Regulator Of Chromosome Condensation 2 Research Articles

Abstract 3062: Unraveling the role of RCC2 in glioblastoma by mutational analysis and a murine model of glioma

Abstract Glioblastoma (GBM) is a brain malignancy characterized by being highly aggressive, lethal, yet treatment modalities remain limited. Recent studies have implicated Regulator of Chromosome Condensation 2 (RCC2), a guanine nucleotide exchange factor (GEF) for RalA, as a potential regulator in glioblastoma pathogenesis. RCC2 has been shown to play a role in promoting glucose metabolism through BACH1-dependent upregulation of hexokinase II (HK2)(1). In this study, we investigated the functional mechanism of RCC2 in gliomagenesis using two complementary approaches. First, the relevance of RCC2 structural regions on its downstream functions were examined. By superimposing the three-dimensional crystal structure of RCC2 onto RCC1 (Regulator of Chromosome Condensation 1), a related GEF, we identified several unique regions within the RCC1-like domain of RCC2. A panel of point, substitution, and deletion mutants were generated, and pulldown experiments were performed to assess their relative binding affinity with BACH1. Additional assays to assess their relative ability to modulate the expression of HK2 and other cellular parameters will be discussed. The structure-functional characterization of distinct regions within RCC2 may identify unique sequences within RCC2 that could have relevance to glucose metabolism, cell proliferation and cell motility. Additionally, we have established a murine glioma model utilizing a Pten heterozygous mice expressing a GFAP-Cre-recombinase transgene (Gfap-cre;Ptenfl/+). Glioma were induced by stereotaxically injected lentiviral constructs expressing either with HRasV12 oncogene alone or together with RCC2. Neurological symptoms were monitored over a period of 7 months, and histopathological analysis was performed to evaluate the morphological and molecular features of the tumors. We showed that co-expression of HRasV12 and RCC2 exhibited earlier emergence of neurological manifests in both male and female groups (Log-rank test, p<0.05) when compared to HRasV12 alone. Overall, our study provides insights into both the structural mechanism of RCC2 in gliomagenesis through mutational analysis of RCC2 and the functional relevance through a murine model of glioma. (1)Liu, T. et. al. Cancer Lett (2022) 549:215914. Funding: This work is supported by a General Research Fund grant (#14113519) from the University Grants Committee of Hong Kong. Citation Format: Wayne L.H. Ho, Penelope M.Y. Or, Andrew M. Chan. Unraveling the role of RCC2 in glioblastoma by mutational analysis and a murine model of glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3062.

Identification of Potential Biomarkers in Stomach Adenocarcinoma using Machine Learning Approaches

Background: Stomach adenocarcinoma (STAD) is a common cancer with poor clinical outcomes globally. Due to a lack of early diagnostic markers of disease, the majority of patients are diagnosed at an advanced stage. Objective: The aim of the present study is to provide some new insights into the available biomarkers for patients with STAD using bioinformatics. Methods: RNA-Sequencing and other relevant data of patients with STAD from The Cancer Genome Atlas (TCGA) database were evaluated to identify differentially expressed genes (DEGs). Then, Machine Learning algorithms were undertaken to predict biomarkers. Additionally, Kaplan–Meier analysis was used to detect prognostic biomarkers. Furthermore, the Gene Ontology and Reactome pathways, protein-protein interactions (PPI), multiple sequence alignment, phylogenetic mapping, and correlation between clinical parameters were evaluated. Results: The results showed 61 DEGs, and the key dysregulated genes associated with STAD are MTHFD1L (Methylenetetrahydrofolate dehydrogenase 1-like), ZWILCH (Zwilch Kinetochore Protein), RCC2 (Regulator of chromosome condensation 2), DPT (Dermatopontin), GCOM1 (GRINL1A complex locus 1), and CLEC3B (C-Type Lectin Domain Family 3 Member B). Moreover, the survival analysis reported ASPA (Aspartoacylase) as a prognostic marker. Conclusion: Our study provides a proof of concept of the potential value of ASPA as a prognostic factor in STAD, requiring further functional investigations to explore the value of emerging markers.

