Human Renal Cancer Cell Lines Research Articles

Comprehensive Pan-Cancer Analysis of Connexin 43 as a Potential Biomarker and Therapeutic Target in Human Kidney Renal Clear Cell Carcinoma (KIRC)

Background and Objectives: Connexin 43 (Cx43) is involved in the transfer of small signaling molecules between neighboring cells, thereby exerting a major influence on the initiation and progression of tumorigenesis. However, there is a lack of systematic research on Cx43 expression and its predictive role in clinical diagnosis and prognosis in pan-cancer. Materials and Methods: Several biological databases were used to evaluate the expression levels of GJA1 (encoding Cx43) and its diagnostic and prognostic significance in pan-cancer. We targeted kidney renal clear cell carcinoma (KIRC) and investigated the relationship between GJA1 expression and different clinical features of KIRC patients. Then, we performed cell-based experiments to partially confirm our results and predicted several proteins that were functionally related to Cx43. Results: The expression of GJA1 has a high level of accuracy in predicting KIRC. High GJA1 expression was remarkably correlated with a favorable prognosis, and this expression was reduced in groups with poor clinical features in KIRC. Cell experiments confirmed the inhibitory effects of increased GJA1 expression on the migratory capacity of human renal cancer (RCC) cell lines, and protein–protein interaction (PPI) analysis predicted that CDH1 and CTNNB1 were closely related to Cx43. Conclusions: GJA1 could be a promising independent favorable prognostic factor for KIRC, and upregulation of GJA1 expression could inhibit the migratory capacity of renal cancer cells.

Abstract 7655: The CXCR4/CXCR7/CXCL12 axis and PBRM1 are involved in everolimus resistance in human renal cancer cell lines

Abstract Purpose: Renal cell carcinoma (RCC) is one of the most common malignancies in the genitourinary system. Combinations of Immunocheckpoints inhibitors plus TKIs represents the actual standard of therapy for metastatic RCC patients, while mTOR inhibitors play a role in the second-line treatment of mRCC in combination with lenvatinib. Thus characterizing resistance mechanism to mTOR inhibitors is unmet need. Experimental Design: A498 RAD-resistant renal cell carcinoma (RCC) cells were generated through increasing doses of RAD001 (RAD1-5-10 μM). Gene expression was assessed by qRT-PCR. pERK and transwell migration were evaluated in A498, RAD10 and RAD10shP. PBRM1sh was obtained in RAD10 with lentiviral particles while CXCR4 transfection in RAD10 was realized using pCMV6-AC-h-CXCR4-GFP. ChIP assay was assessed using Chromata ChIP kit. Results: Gene expression analysis revealed reduction in CXCR4/CXCR7 expression in A498 RAD 1-5-10-resistant cells (RAD1-5-10) (CXCR4:32.8, 37, 40.7-fold and CXCR7:2.3, 2.3, 2-fold, respectively), while PBRM1 and CXCL12 were overexpressed as compared to A498 (PBRM1: 2.2, 2.34 and 3.32-fold, and CXCL12: 7.2-, 5.6-, and 10-fold, respectively). The axis CXCL12-CXCR4 was functionally inhibited in RAD10 as demonstrated by inhibition in CXCL12- pERK and migration. YY1, already described as CXCR4 and CXCR7 negative regulator, was upregulated in RAD10 cells. ChIP confirmed higher binding of YY1 on CXCR4 and CXCR7 promoters in RAD10 as compared to A498. To address CXCR4 role, human CXCR4 was transfected in RAD10 cells (RAD10 CXCR4+) and RAD001 cytotoxicity was evaluated; interestingly, RAD10 CXCR4+ entirely restored RAD001 sensitivity in RAD10 cells. PBRM1 silencing in RAD10 (RAD10shP) downregulated mTOR and 4EBP1 (1.54 and 3-fold, compared to RAD10). As described, CXCR4 decreased in RAD10 and PBRM1 silencing did not further affect it, while CXCR7 increased (20-fold) in RAD10shP, indicating that PBRM1 regulates CXCR7 expression rather than CXCR4 in RAD001 resistant cells. RAD10shP migrated more towards CXCL11 than CXCL12 according to the overexpression of CXCR7 in RAD10shP cells. YY1 expression in RAD10 was higher than WT (2.6-fold) and was reduced in RAD10shP compared to RAD10 (1.6-fold) displaying a possible role of PBRM1 in regulating CXCR7 in RAD001-resistant RCC through YY1. CXCR4/CXCL12 signaling in RAD10shP cells more actively induced pERK and pAKT following CXCL12 or CXCL11-mediated as compared to RAD10 demonstrating that PBRM1 silencing restored a sensitive-like phenotype; however, PBRM1 silencing partially restored RAD001 sensitivity in RAD10 (RAD10 were 48-fold more resistant than A498, RAD10shP were 8.8-fold more resistant than A498). Conclusion: Taken together, these results demonstrate that both PBRM1 and CXCR4 are responsible for RAD001 resistance with PBRM1 regulating CXCR7 and mTOR shedding light on the new mechanistic roles of PBRM1 in RCC Citation Format: Federica Auletta, Caterina Ieranò, Giuseppina Rea, Maria Napolitano, Francesca Galdiero, Anna Maria Bello, Pierfrancesco Tassone, Maria Teresa Di Martino, Stefania Scala. The CXCR4/CXCR7/CXCL12 axis and PBRM1 are involved in everolimus resistance in human renal cancer cell lines [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 7655.

