Human ccRCC Cell Research Articles

Abstract A24: PBRM1 alteration in clear cell renal cell carcinoma increases tumorigenicity through ALDH1A1 upregulation

Abstract In this study, we investigated the mechanisms by which PBRM1 functions as a tumor suppressor in clear cell renal cell carcinoma (ccRCC). PBRM1, also known as BAF180 or Polybromo, is a member of the PBAF SWI/SNF chromatin remodeling complex. Cancer sequencing studies have revealed that SWI/SNF components are widely mutated in cancer. PBRM1 mutations in particular are found in ~40% of ccRCC tumors, making it the second most highly mutated gene in ccRCC (behind VHL). Although many recent studies have looked at how other SWI/SNF components function in cancer control, relatively little is known about the tumor suppressive mechanism of PBRM1 in ccRCC. To investigate genes under PBRM1 control, we first generated mouse embryonic fibroblasts in which Pbrm1 was floxed and could be conditionally deleted. Using human ccRCC cell lines, we also stably knocked-down PBRM1 in the wild-type setting, or stably re-expressed it in the mutant setting. We then used gene expression profiling on these lines to look for genes that were consistently altered with PBRM1 loss. Ten genes were significantly altered across all lines, although only six (IGFBP3, ALDH1A1, EFNA3, WBP5, ESCO2, and PBRM1) changed reliably in the same direction. Further expression analysis using two different human primary tumor datasets revealed that only one of these genes was consistently altered in PBRM1-mutant tumors: ALDH1A1. ALDH1A1, or aldehyde dehydrogenase 1, is involved in the retinoic acid metabolic pathway and irreversibly converts retinaldehyde to retinoic acid. In addition to known roles in hematopoietic stem cell development, white vs. brown fat programming, and insulin signaling, ALDH1A1 is marker of tumor-initiating cells in various cancers. We confirmed that stable knockdown of PBRM1 in ccRCC cell lines resulted in higher expression and activity of ALDH1A1. Alternatively, re-expression of wild-type, but not cancer-associated mutant, PBRM1 lowered ALDH1A1 expression and activity in a PBRM1-mutant line. As ALDH1A1 is a tumor-initiating cell marker, we next investigated PBRM1's effects on tumorigenic potential. In xenograft studies, PBRM1 knockdown cells formed significantly larger tumors. We then used the tumorsphere formation assay as a surrogate measure of tumor-initiating capacity. We confirmed that PBRM1 knockdown cells can form more free-floating tumorspheres. These cells also formed more colonies in soft-agar. This increased tumorigenic potential was at least partly dependent upon ALDH1A1, as revealed by chemical inhibition or siRNA knockdown of ALDH1A1. Additionally, we found that another PBAF-specific component, ARID2, is required for the pro-tumorigenic changes observed as a result of PBRM1 depletion. Knockdown of PBRM1 resulted in increased levels of ARID2, while re-expression of wild-type, but not mutant, PBRM1 lowered ARID2 levels. The primary tumor datasets confirmed increased ARID2 expression in PBRM1-mutant tumors. Knockdown of ARID2 in the context of PBRM1 loss reversed the increase in ALDH1A1 levels and ability to form tumorspheres described above. Interestingly, other SWI/SNF components, including SNF5, BRG1, and BRM, were dispensable for these changes. These findings suggest that one key tumor suppressor function of PBRM1 in ccRCC is to repress ALDH1A1 levels, and that mutation of PBRM1 increases a cell's tumorigenic potential through upregulated ALDH1A1. Citation Format: David Schoenfeld, William Su, Sakellarios Zairis, Deepti Mathur, Raul Rabadan, Ramon Parsons. PBRM1 alteration in clear cell renal cell carcinoma increases tumorigenicity through ALDH1A1 upregulation. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A24.

