIP2 expression in hepatocellular carci-noma patients with HCV infectionrdquo Oncology Reports vol 18no 3 pp 545ndash552 2007

[15] X B Xu J Qin andW Y Liu ldquoCurcumin inhibits the invasionof thyroid cancer cells via down-regulation of PI3KAkt signal-ing pathwayrdquo Gene vol 546 no 2 pp 226ndash232 2014

[16] J J Wheler S L Moulder A Naing et al ldquoAnastrozole andeverolimus in advanced gynecologic and breast malignanciesactivity and molecular alterations in the PI3KAKTmTORpathwayrdquo Oncotarget vol 5 no 10 pp 3029ndash3038 2014

Disease Markers 7

[17] S Giuriato D Blero B Robaye C Bruyns B Payrastre andC Erneux ldquoSHIP2 overexpression strongly reduces the prolif-eration rate of K562 erythroleukemia cell linerdquo Biochemical andBiophysical Research Communications vol 296 no 1 pp 106ndash110 2002

[18] T Sasaoka K Kikuchi TWada et al ldquoDual role of SRC homol-ogy domain 2-containing inositol phosphatase 2 in the reg-ulation of platelet-derived growth factor and insulin-likegrowth factor I signaling in rat vascular smooth muscle cellsrdquoEndocrinology vol 144 no 9 pp 4204ndash4214 2003

[19] N K Prasad M Tandon A Handa et al ldquoHigh expressionof obesity-linked phosphatase SHIP2 in invasive breast cancercorrelates with reduced disease-free survivalrdquo Tumor Biologyvol 29 no 5 pp 330ndash341 2008

[20] X Zhou Y Liu andG Tan ldquoPrognostic value of elevated SHIP2expression in laryngeal squamous cell carcinomardquo Archives ofMedical Research vol 42 no 7 pp 589ndash595 2011

[21] N K Prasad ldquoSHIP2 phosphoinositol phosphatase positivelyregulates EGFR-Akt pathway CXCR4 expression and cellmigration in MDA-MB-231 breast cancer cellsrdquo InternationalJournal of Oncology vol 34 no 1 pp 97ndash105 2009

[22] F ObroceaM Sajin E CMarinescu andD Stoica ldquoColorectalcancer and the 7th revision of the TNM staging system reviewof changes and suggestions for uniform pathologic reportingrdquoRomanian Journal of Morphology and Embryology vol 52 no2 pp 537ndash544 2011

[23] M Fu W Fan X Pu et al ldquoElevated expression of SHIP2 cor-relates with poor prognosis in non-small cell lung cancerrdquoInternational Journal of Clinical and Experimental Pathologyvol 6 no 10 pp 2185ndash2191 2013

[24] M Fu X Gu H Ni et al ldquoHigh expression of inositol pol-yphosphate phosphatase-like 1 associates with unfavorablesurvival in hepatocellular carcinomardquo International Journal ofClinical and Experimental Pathology vol 6 no 11 pp 2515ndash2522 2013

[25] V Taylor M Wong C Brandts et al ldquo51015840 Phospholipid phos-phatase SHIP-2 causes protein kinase B inactivation and cellcycle arrest in glioblastoma cellsrdquoMolecular amp Cellular Biologyvol 20 no 18 pp 6860ndash6871 2000

[26] A Suwa T Kurama and T Shimokawa ldquoSHIP2 and its involve-ment in various diseasesrdquo Expert Opinion on Therapeutic Tar-gets vol 14 no 7 pp 727ndash737 2010

[27] B Ducarouge M Pelissier-Rota M Laine et al ldquoCRF2 sig-naling is a novel regulator of cellular adhesion and migrationin colorectal cancer cellsrdquo PLoS ONE vol 8 no 11 Article IDe79335 2013

[28] N Vassos T Rau S Merkel et al ldquoPrognostic value of 1205731integrin expression in colorectal liver metastasesrdquo InternationalJournal of Clinical and Experimental Pathology vol 7 no 1 pp288ndash300 2014

[29] J F Groulx V Giroux M Beausejour et al ldquoIntegrin alpha6Asplice variant regulates proliferation and the Wntbeta-cateninpathway in human colorectal cancer cellsrdquo Carcinogenesis vol35 no 6 pp 1217ndash1227 2014

[30] J M Haugh F Codazzi M Teruel and T Meyer ldquoSpatialsensing in fibroblasts mediated by 31015840 phosphoinositidesrdquo TheJournal of Cell Biology vol 151 no 6 pp 1269ndash1280 2000

[31] A Y Alexandrova ldquoEvolution of cell interactions with extracel-lular matrix during carcinogenesisrdquo Biochemistry vol 73 no 7pp 733ndash741 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

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Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 3

10

8

6

4

2

0CRC Noncancerous

SHIP

2 ex

pres

sion

norm

aliz

ed b

y G

APD

Hlowast

P = 0004

Figure 1One-step quantitative real-time polymerase chain reaction(qPCR) test was employed to evaluate the expression of SHIP2mRNA in colorectal cancer (CRC) and tumor-adjacent noncancer-ous tissues When normalized to GAPDH the expression of SHIP2mRNA in CRC tissues (511 plusmn 0419) was significantly higher thanthat in matched noncancerous tissues (348 plusmn 0295) (119875 = 0004)

method For all tests the significance level for statisticalanalysis was set at 119875 lt 005

3 Results

31 Summarization of the Characteristics of 102 CRC PatientsA total of 102 CRC cases were enrolled from 63 men and 39women in this present study The mean age of all patients atthe time of diagnosis was 6308 plusmn 10852 years The tumordiameters of 44 cases were ge5 cm and of 58 cases were lt5 cmThe tumors of 87 cases were located in colon 11 cases in rec-tum and 4 cases in ileocecal junction The histological typeof tumor in 99 cases was adenocarcinoma whereas the other3 cases were identified as nonadenocarcinoma Four patientssuffered from a well-differentiated tumor 82 had moderatetumor differentiation and 7 had poor tumor differentiationThere were 11 cases with serum CEA level ge 15 ngmL and59 cases with serum CEA level lt 15 ngmL Positive lymphnode metastasis was noticed in 34 patients while distantmetastasis was observed in 19 patients According to TNMstaging system 63 patients were in stages I-II and 39 patientswere in stages III-IV Of all the 102 cases 61 patients survivedwhile 41 patients died

32 Evaluation of SHIP2 mRNA Expression by qPCR TestTotal RNA was extracted from 15 CRC tissues and matchednoncancerous tissues and subsequently subjected to one-step qPCR for the evaluation of the expression of SHIP2mRNA When normalized to GAPDH the means of SHIP2mRNA expression in CRC tissues and the correspondingnoncancerous tissues were 511 plusmn 0419 and 348 plusmn 0295respectively (119905 = 3174 119875 = 0004) SHIP2 mRNA expres-sion averaged 147-fold higher in CRC samples than that innoncancerous tissue samples (Figure 1)

A1

B1

C1

D1

Figure 2 Representative images of SHIP2 protein expression incolorectal cancer (CRC) tissues and corresponding noncanceroustissues with tissue microarray (TMA) A1 A2 and A3 highimmunohistochemical (IHC) staining of SHIP2 protein expressionin CRC tissue samples B1 B2 and B3 low IHC staining of SHIP2protein expression in CRC tissue samples C1 C2 and C3 highIHC staining of SHIP2 protein expression in noncancerous tissuesamples D1 D2 and D3 low IHC staining of SHIP2 proteinexpression in noncancerous tissue samples Original magnificationtimes40 in A1 B1 C1 and D1 times200 in A2 B2 C2 and D2 times400 in A3B3 C3 and D3

33 Evaluation of SHIP2 Protein Expression by IHC AnalysisIHC analysis was further performed to detect the SHIP2protein expression in CRC High SHIP2 expression wasdetected in 51 (500) out of 102 CRC tissues and in 24(235) out of 102 tumor-adjacent noncancerous tissues Theresults exhibited significant difference (119875 lt 005) and wereconsistent with results of SHIP2 mRNA expression in qPCRtest Positive staining was mainly localized in the cytoplasmof CRC cells Typical IHC staining patterns for SHIP2 in CRCare shown in Figure 2

34 Relationships between SHIP2 Protein Expression andClinicopathological Items The relationships between highSHIP2 protein expression and selected clinicopathologicalitems were shown in Table 1 High SHIP2 protein expressionwas significantly related to lymph node metastasis (119875 =0036) distant metastasis (119875 = 0001) and overall survival(119875 = 0009) (Table 1)

35 Survival Analysis In order to evaluate the prognosticrole of SHIP2 survival analyses were subsequently executedIn univariate analysis 4 items including high SHIP2 proteinexpression (119875 = 0017) lymph node metastasis (119875 = 0001)

