Khyati N. Shah1*, Elizabeth A. Wilson1*, Ritu Malla , Howard L. Elford and Jesika … · 2015. 9....
Transcript of Khyati N. Shah1*, Elizabeth A. Wilson1*, Ritu Malla , Howard L. Elford and Jesika … · 2015. 9....
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Targeting ribonucleotide reductase M2 and NF-kB activation with Didox to circumvent tamoxifen resistance in breast cancer
Khyati N. Shah1*, Elizabeth A. Wilson1*, Ritu Malla1, Howard L. Elford2 and Jesika S. Faridi1
1Department of Physiology and Pharmacology, Thomas J. Long School of Pharmacy & Health
Sciences, University of the Pacific, Stockton CA 95211
2 Molecules for Health, Inc., Richmond, VA 23219, USA.
*K.N. Shah and E.A. Wilson are co–first authors who contributed equally to this article.
Running Title: Didox circumvents tamoxifen resistance
Keyords: RRM2, Didox, Tamoxifen
Grant Support: This work was supported, in part, by a Graduate Student Research
Grant (K.N. Shah), a SAAG intramural fellowship (J.S. Faridi), and the
Thomas J. Long School of Pharmacy and Health Sciences, University of the
Pacific.
Corresponding Author: Jesika S. Faridi, PhD Thomas J. Long Long School of Pharmacy
University of the Pacific
751 Brookside Road
Stockton CA 95211
Phone: (209) 946-2964
Fax: (209) 946-2857
Email: [email protected]
Disclosure of Potential Conflicts of Interest: Dr. Howard L. Elford is an employee and shareholder in Molecules for Health Inc.
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Abstract: Tamoxifen is widely used as an adjuvant therapy for patients with estrogen receptor (ER-α)
positive tumors. However, the clinical benefit is often limited due to the emergence of drug
resistance. In this study, overexpression of ribonucleotide reductase M2 (RRM2) in MCF-7 breast
cancer cells resulted in a reduction in the effectiveness of tamoxifen, through downregulation of ER-
α66 and upregulation of the 36 kDa variant of ER (ER-α36). We identified that NF-κB, HIF-1α, and
MAPK/JNK are the major pathways that are affected by RRM2 overexpression and result in
increased NF-κB activity and increased protein levels of EGFR, HER2, IKK’s, Bcl-2, RelB, and p50.
RRM2 overexpressing cells also exhibited higher migratory and invasive properties. Through time-
lapse microscopy and protein profiling studies of tamoxifen-treated MCF-7 and T-47D cells, we have
identified that RRM2 along with other key proteins is altered during the emergence of acquired
tamoxifen resistance. Inhibition of RRM2 using siRRM2 or the ribonucleotide reductase (RR)
inhibitor Didox not only eradicated and effectively prevented the emergence of tamoxifen resistant
populations, but also led to the reversal of many of the proteins altered during the process of acquired
tamoxifen resistance. Since Didox also appears to be a potent inhibitor of NF-κB activation,
combining Didox with tamoxifen treatment cooperatively reverses ER-α alterations and inhibits NF-
κB activation. Finally, inhibition of RRM2 by Didox reversed tamoxifen-resistant in vivo tumor
growth and decreased in vitro migratory and invasive properties, revealing a beneficial effect of
combination therapy that includes RRM2 inhibition to delay or abrogate tamoxifen resistance.
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Introduction: Estrogen receptor-α (ER-α) plays a fundamental role in the etiology and the progression of
human breast cancer (1). Many therapies have been designed to inhibit the tumor-promoting effects
of ER-α; however, tamoxifen has been the drug of choice for all stages of ER-positive breast cancer.
As adjuvant therapy, tamoxifen reduces the risk of recurrence and improves overall survival (OS) in
early breast cancer patients (2). Tamoxifen arrests cells in G0/G1 and decreases expression of several
ERα target genes (3). Apart from blocking classical ER action, tamoxifen also induces DNA damage
and apoptosis in ER-positive cells (4). Despite these benefits, some tumors recur due to acquired
tamoxifen resistance giving rise to a sub-population of unresponsive cells (5). Several mechanisms of
acquired tamoxifen resistance have been reported including down-regulation of ER-α expression (6).
Tamoxifen resistance has also been linked to crosstalk between ER-α and signaling pathways
involving Epidermal Growth Factor receptor (EGFR), HER2/ERBB2, or insulin-like growth factor
receptor-I (IGFI-R) (7).
