Analysis of the characteristics of chemotherapy-resistant renal cell carcinomas based on global transcriptional analysis of their tissues and cell lines.
ABSTRACT: Starvation-resistant renal cell carcinoma (RCC) cell lines are considered dormant-state cells that survive even under glucose starvation. The cellular biological and global transcriptional analysis using these cells identified potential markers of chemotherapy-resistant RCC and therapeutic agent candidates. Recently, we showed that ARL4C was a predictive biomarker for poor prognosis in patients with chemotherapy-resistant RCC by the global transcriptional analysis of patient primary tissues. The objective of this study was to identify the characteristics of chemotherapy-resistant RCC by the global transcriptional analysis of primary tissues of patients with RCC and RCC cell lines. The connective global transcriptional analysis showed that two starvation-resistant RCC cell lines, SW839 and KMRC-1, were strongly correlated to tissues of patients with chemotherapy-resistant RCC and showed high expressions of invasive- and proliferation-related genes. We found fibronectin (FN1) expression was a predictive biomarker in some patients with chemotherapy-resistant RCC, which especially correlated with two starvation-resistant RCC cell lines. These results indicate these cell lines emulate chemotherapy-resistant RCC and might be useful in the search for markers to predict poor prognosis and in the development of therapeutic agents and their index markers for chemotherapy-resistant RCCs.
Project description:Renal cell carcinomas (RCC) have two types of cells for carbon metabolism and for cell signaling under nutrient-deprivation conditions, namely starvation-resistant and starvation-sensitive cells. Here, we evaluated the mitochondrial characteristics of these cell types and found that the resistant type possessed higher activities for both mitochondrial oxidative phosphorylation and glycolysis than the sensitive types. These higher activities were supported by the stored carbon, lipid and carbohydrate sources, and by a low level of mitochondrial reactive oxygen species (ROS) due to sustained SOD2 expression in the resistant RCC cells. In metastatic RCC cases, higher SOD2 expression was associated with a significantly shorter survival period. We found that treatment with the drugs etomoxir and buformin significantly reduced mitochondrial oxidative phosphorylation and induced cell death under glucose-deprivation conditions in starvation-resistant RCC cells. Our data suggest that inhibitory targeting of mitochondria might offer an effective therapeutic option for metastatic RCC that is resistant to current treatments.
Project description:Metastatic renal cell carcinoma (RCC) is a molecularly heterogeneous disease that is intrinsically resistant to chemotherapy and radiotherapy. Although therapies targeted to the molecules vascular endothelial growth factor and mammalian target of rapamycin have shown clinical effectiveness, their effects are variable and short-lived, underscoring the need for improved treatment strategies for RCC. Here, we used quantitative phosphoproteomics and immunohistochemical profiling of 346 RCC specimens and determined that Src kinase signaling is elevated in RCC cells that retain wild-type von Hippel-Lindau (VHL) protein expression. RCC cell lines and xenografts with wild-type VHL exhibited sensitivity to the Src inhibitor dasatinib, in contrast to cell lines that lacked the VHL protein, which were resistant. Forced expression of hypoxia-inducible factor (HIF) in RCC cells with wild-type VHL diminished Src signaling output by repressing transcription of the Src activator protein tyrosine phosphatase 1B (PTP1B), conferring resistance to dasatinib. Our results suggest that a HIF-regulated VHL-PTP1B-Src signaling pathway determines the sensitivity of RCC to Src inhibitors and that stratification of RCC patients with antibody-based profiling may identify patients likely to respond to Src inhibitors in RCC clinical trials.
Project description:Although cancers are widely considered to be maintained by stem cells, the existence of stem cells in renal cell carcinoma (RCC) has seldom been reported, in part due to the lack of unique surface markers. We here identified cancer stem cell-like cells with side population (SP) phenotype in five human RCC cell lines. Flow cytometry analysis revealed that 769P, a human clear cell RCC cell line, contained the largest amount of SP cells as compared with other four cell lines. These 769P SP cells possessed characteristics of proliferation, self-renewal, and differentiation, as well as strong resistance to chemotherapy and radiotherapy that were possibly related to the ABCB1 transporter. In vivo experiments with serial tumor transplantation in mice also showed that 769P SP cells formed tumors in NOD/SCID mice. Taken together, these results indicate that 769P SP cells have the properties of cancer stem cells, which may play important roles in tumorigenesis and therapy-resistance of RCC.