Comprehensive analysis of the expression profile and clinical implications of regulator of chromosome condensation 2 in pan-cancers.

The Regulator of Chromosome Condensation 2 (RCC2) is an important gene that regulates mitosis and cytoplasmic division in the cell cycle. Although there have been reported in several individual tumors, an integrative analysis of RCC2 and its clinical significance across diverse cancer types is poorly elucidated. In this study, we performed integrative bioinformatics analyses to profile the expression landscape and assess the prognostic value of RCC2 in pan-cancers. Correlations between RCC2 expression and tumor-infiltrating immune cells, tumor mutation burden (TMB), microsatellite instability (MSI), chemokine and their receptors were analyzed using TCGA, ESTIMATE algorithm, and TISIDB database. We also explored the potential molecular functions of RCC2 through functional enrichment analysis and protein interaction networks. We discovered that RCC2 was highly expressed in various tumor tissues and was closely associated with cancer prognosis. Different RCC2-associated immune infiltration patterns were exhibited in different tumor-infiltrating immune cells. In addition, the RCC2 had a potential role in regulating the tumor immune microenvironment and the formation of cancer-associated fibroblasts (CAFs). Meanwhile, RCC2 showed a significant correlation with TMB, MSI, chemokines and their receptors in different tumor types. The role of RCC2 as a clinical therapeutic target was further revealed from the perspective of the immune microenvironment. In conclusion, RCC2 is closely associated with tumorigenesis and cancer-immune infiltration, and could be a promising prognostic and therapeutic biomarker in diverse cancers.

A pan‑cancer analysis of RCC2 and its interaction with HMGA2 protein in an invitro model of colorectal cancer cells.

Regulator of chromosome condensation 2 (RCC2) is highly involved in the development of tumor malignancies. The underlying mechanisms remain to be elucidated. The present study aimed to explore the role of RCC2 in the development of tumor malignancies and explore the underlying mechanisms in colorectal cancer (CRC). RCC2 expression and survival analysis were performed in human pan-cancer. The results of searching its mRNA expression in The Cancer Genome Atlas (TCGA) database showed that RCC2 was highly expressed in different types of cancer. High RCC2 expression levels were significantly correlated with poor survival outcomes by the Kaplan-Meier analysis in the TCGA database. Immunohistochemistry revealed that RCC2 was higher expressed in 36 CRC tissues than in adjacent normal tissues. Co-immunoprecipitation revealed that RCC2 bound to high mobility group A2 (HMGA2). Ectopic expression of RCC2 promoted cell proliferation, migration and invasion, whereas knockdown of HMGA2 exerted the opposite effects. Collectively, the data provided a novel biomarker of RCC2 in various types of cancer. High RCC2 expression levels were correlated with poor prognosis in different types of cancer. In addition, RCC2 may combine with HMGA2 to promote CRC malignancy.

Roles of regulator of chromosome condensation 2 in cancer: Beyond its regulatory function in cell cycle.

Regulator of chromosome condensation 2 (RCC2) is an essential protein in order for mitosis to proceed properly. It localizes in the centrosome of chromosomes where is involved in chromosome segregation and cytokinesis. Furthermore, RCC2 associates with integrin networks at the plasma membrane where participates in the control of cell movement. Because of its known role in cell cycle, RCC2 has been linked with cancer progression. Several reports show that RCC2 induces cancer hallmarks, but the mechanisms explaining how RCC2 exerts these roles are widely unknown. Here, we aim to summarize the main findings explaining the roles and mechanisms of RCC2 in cancer promotion. RCC2 is overexpressed in different cancers, including glioblastoma, lung, ovarian, and esophageal which is related to proliferation, migration, invasion promotion in vitro and tumor progression and metastasis in vivo. Besides, RCC2 overexpression induces epithelial-mesenchymal transition and causes poorer prognosis in cancer patients. RCC2 overexpression has also been linked with resistance development to chemotherapy and radiotherapy by inhibiting apoptosis and activating cancer-promoting transcription factors. Unfortunately, not RCC2 inhibitors are currently available for further pre-clinical and clinical assays. Therefore, these findings emphasize the potential use of RCC2 as a targetable biomarker in cancer and highlight the importance for designing RCC2 chemical inhibitors to evaluate its efficacy in animal studies and clinical trials.