ER-α66 is the classical ER-α of 66-kDa and is often referred to as simply “ER”. Cells which
have high levels of ER-α66 are often termed ER-positive, while those lacking ER-α66 are called ER-
negative. Clinical evidence suggests that approximately 40% of ER-α66 positive breast cancers also
express a 36-kDa variant of ER-α (ER-α36), and this subset of patients is less likely to benefit from
tamoxifen treatment (8, 9). Alternatively, endocrine resistant cells can develop a compensatory
signaling pathway downstream of ER which results in hyperproliferation and increased cancer cell
survival. In this type of resistance, ER-α36 may promote downstream signaling, such as the
phosphatidylinositol 3-kinase (PI3K) pathway (10). We have previously shown that breast cancer
cells overexpressing activated AKT exhibit tamoxifen-stimulated cell proliferation and enhanced cell
motility. Moreover, we identified that RRM2 was a key contributor to AKT-induced tamoxifen
resistance (11).
Tamoxifen chemotherapy initially arrests tumor growth, but upon acquiring resistance, DNA
synthesis is reactivated. The first step in DNA synthesis is conversion of ribonucleotides to their
corresponding deoxyribonucleotides, catalyzed by the enzyme ribonucleotide reductase (RR) (12).
This reaction is also the rate limiting step in DNA synthesis and cell division, and its activity is
closely correlated with tumor growth rate and cell division (12). RR is composed of RRM1 and
RRM2. Although the levels of the RRM1 protein does not change substantially during the cell cycle,
there is an S-phase correlated increase in the RRM2 protein (13). The activity of RR, and therefore
DNA synthesis and cell proliferation, are controlled by RRM2. In contrast, p53R2 (RRM2B) is
involved in supplying dNTPs for DNA repair during mitochondrial DNA synthesis in the G0/G1
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phase of the cell cycle (14). Overexpression of the RRM2 subunit has been associated with malignant
transformation and confers gemcitabine resistance (15). RRM2 functions in coordination with the S
phase checkpoint to regulate DNA damage, replication stress, and genomic instability including
mutagenesis (16, 17). Since chronic incubation of tamoxifen induces DNA damage, cells may
upregulate RRM2 in an attempt to repair the DNA damage and are thus able to survive as a resistant
population.
Due to the DNA-damaging property of tamoxifen, combination therapies that enhance
tamoxifen activity are of clinical benefit. RRM2 is an attractive target for combination therapy as it is
overexpressed in breast cancer and contributes to development of resistance (11). Didox (3, 4-
dihydroxybenzohydroxamic acid), is a strong inhibitor of RR that interferes with DNA synthesis and
repair by blocking the production of deoxyribonucleotides and has demonstrated anti-tumor effects
for decades (12, 18-20). Phase I/II clinical trial studies in cancer patients showed minimal toxicity
and determined that the maximum tolerated dose of Didox is 6 g/m2, yielding peak plasma levels of
425 μM (21-22). Didox can be tolerated by cancer patients at high dosages without major side
effects, making Didox attractive for use in clinical applications. We previously reported that the
combination of Didox and tamoxifen significantly reduced cell proliferation in AKT-induced
tamoxifen resistance (11).
Here, using gain and loss of RRM2, we show that RRM2 is sufficient to confer tamoxifen
resistance in breast cancer cells. We demonstrate that RRM2 is associated with increased NF-κB
activity, ER-α alterations, HER2 and EGFR upregulation, and increases in anti-apoptotic pathways.
Finally, Didox significantly inhibits tamoxifen induced in vivo tumor growth. Our results show that
Didox works synergistically with tamoxifen for the treatment and prevention of resistant breast
cancer cells. Our data provide a preclinical rationale for evaluating tamoxifen in combination with
Didox for breast cancer treatment.
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Materials and methods Cell culture and treatment
MCF-7, T47D, HCC1428, BT483, ZR-75-30, ZR-75-1, SKBR3, BT20, BT549,
HCC2157, MDA-MB-468, and MDA-MB-231 cells were purchased from ATCC between July
and December 2012. Revived cells were used within 15 passages and cultured for less than 6
months. Cells were maintained in DMEM/F12 supplemented with 10% FBS, streptomycin, and
penicillin (Life Technologies). Didox (D) was supplied by Molecules for Health. For experiments,
cells were treated with phenol-red-free, serum-free DMEM/F12 containing 0.1µM ethanol as vehicle
(C), 0.1µM 17β-estradiol (E, Sigma), 1µM 4-hydroxy-tamoxifen (T, Sigma), 30µM D or a
combination of 1µM T + 30µM Didox (T+D) for 24 hours.
Western blot analyses Western blotting was performed as previously described (11, 23). Briefly, cells were
disrupted in RIPA buffer (Sigma) or tumors were homogenized in lysis buffer (Cell Signaling)
supplemented with aprotinin, leupeptin, and okadaic acid (Sigma). Lysates were clarified by
centrifugation, and equal protein (75μg) was used for Western blotting. RRM1, RRM2, RRM2B
(Sigma), p53, ER-α66 (Santa Cruz), ER-α36 (Alpha Diagnostics), GAPDH, EGFR, HER2, Bcl-2,
pAKT, pER (S167) pERK, total ERK, total γH2AX, IKK sampler kit, NF-κB sampler kit, PARP,
Caspase-9 (Cell Signaling), and pγ-H2AX (Millipore) were used for immunoblotting with secondary
antibodies conjugated with IRDye 800CW or 680RD (LI-COR Biosciences) and visualized with a
LI-COR Odyssey Imager.