Project description:BACKGROUND: Human renal cell carcinoma (RCC) is very resistant to chemotherapy. ABT-737 is a novel inhibitor of anti-apoptotic proteins of the Bcl-2 family that has shown promise in various preclinical tumour models. RESULTS: We here report a strong over-additive pro-apoptotic effect of ABT-737 and etoposide, vinblastine or paclitaxel but not 5-fluorouracil in cell lines from human RCC. ABT-737 showed very little activity as a single agent but killed RCC cells potently when anti-apoptotic Mcl-1 or, unexpectedly, A1 was targeted by RNAi. This potent augmentation required endogenous Noxa protein since RNAi directed against Noxa but not against Bim or Puma reduced apoptosis induction by the combination of ABT-737 and etoposide or vinblastine. At the level of mitochondria, etoposide-treatment had a similar sensitizing activity and allowed for ABT-737-induced release of cytochrome c. CONCLUSIONS: Chemotherapeutic drugs can overcome protection afforded by Mcl-1 and A1 through endogenous Noxa protein in RCC cells, and the combination of such drugs with ABT-737 may be a promising strategy in RCC. Strikingly, A1 emerged in RCC cell lines as a protein of similar importance as the well-established Mcl-1 in protection against apoptosis in these cells.
Project description:Background: Abnormal transcriptional upregulation of telomerase reverse transcriptase (TERT) plays a dominant role in telomerase activation in various cancers. TERT promoter mutations (TPMs) have been identified as a key mechanism in TERT upregulation. However, the mechanism of TERT upregulation in cancers with low frequency of TPMs are not fully elucidated so far. Methods: The expression of PUF60 and TERT was detected by real-time PCR, western blot and immunohistochemistry. TERT promoter binding proteins were identified by streptavidin-agarose pulldown assay and mass spectrum (MS) analysis. The role of PUF60/TERT in renal cancer was evaluated on cell growth in vitro and in vivo. Results: In this study, we identify the regulation mechanism of TERT in renal cell carcinoma (RCC) cells which have rare TPMs but exert significant upregulation of TERT. We found that TERT was highly expressed in RCC tumor tissues, and elevated TERT expression was associated with poor prognosis for patients. We also detected the relatively rare TPM status in both RCC tumor tissues and RCC cell lines. Mechanistically, PUF60, a RNA binding protein, was identified as a novel TERT regulator which bound to the TERT and transcriptionally upregulated TERT expression in RCC cells. The in vitro and in vivo experiments also demonstrated that PUF60 could promote RCC cell growth through activation of TERT expression in a TPM status independent way. Furthermore, we showed that there was a strong correlation of the expression of PUF60 and TERT in RCC tumor tissues and RCC cell lines, and the patients with high expression of PUF60 and TERT had significantly shorter survival. Conclusions: Collectively, these results indicated that PUF60 transcriptionally upregulated TERT expression to promote RCC growth and progression in a TPM status independent way, suggesting that the PUF60/TERT signaling pathway may serve as potential prognostic biomarkers and therapeutic targets for RCC.
Project description:Dormant cancer cells are starvation-resistant leading to problems in the management of cancer. In renal cell carcinomas (RCCs), starvation-resistant cells are resistant to various currently available therapies. However, targeting hypoxia inducible factor 2-alpha (HIF2-alpha) induces cell death in dormant-like/starvation-resistant RCCs. This study showed that the apoptotic cell death caused by tumor necrosis factor (TNF)-related apoptosis-induced ligand (TNFSF10/TRAIL) was attenuated by CASP8 and FADD-like apoptosis regulator (CFLAR/c-FLIP) following HIF2-alpha activation, despite the high expression of TRAIL in such RCCs. Knockdowns of TRAIL averted apoptotic cell death caused by HIF2-alpha inhibition in starvation-resistant RCCs. Knockdowns of both HIF2-alpha and c-FLIP augmented apoptotic cell death, whereas overexpression of c-FLIP completely averted apoptosis. In addition, high abundance of TRAIL was correlated with poor prognosis in patients with RCC, suggesting that TRAIL, followed by HIF2-alpha and c-FLIP, play a role in the survival and/or progression of malignant RCCs.
Project description:Renal cell carcinoma (RCC) has a high mortality rate among urological malignancies, and its underlying mechanisms remain unclear. Steroid receptor RNA coactivator (SRA) belongs to the long non-coding RNAs (lncRNAs) and has been demonstrated to be closely related to various types of cancer. In the present study, the decreased expression level of SRA was first confirmed in RCC tissues and cell lines by RT-qPCR. Using knockdown or overexpression systems, it was then found that SRA inhibited the proliferation of RCC cell lines and promoted their apoptosis. In addition, SRA suppressed the migration and invasion, and altered EMT-related markers in RCC cells. More importantly, it was demonstrated that SRA reduced percentage of CD44+/CD24? cells and the sphere-forming efficiency. SRA also attenuated the expression levels of CD44, SOX-2, ABCG2 and OCT-4, which are all associated with cancer cell stemness characteristics. Although SRA increased the phosphorylation of extracellular-regulated protein kinase (ERK), the ERK1/2 pathway could not further interfere with the alteration of EMT-related markers mediated by SRA. Notably, the ERK inhibitor, PD98059, abolished ERK1/2 phosphorylation, whereas it did not exert any marked effects on cell proliferation and EMT-related markers mediated by SRA. Taken together, the findings of the present study indicate that SRA is an important molecule that inhibits the migration, invasion and stem cell characteristics of RCC cells; the ERK signaling pathway may not be involved in this process.