Abstract B189: Synergistic effect of c-Met inhibitor savolitinib in combination with a VEGFR inhibitor fruquintinib in clear cell renal cell carcinoma xenograft models

Abstract Renal cell carcinoma (RCC) is the most common type of kidney tumor in human. Approximately 80∼85% of RCC is clear cell renal cell carcinoma (ccRCC).[1] Although VEGF/VEGFR targeted therapies bring significant advances in the treatment of RCC, ultimate resistance occurs in most cases following a transient period of clinical benefit. [2] The hepatocyte growth factor (HGF) receptor c-Met activation emerges as one of the mechanisms for resistance to anti-VEGF/VEGFR therapies in ccRCC,[3] implying that a combinational inhibition of c-Met and VEGFR pathways may induce a synergistic anti-tumor effect and could produce additional clinical benefit. The aim of this study was to assess the effect of a combination strategy targeting the VEGFR and c-Met pathways in ccRCC xenograft models. Savolitinib (AZD6094, HMPL-504) is a highly selective inhibitor against c-Met. Fruquintinib (HMPL-013) strongly inhibits VEGFR1, 2 and 3. Both of them were discovered by Hutchison MediPharma and are currently being evaluated in clinical trials for the treatment of various cancers. Several subcutaneous xenograft models were established in nude mice with human ccRCC cell lines or patient derived tumors (PDX) to investigate the anti-tumor effect of combination of savolitinib with fruquntinib. Treatment with savolitinib or fruquintinib at clinically relevant dose only exhibited mild to moderate tumor growth inhibition as a single agent in all of tested models, but significantly increased anti-tumor effect was observed in all of tested models for the combination group. It seemed that the enhanced anti-tumor effect was associated with c-Met inhibition. In a ccRCC PDX model KIN1T1342, the increased anti-tumor effect was correlated with dose increment of savolitinib. Immunohistochemistry (IHC) analysis revealed that combination treatment produced stronger inhibition on tumor proliferation marker Ki67 and angiogenesis marker CD31, compared to either savolitinib or fruquntinib alone, indicating that the observed synergistic effect might be attributed to the dual inhibition on tumor signaling and tumor microenvironment. C-Met expression was observed in all tested models, and treatment with savolitinib effectively suppressed phospho-Met. To evaluate c-Met expression in Chinese ccRCC patients, Formalin-Fixed and Parrffin-Embedded (FFPE) tumor sections were collected from sixty-two treatment-naive patients during surgical resection. Positive c-Met expression was found in 69% (43/62) of ccRCC samples under IHC staining.Overall our data demonstrated that c-Met was widely expressed in Chinese ccRCC patients and provided a rationale to test the combined HGF/c-Met and VEGF/VEGFR pathway blockade in the treatment of ccRCC in the clinical trials.

Primary clear cell renal carcinoma cells display minimal mitochondrial respiratory capacity resulting in pronounced sensitivity to glycolytic inhibition by 3-Bromopyruvate.

Changes of cellular metabolism are an integral property of the malignant potential of most cancer cells. Already in the 1930s, Otto Warburg observed that tumor cells preferably utilize glycolysis and lactate fermentation for energy production, rather than the mitochondrial oxidative phosphorylation dominating in normal cells, a phenomenon today known as the Warburg effect. Even though many tumor types display a high degree of aerobic glycolysis, they still retain the activity of other energy-producing metabolic pathways. One exception seems to be the clear cell variant of renal cell carcinoma, ccRCC, where the activity of most other pathways than that of glycolysis has been shown to be reduced. This makes ccRCC a promising candidate for the use of glycolytic inhibitors in treatment of the disease. However, few studies have so far addressed this issue. In this report, we show a strikingly reduced mitochondrial respiratory capacity of primary human ccRCC cells, resulting in enhanced sensitivity to glycolytic inhibition by 3-Bromopyruvate (3BrPA). This effect was largely absent in established ccRCC cell lines, a finding that highlights the importance of using biologically relevant models in the search for new candidate cancer therapies. 3BrPA markedly reduced ATP production in primary ccRCC cells, followed by cell death. Our data suggest that glycolytic inhibitors such as 3BrPA, that has been shown to be well tolerated in vivo, should be further analyzed for the possible development of selective treatment strategies for patients with ccRCC.

The role of HIF1α in renal cell carcinoma tumorigenesis.