CD56 polysialylation promotes the tumorigenesis and progression via the Hedgehog and Wnt/β-catenin signaling pathways in clear cell renal cell carcinoma

BackgroundCD56 has been observed in malignant tumours exhibiting neuronal or neuroendocrine differentiation, such as breast cancer, small-cell lung cancer, and neuroblastoma. Abnormal glycosylation modifications are thought to play a role in regulating tumour cell proliferation, migration, and invasion. Nevertheless, the exact roles and molecular mechanisms of CD56 and polysialylated CD56 (PSA-CD56) in the development and progression of clear cell renal cell carcinoma (ccRCC) remain elusive. Here we unveil the biological significance of CD56 and PSA-CD56 in ccRCC.MethodsIn this study, we employed various techniques, including immunohistochemistry (IHC), RT-qPCR, and western blot, to examine the mRNA and protein expression levels in both human ccRCC tissue and cell lines. Lentivirus infection and CRISPR/Cas9 system were utilized to generate overexpression and knockout cell lines of CD56. Additionally, we conducted several functional assays, such as CCK-8, colony formation, cell scratch, and transwell assays to evaluate cell growth, proliferation, migration, and invasion. Furthermore, we established a xenograft tumor model to investigate the role of CD56 in ccRCC in vivo. To gain further insights into the molecular mechanisms associated with CD56, we employed the Hedgehog inhibitor JK184 and the β-catenin inhibitor Prodigiosin.ResultsCD56 was significantly overexpressed in both human ccRCC tissues and renal cancer cell lines compared to adjacent normal tissues and normal renal epithelial cells. In vitro and in vivo experiments revealed that the knockout of CD56 inhibited the proliferation, migration, and invasion capabilities of ccRCC cells, whereas the overexpression of PSA-CD56 promoted these capacities. Finally, PSA-CD56 overexpression was found to activate both the Hedgehog and Wnt/β-catenin signaling pathways.ConclusionOur findings demonstrate that the oncogenic function of CD56 polysialylation plays a vital role in the tumorigenesis and progression of ccRCC, implying that targeting PSA-CD56 might be a feasible treatment target for ccRCC.

The expression pattern of pyruvate dehydrogenase kinases predicts prognosis and correlates with immune exhaustion in clear cell renal cell carcinoma

Renal cancer cells constitute a paradigm of tumor cells with a glycolytic reprogramming which drives metabolic alterations favouring cell survival and transformation. We studied the expression and activity of pyruvate dehydrogenase kinases (PDK1-4), key enzymes of the energy metabolism, in renal cancer cells. We analysed the expression, subcellular distribution and clinicopathological correlations of PDK1-4 by immunohistochemistry of tumor tissue microarray samples from a cohort of 96 clear cell renal cell carcinoma (ccRCC) patients. Gene expression analysis was performed on whole tumor tissue sections of a subset of ccRCC samples. PDK2 and PDK3 protein expression in tumor cells correlated with lower patient overall survival, whereas PDK1 protein expression correlated with higher patient survival. Gene expression analysis revealed molecular association of PDK2 and PDK3 expression with PI3K signalling pathway, as well as with T cell infiltration and exhausted CD8 T cells. Inhibition of PDK by dichloroacetate in human renal cancer cell lines resulted in lower cell viability, which was accompanied by an increase in pAKT. Together, our findings suggest a differential role for PDK enzymes in ccRCC progression, and highlight PDK as actionable metabolic proteins in relation with PI3K signalling and exhausted CD8 T cells in ccRCC.

Abstract 6792: Targeting immunosuppressive adenosine to enhance vaccinia virus renal cancer oncolysis in vivo

