Inhibition of ribonucleotide reductase subunit M2 enhances the radiosensitivity of metastatic pancreatic neuroendocrine tumor.

Ribonucleotide Reductase (RNR) is a rate-limiting enzyme in the production of deoxyribonucleoside triphosphates (dNTPs), which are essential substrates for DNA repair after radiation damage. We explored the radiosensitization property of RNR and investigated a selective RRM2 inhibitor, 3-AP, as a radiosensitizer in the treatment of metastatic pNETs. We investigated the role of RNR subunit, RRM2, in pancreatic neuroendocrine (pNET) cells and responses to radiation in vitro. We also evaluated the selective RRM2 subunit inhibitor, 3-AP, as a radiosensitizer to treat pNET metastases in vivo. Knockdown of RNR subunits demonstrated that RRM1 and RRM2 subunits, but not p53R3, play significant roles in cell proliferation. RRM2 inhibition activated DDR pathways through phosphorylation of ATM and DNA-PK protein kinases but not ATR. RRM2 inhibition also induced Chk1 and Chk2 phosphorylation, resulting in G1/S phase cell cycle arrest. RRM2 inhibition sensitized pNET cells to radiotherapy and induced apoptosis in vitro. In vivo, we utilized pNET subcutaneous and lung metastasis models to examine the rationale for RNR-targeted therapy and 3-AP as a radiosensitizer in treating pNETs. Combination treatment significantly increased apoptosis of BON (human pNET) xenografts and significantly reduced the burden of lung metastases. Together, our results demonstrate that selective RRM2 inhibition induced radiosensitivity of metastatic pNETs both in vitro and in vivo. Therefore, treatment with the selective RRM2 inhibitor, 3-AP, is a promising radiosensitizer in the therapeutic armamentarium for metastatic pNETs.

Cytoplasmic RRM1 activation as an acute response to gemcitabine treatment is involved in drug resistance of pancreatic cancer cells.

BACKGROUND: RRM1 is functionally associated with DNA replication and DNA damage repair. However, the biological activity of RRM1 in pancreatic cancer remains undetermined. METHODS: To determine relationships between RRM1 expression and the prognosis of pancreatic cancer, and to explore RRM1 function in cancer biology, we investigated RRM1 expression levels in 121 pancreatic cancer patients by immunohistochemical staining and performed in vitro experiments to analyze the functional consequences of RRM1 expression. RESULTS: Patients with high RRM1 expression had significantly poorer clinical outcomes (overall survival; p = 0.006, disease-free survival; p = 0.0491). In particular, high RRM1 expression was also associated with poorer overall survival on adjuvant chemotherapy (p = 0.008). We found that RRM1 expression was increased 24 hours after exposure to gemcitabine and could be suppressed by histone acetyltransferase inhibition. RRM1 activation in response to gemcitabine exposure was induced mainly in the cytoplasm and cytoplasmic RRM1 activation was related to cancer cell viability. In contrast, cancer cells lacking cytoplasmic RRM1 activation were confirmed to show severe DNA damage. RRM1 inhibition with specific siRNA or hydroxyurea enhanced the cytotoxic effects of gemcitabine for pancreatic cancer cells. CONCLUSIONS: Cytoplasmic RRM1 activation is involved in biological processes related to drug resistance in response to gemcitabine exposure and could be a potential target for pancreatic cancer treatment.

Identification of theranostic factors for patients developing metastasis after surgery for early-stage lung adenocarcinoma.

Rationale: Lung adenocarcinoma (LUAD) is an aggressive disease with high propensity of metastasis. Among patients with early-stage disease, more than 30% of them may relapse or develop metastasis. There is an unmet medical need to stratify patients with early-stage LUAD according to their risk of relapse/metastasis to guide preventive or therapeutic approaches. In this study, we identified 4 genes that can serve both therapeutic and diagnostic (theranostic) purposes. Methods: Three independent datasets (GEO, TCGA, and KMPlotter) were used to evaluate gene expression profile of patients with LUAD by unbiased screening approach. Upon significant genes uncovered, functional enrichment analysis was carried out. The predictive power of their expression on patient prognosis were evaluated. Once confirmed their theranostic roles by integrated bioinformatics, we further conducted in vitro and in vivo validation. Results: We found that four genes (ADAM9, MTHFD2, RRM2, and SLC2A1) were associated with poor patient outcomes with an increased hazard ratio in LUAD. Knockdown of them, both separately and simultaneously, suppressed lung cancer cell proliferation and migration ability in vitro and prolonged survival time in metastatic tumor mouse models. Moreover, these four biomarkers were found to be overexpressed in tumor tissues from LUAD patients, and the total immunohistochemical staining scores correlated with poor prognosis. Conclusions: These results suggest that these four identified genes could be theranostic biomarkers for stratifying high-risk patients who develop relapse/metastasis in early-stage LUAD. Developing therapeutic approaches for the four biomarkers may benefit early-stage LUAD patients after surgery.