Co‐Delivery of Paclitaxel and shMCL‐1 by Folic Acid‐Modified Nonviral Vector to Overcome Cancer Chemotherapy Resistance

Acquired chemoresistance presents a major clinical impediment, which is an urgent problem to be solved. Interestingly, myeloma cell leukemia-1 (MCL-1) and folate receptor expression levels are higher in chemotherapy-resistant patients than in pretreatment patients. In this study, a multifunctional folic acid (FA)-targeting core-shell structure is presented for simultaneous delivery of shMCL-1 and paclitaxel (PTX). The transfection efficiency of shMCL-1 with the FA-targeting delivery system is higher than with a nontargeting delivery system in Skov3 and A2780T cells. The FA-targeting system significantly inhibits cell growth, blocks cell cycles, and promotes apoptosis of cancer cells in vitro. The mechanisms involved in inhibiting growth are related to Bcl-2/Bax and cdc2/Cyclin B1 pathways. An analysis of RNA sequencing suggests that shMCL-1 reverses chemoresistance through regulating genes such as regulator of chromosome condensation 2 (RCC2). The synergetic effect of shMCL-1 and PTX effectively inhibits tumor growth in both PTX-resistant and normal cancer models by inducing tumor apoptosis, inhibiting proliferation, and limiting tumor angiogenesis. The study results indicate that a FA-targeting delivery system combining shMCL-1 with PTX can simultaneously target tumor sites and restore the sensitivity of chemotherapy-resistant cancer to PTX. These findings have important implications for patients with normal or PTX-resistant cancer.

Regulator of Chromosome Condensation 2 Modulates Cell Cycle Progression, Tumorigenesis, and Therapeutic Resistance.

Accurate regulation of cell cycle is important for normal tissue development and homeostasis. RCC2 (Regulator of Chromosome Condensation 2) play a role as chromosomal passenger complex (CPC) implicated in all cell cycle phases. RCC2 was initially identified as Ran guanine exchange factor (GEF) for small G proteins. Therefore, RCC2 plays a key role in oncogenesis of most cancers. RCC2 is implicated in Colorectal Cancer (CRC), Lung Adenocarcinoma (LUAD), breast cancer, and ovarian cancer. Expression level of RCC2 protein determines regulation of tumor cell proliferation, invasion, metastasis, and radio-chemotherapeutic resistance. In this review, we explored proteins that interact with RCC2 to modulate tumor development and cancer therapeutic resistance by regulation of cell cycle process through various signaling pathways.

RCC2 Expression Stimulates ER-Positive Breast Tumorigenesis.

Objective Regulator of chromosome condensation 2 (RCC2) has been reported to be involved in the regulation of cell cleavage. This study investigated the effect of RCC2 expression on breast tumorigenesis. Methods MCF-7 cells originating from estrogen receptor-positive (ER+) breast cancer were transfected with anti-RCC2 siRNA or RCC2-expressing plasmids. Cell proliferation, apoptosis, migration, and cytokine production in the transfected cells were examined using the CCK-8 assay, wound healing assay, and flow cytometry, respectively. PCR array was used to investigate the tumorigenic pathway of RCC2 in MCF-7 cells transfected with the anti-RCC2 siRNA. MCF-7 cells were also transfected with lentivirus-containing anti-RCC2 short hairpin RNA and were injected into BALB/c nude mice to generate tumor-bearing mice. Tumor growth in the mouse model was examined using magnetic resonance imaging by diffusion-weighted imaging analysis. Results Western blotting and immunohistochemistry detected significantly increased expression of RCC2 in ER + breast tumor tissues compared with breast fibroadenoma samples. Inhibiting RCC2 expression decreased cell migration and stimulated apoptosis in MCF-7 cells, while overexpressing RCC2 stimulated cell migration and inhibited apoptosis. The inhibition of RCC2 expression significantly decreased breast tumor growth and IL-6 levels in the tumor-bearing mice. PCR array demonstrated that inhibiting RCC2 expression significantly decreased the expression of IGF1 and TWIST1, two well-known tumor-enhancing genes, in MCF-7 cells; conversely, overexpressing RCC2 increased the expression levels of these two genes in the transfected cells. This result was verified in the mouse model following inhibition of RCC2 expression in MCF-7 cells. Additionally, estradiol-17β suppressed MCF-7 cell apoptosis, stimulated cell proliferation and cell migration, and increased RCC2, IGF1, and TWIST1 expression. The siRNA-mediated inhibition of RCC2 expression alleviated the inhibitory effects of estrogen on apoptosis in MCF-7 cells, while overexpressing RCC2 enhanced the estrogen-driven inhibition of apoptosis. Modifying RCC2 expression had no impact on MCF-7 cell proliferation in the presence or absence of estradiol-17β. Conclusions Our results suggest that estrogen-induced RCC2 expression prompts IGF1, TWIST1, and IL-6 expression, stimulates cell migration, and inhibits apoptosis to contribute to ER + breast tumorigenesis.

