Ribonucleotide Reductase Small Subunit M2 Research Articles

Abstract 6202: 6-Phosphofructo-2-kinase in regulating DNA damage and repair in EGFR-driven lung cancer

Abstract Activating mutations of epidermal growth factor receptor (mutEGFR) are established drivers of lung tumor development, aggressiveness, and resistance to targeted therapies. While targeting mutEGFR with small-molecule tyrosine kinase inhibitors (EGFR-TKIs) dramatically improved progression-free survival in patients with non-small cell lung cancers (NSCLCs), intrinsic or acquired resistance to TKI therapy limits the duration of an effective response to TKIs. Alteration in DNA damage response (DDR) plays a vital role in genomic stability and cancer progression and helps cells to escape apoptosis, limiting the efficacy of targeted therapies. Interestingly, lung cancer cells exposed to EGFR-TKIs demonstrate attenuated glycolytic flux - one of the major providers of precursors for de novo nucleotide synthesis. A key regulator of glycolysis is the enzyme 6-phosphofructo-2-kinase (PFKFB3). PFKFB3 synthesizes fructose 2,6-bisphosphate (F2,6BP), which is a potent allosteric activator of the glycolytic rate-limiting enzyme, 6-phosphofructo-1-kinase (PFK1). Accordingly, F2,6BP controls flux throughout the entire glycolytic pathway. In preliminary studies, we provide evidence that PFKFB3 plays multiple roles in regulating the efficacy of DDR in lung cancer cells exposed to EGFR-TKIs. We show that PFKFB3 is required for EGFR-driven glucose metabolism, which provides a list of intermediates utilized in nucleotide synthesis. PFKFB3 regulates the expression of ribonucleotide reductase small subunit M2 (RRM2) of the ribonucleotide reductase (RNR), which is important for de novo onsite synthesis of deoxynucleotide triphosphate (dNTP) during DNA replication and repair. Moreover, a PFKFB3 inhibitor, PFK-158, increases oxidative stress in mutEGFR lung cancer cell lines and, as a result, promotes DNA damage. Importantly, we found that PFKFB3 plays an important role in the chromatin binding of scaffold proteins regulating the assembly of DNA repair complex. Our studies suggest that PFKFB3 plays an important role in DDR and provide a clear rationale to propose the use of PFKFB3 inhibitors in combination with EGFR inhibitors to increase the efficiency and/or overcome resistance to EGFR-TKIs. Citation Format: Nadiia Lypova, Lilibeth Lanceta, Susan Dougherty, Jason Chesney, Yoannis Imbert-Fernandez. 6-Phosphofructo-2-kinase in regulating DNA damage and repair in EGFR-driven lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6202.

Comprehensive bioinformatics analysis of ribonucleoside diphosphate reductase subunit M2(RRM2) gene correlates with prognosis and tumor immunotherapy in pan-cancer.

Ribonucleotide reductase (RNR) small subunit M2 (RRM2) levels are known to regulate the activity of RNR, a rate-limiting enzyme in the synthesis of deoxyribonucleotide triphosphates (dNTPs) and essential for both DNA replication and repair. The high expression of RRM2 enhances the proliferation of cancer cells, thereby implicating its role as an anti-cancer agent. However, little research has been performed on its role in the prognosis of different types of cancers. This pan-cancer study aimed to evaluate the effect of high expression of RRM2 the tumor prognosis based on clinical information collected from The Cancer Genome Atlas (TCGA) and The Genotype-Tissue Expression (GTEx) databases. We found RRM2 gene was highly expressed in 30 types of cancers. And we performed a pan-cancer analysis of the genetic alteration status and methylation of RRM2. Results indicated that RRM2 existed hypermethylation, associated with m6A, m1A, and m5C related genes. Subsequently, we explored the microRNAs (miRNA), long non-coding RNAs (lncRNA), and the transcription factors responsible for the high expression of RRM2 in cancer cells. Results indicated that has-miR-125b-5p and has-miR-30a-5p regulated the expression of RRM2 along with transcription factors, such as CBFB, E2F1, and FOXM. Besides, we established the competing endogenous RNA (ceRNA) diagram of lncRNAs-miRNAs-circular RNAs (circRNA) involved in the regulation of RRM2 expression. Meanwhile, our study demonstrated that high-RRM2 levels correlated with patients’ worse prognosis survival and immunotherapy effects through the consensus clustering and risk scores analysis. Finally, we found RRM2 regulated the resistance of immune checkpoint inhibitors through the PI3K-AKT single pathways. Collectively, our findings elucidated that high expression of RRM2 correlates with prognosis and tumor immunotherapy in pan-cancer. Moreover, these findings may provide insights for further investigation of the RRM2 gene as a biomarker in predicting immunotherapy’s response and therapeutic target.