Meta-analysis of Breast Cancer Datasets RRM2 expression profiles and clinico-pathologic data of breast cancer patients were obtained
from publicly available breast cancer microarray (25-29) and NCBI GEO (30-34) datasets. Data
from tamoxifen-treated ER-positive patients were classified as tamoxifen-resistant if metastasis was
indicated. Oncomine was used for data collection and analyses. P
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Generation of Stable RRM2 overexpressing MCF-7 breast cancer cell lines MCF-7 cells were transfected with pCMV6-RRM2-myc-DDK or vector (Origene) using
Fugene HD (Roche) and grown under geneticin selection after 48 hours. Clones overexpressing
RRM2 were expanded to generate stable expressing clones MCF-7/R2.1, MCF-7/R2.3 and
population MCF-7/R2.pool. The population MCF-7-VC cells stably express vector. Stable cells were
routinely tested and authenticated according to the ATCC guidelines.
Transcription factor reporter assays MCF-7/R2.1 and MCF-7 VC cells were reverse transfected onto the Cignal Finder 10-
Pathway Reporter Array or the Cignal NF-κB Luciferase Reporter using SureFECT according to
manufacturer’s protocol (Qiagen). Cells were treated for 24 hours with C, T, D, or T+D, as
indicated, in phenol-red-free, serum-free DMEM/F12. Relative transcriptional activity was measured
using the Dual Luciferase Assay (Promega). Three independent transfections were performed.
siRNA-mediated suppression of RRM2 siRNA oligos targeting RRM2 (siRRM2) were designed and synthesized at Genentech Inc.
Nontargeting siRNA and Dharmafect-1 were purchased (Dharmacon). Cells were transfected with
siRNA by reverse transfection according to the manufacturers' directions. Transfection efficiencies
were evaluated relative to nontargeting control by RT-qPCR, and the suppression of RRM2
expression was sustained through day 7 for all breast cancer cell lines tested.
Pathway-specific expression arrays Total RNA was isolated using RNeasy according to the manufacturer’s protocol
(Qiagen). All RNA samples were examined for their concentration, purity, and integrity. The human
Breast Cancer PCR array and the ECM and Adhesion Molecule (SABioscience) was used to assess
the expression of 84 breast cancer genes according to the manufacturer’s instructions. Data shown
represent the average of two replicates and were normalized using the previously validated
housekeeping gene RPL13A levels (23).
Establishment and treatment of acquired tamoxifen resistant cells
MCF7 and T-47D were treated with 1μM tamoxifen and dose was increased every 10 days
until 5μM. Once established, TamR resistant cells were maintained in continuous culture with 1μM
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tamoxifen. For protein profiling, replicate plates were treated with tamoxifen in this manner and
lysates were collected over 30 days. Control lysates were collected after 3 days of treatment with
vehicle. TamR lysates were collected with continuous treatment with 1μM tamoxifen alone or
combined with Didox for five days.
Cell proliferation and motility assays
For proliferation, 1000 cells/well were plated in phenol-red-free, DMEM-F12 medium with
2% CSS. After 24 hours, treatment media were replenished on alternate days. On assay days,
CellTiter-96 Aqueous One Solution (Promega) was added, incubated for 1 hour, and measured at
490nm. For the colony formation assay, 3000 cells/well were cultured in 5% FBS phenol red-free
DMEM. The following day, the cells were treated and allowed to grow for 14 days. Colonies were
stained with crystal violet and analyzed. A modified scratch assay was performed by plating 20,000
cells in wells containing inserts (IbIDI Martinsried). After 24 hours inserts were removed, and the
percent open area was calculated after 20 additional hours. Cell migration and invasion experiments
were carried out using the QCM™ 24-well Colorimetric Cell Migration kit or the Invasion Assay kit
(Chemicon) according to manufacturer’s protocols. These experiments were conducted in triplicate
and data is shown as the mean ± SEM.
Synergy determinations The IC50 value of tamoxifen and Didox was determined for MCF-7 VC, MCF-7TamR and
RRM2 overexpressing MCF-7 cells (MCF-7/R2.1). Based on the IC50 value, fixed dose ratios were
used to determine five different drug combinations (i.e. 2X, 1X, X, X/2, X/4, where X: IC50 of an
individual drug). Synergistic, additive, or antagonistic effects were determined using the combination
index (CI) method developed by Chou and Talalay. Synergy, additivity, and antagonism are
indicated by CI values of 1, respectively (24).
