scholarly journals Development of a highly pulmonary metastatic orthotopic renal cell carcinoma murine model

Biology Open ◽  
2021 ◽  
Vol 10 (4) ◽  
Author(s):  
Jee Soo Park ◽  
Myung Eun Lee ◽  
Seung Hwan Kim ◽  
Won Sik Jang ◽  
Won Sik Ham
Keyword(s):  
Renal Cell Carcinoma ◽  
Tumor Progression ◽  
Cell Carcinoma ◽  
Pulmonary Metastasis ◽  
Renal Cell ◽  
Molecular Mechanisms ◽  
Lung Lesions ◽  
In Vivo Selection ◽  
Metastatic Lung

ABSTRACT The incidence of renal cell carcinoma (RCC) is high, and its outcomes remain poor. Mortality is attributable largely to metastatic disease and a dearth of effective therapeutic interventions. The lungs are the most common metastatic site. To elucidate the biological mechanisms underlying pulmonary metastasis and identify superior therapeutic strategies, we developed a novel and clinically relevant murine RCC model exhibiting enhanced pulmonary metastasis. Mice underwent intrarenal implantation using luciferase-expressing Renca, a murine renal adenocarcinoma cell line. Primary renal tumor progression and development of metastatic lung lesions were monitored in live mice using bioluminescent imaging, followed by post-mortem organ assessment. Cells were isolated from pulmonary metastases for reimplantation, followed by repeat monitoring and assessment. This process was repeated once more for a total of two in vivo passages to select for pulmonary metastatic Renca cell subpopulations. However, a single round of in vivo selection was sufficient to produce a near-maximally metastatic subpopulation. Relative to Renca cell-implanted mice, subpopulation-implanted mice exhibited shorter implantation-metastasis intervals (5 days), shorter implantation-moribundity intervals (sacrificed at 18.6±2.9 versus 22.3±1.1 days), a higher number of metastatic lung lesions at 23 days (183.9±39.0 versus 172.6±38.2) and poorer survival. Implantation of cells derived from the second round of in vivo selection produced no further significant differences in the above metrics. This model consistently and efficiently recapitulates RCC pulmonary metastasis while allowing in vivo monitoring of tumor progression, thereby facilitating elucidation of the molecular mechanisms underlying pulmonary metastasis and evaluation of therapeutic modalities.

scholarly journals CircAGAP1 promotes tumor progression by sponging miR-15-5p in clear cell renal cell carcinoma

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Qi Lv ◽  
Gangmin Wang ◽  
Yinan Zhang ◽  
Aijun Shen ◽  
Junjun Tang ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Tumor Progression ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Molecular Mechanisms ◽  
Clear Cell ◽  
Luciferase Reporter ◽  
Clinical Samples ◽  
Cell Renal Cell Carcinoma

Abstract Background Accumulating evidence has revealed that circular RNAs (circRNAs), as novel noncoding RNAs, play critical roles in carcinogenesis and tumor progression. However, the functions and molecular mechanisms of circRNAs in clear cell renal cell carcinoma (ccRCC) are largely unknown. Methods The expression and functions of circAGAP1 were identified in clinical samples, ccRCC cells and in vivo animal models. The molecular mechanism of circAGAP1 was investigated by fluorescence in situ hybridization, RNA immunoprecipitation and luciferase assays. Results circAGAP1 (circ0058792) expression was significantly upregulated in ccRCC tissues compared to adjacent nontumor tissues. Moreover, the expression of circAGAP1 was closely related to the tumor size, nuclear grade and clinical stage of ccRCC in patients. Mechanistic studies demonstrated that cytoplasmic circAGAP1 targeted miR-15-5p in an RNA-induced silencing complex. Additionally, miR-15-5p expression was downregulated in ccRCC. Luciferase reporter assays showed that E2F transcription factor 3 (E2F3) was a target of miR-15-5p, and upregulated E2F3 expression was positively correlated with circAGAP1 in ccRCC. Furthermore, the tumor-promoting functions of circAGAP1 could be alleviated by miR-15-5p mimics in vitro and in vivo. Conclusion Our results clarify that circAGAP1 exerts its oncogenic functions as a competitive endogenous RNA (ceRNA) by sponging miR-15-5p, which promotes E2F3 expression. Targeting circAGAP1 might be a new attractive therapeutic strategy in ccRCC.

