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Original Article

Is Alpha-B Crystallin an Independent Marker forPrognosis in Lung Cancer?

Andrew J.M. Campbell-Lloyd MBBS a, Julie Mundy FRACS a, Rajeev Deva MBBS a,∗,Guy Lampe FRCPA a, Carmel Hawley FRACP a,c, Glen Boyle b,

Rayleene Griffin RN a, Charles Thompson c and Pallav Shah, FRACS a

a Princess Alexandra Hospital, Australiab Queensland Institute of Medical Research, Australia

c University of Queensland, Australia

Background: Alpha B-crystallin (CRYAB) is an oncogene that increases tumour survival by promoting angiogenesisand preventing apoptosis. CRYAB is an independent prognostic marker in epithelial tumours including head and necksquamous cell carcinoma and breast cancer where it is predictive of nodal status and associated with poor outcome. Weexplored the role of CRYAB in non-small-cell lung cancer (NSCLC).

Methods: Immunohistochemical analysis was performed on 50 samples. Following staining with anti-alpha-B crystallinantibody, a blinded pathologist scored samples for nuclear (N) and cytoplasmic (C) staining intensity. Analysis wasperformed using Cox’s proportional hazards model.

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Results: There were 32 adenocarcinomas and 18 squamous cell carcinomas. The median tumour size was T2, grade 2oderately differentiated, and 10 patients had nodal spread. Recurrence was seen in 22 patients (46%). Mortality was

8%, with median time to mortality 871 days. N staining was detected in eight samples (16%), and C staining in 20 (40%),ith both N and C staining positive in five (10%). Staining for CRYAB predicted neither recurrence (N stain p = 0.78, C

tain p = 0.38) nor mortality (N stain p = 0.86, C stain p = 0.66).Conclusion: CRYAB did not predict outcomes in patients treated for NSCLC. Larger studies are required to validate

his finding.(Heart, Lung and Circulation 2013;22:759–766)

Crown Copyright © 2013 Published by Elsevier Inc. on behalf of Australian and New Zealand Society of Cardiac andThoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ). All rights reserved.

eywords. Lung cancer; Markers; Outcomes

ntroduction

alignancies of the lung, particularly non-small celllung cancer (NSCLC), remain the most common

ause of death from cancer and have a ratio of mortality-to-ncidence of 0.87 [1]. It has been suggested that the futuref treatment of lung cancer lies in the greater progno-tic specificity that can be afforded by molecular analysisased staging [2]. Molecular analysis may allow subtyp-

ng of patients with what have traditionally been thoughtf as low grade tumours, thus prompting more aggres-ive treatment in the first instance to prevent recurrencend nodal spread [3]. It is proposed that clinical practiceill soon entail integration of molecular analysis with tra-itional clinical features as outlined in the TNM system.dditionally, our knowledge of the prognostic impact of

eceived 22 November 2012; received in revised form 10 January013; accepted 29 January 2013; available online 10 April 2013

Corresponding author at: Department of Cardiothoracicurgery, Princess Alexandra Hospital, Woolloongabba, Brisbane,LD 4102, Australia. Tel.: +61 7 3240 7675; fax: +61 7 3240 6954.

-mail address: rdeva3@hotmail.com (R. Deva).

additional clinical factors, in particular pre-morbid per-formance scores, age and gender [4,5], continues to grow,allowing greater prognostic accuracy in conjunction withthe current TNM system.

Alpha B-crystallin (CRYAB) is a molecule involved ina wide range of human epithelial malignancies. There isincreasing understanding of the normal and pathologicalrole this molecular chaperone and member of the smallheat shock protein (sHSP) family plays. There appearsto be oncogenic transformation associated up-regulationof CRYAB expression in certain tumours. Current datasuggest that it may be a prognostic marker in breast,renal, thyroid, head and neck, nasopharyngeal, hepa-tocellular and potentially lung cancers. Further, thereis now a solid understanding of the role of CRYAB asan anti-apoptotic molecule, accounting for its apparentoncogenicity.

