Expression of angiostatin cDNA in a murine renal cell carcinoma suppresses tumor growth in vivo

592 Background: Zinc finger protein 395 (ZNF395) is frequently altered in several tumor types. However, the role of ZNF395 remains poorly studied in patients with clear cell renal cell carcinoma (RCC). In this study, we investigated the in vitro and in vivo role of ZNF395 in ccRCC. Methods: cBioPortal For Cancer Genomics was used to correlate the expression of ZNF395 with RCC patient clinical, pathological and molecular profiles. ZNF395 protein and mRNA levels were studied in several RCC cell lines in vitro. Subsequently, ZNF395 knockdown was performed in 786-O and UMRC3 RCC cells and overexpression was done in Caki-1 and 769-P RCC cells. We then evaluated ZNF395 modulation in these cell lines by in vitro MTT, migration and invasion assays. Finally, we studied the effect of ZNF395 knockout and overexpression in vivo using SCID xenograft models. Results: Patients with higher expression of ZNF395 experienced longer disease-free survival and overall survival. Using in vitro models, we confirmed that knockdown of ZNF395 decreased ZNF395 expression, and increased proliferation, migration and invasiveness of 786-O and UMRC3, while overexpression of ZNF395 increased ZNF395 expression, and reduced proliferation, migration and invasiveness of Caki-1 and 769-P. Using in vivo mouse models, knockdown of ZNF395 expression in 786-O promoted tumor growth while its overexpression in Caki-1 resulted in tumor growth inhibition. We are currently performing experiments to understand the process by which ZNF395 regulates ccRCC pathogenesis. Conclusions: Our data support the role of ZNF395 as an important tumor suppressor gene in the pathogenesis of RCC.

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Overexpression of PFKFB3 Promotes Cell Glycolysis and Proliferation in Renal Cell Carcinoma

10.21203/rs.3.rs-255023/v1 ◽

2021 ◽

Author(s):

Jun Li ◽

Shiqiang Zhang ◽

Dingzhun Liao ◽

Qian Zhang ◽

Chujie Chen ◽

...

Keyword(s):

Cell Cycle ◽

Renal Cell Carcinoma ◽

Tumor Growth ◽

Cell Carcinoma ◽

Cell Lines ◽

Renal Cell ◽

Aerobic Glycolysis ◽

Xenograft Model

Abstract Background: Cancer cells prefer aerobic glycolysis to increase their biomass and sustain uncontrolled proliferation. As a key glycolytic activator, phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) has been implicated in the progression of multiple types of tumors. However, the specific function and clinical significance of PFKFB3 in renal cell carcinoma (RCC) remain unclear. In the present study, we explored the role of PFKFB3 in RCC.Methods: We analyzed the expression of PFKFB3 in clear cell renal cell carcinoma (ccRCC) tissues and its relationship with clinical characteristics of ccRCC. Real-time PCR and Western blot analysis were used to detect PFKFB3 expression levels in different RCC cell lines. Furthermore, we determined the glycolytic activity by glucose uptake, lactate secretion assay and ECAR analysis. CCK-8 assay, clone formation assay, flow cytometry and EdU assay were performed to monitor cancer cell proliferation and cell cycle distribution. In addition, nude mice xenograft model was used to investigate the role of PFKFB3 in tumor growth in vivo.Results: In this study, we found that PFKFB3 was significantly up-regulated in RCC tissues and cell lines compared with normal control. Overexpression of PFKFB3 was positively associated with advanced TNM stage and could predict poor prognosis of ccRCC patients. Furthermore, knockdown of PFKFB3 suppresses cell glycolysis, proliferation and cell cycle G1/S transition in RCC cells. Importantly, in vivo experiments confirmed that PFKFB3 knockdown delayed tumor growth derived from the ACHN cell line.Conclusion: Our results suggest that PFKFB3 plays an important role in the progression of RCC via mediating glycolysis and proliferation, and provides a potential therapeutic target for RCC.

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Chromodomain Helicase DNA-Binding Protein 5 Inhibits Renal Cell Carcinoma Tumorigenesis by Activation of the p53 and RB Pathways

BioMed Research International ◽

10.1155/2020/5425612 ◽

2020 ◽

Vol 2020 ◽

pp. 1-12

Author(s):

Sheng Huang ◽

Qitao Yan ◽

Shilin Xiong ◽

Yiqi Peng ◽

Rui Zhao ◽

...