scholarly journals Targeting POLE2 Creates a Novel Vulnerability in Renal Cell Carcinoma via Modulating Stanniocalcin 1

2021 ◽  
Vol 9 ◽  
Author(s):  
Chuanjie Zhang ◽  
Yan Shen ◽  
Lili Gao ◽  
Xiaojing Wang ◽  
Da Huang ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Western Blot ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Molecular Mechanisms ◽  
Cancer Genome ◽  
Clinicopathological Parameters ◽  
Expression Levels

ObjectiveThe aim of this study is to investigate the biological functions and the underlying mechanisms of DNA polymerase epsilon subunit 2 (POLE2) in renal cell carcinoma (RCC).MethodsThe datasets of POLE2 expression in The Cancer Genome Atlas Kidney Clear Cell Carcinoma (TCGA-KIRC) and International Cancer Genome Consortium (ICGC) databases was selected and the correlation between POLE2 and various clinicopathological parameters was analyzed. The POLE2 expression in RCC tissues was examined by immunohistochemistry. The POLE2 knockdown cell lines were constructed. In vitro and in vivo experiments were carried out to investigate the function of POLE2 on cellular biology of RCC, including cell viability assay, clone formation assay, flow cytometry, wound-healing assay, Transwell assay, qRT-PCR, Western blot, etc. Besides, microarray, co-immunoprecipitation, rescue experiment, and Western blot were used to investigate the molecular mechanisms underlying the functions of POLE2.ResultsPOLE2 was overexpressed in RCC tissues, and high expression of POLE2 was correlated with poor prognosis of RCC. Furthermore, knockdown of POLE2 significantly inhibited cell proliferation, migration, and facilitated apoptosis in vitro. In vivo experiments revealed that POLE2 attenuated RCC tumorigenesis and tumor growth. we also illuminated that stanniocalcin 1 (STC1) was a downstream gene of POLE2, which promoted the occurrence and development of RCC. Besides, knockdown of POLE2 significantly upregulated the expression levels of Bad and p21 while the expression levels of HSP70, IGF-I, IGF-II, survivin, and sTNF-R1 were significantly downregulated. Western blot analysis also showed that knockdown of POLE2 inhibited the expression levels of Cancer-related pathway proteins including p-Akt, CCND1, MAPK9, and PIK3CA.ConclusionKnockdown of POLE2 attenuates RCC cells proliferation and migration by regulating STC1, suggesting that POLE2-STC1 may become a potential target for RCC therapy.

scholarly journals SIRT5 Functions as a Tumor Suppressor via Reversing Warburg Effect in Renal Cell Carcinoma

2021 ◽  
Author(s):  
Liu Yihan ◽  
Wang Xiaojing ◽  
Liu Ao ◽  
Zhang Chuanjie ◽  
Wang Haofei ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Western Blot ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Warburg Effect ◽  
Molecular Mechanisms ◽  
Clear Cell ◽  
Clinicopathological Parameters

Abstract Background: The aim of this study is to investigate the biological functions and the underlying mechanisms of SIRT5 in clear cell renal cell carcinoma (ccRCC).Methods: The datasets of SIRT5 expression in The Cancer Genome Atlas Kidney Clear Cell Carcinoma (TCGA-KIRC) was selected and the correlation between SIRT5 and various clinicopathological parameters was analyzed. The SIRT5 expression in RCC tissues was examined by immunohistochemistry. The SIRT5 knockdown cell lines were constructed. In vitro and in vivo experiments were carried out to investigate the function of SIRT5 on cellular biology of RCC, including cell viability assay, wound-healing assay, soft agar colony formation assay, Transwell invasion assay, qRT-PCR, Western blot, etc. Besides, microarray, rescue experiment and Western blot were used to investigate the molecular mechanisms underlying the functions of SIRT5.Results: SIRT5 expression was downregulated in RCC tissues, and low expression of SIRT5 was correlated with poor prognosis of RCC. Knockdown of SIRT5 significantly prompted cell proliferation, migration, and facilitated invasion in vitro. In vivo experiments revealed that knocking down SIRT5 prompted ccRCC tumorigenesis and metastasis. SIRT5 deglycosylated PDHA1 at K351 and increased the activity of PDC, thus changing the metabolic pathway to the TCA cycle and inhibiting the Warburg effect. The overexpression of SIRT5 was related to the low succinylation of PDHA1.Conclusion: SIRT5 correlated with PDHA1 hyposuccinylation and progression in ccRCC, which suggested that SIRT5 might become a potential target for ccRCC therapy.