The transcription factor HIF1α is implicated in the development of clear cell renal cell carcinoma (ccRCC). Although HIF1α was initially believed to be essential for ccRCC development, recent studies hypothesize an oncogenic role for HIF2α in ccRCC, but a tumor suppressor role for HIF1α, leading to uncertainty as to the precise roles of the different HIF transcription factors in this disease. Using evidence available from studies with human ccRCC cell lines, mouse xenografts, murine models of ccRCC, and human ccRCC specimens, we evaluate the roles of HIF1α and HIF2α in the pathogenesis of ccRCC. We present a convergence of clinical and mechanistic data supporting an important role for HIF1α in promoting tumorigenesis in a clinically important and large subset of ccRCC. This indicates that current understanding of the exact roles of HIF1α and HIF2α is incomplete and that further research is required to determine the diverse roles of HIF1α and HIF2α in ccRCC. The TRACK mouse ccRCC model with constitutively active HIF1α but not HIF2α expressed in proximal tubules develops RCC. HIF1α protein is expressed in the majority of human ccRCC specimens. Elevated HIF1α in ccRCC correlates with a worse prognosis. Many publications do not support a tumor suppressor role for HIF1α in ccRCC. HIF1α, but not HIF2α, is expressed in some types of cancer stem cells.

Pairwise comparison of 89Zr- and 124I-labeled cG250 based on positron emission tomography imaging and nonlinear immunokinetic modeling: in vivo carbonic anhydrase IX receptor binding and internalization in mouse xenografts of clear-cell renal cell carcinoma

The PET tracer, (124)I-cG250, directed against carbonic anhydrase IX (CAIX) shows promise for presurgical diagnosis of clear-cell renal cell carcinoma (ccRCC) (Divgi et al. in Lancet Oncol 8:304-310, 2007; Divgi et al. in J Clin Oncol 31:187-194, 2013). The radiometal (89)Zr, however, may offer advantages as a surrogate PET nuclide over (124)I in terms of greater tumor uptake and retention (Rice et al. in Semin Nucl Med 41:265-282, 2011). We have developed a nonlinear immunokinetic model to facilitate a quantitative comparison of absolute uptake and antibody turnover between (124)I-cG250 and (89)Zr-cG250 using a human ccRCC xenograft tumor model in mice. We believe that this unique model better relates quantitative imaging data to the salient biological features of tumor antibody-antigen binding and turnover. We conducted experiments with (89)Zr-cG250 and (124)I-cG250 using a human ccRCC cell line (SK-RC-38) to characterize the binding affinity and internalization kinetics of the two tracers in vitro. Serial PET imaging was performed in mice bearing subcutaneous ccRCC tumors to simultaneously detect and quantify time-dependent tumor uptake in vivo. Using the known specific activities of the two tracers, the equilibrium rates of antibody internalization and turnover in the tumors were derived from the PET images using nonlinear compartmental modeling. The two tracers demonstrated virtually identical tumor cell binding and internalization but showed markedly different retentions in vitro. Superior PET images were obtained using (89)Zr-cG250, owing to the more prolonged trapping of the radiolabel in the tumor and simultaneous washout from normal tissues. Estimates of cG250/CAIX complex turnover were 1.35 - 5.51 × 10(12) molecules per hour per gram of tumor (20 % of receptors internalized per hour), and the ratio of (124)I/(89)Zr atoms released per unit time by tumor was 17.5. Pairwise evaluation of (89)Zr-cG250 and (124)I-cG250 provided the basis for a nonlinear immunokinetic model which yielded quantitative information about the binding and internalization of radioantibody bound to CAIX on tumor cells in vivo. (89)Zr-cG250 is likely to provide high-quality PET images and may be a useful tool to quantify CAIX/cG250 receptor turnover and cG250-accessible antigen density noninvasively in humans.

The von Hippel–Lindau tumor suppressor regulates programmed cell death 5-mediated degradation of Mdm2