Xenograft studies All animal procedures were approved by the Institutional Animal Care and Use Committee of
the University of the Pacific. RRM2 overexpressing MCF-7 cells (4 x 106/site) were subcutaneously
injected into the flank of ncr nude female ovariectomized nude mice (6-15 tumors/group). Mice
were implanted with estrogen (0.72 mg/pellet, Innovative Research of America, (IRA)) and
tamoxifen as indicated (5 mg/pellet, IRA). Beginning day 7 as indicated, daily Didox (N, 3,4-
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Dihydroxybenzohydroxamic, 200 or 425 mg/kg/day) was injected intraperitoneally after compound
was freshly dissolved in water and filtered. The in vivo tumor volume was approximated using the
formula for an ellipsoid (4/3π(r1)2(r2)) where r1
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Results: RRM2 expression inversely correlates with ER expression in breast cancer RRM2 expression is upregulated in cancer and is associated with increased tumor
aggressiveness, poor prognosis, and chemoresistance (11, 14-16). Here, we demonstrate that RRM2
expression is significantly upregulated in ER-negative as compared to ER-positive breast cancer
cells, as is a 36kDa variant of ER (ER-α36) (Fig. 1A). Interestingly, of the ER-positive cells, ZR-75-
1 alone has high levels of RRM2 and ER-α36. We have previously shown that ZR-75-1 cells exhibit
tamoxifen-resistant cell proliferation and that inhibition of RRM2 using siRRM2 not only restores
tamoxifen sensitivity, but it also represses the DNA repair enzymes that protect these cells from
tamoxifen-induced apoptosis (11).
Meta-analysis of five different Oncomine datasets revealed that RRM2 is highly expressed in
ER-negative tumors (Fig. 1B, Supplementary Table S1A) (25-29). Using survival data from
tamoxifen-treated ER-positive patients, RRM2 mRNA expression was determined to be significantly
higher in patients who experienced tumor relapse (Fig. 1C, Supplementary Table S1B) (30-34).
Kaplan-Meier survival plots (KMplots) of tamoxifen-treated ER-positive patients indicate that
increased RRM2 expression is strongly correlated with reduced RFS and OS (Fig. 1D, 1E) (35).
Stable overexpression of RRM2 reduces tamoxifen sensitivity in MCF-7 cells We previously reported that inhibition of RRM2 reverses tamoxifen resistance in breast
cancer cells (11). To strengthen the association between RRM2 and tamoxifen resistance, we
transiently overexpressed RRM2 in ER-positive, tamoxifen-sensitive MCF-7 and T-47D cells and
confirmed RRM2 expression by Western blot analysis (Supplementary Fig. S1). Relative to vector
control cells (VC), RRM2 overexpressing MCF-7 and T-47D cells exhibit reduced tamoxifen
sensitivity and higher IC50 values resulting in resistance ratios of 3.2-3.3. Stable RRM2
overexpressing (clones MCF-7/R2.1, MCF/R2.2, and pool population MCF-7/R2.Pool) and vector
control (MCF-7 VC) MCF-7 cells were then generated (Supplementary Fig. S2). As expected, MCF-
7 VC cell proliferation was significantly inhibited with 1µM tamoxifen. In contrast, RRM2
overexpressing MCF-7 cells demonstrated a loss of sensitivity to tamoxifen. Treatment with the RR
inhibitor Didox inhibited the cell proliferation of MCF-7/R2 cells when given alone and was even
more potent when given in combination with tamoxifen. Interestingly, RRM2 overexpressing cells
had a slightly diminished estrogen response as compared to MCF-7 VC cells. Since all three RRM2
overexpressing stable lines had similar properties, we used MCF-7/R2.1 cells for subsequent
experiments.
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RRM2 overexpression increases NF-κB activity and gene transcription in breast cancer cells RRM2 overexpression specifically upregulates the NF-κB, HIF1-α, and MAPK/JNK
proliferation and differentiation pathways (Fig. 2A). Interestingly, Didox significantly inhibited NF-
κB (p
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S536 indicating increased NF-κB activity. When examining apoptotic proteins, we observed an
initial spike (days 1- 5) then decrease (day 10) in cleaved PARP and Caspase-9 leading to the
increase in the anti-apoptotic factor Bcl-2 (Fig. 3D). Finally, the emergence of tamoxifen resistant
populations occurred around day 15 which coincides with the activation of pERK1/2 and pAKT
(S473) kinases.
Combining tamoxifen and Didox circumvents resistance via cooperatively reducing ER alterations and NF-κB signaling
To evaluate the efficacy of tamoxifen and Didox (T+D) combination therapy on the
emergence of tamoxifen resistance, time-lapsed images were captured and significant cell rounding
(indicative of cell death) was observed as early as day 1. Treatment with T+D prevented the
emergence of tamoxifen resistance in parental cells and successfully eradicated TamR lines (Fig. 4A,
4B). Didox reduced RRM2, ER-α36 and S167 phosphorylation of ERα, and simultaneously
increased ER-α66 levels (Fig. 4C, 4D). Moreover, combination therapy induced apoptosis as
indicated by increased S139 phosphorylation of γ-H2AX, increased cleavage of PARP and Caspase-
9, reduced levels of the anti-apoptotic Bcl-2, resensitizing RRM2 overexpressing and TamR cells to
tamoxifen-induced cell death (Supplementary Fig. S3). Furthermore, Didox reduced the expression
of EGFR, HER2, pERK, and pAKT in T-47DTamR (Fig. 4D), and all but pAKT in MCF-7TamR
cells (Fig. 4B).