At present, knowledge of specific molecular markersof prognosis in lung cancer is limited. We have exploredthe prognostic impact of CRYAB in a series of early stageNSCLC patients by investigating the role of CRYAB as amarker of recurrence (R) and mortality (M).

rown Copyright © 2013 Published by Elsevier Inc. on behalf of Aus-ralian and New Zealand Society of Cardiac and Thoracic SurgeonsANZSCTS) and the Cardiac Society of Australia and New Zealand 1443-9506/04/$36.00

CSANZ). All rights reserved. http://dx.doi.org/10.1016/j.hlc.2013.01.014

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760 Campbell-Lloyd et al. Heart, Lung and CirculationAlpha-B crystallin in lung cancer 2013;22:759–766

Figure 1. Intense nuclear staining (left) and focally intense cytoplasmic staining (right).

Patients

Between 2002 and 2006, 48 patients were treated by themultidisciplinary Lung Cancer Unit at Princess Alexan-dra Hospital in Brisbane, Australia. We excluded anypatient with malignancies other than NSCLC. Histologicalor cytological diagnosis was available for all patients, withpertinent radiological findings from computed tomogra-phy.

We resected 50 NSCLC specimens from 48 patients.Patients underwent wedge resection, lobectomy or pneu-monectomy following clinical staging. Following resectionof specimens, tissue samples were processed routinely forhistological assessment and complete pathological stag-ing as determined by the 7th edition of the World HealthOrganisation TNM classification. In addition, tumourswere graded in the standard fashion, on a 4-point scalefrom well differentiated to undifferentiated. TNM stagingwas complete for all patients, and histological grading wasavailable for all samples.

Mean age was 68 (range 51–78 years), with a male-to-female ratio of 5:3. There were 20 patients over 70 years ofage.

Methods

Following approval by our institutional Human Research

(divided into quartiles) and this was done for both nuclearand cytoplasmic staining. Further, in order to assess theinteraction of CRYAB staining and nodal status, a com-posite score was generated for nodal positivity and stainpositivity. However due to a small sample size, the studywas underpowered to assess so many variables, so ourfindings were converted to a series of binomial variables.For the final analysis, the nuclear and cytoplasmic stain-ing was scored as negative or positive, with an additionalvariable assessing the impact of any stain (i.e. either Nor C staining). These findings were then correlated withtumour type, histopathological findings, nodal status andsurvival data.

Statistics

Analyses were primarily descriptive in view of thelimited number of patients, particularly the limitednumber of patients with positive staining. Chi-squareor Fisher’s exact tests were used to analyse differ-ences between groups. Only univariate analyses wereperformed. Time to mortality and time to recurrencewere performed as time-to-event analyses using theKaplan–Meier method incorporating univariate Cox pro-portional hazards method to calculate the unadjustedhazard ratio associated with predictors of interest. A pvalue of <0.05 was regarded as significant a priori.

and Ethics Committee, we used previously obtained spec-imens for immunohistochemical analysis. The requiredtissue blocks were retrieved and sections (5 �m) offormalin-fixed and paraffin-embedded tissues weredewaxed, and incubated with a rabbit anti-�B crys-tallin polyclonal antibody (Stressgen Biotechnologies,Ann Arbor, MI) at 1:250 dilution overnight at room temper-ature. Normal serum was used as a negative control andall slides were then counterstained with haematoxylin.

Following staining, a blinded pathologist scored thesamples for nuclear (N) and cytoplasmic (C) staining(Fig. 1). Initial scoring was performed for intensity (mild,moderate or intense) and total percentage of staining

Follow up data were obtained by chart review and clini-cal follow up. Patients were followed until death, or the endof the study period (August 2009). Mean follow up for allpatients was 40 months (range 2–67 months). All patientsin the study were followed up.

Results

Based on previous histological assessment, there wereknown to be 32 adenocarcinomas and 18 squamous cellcarcinomas (SCC). Synchronous tumours were found inthree patients, one of whom had a synchronous metastaticcancer to the lung, which was not included in the study.