Functional loss of the von Hippel-Lindau (VHL) tumor suppressor protein (pVHL), which is part of an E3-ubiquitin ligase complex, initiates most inherited and sporadic clear-cell renal cell carcinomas (ccRCC). Genetic inactivation of the TP53 gene in ccRCC is rare, suggesting that an alternate mechanism alleviates the selective pressure for TP53 mutations in ccRCC. Here we use a zebrafish model to describe the functional consequences of pVHL loss on the p53/Mdm2 pathway. We show that p53 is stabilized in the absence of pVHL and becomes hyperstabilized upon DNA damage, which we propose is because of a novel in vivo interaction revealed between human pVHL and a negative regulator of Mdm2, the programmed cell death 5 (PDCD5) protein. PDCD5 is normally localized at the plasma membrane and in the cytoplasm. However, upon hypoxia or loss of pVHL, PDCD5 relocalizes to the nucleus, an event that is coupled to the degradation of Mdm2. Despite the subsequent hyperstabilization and normal transcriptional activity of p53, we find that zebrafish vhl(-/-) cells are still as highly resistant to DNA damage-induced cell cycle arrest and apoptosis as human ccRCC cells. We suggest this is because of a marked increase in expression of birc5a, the zebrafish homolog of Survivin. Accordingly, when we knock down Survivin in human ccRCC cells we are able to restore caspase activity in response to DNA damage. Taken together, our study describes a new mechanism for p53 stabilization through PDCD5 upon hypoxia or pVHL loss, and reveals new clinical potential for the treatment of pathobiological disorders linked to hypoxic stress.

Prognostic implication of TIM-3 in clear cell renal cell carcinoma

Clear cell renal carcinoma (ccRCC) is the most common tumor of the kidney.in adults. The prognosis of ccRCC remains unsatisfactory. Recently, T cell immunoglobulin mucin-3 (TIM-3), a novel transmembrane protein, has been implicated in tumor biology. Its role in ccRCC remains unknown. The aim of this study was to investigate the roles of TIM-3 as a prognostic marker in patients with ccRCC.TIM-3 protein expression was determined by immunohistochemistry and Western blot from 137 ccRCC tumor samples and adjacent normal renal tissues. We performed also the cell proliferation assay using 3-(4,5-dimethylthiazol -2yl)-2,5-diphenyl-2H tetrazolium bromide (MTT) and cell invasion assay. The effects of small interfering RNA (siRNA)-mediated knockdown of TIM-3 (TIM-3 siRNA) in two human ccRCC cell lines were evaluated. TIM-3 expression was higher in ccRCC tissue than in the adjacent normal renal tissue (P<0.001). High TIM-3 expression was an independent predictor of both cancer-specific survival and progression-free survival. TIM-3 protein was expressed in both ccRCC cell lines. Knockdown of TIM-3 suppressed the proliferation and invasion capacity of ccRCC cell lines.TIM-3 expression was associated with poor prognosis in ccRCC. Taken together, TIM-3 is a potential prognostic marker in ccRCC.

Tpl2 Kinase Impacts Tumor Growth and Metastasis of Clear Cell Renal Cell Carcinoma

Due to the innate high metastatic ability of renal cell carcinoma (RCC), many patients with RCC experience local or systemic relapses after surgical resection. A deeper understanding of the molecular pathogenesis underlying advanced RCC is essential for novel innovative therapeutics. Tumor progression locus 2 (Tpl2), upregulated in various tumor types, has been reported to be associated with oncogenesis and metastatic progression via activation of the MAPK signaling pathway. Herein, the relevance of Tpl2 in tumor growth and metastasis of RCC is explored. Inspection of The Cancer Genome Atlas (TCGA) indicated that Tpl2 overexpression was significantly related to the presence of metastases and poor outcome in clear cell RCC (ccRCC), which is the most aggressive subtype of RCC. Moreover, expression of Tpl2 and CXCR4 showed a positive correlation in ccRCC patients. Depletion of Tpl2 by RNAi or activity by a Tpl2 kinase inhibitor in human ccRCC cells remarkably suppressed MAPK pathways and impaired in vitro cell proliferation, clonogenicity, anoikis resistance, migration, and invasion capabilities. Similarly, orthotopic xenograft growth and lung metastasis were significantly inhibited by Tpl2 silencing. Furthermore, Tpl2 knockdown reduced CXCL12-directed chemotaxis and chemoinvasion accompanied with impaired downstream signaling, indicating potential involvement of Tpl2 in CXCR4-mediated metastasis. Taken together, these data indicate that Tpl2 kinase is associated with and contributes to disease progression of ccRCC. Tpl2 kinase activity has prognostic and therapeutic targeting potential in aggressive clear cell renal cell carcinoma.