scholarly journals A novel mechanism of HIF2-dependent PLK1-mediated metastasis and drug resistance of clear cell Renal Cell Carcinoma

2020 ◽  
Author(s):  
Maeva Dufies ◽  
Annelies Verbiest ◽  
Lindsay S Cooley ◽  
Papa Diogop Ndiaye ◽  
Julien Viotti ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Drug Resistance ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Molecular Mechanisms ◽  
Cancer Metastasis ◽  
Clear Cell ◽  
Cell Renal Cell Carcinoma ◽  
Plk1 Expression

AbstractPolo-Like Kinase 1 (Plk1) expression is inversely correlated with survival advantages in many cancers. However, molecular mechanisms that underlie Plk1 expression are poorly understood. Here, we uncover a novel hypoxia-regulated mechanism of Plk1-mediated cancer metastasis and drug resistance. We demonstrated that a new HIF-2-dependent regulatory pathway drives Plk1 expression in clear cell renal cell carcinoma (ccRCC). Mechanistically, HIF-2 transcriptionally targets the hypoxia response element of the Plk1 promoter. In ccRCC patients, high expression of Plk1 was correlated to poor disease-free survival and overall survival. Loss-of-function of Plk1 in vivo markedly attenuated ccRCC growth and metastasis. High Plk1 expression conferred a resistant phenotype of ccRCC to targeted therapeutics such as sunitinib, in vitro, in vivo and in metastatic ccRCC patients. Importantly, high Plk1 expression was defined in a subpopulation of ccRCC patients that are refractory to current therapies. Hence, we propose a therapeutic paradigm for improving outcomes of ccRCC patients.

scholarly journals Ubiquitin specific peptidase 53 inhibits the occurrence and development of clear cell renal cell carcinoma through NF-κB pathway inactivation

2020 ◽  
Author(s):  
Dingwen Gui ◽  
Zhufeng Dong ◽  
Wei Peng ◽  
Weidong Jiang ◽  
Geng Huang ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Molecular Mechanisms ◽  
Clear Cell ◽  
Tumor Xenograft ◽  
Deubiquitinating Enzymes ◽  
Cell Renal Cell Carcinoma ◽  
Clinical Prognosis

Abstract Background Clear cell renal cell carcinoma (ccRCC) is one of the most prevalent malignant diseases in the urinary system with more than 140,000 related deaths annually. The lack of effective tumor biomarkers and understanding of the molecular mechanisms of this disease make the difficulty in the diagnosis and treatment. Ubiquitination-deubiquitination homeostasis is an important factor in ccRCC progression, ubiquitin specific peptidase 53 (USP53) belongs to the family of deubiquitinating enzymes, but its functions are rarely reported. Methods Databases obtained from GEO and TCGA were analyzed to reveal the role of USP53 in ccRCC. CCK-8/BrdU and EDU assays were used to detect the proliferation of ccRCC after USP53 overexpression or knockdown. Tumor xenograft experiment was used to verify the effect of clone formation of ccRCC after USP53 knockdown. Transwell assays were used to detect the metastasis of ccRCC after USP53 overexpression or knockdown. RNA sequencing and western blot analysis were employed to detect the change of genes after USP53 overexpression and knockdown. Results USP53 expression was down-regulated in ccRCC tissues and USP53 expression was significantly negative correlated with the tumor progression and clinical prognosis. The ability of growth and metastasis of ccRCC was inhibited after USP53 overexpression. And USP53 knockdown promoted ccRCC growth and metastasis. Moreover USP53 knockdown promoted the ability of clone formation of ccRCC in vivo . NF-κB signaling pathway significantly enriched and down regulated in USP53 overexpressed cells. And genes in the NF-κB pathway (such as IL1B, CXCL1-3, RELA, RELB, etc.) were obviously down regulated in USP53 overexpressed cells. USP53 overexpression decreased the phosphorylation of IKKβ and P65 in both Caki-1 and 786-O cells. And the expression of IκBα was increased. Phosphorylation of IKKβ and P65 was increased in both Caki-1 and 786-O cells after USP53 knockdown. Conclusion In summary, our data showed that USP53 inhibit ccRCC proliferation and metastasis through NF-κB pathway inactivation. The overexpression of USP53 is accompanied by changes in many inflammatory genes in the NF pathway. These findings may help better understand the pathogenesis of ccRCC and introduce new potential therapeutic targets for kidney cancer patients.