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Table 1. Staining Characteristics from Valid Samples.

Cytoplasmic + Cytoplasmic − p value Nuclear + Nuclear − p value

Tumour type (n = 48) 0.04 0.45

Adeno (30) 30.0% 70.0% 13.3% 86.7%

SCC (18) 61.1% 38.9% 22.2% 77.8%

Tumour size (n = 48) 0.60 0.77

T1 (17) 35.3% 64.7% 23.5% 76.5%

T2 (29) 44.8% 55.2% 13.8% 86.2%

T3 (1) 0% 100% 0.0% 100%

T4 (1) 100% 0% 0.0% 100%

Tumour grade (n = 48) 0.60 0.40

Grade 1 (1) 0% 100% 0% 100%

Grade 2 (28) 39.3% 60.7% 11.5% 88.5%

Grade 3 (19) 47.4% 52.6% 26.3% 73.7%

Grade 4 (0) 0% 0% 0% 0%

Nodal status (n = 48) 0.40 0.84

N0 (37) 43.2% 56.8% 18.9% 81.1%

N1 (8) 37.5% 62.5% 12.5% 87.5%

N2 (2) 0% 100.0% 0% 100.0%

N3 (1) 100% 0% 0% 100%

Recurrence (n = 48) 1.00 0.70

Yes (22) 40.9% 59.1% 18.2% 81.8%

No (26) 43.5% 56.5% 13.0% 87.0%

Mortality (n = 48) 1.00 1.00

Yes (23) 39.1% 60.9% 17.4% 82.6%

No (25) 38.1% 61.9% 14.3% 85.7%

Age (n = 48) 1.00 0.06

>70 (20) 40.0% 60.0% 5.0% 95.0%

<70 (28) 44.0% 56.0% 28.0% 72.0%

Based on TNM staging, median tumour size was T2. Nodaldeposits were detected in 10 (21%) patients. The mediantumour grade was grade 2 (moderately differentiated).

From the 50 samples, 92% were T1 or T2 (17 T1, 29 T2).Histological grade was grade 2 or 3 in 94% (28 grade 2, 19grade 3).

Recurrence (R) was identified in 22 patients (46%), withan annual rate of 14% and a median time to recurrence of404 days. Mortality (M) was 48% (23 patients), with mediantime to mortality 871 days. Recurrence accounted for mor-tality in 22 cases. Within the study period, 17 patients withadenocarcinoma (53%) died (16 patients with recurrence),whilst six patients with SCC (33%) died (all six with recur-rence).

All samples were initially scored for nuclear (N) andcytoplasmic (C) staining intensity and the percentage ofcells stained. Staining was satisfactory in 48 samples (96%).

In the initial analysis (see Table 1) of staining, 41 samples(85%) displayed some staining of the nucleus. However, amajority stained only very mildly, in a small percentage ofcells, with a distribution that did not differentiate betweentumour and surrounding parenchyma. Only those cells

that demonstrated moderate or intense staining (Fig. 1)demonstrated tumour-parenchyma differentiation. Sub-sequently, in the final analysis, only cells that stained withintensity of moderate or intense were considered positive.In the final analysis (see Table 1), N staining was scoredpositive in eight samples in total (17%), including fouradenocarcinomas (13%) and four squamous cell carcino-mas (22%) (Fig. 2). We noticed a trend towards increasednuclear staining in higher grade tumours (12% N+ in grade2 tumours vs. 26% N+ in grade 3 tumours) however this didnot prove significant. Similarly we noticed a difference innuclear staining when considering patient age (tumoursfrom patients <70 years, N+ in 5% vs. in tumours frompatients >70 years N+ in 28%). Positive N staining was seenin four cases where recurrence was subsequently diag-nosed (18%), and in four cases where patients eventuallydied (17%) (Figs. 3 and 4).

The initial analysis (Table 1) yielded similar results forcytoplasmic staining as nuclear staining. Staining waspresent to some degree in 47 of 48 (98%) valid samples.C staining tended to be more widespread than nuclearstaining: diffuse staining of 75–100% of cells was seen in 16

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Figure 2. Staining by tumour type.