Osalmid, a Novel Identified RRM2 Inhibitor, Enhances Radiosensitivity of Esophageal Cancer

Esophageal cancer (EC) is an aggressive malignancy and is often resistant to currently available therapies. Inhibition of ribonucleotide reductase small subunit M2 (RRM2) in tumors is speculated to mediate chemosensitization. Previous studies have reported that Osalmid could act as an RRM2 inhibitor. We explored whether RRM2 was involved in radioresistance and the antitumor effects of Osalmid in EC. RRM2 expression was detected by immunohistochemistry in EC tissues. The effects of Osalmid on cell proliferation, apoptosis, and cell cycle were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphhenyl tetrazolium, colony formation, and flow cytometry assays. DNA damage, cell apoptosis, and senescence induced by Osalmid or ionizing radiation (IR) alone, or both, were detected with immunofluorescence, flow cytometry, Western blot, and β-galactosidase staining. A xenograft mouse model of EC was used to investigate the potential synergistic effects of Osalmid and IR invivo. The expression of RRM2 in treatment-resistant EC tissues is much higher than in treatment-sensitive EC, and strong staining of RRM2 was correlated with shorter overall survival. We observed direct cytotoxicity of Osalmid in EC cells. Osalmid also produced inhibition of the ERK1/2 signal transduction pathway and substantially enhanced IR-induced DNA damage, apoptosis, and senescence. Furthermore, treatment with Osalmid and IR significantly suppressed tumor growth in xenograft EC models without additional toxicity to the hematologic system and internal organs. Our study revealed that RRM2 played a vital role in radioresistance in EC, and Osalmid synergized with IR to exert its antitumor effects both invitro and invivo.

Downregulation of RRM2 Attenuates Retroperitoneal Liposarcoma Progression via the Akt/mTOR/4EBP1 Pathway: Clinical, Biological, and Therapeutic Significance.

BackgroundRetroperitoneal liposarcoma (RLPS) is a rare tumor with high recurrence rate. Ribonucleotide reductase small subunit M2 (RRM2) protein is essential for DNA synthesis and replication. Our previous study has demonstrated that RRM2 downregulation inhibited the proliferation of RLPS cells, but further association between RRM2 and RLPS and relevant mechanisms remains to be explored.MethodsRRM2 expression was evaluated in RLPS tumor tissues and cell lines by using real-time PCR and immunohistochemical analysis. The effect of RRM2 downregulation on cell proliferation, apoptosis, cell cycle, cell migration and invasion was tested by lentivirus. The effect of RRM2 inhibition on tumor growth in vivo was assessed by using patient-derived tumor xenograft (PDX) of RLPS and RRM2 inhibitor. The underlying mechanisms of RRM2 in RLPS were explored by protein microarray and Western blotting.ResultsThe results showed that RRM2 mRNA expression was higher in RLPS tissues than in normal fatty tissues (P<0.001). RRM2 expression was higher in the dedifferentiated, myxoid/round cell, and pleomorphic subtypes (P=0.027), and it was also higher in the high-grade RLPS tissues compared to that in the low-grade RLPS tissues (P=0.004). There was no correlation between RRM2 expression and overall survival (OS) or disease-free survival (DFS) in this group of RLPS patients (P>0.05). RRM2 downregulation inhibited cell proliferation, promoted cell apoptosis, facilitated cell cycle from G1 phase to S phase and inhibited cell migration and invasion. Inhibition of RRM2 suppressed tumor growth in NOD/SCID mice. Protein microarray and Western blot verification showed that activity of Akt/mammalian target of rapamycin/eukaryotic translation initiation factor 4E binding protein 1 (Akt/mTOR/4EBP1) pathway was downregulated along with RRM2 downregulation.ConclusionRRM2 was overexpressed in RLPS tissues, and downregulation of RRM2 could inhibit RLPS progression. In addition, suppression of RRM2 is expected to be a promising treatment for RLPS patients.

Ribonucleotide reductase small subunit M2 is a master driver of aggressive prostate cancer.

Although there are molecularly distinct subtypes of prostate cancer, no molecular classification system is used clinically. The ribonucleotide reductase small subunit M2 (RRM2) gene plays an oncogenic role in many cancers. Our previous study elucidated comprehensive molecular mechanisms of RRM2 in prostate cancer (PC). Given the potent functions of RRM2, we set out to determine whether the RRM2 signature can be used to identify aggressive subtypes of PC. We applied gene ontology and pathway analysis in RNA‐seq datasets from PC cells overexpressing RRM2. We refined the RRM2 signature by integrating it with two molecular classification systems (PCS and PAM50 subtypes) that define aggressive PC subtypes (PCS1 and luminal B) and correlated signatures with clinical outcomes in six published cohorts comprising 4000 cases of PC. Increased expression of genes in the RRM2 signature was significantly correlated with recurrence, high Gleason score, and lethality of PC. Patients with high RRM2 levels showed higher PCS1 score, suggesting the aggressive PC feature. Consistently, RRM2‐regulated genes were highly enriched in the PCS1 signature from multiple PC cohorts. A simplified RRM2 signature (12 genes) was identified by intersecting the RRM2 signature, PCS1 signature, and the PAM50 classifier. Intriguingly, inhibition of RRM2 specifically targets PCS1 and luminal B genes. Furthermore, 11 genes in the RRM2 signature were correlated with enzalutamide resistance by using a single‐cell RNA‐seq dataset from PC circulating tumor cells. Finally, high expression of RRM2 was associated with an immunosuppressive tumor‐immune microenvironment in both primary prostate cancer and metastatic prostate cancer using CIBERSORT analysis and LM22, a validated leukocyte gene signature matrix. These data demonstrate that RRM2 is a driver of aggressive prostate cancer subtypes and contributes to immune escape, suggesting that RRM2 inhibition may be of clinical benefit for patients with PC.