Since there was an observed increase in NF-κB signaling in TamR cells, we sought to
examine the effect of T+D in this pathway. Didox reduces expression of the NF-κB related IKK’s,
p50, RelB, pIKK, p-IκBα, and the S536 phosphorylation of p65 (Fig. 4B, 4D). Using an NF-κB
reporter assay, we found that Didox alone, and more significantly, T+D downregulates NF-κB
activity in MCF-7/R2.1, MCF-7TamR, and T-47DTamR cells (Fig. 4E).
Didox cooperates with tamoxifen to reduce cell proliferation and tumor growth To evaluate the combined effects of T+D, cellular viability was evaluated using the
combination index (CI) (24). Compared to single-agent treatments, the combination of T+D
significantly reduced cell viability in MCF-7/R2.1 and MCF-7TamR cells (Supplementary Fig. S4).
Combination index values of 0.75 and 0.47 indicate synergistic actions of 1.25µM tamoxifen and
37.5µM Didox in MCF-7/R2.1 and MCF-7TamR (but not parental MCF-7) cells respectively on
reducing cell proliferation. Cell viability and toxicity studies indicate that with tamoxifen treatment,
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MCF7/R2.1 and MCF7TamR cells exhibit higher viability, lower toxicity, and higher IC50 than
parental MCF-7 cells (Supplementary Fig. S5).
To determine the in vivo efficacy tamoxifen and Didox (T+D) combination therapy in RRM2
overexpressing breast tumors, MCF-7/R2.1 cells were subcutaneously injected in mice treated with
tamoxifen as indicated. Daily Didox (200 or 425 mg/kg/day) treatment was begun as indicated seven
days after cell injection. Mice treated with tamoxifen exhibited significantly greater tumor growth as
compared to mice treated with the combination of T+D200 (p
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Discussion: Tamoxifen effectively blocks estrogen stimulated tumor growth by inhibiting the activity of
ER-α66 in breast cancer cells (2). Overexpression of tyrosine kinase signaling pathways and
subsequent downregulation of ER-α66 after long term tamoxifen treatment is responsible for the
development of acquired tamoxifen resistance in ER-α66-positive primary tumors (3, 5-8). As well,
tamoxifen resistance can contribute to greater breast tumor aggressiveness (37). Although the
mechanisms underlying acquired tamoxifen resistance are largely unknown, we have demonstrated
that RRM2 is upregulated in AKT-induced tamoxifen resistant breast cancer cells and that inhibition
of RRM2 by siRNA significantly overturns this resistance (11). This current study shows that RRM2
overexpression alone is sufficient to promote tamoxifen resistance, is expressed during the
emergence of de novo tamoxifen resistance (Fig. 3C), and downregulates ESR1 (ER-α) gene and
protein expression (Fig. 2B, 3C). Using in vitro breast cancer cells, patient datasets, tissue
microarrays, and tumor xenografts, we report for the first time that there is an inverse correlation
between RRM2 and ER-α66 and a direct correlation with ER-α36. By inhibiting RRM2 using
Didox, we show that the emergence of acquired tamoxifen resistance is circumvented, while the
downregulation of ER-α66 and upregulation of ER-α36 is reversed.
Our data suggest that ER-α66 downregulation in RRM2 overexpressing breast cancers may
occur through increases in NF-κB activation and signaling. Transrepression of ER by NF-κB has
been proposed as a mechanism by which ER-positive breast tumor cells lose ER expression and,
hence, give rise to a subpopulation of tumor cells that are resistant to endocrine treatment (38, 39).
Furthermore, we provide evidence that increased NF-κB signaling leading to ERα alterations are
mediated via RRM2 overexpression and can be reversed using Didox through its ability to inhibit
NF-κB activation (Fig. 4E). We have shown that the inhibition of RRM2 by Didox was able to
eradicate MCF-7TamR and T-47DTamR populations by day 15, supporting the use of Didox to
resensitize breast cancer cells to tamoxifen therapy. More importantly, Didox prevented the
emergence of tamoxifen resistance in two models, suggesting the therapeutic use of Didox to also
prevent the emergence of tamoxifen resistance (Fig. 4A, 4B).