Figure 3. Staining vs. mortality.

(33%) cases. However, 56% of the samples demonstratedmild staining only, including 100% of those samples whichstained diffusely (75–100%). Intense C staining was seenin 14 (29%) valid samples, and of those 93% (13/14 samplesscored “intense”) did so only very focally (1st quar-tile, 1–25% of cells stained). It would seem that sampleswere either diffusely, mildy positive, or focally, intenselypositive (Fig. 1), with very little in between and low inten-sity staining did not differentiate between tumour andparenchyma. In the final analysis (Table 1), C staining wasscored as for nuclear staining. C staining was detected in20 cases (41.7%) including nine adenocarcinomas (28%)and 11 squamous cell carcinomas (61%) (Fig. 2). C staining

Figure 4. Staining vs. recurrence.

was identified in nine cases that progressed to recur-rence (41%) and nine cases that ended in death (39%)(Figs. 3 and 4). Both N and C staining were positive infive samples (10%).

In general, we noticed increased staining in SCC com-pared with adenocarcinoma (Fig. 2). C+ staining wasnoticed in 61% of SCC as compared to 30% of adenocarci-nomas. N+ staining was noticed in 22% of SCC vs. 13% ofadenocarcinomas. Whilst trends were noticed in stainingproperties for higher grade tumours and for older patients,there was no trend for mortality or recurrence.

Using the univariate and multivariate Cox propor-tional hazards model, we assessed the predictive value ofCRYAB. We determined the most significant variable onunivariate analysis and this was subsequently included inthe multivariate model with CRYAB. For recurrence, agewas the most significant predictive variable (p = 0.01). Formortality, none of the variables assessed were significanton univariate analysis.

In this study, positive staining for CRYAB failed to pre-dict both recurrence (N stain p = 0.78, C stain p = 0.38,any stain p = 0.08) and mortality (N stain p = 0.86, C stainp = 0.66, any stain p = 0.21) based on univariate and mul-tivariate analysis (see Table 2). Kaplan–Meier analysesrevealed no survival difference detected between patientswhose tumours were CRYAB+ and those whose tumourswere CRYAB (Figs. 7 and 8). This result is contrary to the

findings of other investigators, but remains biologicallyplausible.

Discussion

Our understanding of the molecular basis of tumouri-genesis, the mechanism of action of anti-cancer drugs,and in particular, an appreciation for the impact of avariety of mutations commonly encountered in humancancers on treatment response has rapidly expanded inthe last decade. There has been a corresponding increasein the cancer literature describing genetic, and pro-tein based markers which have prognostic potential andare predictive of responses to specific agents. The fur-ther development of prognostic molecular markers andtherapeutic agents is the next frontier of cancer treat-ment.

CRYAB is one of 10 members of the small heat shock pro-tein (sHSP) family, all of which share a common C terminalmotif, the alpha crystallin domain. CRYAB is a ubiquitousstress inducible molecular chaperone, the expression ofwhich is upregulated by heat, radiation, oxidative stressand anticancer drugs [6]. Apoptosis may be induced by avariety of mechanisms, and it appears that CRYAB displayssome redundancy in interacting with multiple pathways,at multiple levels. These include and are not limited to thecaspase pathway, death receptors, p53 pathway, MAPKpathway and tumour angiogenesis, some of which havebeen successfully targeted for anticancer therapy.

The caspase cascade is an endpoint of several apoptoticpathways and may be activated by the development ofmitochondrial permeability, as well as the binding of spe-cific ligands to death receptors. CRYAB has been shown

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Table 2. Univariate and Multivariate Analysis.