A Novel Mechanism Driving Poor-Prognosis Prostate Cancer: Overexpression of the DNA Repair Gene, Ribonucleotide Reductase Small Subunit M2 (RRM2).

Defects in genes in the DNA repair pathways significantly contribute to prostate cancer progression. We hypothesize that overexpression of DNA repair genes may also drive poorer outcomes in prostate cancer. The ribonucleotide reductase small subunit M2 (RRM2) is essential for DNA synthesis and DNA repair by producing dNTPs. It is frequently overexpressed in cancers, but very little is known about its function in prostate cancer. The oncogenic activity of RRM2 in prostate cancer cells was assessed by inhibiting or overexpressing RRM2. The molecular mechanisms of RRM2 function were determined. The clinical significance of RRM2 overexpression was evaluated in 11 prostate cancer clinical cohorts. The efficacy of an RRM2 inhibitor (COH29) was assessed in vitro and in vivo. Finally, the mechanism underlying the transcriptional activation of RRM2 in prostate cancer tissue and cells was determined. Knockdown of RRM2 inhibited its oncogenic function, whereas overexpression of RRM2 promoted epithelial mesenchymal transition in prostate cancer cells. The prognostic value of RRM2 RNA levels in prostate cancer was confirmed in 11 clinical cohorts. Integrating the transcriptomic and phosphoproteomic changes induced by RRM2 unraveled multiple oncogenic pathways downstream of RRM2. Targeting RRM2 with COH29 showed excellent efficacy. Thirteen putative RRM2-targeting transcription factors were bioinformatically identified, and FOXM1 was validated to transcriptionally activate RRM2 in prostate cancer. We propose that increased expression of RRM2 is a mechanism driving poor patient outcomes in prostate cancer and that its inhibition may be of significant therapeutic value.

Abstract LB-275: Targeting poor-prognosis subtypes of prostate cancer by inhibition of DNA repair gene ribonucleotide reductase small subunit M2

Abstract Studies reveal defective DNA repair contribute to prostate cancer (PC) progression. We hypothesize that overexpression of DNA repair genes could also contribute to poorer outcomes in PC. The nucleotide metabolism enzyme ribonucleotide reductase (RNR) is essential for DNA synthesis and DNA repair by producing dNTPs. The small subunit M2 (RRM2), as the rate-limiting RNR subunit, is frequently up-regulated in cancers. Clinically, targeting of RRM2 with small molecules is being tested in multiple cancers, but there is little knowledge of RRM2 function in PC. The analysis of multiple PC clinical cohorts (total include 1602 cases) revealed that high RRM2 level was associated with poor clinical outcomes, including a higher likelihood of metastasis (p&amp;lt;0.001), biochemical recurrence (p&amp;lt;0.001), and lethality (p&amp;lt;0.0001). In PC cells, knockdown of RRM2 inhibited dNTP production and induced DNA damage, which led to significant cell growth inhibition, major S phase arrest, and apoptosis. Overexpression of RRM2 promoted epithelial-mesenchymal transition (EMT) by increasing the expression of multiple EMT markers. Furthermore, the small molecule RRM2 inhibitor (COH29) induced a similar phenotype as knocking down RRM2 in PC cells. RNA-Seq analysis in siRRM2 or COH29 treated PC cells provided a global assessment of RRM2-regulated transcriptome changes. GSEA analysis revealed that inhibition of RRM2 could activate biological processes including cell cycle checkpoint, DNA damage response, and apoptotic signaling. COH29 treatment could target genes highly enriched in PC. We further applied an RRM2-regulated gene signature (from RNA-Seq datasets) to TCGA and Taylor cohorts. Intriguingly, the RRM2 signature was highly correlated with metastasis and disease free survival (p&amp;lt;0.001). Furthermore, inhibition of RRM2 specifically targets poor prognostic luminal subtypes (PCS1 subtype; Lum B in PAM50 classifier) recently reported. Besides transcriptome changes, protein kinase arrays showed that AKT/mTOR and SFK-STAT signaling were repressed by inhibition of RRM2. These oncogenic signaling pathways are crucial for EMT program. Amplification of RRM2 is rare in PC and transcriptional activation of RRM2 may play a major role in overexpression of RRM2. H3K27ac ChIP-Seq from tissues revealed more activated RRM2 promoter in PC than in normal prostate. 13 potential RRM2-targeting transcription factors (TFs) were identified by integrating clinical cohorts and a TF database. They showed a positive correlation with RRM2 expression in PC cohorts. Among these TFs, FOXM1 was reported to be the master driver of the aggressive luminal subtype of PC. We revealed that FOXM1 expression was associated with clinical outcomes. The ChIP-PCR and luciferase reporter assays provided evidence of physical binding of FOXM1 to the RRM2 promoter in PC cells. Knockdown of FOXM1 significantly repressed RRM2 mRNA and protein levels. Altogether, FOXM1-regulated transcriptional activation contributes to overexpression of RRM2. Intriguingly, COH29 can also repress FOXM1 expression, which leads to transcription repression of RRM2. Altogether, our study elucidated the molecular mechanisms underlying RRM2 oncogenic functions and the transcriptional regulation of RRM2 in PC cells. We suggest that RRM2 can be a novel therapeutic target for PC treatment. Citation Format: Ying Z. Mazzu, Joshua Armenia, Goutam Chakraborty, Yuki Yoshikawa, Travis A. Gerke, Si Ana A. Coggins, Xintao Qiu, Mohammad Atiq, Konrad H. Stopsack, Gwo-Shu Mary Lee, Henry W. Long, Baek Kim, Matthew L. Freedman, Mark M. Pomerantz, Lorelei A. Mucci, Philip W. Kantoff. Targeting poor-prognosis subtypes of prostate cancer by inhibition of DNA repair gene ribonucleotide reductase small subunit M2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-275.

