Khyati N. Shah1, Elizabeth A. Wilson1, Ritu Malla , Howard L. Elford and Jesika … · 2015. 9. 2. · 1 Targeting ribonucleotide reductase M2 and NF-kB activation with Didox to

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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.

on June 4, 2021. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 2, 2015; DOI: 10.1158/1535-7163.MCT-14-0689

http://mct.aacrjournals.org/

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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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20

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

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)




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




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




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




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




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




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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Khyati N. Shah1*, Elizabeth A. Wilson1*, Ritu Malla , Howard L. Elford and Jesika … · 2015. 9. 2. · 1 Targeting ribonucleotide reductase M2 and NF-kB activation with Didox to

Khyati N. Shah1, Elizabeth A. Wilson1, Ritu Malla , Howard L. Elford and Jesika … · 2015. 9. 2. · 1 Targeting ribonucleotide reductase M2 and NF-kB activation with Didox to