Risk factors Univariate Multivariate

HR 95% CI p value HR 95% CI p value

Recurrence

Adenocarcinoma 1.63 0.51–5.24 0.41

T score 1.88 0.74–4.76 0.19

N score 0.98 0.44–2.19 0.97

Nuclear stain 0.81 0.19–3.49 0.78 0.68 0.15–3.12 0.78

Cytoplasmic stain 0.68 0.29–1.60 0.38 0.74 0.31–1.82 0.51

Any stain 0.18 0.18–1.09 0.08 0.57 0.22–1.48 0.25

Mortality

Adenocarcinoma 2.63 0.82–8.44 0.1

T score 1 0.6–1.6 0.97

N score 0.69 0.3–1.58 0.38

Nuclear stain 0.93 0.42–2.15 0.86 0.99 0.42–2.39 1.0

Cytoplasmic stain 0.82 0.34–1.96 0.66 0.90 0.35–2.29 0.82

Any stain 0.54 0.21–1.40 0.21 0.64 0.21–1.69 0.37

to inhibit the proximal execution element in the caspasecascade, thereby promoting cell survival [9,10].

Death receptors bind various ligands, one of which isTRAIL (TNF related apoptosis inducing ligand). TRAILpreferentially induces apoptosis in transformed (i.e.malignant) cells, which itself hints at potential therapeu-tic roles. Many neoplasms are resistant to TRAIL inducedapoptosis. CRYAB correlates with TRAIL resistance: as acorollary, selective inhibition of CRYAB sensitises cancercells to TRAIL and is anti-neoplastic, and this supports apotential therapeutic role [11].

CRYAB induced inhibition of apoptosis may also arisedue to sequestration of p53 in the cytoplasm. By preven-ting mitochondrial translocation, the pro-apoptotic actionof p53 is suppressed [12]. Further, given that p53 is upre-gulated by ERK (part of the MAP kinase cascade) [13], itwould appear that again CRYAB plays a role at multiplelevels of a single pathway, in this instance by also preven-ting RAS, and hence ERK activation [14]. The oncogenicpotential of CRYAB is reinforced by the fact that selectiveinhibitors of the MAP kinase pathway appear to hold somepromise as viable anti-cancer agents [14–16].

Many recent revelations in tumourigenesis haveexplored the role of angiogenesis and neovascularisation[18]. It appears that here too, CRYAB is involved. VEGF(vascular endothelial growth factor) expression can betriggered in tumourigenesis by the tumour environment(mtaFiecpm

Dimberg et al. demonstrated that CRYAB was expressedin tumour vessels in lung cancer, but not in normal lungvessels. Furthermore, knockout of CRYAB function wasassociated with inhibition of tubular morphogenesis, andas a result he new vessels were not able to maintain thetumour.

Unfortunately, not every aspect of the role of CRYAB isclear. Lin et al. have demonstrated that CRYAB is part ofthe E3 ligase responsible for the determination of speci-ficity of the ubiquitin protein. They have shown that lossof the E3 ligase (i.e. CRYAB) is oncogenic, specifically inthose breast cancer cell lines that over-express cyclin D1[19]. This confuses the picture somewhat, in suggestingthat the role of CRYAB is sufficiently diverse as to allowoncogenicity by virtue of its own action, as well as tumoursuppression, depending on the interaction of that role withother cellular mechanics. This diversity would account forsome of the inconsistent findings related to CRYAB in var-ious cancers, including our own findings in lung cancer.

Cherneva et al. have recently examined the expres-sion of CRYAB in a series of patients with NSCLC [30].They used a tissue microarray with samples from 146patients with NSCLC and concluded that there was a dis-tinctive expression profile for adenocarcinoma and SCC.Further, they suggest that nuclear staining was more com-monly detected in advanced stages (p = 0.08) and was abiomarker of aggressive biology (p = 0.042). Nuclear stain-iamofnsano

e.g. hypoxia) or by genetic mutations (including K-rasutations, which are common in lung cancer). We know

hat VEGF can be targeted and blocked with a monoclonalntibody such as bevacizumab (Avastin; Genentech, Sanrancisco CA), with prolongation of life in many cancers,ncluding lung cancer [17]. However, there appear to bescape mechanisms by which tumour angiogenesis canontinue in spite of VEGF blockade, CRYAB appears tolay a downstream role in neovascularisation, and thusay present a new opportunity to prevent tumour growth.