Here, by overexpressing RRM2, we were able to reproduce the tamoxifen-resistant
phenotype and have identified that NF-κB, HIF-1α, and MAPK/JNK are the major pathways that are
affected (Fig.2A). These pathways have been shown to play a significant role in mammary-
carcinogenesis and have been implicated in tumor resistance. In particular, the NF-κB pathway has
been shown to regulate cell proliferation, differentiation, and invasion (39). Similar to our study, the
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NF-κB pathway was reported to be increased by RRM2 and inhibited by Didox, strengthening the
findings that RRM2 is a key regulator of the NF-κB pathway (40, 41). Although Didox exerts its
activity by destabilizing RR through its free radical scavenging and iron chelating properties, Didox
has also been shown to have strongly inhibit NF-κB activation (42).
Due to tamoxifen’s ability to induce cell death by causing DNA damage, we sought to
determine whether RRM2 plays in role in protecting breast cancer cells from tamoxifen-induced cell
death (4). It is likely that cells having high RRM2 (cells in S-phase or in drug induced upregulation
of RRM2) in a heterogeneous population survive and emerge as a resistant population that is
protected from cell death due to DNA damage. Others have shown that RRM2 regulates anti-
apoptotic proteins such as Bcl-2 in certain cancers (43). Here, we show that RRM2 overexpression
protects against tamoxifen-induced apoptosis and results in lowered S139 γH2AX phosphorylation,
reduced PARP and Caspase-9 cleavage, and increased Bcl-2 levels, which are well-established
mechanisms of chemoresistance (44). These RRM2-induced protective effects were reversed by the
combination of tamoxifen and Didox treatment (Fig. 2D). This is the first report that Didox can
resensitize breast cancer cells to tamoxifen-induced cell death. Since high RRM2 expression is
correlated with poor RFS and OS in tamoxifen-treated patients (Fig. 1D and 1E), RR inhibitors used
in combination with tamoxifen may improve RFS and OS.
Recently, there has been interest in drug combinations with greater efficacy and reduced
toxicity. To date, no serious side effects have been reported with Didox treatment even when
administered for long periods (21). Didox has been shown to inhibit proliferation of several cancer
cell types (18-20, 45) and modulate other cancer chemotherapeutic agents resulting in elevated
apoptosis (42). Didox was also shown to synergize with temozolomide in brain tumors and with
doxorubicin in liver cancer cells (46, 47). Our study indicates that combining Didox with tamoxifen
synergistically reduced cell in RRM2 overexpressing MCF-7, MCF-7TamR, and T-47DTamR cells
(Fig. 5, Supplementary Fig. S4). Additionally, our study reports that Didox inhibits RRM2 induced
cell motility, migration, and invasion (Fig. 6). Our data suggest that combining Didox with tamoxifen
may offer significant benefits to patients with ER-positive breast cancer with high RRM2 expression
or patients who have relapsed after long-term tamoxifen treatment. Similarly, RRM2 was recently
suggested as a prognostic marker associated with poor survival and tamoxifen resistance, which
supports our findings that RRM2 is an important contributor on the pathway to tamoxifen resistance
(48).
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In summary, our findings strongly complement the current knowledge regarding the
molecular mechanisms underlying acquired tamoxifen resistance in breast cancer and also provide
strong evidence that RRM2 is important in regulating ER-α expression, hence responsiveness to
tamoxifen. Our data demonstrate for the first time that overexpression of RRM2 in MCF-7 breast
cancer cells leads to upregulation of EGFR and NF-κB signaling, downregulation of ER-α66
expression, and upregulation of ER-α36, contributing to the generation of acquired tamoxifen
resistance. Overexpression of RRM2 also enhances the proliferative capacity and the migratory and
invasive ability of MCF-7 breast cancer cells. Furthermore, co-treatment of tamoxifen and Didox
resulted in reduced proliferation rates with decreased in vitro migratory and invasive properties.
Most importantly, the combination treatment produced significant tumor inhibition, suggesting a
critical role of RRM2 in maintaining a malignant phenotype in breast cancer. These findings indicate
that RRM2 may be potentially used as a prognostic factor in breast cancer patients undergoing
tamoxifen therapy and can be considered a potential therapeutic target in tumors that have acquired
resistance to tamoxifen. Further study of the molecular mechanisms by which RRM2 is activated
during the development of acquired tamoxifen resistance in breast cancer will provide more detailed
insights regarding the biological function of RRM2. Lastly, these data provide a rationale for the
combination of tamoxifen and Didox therapy to circumvent or prevent tamoxifen resistance in breast
cancer.
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List of Abbreviations Abbreviations
ER-α, Estrogen receptor-α; C, Vehicle control; E, Estrogen; T, Tamoxifen; Tam,
Tamoxifen; D, Didox; HBSS, Hank’s buffered salt solution; FBS, Fetal bovine serum; CSS,
Charcoal-stripped-serum; RT-qPCR, Reverse Transcription Quantitative PCR Polymerase Chain
Reaction; SEM, standard error of the mean; OS, overall survival; RFS, relapse free survival; DMFS,
Distant Metastasis Free Survival; RR, ribonucleotide reductase; RRM2, ribonucleotide reductase
M2; KMplots, Kaplan-Meier survival plots; SEM, standard error of the mean; SD, standard
deviation. Acknowledgements
The authors thank Dr. Arturo Cardounel and Dr. Murugesan Velayutham (University of
Pittsburgh) for their clinical expertise and assistance with the tissue microarrays. The authors thank
Mr. Mike Manzer (UC Davis, VMTH) for his immunohistological and digital imaging assistance.