Project description:Methionine adenosyltransferase 2A (MAT2A) is an enzyme that catalyzes the formation of S-adenosylmethionine (SAMe) by joining methionine and ATP. SAMe is a methyl donor for transmethylation and has an important role for DNA and/or protein methylation. MAT2A is expressed widely in many tissues especially in kidney. Several studies have demonstrated that there are abnormal expressions of MAT2A in several kinds of cancers such as liver and colon cancers. But the relationship of MAT2A between renal cell carcinomas (RCC) is less understood.The mRNA expression level of the MAT2A gene was determined in 24 RCC patients and 4 RCC cell lines, using real-time quantitative-polymerase chain reaction (RT-PCR). The MAT2A protein content was measured by western blotting and immunohistochemical analysis in 55 RCC patients. The mRNA levels of heme oxygenase-1 (HO-1) and cyclooxygenase-2 (COX-2) were also analysized in patients using RT-PCR. The correlations between the MAT2A and HO-1 as well as COX-2 were analyzed with nonparametric Spearman method.MAT2A transcript was significantly downregulated in cancer tissues compared to normal tissues (P < 0.05). Immunohistochemical analysis and western blotting indicated that level of MAT2A protein was decreased in cancer tissues. The statistical analysis reveals a negative correlation between MAT2A and HO-1 expression in RCC patients and cell lines (P < 0.01).This study demonstrated that MAT2A was lower expression in cancer tissues, suggesting that it may be involved in the development of RCC. MAT2A is a transcriptional corepressor for HO-1 expression by supplying SAM for methyltransferases, which may be one of potential mechanism of MAT2A as tumor suppressor in kidney carcinogenesis.
Project description:Background: Long noncoding RNAs (lncRNAs) have been demonstrated to play essential roles in renal cell carcinoma (RCC). However, the role of lncRNA KCNQ1DN in RCC remains unclear. Methods: The expression of KCNQ1DN in RCC and the corresponding adjacent tissues was measured by qPCR. RNA fluorescence in situ hybridization (FISH) assay, methylation analysis, reporter gene assays and functional tests were performed to reveal the effects of KCNQ1DN on RCC. Results: In the present study, we found that lncRNA KCNQ1DN was notably decreased in RCC tissues and cell lines. RNA FISH assay showed that KCNQ1DN mainly localized to the cytoplasm. Methylation analysis revealed that the proximal region of KCNQ1DN promoter was hypermethylated in RCC tissues relative to the adjacent normal ones. Functional studies clarified that KCNQ1DN repressed the RCC cell growth and cell cycle progression. Mechanistically, KCNQ1DN inhibited the expression of c-Myc, which might further upregulate cyclin D1 and suppress p27 at mRNA and protein levels in RCC cells. Reporter gene assays revealed that the transcriptional activity of c-Myc promoter was inhibited by KCNQ1DN. The in vivo experiments in nude mice showed that KCNQ1DN overexpression dramatically repressed the growth of xenograft tumors and the expression of corresponding c-Myc. Conclusion: These results indicated that KCNQ1DN inhibit the growth of RCC cells in vitro and in vivo through repressing the oncogene c-myc, suggesting that KCNQ1DN may serve as a novel target for the treatment of RCC.
Project description:Renal cell carcinoma (RCC) is the most common urological cancer with steadily increasing incidence. A series of tumor suppressor genes (TSGs) have been identified methylated in RCC as potential epigenetic biomarkers. We identified a 1p36.3 TSG candidate CHD5 as a methylated target in RCC through epigenome study. As the role of CHD5 in RCC pathogenesis remains elusive, we further studied its expression and molecular functions in RCC cells. We found that CHD5 was broadly expressed in most normal genitourinary tissues including kidney, but frequently silenced or downregulated by promoter CpG methylation in 78% of RCC cell lines and 44% (24/55) of primary tumors. In addition, CHD5 mutations appear to be rare in RCC tumors through genome database mining. In methylated/silenced RCC cell lines, CHD5 expression could be restored with azacytidine demethylation treatment. Ectopic expression of CHD5 in RCC cells significantly inhibited their clonogenicity, migration and invasion. Moreover, we found that CHD5, as a chromatin remodeling factor, suppressed the expression of multiple targets including oncogenes (MYC, MDM2, STAT3, CCND1, YAP1), epigenetic master genes (Bmi-1, EZH2, JMJD2C), as well as epithelial-mesenchymal transition and stem cell markers (SNAI1, FN1, OCT4). Further chromatin immunoprecipitation (ChIP) assays confirmed the binding of CHD5 to target gene promoters. Thus, we demonstrate that CHD5 functions as a novel TSG for RCC, but is predominantly inactivated by promoter methylation in primary tumors.