Prognostic and therapeutic significance of ribonucleotide reductase small subunit M2 in prostate cancer.

240 Background: DNA-repair defects are common in advanced prostate cancer (PC) and high-risk localized tumors. Besides deficiency of DNA repair, overexpression of DNA repair genes could also contribute to poorer outcomes for PC patients. The nucleotide metabolism enzyme ribonucleotide reductase (RNR) plays the key role in DNA synthesis and repair. RRM2, the rate-limiting RNR subunit, is frequently up-regulated in cancer, and its overexpression leads to chemoresistance. Although targeting RRM2 by siRNA and small molecules has been applied in clinical trials in multiple cancers, limited knowledge of RRM2 function in PC delays potential clinical application of RRM2 inhibition. Methods: We leveraged publically available PC clinical cohorts to examine RRM2 levels and clinical outcomes. siRNAs was applied to knockdown of RRM2 in multiple PC cell lines. Cell growth, cell cycle and apoptosis were analyzed to determine siRRM2 or RRM2 inhibitor (COH29)-induced phenotypes. RNA-seq and protein array were performed to identify downstream targets of RRM2. Immunohistochemistry staining was applied to determine prevalence of RRM2 protein expression in PC tissues microarrays (TMAs). Results: In PC cohorts, increased RRM2 expression was associated with a higher likelihood of metastasis, poorer disease-free survival, and increased risk of development of lethal disease (N = 1200, PHS/HPFS cohorts). In PC cells, Inhibition of RRM2 induced remarkable cell growth inhibition, cell cycle arrest (at S phase) and apoptosis. DNA damage was observed in siRRM2/COH29-treated PC cells with increased activation of DNA damage markers. GSEA analysis of the RNA-seq dataset revealed multiple biological processes were affected by inhibition of RRM2, such as cell cycle, apoptosis, and DNA damage response. Intriguingly, MYC oncogenic signaling is the major downstream targets of RRM2. Furthermore, inhibition of RRM2 can block multiple oncogenic signaling including mTOR/AKT, SFK, and STAT signaling by repressing the key phospho-kinases in PC cells. Among 121 cases on the PC TMAs, 20% showed strong RRM2 protein expression. Conclusions: RRM2 may serve as a prognostic biomarker and novel therapeutic target in PC.

SATB1 promotes the malignant of human non-Hodgkin lymphoma by activating the ribonucleotide reductase small subunit M2

To explore the role of the special AT rich sequence binding protein-1 (SATB1) and ribonucleotide reductase M2 (RRM2) in enhancing malignant progression of non-Hodgkin lymphoma (NHL). Methods: A total of 42 NHL and 42 chronic lymphadenitis patients were recruited. The protein expressions of SATB1 and RRM2 in cervical lymph nodes were determined by Western blot. After overexpression of SATB1, siSATB1 or siRRM2, the mRNA levels of SATB1 and RRM2 in cells were analyzed via RT-PCR, the cell proliferation was evaluated via MTT and EdU assays, while the migration and invasion of cells were assessed by transwell assays. Results: Compared with chronic lymphadenitis, the expressions of SATB1 and RRM2 in NHL patients were up-regulated. There was positive correlation between SATB1 and RRM2 in NHL patients. RRM2 mRNA level was up-regulated after transfection of SATB1 and down-regulated after transfection of siSATB1. Overexpression of SATB1 increased tumor cell proliferation, migration and invasion, while knockdown of RRM2 reversed those phenomena. Conclusion: SATB1 functions as an oncogene and promotes tumor cell proliferation, migration and invasion by up-regulation of RRM2 in NHL.