4 Disease Markers

Table 1 The relationship between SHIP2 expression and clinicalattributes of 102 CRC patients

Groups Number SHIP21205942119875 value

+ Gender

Male 63 31 492 004 0839Female 39 20 513

Age (years)ge60 68 34 500 000 1000lt60 34 17 500

Tumor size (cm)ge5 44 21 477 016 0689lt5 58 30 517

Tumor locationColon 87 43 494

110 0576Rectum 11 5 455Ileocecal junction 4 3 750

Histological typeAdenocarcinoma 99 48 485 309 0079Nonadenocarcinoma 3 3 1000

Tumor differentiationWell 4 1 250

132 0518Moderately 82 44 537Poorly 7 4 571Insufficient data 9 2

Serum CEA level (ngmL)ge15 11 6 545

030 0581lt15 59 25 424Insufficient data 32 20

Lymph node metastasisPositive 34 22 647 441 0036lowastNegative 68 29 426

Distant metastasisPositive 19 17 895 1455 0001lowastNegative 83 34 410

TNM stageStages I II 63 27 429 336 0067Stages III IV 39 24 615

Overall survivalSurvival 61 24 393 689 0009lowastDeath 41 27 659

lowast119875 lt 005

distant metastasis (119875 = 0001) and TNM stage (119875 = 0001)showed a significant correlation with the overall survivalrate of 102 CRC patients Furthermore multivariate analysisconfirmed that high SHIP2 expression (119875 = 0040) anddistant metastasis (119875 = 0001) were two independentprognostic factors for CRC (Table 2) Kaplan-Meier survivalcurves demonstrated that CRC patients with high SHIP2expression and positive distant metastasis encountered asignificantly poorer survival time (Figure 3)

4 Discussion

In insulin signaling SHIP2 plays a substantial role in thenegative regulation of insulin sensitivity [14] SHIP2 hydro-lyzes the PI3K product PIP3 to PIP2 and inhibits theperformance of PI3KAkt signaling simultaneously [25]In turn SHIP2 suppresses cell growth through regulatingintracellular insulin sensitivity On the other hand SHIP2also regulates cell adhesion pathways which take great effectsin the movement of cells Considering the fact that theSHIP2 may function diversely in adhesion signaling bycomparing to its role in insulin signaling it is highly possiblethat abnormal expression of SHIP2 in some pathologicalconditions may impart its capability on cancer developmentfrom insulin regulation in normal physiological situations[12] Accordingly a review by Suwa et al states that SHIP2shows both prooncogenic effects and anticancer effects forSHIP2 function may differ based on interactive and imbal-ancedmodulation by upstream stimulators including insulinEGF and IGF-I and downstream molecules including Akt2tyrosine kinase receptor and focal adhesion adaptor proteins[26] For CRC a great number of studies pointed the crucialrelationships in the expression of adhesion proteins andcatenin pathway with CRC development [27ndash29] the exactrole of SHIP2 in CRC is barely explored

In this study the qPCR result showed that the SHIP2mRNA expressions in CRC tissues were higher than those innormal noncancerous tissues Furthermore TMA with CRCsamples was constructed and IHC analysis was performed toconfirm that higher SHIP2 protein expression was detectedin CRC samples than that in matched noncancerous samplesThe data elucidated the oncogenic role of SHIP2 in tumorige-nesis in CRC and were consistent with previous studies thatindicated high expression of SHIP2 in cancer tissues [19 20]Besides high SHIP2 expression in CRC was correlated withseveral clinical attributes including lymph node metastasisdistant metastasis and overall survival The results showingthe prooncogenic effect of SHIP2 were in accord with ourlatest study concerning SHIP2 expression in lung cancer[23] Similarly a number of mechanical speculations havebeen raised to explore the characteristics of SHIP2 in cancerdevelopment SHIP2 may regulate cell spreading and inducegeneration of local pools of PIP2 which play critical partsin the actin remodeling involved in the movement of cells[30] SHIP2 may perform as a scaffolding protein to promotespecific interaction with substantial cytoskeleton regulatorsthereby subsequently controlling actin remodeling [26 31]To summarize SHIP2 is believed to modulate the ability ofcells to migrate and attach via maintenance and remodelingof the actin cytoskeleton Hence the loss of focal adhesionleading to inhibitory spreading ability indicates defective celllocomotion which in turn regulates cancer cell migrationand invasion during metastasis Our data of the associationsbetween SHIP2 expression and clinicopathologic items inCRC in this present study were also in agreement with theabove reports concerning the prooncogenic effects of SHIP2

In addition high SHIP2 protein expression lymph nodemetastasis distant metastasis and TNM stage were revealedto be correlated with overall survival of CRC patients by

Disease Markers 5

Table 2 Univariate and multivariate analyses of prognostic factors in 102 CRC patients for overall survival

Univariate analysis Multivariate analysisHR 119875 gt |119911| 95 CI HR 119875 gt |119911| 95 CI

SHIP2 expressionHigh versus low 220 0017lowast 1151ndash4189 205 0040lowast 1031ndash3820

GenderMale versus female 144 0281 0744ndash2774

Age (years)ge60 versus lt60 083 0562 0439ndash1565

Tumor size (cm)ge5 versus lt5 129 0410 0701ndash2388

Tumor locationColon versus rectum versus ileocecal junction 068 0354 0306ndash1528

Histological typeAdenocarcinoma versus nonadenocarcinoma 105 0960 0145ndash7662

Tumor differentiationWell and moderately versus poorly 150 0413 0569ndash3943

Serum CEA level (ngmL)ge15 versus lt15 233 0057 0976ndash5573

Lymph node metastasisPositive versus negative 291 0001lowast 1573ndash5408 126 0668 0443ndash3568

Distant metastasisPositive versus negative 706 0001lowast 3687ndash13521 509 0001lowast 2034ndash12736

TNM stageStages I-II versus stages III-IV 025 0001lowast 0129ndash0466 072 0638 0188ndash2789

lowast119875 lt 005

10

08

06

04

02

00

000 10000 120004000 6000 80002000

Cum

surv

ival

SHIP2 expression

High expressionLow expression

High expression-censoredLow expression-censored

Analysis time (months)

(a) Kaplan-Meier survival estimates by SHIP2 expression

10

08

06

04

02

00

000 10000 120004000 6000 80002000

Cum

surv

ival

Analysis time (months)

Distant metastasisNegative distant metastasis

Positive distant metastasis Positive distant metastasis-

Negative distant metastasis-censored

censored(b) Kaplan-Meier survival estimates by distant metastasis

Figure 3 Survival analysis of colorectal cancer (CRC) patients by Kaplan-Meier method (a) Overall survival rate in patients with high SHIP2protein expression (green line) was significantly lower than that in patients with low SHIP2 expression (blue line) (b) Overall survival rate inpatients with positive distant metastasis (green line) was significantly lower than that in patients with negative distant metastasis (blue line)

6 Disease Markers

univariate analysis while high SHIP2 expression and distantmetastasis were further authenticated as independent predic-tors for unfavorable overall survival of CRC by multivariateanalysis Kaplan-Meier analysis finally demonstrated thatthe life span of patients with high SHIP2 expression waslonger than that of patients with negative expression Severalstudies including two researches of ourselves support ourdata announcing the prognostic role of SHIP2 in cancer[19 20 23 24] Hence it is rational to presume that SHIP2plays oncogenic role dominantly in CRC based on thispresent research Detailed studies of PI3KAkt signaling andadhesion protein interacting with SHIP2 in various cell typesor tissues are necessary to clarify the potential role of SHIP2in procarcinogenesis

In conclusion we identified for the first time that ahigher expression of SHIP2 in CRC tissues compared to thenoncancerous tissues and SHIP2 might play an essential roleas a prognostic marker of survival in patients with CRC Ourstudy is helpful in understanding the characteristics of SHIP2in CRC development

Ethical Approval

The study protocol was approved by the Ethics Committee ofNanjing Medical University and each local hospital Writteninformed consent was acquired from all of the patients thatwere enrolled in this paper

Conflict of Interests

The authors declare no conflict of interests

Authorsrsquo Contribution

Jun Wang and Xuefeng Gu conceived and designed theexperiments Yaoguang Ding Yajing Weng Huihui Ni WeiZhang Feng Jin Ning Xu Jiang Li and Liang Qiu performedthe experiments Ju Yang Maoying Fu Weifei Fan XiaolinPu Zhijun Ge and Feng Zhan collected the data Ju YangMaoying Fu Jiang Li and Liang Qiu analyzed the dataXuefeng Gu wrote the paper Jun Wang and Xuefeng Gusupervised the study Ju Yang and Maoying Fu contributedequally to this work

