Ribonucleotide Reductase Subunit M1 Research Articles

The tailored chemotherapy based on RRM1 immunohistochemical expression in patients with advanced non-small cell lung cancer

Ribonucleotide reductase subunit M1 (RRM1) has emerged as a promising biomarker to predict the efficacy of gemcitabine. The purpose of the study was to evaluate whether the tailored chemotherapy based on RRM1 immunohistochemical (IHC) expression had any benefit for patients with advanced non-small cell lung cancer (NSCLC). A single-institution study was conducted in patients with advanced NSCLC. In personalized therapy group, patients received chemotherapy based on RRM-1 IHC expression levels. Low RRM1 group received gemcitabine or gemcitabine/cisplatin, high RRM1 group received docetaxel or docetaxel/ cisplatin. In standard therapy group, non-customized chemotherapy was delivered. In this trial, Patients aged ⩾ 70 years received single agent chemotherapy, whereas patients below 70 had platinum-based chemotherapy. There were statistically significant improvements between the personalized therapy group versus the standard therapy group in disease control rate (82.9% vs 55.3%, P=0.004), and PFS (median: 5.5 months vs 3.0 months, P=0.005). Besides, the OS had a tendency to become more prolonged (median: 16.0 months vs 12.4 months, P=0.286). The subgroup analysis suggested the survival benefit in the elderly patients was more obvious. RRM1 IHC expression tailored selection of first-line therapy could improve therapeutic outcomes in patients with advanced NSCLC.

A phase II trial of sequential ribonucleotide reductase inhibition in aggressive myeloproliferative neoplasms

Myeloproliferative neoplasms are a varied group of disorders that can have prolonged chronic phases, but eventually accelerate and can transform into a secondary acute myeloid leukemia that is ultimately fatal. Triapine is a novel inhibitor of the M2 subunit of ribonucleotide reductase. Sequential inhibition of ribonucleotide reductase with triapine and an M1 ribonucleotide reductase inhibitor (fludarabine) was noted to be safe, and led to a 29% complete plus partial response rate in myeloproliferative neoplasms. This article reports the findings of a phase II trial of triapine (105 mg/m(2)/day) followed by fludarabine (30 mg/m(2)/day) daily for 5 consecutive days in 37 patients with accelerated myeloproliferative neoplasms and secondary acute myeloid leukemia. The overall response rate was 49% (18/37), with a complete remission rate of 24% (9/37). Overall response rates and complete remissions were seen in all disease subsets, including secondary acute myeloid leukemia, in which the overall response rate and complete remission rate were 48% and 33%, respectively. All patients with known JAK2 V617F mutations (6/6) responded. The median overall survival of the entire cohort was 6.9 months, with a median overall survival of both overall responders and complete responders of 10.6 months. These data further demonstrate the promise of sequential inhibition of ribonucleotide reductase in patients with accelerated myeloproliferative neoplasms and secondary acute myeloid leukemia. This study was registered with clinicaltrials.gov (NCT00381550).

Gene aberrations of RRM1 and RRM2B and outcome of advanced breast cancer after treatment with docetaxel with or without gemcitabine

BackgroundThe purpose of the present study was to retrospectively evaluate whether copy number changes of the genes encoding the ribonucleotide reductase subunit M1 (RRM1) and/or subunit M2B (RRM2B) predict sensitivity to gemcitabine administered in combination with docetaxel compared to single agent docetaxel in advanced breast cancer patients.MethodsPrimary tumor samples from patients randomly assigned to gemcitabine plus docetaxel or docetaxel alone were analyzed for RRM1 and RRM2B copy number changes using Fluorescence In Situ Hybridization (FISH) technology with probes covering respectively RRM1 at 11p15.5 and a reference probe covering the centromere of chromosome 11 (CEN-11), and RRM2B at 8q22.3 and a reference probe covering the centromere of chromosome 8 (CEN-8). The assays were validated in a material of 60 normal breast samples. Time to progression (TTP) was the primary endpoint. Overall survival (OS) and response rate (RR) were secondary endpoints. Associations between RRM1/CEN-11 and/or RRM2B/CEN-8 ratios and time-to-event endpoints were analyzed by unadjusted and adjusted Cox proportional hazards regression models. Heterogeneity of treatment effects on TTP and OS according to gene status were investigated by subgroup analyses, and the Wald test was applied. All statistical tests were two-sided.ResultsFISH analysis for both RRM1 and RRM2B was successful in 251 patients. RRM1 and RRM2B aberrations (deletions and amplifications) were observed in 15.9% and 13.6% of patients, respectively. RRM1 aberrations were associated with a decreased OS in the time interval 1.5-7.4 years (hazard ratio = 1.72, 95% confidence interval = 1.05-2.79, P = 0.03). RRM2B aberrations alone or in combination with RRM1 aberrations had no prognostic impact in terms of TTP or OS. RR was not different by gene status. No significant differences were detected in TTP or OS within subgroups according to gene status and chemotherapy regimen.ConclusionsThis study demonstrated the presence of RRM1 and RRM2B copy number changes in primary breast tumor specimens. Nevertheless, we found no support of the hypothesis that aberrations of RRM1 or RRM2B, neither individually nor in combination, are associated with an altered clinical outcome following chemotherapy with gemcitabine in combination with docetaxel compared to docetaxel alone in advanced breast cancer patients.