Finally, the authors thank Dr. John Livesey and Dr. Timothy Smith (Department of Physiology and
Pharmacology, University of the Pacific, Thomas J. Long School of Pharmacy and Health
Sciences) and Dr. Lisa Wrischnik (Department of Biological Sciences) for critical reading and
constructive comments during the preparation of this manuscript.
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Figure Legends
Fig. 1: RRM2 inversely correlates with ER-α66 expression. (A) Western blot analysis of RRM2 and
ER levels in ER-positive and ER-negative cells. (B) Meta-analysis demonstrates higher RRM2
expression in ER-negative (dark box) as compared to ER-positive cancer patients (white box). (C)
Tamoxifen-resistant patients (dark box) have higher RRM2 than tamoxifen-sensitive patients (white
box. Fold change and p-values given in Supplementary Table S1. KMplots demonstrate
overexpression of RRM2 is predictive of (D) lower RFS (p=4.9e-5) and (E) lower OS (p=0.00025) in
tamoxifen-treated ER-positive patients.
Fig. 2: RRM2 overexpression modulates NF-κB activity and gene transcription. (A) Reporter array shows NF-κB (p
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21
mg/kg/day (D), n=6-15. Didox treatment was initiated at day 7 post-cell injection when RRM2
overexpressing MCF-7 xenografts reach maximum tumor volumes (30 mm3) in the absence of
tamoxifen. Mean tumor volume ± SEM is shown ** p
-
RRM2
ER-α36
ER-α66
GAPDH
ER-Positive ER-Negative
A B
-2
0
2
4
6
8
Re
lati
ve m
RN
A e
xpre
ssio
n
25 26 27 28 29
ER-Positive ER-Negative
C OS in Tam treated ER Positive patients D E
-2
0
2
4
6
8
10
Re
lati
ve m
RN
A e
xpre
ssio
n
30 31 32 33 34
Tam-resistant Tam-sensitive
Low RRM2
High RRM2
Pro
bab
ilit
y o
f R
FS
Low RRM2
High RRM2
Pro
bab
ilit
y o
f O
S
RFS in Tam treated ER positive patient
Fig. 1
HR=1.96 (1.41 – 2.73)
P=4.9e-05
HR=2.23 (1.43 – 3.46)
P=0.00025
Time (months) Time (months)
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-
RRM2
RRM1
ER-α66
ER-α36
EGFR
HER2
GAPDH
RRM2B
Bcl-2
p53
IKK-δ
IKK-β
IKK-α
IKK-ε
IKK-γ
p52
p50
p65
RelB
C-Rel
PARP
Cl-PARP
Caspase9
Cl-Caspase9
C T D T+D C T D T+D
MCF-7 MCF-7/R2.1
pγ-H2AX (S139)
γ-H2AX
GAPDH
-40
-20
0
20
40
BC
L2 IL6
PA
RP
BIR
C5
CC
ND
2
EGFR
ERB
B2
MY
C
IGF1
VEG
FA
MM
P9
CC
ND
1
TWIS
T1
MG
MT
JUN
EGF
IGF1
R
AR
CD
H1
TP5
3
AP
C
CD
KN
1A
BA
D
ESR
1
CD
KN
2A
Fold
Ch
ange
-7 -4 -1 2 5 8
NFKB
HIF1-α
MAPK/ERK
MAPK/JNK
Wnt
Myc/max
TGF-B
pRB
Notch
p53
Log2 (Fold Change)
RRM2 +Didox
RRM2 -Didox
Increase Decrease
* *
*
*
*
Fig. 2
A B
C
D
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-
ER-α66
ER-α36
ER-α66 (pS167)
ER-β
ESTROGEN RECEPTOR SIGNALING
PANEL
p52
p50
p65
RelB
p p65 (S53)
GAPDH
C-Rel
NF-κB SIGNALING PANEL
p53
EGFR
HER2
pERK1/2
Total ERK1/2
pAKT (S473)
Total AKT
GROWTH RECEPTOR SIGNALING
PANEL
MCF-7 1 5 10 15 20 30 Days
IKK-δ
pIKK-α/β (S176/180)
IKK-β
IKK-α
pIkBα (S32)
Total IkBα
IKK-ε
IKK-γ
IKK SIGNALING PANEL