CREB1 directly activates the transcription of ribonucleotide reductase small subunit M2 and promotes the aggressiveness of human colorectal cancer.

As the small subunit of Ribonucleotide reductase (RR), RRM2 displays a very important role in various critical cellular processes such as cell proliferation, DNA repair, and senescence, etc. Importantly, RRM2 functions like a tumor driver in most types of cancer but little is known about the regulatory mechanism of RRM2 in cancer development. In this study, we found that the cAMP responsive element binding protein 1 (CREB1) acted as a transcription factor of RRM2 gene in human colorectal cancer (CRC). CREB1 directly bound to the promoter of RRM2 gene and induced its transcriptional activation. Knockdown of CREB1 decreased the expression of RRM2 at both mRNA and protein levels. Moreover, knockdown of RRM2 attenuated CREB1-induced aggressive phenotypes of CRC cells in vitro and in vivo. Analysis of the data from TCGA database and clinical CRC specimens with immunohistochemical staining also demonstrated a strong correlation between the co-expression of CREB1 and RRM2. Decreased disease survivals were observed in CRC patients with high expression levels of CREB1 or RRM2. Our results indicate CREB1 as a critical transcription factor of RRM2 which promotes tumor aggressiveness, and imply a significant correlation between CREB1 and RRM2 in CRC specimens. These may provide the possibility that CREB1 and RRM2 could be used as biomarkers or targets for CRC diagnosis and treatment.

ATR–CHK1–E2F3 signaling transactivates human ribonucleotide reductase small subunit M2 for DNA repair induced by the chemical carcinogen MNNG

BackgroundN-methyl-N′-nitro-N-nitrosoguanidine (MNNG), an alkylating agent and an environmental carcinogen, causes DNA lesions and even carcinomas. DNA damage responses induced by MNNG activate various DNA repair genes and related signaling pathways. The present study aimed to investigate the regulatory mechanisms of human RR small subunit M2 (hRRM2) in response to MNNG. ResultsIn this study, we demonstrated that the RRM2 gene was transactivated by MNNG exposure more strongly than the other small subunit, p53R2. The upregulated RRM2 translocated to the nucleus for DNA repair. Further study showed that E2F3 transactivated RRM2 expression by directly binding to its promoter after MNNG exposure. The transactivation was enhanced by the upregulation of NFY, which bound to the RRM2 promoter adjacent to the E2F3 binding site and interacted with E2F3. In response to MNNG treatment, E2F3 accumulated mainly through its phosphorylation at S124 and was dependent on ATR–CHK1 signaling. In comparison, p53R2 played a relatively weaker role in the MNNG-induced DNA damage response, and its transcription was regulated by the ATR–CHK2–E2F1/p53 pathway. ConclusionsWe suggest that MNNG-stimulated ATR/CHK1 signaling stabilizes E2F3 by S124 phosphorylation, and then E2F3 together with NFY co-transactivate RRM2 expression for DNA repair. General significanceWe propose a new mechanism for RRM2 regulation to maintain genome stability in response to environmental chemical carcinogens.

Inhibition of hepatitis B virus replication by targeting ribonucleotide reductase M2 protein

Chronic hepatitis B virus (HBV) infection is a key factor for hepatocellular carcinoma worldwide. Ribonucleotide reductase (RR) regulates the deoxyribonucleoside triphosphates biosynthesis and serves as a target for anti-cancer therapy. Here, we demonstrate that RR is essential for HBV replication and the viral covalently-closed-circular DNA (cccDNA) synthesis in host liver cells. By performing computer-assisted virtual screening against the crystal structure of RR small subunit M2 (RRM2), osalmid, was identified as a potential RRM2-targeting compound. Osalmid was shown to be 10-fold more active in inhibiting RR activity than hydroxyurea, and significantly inhibited HBV DNA and cccDNA synthesis in HepG2.2.15 cells. In contrast, hydroxyurea and the RR large subunit (RRM1)-inhibitory drug gemcitabine showed little selective activity against HBV replication. In addition, osalmid also was shown to possess potent activity against a 3TC-resistant HBV strain, suggesting utility in treating drug-resistant HBV infections. Interestingly, osalmid showed synergistic effects with lamivudine (3TC) in vitro and in vivo without significant toxicity, and was shown to inhibit RR activity in vivo, thus verifying its in vivo function. Furthermore, 4-cyclopropyl-2-fluoro-N-(4-hydroxyphenyl) benzamide (YZ51), a novel derivative of osalmid, showed higher efficacy than osalmid with more potent RR inhibitory activity. These results suggest that RRM2 might be targeted for HBV inhibition, and the RRM2-targeting compound osalmid and its derivative YZ51 could be a novel class of anti-HBV candidates with potential use for hepatitis B and HBV-related HCC treatment.