A comprehensive review of hydroxyurea for ß-haemoglobinopathies: the role revisited during COVID-19 pandemic.

BACKGROUND: Hydroxyurea is one of the earliest drugs that showed promise in the management of haemoglobinopathies that include ß-thalassaemia and sickle cell disease. Despite this, many aspects of hydroxyurea are either unknown or understudied; specifically, its usefulness in ß-thalassaemia major and haemoglobin E ß-thalassaemia is unclear. However, during COVID-19 pandemic, it has become a valuable adjunct to transfusion therapy in patients with ß-haemoglobinopathies. In this review, we aim to explore the available in vitro and in vivo mechanistic data and the clinical utility of hydroxyurea in ß-haemoglobinopathies with a special emphasis on its usefulness during the COVID-19 pandemic. MAIN BODY: Hydroxyurea is an S-phase-specific drug that reversibly inhibits ribonucleoside diphosphate reductase enzyme which catalyses an essential step in the DNA biosynthesis. In human erythroid cells, it induces the expression of γ-globin, a fetal globin gene that is suppressed after birth. Through several molecular pathways described in this review, hydroxyurea exerts many favourable effects on the haemoglobin content, red blood cell indices, ineffective erythropoiesis, and blood rheology in patients with ß-haemoglobinopathies. Currently, it is recommended for sickle cell disease and non-transfusion dependent ß-thalassaemia. A number of clinical trials are ongoing to evaluate its usefulness in transfusion dependent ß-thalassaemia. During the COVID-19 pandemic, it was widely used as an adjunct to transfusion therapy due to limitations in the availability of blood and logistical disturbances. Thus, it has become clear that hydroxyurea could play a remarkable role in reducing transfusion requirements of patients with haemoglobinopathies, especially when donor blood is a limited resource. CONCLUSION: Hydroxyurea is a well-tolerated oral drug which has been in use for many decades. Through its actions of reversible inhibition of ribonucleoside diphosphate reductase enzyme and fetal haemoglobin induction, it exerts many favourable effects on patients with ß-haemoglobinopathies. It is currently approved for the treatment of sickle cell disease and non-transfusion dependent ß-thalassaemia. Also, there are various observations to suggest that hydroxyurea is an important adjunct in the treatment of transfusion dependent ß-thalassaemia which should be confirmed by randomised clinical trials.

Inhibiting RRM2 to enhance the anticancer activity of chemotherapy.

RRM2, the small subunit of ribonucleotide reductase, is identified as a tumor promotor and therapeutic target. It is common to see the overexpression of RRM2 in chemo-resistant cancer cells and patients. RRM2 mediates the resistance of many chemotherapeutic drugs and could become the predictor for chemosensitivity and prognosis. Therefore, inhibition of RRM2 may be an effective means to enhance the anticancer activity of chemotherapy. This review tries to discuss the mechanisms of RRM2 overexpression and the role of RRM2 in resistance to chemotherapy. Additionally, we compile the studies on small interfering RNA targets RRM2, RRM2 inhibitors, kinase inhibitors, and other ways that could overcome the resistance of chemotherapy or exert synergistic anticancer activity with chemotherapy through the expression inhibition or the enzyme inactivation of RRM2.

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

PURPOSE: 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. METHODS AND MATERIALS: 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 ß-galactosidase staining. A xenograft mouse model of EC was used to investigate the potential synergistic effects of Osalmid and IR in vivo. RESULTS: 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. CONCLUSIONS: Our study revealed that RRM2 played a vital role in radioresistance in EC, and Osalmid synergized with IR to exert its antitumor effects both in vitro and in vivo.

Identification of osalmid metabolic profile and active metabolites with anti-tumor activity in human hepatocellular carcinoma cells.