The prognostic value of ERCC1 and RRM1 gene expression in completely resected non-small cell lung cancer: tumor recurrence and overall survival

BackgroundThe roles of excision repair cross-complementing group 1 gene (ERCC1) expression and ribonucleotide reductase subunit M1 gene (RRM1) expression in completely resected non-small cell lung cancer (NSCLC) are still debatable. Previous studies have shown that both genes affected the overall survival and outcomes of patients who received platinum-based chemotherapy; however, some studies did not show this correlation. The aim of this study was to evaluate the prognostic values of ERCC1 and RRM1 gene expression in predicting tumor recurrence and overall survival in patients with completely resected NSCLC who received adjuvant chemotherapy and in those who did not.Patients and methodsA retrospective cohort study was conducted in 247 patients with completely resected NSCLC. All patients had been treated with anatomic resection (lobectomy or pneumonectomy) with systematic mediastinal lymphadenectomy between January 2002 and December 2011 at Chiang Mai University Hospital, Chiang Mai, Thailand. They were divided into two groups: recurrence and no recurrence. Protein expression of ERCC1 and RRM1 was determined by immunohistochemistry. Correlations between clinicopathologic variables, including ERCC1 and RRM1 expression and tumor recurrence, were analyzed. Univariate and multivariate Cox proportional hazards regression analysis stratified by nodal involvement, tumor staging, intratumoral blood vessel invasion, intratumoral lymphatic invasion, and tumor necrosis was used to identify the prognostic roles of ERCC1 and RRM1.ResultsERCC1 and RRM1 expression did not demonstrate prognostic value for tumor recurrence and overall survival in patients with completely resected NSCLC. In patients who did not receive adjuvant chemotherapy treatment, those with high ERCC1 and high RRM1 expression seemed to have greater potential for tumor recurrence and shorter overall survival than did those who had low ERCC1 and low RRM1 (hazard ratio [HR] =1.7, 95% confidence interval [CI] =0.6–4.3, P=0.292 and HR =1.6, 95% CI =0.5–4.5, P=0.411, respectively). In contrast, in patients who received adjuvant chemotherapy treatment, those with high ERCC1 and high RRM1 expression seemed to have benefited from adjuvant chemotherapy and showed good overall survival compared with those who had low ERCC1 and low RRM1 (HR =0.8, 95% CI = 0.4–1.8, P=0.612 and HR = 0.4, 95% CI = 0.1–2.4, P=0.325, respectively). Subgroup analysis in patients whose first-line metastatic chemotherapy failed demonstrated that ERCC1 expression and RRM1 expression were not prognostic factors for tumor recurrence and overall survival; however, patients who had high ERCC1 and high RRM1 expression seemed to have benefited from first-line chemotherapy treatment (HR =0.7, 95% CI =0.3–1.8, P=0.458).ConclusionERCC1 expression and RRM1 expression were not prognostic of tumor recurrence and overall survival in patients with completely resected NSCLC, either with or without adjuvant chemotherapy. Prospective studies that include a larger number of patients are needed for definite conclusions.

In vitro effects of lapatinib with gemcitabine for pancreatic cancer cells.

We investigated whether lapatinib plus gemcitabine has synergistic or antagonistic effects on the pancreatic cancer cell lines MiaPaca-2 and PANC-1. Furthermore, the changes of gemcitabine sensitivity-related genes by lapatinib treatment were examined. The effects of lapatinib, gemcitabine, and combined treatment with both agents on cell viability were examined by methyl thiazolyl tetrazolium analysis. Synergy between lapatinib and gemcitabine was assessed by median effect analysis. The mRNA amounts of human equilibrative nucleoside transporter (hENT1), deoxycytidine kinase (dCK) and ribonucleotide reductase subunit M1 (RRM1) genes were measured by quantitative real-time polymerase chain reaction in cells exposed to lapatinib for 48 h, as compared with untreated cells. No synergistic effects were observed with combined treatment in either cell line. In contrast, antagonistic effects occurred on MiaPaca-2 cells with the two agents. Specific changes in gemcitabine sensitivity-related genes induced by lapatinib were not detected in either MiaPaca-2 or PANC-1. Lapatinib may not enhance the anti-tumor effects of gemcitabine for pancreatic cancer.