scholarly journals Plk1, upregulated by HIF-2, mediates metastasis and drug resistance of clear cell renal cell carcinoma

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Maeva Dufies ◽  
Annelies Verbiest ◽  
Lindsay S. Cooley ◽  
Papa Diogop Ndiaye ◽  
Xingkang He ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Drug Resistance ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Molecular Mechanisms ◽  
Cancer Metastasis ◽  
Clear Cell ◽  
Cell Renal Cell Carcinoma ◽  
Plk1 Expression

AbstractPolo-like kinase 1 (Plk1) expression is inversely correlated with survival advantages in many cancers. However, molecular mechanisms that underlie Plk1 expression are poorly understood. Here, we uncover a hypoxia-regulated mechanism of Plk1-mediated cancer metastasis and drug resistance. We demonstrated that a HIF-2-dependent regulatory pathway drives Plk1 expression in clear cell renal cell carcinoma (ccRCC). Mechanistically, HIF-2 transcriptionally targets the hypoxia response element of the Plk1 promoter. In ccRCC patients, high expression of Plk1 was correlated to poor disease-free survival and overall survival. Loss-of-function of Plk1 in vivo markedly attenuated ccRCC growth and metastasis. High Plk1 expression conferred a resistant phenotype of ccRCC to targeted therapeutics such as sunitinib, in vitro, in vivo, and in metastatic ccRCC patients. Importantly, high Plk1 expression was defined in a subpopulation of ccRCC patients that are refractory to current therapies. Hence, we propose a therapeutic paradigm for improving outcomes of ccRCC patients.

scholarly journals METTL13 Inhibits Progression of Clear Cell Renal Cell Carcinoma with Repression on PI3K/AKT/mTOR/HIF-1α Pathway and c-Myc Expression

2021 ◽  
Author(s):  
Zhuonan Liu ◽  
Tianshui Sun ◽  
Chiyuan Piao ◽  
Zhe Zhang ◽  
Chuize Kong
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Molecular Mechanisms ◽  
Clear Cell ◽  
Epithelial Mesenchymal Transition ◽  
Cell Renal Cell Carcinoma ◽  
Mesenchymal Transition

Abstract Background: Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive type of renal malignancy. Methyltransferase like 13 (METTL13) functions as an oncogene in most of human cancers, but its function and mechanism in ccRCC remain unreported. Methods: qRT-PCR, western blot and immunohistochemistry were used to detect METTL13’s expressions in tissues. The effects of METTL13 on ccRCC cells’ growth and metastasis were determined by both functional experiments and animal experiments. Weighted gene co-expression network analysis (WGCNA) was performed to annotate METTL13’s functions and co-immunoprecipitation (co-IP) was used to determine the interaction between two proteins. Results: METTL13 was lowly expressed in ccRCC tissues compared to normal kidney tissues and its low expression predicted poor prognosis for ccRCC patients. In vitro study indicated METTL13’s inhibition on ccRCC cells’ proliferation, viability, migratory ability and invasiveness as well as epithelial-mesenchymal transition (EMT). Bioinformatic analyses showed various biological functions and pathways METTL13 was involved in. In ccRCC cells, we observed that METTL13 could negatively regulate PI3K/AKT/mTOR/HIF-1α pathway and that it combined to c-Myc and inhibited c-Myc expression. In vivo experiment confirmed that METTL13 inhibited ccRCC cell growth and metastasis. Conclusions: In general, our finding suggests that associated with favorable prognosis of ccRCC patients, METTL13 can inhibit growth and metastasis of ccRCC cells with multiple potential molecular mechanisms. Therefore, it’s likely for METTL13 to serve as a new diagnostic and therapeutic target for ccRCC in the future.