BACKGROUNDS: Ribonucleotide reductase (RR) catalyzes the essential step in the formation of all four deoxynucleotides. Upregulated activity of RR plays an active role in tumor progression. As the regulatory subunit of RR, ribonucleotide reductase subunit M2 (RRM2) is regarded as one of the effective therapeutic targets for DNA replication-dependent diseases, such as cancers. Recent studies have revealed that osalmid significantly inhibits the activity of RRM2, but the metabolic profile of osalmid remains unknown. OBJECTIVE: The aim of this study was to clarify the metabolic profile including metabolites, isoenzymes and metabolic pathways of osalmid. The anti-human hepatocellular carcinoma activity and mechanism of metabolites were further investigated. MATERIALS AND METHODS: Ultra high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) was used for identifying metabolites and for characterizing phase I and phase II metabolic pathways with recombinant enzymes or in human liver microsomes of osalmid. The eHiTS docking system was used for potential RRM2 inhibitor screening among metabolites. Cytotoxicity assays were performed for evaluating cell proliferation inhibitory activity of metabolites. Cell cycle assays and cell apoptosis assays were assessed by flow cytometry. Western blotting analysis of RRM2, cyclin D1, p21, p53, phosphorylated p53, Bcl-2 and Bax was performed to explore the anti-hepatocellular carcinoma mechanism of the active metabolites. RESULTS: Ten metabolites of osalmid were identified, and none of them have been reported previously. Hydroxylation, glucuronidation, sulfonation, acetylation and degradation were recognized as the main metabolic processes of osalmid. Isozymes of CYP1A2, CYP2C9, UGT1A1, UGT1A6, UGT1A9, UGT2B7 and UGT2B15 were involved in phase I and phase II metabolism of osalmid. Metabolites M7, M8 and M10 showed higher binding affinities with the RRM2 active site than osalmid. Metabolite M7 exhibited potent inhibitory activity to hepatocellular carcinoma cell lines by both competitive inhibition and down-regulation of RRM2. Moreover, M7 significantly induced cell cycle arrest and apoptosis by activating p53-related pathways. CONCLUSIONS: The metabolic profile of osalmid was identified. M7 significantly inhibited human hepatocellular carcinoma progression by inhibiting RRM2 activity. Furthermore, M7 induced cell cycle arrest and apoptosis by activating p53-related signaling pathways.

Acquired tamoxifen resistance is surmounted by GW8510 through ribonucleotide reductase M2 downregulation-mediated autophagy induction.

Tamoxifen resistance is a major roadblock in the treatment of patients with breast cancer. Ribonucleotide reductase M2 (RRM2) was found to be involved in acquired resistance of breast cancer cells (BCCs) to tamoxifen. Here, we used GW8510, which has been identified as a potential RRM2 inhibitor, to evaluate the effect of RRM2 inhibition on reversing resistance of BCCs to tamoxifen and investigate its mechanisms. We showed that RRM2 overexpression played a key role in the development of acquired tamoxifen resistance in BCCs through downregulation of autophagy level. Combination treatment with tamoxifen and GW8510 significantly inhibited survival of the tamoxifen-resistant BCCs through induction of autophagic cell death compared to either of the two drugs. Furthermore, combination of tamoxifen and GW8510 resulted in marked growth inhibition of tamoxifen-resistant BBC xenograft tumor in vivo compared to tamoxifen or GW8510 alone. In conclusion, tamoxifen in combination with GW8510 can overcome acquired tamoxifen resistance in BCCs and may be a rational therapeutic approach against breast cancer with high RRM2 expression.

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.

Cancer Fighting SiRNA-RRM2 Loaded Nanorobots.

BACKGROUND: Silencing of several genes is critical for cancer therapy. These genes may be apoptotic gene, cell proliferation gene, DNA synthesis gene, etc. The two subunits of Ribonucleotide Reductase (RR), RRM1 and RRM2, are critical for DNA synthesis. Hence, targeting the blockage of DNA synthesis at tumor site can be a smart mode of cancer therapy. Specific targeting of blockage of RRM2 is done effectively by SiRNA. The drawbacks of siRNA delivery in the body include the poor uptake by all kinds of cells, questionable stability under physiological condition, non-target effect and ability to trigger the immune response. These obstacles may be overcome by target delivery of siRNA at the tumor site. This review presents a holistic overview regarding the role of RRM2 in controlling cancer progression. The nanoparticles are more effective due to specific characteristics like cell membrane penetration capacity, less toxicity, etc. RRM2 have been found to be elevated in different types of cancer and identified as the prognostic and predictive marker of the disease. Reductase RRM1 and RRM2 regulate the protein and gene expression of E2F, which is critical for protein expression and progression of cell cycle and cancer. The knockdown of RRM2 leads to apoptosis via Bcl2 in cancer. Both Bcl2 and E2F are critical in the progression of cancer, hence a gene that can affect both in regulating DNA replication is essential for cancer therapy. AIM: The aim of the review is to identify the related gene whose silencing may inhibit cancer progression. CONCLUSION: In this review, we illuminate the critical link between RRM-E2F, RRM-Bcl2, RRM-HDAC for the therapy of cancer. Altogether, this review presents an overview of all types of SiRNA targeted for cancer therapy with special emphasis on RRM2 for controlling the tumor progression.