Acknowledgments

This work was supported by a grant from Natural ScienceFoundation of Jiangsu Province (Grant no BK20130895)and Natural Science Research Program for Colleges andUniversities in Jiangsu Province (Grant no 11KJB320006)

References

[1] C-W Huang H-L Tsai Y-T Chen et al ldquoThe prognostic val-ues of EGFR expression and KRAS mutation in patients withsynchronous or metachronous metastatic colorectal cancerrdquoBMC Cancer vol 13 article 599 2013

[2] M Yasunaga and YMatsumura ldquoRole of SLC6A6 in promotingthe survival and multidrug resistance of colorectal cancerrdquoScientific Reports vol 4 article 4852 2014

[3] T-W Ke H-L Hsu Y-H Wu W T-L Chen Y-W Chengand C-W Cheng ldquoMicroRNA-224 suppresses colorectal cancercell migration by targeting Cdc42rdquo Disease Markers vol 2014Article ID 617150 11 pages 2014

[4] F-T Duan F Qian K Fang K-Y Lin W-T Wang and Y-QChen ldquomiR-133b a muscle-specific microRNA is a novel prog-nosticmarker that participates in the progression of human col-orectal cancer via regulation of CXCR4 expressionrdquo MolecularCancer vol 12 no 1 article 164 2013

[5] S-C ZhangW JinH Liu et al ldquoRPSAgenemutants associatedwith risk of colorectal cancer among the Chinese populationrdquoAsian Pacific Journal of Cancer Prevention vol 14 no 12 pp7127ndash7131 2013

[6] MC SWong J Y L ChingHHHirai et al ldquoPerceived obsta-cles of colorectal cancer screening and their associated factorsamong 10078 Chinese participantsrdquo PLoS ONE vol 8 no 7Article ID e70209 2013

[7] X Lin Z Yi J Diao et al ldquoShaoYao decoction amelioratescolitis-associated colorectal cancer by downregulating proin-flammatory cytokines and promoting epithelial-mesenchymaltransitionrdquo Journal of Translational Medicine vol 12 no 1article 105 2014

[8] W S Atkin R Edwards I Kralj-Hans et al ldquoOnce-only flexiblesigmoidoscopy screening in prevention of colorectal cancer amulticentre randomised controlled trialrdquo The Lancet vol 375no 9726 pp 1624ndash1633 2010

[9] Y-F Yang X-Y ZhangMYang et al ldquoPrognostic role of nucle-ophosmin in colorectal carcinomasrdquo Asian Pacific Journal ofCancer Prevention vol 15 no 5 pp 2021ndash2026 2014

[10] Y Li JWei C Xu Z Zhao and T You ldquoPrognostic significanceof cyclin D1 expression in colorectal cancer a meta-analysisof observational studiesrdquo PLoS ONE vol 9 no 4 Article IDe94508 2014

[11] W-H Leung and S Bolland ldquoThe inositol 51015840-phosphataseSHIP-2 negatively regulates IgE-induced mast cell degranula-tion and cytokine productionrdquoThe Journal of Immunology vol179 no 1 pp 95ndash102 2007

[12] N K Prasad M Tandon S Badve P W Snyder and H Nak-shatri ldquoPhosphoinositol phosphatase SHIP2 promotes cancerdevelopment and metastasis coupled with alterations in EGFreceptor turnoverrdquo Carcinogenesis vol 29 no 1 pp 25ndash342008

[13] A Koch AMancini O El Bounkari and T Tamura ldquoThe SH2-domian-containing inositol 5-phosphatase (SHIP)-2 binds to c-Met directly via tyrosine residue 1356 and involves hepatocytegrowth factor (HGF)-induced lamellipodium formation cellscattering and cell spreadingrdquo Oncogene vol 24 no 21 pp3436ndash3447 2005

[14] S Sumie T Kawaguchi M Komuta et al ldquoSignificance of glu-cose intolerance and SHIP2 expression in hepatocellular carci-noma patients with HCV infectionrdquo Oncology Reports vol 18no 3 pp 545ndash552 2007

[15] X B Xu J Qin andW Y Liu ldquoCurcumin inhibits the invasionof thyroid cancer cells via down-regulation of PI3KAkt signal-ing pathwayrdquo Gene vol 546 no 2 pp 226ndash232 2014

[16] J J Wheler S L Moulder A Naing et al ldquoAnastrozole andeverolimus in advanced gynecologic and breast malignanciesactivity and molecular alterations in the PI3KAKTmTORpathwayrdquo Oncotarget vol 5 no 10 pp 3029ndash3038 2014

Disease Markers 7

[17] S Giuriato D Blero B Robaye C Bruyns B Payrastre andC Erneux ldquoSHIP2 overexpression strongly reduces the prolif-eration rate of K562 erythroleukemia cell linerdquo Biochemical andBiophysical Research Communications vol 296 no 1 pp 106ndash110 2002

[18] T Sasaoka K Kikuchi TWada et al ldquoDual role of SRC homol-ogy domain 2-containing inositol phosphatase 2 in the reg-ulation of platelet-derived growth factor and insulin-likegrowth factor I signaling in rat vascular smooth muscle cellsrdquoEndocrinology vol 144 no 9 pp 4204ndash4214 2003

[19] N K Prasad M Tandon A Handa et al ldquoHigh expressionof obesity-linked phosphatase SHIP2 in invasive breast cancercorrelates with reduced disease-free survivalrdquo Tumor Biologyvol 29 no 5 pp 330ndash341 2008

[20] X Zhou Y Liu andG Tan ldquoPrognostic value of elevated SHIP2expression in laryngeal squamous cell carcinomardquo Archives ofMedical Research vol 42 no 7 pp 589ndash595 2011

[21] N K Prasad ldquoSHIP2 phosphoinositol phosphatase positivelyregulates EGFR-Akt pathway CXCR4 expression and cellmigration in MDA-MB-231 breast cancer cellsrdquo InternationalJournal of Oncology vol 34 no 1 pp 97ndash105 2009

[22] F ObroceaM Sajin E CMarinescu andD Stoica ldquoColorectalcancer and the 7th revision of the TNM staging system reviewof changes and suggestions for uniform pathologic reportingrdquoRomanian Journal of Morphology and Embryology vol 52 no2 pp 537ndash544 2011

[23] M Fu W Fan X Pu et al ldquoElevated expression of SHIP2 cor-relates with poor prognosis in non-small cell lung cancerrdquoInternational Journal of Clinical and Experimental Pathologyvol 6 no 10 pp 2185ndash2191 2013

[24] M Fu X Gu H Ni et al ldquoHigh expression of inositol pol-yphosphate phosphatase-like 1 associates with unfavorablesurvival in hepatocellular carcinomardquo International Journal ofClinical and Experimental Pathology vol 6 no 11 pp 2515ndash2522 2013

[25] V Taylor M Wong C Brandts et al ldquo51015840 Phospholipid phos-phatase SHIP-2 causes protein kinase B inactivation and cellcycle arrest in glioblastoma cellsrdquoMolecular amp Cellular Biologyvol 20 no 18 pp 6860ndash6871 2000

[26] A Suwa T Kurama and T Shimokawa ldquoSHIP2 and its involve-ment in various diseasesrdquo Expert Opinion on Therapeutic Tar-gets vol 14 no 7 pp 727ndash737 2010

[27] B Ducarouge M Pelissier-Rota M Laine et al ldquoCRF2 sig-naling is a novel regulator of cellular adhesion and migrationin colorectal cancer cellsrdquo PLoS ONE vol 8 no 11 Article IDe79335 2013

[28] N Vassos T Rau S Merkel et al ldquoPrognostic value of 1205731integrin expression in colorectal liver metastasesrdquo InternationalJournal of Clinical and Experimental Pathology vol 7 no 1 pp288ndash300 2014

[29] J F Groulx V Giroux M Beausejour et al ldquoIntegrin alpha6Asplice variant regulates proliferation and the Wntbeta-cateninpathway in human colorectal cancer cellsrdquo Carcinogenesis vol35 no 6 pp 1217ndash1227 2014

[30] J M Haugh F Codazzi M Teruel and T Meyer ldquoSpatialsensing in fibroblasts mediated by 31015840 phosphoinositidesrdquo TheJournal of Cell Biology vol 151 no 6 pp 1269ndash1280 2000

[31] A Y Alexandrova ldquoEvolution of cell interactions with extracel-lular matrix during carcinogenesisrdquo Biochemistry vol 73 no 7pp 733ndash741 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Disease Markers

Table 1 The relationship between SHIP2 expression and clinicalattributes of 102 CRC patients