ng was associated with shorter overall survival (p = 0.002)nd they finally conclude that CRYAB is an independentarker of prognosis on multivariate analysis. The results

f this study contrast strongly with ours. Cherneva et al.ound that nearly all of the studied tumours displayeduclear staining, with an almost equal level of cytoplasmictaining. They found that most tumours stained diffuselynd homogenously. In our final analysis, we found thatuclear staining of tumour cells was uncommon (16%f samples), and that cytoplasmic staining, whilst more

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Figure 5. Staining vs. tumour size.

common (40% of samples), tended to be focally intense.The tumours studied were primarily SCC (66%) as com-pared to adenocarcinoma (24%) whereas our populationincluded primarily adenocarcinoma. In our analysis wedid note an increased tendency for SCC to demonstratepositive staining. Their sample included patients withstage III (i.e. >T3, >N2) disease in 45%. Thirty-five of ourpatients (73%) were N0, and 92% of samples in our studywere from T1 or T2 tumours (Fig. 5). It is certainly possiblethat given the significant differences in the studied popu-lations, that these results were influenced accordingly. Wehave studied a group of predominantly stage I–II patients.It would be interesting to compare our results to otherlimited subsets of early stage tumours. We believe thatthe prognostic value and the benefit to the patients bypotentially “up-staging” their tumours, leading to moreaggressive treatment of early stage tumours, is only pos-sible to assess by studying a group of patients as we have,with early stage, limited disease.

Our data has indicated that there may be trends towardsincreased levels of CRYAB in higher grade lung cancers(Fig. 6). Further, tumours from older patients appear toshow greater propensity to staining. It is possible thereforethat CRYAB is associated with aggressive tumour biology,as Cherneva et al. have suggested. These findings, despiteappreciable numerical differences, were not significant inour study. The main limitation of our study is the small

Figure 7. Kaplan–Meier curve for recurrence (any stain).

larger populations will hopefully lead to more definiteconclusions.

Given the evidence for the mechanisms of action ofCRYAB, it is little surprise that it is involved in a broadrange of human epithelial carcinomas [20–22]. CRYABappears to be associated with renal [22], breast [14,24],thyroid [25], nasopharyngeal [26], head and neck [27,28]and hepatocellular [29] carcinomas. Recent reports havealso suggested that CRYAB is associated with local tumourinvasion and possibly overall survival in certain lungcancer patients, contrary to our own findings [30]. Withrespect to these tumours, it appears that CRYAB mayrepresent a prognostic factor, it may be associated withmore aggressive tumour growth, it may predict nodalinvolvement, and it may also account for resistance tochemotherapy. Breast cancer has been well studied in rela-tion to CRYAB. Chelouche-Lev et al. showed from a largestudy of more than 600 patients that there is a stronglysignificant association between CRYAB expression andlymph node involvement. Further, the intensity of stainingwas significantly correlated with shorter survival, whichraises the prospect of a truly quantitative impact of CRYABin tumourigenesis and metastatic spread [24].

sample size, which has limited our analysis. Studies with

Figure 6. Staining vs. histological grade.

Figure 8. Kaplan–Meier curve for mortality (any stain).

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The future of lung cancer treatment, and indeed thatof nearly all human malignancies, would appear to bein the use of molecular staging and targeted therapy.Newly identified markers such as TIMP-3 (tissue inhibitorof metalloproteinase-3) have been suggested as progno-stic markers in lung cancer [31]. The absence of TIMP-3appears to be correlated with, in particular, nodal involve-ment, which would logically follow from the role of MMPs(matrix metalloproteinases) in the metastatic cascade ofhuman cancer. Potti et al. have also recently described thedevelopment of a lung “metagene” model which describesa variety of gene expression profiles in lung cancer. Themetagene model has been shown to consistently, andsignificantly outperform current models predicated onclinical data by almost 30% in terms of prognostic accuracy[32].