Sorafenib Inhibits Ribonucleotide Reductase Regulatory Subunit M2 (RRM2) in Hepatocellular Carcinoma Cells.

The main curative treatments for hepatocellular carcinoma (HCC) are surgical resection and liver transplantation, which only benefits 15% to 25% of patients. In addition, HCC is highly refractory and resistant to cytotoxic chemotherapy. Although several multi-kinase inhibitors, such as sorafenib, regorafenib, and lenvatinib, have been approved for treating advanced HCC, only a short increase of median overall survival in HCC patients was achieved. Therefore, there is an urgent need to design more effective strategies for advanced HCC patients. Human ribonucleotide reductase is responsible for the conversion of ribonucleoside diphosphate to 2'-deoxyribonucleoside diphosphate to maintain the homeostasis of nucleotide pools. In this study, mining the cancer genomics and proteomics data revealed that ribonucleotide reductase regulatory subunit M2 (RRM2) serves as a prognosis biomarker and a therapeutic target for HCC. The RNA sequencing (RNA-Seq) analysis and public microarray data mining found that RRM2 was a novel molecular target of sorafenib in HCC cells. In vitro experiments validated that sorafenib inhibits RRM2 expression in HCC cells, which is positively associated with the anticancer activity of sorafenib. Although both RRM2 knockdown and sorafenib induced autophagy in HCC cells, restoration of RRM2 expression did not rescue HCC cells from sorafenib-induced autophagy and growth inhibition. However, long-term colony formation assay indicated that RRM2 overexpression partially rescues HCC cells from the cytotoxicity of sorafenib. Therefore, this study identifies that RRM2 is a novel target of sorafenib, partially contributing to its anticancer activity in HCC cells.

Functional Genomic Screen in Mesothelioma Reveals that Loss of Function of BRCA1-Associated Protein 1 Induces Chemoresistance to Ribonucleotide Reductase Inhibition.

Loss of function of BRCA1-associated protein 1 (BAP1) is observed in about 50% of malignant pleural mesothelioma (MPM) cases. The aim of this study was to investigate whether this aspect could be exploited for targeted therapy. A genetically engineered model was established expressing either functional or nonfunctional BAP1, and whole-genome siRNA synthetic lethality screens were performed assessing differentially impaired survival between the two cell lines. The whole-genome siRNA screen unexpectedly revealed 11 hits (FDR < 0.05) that were more cytotoxic to BAP1-proficient cells. Two actionable targets, ribonucleotide reductase (RNR) catalytic subunit M1 (RRM1) and RNR regulatory subunit M2 (RRM2), were validated. In line with the screen results, primary mesothelioma (BAP1 +/-) overexpressing BAP1 C91A (catalytically dead mutant) was more resistant to RNR inhibition, while BAP1 knockdown in the BAP1-proficient cell lines rescued the cells from their vulnerability to RNR depletion. Gemcitabine and hydroxyurea were more cytotoxic in BAP1-proficient cell line-derived spheroids compared with BAP1 deficient. Upregulation of RRM2 upon gemcitabine and hydroxyurea treatment was more profound in BAP1 mut/del cell lines. Increased lethality mediated by RNR inhibition was observed in NCI-H2452 cells reconstituted with BAP1-WT but not with BAP1 C91A. Upregulation of RRM2 in NCI-H2452-BAP1 WT spheroids was modest compared with control or C91A mutant. Together, we found that BAP1 is involved in the regulation of RNR levels during replication stress. Our observations reveal a potential clinical application where BAP1 status could serve as predictive or stratification biomarker for RNR inhibition-based therapy in MPM.