Keyword(s):

Renal Cell Carcinoma ◽

Dna Binding ◽

Tumor Growth ◽

Cell Carcinoma ◽

Renal Cell ◽

Binding Protein ◽

Dna Binding Protein ◽

Migration And Invasion ◽

Fuhrman Grade

Chromodomain helicase DNA-binding protein 5 (CHD5) plays a crucial tumor suppressor role in multiple types of tumors. For this study, we investigated its clinical significance and the molecular mechanism(s) underlying tumorigenesis in renal cell carcinoma (RCC). Initially, CHD5 expression was assessed in primary tumor tissue and in tissue array. Correlations among CHD5 expression and clinicopathological characteristics were analyzed. Next, lentivirus-mediated CHD5 overexpression in the ACHN and 769-P cells was used to assess effects on proliferation, migration, invasion ability, and the regulation of the p14ARF/p53 and p16INK4a/RB signaling pathways. Finally, a xenograft mouse model was used to verify its impact on tumor growth in vivo. Results demonstrated that CHD5 was downregulated in tumor tissues and that low CHD5 expression was correlated with advanced TNM stage, high Fuhrman grade, lymph node metastasis, and poor survival. Overexpression of CHD5 inhibited proliferation, migration, and invasion in vitro; prompted cell cycle G1 phase arrest; induced apoptosis; and suppressed tumor growth in vivo. Furthermore, we confirmed that CHD5 activates the p53 and RB pathways to inhibit tumorigenesis in RCC. In summary, CHD5 is involved in the initiation and progression of RCC and may serve as a diagnostic biomarker and a potential therapeutic target for RCC.

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The Role of Interleukin-6 in the Induction of Hypercalcemia in Renal Cell Carcinoma Transplanted into Nude Mice

Endocrinology ◽

10.1210/endo.138.5.5095 ◽

1997 ◽

Vol 138 (5) ◽

pp. 1879-1885 ◽

Cited By ~ 7

Author(s):

Max G. Weissglas ◽

Denis H. J. Schamhart ◽

Clemens W. G. M. Löwik ◽

Socrates E. Papapoulos ◽

Harry M. Theuns ◽

...

Keyword(s):

Renal Cell Carcinoma ◽

Tumor Growth ◽

Cell Carcinoma ◽

Serum Calcium ◽

Nude Mice ◽

Renal Cell ◽

Messenger Rna ◽

Neutralizing Antibodies ◽

Human Renal Cell Carcinoma

Abstract Hypercalcemia is a well known complication of renal cell carcinoma (RCC). As RCCs can produce IL-6, and IL-6 may stimulate bone resorption and cause mild hypercalcemia, we examined whether IL-6 is involved in renal cancer-associated hypercalcemia in vivo. Three human renal cell carcinoma tumor lines (RC-8, RC-9, and NC-65) growing in nude mice were studied. Tumors were implanted sc, and parameters of bone metabolism and serum human IL-6 levels were determined in relation to tumor volume (TV). All three tumor lines secreted human IL-6, although in different quantities. The maximum level of IL-6 in RC-8 was 434 pg/ml (TV, 200 mm3), that in RC-9 was 81 pg/ml (TV, 1800 mm3), and that in NC-65 was 2368 pg/ml (TV, 1800 mm3). Hypercalcemia developed in RC-8 and RC-9 tumor-bearing animals, but not in NC-65-bearing animals. The hypercalcemia in both RC-8 and RC-9 tumor lines was associated with elevated levels of PTH-related peptide (PTHrP) and loss of trabecular bone volume. Serum calcium and phosphate concentrations showed an almost linear relationship with plasma PTHrP independently of the tumor line and serum IL-6 levels. No hypercalcemia occurred in the NC-65 animals, which had the highest levels of IL-6, but no detectable plasma PTHrP and PTHrP messenger RNA expression in the tumor. Administration of neutralizing antibodies to IL-6 to RC-8 animals normalized serum calcium concentrations and PTHrP values and induced a significant inhibition of tumor growth. No such effect on tumor growth of anti-IL-6 was seen in the other two tumor lines. The normalization of serum calcium in RC-8 mice is most likely attributed to the growth-inhibiting effect of anti-IL-6 on RC-8 tumor. We conclude that IL-6 secreted by RCC does not contribute directly to hypercalcemia, but may enhance hypercalcemia by stimulating the tumor growth of a subpopulation of PTHrP-secreting carcinomas.