DNA repair initiation induces expression of ribonucleotide reductase in human chronic lymphocytic leukemia cells

Mammalian ribonucleotide reductase (RR) is a heterodimer enzyme responsible for maintaining levels of deoxynucleotides needed for DNA replication. The M2 subunit of RR is known to increase in tandem with progression of cells into S phase, whereas the M1 subunit is expressed at steady-state. Since the expression level of the M2 subunit increases because the cells need deoxyribonucleoside triphosphates (dNTPs) for replication, it is logical to hypothesize that the same increase will be seen during DNA repair. To test this, we used chronic lymphocytic leukemia (CLL) cells, which are replicationally quiescent and have low endogenous levels of RR and dNTPs. Cyclophosphamide was selected as the DNA damaging agent because of its clinical use in the treatment of CLL. DNA repair, measured by [3H]thymidine incorporation after 4 h treatment with 4-hydroperoxycyclophosphamide, increased in a dose-dependent manner at 3, 10 and 50 μM. The induction of DNA repair concomitantly increased the mRNA and protein levels of M2 subunit (median 1.6-fold; range 0.9–5.3). Maximum induction occurred at 10 μM after 4 h and correlated with [3H]thymidine incorporation (p = 0.02). In contrast, no change was observed in mRNA or protein levels of M1 subunit. We conclude that RR is regulated not only during replication but also during DNA repair, and in both cases M2 subunit expression is increased.

RRM2 Regulates Bcl-2 in Head and Neck and Lung Cancers: A Potential Target for Cancer Therapy

Ribonucleotide reductase subunit M2 (RRM2) plays an active role in tumor progression. Recently, we reported that depletion of RRM2 by systemic delivery of a nanoparticle carrying RRM2-specific siRNA suppresses head and neck tumor growth. The aim of this study is to clarify the underlying mechanism by which RRM2 depletion inhibits tumor growth. siRNA-mediated gene silencing was carried out to downregulate RRM2. Immunoblotting, reverse-transcriptase PCR, confocal microscopy, tissue fractionation, gene overexpression and knockdown were employed to analyze critical apoptosis signaling. Conventional immunohistochemistry and quantum dot-based immunofluorescence were applied to detect RRM2 and Bcl2 expression and localization in tissue samples from patients and mice. Knockdown of RRM2 led to apoptosis through the intrinsic pathway in head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC) cell lines. We showed that Bcl-2 is a key determinant controlling apoptosis, both in vitro and in vivo, and that RRM2 depletion significantly reduces Bcl-2 protein expression. We observed that RRM2 regulates Bcl-2 protein stability, with RRM2 suppression leading to increased Bcl-2 degradation, and identified their colocalization in HNSCC and NSCLC cells. In a total of 50 specimens each from patients with HNSCC and NSCLC, we identified the colocalization of Bcl-2 and RRM2 and found a significant positive correlation between their expression in HNSCC (R = 0.98; P < 0.0001) and NSCLC (R = 0.92; P < 0.0001) tumor tissues. Our novel findings add to the knowledge of RRM2 in regulating expression of the antiapoptotic protein Bcl-2 and reveal a critical link between RRM2 and Bcl-2 in apoptosis signaling.

Prognostic predictive values of gemcitabine sensitivity-related gene products for unresectable or recurrent biliary tract cancer treated with gemcitabine alone

BackgroundGemcitabine is a pyrimidine nucleoside analog that is a commonly used chemotherapeutic agent for unresectable or recurrent biliary tract cancer (BTC). Several molecules involved in gemcitabine metabolism, including human equilibrative nucleoside transporter (hENT1), deoxycytidine kinase (dCK), and ribonucleotide reductase subunit M1 (RRM1), have been investigated as predictive biomarkers of gemcitabine efficacy, mostly in pancreatic cancer. The aim of this study is to clarify which biomarker is the most reliable among hENT1, dCK, and RRM1 to predict survival in patients with advanced BTC treated with gemcitabine alone.MethodsThe analysis was performed on samples from 28 patients with unresectable or recurrent BTC who were treated with gemcitabine alone as first-line therapy. The starting date of overall survival (OS) and progression-free survival (PFS) was defined as the date of first treatment with gemcitabine. Intratumoral hENT1, dCK, and RRM1 expressions were examined by immunohistochemistry.ResultsThe expressions of hENT1, dCK, and RRM1 had no significant relationships with age, gender, primary tumor site, recurrence/unresectable, or histological type. Among the three molecules, only hENT1 expression was a significant factor affecting OS and PFS in univariate analysis; OS was 11.4 months for high hENT1 expression versus 5.7 months for low, P = 0.0057; PFS was 7.7 months for high versus 2.5 months for low, P = 0.0065. Multivariate analyses also identified hENT1 expression as an independent predictive factor for OS.ConclusionshENT1 is the most reliable predictive marker of survival in patients with advanced BTC treated with gemcitabine.