RCC2 Interacts with Small GTPase RalA and Regulates Cell Proliferation and Motility in Gastric Cancer.

BackgroundRegulator of chromosome condensation 2 (RCC2), also known as TD-60, is associated with various human malignant cancers. RCC2 has been shown to exhibit guanine exchange factor (GEF) activity and contribute to early mitosis. However, the role and mechanism of RCC2 in gastric cancer remain unclear.Materials and MethodsRCC2 expression in gastric cancer was studied using qPCR, Western blotting and immunochemistry staining of clinical specimens, and its roles in the cytobiology, mouse model and related molecular pathways were evaluated using gastric cell lines.ResultsRCC2 was frequently overexpressed in gastric cancer. RCC2 knockdown significantly inhibited cell proliferation, migration and invasion in vitro, which was further confirmed by the RCC2 overexpression results in gastric cancer cells. Moreover, RCC2 knockdown inhibited tumor progression in vivo. Further study revealed the interaction between RCC2 and RalA. The level of RalA-GTP was decreased in gastric cancer cells after RCC2 knockdown, while an increased phosphorylation level in MAPK/JNK was found. Furthermore, the changes in the level of RalA-GTP as well as cell proliferation, migration and invasion abilities were further confirmed using RBC8, a specific small-molecule inhibitor of the intracellular actions of Ral GTPases, in gastric cancer cells.ConclusionRCC2 plays an important role in gastric cancer. RCC2 knockdown inhibits cell growth, cell motility and tumor progression, which may act through RalA and affect the MAPK/JNK pathway.

RCC2 contributes to tumor invasion and chemoresistance to cisplatin in hepatocellular carcinoma.

Tumor metastasis and chemoresistance are the main causes of treatment failure and high mortality in hepatocellular carcinoma (HCC). Therefore, it is critical to clarify the biological action and potential mechanisms in HCC cells to develop novel therapeutics. The regulator of chromosome condensation 2 (RCC2), a component of the chromosomal passenger complex, was shown to have important roles in tumor development andradio-chemotherapyresistance.However, its role in theaggressive phenotypes and cisplatin (DDP)-resistance of HCC is not known. Therefore, this study aimed to investigate the role of RCC2 in HCC pathogenesis. Interestingly, we found that RCC2 was upregulated in HCC patient specimens and HCC cell lines and was correlated with the pathological grade of HCC. To evaluate the function of RCC2 in HCC cell,lentivirus vector-based shRNAs were transfected into HCC cells. Silencing RCC2 inhibited the HCC cell proliferation, migration, invasion, and increased the apoptosis rate upon DDP treatment. Further analysis showed that RCC2-mediated downregulation of the expression of survival proteins occurred via the AKTand Bcl2pathways.Our results suggest that RCC2 might act as an oncogenic protein promoting metastatic behaviors andcisplatinresistancein HCC cells, and thereby could be a potential prognostic biomarker and therapeutic target for HCC.