Groups Number SHIP21205942119875 value

+ Gender

Male 63 31 492 004 0839Female 39 20 513

Age (years)ge60 68 34 500 000 1000lt60 34 17 500

Tumor size (cm)ge5 44 21 477 016 0689lt5 58 30 517

Tumor locationColon 87 43 494

110 0576Rectum 11 5 455Ileocecal junction 4 3 750

Histological typeAdenocarcinoma 99 48 485 309 0079Nonadenocarcinoma 3 3 1000

Tumor differentiationWell 4 1 250

132 0518Moderately 82 44 537Poorly 7 4 571Insufficient data 9 2

Serum CEA level (ngmL)ge15 11 6 545

030 0581lt15 59 25 424Insufficient data 32 20

Lymph node metastasisPositive 34 22 647 441 0036lowastNegative 68 29 426

Distant metastasisPositive 19 17 895 1455 0001lowastNegative 83 34 410

TNM stageStages I II 63 27 429 336 0067Stages III IV 39 24 615

Overall survivalSurvival 61 24 393 689 0009lowastDeath 41 27 659

lowast119875 lt 005

distant metastasis (119875 = 0001) and TNM stage (119875 = 0001)showed a significant correlation with the overall survivalrate of 102 CRC patients Furthermore multivariate analysisconfirmed that high SHIP2 expression (119875 = 0040) anddistant metastasis (119875 = 0001) were two independentprognostic factors for CRC (Table 2) Kaplan-Meier survivalcurves demonstrated that CRC patients with high SHIP2expression and positive distant metastasis encountered asignificantly poorer survival time (Figure 3)

4 Discussion

In insulin signaling SHIP2 plays a substantial role in thenegative regulation of insulin sensitivity [14] SHIP2 hydro-lyzes the PI3K product PIP3 to PIP2 and inhibits theperformance of PI3KAkt signaling simultaneously [25]In turn SHIP2 suppresses cell growth through regulatingintracellular insulin sensitivity On the other hand SHIP2also regulates cell adhesion pathways which take great effectsin the movement of cells Considering the fact that theSHIP2 may function diversely in adhesion signaling bycomparing to its role in insulin signaling it is highly possiblethat abnormal expression of SHIP2 in some pathologicalconditions may impart its capability on cancer developmentfrom insulin regulation in normal physiological situations[12] Accordingly a review by Suwa et al states that SHIP2shows both prooncogenic effects and anticancer effects forSHIP2 function may differ based on interactive and imbal-ancedmodulation by upstream stimulators including insulinEGF and IGF-I and downstream molecules including Akt2tyrosine kinase receptor and focal adhesion adaptor proteins[26] For CRC a great number of studies pointed the crucialrelationships in the expression of adhesion proteins andcatenin pathway with CRC development [27ndash29] the exactrole of SHIP2 in CRC is barely explored

In this study the qPCR result showed that the SHIP2mRNA expressions in CRC tissues were higher than those innormal noncancerous tissues Furthermore TMA with CRCsamples was constructed and IHC analysis was performed toconfirm that higher SHIP2 protein expression was detectedin CRC samples than that in matched noncancerous samplesThe data elucidated the oncogenic role of SHIP2 in tumorige-nesis in CRC and were consistent with previous studies thatindicated high expression of SHIP2 in cancer tissues [19 20]Besides high SHIP2 expression in CRC was correlated withseveral clinical attributes including lymph node metastasisdistant metastasis and overall survival The results showingthe prooncogenic effect of SHIP2 were in accord with ourlatest study concerning SHIP2 expression in lung cancer[23] Similarly a number of mechanical speculations havebeen raised to explore the characteristics of SHIP2 in cancerdevelopment SHIP2 may regulate cell spreading and inducegeneration of local pools of PIP2 which play critical partsin the actin remodeling involved in the movement of cells[30] SHIP2 may perform as a scaffolding protein to promotespecific interaction with substantial cytoskeleton regulatorsthereby subsequently controlling actin remodeling [26 31]To summarize SHIP2 is believed to modulate the ability ofcells to migrate and attach via maintenance and remodelingof the actin cytoskeleton Hence the loss of focal adhesionleading to inhibitory spreading ability indicates defective celllocomotion which in turn regulates cancer cell migrationand invasion during metastasis Our data of the associationsbetween SHIP2 expression and clinicopathologic items inCRC in this present study were also in agreement with theabove reports concerning the prooncogenic effects of SHIP2

In addition high SHIP2 protein expression lymph nodemetastasis distant metastasis and TNM stage were revealedto be correlated with overall survival of CRC patients by

Disease Markers 5

Table 2 Univariate and multivariate analyses of prognostic factors in 102 CRC patients for overall survival

Univariate analysis Multivariate analysisHR 119875 gt |119911| 95 CI HR 119875 gt |119911| 95 CI

SHIP2 expressionHigh versus low 220 0017lowast 1151ndash4189 205 0040lowast 1031ndash3820

GenderMale versus female 144 0281 0744ndash2774

Age (years)ge60 versus lt60 083 0562 0439ndash1565

Tumor size (cm)ge5 versus lt5 129 0410 0701ndash2388

Tumor locationColon versus rectum versus ileocecal junction 068 0354 0306ndash1528

Histological typeAdenocarcinoma versus nonadenocarcinoma 105 0960 0145ndash7662

Tumor differentiationWell and moderately versus poorly 150 0413 0569ndash3943

Serum CEA level (ngmL)ge15 versus lt15 233 0057 0976ndash5573

Lymph node metastasisPositive versus negative 291 0001lowast 1573ndash5408 126 0668 0443ndash3568

Distant metastasisPositive versus negative 706 0001lowast 3687ndash13521 509 0001lowast 2034ndash12736

TNM stageStages I-II versus stages III-IV 025 0001lowast 0129ndash0466 072 0638 0188ndash2789

lowast119875 lt 005

10

08

06

04

02

00

000 10000 120004000 6000 80002000

Cum

surv

ival

SHIP2 expression

High expressionLow expression

High expression-censoredLow expression-censored

Analysis time (months)

(a) Kaplan-Meier survival estimates by SHIP2 expression

10

08

06

04

02

00

000 10000 120004000 6000 80002000

Cum

surv

ival

Analysis time (months)

Distant metastasisNegative distant metastasis

Positive distant metastasis Positive distant metastasis-

Negative distant metastasis-censored

censored(b) Kaplan-Meier survival estimates by distant metastasis

Figure 3 Survival analysis of colorectal cancer (CRC) patients by Kaplan-Meier method (a) Overall survival rate in patients with high SHIP2protein expression (green line) was significantly lower than that in patients with low SHIP2 expression (blue line) (b) Overall survival rate inpatients with positive distant metastasis (green line) was significantly lower than that in patients with negative distant metastasis (blue line)

6 Disease Markers

univariate analysis while high SHIP2 expression and distantmetastasis were further authenticated as independent predic-tors for unfavorable overall survival of CRC by multivariateanalysis Kaplan-Meier analysis finally demonstrated thatthe life span of patients with high SHIP2 expression waslonger than that of patients with negative expression Severalstudies including two researches of ourselves support ourdata announcing the prognostic role of SHIP2 in cancer[19 20 23 24] Hence it is rational to presume that SHIP2plays oncogenic role dominantly in CRC based on thispresent research Detailed studies of PI3KAkt signaling andadhesion protein interacting with SHIP2 in various cell typesor tissues are necessary to clarify the potential role of SHIP2in procarcinogenesis

In conclusion we identified for the first time that ahigher expression of SHIP2 in CRC tissues compared to thenoncancerous tissues and SHIP2 might play an essential roleas a prognostic marker of survival in patients with CRC Ourstudy is helpful in understanding the characteristics of SHIP2in CRC development

Ethical Approval

The study protocol was approved by the Ethics Committee ofNanjing Medical University and each local hospital Writteninformed consent was acquired from all of the patients thatwere enrolled in this paper

Conflict of Interests

The authors declare no conflict of interests

Authorsrsquo Contribution

Jun Wang and Xuefeng Gu conceived and designed theexperiments Yaoguang Ding Yajing Weng Huihui Ni WeiZhang Feng Jin Ning Xu Jiang Li and Liang Qiu performedthe experiments Ju Yang Maoying Fu Weifei Fan XiaolinPu Zhijun Ge and Feng Zhan collected the data Ju YangMaoying Fu Jiang Li and Liang Qiu analyzed the dataXuefeng Gu wrote the paper Jun Wang and Xuefeng Gusupervised the study Ju Yang and Maoying Fu contributedequally to this work

Acknowledgments

This work was supported by a grant from Natural ScienceFoundation of Jiangsu Province (Grant no BK20130895)and Natural Science Research Program for Colleges andUniversities in Jiangsu Province (Grant no 11KJB320006)