A multicenter phase II randomized study of customized neoadjuvant therapy versus standard chemotherapy (CT) in non-small cell lung cancer (NSCLC) patients with resectable stage IIIA(N2) disease (CONTEST trial).

TPS7606 Background: Stage IIIA NSCLC constitutes 30% of all NSCLC patients (pts). The most powerful prognostic factor that has been identified in this stage is clearance of mediastinal lymph nodes and pathologic complete response (pCR). A pCR is obtained in 5-15% of pts with a significant survival prolongation. The identification of molecular biomarkers, such as excision repair cross-complementation 1 (ERCC1), ribonucleotide reductase subunit M1 (RRM1), and thymidylate synthase (TS), may predict response to CT. Similarly, EGFR mutations may predict response to EGFR inhibitors. Methods: CONTEST, a multicenter (19 Italian centers), randomized (2:1) 2-arm phase II study, recruits pts with resectable stage IIIA (N2) NSCLC. Pts will be randomized to receive before resection either standard CT with Cisplatin (CDDP) 75 mg/m2 + Docetaxel (Doc) 75 mg/m2 on day (d) 1 q 21 d for 3 cycles (cys) or customized therapy using predetermined values for ERCC1, RRM1, TS, and EGFR mutations. The choices of customized arms are as follows: -EGFR+: Gefitinib 250 mg/d for 8 weeks. -EGFR-/non-squamous (NS)/TS-/ERCC1-: CDDP 75 mg/m2 + Pemetrexed 500 mg/m2 d 1 q 21 d for 3 cys. -EGFR-/squamous (S) or NS TS+/ERCC1-/RRM1+: CDDP 75 mg/m2 + Doc 75 mg/m2 d 1 q 21 d for 3 cys. -EGFR-/S or NS TS+/ERCC1-/RRM1-: CDDP 75 mg/m2 d 1+ Gemcitabine (Gem) 1250 mg/m2 d 1,8 q 21 d for 3 cys. -EGFR-/S or NS TS+/ERCC1+/RRM1+: Doc 75 mg/m2 d 1 + Vinorelbine 20 mg/m2 d 1,8 q 21 d for 3 cys. -EGFR-/S or NS TS+/ERCC1+/RRM1-: Doc 40 mg/m2 y 1, 8 + Gem 1200 mg/m2 d 1,8 q 21 d for 3 cys. The primary end point will be obtained by comparing the pCR in all randomized pts based on treatment arm. Because pCR is a surrogate endpoint and given the expected proportion of pCR's in the control group pc= 5%, the minimal clinically worthwhile effect of this customized treatment is an increase in this proportion to 20%. To detect such an effect at the 0.05 (1-sided) significance level with 80% power, a total of 168 pts will be enrolled. This study is open for accrual; further details can be found on ClinicalTrials.gov (NCT01784549). Funded by Italian Ministry of Health – RF 2009-1530324. Clinical trial information: NCT01784549.

Perturbation of nucleotide metabolism - the driving force of oncogene-induced senescence