Prediction of relapse-free survival according to adjuvant chemotherapy and regulator of chromosome condensation 2 (RCC2) expression in colorectal cancer

BackgroundThere is a need for improved selection of patients for adjuvant chemotherapy after resection of non-metastatic colorectal cancer (CRC). Regulator of chromosome condensation 2 (RCC2) is a potential prognostic biomarker. We report on the establishment of a robust protocol for RCC2 expression analysis and prognostic tumour biomarker evaluation in patients who did and did not receive adjuvant chemotherapy.Materials and methodsRCC2 was analysed in 2916 primary CRCs from the QUASAR2 randomised trial and two single-hospital Norwegian series. A new protocol using fluorescent antibody staining and digital image analysis was optimised. Biomarker value for 5-year relapse-free survival was analysed in relation to tumour stage, adjuvant chemotherapy and the molecular markers microsatellite instability, KRAS/BRAFV600E/TP53 mutations and CDX2 expression.ResultsLow RCC2 expression was scored in 41% of 2696 evaluable samples. Among patients with stage I–III CRC who had not received adjuvant chemotherapy, low RCC2 expression was an independent marker of inferior 5-year relapse-free survival in multivariable Cox models including clinicopathological factors and molecular markers (HR 1.45, 95% CI 1.09 to 1.94, p=0.012, N=521). RCC2 was not prognostic in patients who had received adjuvant chemotherapy, neither in QUASAR2 nor the pooled Norwegian series. The interaction between RCC2 and adjuvant chemotherapy for prediction of patient outcome was significant in stage III, and strongest among patients with microsatellite stable tumours (pinteraction=0.028).ConclusionsLow expression of RCC2 is a biomarker for poor prognosis in patients with stage I–III CRC and seems to be a predictive biomarker for effect of adjuvant chemotherapy.

RCC2 promotes proliferation and radio-resistance in glioblastoma via activating transcription of DNMT1

Regulator of chromosome condensation 2 (RCC2) is a regulator of cell-cycle progression linked in multiple cancers to pro-tumorigenic phenomena including promotion of tumor growth, tumor metastases and poorer patient prognoses. However, the role of RCC2 in GBM remains under-investigated. Here, we sought to determine the relevance of RCC2 in GBM, as well as its roles in GBM development, progression and prognosis. Initial clinical evaluation determined significant RCC2 enrichment in GBM when compared to normal brain tissue, and elevated expression was closely associated with a poorer prognosis in glioma patients. Via shRNA inhibition, we determined that RCC2 is essential to tumor proliferation and tumorigenicity in vitro and in vivo. Additionally, RCC2 was determined to promote radioresistance of GBM tumor cells. Investigation of the underlying mechanisms implicated DNA mismatch repair, JAK-STAT pathway and activated transcription of DNA methyltransferase 1 (DNMT1). For validation, pharmacologic inhibition via administration of a DNMT1 inhibitor demonstrated attenuated GBM tumor growth both in vitro and in vivo. Collectively, this study determined a novel therapeutic target for GBM in the form of RCC2, which plays a pivotal role in GBM proliferation and radio-resistance via regulation of DNMT1 expression in a p-STAT3 dependent manner.

RCC2 promotes breast cancer progression through regulation of Wnt signaling and inducing EMT.

Regulator of chromosome condensation 2 (RCC2), also known as TD-60, is an RCC1 family member and plays an essential role in mitosis. However, the roles of RCC2 in breast cancer are still unclear. In this study, RCC2 was found to exert oncogenic activities in breast cancer. Samples of breast cancer tissue revealed an increased level of RCC2 and a high level of RCC2 was associated with poor overall survival rate of breast cancer patients. Overexpression of RCC2 significantly enhanced cell proliferation and migration abilities of breast cancer cells in vitro and in vivo. Mechanistically, RCC2 induced epithelial-mesenchymal transition (EMT) through the activation of Wnt signaling pathway. Collectively, our study indicates that RCC2 contributes to breast cancer progression and functions as an important regulator of EMT through the activation of Wnt signaling.

MicroRNA-331-3p inhibits proliferation and metastasis of ovarian cancer by targeting RCC2.