Abstract Background: The oncolytic vaccinia virus (VV) is a promising biotherapy undergoing clinical development in human cancer, including renal cell carcinoma (RCC). While VV induces potent tumor oncolysis and immunomodulation, clinical experience has shown that OV therapy alone leads to limited benefit. Moreover, OVs and checkpoint inhibitor combinations, do not significantly improve clinical responses, underscoring the need to identify new tumor microenvironment targets to overcome OV resistance in vivo. The adenosine pathway is a biologically and clinically relevant target in RCC and is associated with adaptive resistance to immunotherapy. The objectives of this study are to characterize the role of the adenosine axis in tumor escape mechanisms to vaccinia virus in RCC, and to determine the efficacy of novel OV-immunotherapy combinations targeting adenosine. Methods: The in vitro oncolytic effects of JX-594 and mJX-594 (oncolytic VVs expressing human or murine GMCSF, respectively) in human (786-0, ACHN, CAKi-1, UOK262) and murine (RENCA) renal cancer cell lines were assessed. Proteomic analysis of 786-0 lysates treated with JX-594 was performed for mechanistic studies. The effects of JX-594 and mJX 594 on CD73 and CD39 expression were determined in human and murine RCC. In vivo antitumor effects of mJX-594 alone, and in combination with A2AR, A2BR and Dual Adenosine receptor inhibition was determined in the RENCA model in immunocompetent mice. Results: Potent in vitro oncolysis and efficient viral replication were observed in human and murine RCC cells after infection with vaccinia virus. VV oncolysis was associated with significant modulation of proliferation, survival, and ER stress pathways, as determined by functional proteomics. JX-594 and mJX-594 treatment induced increased expression of CD39 and CD73 in RCC cell lines and in renal tumors in vivo. While no significant enhancement of vaccinia oncolysis was observed with combinations with A2AR and A2BR inhibitors in vitro, a significant improvement on in vivo tumor control and survival were observed when mJX594 given intravenously was combined with A2AR and A2BR inhibitors (triplet), at a higher degree than single agents or doublets (mJX-594 in combination with single A2AR or A2BR inhibitors). Characterization of treatment mediated changes in immune signatures (nanostring) is underway and will be presented at the meeting. Conclusion: Vaccinia virus potently induces in vitro oncolysis in RCC cell lines, while also increasing expression of adenosine rate limiting enzymes in vitro and in vivo, which could explain tumor escape to oncolytic VV. MJX-594 combination with A2AR and A2BR inhibition safely and significantly improves renal cancer oncolysis and tumor control in vivo. Our studies uncover a novel, translationally relevant strategy to improve OV efficacy in vivo, in RCC and other cancers. Citation Format: Valery A. Chavez, Floritza Bustamante, Carolina Merchan-Mendes, Shannon Saigh, Patricia Guevara, Eric Wieder, Jaime Merchan. Targeting immunosuppressive adenosine to enhance vaccinia virus renal cancer oncolysis in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6792.

Diallyl Trisulfide Suppresses the Renal Cancer Stem-like Cell Properties via Nanog

Cancer stem-like cells (CSCs), which play an important role in tumor initiation and progression, have been identified in many cancers. Diallyl trisulfide (DATS) is an organosulfur compound extracted from garlic with anticancer activities. Nanog is a transcription factor responsible for maintaining the stemness of CSCs, but its role in the DATS-induced attenuation of renal CSC properties is unknown. In this study, renal CSCs were enriched from human renal cancer cell lines 786-O and ACHN cultured in a serum-free medium (SFM). The properties of CSCs were analyzed by evaluating the ability of the cells in sphere formation and measuring the expression of stem cell markers. We found that downregulation of Nanog inhibited renal CSC properties. DATS suppressed renal CSC activities by reducing tumorsphere formation, decreasing stem cell markers including Nanog, CD44, ALDH1A1, and Oct4, inhibiting cell proliferation and promoting apoptosis. We further revealed that overexpression of Nanog reversed the suppressive effects of DATS on renal CSCs. Taken together, our results demonstrated that DATS inhibited renal CSCs by suppressing Nanog. These novel findings suggested that, through Nanog targeting, DATS can potentially be used as an anti-tumor agent for renal cancer.

Characterization of the basement membrane in kidney renal clear cell carcinoma to guide clinical therapy.

Renal cell carcinoma (RCC) is the most common kidney cancer in adults. According to the histological features, it could be divided into several subtypes, of which the most common one is kidney renal clear cell carcinoma (KIRC), which contributed to more than 90% of cases for RCC and usually ends with a dismal outcome. Previous studies suggested that basement membrane genes (BMGs) play a pivotal role in tumor development. However, the significance and prognostic value of BMGs in KIRC still wrap in the mist. KIRC data were downloaded from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. A prognostic risk score (PRS) model based on BMGs was established using univariate and least absolute shrinkage and selection operator (LASSO) and the Cox regression analysis was performed for prognostic prediction. The Kaplan-Meier analysis, univariate Cox regression, multivariate Cox regression, receiver operating characteristic (ROC) curves, nomogram, and calibration curves were utilized to evaluate and validate the PRS model. All KIRC cases were divided into the high-risk score (HRS) group and the low-risk score (LRS) group according to the median risk scores. In addition, single-sample gene set enrichment analysis (ssGSEA), immune analysis, tumor microenvironment (TME) analysis, principal component analysis (PCA), and half-maximal inhibitory concentration (IC50) were also applied. Expression levels of BMGs were confirmed by qRT-PCR in both human renal cancer cell lines and tissues. We established the BMGs-based prognostic model according to the following steps. Within the TCGA cohort, patients' prognosis of the HRS group was significantly worse than that of the LRS group, which was consistent with the analysis results of the GEO cohort. PCA patterns were significantly distinct for LRS and HRS groups and pathological features of the HRS group were more malignant compared with the LRS group. Correlation analysis of the PRS model and TME features, such as immune cell scores, stromal cell scores, and ESTIMATE values, revealed a higher immune infiltration in the HRS group compared with the LRS group. The chemotherapeutic response was also evaluated in KIRC treatment. It showed that the HRS group exhibited stronger chemoresistance to chemotherapeutics like FR-180204, GSK1904529A, KIN001-102, and YM201636. The therapeutic reactivity of the other 27 chemotherapeutic agents was summarized as well. Furthermore, the FREM2 level was measured in both human kidney tissues and associated cell lines, which suggested that lower FREM2 expression prompts a severer pathology and clinical ending. Our study showed that KIRC is associated with a unique BMG expression pattern. The risk scores related to the expression levels of 10 BMGs were assessed by survival status, TME, pathological features, and chemotherapeutic resistance. All results suggested that FREM2 could be a potential candidate for KIRC prognosis prediction. In this study, we established a valid model and presented new therapeutic targets for the KIRC prognosis prediction as well as the clinical treatment recommendation, and finally, facilitated precision tumor therapy for every single individual.