E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2

As the ribonucleotide reductase small subunit, the high expression of ribonucleotide reductase small subunit M2 (RRM2) induces cancer and contributes to tumor growth and invasion. In several colorectal cancer (CRC) cell lines, we found that the expression levels of RRM2 were closely related to the transcription factor E2F1. Mechanistic studies were conducted to determine the molecular basis. Ectopic overexpression of E2F1 promoted RRM2 transactivation while knockdown of E2F1 reduced the levels of RRM2 mRNA and protein. To further investigate the roles of RRM2 which was activated by E2F1 in CRC, CCK-8 assay and EdU incorporation assay were performed. Overexpression of E2F1 promoted cell proliferation in CRC cells, which was blocked by RRM2 knockdown attenuation. In the migration and invasion tests, overexpression of E2F1 enhanced the migration and invasion of CRC cells which was abrogated by silencing RRM2. Besides, overexpression of RRM2 reversed the effects of E2F1 knockdown partially in CRC cells. Examination of clinical CRC specimens demonstrated that both RRM2 and E2F1 were elevated in most cancer tissues compared to the paired normal tissues. Further analysis showed that the protein expression levels of E2F1 and RRM2 were parallel with each other and positively correlated with lymph node metastasis (LNM), TNM stage and distant metastasis. Consistently, the patients with low E2F1 and RRM2 levels have a better prognosis than those with high levels. Therefore, we suggest that E2F1 can promote CRC proliferation, migration, invasion and metastasis by regulating RRM2 transactivation. Understanding the role of E2F1 in activating RRM2 transcription will help to explain the relationship between E2F1 and RRM2 in CRC and provide a novel predictive marker for diagnosis and prognosis of the disease.

Prognostic and therapeutic significance of ribonucleotide reductase small subunit M2 in estrogen-negative breast cancers.

BackgroundRibonucleotide reductase (RR) is an essential enzyme involved in DNA synthesis. We hypothesized that RR subunit M2 (RRM2) might be a novel prognostic and predictive biomarker for estrogen receptor (ER)-negative breast cancers.MethodsIndividual and pooled survival analyses were conducted on six independent large-scale breast cancer microarray data sets; and findings were validated on a human breast tissue set (ZJU set).ResultsGene set enrichment analysis revealed that RRM2-high breast cancers were significantly enriched for expression of gene sets that increased in proliferation, invasiveness, undifferentiation, embryonic stem/progenitor-like phenotypes, and poor patient survival (p < 0.01). Independent and pooled analyses verified that increased RRM2 mRNA levels were associated with poor patient outcome in a dose-dependent manner. The prognostic power of RRM2 mRNA was comparable to multiple gene signatures, and it was superior to TNM stage. In ER-negative breast cancers, RRM2 showed more prognostic power than that in ER-positive breast cancers. Further analysis indicated that RRM2 was a more accurate prognostic biomarker for ER-negative breast cancers than the pathoclinical indicators and uPA. A new RR inhibitor, COH29, could significantly enhance the chemosensitivity to doxorubicin in ER-negative MDA-MB-231 cells, but not in ER-positive MCF-7 cells.ConclusionRRM2 appears to be a promising prognostic biomarker and therapeutic target for ER-negative breast cancer patients.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-664) contains supplementary material, which is available to authorized users.

The Conserved Lys-95 Charged Residue Cluster Is Critical for the Homodimerization and Enzyme Activity of Human Ribonucleotide Reductase Small Subunit M2

Ribonucleotide reductase (RR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides for DNA synthesis. Human RR small subunit M2 exists in a homodimer form. However, the importance of the dimer form to the enzyme and the related mechanism remain unclear. In this study, we tried to identify the interfacial residues that may mediate the assembly of M2 homodimer by computational alanine scanning based on the x-ray crystal structure. Co-immunoprecipitation, size exclusion chromatography, and RR activity assays showed that the K95E mutation in M2 resulted in dimer disassembly and enzyme activity inhibition. In comparison, the charge-exchanging double mutation of K95E and E98K recovered the dimerization and activity. Structural comparisons suggested that a conserved cluster of charged residues, including Lys-95, Glu-98, Glu-105, and Glu-174, at the interface may function as an ionic lock for M2 homodimer. Although the measurements of the radical and iron contents showed that the monomer (the K95E mutant) was capable of generating the diiron and tyrosyl radical cofactor, co-immunoprecipitation and competitive enzyme inhibition assays indicated that the disassembly of M2 dimer reduced its interaction with the large subunit M1. In addition, the immunofluorescent and fusion protein-fluorescent imaging analyses showed that the dissociation of M2 dimer altered its subcellular localization. Finally, the transfection of the wild-type M2 but not the K95E mutant rescued the G1/S phase cell cycle arrest and cell growth inhibition caused by the siRNA knockdown of M2. Thus, the conserved Lys-95 charged residue cluster is critical for human RR M2 homodimerization, which is indispensable to constitute an active holoenzyme and function in cells.