References

[1] C-W Huang H-L Tsai Y-T Chen et al ldquoThe prognostic val-ues of EGFR expression and KRAS mutation in patients withsynchronous or metachronous metastatic colorectal cancerrdquoBMC Cancer vol 13 article 599 2013

[2] M Yasunaga and YMatsumura ldquoRole of SLC6A6 in promotingthe survival and multidrug resistance of colorectal cancerrdquoScientific Reports vol 4 article 4852 2014

[3] T-W Ke H-L Hsu Y-H Wu W T-L Chen Y-W Chengand C-W Cheng ldquoMicroRNA-224 suppresses colorectal cancercell migration by targeting Cdc42rdquo Disease Markers vol 2014Article ID 617150 11 pages 2014

[4] F-T Duan F Qian K Fang K-Y Lin W-T Wang and Y-QChen ldquomiR-133b a muscle-specific microRNA is a novel prog-nosticmarker that participates in the progression of human col-orectal cancer via regulation of CXCR4 expressionrdquo MolecularCancer vol 12 no 1 article 164 2013

[5] S-C ZhangW JinH Liu et al ldquoRPSAgenemutants associatedwith risk of colorectal cancer among the Chinese populationrdquoAsian Pacific Journal of Cancer Prevention vol 14 no 12 pp7127ndash7131 2013

[6] MC SWong J Y L ChingHHHirai et al ldquoPerceived obsta-cles of colorectal cancer screening and their associated factorsamong 10078 Chinese participantsrdquo PLoS ONE vol 8 no 7Article ID e70209 2013

[7] X Lin Z Yi J Diao et al ldquoShaoYao decoction amelioratescolitis-associated colorectal cancer by downregulating proin-flammatory cytokines and promoting epithelial-mesenchymaltransitionrdquo Journal of Translational Medicine vol 12 no 1article 105 2014

[8] W S Atkin R Edwards I Kralj-Hans et al ldquoOnce-only flexiblesigmoidoscopy screening in prevention of colorectal cancer amulticentre randomised controlled trialrdquo The Lancet vol 375no 9726 pp 1624ndash1633 2010

[9] Y-F Yang X-Y ZhangMYang et al ldquoPrognostic role of nucle-ophosmin in colorectal carcinomasrdquo Asian Pacific Journal ofCancer Prevention vol 15 no 5 pp 2021ndash2026 2014

[10] Y Li JWei C Xu Z Zhao and T You ldquoPrognostic significanceof cyclin D1 expression in colorectal cancer a meta-analysisof observational studiesrdquo PLoS ONE vol 9 no 4 Article IDe94508 2014

[11] W-H Leung and S Bolland ldquoThe inositol 51015840-phosphataseSHIP-2 negatively regulates IgE-induced mast cell degranula-tion and cytokine productionrdquoThe Journal of Immunology vol179 no 1 pp 95ndash102 2007

[12] N K Prasad M Tandon S Badve P W Snyder and H Nak-shatri ldquoPhosphoinositol phosphatase SHIP2 promotes cancerdevelopment and metastasis coupled with alterations in EGFreceptor turnoverrdquo Carcinogenesis vol 29 no 1 pp 25ndash342008

[13] A Koch AMancini O El Bounkari and T Tamura ldquoThe SH2-domian-containing inositol 5-phosphatase (SHIP)-2 binds to c-Met directly via tyrosine residue 1356 and involves hepatocytegrowth factor (HGF)-induced lamellipodium formation cellscattering and cell spreadingrdquo Oncogene vol 24 no 21 pp3436ndash3447 2005

[14] S Sumie T Kawaguchi M Komuta et al ldquoSignificance of glu-cose intolerance and SHIP2 expression in hepatocellular carci-noma patients with HCV infectionrdquo Oncology Reports vol 18no 3 pp 545ndash552 2007

[15] X B Xu J Qin andW Y Liu ldquoCurcumin inhibits the invasionof thyroid cancer cells via down-regulation of PI3KAkt signal-ing pathwayrdquo Gene vol 546 no 2 pp 226ndash232 2014

[16] J J Wheler S L Moulder A Naing et al ldquoAnastrozole andeverolimus in advanced gynecologic and breast malignanciesactivity and molecular alterations in the PI3KAKTmTORpathwayrdquo Oncotarget vol 5 no 10 pp 3029ndash3038 2014

Disease Markers 7

[17] S Giuriato D Blero B Robaye C Bruyns B Payrastre andC Erneux ldquoSHIP2 overexpression strongly reduces the prolif-eration rate of K562 erythroleukemia cell linerdquo Biochemical andBiophysical Research Communications vol 296 no 1 pp 106ndash110 2002

[18] T Sasaoka K Kikuchi TWada et al ldquoDual role of SRC homol-ogy domain 2-containing inositol phosphatase 2 in the reg-ulation of platelet-derived growth factor and insulin-likegrowth factor I signaling in rat vascular smooth muscle cellsrdquoEndocrinology vol 144 no 9 pp 4204ndash4214 2003

[19] N K Prasad M Tandon A Handa et al ldquoHigh expressionof obesity-linked phosphatase SHIP2 in invasive breast cancercorrelates with reduced disease-free survivalrdquo Tumor Biologyvol 29 no 5 pp 330ndash341 2008

[20] X Zhou Y Liu andG Tan ldquoPrognostic value of elevated SHIP2expression in laryngeal squamous cell carcinomardquo Archives ofMedical Research vol 42 no 7 pp 589ndash595 2011

[21] N K Prasad ldquoSHIP2 phosphoinositol phosphatase positivelyregulates EGFR-Akt pathway CXCR4 expression and cellmigration in MDA-MB-231 breast cancer cellsrdquo InternationalJournal of Oncology vol 34 no 1 pp 97ndash105 2009

[22] F ObroceaM Sajin E CMarinescu andD Stoica ldquoColorectalcancer and the 7th revision of the TNM staging system reviewof changes and suggestions for uniform pathologic reportingrdquoRomanian Journal of Morphology and Embryology vol 52 no2 pp 537ndash544 2011

[23] M Fu W Fan X Pu et al ldquoElevated expression of SHIP2 cor-relates with poor prognosis in non-small cell lung cancerrdquoInternational Journal of Clinical and Experimental Pathologyvol 6 no 10 pp 2185ndash2191 2013

[24] M Fu X Gu H Ni et al ldquoHigh expression of inositol pol-yphosphate phosphatase-like 1 associates with unfavorablesurvival in hepatocellular carcinomardquo International Journal ofClinical and Experimental Pathology vol 6 no 11 pp 2515ndash2522 2013

[25] V Taylor M Wong C Brandts et al ldquo51015840 Phospholipid phos-phatase SHIP-2 causes protein kinase B inactivation and cellcycle arrest in glioblastoma cellsrdquoMolecular amp Cellular Biologyvol 20 no 18 pp 6860ndash6871 2000

[26] A Suwa T Kurama and T Shimokawa ldquoSHIP2 and its involve-ment in various diseasesrdquo Expert Opinion on Therapeutic Tar-gets vol 14 no 7 pp 727ndash737 2010

[27] B Ducarouge M Pelissier-Rota M Laine et al ldquoCRF2 sig-naling is a novel regulator of cellular adhesion and migrationin colorectal cancer cellsrdquo PLoS ONE vol 8 no 11 Article IDe79335 2013

[28] N Vassos T Rau S Merkel et al ldquoPrognostic value of 1205731integrin expression in colorectal liver metastasesrdquo InternationalJournal of Clinical and Experimental Pathology vol 7 no 1 pp288ndash300 2014

[29] J F Groulx V Giroux M Beausejour et al ldquoIntegrin alpha6Asplice variant regulates proliferation and the Wntbeta-cateninpathway in human colorectal cancer cellsrdquo Carcinogenesis vol35 no 6 pp 1217ndash1227 2014

[30] J M Haugh F Codazzi M Teruel and T Meyer ldquoSpatialsensing in fibroblasts mediated by 31015840 phosphoinositidesrdquo TheJournal of Cell Biology vol 151 no 6 pp 1269ndash1280 2000

[31] A Y Alexandrova ldquoEvolution of cell interactions with extracel-lular matrix during carcinogenesisrdquo Biochemistry vol 73 no 7pp 733ndash741 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 5

Table 2 Univariate and multivariate analyses of prognostic factors in 102 CRC patients for overall survival

Univariate analysis Multivariate analysisHR 119875 gt |119911| 95 CI HR 119875 gt |119911| 95 CI

SHIP2 expressionHigh versus low 220 0017lowast 1151ndash4189 205 0040lowast 1031ndash3820

GenderMale versus female 144 0281 0744ndash2774

Age (years)ge60 versus lt60 083 0562 0439ndash1565

Tumor size (cm)ge5 versus lt5 129 0410 0701ndash2388

Tumor locationColon versus rectum versus ileocecal junction 068 0354 0306ndash1528