Organic cation transporter 3 mediates the non-norepinephrine transporter driven uptake of meta-[211At]astato-benzylguanidine

IntroductionMeta-[211At]astato-benzylguanidine ([211At]MABG) accumulates in pheochromocytoma via norepinephrine transporter (NET) and leads to a strong antitumor effect, but it also distributed in normal tissues non-specifically. Meta-[131I]iodo-benzylguanidine ([131I]MIBG), an iodine-labeled analog of [211At]MABG, is known to be transported by not only NET but also organic cation transporter (OCT). The involvement of OCT in [211At]MABG uptake is still largely unknown. We investigated the involvement of OCT in the non-NET-driven uptake of [211At]MABG both in vitro and in vivo. Methods[123I]MIBG and [211At]MABG uptake was investigated in PC-12 (rat pheochromocytoma cell line), NIH/3T3 (mouse fibroblasts cell line), ACHN (human renal cancer cell line), and BxPC-3 (human pancreatic cancer cell line). Herein, we used desipramine and dl-norepinephrine to inhibit NET, and we used steroids (hydrocortisone and prednisolone) to inhibit OCT3. The [211At]MABG uptake in OCT3-knockdown cells established with OCT3-selective siRNA was also investigated. We investigated the biodistribution of [211At]MABG in PC-12 tumor-bearing mice after a preloading of phosphate-buffered saline (PBS) or hydrocortisone solution. ResultsThe uptake of both [123I]MIBG and [211At]MABG was significantly inhibited by desipramine in PC-12 cells but not the other cell lines. The expression of OCT3 was relatively higher than those of the other OCT subtypes in ACHN and BxPC-3 cells. The expression of OCTs was not observed in NIH/3T3 cells. The uptake of both [123I]MIBG and [211At]MABG in ACHN and BxPC-3 cells was significantly inhibited by the steroid treatments. The [211At]MABG uptake was also reduced in OCT3-knockdown cells (p < 0.001). The radioactivity of [211At]MABG was significantly reduced in normal tissues by the preloading of hydrocortisone. In contrast, there was an increasing trend of [211At]MABG uptake in the PC-12 tumors. The tumor-to-normal tissue ratio was significantly increased by the preloading of hydrocortisone compared to that of PBS. ConclusionOur results suggest that OCT3 is involved in non-NET-driven [211At]MABG uptake. The preloading of hydrocortisone selectively reduced [211At]MABG accumulation in normal organs in vivo. OCT3 inhibition may therefore be beneficial for a reduction of the radiation risk in healthy organs in the treatment of malignant pheochromocytomas.

Abstract 95: In vitro, in vivo and molecular effects of triptolide and minnelide in renal cell carcinoma