We await with interest the outcomes of newer studiesincluding those using the lung metagene model designedto determine whether there is benefit in treating stage 1patients with high metagene scores. The other project ofinterest at present is the BATTLE trial (biomarker basedapproaches of targeted therapy for lung cancer elim-ination) which involves the adaptive randomisation ofpatients based on an 11 marker profile.

Conclusion

Iwpatghts

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[9] Kamradt MC, Chen F, Cryns VL. The small heat shock proteinalpha B-crystallin negatively regulates cytochrome c- andcaspase-8-dependent activation of caspase-3 by inhibiting itsautoproteolytic maturation. J Biol Chem 2001;276:16059–63.

[10] Kamradt MC, Chen F, Sam S, Cryns VL. The small heatshock protein alpha B-crystallin negatively regulates apopto-sis during myogenic differentiation by inhibiting caspase-3activation. J Biol Chem 2002;277:38731–6.

[11] Kamradt MC, Lu M, Werner ME, Kwan T, Chen F, StroheckerA, et al. The small heat shock protein alpha B-crystallinis a novel inhibitor of TRAIL-induced apoptosis that sup-presses the activation of caspase-3. J Biol Chem 2005;280:11059–66.

[12] Liu S, Li J, Tao Y, Xiao X. Small heat shock protein alphaB-crystallin binds to p53 to sequester its translocation tomitochondria during hydrogen peroxide-induced apoptosis.Biochem Biophys Res Commun 2007;354:109–14.

[13] Li DW, Liu JP, Mao YW, Xiang H, Wang J, Ma WY, et al.Calcium-activated RAF/MEK/ERK signaling pathway medi-ates p53-dependent apoptosis and is abrogated by alphaB-crystallin through inhibition of RAS activation. Mol BiolCell 2005;16:4437–53.

[14] Moyano JV, Evans JR, Chen F, Lu M, Werner ME, YehielyF, et al. AlphaB-crystallin is a novel oncoprotein that pre-dicts poor clinical outcome in breast cancer. J Clin Invest2006;116:261–70.

[15] Gruvberger-Saal SK, Parsons R. Is the small heat shockprotein alphaB-crystallin an oncogene? J Clin Invest2006;116:30–2.

[16] Sebolt-Leopold JS. Advances in the development of

t seems likely that CRYAB interacts with such redundancyith the machinery of cell death that the influence of itsresence or absence may depend on other molecular char-cteristics of particular tumour cells. At present, attemptso identify over-expression of CRYAB as a potential pro-nostic marker are the main thrust of various studies,owever the potential for these death-preventing proteins

o become the targets of therapy remains to be demon-trated.

eferences

[1] Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics,2002. CA Cancer J Clin 2005;55:74–108.

[2] West H, Lilenbaum R, Harpole D, Wozniak A, Sequist L.Molecular analysis-based treatment strategies for the man-agement of non-small cell lung cancer. J Thorac Oncol2009;4:s1029–39.

[3] Sienel W, Dango S, Ehrhardt P, Eggeling S, Kirschbaum A,Passlick B. The future in diagnosis and staging of lung cancer.Molecular techniques. Respiration 2006;73:575–80.

[4] Blanchon F, Grivaux M, Asselain B, Lebas FX, Orlando JP,Piquet J, et al. 4-Year mortality in patients with non-small-cell lung cancer: development and validation of a prognosticindex. Lancet Oncol 2006;7:829–36.

[5] Sculier JP, Chansky K, Crowley JJ, Van Meerbeeck J, Gold-straw P. The impact of additional prognostic factors onsurvival and their relationship with the anatomical extentof disease expressed by the 6th edition of the TNM classi-fication of malignant tumors and the proposals for the 7thedition. J Thorac Oncol 2008;3:457–66.

[6] Parcellier A, Schmitt E, Brunet M, Hammann A, SolaryE, Garrido C. Small heat shock proteins HSP27 andalphaB-crystallin: cytoprotective and oncogenic functions.Antioxid Redox Signal 2005;7:404–13.

cancer therapeutics directed against the RAS-mitogen-activated protein kinase pathway. Clin Cancer Res 2008;14:3651–6.