SiRNA-Mediated RRM2 Gene Silencing Combined with Cisplatin in the Treatment of Epithelial Ovarian Cancer In Vivo: An Experimental Study of Nude Mice.

Introduction: We aimed to explore small interfering (si)RNA silencing of ribonucleotide reductase M2 (RRM2) gene combined with cisplatin for the treatment of human ovarian cancer in nude mice models of subcutaneous transplantation of tumor cells. Methods: After conventional cultivation of human ovarian cancer cell line SKOV3 in vitro, SKOV3 cells were injected into the right back of nude mice by subcutaneous injection to establish the subcutaneous tumor models. Twenty-four tumor-burdened rats were randomly divided into four groups (n=6): siRNA group, siRNA in combination with cisplatin group, cisplatin group, and control group. Intraperitoneal injection of cisplatin and subcutaneous injection of siRNA were performed weekly. Tumor volume was measured, and tumor growth inhibition rate was calculated. RRM2 expression at the mRNA and protein levels was detected by reverse transcription-polymerase chain reaction and immunohistochemistry. Results: In the siRNA group, the tumor volume and tumor growth inhibition rate were 249.60±20.46 mm³ and 36.39%, respectively. The tumor growth inhibition rate and tumor volume were significantly different between the siRNA and control groups (p<0.05). In the cisplatin group, the tumor volume and tumor growth inhibition rate were 249.86±12.46 mm³ and 41.10%, respectively. The tumor growth inhibition rate and tumor volume were significantly different between the cisplatin and control groups (p<0.05). In the siRNA + cisplatin group, the tumor volume reduced to 180.84±16.25 mm³ and the tumor growth inhibition rate was increased to 64.33%, which were significantly different compared with the control group (p<0.01). Significant downregulation of RRM2 mRNA and protein expression in the tumor tissues was detected by reverse transcription polymerase chain reaction and immunohistochemistry assay (p<0.05). Discussion: siRNA alone or combined with cisplatin can effectively inhibit the growth of human ovarian cancer in nude mice models of subcutaneous transplantation of tumor cells. RRM2 gene silencing may be a potential treatment regimen for ovarian cancer in future.

EZH2 cooperates with E2F1 to stimulate expression of genes involved in adrenocortical carcinoma aggressiveness.

BACKGROUND: EZH2 is overexpressed and associated with poor prognosis in adrenocortical carcinoma (ACC) and its inhibition reduces growth and aggressiveness of ACC cells in culture. Although EZH2 was identified as the methyltransferase that deposits the repressive H3K27me3 histone mark, it can cooperate with transcription factors to stimulate gene transcription. METHODS: We used bioinformatics approaches on gene expression data from three cohorts of patients and a mouse model of EZH2 ablation, to identify targets and mode of action of EZH2 in ACC. This was followed by ChIP and functional assays to evaluate contribution of identified targets to ACC pathogenesis. RESULTS: We show that EZH2 mostly works as a transcriptional inducer in ACC, through cooperation with the transcription factor E2F1 and identify three positive targets involved in cell cycle regulation and mitosis i.e., RRM2, PTTG1 and ASE1/PRC1. Overexpression of these genes is associated with poor prognosis, suggesting a potential role in acquisition of aggressive ACC features. Pharmacological and siRNA-mediated inhibition of RRM2 blocks cell proliferation, induces apoptosis and inhibits cell migration, suggesting that it may be an interesting target in ACC. CONCLUSIONS: Altogether, these data show an unexpected role of EZH2 and E2F1 in stimulating expression of genes associated with ACC aggressiveness.

Knockdown of ribonucleotide reductase regulatory subunit M2 increases the drug sensitivity of chronic myeloid leukemia to imatinib­based therapy.

Imatinib­based targeted treatment is the standard therapy for chronic myeloid leukemia (CML); however, drug resistance is an inevitable issue for imatinib­based CML treatment. Imatinib resistance can be ascribed to Bcr­Abl­dependent and independent resistance. In the present study, peripheral blood samples were collected from imatinib­sensitive (IS) and imatinib­resistant (IR) CML patients and transcriptome sequencing was carried out. From the RNA­seq data, a significantly altered IR­related gene (IRG), ribonucleotide reductase regulatory subunit M2 (RRM2) was identified. Using real­time quantitative fluorescence PCR (qF­PCR), we found that RRM2 was elevated in both IR CML patients and an IR cell line. Using reverse­transcription PCR (RT­PCR) and western blot analysis, we indicated that imatinib can increase RRM2 level in a dose­dependent manner in IR cells. We also demonstrated that RRM2 is involved in the Bcl­2/caspase cell apoptotic pathway and in the Akt cell signaling pathway, and therefore affects the cell survival following imatinib therapy. The present study, for the first time, indicates that RRM2 is responsible for drug resistance in imatinib­based therapy. Therefore, RRM2 gene can be considered as a potential therapeutic target in the clinical treatment of CML.