scholarly journals Inhibition of BRD4 Suppresses Cell Proliferation and Induces Apoptosis in Renal Cell Carcinoma

2017 ◽  
Vol 41 (5) ◽  
pp. 1947-1956 ◽  
Author(s):  
Xinchao Wu ◽  
Dong Liu ◽  
Xuemei Gao ◽  
Fei Xie ◽  
Dan Tao ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Proliferation ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Tumor Stage ◽  
Promising Target

Background/Aims: Renal cell carcinoma (RCC) remains an intractable genitourinary malignancy. Resistance to chemotherapy or targeted therapies in RCC is presumably due to the complicated underlying molecular mechanisms and insufficient understanding. The aim of this research was to assess the expression and role of bromodomain-4 protein (BRD4) in RCC and evaluate the effects of BRD4 inhibitor JQ1 for RCC treatment. Methods: BRD4 expressionlevels were assessed by qRT-PCR and western blot in RCC tissues and cells. The effects of BRD4 knockdown or JQ1 on RCC cells were assessed by MTT assay and flow cytometry. The effects of in vivo treatment were evaluated through xenograft experiments. Results: BRD4 is significantly overexpressed in RCC, and is related to tumor stage and lymph node metastasis. Inhibition of BRD4 suppressed RCC cell proliferation, induced cell apoptosis in vitro and repressed tumor growth in vivo. Inhibition of BRD4 decreased BCL2 and C-MYC expression while increased BAX and cleaved caspase3 expression, and strikingly diminished the recruitment of BRD4 to BCL2 promoter. Conclusions: Our research reveals that BRD4 probably play a critical role in RCC progression, and is a new promising target for pharmacological treatment directed against this intractable disease.

scholarly journals METTL13 inhibits progression of clear cell renal cell carcinoma with repression on PI3K/AKT/mTOR/HIF-1α pathway and c-Myc expression

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Zhuonan Liu ◽  
Tianshui Sun ◽  
Chiyuan Piao ◽  
Zhe Zhang ◽  
Chuize Kong
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Molecular Mechanisms ◽  
Clear Cell ◽  
Epithelial Mesenchymal Transition ◽  
Cell Renal Cell Carcinoma ◽  
Mesenchymal Transition

Abstract Background Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive type of renal malignancy. Methyltransferase like 13 (METTL13) functions as an oncogene in most of human cancers, but its function and mechanism in ccRCC remains unreported. Methods qRT-PCR, western blotting and immunohistochemistry were used to detect METTL13’s expression in tissues. The effects of METTL13 on ccRCC cells’ growth and metastasis were determined by both functional experiments and animal experiments. Weighted gene co-expression network analysis (WGCNA) was performed to annotate METTL13’s functions and co-immunoprecipitation (co-IP) was used to determine the interaction between METTL13 and c-Myc. Results METTL13 was underexpressed in ccRCC tissues compared to normal kidney tissues and its low expression predicted poor prognosis for ccRCC patients. The in vitro studies showed that knockdown and overexpression of METTL13 respectively led to increase and decrease in ccRCC cells’ proliferation, viability, migratory ability and invasiveness as well as epithelial-mesenchymal transition (EMT). The in vivo experiment demonstrated the inhibitory effect that METTL13 had on ccRCC cells’ growth and metastasis. Bioinformatic analyses showed various biological functions and pathways METTL13 was involved in. In ccRCC cells, we observed that METTL13 could negatively regulate PI3K/AKT/mTOR/HIF-1α pathway and that it combined to c-Myc and inhibited c-Myc protein expression. Conclusions In general, our finding suggests that high expression of METTL13 is associated with favorable prognosis of ccRCC patients. Meanwhile, METTL13 can inhibit growth and metastasis of ccRCC cells with participation in multiple potential molecular mechanisms. Therefore, we suggest METTL13 can be a new diagnostic and therapeutic target for ccRCC in the future.

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