[17] Jain RK, Duda DG, Clark JW, Loeffler JS. Lessons from phaseIII clinical trials on anti-VEGF therapy for cancer. Nat ClinPract Oncol 2006;3:24–40.

[18] Dimberg A, Rylova S, Dieterich LC, Olsson AK, SchillerP, Wikner C, et al. AlphaB-crystallin promotes tumorangiogenesis by increasing vascular survival during tubemorphogenesis. Blood 2008;111:2015–23.

[19] Lin DI, Barbash O, Kumar KG, Weber JD, Harper JW, Klein-Szanto AJ, et al. Phosphorylation-dependent ubiquitinationof cyclin D1 by the SCF(FBX4-alphaB crystallin) complex. MolCell 2006;24:355–66.

[20] Iwaki T, Kume-Iwaki A, Goldman JE. Cellular distributionof alpha B-crystallin in non-lenticular tissues. J HistochemCytochem 1990;38:31–9.

[21] Klemenz R, Andres AC, Frohli E, Schafer R, Aoyama A.Expression of the murine small heat shock proteins hsp 25and alpha B crystallin in the absence of stress. J Cell Biol1993;120:639–45.

[22] Pinder SE, Balsitis M, Ellis IO, Landon M, Mayer RJ, Lowe J.The expression of alpha B-crystallin in epithelial tumours: auseful tumour marker? J Pathol 1994;174:209–15.

[24] Chelouche-Lev D, Kluger HM, Berger AJ, Rimm DL,Price JE. AlphaB-crystallin as a marker of lymph nodeinvolvement in breast carcinoma. Cancer 2004;100:2543–8.

[25] Mineva I, Gartner W, Hauser P, Kainz A, Loffler M, Wolf G,et al. Differential expression of alphaB-crystallin and Hsp27-1 in anaplastic thyroid carcinomas because of tumor-specificalphaB-crystallin gene (CRYAB) silencing. Cell Stress Chap-erones 2005;10:171–84.

[26] Lung HL, Lo CC, Wong CC, Cheung AK, Cheong KF, WongN, et al. Identification of tumor suppressive activity byirradiation microcell-mediated chromosome transfer and

OR

IGIN

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AR

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766 Campbell-Lloyd et al. Heart, Lung and CirculationAlpha-B crystallin in lung cancer 2013;22:759–766

involvement of alpha B-crystallin in nasopharyngeal carci-noma. Int J Cancer 2008;122:1288–96.

[27] Boslooper K, King-Yin Lam A, Gao J, Weinstein S, Johnson N.The clinicopathological roles of alpha-B-crystallin and p53expression in patients with head and neck squamous cellcarcinoma. Pathology 2008;40:500–4.

[28] Chin D, Boyle GM, Williams RM, Ferguson K, Pandeya N,Pedley J, et al. Alpha B-crystallin, a new independent markerfor poor prognosis in head and neck cancer. Laryngoscope2005;115:1239–42.

[29] Tang Q, Liu YF, Zhu XJ, Li YH, Zhu J, Zhang JP, et al.Expression and prognostic significance of the alpha B-crystallin gene in human hepatocellular carcinoma. HumPathol 2009;40:300–5.

[30] Cherneva R, Petrov D, Georgiev O, Slavova Y, TonchevaD, Stamenova M, et al. Expression profile of the smallheat-shock protein alpha-B-crystallin in operated-on non-small-cell lung cancer patients: clinical implication. Eur JCardiothorac Surg 2010;37:44–50.

[31] Mino N, Takenaka K, Sonobe M, Miyahara R, Yanagi-hara K, Otake Y, et al. Expression of tissue inhibitor ofmetalloproteinase-3 (TIMP-3) and its prognostic signifi-cance in resected non-small cell lung cancer. J Surg Oncol2007;95:250–7.

[32] Potti A, Mukherjee S, Petersen R, Dressman HK, Bild A,Koontz J, et al. A genomic strategy to refine prognosisin early-stage non-small-cell lung cancer. N Engl J Med2006;355:570–80.