Histological typeAdenocarcinoma versus nonadenocarcinoma 105 0960 0145ndash7662

Tumor differentiationWell and moderately versus poorly 150 0413 0569ndash3943

Serum CEA level (ngmL)ge15 versus lt15 233 0057 0976ndash5573

Lymph node metastasisPositive versus negative 291 0001lowast 1573ndash5408 126 0668 0443ndash3568

Distant metastasisPositive versus negative 706 0001lowast 3687ndash13521 509 0001lowast 2034ndash12736

TNM stageStages I-II versus stages III-IV 025 0001lowast 0129ndash0466 072 0638 0188ndash2789

lowast119875 lt 005

10

08

06

04

02

00

000 10000 120004000 6000 80002000

Cum

surv

ival

SHIP2 expression

High expressionLow expression

High expression-censoredLow expression-censored

Analysis time (months)

(a) Kaplan-Meier survival estimates by SHIP2 expression

10

08

06

04

02

00

000 10000 120004000 6000 80002000

Cum

surv

ival

Analysis time (months)

Distant metastasisNegative distant metastasis

Positive distant metastasis Positive distant metastasis-

Negative distant metastasis-censored

censored(b) Kaplan-Meier survival estimates by distant metastasis

Figure 3 Survival analysis of colorectal cancer (CRC) patients by Kaplan-Meier method (a) Overall survival rate in patients with high SHIP2protein expression (green line) was significantly lower than that in patients with low SHIP2 expression (blue line) (b) Overall survival rate inpatients with positive distant metastasis (green line) was significantly lower than that in patients with negative distant metastasis (blue line)

6 Disease Markers

univariate analysis while high SHIP2 expression and distantmetastasis were further authenticated as independent predic-tors for unfavorable overall survival of CRC by multivariateanalysis Kaplan-Meier analysis finally demonstrated thatthe life span of patients with high SHIP2 expression waslonger than that of patients with negative expression Severalstudies including two researches of ourselves support ourdata announcing the prognostic role of SHIP2 in cancer[19 20 23 24] Hence it is rational to presume that SHIP2plays oncogenic role dominantly in CRC based on thispresent research Detailed studies of PI3KAkt signaling andadhesion protein interacting with SHIP2 in various cell typesor tissues are necessary to clarify the potential role of SHIP2in procarcinogenesis

In conclusion we identified for the first time that ahigher expression of SHIP2 in CRC tissues compared to thenoncancerous tissues and SHIP2 might play an essential roleas a prognostic marker of survival in patients with CRC Ourstudy is helpful in understanding the characteristics of SHIP2in CRC development

Ethical Approval

The study protocol was approved by the Ethics Committee ofNanjing Medical University and each local hospital Writteninformed consent was acquired from all of the patients thatwere enrolled in this paper

Conflict of Interests

The authors declare no conflict of interests

Authorsrsquo Contribution

Jun Wang and Xuefeng Gu conceived and designed theexperiments Yaoguang Ding Yajing Weng Huihui Ni WeiZhang Feng Jin Ning Xu Jiang Li and Liang Qiu performedthe experiments Ju Yang Maoying Fu Weifei Fan XiaolinPu Zhijun Ge and Feng Zhan collected the data Ju YangMaoying Fu Jiang Li and Liang Qiu analyzed the dataXuefeng Gu wrote the paper Jun Wang and Xuefeng Gusupervised the study Ju Yang and Maoying Fu contributedequally to this work

Acknowledgments

This work was supported by a grant from Natural ScienceFoundation of Jiangsu Province (Grant no BK20130895)and Natural Science Research Program for Colleges andUniversities in Jiangsu Province (Grant no 11KJB320006)

References

[1] C-W Huang H-L Tsai Y-T Chen et al ldquoThe prognostic val-ues of EGFR expression and KRAS mutation in patients withsynchronous or metachronous metastatic colorectal cancerrdquoBMC Cancer vol 13 article 599 2013

[2] M Yasunaga and YMatsumura ldquoRole of SLC6A6 in promotingthe survival and multidrug resistance of colorectal cancerrdquoScientific Reports vol 4 article 4852 2014

[3] T-W Ke H-L Hsu Y-H Wu W T-L Chen Y-W Chengand C-W Cheng ldquoMicroRNA-224 suppresses colorectal cancercell migration by targeting Cdc42rdquo Disease Markers vol 2014Article ID 617150 11 pages 2014

[4] F-T Duan F Qian K Fang K-Y Lin W-T Wang and Y-QChen ldquomiR-133b a muscle-specific microRNA is a novel prog-nosticmarker that participates in the progression of human col-orectal cancer via regulation of CXCR4 expressionrdquo MolecularCancer vol 12 no 1 article 164 2013

[5] S-C ZhangW JinH Liu et al ldquoRPSAgenemutants associatedwith risk of colorectal cancer among the Chinese populationrdquoAsian Pacific Journal of Cancer Prevention vol 14 no 12 pp7127ndash7131 2013

[6] MC SWong J Y L ChingHHHirai et al ldquoPerceived obsta-cles of colorectal cancer screening and their associated factorsamong 10078 Chinese participantsrdquo PLoS ONE vol 8 no 7Article ID e70209 2013

[7] X Lin Z Yi J Diao et al ldquoShaoYao decoction amelioratescolitis-associated colorectal cancer by downregulating proin-flammatory cytokines and promoting epithelial-mesenchymaltransitionrdquo Journal of Translational Medicine vol 12 no 1article 105 2014

[8] W S Atkin R Edwards I Kralj-Hans et al ldquoOnce-only flexiblesigmoidoscopy screening in prevention of colorectal cancer amulticentre randomised controlled trialrdquo The Lancet vol 375no 9726 pp 1624ndash1633 2010

[9] Y-F Yang X-Y ZhangMYang et al ldquoPrognostic role of nucle-ophosmin in colorectal carcinomasrdquo Asian Pacific Journal ofCancer Prevention vol 15 no 5 pp 2021ndash2026 2014

[10] Y Li JWei C Xu Z Zhao and T You ldquoPrognostic significanceof cyclin D1 expression in colorectal cancer a meta-analysisof observational studiesrdquo PLoS ONE vol 9 no 4 Article IDe94508 2014

[11] W-H Leung and S Bolland ldquoThe inositol 51015840-phosphataseSHIP-2 negatively regulates IgE-induced mast cell degranula-tion and cytokine productionrdquoThe Journal of Immunology vol179 no 1 pp 95ndash102 2007

[12] N K Prasad M Tandon S Badve P W Snyder and H Nak-shatri ldquoPhosphoinositol phosphatase SHIP2 promotes cancerdevelopment and metastasis coupled with alterations in EGFreceptor turnoverrdquo Carcinogenesis vol 29 no 1 pp 25ndash342008

[13] A Koch AMancini O El Bounkari and T Tamura ldquoThe SH2-domian-containing inositol 5-phosphatase (SHIP)-2 binds to c-Met directly via tyrosine residue 1356 and involves hepatocytegrowth factor (HGF)-induced lamellipodium formation cellscattering and cell spreadingrdquo Oncogene vol 24 no 21 pp3436ndash3447 2005

[14] S Sumie T Kawaguchi M Komuta et al ldquoSignificance of glu-cose intolerance and SHIP2 expression in hepatocellular carci-noma patients with HCV infectionrdquo Oncology Reports vol 18no 3 pp 545ndash552 2007

[15] X B Xu J Qin andW Y Liu ldquoCurcumin inhibits the invasionof thyroid cancer cells via down-regulation of PI3KAkt signal-ing pathwayrdquo Gene vol 546 no 2 pp 226ndash232 2014

[16] J J Wheler S L Moulder A Naing et al ldquoAnastrozole andeverolimus in advanced gynecologic and breast malignanciesactivity and molecular alterations in the PI3KAKTmTORpathwayrdquo Oncotarget vol 5 no 10 pp 3029ndash3038 2014

Disease Markers 7

[17] S Giuriato D Blero B Robaye C Bruyns B Payrastre andC Erneux ldquoSHIP2 overexpression strongly reduces the prolif-eration rate of K562 erythroleukemia cell linerdquo Biochemical andBiophysical Research Communications vol 296 no 1 pp 106ndash110 2002

[18] T Sasaoka K Kikuchi TWada et al ldquoDual role of SRC homol-ogy domain 2-containing inositol phosphatase 2 in the reg-ulation of platelet-derived growth factor and insulin-likegrowth factor I signaling in rat vascular smooth muscle cellsrdquoEndocrinology vol 144 no 9 pp 4204ndash4214 2003