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circTLK1 facilitates the proliferation and metastasis of renal cell carcinoma by regulating miR-495-3p/CBL axis

Open Life Sciences ◽

10.1515/biol-2021-0041 ◽

2021 ◽

Vol 16 (1) ◽

pp. 362-374

Author(s):

Xiangli Lei ◽

Meiling Yang ◽

Zhifang Xiao ◽

Heng Zhang ◽

Shuai Tan

Keyword(s):

Renal Cell Carcinoma ◽

Tumor Growth ◽

Western Blot ◽

Cell Carcinoma ◽

Renal Cell ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Circular Rnas ◽

Proliferation And Metastasis

Abstract Renal cell carcinoma (RCC) is a common urological malignancy. Circular RNAs (circRNAs) have been confirmed to play an important regulatory role in various cancers. This study aimed to investigate the role and potential mechanism of circTLK1 (hsa_circ_0004442) in RCC. The levels of circTLK1, Cbl proto-oncogene (CBL), and microRNA-495-3p (miR-495-3p) were detected by quantitative reverse transcription polymerase chain reaction or western blot. Cell proliferation, cycle arrest and apoptosis, migration, and invasion were assessed by colony formation, flow cytometry, scratch, and transwell assays. The levels of E-cadherin and Vimentin were measured by western blot. The targeting relationship between miR-495-3p and miR-495-3p or CBL was verified by dual-luciferase reporter assay. Tumor growth in vivo was evaluated by xenograft assay. The results found that circTLK1 and CBL were up-regulated in RCC tissues and cells. Silencing of circTLK1 or CBL inhibited proliferation and metastasis and accelerated apoptosis in RCC cells. In addition, circTLK1 directly bound to miR-495-3p, and CBL was the target of miR-495-3p. circTLK1 sponged miR-495-3p to increase CBL expression. Moreover, knockdown of circTLK1 suppressed tumor growth in vivo. In conclusion, down-regulation of circTLK1 restrained proliferation and metastasis and promoted apoptosis in RCC cells by modulating miR-495-3p/CBL axis.

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RUNX1/miR-582-5p Pathway Regulates the Tumor Progression in Clear Cell Renal Cell Carcinoma by Targeting COL5A1

Frontiers in Oncology ◽

10.3389/fonc.2021.610992 ◽

2021 ◽

Vol 11 ◽

Author(s):

Jianxin Xue ◽

Shenhao Zhu ◽

Feng Qi ◽

Kai Zhu ◽

Pu Cao ◽

...

Keyword(s):

Renal Cell Carcinoma ◽

Tumor Growth ◽

Cell Carcinoma ◽

Renal Cell ◽

Clear Cell ◽

Negative Regulator ◽

Expression Level ◽

Cell Renal Cell Carcinoma

Recent evidences indicated that miRNAs played core role in the progression of clear cell renal cell carcinoma (ccRCC). However, its molecular mechanism in ccRCC is still remained unclear. The study was designed to identify the role and regulatory mechanism of miR-582-5p in ccRCC. In this study, the low expression level of miR-582-5p were detected by qRT-PCR in ccRCC patient tumor samples and ccRCC cell lines, respectively. The expression level of miR-582-5p was associated with tumor stage and metastasis. In vivo and in vitro experiments found miR-582-5p inhibit tumor growth via suppressing COL5A1 expression. Additionally, RUNX1 was identified as the negative regulator of miR-582-5p through database prediction and chromatin immunoprecipitation. Finally, the negative relation of RUNX1 and miR-582-5p was verified through rescue experiment both in vitro and in vivo. In summary, miR-582-5p, which was regulated by RUNX1, inhibited tumor growth and invasion by targeting COL5A1, indicating that miR-582-5p may act as a biomarker and that the RUNX1/miR-582-5p/COL5A1 axis could be a potential therapeutic target for ccRCC.

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Preclinical efficacy of dual mTORC1/2 inhibitor AZD8055 in renal cell carcinoma harboring a TFE3 gene fusion

BMC Cancer ◽

10.1186/s12885-019-6096-0 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 1

Author(s):

Eric C. Kauffman ◽

Martin Lang ◽

Soroush Rais-Bahrami ◽

Gopal N. Gupta ◽

Darmood Wei ◽

...

Keyword(s):