Induction of premature (stress induced) cellular senescence of normal cells provides the mechanism eliminating proliferative capability of potentially carcinogenic cells and thereby is considered to be a barrier preventing neoplastic tranformation [1]. On the other hand, induction of senescence in tumor cells provides a therapeutic opportunity to treat cancers that may be potentially susceptible [2]. Among factors inducing senescence are oxidative DNA damage leading to persistent replication stress, activation of oncogenes and loss of tumor suppressor genes [1]. It was recently reported however that perturbation of the nucleotide metabolism can be added into these already “classic” inducers. The early observations by Mannava et al.,[3]and Liu et al [4], established evidence of a cause-effect relationship between C-MYC, the oncogene that is capable of suppressing senescence and nucleotide metabolism. The authors reported that downregulation of the C-MYC in several human metastatic melanoma lines via lentivirus-based shRNA led to repression of several genes encoding enzymes related to dNTP metabolism such as thymidylate synthase (TS), inosine monophosphate dehydrogenase 2 (IMPDH2) and phosphoribosyl pyrophosphate synthetase 2 (PRPS2). This observation provided a rationale to explore a potential link between dNTP metabolism, DNA damage and the oncogene induced senescence (OIS). Additional evidence on the role of perturbed dNTP pools in induction of DNA damage and replication stress was provided by Bester et al.,[5]. Their studies were focused on oncogenes regulating cell cycle progression, specifically the Rb-E2F pathway. The authors observed that aberrant activation of this pathway by HPV-16 E6/E7 or cyclin E oncogenes led to a reduction of cellular nucleotide levels. Interestingly, when C-MYC was coexpressed with cyclin E, its presence led to an increase in transcription of nucleotide biosynthesis genes, prevented the reduction of nucleotide pool and rescued the replication-stress induced DNA damage [5]. In the subsequent studies Mannava et al.,[6] provided direct evidence that down-regulation of nucleoside pools is a cause of DNA damage and senescence in normal human fibroblasts (NHFs) overexpressing HRASG12V, a classical model of oncogene-induced senescence. Among the enzymes down-regulated by activated HRAS were TS, ribonucleotide reductase subunits M1 and M2 (RRM1 and RRM2). Ectopic coexpression of TS, RRM1 and RRM2 or addition of deoxyribonucleosides to a large extent prevented DNA damage, decreased expression of senescence-associated phenotypes as well as attenuated the extent of proliferation arrest. Interestingly, overexpression of TS, RRM1 and RRM2 in quiescent NHFs had no effect on their proliferation arrest suggesting that unlike the OIS, quiescence is not induced by perturbation of dNTP pools. Moreover, in a separate study, Mannava et al.,[7]demonstrated that shRNA-mediated depletion of RRM2 and TS was sufficient to induce senescence in melanoma cells. The observation that knockdown of RRM2 and TS in cells with ctivated oncogenes drives them to senescence provides further evidence that abundance of dNTPs promotes melanoma by suppressing cellular senescence. Most of the above findings were reproduced in a study of Aird et al.,[8] on a role of suppressed nucleotide pools in DNA damage and oncogene-induced senescence. Additionally the authors reported that RRM2 repression correlated with senescent status of melanocytes in human benign nevi and melanoma harboring oncogenic BRAF or NRAS. Furthermore, high expression of RRM2 correlates with poor survival of melanoma patiens[8]. The seminal findings on different cellular and oncogene models that perturbation of dNTP pools leads to DNA damage, replication stress, irreversible senescence [5-8],may have important clinical implications. The example that with similar oncogene activation the senescence of melanocytes induced by shortage of dNTPs results in nevi remaining benign for years whereas abundance of dNTPs leads to malignant melanoma, opens new possibilities to explore potential role of dNTPs as the target in cancer treatment. In light of these findings the commonly used anticancer drug hydroxyurea which targets dNTPs or 5-fluorouracil that affects thymidylates should be revaluated in terms of its possible senescence-promoting efficiency. For years, acceptance of antitumor drugs by the FDA was based on response criteria assessed by tumor shrinkage that most likely occurs as a result of rapid cell death by apoptosis. Assessment of the tumor response by its size reduction criteria precluded the detection of potential treatment benefits that could be mediated by induction of senescence of tumor cells which may remain alive but reproductively dead. It is tempting to explore whether a combined treatment of cancer initially by cytotoxic regimen, relying on induction of apoptosis, be followed by prolonged treatment promoting senescence of cancer cells, is more effective than the traditional chemotherapy.

Small Interfering RNA (siRNA)-Mediated Silencing of the M2 Subunit of Ribonucleotide Reductase

To investigate the effects of small interfering RNA (siRNA)-mediated silencing of the ribonucleotide reductase M2 subunit (RRM2) on the apoptosis and the drug sensitivity of cisplatin-resistant SKOV3/DDP cells. Small interfering RNA transfection was mediated by lipofectamine 2000 to silence RRM2 gene. Messenger RNA (mRNA), and protein expression levels of RRM2 were evaluated by real-time polymerase chain reaction and Western blot after transfection. The cell growth inhibition rate was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The cellular apoptosis and cycling was identified by flow cytometry (FCM). The messenger RNA and protein expression levels of RRM2 markedly decreased after the RRM2 siRNA transfection. The half inhibition concentration of cisplatin in RRM2-RNA interference cells (interference group) was lower than that in RRM2-negative cells (noninterference group) and the SKOV3/DDP cells (blank control group) (P = 0.032). Small interfering RNA-mediated inhibition of RRM2 effectively induced G1/S-phase cell cycle arrest and increased drug (gemcitabine and cisplatin)-induced apoptotic fraction at 72 hours ( (96% ± 3.0)%) after transfection (P < 0.05). Small interfering RNA-mediated RRM2 knockdown significantly reversed SKOV3/DDP cell resistance to cisplatin. RNA interference technology combined with gemcitabine and cisplatin can effectively improve the apoptosis rate of the cisplatin-resistant ovarian cancer cell, which is expected to become the first-line treatment options for the cisplatin-resistant ovarian cancer.