[19] N K Prasad M Tandon A Handa et al ldquoHigh expressionof obesity-linked phosphatase SHIP2 in invasive breast cancercorrelates with reduced disease-free survivalrdquo Tumor Biologyvol 29 no 5 pp 330ndash341 2008

[20] X Zhou Y Liu andG Tan ldquoPrognostic value of elevated SHIP2expression in laryngeal squamous cell carcinomardquo Archives ofMedical Research vol 42 no 7 pp 589ndash595 2011

[21] N K Prasad ldquoSHIP2 phosphoinositol phosphatase positivelyregulates EGFR-Akt pathway CXCR4 expression and cellmigration in MDA-MB-231 breast cancer cellsrdquo InternationalJournal of Oncology vol 34 no 1 pp 97ndash105 2009

[22] F ObroceaM Sajin E CMarinescu andD Stoica ldquoColorectalcancer and the 7th revision of the TNM staging system reviewof changes and suggestions for uniform pathologic reportingrdquoRomanian Journal of Morphology and Embryology vol 52 no2 pp 537ndash544 2011

[23] M Fu W Fan X Pu et al ldquoElevated expression of SHIP2 cor-relates with poor prognosis in non-small cell lung cancerrdquoInternational Journal of Clinical and Experimental Pathologyvol 6 no 10 pp 2185ndash2191 2013

[24] M Fu X Gu H Ni et al ldquoHigh expression of inositol pol-yphosphate phosphatase-like 1 associates with unfavorablesurvival in hepatocellular carcinomardquo International Journal ofClinical and Experimental Pathology vol 6 no 11 pp 2515ndash2522 2013

[25] V Taylor M Wong C Brandts et al ldquo51015840 Phospholipid phos-phatase SHIP-2 causes protein kinase B inactivation and cellcycle arrest in glioblastoma cellsrdquoMolecular amp Cellular Biologyvol 20 no 18 pp 6860ndash6871 2000

[26] A Suwa T Kurama and T Shimokawa ldquoSHIP2 and its involve-ment in various diseasesrdquo Expert Opinion on Therapeutic Tar-gets vol 14 no 7 pp 727ndash737 2010

[27] B Ducarouge M Pelissier-Rota M Laine et al ldquoCRF2 sig-naling is a novel regulator of cellular adhesion and migrationin colorectal cancer cellsrdquo PLoS ONE vol 8 no 11 Article IDe79335 2013

[28] N Vassos T Rau S Merkel et al ldquoPrognostic value of 1205731integrin expression in colorectal liver metastasesrdquo InternationalJournal of Clinical and Experimental Pathology vol 7 no 1 pp288ndash300 2014

[29] J F Groulx V Giroux M Beausejour et al ldquoIntegrin alpha6Asplice variant regulates proliferation and the Wntbeta-cateninpathway in human colorectal cancer cellsrdquo Carcinogenesis vol35 no 6 pp 1217ndash1227 2014

[30] J M Haugh F Codazzi M Teruel and T Meyer ldquoSpatialsensing in fibroblasts mediated by 31015840 phosphoinositidesrdquo TheJournal of Cell Biology vol 151 no 6 pp 1269ndash1280 2000

[31] A Y Alexandrova ldquoEvolution of cell interactions with extracel-lular matrix during carcinogenesisrdquo Biochemistry vol 73 no 7pp 733ndash741 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Disease Markers

univariate analysis while high SHIP2 expression and distantmetastasis were further authenticated as independent predic-tors for unfavorable overall survival of CRC by multivariateanalysis Kaplan-Meier analysis finally demonstrated thatthe life span of patients with high SHIP2 expression waslonger than that of patients with negative expression Severalstudies including two researches of ourselves support ourdata announcing the prognostic role of SHIP2 in cancer[19 20 23 24] Hence it is rational to presume that SHIP2plays oncogenic role dominantly in CRC based on thispresent research Detailed studies of PI3KAkt signaling andadhesion protein interacting with SHIP2 in various cell typesor tissues are necessary to clarify the potential role of SHIP2in procarcinogenesis

In conclusion we identified for the first time that ahigher expression of SHIP2 in CRC tissues compared to thenoncancerous tissues and SHIP2 might play an essential roleas a prognostic marker of survival in patients with CRC Ourstudy is helpful in understanding the characteristics of SHIP2in CRC development

Ethical Approval

The study protocol was approved by the Ethics Committee ofNanjing Medical University and each local hospital Writteninformed consent was acquired from all of the patients thatwere enrolled in this paper

Conflict of Interests

The authors declare no conflict of interests

Authorsrsquo Contribution

Jun Wang and Xuefeng Gu conceived and designed theexperiments Yaoguang Ding Yajing Weng Huihui Ni WeiZhang Feng Jin Ning Xu Jiang Li and Liang Qiu performedthe experiments Ju Yang Maoying Fu Weifei Fan XiaolinPu Zhijun Ge and Feng Zhan collected the data Ju YangMaoying Fu Jiang Li and Liang Qiu analyzed the dataXuefeng Gu wrote the paper Jun Wang and Xuefeng Gusupervised the study Ju Yang and Maoying Fu contributedequally to this work

Acknowledgments

This work was supported by a grant from Natural ScienceFoundation of Jiangsu Province (Grant no BK20130895)and Natural Science Research Program for Colleges andUniversities in Jiangsu Province (Grant no 11KJB320006)

References

[1] C-W Huang H-L Tsai Y-T Chen et al ldquoThe prognostic val-ues of EGFR expression and KRAS mutation in patients withsynchronous or metachronous metastatic colorectal cancerrdquoBMC Cancer vol 13 article 599 2013

[2] M Yasunaga and YMatsumura ldquoRole of SLC6A6 in promotingthe survival and multidrug resistance of colorectal cancerrdquoScientific Reports vol 4 article 4852 2014

[3] T-W Ke H-L Hsu Y-H Wu W T-L Chen Y-W Chengand C-W Cheng ldquoMicroRNA-224 suppresses colorectal cancercell migration by targeting Cdc42rdquo Disease Markers vol 2014Article ID 617150 11 pages 2014

[4] F-T Duan F Qian K Fang K-Y Lin W-T Wang and Y-QChen ldquomiR-133b a muscle-specific microRNA is a novel prog-nosticmarker that participates in the progression of human col-orectal cancer via regulation of CXCR4 expressionrdquo MolecularCancer vol 12 no 1 article 164 2013

[5] S-C ZhangW JinH Liu et al ldquoRPSAgenemutants associatedwith risk of colorectal cancer among the Chinese populationrdquoAsian Pacific Journal of Cancer Prevention vol 14 no 12 pp7127ndash7131 2013

[6] MC SWong J Y L ChingHHHirai et al ldquoPerceived obsta-cles of colorectal cancer screening and their associated factorsamong 10078 Chinese participantsrdquo PLoS ONE vol 8 no 7Article ID e70209 2013

[7] X Lin Z Yi J Diao et al ldquoShaoYao decoction amelioratescolitis-associated colorectal cancer by downregulating proin-flammatory cytokines and promoting epithelial-mesenchymaltransitionrdquo Journal of Translational Medicine vol 12 no 1article 105 2014

[8] W S Atkin R Edwards I Kralj-Hans et al ldquoOnce-only flexiblesigmoidoscopy screening in prevention of colorectal cancer amulticentre randomised controlled trialrdquo The Lancet vol 375no 9726 pp 1624ndash1633 2010

[9] Y-F Yang X-Y ZhangMYang et al ldquoPrognostic role of nucle-ophosmin in colorectal carcinomasrdquo Asian Pacific Journal ofCancer Prevention vol 15 no 5 pp 2021ndash2026 2014

[10] Y Li JWei C Xu Z Zhao and T You ldquoPrognostic significanceof cyclin D1 expression in colorectal cancer a meta-analysisof observational studiesrdquo PLoS ONE vol 9 no 4 Article IDe94508 2014

[11] W-H Leung and S Bolland ldquoThe inositol 51015840-phosphataseSHIP-2 negatively regulates IgE-induced mast cell degranula-tion and cytokine productionrdquoThe Journal of Immunology vol179 no 1 pp 95ndash102 2007

[12] N K Prasad M Tandon S Badve P W Snyder and H Nak-shatri ldquoPhosphoinositol phosphatase SHIP2 promotes cancerdevelopment and metastasis coupled with alterations in EGFreceptor turnoverrdquo Carcinogenesis vol 29 no 1 pp 25ndash342008

[13] A Koch AMancini O El Bounkari and T Tamura ldquoThe SH2-domian-containing inositol 5-phosphatase (SHIP)-2 binds to c-Met directly via tyrosine residue 1356 and involves hepatocytegrowth factor (HGF)-induced lamellipodium formation cellscattering and cell spreadingrdquo Oncogene vol 24 no 21 pp3436ndash3447 2005