IntroductionEpithelial ovarian carcinoma (EOC) is one of the most lethal gynecologic malignancies, with a poor 5-year survival rate. Numerous studies have shown that microRNAs participate in the malignant behavior of ovarian cancer cells by directly targeting multiple oncogenes or tumor suppressor genes.Material and methodsReverse transcription-PCR was used to determine the level of miR-331-3p in EOC. Cells proliferation was measured with the Cell Counting Kit-8. Cell mobility were measured by wound-healing assay. Cell migration and invasion were measured by transwell assay. Luciferase assays were used to demonstrate that RCC2 was a directed target of miR-331-3p in EOC. Western blots were used to measure the protein expression.ResultsWe found that the expression of microRNA-331-3p (miR-331-3p) in ovarian cancer cell lines is reduced (p < 0.01), and an increase of expression of miR-331-3p in ovarian cancer cells significantly inhibits cell proliferation (p < 0.001). Transwell and wound-healing assays showed that miR-331-3p inhibits the cell motility of ovarian cancer cells (p < 0.001). Regulator of chromosome condensation 2 (RCC2) was predicted to be a novel target for miR-331-3p. Our luciferase activity assay confirmed that RCC2 is directly targeted by miR-331-3p. RCC2 was negatively regulated by miR-331-3p (p < 0.001), and overexpression of RCC2 could restore the malignant behaviors of ovarian cancer cells, which was suppressed by miR-331-3p.ConclusionsThese data indicate that miR-331-3p can inhibit proliferation, migration, and invasion of ovarian cancer cells via directly targeting RCC2. Our study provides potential therapeutic targets for the treatment of ovarian cancer.

RCC2 over-expression in tumor cells alters apoptosis and drug sensitivity by regulating Rac1 activation

BackgroundSmall GTP binding protein Rac1 is a component of NADPH oxidases and is essential for superoxide-induced cell death. Rac1 is activated by guanine nucleotide exchange factors (GEFs), and this activation can be blocked by regulator of chromosome condensation 2 (RCC2), which binds the switch regions of Rac1 to prevent access from GEFs.MethodsThree cancer cell lines with up- or down-regulation of RCC2 were used to evaluate cell proliferation, apoptosis, Rac1 signaling and sensitivity to a group of nine chemotherapeutic drugs. RCC2 expression in lung cancer and ovarian cancer were studied using immunochemistry stain of tumor tissue arrays.ResultsForced RCC2 expression in tumor cells blocked spontaneous- or Staurosporine (STS)-induced apoptosis. In contrast, RCC2 knock down in these cells resulted in increased apoptosis to STS treatment. The protective activity of RCC2 on apoptosis was revoked by a constitutively activated Rac1, confirming a role of RCC2 in apoptosis by regulating Rac1. In an immunohistochemistry evaluation of tissue microarray, RCC2 was over-expressed in 88.3% of primary lung cancer and 65.2% of ovarian cancer as compared to non-neoplastic lung and ovarian tissues, respectively. Because chemotherapeutic drugs can kill tumor cells by activating Rac1/JNK pathway, we suspect that tumors with RCC2 overexpression would be more resistant to these drugs. Tumor cells with forced RCC2 expression indeed had significant difference in drug sensitivity compared to parental cells using a panel of common chemotherapeutic drugs.ConclusionsRCC2 regulates apoptosis by blocking Rac1 signaling. RCC2 expression in tumor can be a useful marker for predicting chemotherapeutic response.

Overexpression of RCC2 Enhances Cell Motility and Promotes Tumor Metastasis in Lung Adenocarcinoma by Inducing Epithelial-Mesenchymal Transition.