Cell Cycle ◽

Renal Cell Carcinoma ◽

Tumor Growth ◽

Cell Carcinoma ◽

Renal Cell ◽

Gene Fusion ◽

Mtor Pathway ◽

Preclinical Efficacy ◽

Ccrcc Cell

Abstract Background Renal cell carcinomas (RCC) harboring a TFE3 gene fusion (TfRCC) represent an aggressive subset of kidney tumors. Key signaling pathways of TfRCC are unknown and preclinical in vivo data are lacking. We investigated Akt/mTOR pathway activation and the preclinical efficacy of dual mTORC1/2 versus selective mTORC1 inhibition in TfRCC. Methods Levels of phosphorylated Akt/mTOR pathway proteins were compared by immunoblot in TfRCC and clear cell RCC (ccRCC) cell lines. Effects of the mTORC1 inhibitor, sirolimus, and the dual mTORC1/2 inhibitor, AZD8055, on Akt/mTOR activation, cell cycle progression, cell viability and cytotoxicity were compared in TfRCC cells. TfRCC xenograft tumor growth in mice was evaluated after 3-week treatment with oral AZD8055, intraperitoneal sirolimus and respective vehicle controls. Results The Akt/mTOR pathway was activated to a similar or greater degree in TfRCC than ccRCC cell lines and persisted partly during growth factor starvation, suggesting constitutive activation. Dual mTORC1/2 inhibition with AZD8055 potently inhibited TfRCC viability (IC50 = 20-50 nM) due at least in part to cell cycle arrest, while benign renal epithelial cells were relatively resistant (IC50 = 400 nM). Maximal viability reduction was greater with AZD8055 than sirolimus (80–90% versus 30–50%), as was the extent of Akt/mTOR pathway inhibition, based on significantly greater suppression of P-Akt (Ser473), P-4EBP1, P-mTOR and HIF1α. In mouse xenograft models, AZD8055 achieved significantly better tumor growth inhibition and prolonged mouse survival compared to sirolimus or vehicle controls. Conclusions Akt/mTOR activation is common in TfRCC and a promising therapeutic target. Dual mTORC1/2 inhibition suppresses Akt/mTOR signaling more effectively than selective mTORC1 inhibition and demonstrates in vivo preclinical efficacy against TFE3-fusion renal cell carcinoma.

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ERCC6L that is up-regulated in high grade of renal cell carcinoma enhances cell viability in vitro and promotes tumor growth in vivo potentially through modulating MAPK signalling pathway

Cancer Gene Therapy ◽

10.1038/s41417-018-0064-8 ◽

2018 ◽

Vol 26 (9-10) ◽

pp. 323-333 ◽

Cited By ~ 5

Author(s):

Gejun Zhang ◽

Zi Yu ◽

Shui Fu ◽

Chengcheng Lv ◽

Qingzhuo Dong ◽

...

Keyword(s):

Renal Cell Carcinoma ◽

Tumor Growth ◽

Cell Viability ◽

Cell Carcinoma ◽

Renal Cell ◽

High Grade ◽

Mapk Signalling Pathway ◽

Mapk Signalling

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A multi-layered systems approach for renal cell carcinoma

10.1101/2020.01.13.904235 ◽

2020 ◽

Author(s):

Lindsay S. Cooley ◽

Justine Rudewicz ◽

Wilfried Souleyreau ◽

Kim Clarke ◽

Francesco Falciani ◽

...

Keyword(s):

Renal Cell Carcinoma ◽

Tumor Growth ◽

Cell Carcinoma ◽

Primary Tumor ◽

Renal Cell ◽

Clonal Selection ◽

Predictive Biomarkers ◽

Response To Therapy ◽

Primary Tumor Growth

AbstractRenal cell carcinoma (RCC) still lacks prognostic and predictive biomarkers to monitor the disease and the response to therapy. The usual strategy in translational research is to start from human samples, to identify molecular markers and gene networks and then to functionally validate them in vitro and in animal models. We devised herein a completely opposite strategy from “mouse to man” by performing an aggressiveness screen and used functional genomics, imaging, clinical data and computational approaches in order to discover molecular pathways and players in renal cancer development and metastasis. Multiple cell lines for primary tumor growth, survival in the blood circulation and lung metastasis or metastatic spread from the primary tumor were generated and analyzed using a multi-layered approach which includes large-scale transcriptome, genome and methylome analyses. Transcriptome and methylome analyses demonstrated distinct clustering in three different groups. Remarkably, DNA sequencing did not show significant genomic variations in the different groups which indicates absence of clonal selection during the in vivo amplification process. Transcriptome analysis revealed distinct signatures of tumor aggressiveness which were validated in patient cohorts. Methylome analysis of full-length DNA allowed clustering of the same groups and revealed clinically relevant signatures. Furthermore, we identified SAA2 and CFB as soluble prognostic and predictive biomarkers of the therapeutic response. We also uncovered IL34 as another soluble prognostic biomarker and key regulator of renal cell carcinoma (RCC) progression. This was also functionally validated in vivo, and a mathematical model of IL34-dependent primary tumor growth and metastasis development was provided. These results indicate that such multilayered analysis in a RCC animal model leads to meaningful results that are of translational significance.One Sentence SummaryAn aggressiveness screen with multilayer systems analysis to identify signatures and biomarkers for renal cell carcinoma aggressiveness.

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