Abstract Background: The worldwide incidence of Renal Cell Carcinoma (RCC) is increasing. Although new therapies have improved outcomes in advanced RCC, most patients eventually fail treatment and succumb to this devastating disease. Systemic therapy strategies have focused on TKI and immunotherapy, alone or in combination. Triptolide, a diterpenoid epoxide extracted from Tripterygium wilfordii Hook f (TWHf), has potent antitumor activity in multiple cancer models. Few studies have focused on the potential of triptolide as a novel therapeutic option in RCC. Objective: the objectives of this study are to characterize the in vitro and in vivo effects of Triptolide (T) and its water-soluble form, Minnelide (M), in models of renal cell carcinoma. Methods: The in vitro effects of triptolide (T) on human and murine RCC (786-0, A498, Caki-1, ACHN, and RENCA) cell proliferation was assessed using cell count and xCelligence assays at 24, 48, 72 and 96h. Molecular and mechanistic characterization of triptolide’s effects in 786-0 cells were analyzed by the (Reverse Phase Protein Array) and validated by western blot analysis. The in vivo effects (tumor progression and survival) of M were assessed in nude mice bearing 786-0 tumors (8x106 cells per mouse). M was administered to tumor-bearing mice at two different doses (0.21mg/kg and 0.42mg/kg daily), intraperitoneally, for 21 days. Correlative studies to explain the in vivo effect of M and the correlation with the molecular changes in vitro seen are being performed and will be shown in the poster. Results: T significantly inhibited, in a dose-response manner, cell proliferation in all human and murine renal cancer cell lines, with IC50 ranging between 12.5 and 25 nM). T was associated with significant negative modulation of proliferation, cell cycle, survival, increased apoptosis, and ER stress pathways in 786-0 cells, as demonstrated by RPPA analysis and validated by western Blot of selected pathway proteins, such as apoptosis (PARP cleavage and Caspase 3 activation), ER stress induction (pEIF2a, CHOP), and down-regulation of survival and proliferation pathways (pAKT). In vivo, M was associated with significant antitumor effects in 786-0 xenografts, with complete responses in the majority of mice while on treatment. These effects were associated with significant prolongation in overall survival in M treated vs. control mice. No significant toxicity or treatment-related deaths were observed. Conclusion: Our results have shown for the first time the potent in vitro and in vivo antitumor effects of T in RCC and the molecular changes associated with these effects. The profound antitumor effects in the aggressive 786-0 RCC xenograft model are highly encouraging and warrant further preclinical studies and potential clinical trials of M this devastating disease. Correlative tumor studies to understand the mechanisms of M in vivo antitumor effects are underway and will be presented at the meeting. Citation Format: Valery A. Chavez, Floritza Bustamante, Abner Murray, Ashok Saluja, Jaime Merchan. In vitro, in vivo and molecular effects of triptolide and minnelide in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 95.

Evaluation of the medicinal potential of a traditionally important plant from Turkey: Cerinthe minor L.

Background and Aims: Cerinthe minor L. has been used for both culinary and medicinal purposes in Turkey. Since the plant has remained uninvestigated pharmacognostically, this study aims to provide data on the phytochemical profile and in vitro biological activities of the ethanol extract prepared from the aerial and root parts of Cerinthe minor L. Methods: The phytochemical profile of the extract was determined by LC-MS/MS. Total phenolic and total flavonoid contents of Cerinthe minor ethanol extract were determined as pyrocatechol, and quercetin equivalents, respectively. Antioxidant activity of the extract was investigated by DPPH, and ABTS cation radical scavenging activity, and CUPRAC activity assays. Enzyme inhibitory activity assays were used to investigate anticholinesterase, antityrosinase, and antiurease activities. Cytotoxic potential of the plant was clarified by using the XTT method, and antimicrobial activity of the extract was established by the microbroth dilution technique. Results: According to the LC-MS/MS results, 11 different constituents were present in the extract, and the major compounds were, malic acid (5392.18±60.93 μg/g extract), fumaric acid (4730.99±58.66 μg/g extract), and rosmarinic acid (2470.07±176.12 μg/g extract). Total phenolic and flavonoid contents were found as 10.39±0.73 μg PEs/mg extract, and 0.75±0.07 μg QEs/mg extract, respectively. The extract demonstrated moderate antioxidant activity, whereas no enzyme inhibitory activity was exerted against the tested enzymes. No cytotoxic activity was observed on human renal (A498, UO-31) or human colon (COLO205, KM12) cancer cell lines. The extract was shown to possess low-moderate antimicrobial activity against C. tropicalis with a MIC value of 78.12 µg/mL. Conclusion: Having several pharmacologically valuable compounds, and not causing any toxicity on studied cell lines, Cerinthe minor is a plant that requires attention with its medicinal potential.

The post-transcriptional inhibition of CXCR4 expression by miR-139 regulates the proliferation of human kidney cancer cells.

The C-X-C chemokine receptor 4 (CXCR4) has been reported to be involved in several cancer related processes. The current study was designed to investigate the role of CXCR4 in human kidney cancer and to unveil the underlying molecular mechanisms. The results showed the expression of CXCR4 to be significantly (P<0.05) upregulated in human renal cancer tissues and cell lines. Silencing of CXCR4 lead to a significant (P<0.05) decline of cell proliferation and colony formation of the Caki-1 and A498 kidney cancer cells. Moreover, the migration and invasion of the Caki-1 and A498 cells was also significantly (P<0.05) inhibited upon CXCR4 silencing. TargetScan analysis and dual luciferase assay revealed that CXCR4 interacts with microRNA-139 (miR-139). The expression of miR-139 was found to be significantly (P<0.05) downregulated in human kidney cancer cells lines. Overexpression of miR-139 caused post-transcriptional suppression of CXCR4 expression and significant (P<0.05) inhibition of the Caki-1 and A498 cell proliferation. Nonetheless, CXCR4 overexpression could nullify the inhibitory effects of miR-139 on the proliferation of Caki-1 and A498 cells. Taken together, the results revealed that CXCR/miR-139 axis regulates the proliferation, migration, and invasion of human kidney cancer cells and may act as a therapeutic target.