Purpose: Investigate the role of regulator of chromosome condensation 2 (RCC2) on lung adenocarcinoma (LUAD) metastasis.Experimental Design: Clinical specimens were used to assess the impact of RCC2 on LUAD metastasis. Mouse models, cytobiology, and molecular biology assays were performed to elucidate the function and underlying mechanisms of RCC2 in LUAD.Results: RCC2 expression was frequently increased in LUADs (88/122, 72.13%). It was confirmed by analysis of a larger cohort of TCGA RNA-seq data containing 488 LUADs and 58 normal lung tissues (P < 0.001). Importantly, increased level of RCC2 was significantly associated with T status of tumor (P = 0.002), lymph node metastasis (P = 0.004), and advanced clinical stage (P = 0.001). Patients with LUAD with higher expression of RCC2 had shorter overall survival. Cox regression analysis demonstrated that RCC2 was an independent poorer prognostic factor for patients with LUAD. Moreover, forced expression of RCC2 promoted intrapulmonary metastasis in vivo and significantly enhanced LUAD cell migration, invasion, and proliferation in vitro Further study found that RCC2 induced epithelial-mesenchymal transition (EMT) and also stimulated the expression of MMP-2 and MMP-9. In addition, RCC2 was able to activate JNK, while inhibition of JNK suppressed the effect of RCC2 on LUAD cell migration, invasion, EMT, and the expression of MMP-2 and MMP-9.Conclusions: RCC2 plays a pivotal role in LUAD metastasis by inducing EMT via activation of MAPK-JNK signaling. Clin Cancer Res; 23(18); 5598-610. ©2017 AACR.

Epigenetically altered miR-1247 functions as a tumor suppressor in pancreatic cancer

Altered expression of microRNAs has been strongly implicated in human cancers, and growing evidence is emerging that a number of miRNAs are downregulated in cancer associated with CpG island hypermethylation. Although pancreatic cancer is one of the most malignant human cancers, the roles of miRNAs underlying the tumorigenesis of pancreatic cancer are still poorly understood. In the present study, we explored the molecular functional role of microRNA-1247 as tumor suppressor associated with epigenetic alteration in pancreatic cancer. CpG islands methylation of miR-1247 is frequently observed in various pancreatic cancer cell lines and in primary pancreatic tumors, but not in normal pancreatic tissue. Ectopic expression of miR-1247 in five pancreatic cancer cell lines results in suppressing of cell growth, proliferation, migration, and invasion in vitro and tumorigenicity of pancreatic cancer cells in vivo. Interestingly, we found one putative target gene of miR-1247, regulator of chromosome condensation 2 (RCC2), harbored miR-1247 target sequences in the 3′ UTR of its mRNA. In functional studies in vitro to understand the interaction between miR-1247 and RCC2, decreasing of RCC2 gene expression by miR-1247 was observed by immunoblotting and immunohistochemistry at both mRNA and protein levels. Moreover, luciferase reporter assay confirmed that RCC2 was a direct target of miR-1247. Taken together, our data suggest that CpG island hypermethylation of miR-1247 is responsible for its downregulation in pancreatic cancer, and ectopic expression of miR-1247 functions as a potential tumor suppressor targeting RCC2 in pancreatic cancer cells.

Regulator of Chromosome Condensation 2 Identifies High-Risk Patients within Both Major Phenotypes of Colorectal Cancer.

Colorectal cancer has high incidence and mortality worldwide. Patients with microsatellite instable (MSI) tumors have significantly better prognosis than patients with microsatellite stable (MSS) tumors. Considerable variation in disease outcome remains a challenge within each subgroup, and our purpose was to identify biomarkers that improve prediction of colorectal cancer prognosis. Mutation analyses of 42 MSI target genes were performed in two independent MSI tumor series (n = 209). Markers that were significantly associated with prognosis in the test series were assessed in the validation series, followed by functional and genetic explorations. The clinical potential was further investigated by immunohistochemistry in a population-based colorectal cancer series (n = 903). We identified the cell-cycle gene regulator of chromosome condensation 2 (RCC2) as a cancer biomarker. We found a mutation in the 5' UTR region of RCC2 that in univariate and multivariate analyses was significantly associated with improved outcome in the MSI group. This mutation caused reduction of protein expression in dual luciferase gene reporter assays. siRNA knockdown in MSI colon cancer cells (HCT15) caused reduced cell proliferation, cell-cycle arrest, and increased apoptosis. Massive parallel sequencing revealed few RCC2 mutations in MSS tumors. However, weak RCC2 protein expression was significantly associated with poor prognosis, independent of clinical high-risk parameters, and stratifies clinically important patient subgroups with MSS tumors, including elderly patients (>75 years), stage II patients, and those with rectal cancer. Impaired RCC2 affects functional and clinical endpoints of colorectal cancer. High-risk patients with either MSI or MSS tumors can be identified with cost-effective routine RCC2 assays.