Gemcitabine resistance mediated by ribonucleotide reductase M2 in lung squamous cell carcinoma is reversed by GW8510 through autophagy induction.

Although chemotherapeutic regimen containing gemcitabine is the first-line therapy for advanced lung squamous cell carcinoma (LSCC), gemcitabine resistance remains an important clinical problem. Some studies suggest that overexpressions of ribonucleotide reductase (RNR) subunit M2 (RRM2) may be involved in gemcitabine resistance. We used a novel RRM2 inhibitor, GW8510, as a gemcitabine sensitization agent to investigate the therapeutic utility in reversing gemcitabine resistance in LSCC. Results showed that the expressions of RRM2 were increased in gemcitabine intrinsic resistant LSCC cells upon gemcitabine treatment. GW8510 not only suppressed LSCC cell survival, but also sensitized gemcitabine-resistant cells to gemcitabine through autophagy induction mediated by RRM2 down-regulation along with decrease in dNTP levels. The combination of GW8510 and gemcitabine produced a synergistic effect on killing LSCC cells. The synergism of the two agents was impeded by addition of autophagy inhibitors chloroquine (CQ) or bafilomycin A1 (Baf A1), or knockdown of the autophagy gene, Bcl-2-interacting protein 1 (BECN1). Moreover, GW8510-caused LSCC cell sensitization to gemcitabine through autophagy induction was parallel with impairment of DNA double-strand break (DSB) repair and marked increase in cell apoptosis, revealing a cross-talk between autophagy and DNA damage repair, and an interplay between autophagy and apoptosis. Finally, gemcitabine sensitization mediated by autophagy induction through GW8510-caused RRM2 down-regulation was demonstrated in vivo in gemcitabine-resistant LSCC tumor xenograft, further indicating that the sensitization is dependent on autophagy activation. In conclusion, GW8510 can reverse gemcitabine resistance in LSCC cells through RRM2 downregulation-mediated autophagy induction, and GW850 may be a promising therapeutic agent against LSCC as it combined with gemcitabine.

Suppression of RRM2 inhibits cell proliferation, causes cell cycle arrest and promotes the apoptosis of human neuroblastoma cells and in human neuroblastoma RRM2 is suppressed following chemotherapy.

Ribonucleotide reductase regulatory subunit M2 (RRM2) is a rate­limiting enzyme for DNA synthesis and repair. RRM2 has vital roles in controlling the progression of cancer. In the present study, we investigated the RRM2 level in neuroblastoma tissues, analyzed its relationship with clinicopathological characteristics of neuroblastoma patients, and explored the effect of RRM2 on the biological functions of neuroblastoma cells. RRM2 levels in 67 pairs of neuroblastoma and matched adjacent non­cancerous tissues were detected by qRT­PCR, and its association with patient clinicopathological features was assessed. Using RRM2 siRNA, the role of RRM2 in cell viability was detected by CCK­8 assay, and the effects on cell cycle distribution and cell apoptosis were detected by flow cytometry. Hoechst 33342 staining was also performed. For RRM2 protein detection in cells and tissues, western blot analyses were employed. Our results revealed that RRM2 expression was significant higher in neuroblastoma tissues than that noted in adjacent non­cancerous tissues at both the mRNA and protein levels. The increased RRM2 level was significantly associated with clinical stage. RRM2 levels were suppressed in stage III and IV tumors in the chemotherapy subgroup, compared with levels noted in tumors in the preoperative non­chemotherapy subgroup. RRM2 siRNA significantly inhibited cell viability in the SH­5Y5Y cells, induced cell arrest in the G0/G1 phase, and enhanced cell apoptosis. Taken together, overexpression of RRM2 is associated with the genesis and progression of neuroblastoma, and may be a potential chemotherapeutic target.

The promoted delivery of RRM2 siRNA to vascular smooth muscle cells through liposome-polycation-DNA complex conjugated with cell penetrating peptides.