The PGRMC1 Antagonist AG-205 Inhibits Synthesis of Galactosylceramide and Sulfatide.

Sulfatide synthesis in the human renal cancer cell line SMKT-R3 was strongly inhibited in the presence of low µM concentrations of AG-205, a progesterone receptor membrane component 1 (PGRMC1) antagonist. This was also the case in Chinese hamster ovary (CHO) cells stably transfected with UDP-galactose: ceramide galactosyltransferase and cerebroside sulfotransferase, the two enzymes required for sulfatide synthesis. In CHO cells synthesizing galactosylceramide but not sulfatide, galactosylceramide was also strongly reduced, suggesting an effect at the level of galactolipid synthesis. Notably, AG-205 inhibited galactosylceramide synthesis to a similar extent in wild type CHO cells and cells that lack PGRMC1 and/or PGRMC2. In vitro enzyme activity assays showed that AG-205 is an inhibitor of UDP-galactose: ceramide galactosyltransferase, but not cerebroside sulfotransferase. This study shows that PGRMC1 is only one of several targets of AG-205 and should be used with caution, especially in studies using cells synthesizing galactosylceramide and sulfatide.

Broussochalcone A Induces Apoptosis in Human Renal Cancer Cells via ROS Level Elevation and Activation of FOXO3 Signaling Pathway.

Broussochalcone A (BCA) is a chalcone compound extracted from the cortex of Broussonetiapapyrifera (L.) Ventenat that exerts various effects, such as potent antioxidant, antiplatelet, and anticancer effects. However, the effects of BCA against cancers have been seldom studied. This study is aimed at demonstrating the apoptotic mechanisms of BCA in A498 and ACHN cells, which are two types of human renal cancer cell lines. MTT, cell counting, and colony formation assays indicated that BCA treatment inhibited cell viability and cell growth. Further, cell cycle analysis revealed that BCA induced cell cycle arrest at the G2/M phase. Annexin V/PI staining and TUNEL assays were performed to determine the apoptotic effects and DNA fragmentation after treatment with BCA. Based on western blot analysis, BCA induced the upregulation of cleaved PARP, FOXO3, Bax, p21, p27, p53, phosphorylated p53 (ser15, ser20, and ser46), and active forms of caspase-3, caspase-7, and caspase-9 proteins, but downregulated the proforms of the proteins. The expression levels of pAkt, Bcl-2, and Bcl-xL were also found to be downregulated. Western blot analysis of nuclear fractionation results revealed that BCA induced the nuclear translocation of FOXO3, which might be induced by DNA damage owing to the accumulation of reactive oxygen species (ROS). Elevated intracellular ROS levels were also found following BCA treatment. Furthermore, DNA damage was detected after BCA treatment using a comet assay. The purpose of this study was to elucidate the apoptotic effects of BCA against renal cancer A498 and ACHN cells. Collectively, our study findings revealed that the apoptotic effects of BCA against human renal cancer cells occur via the elevation of ROS level and activation of the FOXO3 signaling pathway.

Melatonin combined with sorafenib synergistically inhibit the invasive ability through targeting metastasis-associated protein 2 expression in human renal cancer cells.

Objectives:Renal cell carcinoma (RCC) was the most common and lethal urological malignancy with the dismal outcome when distant metastasis. Melatonin was known as a potential oncostatic agent against several types of malignancy and sorafenib had been considered as an agent to treat RCC, but the synergistic effects of melatonin and sorafenib on human RCC have not been elucidated.Materials and Methods:Human renal cancer cell lines (Caki-1 and ACHN) were treated with melatonin combined with sorafenib were detected the cell growth and cell cycle by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and flow cytometry. The ability of cell migration/invasion was performed with in vitro migration and invasion assay. The proteins and mRNA expression of metastasis-associated protein 2 (MTA2) from the RCC cells were measured by quantitative reverse transcription-polymerase chain reaction and western blotting. Clinical significance of MTA2 in RCC tissues was analyzed from The Cancer Genome Atlas database by using TISIDB software.Results:Our results showed that melatonin combined with sorafenib, sorafenib or melatonin-treated alone did not induce the cytotoxic effects or cell cycle arrest in human RCC cells and HK2 cells. Additionally, cotreatment with melatonin and sorafenib synergistically reduced migration and invasion in human Caki-1 and ACHN cells through synergistically suppression of MTA2 expression. Bioinformatics analysis showed that MTA2 expression significantly correlated with overall survival (P < 0.002), tumor grade (P < 0.001) and tumor stage (P < 0.001) in human RCC.Conclusion:Our results demonstrated that concomitantly used melatonin and sorafenib could significantly reduce the abilities of migration and invasion of RCC cells through inhibiting MTA2. We considered that this novel promising combination strategy towards the treatment of RCC, but further studies are warranted.