Abstract 3545: ATM, TS and RRM1 protein expression in nasopharyngeal carcinomas (NPC) treated with curative intent.

Abstract Background: Although NPC is a radiation and chemotherapy sensitive tumour, some patients still relapse with distant metastatic disease. Biomarkers are needed to help identify which patients are at higher risk of relapse and/or who will respond to therapy. Ataxia telangiectasia mutated (ATM) is a putative marker for radiation sensitivity while, thymidylate synthetase (TS), and ribonucleotide reductase subunit M1 (RRM1) levels have been associated with response to 5FU and gemcitabine, two chemotherapy agents often used in NPC. We characterized the protein expression of ATM, TS, and RRM1 in tumours from 146 patients with non-metastatic NPC treated at two Canadian institutions between Jan 2000-Dec 2007 and explored the relationship of each marker with clinical outcomes. Methods: Pre-treatment, formalin-fixed, paraffin-embedded NPC tumour specimens were assembled in a tissue microarray. ATM, TS and RRM1 protein expression were evaluated by quantitative fluorescence immunohistochemistry and with automated quantitative digital image analysis (AQUA) using the Y170, TS106 and 60073-2-Ig monoclonal antibodies respectively. Protein expression levels were assessed in tumour, tumour cytoplasm, tumour nuclear, and non-malignant tumour stroma compartments. The effect of protein expression levels on overall survival (OS) and disease-free survival (DFS) was examined using Cox regression. For all biomarkers, AQUA scores were analyzed in quartiles. Results: Patient characteristics were: mean age=52 years (SD=12.2; range 18 to 85), 67% male, 73% KPS ≥ 90%, WHO type 1/2/3=11%/28%/61%, stage III/IV=65%. Fifty-eight (40%) patients received platinum-based CRT; the remainder was treated with RT alone. With a median follow-up of 50 months (range 3 to 120), the 5 year survival rates were 71% (95% CI=62%-78%) for OS and 48% (95% CI=39%-57%) for DFS. After adjusting for KPS, stage, and WHO type, OS was worse for the group with tumour:stromal (T/S) ATM ratios in the top quartile versus those in the lowest quartile (p=0.049). OS was also worse for tumours with TS levels in the lowest quartile versus the top quartile (p=0.033). RRM1 was not associated with outcomes (p=0.748). There was no significant effect of any of the biomarkers on disease-free survival (ATM T/S p=0.708; TS p=0.978; RRM1 p=0.918). Conclusions: In our cohort of non-metastatic NPC patients, relative overexpression of ATM and low TS protein levels were associated with worse outcomes suggesting further investigation of these potential biomarkers is warranted. Citation Format: Alexander C. Klimowicz, Amanda Jagdis, Tien Phan, Janessa J. Laskin, Harold Y. Lau, Jodi E. Siever, Stephanie K. Petrillo, Thomas A. Thomson, M. Sarah Rose, Anthony M. Magliocco, Desirée Hao. ATM, TS and RRM1 protein expression in nasopharyngeal carcinomas (NPC) treated with curative intent. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3545. doi:10.1158/1538-7445.AM2013-3545

Abstract 4932: Role of ribonucleotide reductase subunit M2 in regulation of head and neck and lung cancer undergoing invasion.