The prognostic value of ribonucleotide reductase small subunit M2 in predicting recurrence for prostate cancers

PurposeTo investigate the prognostic significance of ribonucleotide reductase small subunit M2 (RRM2) in low- and intermediate-risk prostate cancer (PCa). Materials and methodsA retrospective outcome study was conducted on 164 eligible PCa samples from the City of Hope (n = 90) and the Taipei Medical University (n = 74). The RRM2 protein levels were detected by immunohistochemistry. Biochemical recurrence was assessed using Kaplan-Meier and Cox proportional hazard analyses. Cell invasion assays, Ras/Raf, and matrix metallopeptidase 9 activities were determined to evaluate the role of RRM2 on invasiveness of PCa. ResultsExpression of RRM2 was significantly increased in patients with higher Gleason score, who had advanced T stage, and who were margin/capsule positive (P<0.05). Analysis revealed that the expression of RRM2 positively associated with biochemical recurrence of PCa in the City of Hope set (hazard ratio = 5.26; 95% CI 1.50–24.71) and the Taipei Medical University set (hazard ratio = 2.55; 95% CI 1.30–9.22). In stratification analysis, RRM2 was significantly correlated with poor outcome in patients with lower-risk PCa, including those with Gleason score 4 to 7, margin−, capsule−, and stage T1-T2. In patients with Gleason score 4 to 7, the risk of recurrence was proportional to RRM2 protein levels. The prognostic performance of RRM2 was superior to that of pathoclinical factors, including margin/capsule status and T stage. An in vitro study demonstrated that RRM2 could promote tumor invasion activities in PCa cell lines. Suppression of RRM2 reduced the Ras/Raf and matrix metallopeptidase 9 activities. ConclusionRRM2 plays a critical role in proliferation and invasion of PCa. Adding RRM2 as a biomarker in clinical assessments may increase model precision in predicting recurrence in patients with low-risk PCa.

Ribonucleotide reductase small subunit M2 serves as a prognostic biomarker and predicts poor survival of colorectal cancers

The overexpression of RRM2 [RR (ribonucleotide reductase) small subunit M2] dramatically enhances the ability of the cancer cell to proliferate and to invade. To investigate further the relevance of RRM2 and CRCs (colorectal cancers), we correlated the expression of RRM2 with the clinical outcome of CRCs. A retrospective outcome study was conducted on CRCs collected from the COH [(City of Hope) National Medical Center, 217 cases] and ZJU (Zhejiang University, 220 cases). IHC (immunohistochemistry) was employed to determine the protein expression level of RRM2, and quantitative real-time PCR was employed to validate. Multivariate logistic analysis indicated that the adjusted ORs (odds ratios) of RRM2-high for distant metastases were 2.06 [95% CI (confidence interval), 1.01–4.30] and 5.89 (95% CI, 1.51–39.13) in the COH and ZJU sets respectively. The Kaplan–Meier analysis displayed that high expression of RRM2 had a negative impact on the OS (overall survival) and PFS (progress-free survival) of CRC in both sets significantly. The multivariate Cox analysis further demonstrated that HRs (hazard ratios) of RRM2-high for OS were 1.88 (95% CI, 1.03–3.36) and 2.06 (95% CI, 1.10–4.00) in the COH and ZJU sets respectively. Stratification analysis demonstrated that the HR of RRM2 dramatically increased to 12.22 (95% CI, 1.62–258.31) in the MMR (mismatch repair) gene-deficient subgroup in the COH set. Meanwhile, a real-time study demonstrated that down-regulation of RRM2 by siRNA (small interfering RNA) could significantly and specifically reduce the cell growth and adhesion ability in HT-29 and HCT-8 cells. Therefore RRM2 is an independent prognostic factor and predicts poor survival of CRCs. It is also a potential predictor for identifying good responders to chemotherapy for CRCs.

Abstract 706: Ribonucleotide reductase small subunit M2 is a potential prognostic biomarker in human epidermal growth factor receptor 2- positive and triple negative breast cancers