Peripheral vascular disease (PVD) is a prevalent vascular disease that affect a large number of patients. The establishment of optimal treatments to mitigate the intimal hyperplasia (IH)-induced restenosis would help relieve the health burden of the PVD. Ribonucleotide reductase M2 (RRM2) is critical to cellular migration and proliferation. We have previously demonstrated that suppression of RRM2 expression could substantially inhibit hepatocellular carcinoma cell proliferation and migration. We hereby developed RRM2 small interfering RNA (siRNA)-loaded cell penetrating peptides-conjugated liposome-polycation-DNA complex (LPD) (RRM2-CLPD), aiming to inhibit the migration and proliferation of vascular smooth muscle cells (VSMCs) crucial for IH. RRM2-CLPD is of a small size (∼150 nm) and high siRNA encapsulation efficiency (∼90%). Further, we demonstrated that RRM2-CLPD could significantly inhibited RRM2 gene and protein expression by ∼80%. Notably, RRM2-CLPD was able to effectively bind to VSMCs, resulting in significant cellular proliferation and migration inhibition. Taken together, RRM2-CLPD represent a very promising treatment for IH.

Gallium Maltolate Disrupts Tumor Iron Metabolism and Retards the Growth of Glioblastoma by Inhibiting Mitochondrial Function and Ribonucleotide Reductase.

Gallium, a metal with antineoplastic activity, binds transferrin (Tf) and enters tumor cells via Tf receptor1 (TfR1); it disrupts iron homeostasis leading to cell death. We hypothesized that TfR1 on brain microvascular endothelial cells (BMEC) would facilitate Tf-Ga transport into the brain enabling it to target TfR-bearing glioblastoma. We show that U-87 MG and D54 glioblastoma cell lines and multiple glioblastoma stem cell (GSC) lines express TfRs, and that their growth is inhibited by gallium maltolate (GaM) in vitro After 24 hours of incubation with GaM, cells displayed a loss of mitochondrial reserve capacity followed by a dose-dependent decrease in oxygen consumption and a decrease in the activity of the iron-dependent M2 subunit of ribonucleotide reductase (RRM2). IHC staining of rat and human tumor-bearing brains showed that glioblastoma, but not normal glial cells, expressed TfR1 and RRM2, and that glioblastoma expressed greater levels of H- and L-ferritin than normal brain. In an orthotopic U-87 MG glioblastoma xenograft rat model, GaM retarded the growth of brain tumors relative to untreated control (P = 0.0159) and reduced tumor mitotic figures (P = 0.045). Tumors in GaM-treated animals displayed an upregulation of TfR1 expression relative to control animals, thus indicating that gallium produced tumor iron deprivation. GaM also inhibited iron uptake and upregulated TfR1 expression in U-87 MG and D54 cells in vitro We conclude that GaM enters the brain via TfR1 on BMECs and targets iron metabolism in glioblastoma in vivo, thus inhibiting tumor growth. Further development of novel gallium compounds for brain tumor treatment is warranted. Mol Cancer Ther; 17(6); 1240-50. ©2018 AACR.

CHK1 inhibition sensitizes pancreatic cancer cells to gemcitabine via promoting CDK-dependent DNA damage and ribonucleotide reductase downregulation.

Inhibition of checkpoint kinase 1 (CHK1) is a promising therapeutic strategy to increase the effectiveness of DNA-damaging drugs in pancreatic cancer. However, owing to the multiple roles of CHK1 in the DNA damage response (DDR) pathway, the molecular mechanism of chemosensitization by CHK1 inhibitors is not definitive. In the present study, we explored the antitumor mechanism of LY2603618, a specific CHK1 inhibitor, alone or in combination with gemcitabine in 5 pancreatic cancer cell lines. LY2603618 treatment of the pancreatic cancer cell lines resulted in growth inhibition, with IC50 values ranging from 0.89 to 2.75 µM, but limited cell death. Importantly, treatment of pancreatic cancer cell lines with LY2603618 reduced the levels of pCDC25C, pCDK1, and pCDK2, accompanied by DNA damage and RRM1/2 downregulation. Furthermore, LY2603618 synergized with gemcitabine treatment to induce growth inhibition and apoptosis in pancreatic cancer cells. Mechanistic investigations showed that gemcitabine sensitization by CHK1 inhibition was associated with CDK­dependent RRM1/2 downregulation and DNA damage enhancement. These findings provide a basis for further development of combining CHK1 inhibitors and gemcitabine to treat pancreatic cancer.