Abstract Ribonucleotide reductase subunit M2 (RRM2) is a key enzyme in DNA replication which can serve as a potential therapeutic target. RRM2 is also thought to modulate cellular invasiveness but the mechanisms through which RRM2 affects the invasive phenotype have not been elucidated. In this study, we report a mechanism linking RRM2 to invasion which provides a vital clue for the development of novel therapeutics. Since RRM2 is a known prognostic factor and an indicator of poor patient outcome in several cancers, we investigated the correlation between RRM2 and metastasis in tumor tissues from head and neck squamous cell carcinoma (HNSCC) patients. We utilized both quantum dot (QD)-immunofluorescence and conventional immunohistochemistry techniques to stain RRM2 proteins in metastatic (n = 20) and non-metastatic (n = 20) HNSCC tissue samples. In addition, we validated the clinical findings in an in vitro system and explored the underlying mechanism in invasion. The function of RRM2 in cell invasion was examined in different HNSCC cell lines (Tu212, Tu686, 37A, M4e, 886LN) and non-small cell lung cancer (NSCLC) cell lines (A549, H460 and H1299). siRNA was applied to knockdown the RRM2 protein while pcDNA3/RRM2 plasmid was used to establish RRM2 overexpressing cell lines. Cell invasion was examined by Boyden chamber invasion assay. Molecular signaling analysis was performed by Western blotting. Our results showed that RRM2 had a significant positive correlation with metastasis in HNSCC tumor tissues (p value &amp;lt; 0.01). We also identified varying degrees of RRM2 expression levels in HNSCC and NSCLC cell lines, which were correlated with their invasive capability. Highly invasive HNSCC (M4e) and NSCLC (H1299) cell lines that feature epithelial-mesenchymal transition (EMT) expressed the highest level of RRM2 protein and exhibited the greatest invasive ability among the cell lines studied. Upon knockdown of RRM2 by siRNA treatment, we found more than 3-fold reduction of invasive ability of M4e and H1299 cells compared to non-targeted siRNA control (p value &amp;lt; 0.001). In contrast, overexpression of RRM2 enhanced cell invasiveness 2 to 4-fold in poorly invasive HNSCC (Tu212 and Tu686) and NSCLC (A549) cell lines compared to empty vector (p value &amp;lt; 0.001). We determined that RRM2 regulated EMT features and identified molecules, such as E-cadherin, vimentin, Twist1, caveolin-1, Akt, and Erk, which are associated with this biological process. Our study demonstrated that RRM2 substantially promoted cell invasion. Thus, an elevated RRM2 may serve as a potential biomarker for metastasis. Targeting RRM2 and its downstream signaling intermediaries represents a rational approach for developing novel anticancer therapeutics. (This work is supported by grants from NIH U01CA151802, P50CA128613 and DOD W81XWH-07-1-0306) Citation Format: Mohammad Aminur Rahman, A.R.M. R. Amin, Sreenivas Nannapaneni, Zhongliang Hu, Hyunseok Kang, Zhuo (Georgia) Chen, Dong M. Shin. Role of ribonucleotide reductase subunit M2 in regulation of head and neck and lung cancer undergoing invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4932. doi:10.1158/1538-7445.AM2013-4932

Abstract 943: RRM2: a key contributor in tamoxifen resistance in breast cancer.

Abstract Tamoxifen is primarily used for early and advanced ER positive breast cancer patients. Unfortunately about 58% of the patients fail to respond to tamoxifen and eventually relapse with more aggressive tumors. Therefore, understanding the molecular mechanism of resistance is necessary to design new therapeutic strategies to overcome resistance. In this study, we show that Ribonucleotide Reductase M2 subunit is highly expressed in Akt over-expressing breast cancer cell lines and these breast cancer cell lines are highly resistant to tamoxifen treatment In-vitro. In these breast cancer cell lines, RNA interference mediated by small interfering RNA against RRM2 sensitizes these cells to tamoxifen-induced toxicity and reversed the tamoxifen resistance. Also, Using Oncomine database, we demonstrate that RRM2 is overexpressed in breast cancer. This is the first demonstration that investigate role of RRM2 in tamoxifen resistance and could serve as a potential target to restore tamoxifen sensititivity in breast cancer cells. Citation Format: Khyati Niral Shah, David Peterson, Marie Evangelista, Jesika Faridi. RRM2: a key contributor in tamoxifen resistance in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 943. doi:10.1158/1538-7445.AM2013-943

Suppression of Nucleotide Metabolism Underlies the Establishment and Maintenance of Oncogene-Induced Senescence

Oncogene-induced senescence is characterized by a stable cell growth arrest, thus providing a tumor suppression mechanism. However, the underlying mechanisms for this phenomenon remain unknown. Here, we show that a decrease in deoxyribonucleotide triphosphate (dNTP) levels underlies oncogene-induced stable senescence-associated cell growth arrest. The decrease in dNTP levels is caused by oncogene-induced repression of ribonucleotide reductase subunit M2 (RRM2), a rate-limiting protein in dNTP synthesis. This precedes the senescence-associated cell-cycle exit and coincides with the DNA damage response. Consistently, RRM2 downregulation is both necessary and sufficient for senescence. Strikingly, suppression of nucleotide metabolism by RRM2 repression is also necessary for maintenance of the stable senescence-associated cell growth arrest. Furthermore, RRM2 repression correlates with senescence status in benign nevi and melanoma, and its knockdown drives senescence of melanoma cells. These data reveal the molecular basis whereby the stable growth arrest of oncogene-induced senescence is established and maintained through suppression of nucleotide metabolism.