Attenuation of Krüppel-Like Factor 4 Facilitates Carcinogenesis by Inducing G1/S Phase Arrest in Clear Cell Renal Cell Carcinoma

Krüppel-like factor 4 (KLF4) is a transcription factor with diverse functions in various cancer types; however, the function of KLF4 in clear cell renal cell carcinoma (ccRCC) carcinogenesis remains unknown. In this study, we initially examined KLF4 expression by using a cohort of surgically removed ccRCC specimens and cell lines. Results indicated that the transcription and translation of KLF4 were lower in ccRCC tissues than in patient-matched normal tissues. Furthermore, the KLF4 expression was significantly downregulated in the five ccRCC cell lines at protein and mRNA levels compared with that in normal renal proximal tubular epithelial cell lines (HKC). KLF4 downregulation was significantly correlated with tumor stage and tumor diameter. Promoter hypermethylation may contribute to its low expression. In addition, in vitro studies indicated that the KLF4 overexpression significantly inhibited proliferation in human ccRCC cell lines 786-O and ACHN. Moreover, the KLF4 overexpression arrested the cell cycle progress at the G1/S phase transition by upregulating p21WAF1/CIP1 expression and downregulating cyclin D1 expression, KLF4 knockdown in HKC cells did the opposite. In vivo studies confirmed the anti-proliferative effect of KLF4. Our results suggested that KLF4 had an important function in suppressing the growth of ccRCC.

Vasoactive intestinal peptide induces oxidative stress and suppresses metastatic potential in human clear cell renal cell carcinoma

Molecular mechanisms involved in progression of clear-cell renal-cell carcinomas (ccRCCs) are poorly understood. A common genetic mutation found in ccRCC is the loss of the von Hippel–Lindau (VHL) gene, which contributes to cancer progression and metastasis. We investigated VIP effects on metastatic and angiogenic factors in human VHL-null A498 ccRCC and HK2 renal cells. VIP increased adhesion but decreased expression of metalloproteinases, MMP2 and MMP9, as well as cell migration and VEGF expression and secretion in A498 but not in HK2 cells. VIP enhanced ROS levels and decreased nuclear levels of β-catenin and NFκB p50-subunit in A498 cells, suggesting neuropeptide involvement in the observed decrease of metastatic ability in clear-cell carcinoma. VIP effects in A498 cells were blocked by the VPAC1/2-receptor antagonist JV-1-53. In conclusion, present data point to a role of VIP in preventing invasion and metastasis in ccRCCs and support its potential therapeutic usefulness in this disease.

Preliminary analysis of differentially expressed proteins of clear-cell renal cell carcinoma by comparative proteomic technologies

To explore the different expression of proteins between human clear-cell renal cell carcinoma (ccRCC) cell line RLC-310 and normal renal proximal tubule epithelial cell line HK-2, and to search new differentially expressed proteins of RCC. RLC-310 and HK-2 cells were cultured in vitro. The total proteins were separated by ProteomeLab PF 2D protein fractionation system and the differential expression protein fractions of the two cell lines were analyzed and identified by capillary liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS). RT-PCR and Western blot were used to confirm the representative differential expression at mRNA and protein levels. One hundred and ninty-six differentially expressed proteins were identified. These differentially expressed proteins involved in many aspects, including cell proliferation and anti-apoptosis, energy metabolism, mitochondria reduction and oxidation, oxidative stress and resistance, cell signaling, invasion and adhesion, cytoskeleton and motion, neovascularization, etc. Except for previously reported RCC associated proteins: annexin A2, fatty acid-binding protein, vimentin, fibronectin, and so on, Septin-9 was firstly found highly expressed in RLC-310 cells when compared with that in the HK-2 cells. Moreover, the overexpression of Septin-9 was confirmed by RT-PCR and Western blot analysis at both mRNA and protein levels (P < 0.05). The human ccRCC cell line RLC-310 cells display differential protein profiles compared with those of the normal renal cell line HK-2 cells. The identified differential expression proteins are involved in many aspects of RCC development. It is worth further study and elucidate the molecular mechanisms of RCC. The representative differential protein Septin-9 deserves further study its role in the angiogenesis of ccRCC.