Abstract Human epidermal growth factor receptor 2 (HER2)-positive breast cancer and triple negative breast cancer (TNBC) are two subtypes with poor outcome of all breast carcinomas. However, among all the patients in these two subtypes some still yielded relatively better prognosis. Ribonucleotide reductase (RR) small subunits M2 was associated with cancer cell invasiveness and metastasis, which prognoses poor survival in many cancers. Here we investigated whether RRM2 could serve as a prognostic biomarker for breast cancers, especially HER2- positive and TNBC subgroups. One hundred seventy five assessable breast cancers (40 cases were HER2-positive or TNBC) were collected from affiliated hospital of Zhejiang University (ZJU set); and six published microarray data sets (total 1154 cases, 63 cases were identified as HER2- positive or TNBC) with pathoclinical and follow- up information were employed for validation. In the ZJU set, the protein level of RRM2 was determined by IHC. It was indicated that RRM2 was positively correlated with cell proliferative marker Ki-67 (trends P=0.018) and stem/progenitor cell marker CD44+/CD24-/low (P=0.044). Multivariate COX model revealed that RRM2 was an independent prognostic biomarker for both OS (Hazard Ratio, HR=2.79, 95%Cl 1.21-6.75) and PFS (Hazard Ratio, HR=1.82, 95%Cl 1.02-3.26) for all the breast cancers. Moreover, in HER2-positive or TNBC breast cancers, RRM2-high was significantly associated with poor survival (P=0.025). Consequently, none of the patients with RRM2-low died of breast cancer in HER2-positive or TNBC subgroups. Furthermore, above findings were validated in the published data sets. The mRNA level of RRM2 evaluated significantly in highly-invasive subtypes including Luminal B, HER2-positive and TNBC in comparison with Luminal A and unclassified subgroups (P&amp;lt;0.05). Overexpression of RRM2 was positively associated with poor differentiation (P&amp;lt;0.05), advanced AJCC stage (P&amp;lt;0.05) as well as poor survival (P&amp;lt;0.05) in a dose-dependent manner. It was also revealed that high expression of RRM2 prognosed poor survival (HR=5.56, 95% CI 1.14-99.9) in HER2-positive breast cancers or TNBC. Moreover, the RRM2-high (protein or mRNA levels) was associated with better response to chemotherapy comparing to RRM2-low. Taken together, the above findings suggested that RRM2 was a potential biomarker for poor survival in breast cancer, especially in HER2- positive breast cancer and TNBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 706. doi:1538-7445.AM2012-706

Ribonucleotide Reductase Subunits M2 and p53R2 are Potential Biomarkers for Metastasis of Colon Cancer

Ribonucleoside diphosphate reductase plays a key role in converting ribonucleoside diphosphate to 2'-deoxyribonucleoside diphosphate, which is necessary for DNA repair and replication. To determine if human ribonucleotide reductase small subunit M2 (hRRM2) and p53-dependent human ribonucleotide reductase small subunit R2 (p53R2) play roles on invasion ability of cancer cells, the gene transferring technique was used to construct stable hRRM2 and p53R2 overexpression transfectants. Increase of hRRM2 dramatically enhanced the cell migration in KB and PC-3 cells, but p53R2 overexpression reduced cellular invasion potential to 50% and 40% in KB and PC-3 cells, respectively. Furthermore, hRRM2 enhanced cancer cells to induce the cell migration of Human Umbilical Vein Endothelial Cells, but p53R2 reduced this ability in transfectants. To further determine the role of human ribonucleotide reductase subunits on cancer metastasis, a tissue array, including 59 primary and 49 metastatic colon adenocarcinoma samples, was used. Immunohistochemistry was used to evaluate the relationship between human ribonucleotide reductase subunits and metastasis. Univariate and multivariate analysis revealed that p53R2 is negatively related to the metastasis of colon adenocarcinoma samples (odds ratio, 0.23; P < 0.05); hRRM2 increases the risk of metastasis in colon cancer, but did not show significantly. Thus, opposing regulation of hRRM2 and p53R2 in invasion potential might play a critical role in determining the invasion and metastasis phenotype in cancer cells. The expression level of ribonucleotide reductase small subunits could serve as biomarkers to predict the malignancy potential of human cancers in the future.

Metastasis-Suppressing Potential of Ribonucleotide Reductase Small Subunit p53R2 in Human Cancer Cells

Previous gene transfection studies have shown that the accumulation of human ribonucleotide reductase small subunit M2 (hRRM2) enhances cellular transformation, tumorigenesis, and malignancy potential. The latest identified small subunit p53R2 has 80% homology to hRRM2. Here, we investigate the role of p53R2 in cancer invasion and metastasis. The immunohistochemistry was conducted on a tissue array including 49 primary and 59 metastatic colon adenocarcinoma samples to determine the relationship between p53R2 expression and metastasis. A Matrigel invasive chamber was used to sort the highly invasive cells and to evaluate the invasion potential of p53R2. Univariate and multivariate analyses revealed that p53R2 is negatively related to the metastasis of colon adenocarcinoma samples (odds ratio, 0.23; P<0.05). The decrease of p53R2 is associated with cell invasion potential, which was observed in both p53 wild-type (KB) and mutant (PC-3 and Mia PaCa-2) cell lines. An increase in p53R2 expression by gene transfection significantly reduced the cellular invasion potential to 54% and 30% in KB and PC-3 cells, respectively, whereas inhibition of p53R2 by short interfering RNA resulted in a 3-fold increase in cell migration. Opposite regulation of hRRM2 and p53R2 in invasion potential might play a critical role in determining the invasion and metastasis phenotype in cancer cells. The expression level of ribonucleotide reductase small subunits may serve as a biomarker to predict the malignancy potential of human cancers in the future.