Alternative cellular roles for proteins identified using proteomics

The recent report by Grigera et al. [1] provided interesting new data on the ability of the actin-binding protein cortactin to associate with regulator of chromosome condensation-2 (RCC2; also known as TD-60), a component of the mitotic machinery [2]. Owing to the localisation of cortactin to the equatorial plane of dividing cells, the authors inferred that its interaction with RCC2 was functionally important in mitosis. This may be the case, but the data also support an alternative interpretation that RCC2 may play a role in cortactin-mediated branched actin regulation during cell movement. Cortactin promotes actin nucleation and stabilises F-actin, and thus it regulates many cell motility processes, such as focal adhesion assembly, lamellipodial protrusion, vesicle transport and endocytosis [3], although the full gamut of cortactin function is incompletely understood. Cortactin strongly localises to the leading edge of migrating cells, where it is phosphorylated, and signalling to small guanosine triphosphatases (GTPases), such as Rac1, plays an important part in its function [4,5]. Engagement of integrin adhesion receptors leads to activation of small GTPases, which play a central role in the modulation of cell adhesion complexes [6,7]. Using a proteomic approach, we showed that specific integrin adhesion complexes contain RCC2 (Fig. 1) [8–10]. Moreover, we demonstrated that, through precise restriction of Rac1 and Arf6 GTPase activity, RCC2 controls directional cell motility [8,9,11]. Indeed, our findings suggest that, rather than acting as a putative guanine nucleotide exchange factor and activating Rac1, RCC2 limits the activation of Rac1. Fig. 1 Proteomic analysis of molecular interactions can reveal alternative cellular roles for proteins. RCC2 (red circle, or node, in interaction network) is a passenger protein with a role in cell division [2]. Proteomic and bioinformatic analyses of integrin ... Together with the work of Grigera et al. [1], our data indicate an intriguing overlap between molecules implicated in cell adhesion processes and RCC2 function. Thus, the data reported by Grigera et al. [1] do not exclude the possibility that RCC2 interacts with cortactin as a component of the cell migration machinery [8,9] or indeed of integrin adhesion-regulated mitosis [12,13]. A current lack of data reporting the colocalisation of RCC2 with cortactin prevents a complete understanding of the interaction between these molecules, but further study of their contributions to cell behaviour is warranted. The works discussed above exemplify the discovery of unexpected cellular roles for proteins; such examples continue to increase as more data-driven proteomic studies appear in the literature. The consequence, in the postgenomic era, is that a single gene cannot be correlated with a single protein function. Proteins may possess multifunctionality within a single polypeptide chain, known as “moonlighting” [14], or as different splice variants. Therefore, understanding the context of protein interactions is critical to gaining mechanistic insight into protein function. Many MS-based proteomic studies rely on analysis of cell or tissue lysates, in which cellular context (e.g. subcellular localisation or cell type-specific expression) has been eliminated. Furthermore, many analyses of proteomic data use protein interaction networks as a tool for data interrogation. Such analyses lever the wealth of knowledge about all reported protein interactions and can be incredibly informative, including by suggesting alternative functions for proteins [15], but these datasets should be interpreted in the context of their derivation and the methodological limitations. Reported interactions in large-scale interaction networks have usually been amalgamated from data from multiple conditions, cell types and subcellular locations. With a lack of cellular context, proteins can appear to be promiscuous binders, whereas, in a cell, they may bind to distinct partners under restricted conditions. Accordingly, to achieve mechanistic insight into protein function, the analysis of protein interaction networks should rarely be the final interpretation of MS data, but instead be the springboard for further experimentation. Overall, data-driven MS-based proteomic and bioinformatic analyses, within the framework of hypothesis-driven research, provide a valuable opportunity for the discovery of new cellular roles for proteins, and will continue to provide surprising results about biological systems.