Ferroptosis is involved in the anti-tumor effect of lycorine in renal cell carcinoma cells.

Renal cell carcinoma (RCC) is a most common malignant tumor in the genitourinary system. Studies have shown that Lycorine has promising anticancer activities with minor side effects. However, the effect of lycorine on the proliferation of RCC cells and its underlying anti-tumor mechanism have not yet been fully elucidated. The human renal cancer cell lines 786-O, A498 and Caki-1 were cultured and treated with different concentrations of lycorine or ferrostatin-1, a ferroptosis inhibitor. Cell viability and colony formation assays were used to measure cell proliferation. The 5-, 12- and 15-HETE hydroxyeicosatetraenoic acid (HETE) and MDA levels, as well as the reduced to oxidized glutathione (GHS/GSSG) ratio, were analyzed. Western blot analysis was used to detect the expression of glutathione peroxidase 4 (GPX4) and acyl-CoA synthetase long chain family member 4 (ACSL4), which are key markers of ferroptosis. Transmission electron microscopy was used to observe the morphological features associated with ferroptosis. Lycorine was found to inhibit the proliferation of RCC cells. After lycorine treatment, the expression levels of GPX4 in RCC cells decreased, whereas those of ACSL4 increased. Lycorine induced the expression of 5-HETE, 12-HETE, 15-HETE and MDA in RCC cells, and reduced the GSH/GSSG ratio. In addition, ferrostatin-1 could prevent lycorine-induced ferroptosis in RCC cells.

Kaiso Regulates DNA Methylation Homeostasis.

Gain and loss of DNA methylation in cells is a dynamic process that tends to achieve an equilibrium. Many factors are involved in maintaining the balance between DNA methylation and demethylation. Previously, it was shown that methyl-DNA protein Kaiso may attract NCoR, SMRT repressive complexes affecting histone modifications. On the other hand, the deficiency of Kaiso resulted in reduced methylation of ICR in H19/Igf2 locus and Oct4 promoter in mouse embryonic fibroblasts. However, nothing is known about how Kaiso influences DNA methylation at the genome level. Here we show that deficiency of Kaiso led to whole-genome hypermethylation, using Kaiso deficient human renal cancer cell line obtained via CRISPR/CAS9 genome editing. However, Kaiso serves to protect genic regions, enhancers, and regions with a low level of histone modifications from demethylation. We detected hypomethylation of binding sites for Oct4 and Nanog in Kaiso deficient cells. Kaiso immunoprecipitated with de novo DNA methyltransferases DNMT3a/3b, but not with maintenance methyltransferase DNMT1. Thus, Kaiso may attract methyltransferases to surrounding regions and modulate genome methylation in renal cancer cells apart from being methyl DNA binding protein.

The Emerging Role of Poly (ADP-Ribose) Polymerase Inhibitors as Effective Therapeutic Agents in Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is the sixth most common cancer in the US. However, no significant changes in management have occurred since the tyrosine kinase era until the recent breakthrough with checkpoint inhibitors. Therefore, the need for more therapeutic options is paramount. Our objective was to determine whether PARP inhibition represents a novel therapeutic option for RCC. We used publicly available COSMIC, GDC Data Portal, and cBioPortal databases to explore mutations in DNA repair genes in RCC tissues from the TCGA cohort. We treated a human normal renal epithelial cell line RPTEC/TERT1 and two human renal cancer cell lines ACHN and CAKI-2 with PARPi niraparib, olaparib, rucaparib, veliparib, and talazoparib. Cell survival, cell proliferation, clonogenic ability, and apoptosis were assessed. RCC xenografts in SCID mice were treated with PARPi to evaluate their efficacy in vivo. Data mining revealed that ~27-32% of RCC tissues contain mutations in homologous recombination genes. Niraparib and talazoparib were the most effective at reducing cell survival, proliferation, and clonogenic ability in vitro. Niraparib, talazoparib, and rucaparib were the most effective in reducing RCC xenograft growth in vivo. Agents such as PARPi that exploit mutations in DNA damage repair genes may be effective therapeutic options for RCC.

A novel Apigenin derivative suppresses renal cell carcinoma via directly inhibiting wild-type and mutant MET

MET, the receptor of hepatocyte growth factor (HGF), is a driving factor in renal cell carcinoma (RCC) and also a proven drug target for cancer treatment. To improve the activity and to investigate the mechanisms of action of Apigenin (APG), novel derivatives of APG with improved properties were synthesized and their activities against Caki-1 human renal cancer cell line were evaluated. It was found that compound 15e exhibited excellent potency against the growth of multiple RCC cell lines including Caki-1, Caki-2 and ACHN and is superior to APG and Crizotinib. Subsequent investigations demonstrated that compound 15e can inhibit Caki-1 cell proliferation, migration and invasion. Mechanistically, 15e directly targeted the MET kinase domain, decreased its auto-phosphorylation at Y1234/Y1235 and inhibited its kinase activity and downstream signaling. Importantly, 15e had inhibitory activity against mutant MET V1238I and Y1248H which were resistant to approved MET inhibitors Cabozantinib, Crizotinib or Capmatinib. In vivo tumor graft study confirmed that 15e repressed RCC growth through inhibition of MET activation. These results indicate that compound 15e has the potential to be developed as a treatment for RCC, and especially against drug-resistant MET mutations.