MCF-7 1 5 10 15 20 30 Days
Day 1 Day 5 Day 10 Day 15 Day 20 Day 25 Day 30
5uM T
Day 1 Day 5 Day 3
C
APOPTOSIS PANEL
MCF-7 1 5 10 15 20 30 Days
PARP
Cl-PARP
Caspase9
Cl-Caspase9
pγ-H2AX (S139)
y-H2AX
GAPDH
Bcl-2
0
5
10
15
20
0 2 4 6 8
Cel
l N
um
ber
X
Thousa
nds
Days
MCF-7MCF-7 TamR TMCF-7 T
*
Fig. 3
A
RRM2
RRM1
RRM2B
RIBONUCLEOTIDE REDUCTASE PANEL
B
C
D
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-
T-47D
T+D
T-47D
TamR
T+D
Day 1 Day 15 Day 30 Day 1 Day 5 Day 10 Day 15 Day 20 Day 25 Day 30
MCF-7
TamR
T+D
MCF-7
T+D
pIKK-α/β (S176/180)
pAKT (S473)
pIkBα (S32)
p-p65 (S536)
pγ-H2AX (S139)
RRM2
RRM1
RRM2B
RIBONUCLEOTIDE REDUCTASE
ER-α66
ER-α36
pER (S167)
ER-β
ESTROGEN RECEPTOR
p53
EGFR
HER-2
pERK1/2
Total ERK1/2
Total AKT
GROWTH RECEPTOR
p52
p50
p65
RelB
GAPDH
C-Rel
NF-κB SIGNALING
IKK-δ
IKK-β
IKK-α
Total IkBα
IKK-ε
IKK-γ
IKK SIGNALING
Total γ-H2AX
PARP
Cl-PARP
Caspase9
Cl-Caspase9
GAPDH
Bcl-2
APOPTOSIS
IKK-δ
IKK-β
IKK-α
Total IkBα
IKK-ε
IKK-γ
IKK SIGNALING
p52
p50
p65
RelB
p-p65 (S536)
GAPDH
C-Rel
NF-κB SIGNALING
pIKK-α/β (S176/180)
pAKT (S473)
pIkBα (S32) Total γ-H2AX
PARP
Cl-PARP
Caspase9
Cl-Caspase9
GAPDH
Bcl-2
APOPTOSIS
pγ-H2AX (S139)
RRM2
RRM1
RRM2B
RIBONUCLEOTIDE REDUCTASE
ER-α66
ER-α36
ER-β
ESTROGEN RECEPTOR
p53
EGFR
HER-2
pERK1/2
Total ERK1/2
Total AKT
GROWTH RECEPTOR
pER (S167)
-6
-4
-2
0
2
4
6
MCF-7 MCF-7/R2.1 MCF-7 TamR T-47D T-47D TamR
NF-κB reporter activity
(Fold Change)
C T D T+D
*
*
*
Fig. 4 A B
E
C D
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A
C
B
RRM2
ER-a66
ER-a36
Ki67
H&E
T T+D425
D
Fig. 5
● ● ● ● **
** ** **
* * *
0
20
40
60
80
100
120
140
160
7 14 21 28
Me
an T
um
or
Vo
lum
e (
mm
3)
Days
CDTT+D425T+D200
C D T T+D425
RRM2
ER-a66
ER-a36
GAPDH
*
* * **
●
0.4
0.6
0.8
1
1.2
1.4
C D T T+D425
Me
an f
old
Co
ntr
ol
RRM2 ER- 66 ER- 36a a
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-
p=0.00065
p=0.15 p=0.045
-1
1
3
5
7
9
Grade1
Grade2
Grade3
Rel
ativ
e m
RN
A E
xpre
ssio
n
(28)
p=0.0009
p=0.35 p=0.006
-1
1
3
5
7
Grade1
Grade2
Grade3
Rel
ativ
e m
RN
A E
xpre
ssio
n
(26)
A B C
20
70
120%
Op
en a
rea
Cell Motility RRM2 - Didox RRM2 + Didox
0
1
2
O.D
.
Cell Invasion
0
1
2
O.D
.
Cell Migration
E
-40
-30
-20
-10
0
10
20
30
VC
AM
1
MM
P3
SE
LE
MM
P2
PE
CA
M1
FN
1
NC
AM
1
MM
P9
ITG
B3
CN
TN
1
CT
GF
LA
MA
3
CD
H1
ICA
M1
CO
L15A
1
CT
NN
A1
Fo
ld C
han
ge
RRM2 -Didox RRM2 +Didox
F
G RRM2 ER-a36
D
Gra
de
1
Gra
de
2
Gra
de
3
500 mm
0
50
100
150
200
250
300
Normal Grade 1
Grade 2
Grade 3
Ave
rage
RR
M2
sta
inin
g (H
-sco
re)
H
0
50
100
150
200
250
300
Normal Grade1
Grade2
Grade3
Ave
rage
ER
-a3
6 (
H-s
core
)
I
Fig. 6
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Published OnlineFirst September 2, 2015.Mol Cancer Ther Khyati N. Shah, Elizabeth A Wilson, Ritu Malla, et al. with Didox to circumvent tamoxifen resistance in breast cancer
B activationκTargeting ribonucleotide reductase M2 and NF-
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