Differential Processing of let-7a Precursors Influences RRM2 Expression and Chemosensitivity in Pancreatic Cancer: Role of LIN-28 and SET Oncoprotein

Overexpression of ribonucleotide reductase subunit M2 (RRM2), involved in deoxyribonucleotide synthesis, drives the chemoresistance of pancreatic cancer to nucleoside analogs (e.g., gemcitabine). While silencing RRM2 by synthetic means has shown promise in reducing chemoresistance, targeting endogenous molecules, especially microRNAs (miRNAs), to advance chemotherapeutic outcomes has been poorly explored. Based on computational predictions, we hypothesized that the let-7 tumor suppressor miRNAs will inhibit RRM2-mediated gemcitabine chemoresistance in pancreatic cancer. Reduced expression of the majority of let-7 miRNAs with an inverse relationship to RRM2 expression was identified in innately gemcitabine-resistant pancreatic cancer cell lines. Direct binding of let-7 miRNAs to the 3′ UTR of RRM2 transcripts identified post-transcriptional regulation of RRM2 influencing gemcitabine chemosensitivity. Intriguingly, overexpression of human precursor-let-7 miRNAs led to differential RRM2 expression and chemosensitivity responses in a poorly differentiated pancreatic cancer cell line, MIA PaCa-2. Defective processing of let-7a precursors to mature forms, in part, explained the discrepancies observed with let-7a expressional outcomes. Consistently, the ratios of mature to precursor let-7a were progressively reduced in gemcitabine-sensitive L3.6pl and Capan-1 cell lines induced to acquire gemcitabine resistance. Besides known regulators of let-7 biogenesis (e.g., LIN-28), short hairpin RNA library screening identified several novel RNA binding proteins, including the SET oncoprotein, to differentially impact let-7 biogenesis and chemosensitivity in gemcitabine-sensitive versus -resistant pancreatic cancer cells. Further, LIN-28 and SET knockdown in the cells led to profound reductions in cellular proliferation and colony-formation capacities. Finally, defective processing of let-7a precursors with a positive correlation to RRM2 overexpression was identified in patient-derived pancreatic ductal adenocarcinoma (PDAC) tissues. These data demonstrate an intricate post-transcriptional regulation of RRM2 and chemosensitivity by let-7a and that the manipulation of regulatory proteins involved in let-7a transcription/processing may provide a mechanism for improving chemotherapeutic and/or tumor growth control responses in pancreatic cancer.

Gene polymorphisms of prognostic factors in non-small cell lung cancer

Many studies show that excision repair cross-complementing gene 1 (ERCC1),ribonucleotide reductase subunit M1 (RRM1),p53,epidermal growth factor receptor (EGFR),and vascular endothelial growth factor (VEGF) are closely related to the curative effect and prognosis of non-small cell lung cancer (NSCLC).In recent years,many studies have investigated the association between polymorphisms of these factors and the curative effect and prognosis in patients with NSCLC.The polymorphisms of these factors may be potential biomarkers to predict the clinical outcome in patients with NSCLC. Key words: Carcinoma, non-small cell lung; Prognosis; Polymorphism, genetic

Acquired gemcitabine resistance is associated with expression of ribonucleotide reductase subunit M2

Acquired gemcitabine resistance is associated with expression of ribonucleotide reductase subunit M2

The effect of the acid-sensitivity of 4-(N)-stearoyl gemcitabine-loaded micelles on drug resistance caused by RRM1 overexpression

Chemoresistance is a major issue for most gemcitabine-related chemotherapies. The overexpression of ribonucleotide reductase subunit M1 (RRM1) plays a key role in gemcitabine resistance. In this study, we synthesized a new highly acid-sensitive amphiphilic micelle material by conjugating hydrophilic polyethylene glycol with a hydrophobic stearic acid derivative (C18) using a hydrazone bond, which was named as PHC-2. A lipophilic prodrug of gemcitabine, 4-(N)-stearoyl gemcitabine (GemC18), was loaded into micelles prepared with PHC-2, a previously synthesized less acid-sensitive PHC-1, and their acid-insensitive counterpart, PAC. GemC18 loaded in acid-sensitive micelles can overcome gemcitabine resistance, and GemC18 in the highly acid-sensitive PHC-2 micelles was more cytotoxic than in the less acid-sensitive PHC-1 micelles. Mechanistic studies revealed that upon cellular uptake and lysosomal delivery, GemC18 in the acid-sensitive micelles was released and hydrolyzed more efficiently. Furthermore, GemC18 loaded in the highly acid-sensitive PHC-2 micelles inhibited the expression of RRM1 and increased the level of gemcitabine triphosphate (dFdCTP) in gemcitabine resistant tumor cells. The strategy of delivering lipophilized nucleoside analogs using highly acid-sensitive micelles may represent a new platform technology to increase the antitumor activity of nucleoside analogs and to overcome tumor cell resistance to them.

Expression and possible role of ribonucleotide reductase subunit M2 (RRM2) in endometriosis

Ribonucleotide reductase subunit M2 (RRM2), a rate-limiting enzyme in cell replication, has been known to play a critical role in controlling proliferative, invasive and metastatic potential of tumor. However, little is known about the expression and function of RRM2 in endometriosis. The present study aimed to evaluate the expression of RRM2 in endometrium of women with and without endometriosis and to investigate the effect of celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, on RRM2 expression.