Diverse Roles of G‐Protein‐Coupled Receptor 40 (GPR40/FFA1) and GPR120 (FFA4) in the Regulation of Cell Growth and Motile Activities in Renal Cell Carcinoma (RCC)

Renal cell carcinoma (RCC) is the 16th most common cancer worldwide with roughly 400,000 new cases diagnosed annually and approximately half of those leading to mortality. Even though the 5-year survival rate for early-stage RCC is 80%-90%, it is only 5% for the patients with metastasis. Unfortunately, anticancer drugs that are used to treat RCC are proven to be useful for only a small number of patients. Therefore, it is crucial to discover the novel molecular pathways underlying RCC to develop better therapies. The G protein-coupled receptors GPR40 and GPR120, also referred to as free fatty acid receptor 1 (FFA1) and 4 (FFA4), respectively, are activated by medium- to long-chain free fatty acids. Emerging evidence suggests that GPR40 and GPR120 are implicated in the proliferation, invasiveness, and apoptosis of various tumor cells, however, the precise mechanisms of GPCRs signaling events leading to the modifications in cancer cell growth and migration are still scarce, and the roles of these receptors in RCC are unstudied to date. Here, for the first time, we reveal the effects of GPR40 and GPR120 agonism on cell proliferation and migration in metastatic tumor-derived renal cancer cells. The present study used in vitro techniques including cell proliferation assay, wound-healing assay, and transwell migration assay to investigate the functions of GPR40 and GPR120 on the proliferative and migration capacities of the RCC cell line. The human renal cancer cell line ACHN was obtained from the American Type Culture Collection (ATCC). Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis was performed to confirm the presence of GPR40 and GPR120 transcripts. Cell proliferation was assessed by plating 50,000 cells per well, followed by treatment with GPR40 and GPR120 agonists and antagonists as noted for 6 days. Cells were counted every 24 hours. For the wound-healing assay, 100,000 cells were plated on a 48-well plate and a scratch-made through the center. The time to wound healing was assessed at 0, 24, 36, and 48 hours. For migration assays, cells were plated in the top chamber of a transwell plate and cells migrated was counted by crystal violet staining after 16 hr. Using selective GPR40 and GPR120 agonists and antagonists, our results demonstrate that GPR40 enhanced and GPR120 suppressed cell proliferation, suggesting that the opposite effects of GPR120 and GPR40 are involved in the cell growth activity. The wound-healing assay revealed that both GPR40 and GPR120 agonism markedly covered the wounded area compared to vehicle-controls, an effect that is blocked by a selective GPR40 and GPR120 antagonist, respectively. In transwell migration assay, GPR40 negatively and GPR120 positively affect the ability of single cells to respond to the chemoattractant; further confirming their involvement in the acquisition of migratory properties in RCC cell. Our present results suggest that GPR40/120-mediated signaling may be involved in the regulation of renal cancer tumorigenesis and progression on the cellular level, and mechanistic studies are underway to assess the cellular cascades that are influenced by GPR40 and GPR120.

Transferrin-based radiolabeled probe predicts the sensitivity of human renal cancer cell lines to ferroptosis inducer erastin

Ferroptosis induction has been recognized as a novel cancer therapeutic strategy. To effectively apply ferroptosis-targeting cancer therapy to individual patients, a diagnostic indicator for selecting this therapeutic strategy from a number of molecular targeting drugs is needed. However, to date, methods that can predict the efficacy of ferroptosis-targeting treatment have not been established yet. In this study, we focused on the iron metabolic pathway to develop a nuclear imaging technique for diagnosing the susceptibility of cancer cells to ferroptosis. As a nuclear probe, human transferrin (Tf) was labeled with Gallium-68 (68Ga) using 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) as a chelator (68Ga-NOTA-Tf). Western blot assay and clonogenic survival assay with human renal cancer cell lines A498 and 786-O revealed that the protein expression level of transferrin receptor1 (TfR1) and sensitivity to a ferroptosis inducer, erastin, were correlated. A cellular uptake assay with 68Ga-NOTA-Tf revealed that the cancer cells sensitive to erastin highly internalized the 68Ga-NOTA-Tf. Furthermore, treatment with the TfR1 inhibitor ferristatin II reduced the cellular uptake of 68Ga-NOTA-Tf, indicating that the intracellular uptake of the probe was mediated by TfR1. These results suggest that 68Ga-NOTA-Tf can be useful in predicting the sensitivity of cancer cells to ferroptosis inducers.