[14] S Sumie T Kawaguchi M Komuta et al ldquoSignificance of glu-cose intolerance and SHIP2 expression in hepatocellular carci-noma patients with HCV infectionrdquo Oncology Reports vol 18no 3 pp 545ndash552 2007

[15] X B Xu J Qin andW Y Liu ldquoCurcumin inhibits the invasionof thyroid cancer cells via down-regulation of PI3KAkt signal-ing pathwayrdquo Gene vol 546 no 2 pp 226ndash232 2014

[16] J J Wheler S L Moulder A Naing et al ldquoAnastrozole andeverolimus in advanced gynecologic and breast malignanciesactivity and molecular alterations in the PI3KAKTmTORpathwayrdquo Oncotarget vol 5 no 10 pp 3029ndash3038 2014

Disease Markers 7

[17] S Giuriato D Blero B Robaye C Bruyns B Payrastre andC Erneux ldquoSHIP2 overexpression strongly reduces the prolif-eration rate of K562 erythroleukemia cell linerdquo Biochemical andBiophysical Research Communications vol 296 no 1 pp 106ndash110 2002

[18] T Sasaoka K Kikuchi TWada et al ldquoDual role of SRC homol-ogy domain 2-containing inositol phosphatase 2 in the reg-ulation of platelet-derived growth factor and insulin-likegrowth factor I signaling in rat vascular smooth muscle cellsrdquoEndocrinology vol 144 no 9 pp 4204ndash4214 2003

[19] N K Prasad M Tandon A Handa et al ldquoHigh expressionof obesity-linked phosphatase SHIP2 in invasive breast cancercorrelates with reduced disease-free survivalrdquo Tumor Biologyvol 29 no 5 pp 330ndash341 2008

[20] X Zhou Y Liu andG Tan ldquoPrognostic value of elevated SHIP2expression in laryngeal squamous cell carcinomardquo Archives ofMedical Research vol 42 no 7 pp 589ndash595 2011

[21] N K Prasad ldquoSHIP2 phosphoinositol phosphatase positivelyregulates EGFR-Akt pathway CXCR4 expression and cellmigration in MDA-MB-231 breast cancer cellsrdquo InternationalJournal of Oncology vol 34 no 1 pp 97ndash105 2009

[22] F ObroceaM Sajin E CMarinescu andD Stoica ldquoColorectalcancer and the 7th revision of the TNM staging system reviewof changes and suggestions for uniform pathologic reportingrdquoRomanian Journal of Morphology and Embryology vol 52 no2 pp 537ndash544 2011

[23] M Fu W Fan X Pu et al ldquoElevated expression of SHIP2 cor-relates with poor prognosis in non-small cell lung cancerrdquoInternational Journal of Clinical and Experimental Pathologyvol 6 no 10 pp 2185ndash2191 2013

[24] M Fu X Gu H Ni et al ldquoHigh expression of inositol pol-yphosphate phosphatase-like 1 associates with unfavorablesurvival in hepatocellular carcinomardquo International Journal ofClinical and Experimental Pathology vol 6 no 11 pp 2515ndash2522 2013

[25] V Taylor M Wong C Brandts et al ldquo51015840 Phospholipid phos-phatase SHIP-2 causes protein kinase B inactivation and cellcycle arrest in glioblastoma cellsrdquoMolecular amp Cellular Biologyvol 20 no 18 pp 6860ndash6871 2000

[26] A Suwa T Kurama and T Shimokawa ldquoSHIP2 and its involve-ment in various diseasesrdquo Expert Opinion on Therapeutic Tar-gets vol 14 no 7 pp 727ndash737 2010

[27] B Ducarouge M Pelissier-Rota M Laine et al ldquoCRF2 sig-naling is a novel regulator of cellular adhesion and migrationin colorectal cancer cellsrdquo PLoS ONE vol 8 no 11 Article IDe79335 2013

[28] N Vassos T Rau S Merkel et al ldquoPrognostic value of 1205731integrin expression in colorectal liver metastasesrdquo InternationalJournal of Clinical and Experimental Pathology vol 7 no 1 pp288ndash300 2014

[29] J F Groulx V Giroux M Beausejour et al ldquoIntegrin alpha6Asplice variant regulates proliferation and the Wntbeta-cateninpathway in human colorectal cancer cellsrdquo Carcinogenesis vol35 no 6 pp 1217ndash1227 2014

[30] J M Haugh F Codazzi M Teruel and T Meyer ldquoSpatialsensing in fibroblasts mediated by 31015840 phosphoinositidesrdquo TheJournal of Cell Biology vol 151 no 6 pp 1269ndash1280 2000

[31] A Y Alexandrova ldquoEvolution of cell interactions with extracel-lular matrix during carcinogenesisrdquo Biochemistry vol 73 no 7pp 733ndash741 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 7

[17] S Giuriato D Blero B Robaye C Bruyns B Payrastre andC Erneux ldquoSHIP2 overexpression strongly reduces the prolif-eration rate of K562 erythroleukemia cell linerdquo Biochemical andBiophysical Research Communications vol 296 no 1 pp 106ndash110 2002

[18] T Sasaoka K Kikuchi TWada et al ldquoDual role of SRC homol-ogy domain 2-containing inositol phosphatase 2 in the reg-ulation of platelet-derived growth factor and insulin-likegrowth factor I signaling in rat vascular smooth muscle cellsrdquoEndocrinology vol 144 no 9 pp 4204ndash4214 2003

[19] N K Prasad M Tandon A Handa et al ldquoHigh expressionof obesity-linked phosphatase SHIP2 in invasive breast cancercorrelates with reduced disease-free survivalrdquo Tumor Biologyvol 29 no 5 pp 330ndash341 2008

[20] X Zhou Y Liu andG Tan ldquoPrognostic value of elevated SHIP2expression in laryngeal squamous cell carcinomardquo Archives ofMedical Research vol 42 no 7 pp 589ndash595 2011

[21] N K Prasad ldquoSHIP2 phosphoinositol phosphatase positivelyregulates EGFR-Akt pathway CXCR4 expression and cellmigration in MDA-MB-231 breast cancer cellsrdquo InternationalJournal of Oncology vol 34 no 1 pp 97ndash105 2009

[22] F ObroceaM Sajin E CMarinescu andD Stoica ldquoColorectalcancer and the 7th revision of the TNM staging system reviewof changes and suggestions for uniform pathologic reportingrdquoRomanian Journal of Morphology and Embryology vol 52 no2 pp 537ndash544 2011

[23] M Fu W Fan X Pu et al ldquoElevated expression of SHIP2 cor-relates with poor prognosis in non-small cell lung cancerrdquoInternational Journal of Clinical and Experimental Pathologyvol 6 no 10 pp 2185ndash2191 2013

[24] M Fu X Gu H Ni et al ldquoHigh expression of inositol pol-yphosphate phosphatase-like 1 associates with unfavorablesurvival in hepatocellular carcinomardquo International Journal ofClinical and Experimental Pathology vol 6 no 11 pp 2515ndash2522 2013

[25] V Taylor M Wong C Brandts et al ldquo51015840 Phospholipid phos-phatase SHIP-2 causes protein kinase B inactivation and cellcycle arrest in glioblastoma cellsrdquoMolecular amp Cellular Biologyvol 20 no 18 pp 6860ndash6871 2000

[26] A Suwa T Kurama and T Shimokawa ldquoSHIP2 and its involve-ment in various diseasesrdquo Expert Opinion on Therapeutic Tar-gets vol 14 no 7 pp 727ndash737 2010

[27] B Ducarouge M Pelissier-Rota M Laine et al ldquoCRF2 sig-naling is a novel regulator of cellular adhesion and migrationin colorectal cancer cellsrdquo PLoS ONE vol 8 no 11 Article IDe79335 2013

[28] N Vassos T Rau S Merkel et al ldquoPrognostic value of 1205731integrin expression in colorectal liver metastasesrdquo InternationalJournal of Clinical and Experimental Pathology vol 7 no 1 pp288ndash300 2014

[29] J F Groulx V Giroux M Beausejour et al ldquoIntegrin alpha6Asplice variant regulates proliferation and the Wntbeta-cateninpathway in human colorectal cancer cellsrdquo Carcinogenesis vol35 no 6 pp 1217ndash1227 2014

[30] J M Haugh F Codazzi M Teruel and T Meyer ldquoSpatialsensing in fibroblasts mediated by 31015840 phosphoinositidesrdquo TheJournal of Cell Biology vol 151 no 6 pp 1269ndash1280 2000

[31] A Y Alexandrova ldquoEvolution of cell interactions with extracel-lular matrix during carcinogenesisrdquo Biochemistry vol 73 no 7pp 733ndash741 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom