Cell Renal Cell Cancer Research Articles

RAPTOR up-regulation contributes to resistance of renal cancer cells to PI3K-mTOR inhibition.

The outlook for patients with advanced renal cell cancer (RCC) has been improved by targeted agents including inhibitors of the PI3 kinase (PI3K)-AKT-mTOR axis, although treatment resistance is a major problem. Here, we aimed to understand how RCC cells acquire resistance to PI3K-mTOR inhibition. We used the RCC4 cell line to generate a model of in vitro resistance by continuous culture in PI3K-mTOR kinase inhibitor NVP-BEZ235 (BEZ235, Dactolisib). Resistant cells were cross-resistant to mTOR inhibitor AZD2014. Sensitivity was regained after 4 months drug withdrawal, and resistance was partially suppressed by HDAC inhibition, supporting an epigenetic mechanism. BEZ235-resistant cells up-regulated and/or activated numerous proteins including MET, ABL, Notch, IGF-1R, INSR and MEK/ERK. However, resistance was not reversed by inhibiting or depleting these pathways, suggesting that many induced changes were passengers not drivers of resistance. BEZ235 blocked phosphorylation of mTOR targets S6 and 4E-BP1 in parental cells, but 4E-BP1 remained phosphorylated in resistant cells, suggesting BEZ235-refractory mTORC1 activity. Consistent with this, resistant cells over-expressed mTORC1 component RAPTOR at the mRNA and protein level. Furthermore, BEZ235 resistance was suppressed by RAPTOR depletion, or allosteric mTORC1 inhibitor rapamycin. These data reveal that RAPTOR up-regulation contributes to PI3K-mTOR inhibitor resistance, and suggest that RAPTOR expression should be included in the pharmacodynamic assessment of mTOR kinase inhibitor trials.

Abstract 3520: PBRM1 inactivation in renal cell cancer alters master transcription factor hub composition to repress instead of activate epithelial-differentiation

Abstract Clear cell renal cell cancers (RCC) universally inactivate PBRM1 (single allele deletion 100%, bi-allelic deletion/mutation ~40%), however, the mechanisms by which PBRM1 loss contributes to RCC-genesis are unknown. Of ~4000 genes consistently less expressed in RCC than normal renal cortex, >30% were kidney differentiation genes by gene ontology analysis, e.g., 394 (~10%) suppressed genes had specialized renal epithelial functions of cation transport, cell adhesion and excretion (TCGA). Suppression of hundreds of epithelial-differentiation genes suggested disruption to the core transcription factor circuit (protein network hub) activating this program. This master transcription factor hub contains PAX2, PAX8, GATA3 and LHX1, shown by murine knock-outs and human pedigrees with renal defects, and confirmed by our protein interactome studies. Of these master transcription factors, PAX2 and PAX8 expression was preserved in RCC compared to normal kidney, but downstream GATA3 and LHX1 expression was markedly decreased (2 to 4-fold) even though PAX8 localized at these target genes by chromatin immunoprecipitation - this suggested compromised PAX8 transcription activating function. PAX8 protein interactome in RCC cells indicated that PBRM1 is a PAX8 coactivator, and introduction of PBRM1 into PBRM1-haploinsufficient RCC cells (ACHN) by transfection increased activation of GATA3, WT1 and HNF4A, decreased MYC protein, increased p27/CDKN1B protein that mediates cell cycle exits by terminal differentiation, produced morphological changes of epithelial differentiation, and decreased proliferation. Coactivator versus corepressor recruitment by master transcription factors is a dynamic interchange, and the protein interactome studies indicated PBRM1 loss unbalanced stoichiometry of the PAX8-associated protein hub towards corepressors including DNMT1. Depletion of DNMT1 by non-cytotoxic concentrations of decitabine (0.5 µM) produced a remarkable shift from corepressors to coactivators in the PAX8 protein interactome, accompanied by terminal epithelial-differentiation as observed with PBRM1 reintroduction. Cell cycle exits by differentiation do not require p53/p16, and this effect was also produced in vivo in a subcutaneous xenograft model of TP53-mutated refractory/relapsed RCC, especially when decitabine in vivo pharmacology was optimized for non-cytotoxic DNMT1-depletion by avoiding a high Cmax and increasing its half-life and distribution into solid tissue, by administration of low dose in combination with an inhibitor of the enzyme cytidine deaminase that degrades decitabine. PBRM1 loss in RCC thus alters composition of a master transcription factor hub to repress instead of activate terminal epithelial-differentiation, an effect that can be reversed pharmacologically for novel, p53/p16-independent differentiation-restoring therapy. Citation Format: Xiaorong Gu, Francis Enane, Reda Mahfouz, Tomas Radivoyevitch, Bartlomiej Przychodzen, Yvonne Parker, Daniel Lindner, Brian Rini, Yogen Saunthararajah. PBRM1 inactivation in renal cell cancer alters master transcription factor hub composition to repress instead of activate epithelial-differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3520. doi:10.1158/1538-7445.AM2017-3520

MicroRNA-1 and MicroRNA-21 Individually Regulate Cellular Growth of Non-malignant and Malignant Renal Cells.

Due to its poor prognosis, it is increasingly necessary to understand the biology of renal cell cancer (RCC). Therefore, we investigated the role of microRNAs miR-1 and miR-21 in the growth of RCC cells compared to that of non-malignant renal cells. Four malignant cell lines (Caki-1, 786-O, RCC4, A498) were examined regarding their cell growth, microRNA and telomerase expression, and were compared to non-malignant RC-124 renal cells. Inconsistencies appeared in the panel of RCC cells regarding antiproliferative and proliferative properties of miR-1 and miR-21, respectively. Notably, and most likely due to immortaliziation, non-malignant RC-124 cells exhibited telomerase expression and activity. miR-1 and miR-21 functionality in cancer progression, particularly in tumor growth, may be more dependent on the individual cellular context and may reflect RCC heterogeneity. Thus, both microRNAs, in combination with other stratifying biomarkers, may be useful in terms of RCC diagnosis, prognosis, or treatment response.

High Annexin A5 expression promotes tumor progression and poor prognosis in renal cell carcinoma.

AnnexinA5 has been found to act as an oncogenic protein in a variety of cancers. However, its specific biological role and mechanism in renal cell cancer(RCC) remains unknown. Quantitative Real-time PCR and western blotting were used to evaluate the mRNA and protein expression level of AnnexinA5 in human RCC cell lines and tissues. Immunohistochemistry was adopted to measure the AnnexinA5 expression in 123cases of RCC tissues. Survival analysis was performed to explore the association between AnnexinA5 expression and the prognosis of RCC. The effect of AnnexinA5 on RCC growth and metastasis was studied invitro and invivo. AnnexinA5 was frequently highly expressed in both human RCC cells and tissues. High AnnexinA5 expression was associated with higher clinical stage and histological grade. In addition, AnnexinA5 might be used as a predictive factor for the prognosis of RCC. Further research suggested that upregulated AnnexinA5 in RCC cells could significantly promote tumor cell proliferation, migration and invasion invitro. Subcutaneous xenograft tumor model displayed that knockdown of AnnexinA5 could impede tumorigenesis invivo. Moreover, mechanism study exhibited that AnnexinA5 could activate PI3K/Akt/mTOR signaling pathway, promote epithelial-mesenchymal transition(EMT) and the expression of MMP2 and MMP9. AnnexinA5 may be a potential prognostic biomarker in RCC and promotes proliferation, migration and invasion of RCC cells via activating PI3K/Akt/mTOR signaling pathway and regulating EMT process and MMP expression.

Integrin signaling potentiates transforming growth factor-beta 1 (TGF-β1) dependent down-regulation of E-Cadherin expression – Important implications for epithelial to mesenchymal transition (EMT) in renal cell carcinoma

Signal transduction through the transforming growth factor-beta 1 (TGF-β1) pathway affects epithelial to mesenchymal transition (EMT), partly by modulation of E-Cadherin expression. The concurrent impact of extracellular matrix driven regulation of integrin signaling on EMT has not been well characterized. We assessed the cumulative effect and molecular mechanisms of TGF-β1 and integrin signal transduction on E-Cadherin in a renal cell cancer (RCC) model. Stimulation of RCC cells with TGF-β1 demonstrated a three-fold increased expression of integrin αv. A ligand of integrin αv-β3, (cyclopentapeptide containing Arginyl-Glycyl-Aspartic acid motif, RGD), was used to mimic integrin signaling. Treatment of cells with RGD and TGF-β1 demonstrated significantly greater E-cadherin depletion than either ligand alone. This cooperative action on E-Cadherin expression is regulated by transcription factor Snai1 and is followed on a cellular level by increased cellular mobility as evidenced in a wound healing assay. Subsequent silencing of potential downstream mediators of the cumulative action of RGD and TGF-β1 was carried out by small interfering RNA transfection and confirmed by Western blotting and/or RT-PCR. SiRNA mediated silencing of FAK and PINCH1 independently abrogated the cumulative effect of RGD and TGF-β1 on E-Cadherin expression.We have identified a novel mechanism through which extracellular matrix event transduction by integrins further augments TGF-β1 related effects on EMT. Molecular machinery involved in the integrin αv-TGF-β1 interplay may represent a therapeutic target in RCC.

Synergistic combination of YS-1 and adriamycin inhibits human renal cancer through ERK1/2 signaling pathway invitro and invivo.

Adriamycin (ADM) is a principal drug for the treatment of renal cell cancer (RCC). Due to its limited response and high renal and cardiac toxicity, synergistic effects of ADM in combination with other drugs have been widely researched. In this study, we found the combination between YS-1 and ADM, performed higher anticancer activity on 786-O human RCC cells invitro and invivo, than that reported on its anti-angiogenesis effect compared with monotherapy of ADM. Our data showed that when combined with ADM, YS-1 promoted the sensitivity of 786-O cells to ADM. The combination of YS-1 and ADM also inhibited cell proliferation, but without affecting cell apoptosis. We found that ADM monotherapy treatment notably upregulated the activity of extracellular signal-regulated kinase ERK1 and ERK2 (ERK1/2), but when combined with YS-1, the p-ERK1/2 level was reduced; then inhibited the Ras/Raf/MEK pathway. Additionally, the synergistic effects on cell cycle arrest inhibition were eliminated when ERK1/2 was silenced using siRNA. Our combination therapy of YS-1 with ADM showed the strongest antitumor effects invivo (inhibition ratio: 5mg/kg YS-1 combined with 1 mg/kg ADM, 68.19%) in comparison with individual effects (inhibition ratio: 5mg/kg YS-1, 30.07%; 1mg/kg ADM, 50.42%). Collectively, these findings indicated that YS-1 did not only enhance the ability of ADM to inhibit tumor proliferation, but also reduce the renal toxicity to protect the normal renal tissues.

Different Cytokine and Chemokine Expression Patterns in Malignant Compared to Those in Nonmalignant Renal Cells

Objective Cytokines and chemokines are widely involved in cancer cell progression and thus represent promising candidate factors for new biomarkers. Methods Four renal cell cancer (RCC) cell lines (Caki-1, 786-O, RCC4, and A498) and a nonmalignant renal cell line (RC-124) were examined with respect to their proliferation. The cytokine and chemokine expression pattern was examined by a DNA array (Human Cytokines & Chemokines RT2 Profiler PCR Array; Qiagen, Hilden, Germany), and expression profiles were compared. Results Caki-1 and 786-O cells exhibited significantly increased proliferation rates, whereas RCC4 and A498 cells demonstrated attenuated proliferation, compared to nonmalignant RC-124 cells. Expression analysis revealed 52 cytokines and chemokines primarily involved in proliferation and inflammation and differentially expressed not only in malignant and nonmalignant renal cells but also in the four RCC cell lines. Conclusion This is the first study examining the expression of 84 cytokines and chemokines in four RCC cell lines compared to that in a nonmalignant renal cell line. VEGFA, NODAL, and BMP6 correlated with RCC cell line proliferation and, thus, may represent putative clinical biomarkers for RCC progression as well as for RCC diagnosis and prognosis.

YM155 Reverses Statin Resistance in Renal Cancer by Reducing Expression of Survivin.

The purpose of the present study was to clarify whether treatment with YM155, a novel small-molecule inhibitor of survivin, reverses statin resistance in statin-resistant renal cell cancer (RCC). We induced simvastatin resistance in a renal clear cell carcinoma cell line (Caki-1-staR). In vitro and in vivo models were used to test the efficacy of YM155 and simvastatin. Survivin gene expression was significantly stronger in Caki-1-staR cells than in its parent cells (Caki-1). In Caki-1-staR cells, YM155 significantly reduced expression of survivin gene and cell proliferation in a dose-dependent manner. Treatment with YM155 significantly reversed simvastatin resistance in Caki-1-staR cells. YM155 significantly inhibited the growth of Caki-1-staR tumors in a nude mouse tumor xenograft model. Furthermore, YM155 significantly enhanced the antitumor effects of simvastatin on Caki-1-staR tumors. Our results indicate that inhibition of survivin by YM155 overcomes statin resistance in RCC cells.

MiR-206 inhibits renal cell cancer growth by targeting GAK.

Renal cell cancer (RCC) remains one of the most lethal types of cancer in adults. MicroRNAs (miRNAs) play key roles in the pathogenesis of RCC. The role of miR-206 in RCC has not been fully understood. The purpose of this study was to examine the role of miR-206 in the regulation of proliferation and metastasis of RCC and the possible mechanism. miR-206 expression was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in RCC cell lines (786-O and OS-RC-2 cells) and clinical samples. MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] method, colony formation and transwell assay were used to detect the tumor-suppressing ability of miR-206 in RCC. Luciferase assay was performed to verify the precise target of miR-206. The results showed that the expression of miR-206 was significantly down-regulated in RCC tissues and cells. The expression level of cyclin G-associated kinase (GAK), a master regulator of tumor proliferation and metastasis, was up-regulated with the decrease in miR-206 in RCC tissues as well as RCC cell lines. In addition, the miR-206 inhibitor promoted the proliferation, migration and invasion of 786-O and OS-RC-2 cells. Bioinformatics combined with luciferase and Western blot assays revealed that miR-206 inhibited the expression of GAK. Moreover, miR-206 regulates RCC cell growth partly through targeting GAK. Our study indicated that miR-206 functions as a tumor suppressor in regulating the proliferation, migration and invasion of RCC by directly targeting GAK, and it holds promises as a potential therapeutic target for RCC.

Choosing the right cell line for renal cell cancer research.

Cell lines are still a tool of choice for many fields of biomedical research, including oncology. Although cancer is a very complex disease, many discoveries have been made using monocultures of established cell lines. Therefore, the proper use of in vitro models is crucial to enhance our understanding of cancer. Therapeutics against renal cell cancer (RCC) are also screened with the use of cell lines. Multiple RCC in vitro cultures are available, allowing in vivo heterogeneity in the laboratory, but at the same time, these can be a source of errors. In this review, we tried to sum up the data on the RCC cell lines used currently. An increasing amount of data on RCC shed new light on the molecular background of the disease; however, it revealed how much still needs to be done. As new types of RCC are being distinguished, novel cell lines and the re-exploration of old ones seems to be indispensable to create effective in vitro tools for drug screening and more.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0565-8) contains supplementary material, which is available to authorized users.

The Silencing of CCND2 by Promoter Aberrant Methylation in Renal Cell Cancer and Analysis of the Correlation between CCND2 Methylation Status and Clinical Features

Cyclin D2 (CCND2) is a member of the D-type cyclins, which plays a pivotal role in cell cycle regulation, differentiation and malignant transformation. However, its expression status and relative regulation mechanism remains unclear in renal cell cancer (RCC). In our study, the mRNA expression level of CCND2 is down-regulated in 22/23 paired RCC tissues (p<0.05). In addition, its protein expression level is also decreased in 43/43 RCC tumor tissues compared with its corresponding non-malignant tissues (p<0.001). We further detected that CCND2 was down-regulated or silenced in 6/7 RCC cell lines, but expressed in “normal” human proximal tubular (HK-2) cell line. Subsequently, MSP and BGS results showed that the methylation status in CCND2 promoter region is closely associated with its expression level in RCC cell lines. Treatment with 5-Aza with or without TSA restored CCND2 expression in several methylated RCC cell lines. Among the 102 RCC tumors, methylation of CCND2 was detected in 29/102 (28%) cases. Only 2/23 (8.7%) adjacent non-malignant tissues showed methylation. We then analyzed the correlation of clinical features and its promoter methylation. Collectively, our data suggested that loss of CCND2 expression is closely associated with the promoter aberrant methylation.

Triiodothyronine regulates cell growth and survival in renal cell cancer.

Triiodothyronine plays an important role in the regulation of kidney cell growth, differentiation and metabolism. Patients with renal cell cancer who develop hypothyreosis during tyrosine kinase inhibitor (TKI) treatment have statistically longer survival. In this study, we developed cell based model of triiodothyronine (T3) analysis in RCC and we show the different effects of T3 on renal cell cancer (RCC) cell growth response and expression of the thyroid hormone receptor in human renal cell cancer cell lines from primary and metastatic tumors along with human kidney cancer stem cells. Wild-type thyroid hormone receptor is ubiquitously expressed in human renal cancer cell lines, but normalized against healthy renal proximal tube cell expression its level is upregulated in Caki-2, RCC6, SKRC-42, SKRC-45 cell lines. On the contrary the mRNA level in the 769-P, ACHN, HKCSC, and HEK293 cells is significantly decreased. The TRβ protein was abundant in the cytoplasm of the 786-O, Caki-2, RCC6, and SKRC-45 cells and in the nucleus of SKRC-42, ACHN, 769-P and cancer stem cells. T3 has promoting effect on the cell proliferation of HKCSC, Caki-2, ASE, ACHN, SK-RC-42, SMKT-R2, Caki-1, 786-0, and SK-RC-45 cells. Tyrosine kinase inhibitor, sunitinib, directly inhibits proliferation of RCC cells, while thyroid hormone receptor antagonist 1-850 (CAS 251310‑57-3) has less significant inhibitory impact. T3 stimulation does not abrogate inhibitory effect of sunitinib. Renal cancer tumor cells hypostimulated with T3 may be more responsive to tyrosine kinase inhibition. Moreover, some tumors may be considered as T3-independent and present aggressive phenotype with thyroid hormone receptor activated independently from the ligand. On the contrary proliferation induced by deregulated VHL and or c-Met pathways may transgress normal T3 mediated regulation of the cell cycle.

RNA interference-mediated silencing of speckle-type POZ protein promotes apoptosis of renal cell cancer cells.

This study aimed to investigate the effects of silencing the speckle-type POZ protein (SPOP) gene on renal cell cancer (RCC) cells and to explore its possible mechanism. The A498 and ACHN RCC cells were transfected with small interference RNA (siRNA)-SPOP by lipofection methods. The silencing efficiency was monitored by quantitative real-time polymerase chain reaction and Western blot. The effects of SPOP silencing on cell apoptosis, cell viability, colony formation ability, cell migration ability, and chemosensitivity to Sorafenib were assessed by flow cytometry, an MTT assay, a colony formation assay, a trans-well migration assay, and a CCK-8 assay, respectively. Its effects on the expression of several cytokines were determined by a protein microarray. Relevant signaling pathways were also analyzed. Compared with the control group, the cell apoptosis rate was significantly higher; the cell viability, the colony formation, and migration ability were significantly decreased in the siRNA-SPOP group. The protein microarray screening showed that the expression of vascular endothelial growth factor receptor, matrix metallopeptidase-9, vascular cell adhesion molecule-1, and stromal cell-derived factor-1 in the siRNA group was significantly decreased and that the expression of granulocyte–macrophage colony-stimulating factor and E-cadherin was significantly increased (P<0.05). The relevant signaling pathways were the integrin-mediated cell surface interactions pathway and extracellular matrix organization signal pathway. SPOP gene silencing induced cell apoptosis, decreased cell viability, colony formation, and migration ability, and elevated the drug sensitivity in the RCC cells. A possible mechanism is that silencing SPOP induces the differential expression of E-cadherin, vascular endothelial growth factor receptor, matrix metallopeptidase-9, and vascular cell adhesion molecule, which are related to the integrin-mediated cell surface interactions and extracellular matrix organization signaling pathway.

Hormone signaling pathways as treatment targets in renal cell cancer (Review).

Epidemiological, clinical, biochemical and genetic research has revealed that renal cell cancer (RCC) etiology is hormone-related. It was shown that hormone receptors are abnormally expressed in RCC cells. Abnormal endocrine stimulation also plays a significant role in RCC pathophysiology. Cellular proliferation, migration, angiogenesis, and drug resistance in RCC is modulated by para- and autocrine hormonal stimulation. In particular, RCC overexpression of gonadotropin-releasing hormone and its receptor was reported. On the contrary, corticotropin releasing hormone was reported to inhibit RCC cell proliferation and regulate angiogenesis. Overexpression of luteinizing hormone also promotes RCC tumor angiogenesis. Estrogen receptor α overexpression increases the transcriptional factor activity of hypoxia inducible factor HIF-1α, but estrogen receptor β has a cancer suppressive role. Glucocorticoid receptors and androgen receptor are markers of indolent RCC and assigned tumor suppressive activity. Proopiomelanocortin is upregulated in VHL-mutated renal cell carcinoma via Nur77 transcription factor signaling. In RCC, follicle-stimulating hormone receptor promotes angiogenesis and metastatic formation via VEGF release. Mineralocorticoid receptor overexpression promotes cell survival and increases RCC cell proliferation. Vitamin D receptor expression is downregulated or absent in RCC and differentiate subtypes of renal cell tumors. RAR-β promotes tumorigenesis but retinoic acid receptor γ expression correlates negatively with the TNM stage at diagnosis. Finally, progesterone receptor expression is negatively correlated with the cancer stage. Molecular data analysis revealed the possibility of renal cancer cell proliferation induction via hormone activated pathways. Inhibition of hormonal signaling may thus play a putative role in supportive therapies against this cancer type.

Sunitinib activates Axl signaling in renal cell cancer.

Mass spectrometry-based phosphoproteomics provides a unique unbiased approach to evaluate signaling network in cancer cells. The tyrosine kinase inhibitor sunitinib is registered as treatment for patients with renal cell cancer (RCC). We investigated the effect of sunitinib on tyrosine phosphorylation in RCC tumor cells to get more insight in its mechanism of action and thereby to find potential leads for combination treatment strategies. Sunitinib inhibitory concentrations of proliferation (IC50) of 786-O, 769-p and A498 RCC cells were determined by MTT-assays. Global tyrosine phosphorylation was measured by LC-MS/MS after immunoprecipitation with the antiphosphotyrosine antibody p-TYR-100. Phosphoproteomic profiling of 786-O cells yielded 1519 phosphopeptides, corresponding to 675 unique proteins including 57 different phosphorylated protein kinases. Compared to control, incubation with sunitinib at its IC50 of 2 µM resulted in downregulation of 86 phosphopeptides including CDK5, DYRK3, DYRK4, G6PD, PKM and LDH-A, while 94 phosphopeptides including Axl, FAK, EPHA2 and p38α were upregulated. Axl- (y702), FAK- (y576) and p38α (y182) upregulation was confirmed by Western Blot in 786-O and A498 cells. Subsequent proliferation assays revealed that inhibition of Axl with a small molecule inhibitor (R428) sensitized 786-O RCC cells and immortalized endothelial cells to sunitinib up to 3 fold. In conclusion, incubation with sunitinib of RCC cells causes significant upregulation of multiple phosphopeptides including Axl. Simultaneous inhibition of Axl improves the antitumor activity of sunitinib. We envision that evaluation of phosphoproteomic changes by TKI treatment enables identification of new targets for combination treatment strategies.

Immunization of A4galt-deficient mice with glycosphingolipids from renal cell cancers resulted in the generation of anti-sulfoglycolipid monoclonal antibodies.

In this study, we immunized Gb3/CD77 synthase gene (A4galt) knockout (KO) mice with glycosphingolipids (GSLs) extracted from 3 renal cell cancer (RCC) cell lines to raise monoclonal antibodies (mAbs) reactive with globo-series GSLs specifically expressed in RCCs. Although a number of mAbs reactive with globo-series GSLs were generated, they reacted with both RCC cell lines and normal kidney cells. When we analyzed recognized antigens by mAbs that were specifically reactive with RCC, but not with normal kidney cells at least on the cell surface, many of them turned out to be reactive with sulfoglycolipids. Eight out of 11 RCC-specific mAbs were reactive with SM2 alone, and the other 3 mAbs were more broadly reactive with sulfated glycolipids, i.e. SM3 and SM4 as well as SM2. In the immunohistochemistry, these anti-sulfoglycolipids mAbs showed RCC-specific reaction, with no or minimal reaction with adjacent normal tissues. Thus, immunization of A4galt KO mice with RCC-derived GSLs resulted in the generation of anti sulfated GSL mAbs, and these mAbs may be applicable for the therapeutics for RCC patients.

Abstract 4299: MicroRNA 200c regulates cytochrome P450 1B1-mediated docetaxel resistance in renal cell cancers

Abstract Cytochrome P450 1B1 (CYP1B1) is up-regulated in many types of cancer. It has been suggested that CYP1B1 may be a general tumor biomarker and have an important function in cancers. Despite its high protein expression and enzymatic activity in renal cell cancer (RCC), functional significance of CYP1B1 has not been elucidated. Here we explored the functional role and regulatory mechanism of CYP1B1 in RCC. Reduction of CYP1B1 levels fail to prevent in vitro tumorigenicity such as proliferation, apoptosis, and cell cycle progression of RCC cells. Moreover, the expression levels are not associated with tumor type, stage, Fuhrman grade and 5-year survival probability after surgery. Instead, alteration of CYP1B1 expression regulates the chemosensitivity of RCC cells to docetaxel suggesting its critical contribution to the RCC chemoresistance. Additionally, miR-200c, which is significantly down-regulated in RCC tissues and cell lines regulates CYP1B1 mRNA, protein and enzyme activity. An inverse association was also observed between the expression levels of miR-200c and CYP1B1 protein in RCC tissues. Thus, high expression of CYP1B1 could be a result of decreased miR-200c expression in RCC. Finally, alteration of miR-200c levels affects the chemosensitivity of RCC cells. Restoration of docetaxel resistance by exogenous expression of CYP1B1 in miR-200c-over-expressing cells indicates that CYP1B1 is a functional target of miR-200c. These results suggest that CYP1B1 up-regulation mediated by low miR-200c is one of the mechanisms underlying resistance of RCC cells to docetaxel. Therefore, expression of CYP1B1 and miR-200c in RCC may be useful as a prediction for docetaxel response. Citation Format: Inik Chang, Yozo Mitsui, Shinichiro Fukuhara, Ankurpreet Gill, Darryn K. Wong, Soichiro Yamamura, Varahram Shahryari, Z. Laura Tabatabai, Rajvir Dahiya, Dong Min Shin, Yuichiro Tanaka. MicroRNA 200c regulates cytochrome P450 1B1-mediated docetaxel resistance in renal cell cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4299. doi:10.1158/1538-7445.AM2015-4299

Abstract 5315: Hydroxychloroquine inhibits proliferation and S6 phosphorylation in human renal carcinoma cells

Abstract mTOR inhibitors are used to treat metastatic renal cell cancer (RCC), but most patients eventually become resistant. One possible mechanism for resistance is upregulation of autophagy, a pathway which helps recycle intracellular proteins and promotes cell survival. Hydroxychloroquine (HCQ), a potent autophagy inhibitor used to treat malaria and autoimmune disorders, is currently being studied in the context of cancer treatment. Here, we have investigated the effects of HCQ on three different renal carcinoma derived cell lines. We found that HCQ treatment inhibits RCC cell growth, promotes apoptosis, inhibits mitochondrial oxygen consumption, and increases glycolysis rates. To understand the molecular mechanism behind these effects, we examined various nodes in the mTOR pathway and compared the effects of HCQ with the effects of the mTOR inhibitor RAD001. A key downstream readout of the pathway, phospho-S6 protein, was inhibited by both HCQ and RAD001. However, the upstream kinase, P70S6K, was only inhibited by RAD001 and not HCQ, suggesting that the block by HCQ was downstream of the p70S6K. Treatment with the proteasome inhibitor bortezomib, restored phospho-S6 levels, suggesting that the reduction of phospho S6 is caused by increased degradation of phospho, but not total S6. Surprisingly, depletion of the key autophagy regulator Atg7 did not mimic the effects of HCQ, indicating that HCQ regulates S6 phosphorylation by ATG7-independent mechanism. Our finding suggests that HCQ may be useful in the treatment of renal cell carcinoma. Citation Format: Hyung-Ok Lee, Aladdin Mustafa, Gary R. Hudes, Warren D. Kruger. Hydroxychloroquine inhibits proliferation and S6 phosphorylation in human renal carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5315. doi:10.1158/1538-7445.AM2015-5315

Hydroxychloroquine Destabilizes Phospho-S6 in Human Renal Carcinoma Cells.

mTOR inhibitors are used to treat metastatic renal cell cancer (RCC), but most patients eventually become resistant. One possible mechanism for resistance is upregulation of autophagy, a pathway that helps recycle intracellular proteins and promotes cell survival. Hydroxychloroquine (HCQ), a potent autophagy inhibitor used to treat malaria and autoimmune disorders, is currently being studied in the context of cancer treatment. Here, we have investigated the effects of HCQ on three different renal carcinoma derived cell lines. We found that HCQ treatment inhibits RCC cell growth, promotes apoptosis, inhibits mitochondrial oxygen consumption, and increases rates of glycolysis. To understand the molecular mechanism behind these effects, we examined various nodes in the mTOR pathway and compared the effects of HCQ with the effects of the mTOR inhibitor RAD001. A key downstream readout of the pathway, phospho-S6 protein, was inhibited by both HCQ and RAD001. However, the upstream kinase, P70S6K was only inhibited by RAD001 and not HCQ, suggesting that the block by HCQ was downstream of P70S6K. Treatment with the proteasome inhibitor bortezomib restored phospho-S6 levels, suggesting that the reduction of phospho-S6 is caused by increased degradation of phospho-S6, but not total S6. Surprisingly, treatment with other autophagy inhibitors did not exhibit the same effects. Our findings suggest that HCQ causes the down-regulation of phospho-S6 in RCC cell lines via a novel mechanism that is not shared with other autophagy inhibitors.

MP47-05 ANDROGEN RECEPTOR MRNA EXPRESSION IS ASSOCIATED WITH PROGNOSIS IN LOCALIZED RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research II1 Apr 2015MP47-05 ANDROGEN RECEPTOR MRNA EXPRESSION IS ASSOCIATED WITH PROGNOSIS IN LOCALIZED RENAL CELL CARCINOMA Parth Modi, Yun-Sok Ha, Geuntaek Lee, Han-Jong Ahn, Wun-Jae Kim, and Isaac Kim Parth ModiParth Modi More articles by this author , Yun-Sok HaYun-Sok Ha More articles by this author , Geuntaek LeeGeuntaek Lee More articles by this author , Han-Jong AhnHan-Jong Ahn More articles by this author , Wun-Jae KimWun-Jae Kim More articles by this author , and Isaac KimIsaac Kim More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1525AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The incidence of renal cell cancer (RCC) is higher in men than women. While the reason for this is unknown, some have suggested that sex hormone signaling may play a role. Recent studies have demonstrated the presence of androgen receptor (AR) in RCC cell lines and pathologic specimens. However, the role of AR in RCC is still not well understood. This study evaluates the correlation between AR mRNA expression and clinical outcomes in patients undergoing surgical therapy for localized RCC. Additionally, cell culture experiments were performed to evaluate the effects of androgen signaling in RCC cells. METHODS AR mRNA expression was evaluated by quantitative real-time PCR (qPCR) in 115 tumor samples from patients with primary pT1 or T2 RCC and 57 specimens of corresponding normal kidney tissue. In a concurrent cell line study, reverse transcriptase PCR (RT-PCR) and western blot were used to examine the expression of AR in human RCC cell lines. The effects on cellular proliferation were investigated after administering dihydrotestosterone (DHT) to activate and enzalutamide to block androgen signaling in tissue culture. RESULTS AR mRNA expression levels were significantly higher in patients with pT2 tumors than in those with pT1 tumors (P = 0.011). Kaplan-Meier analysis revealed significant differences in the time to progression and cancer specific survival between low- and high-AR mRNA expression groups, independent of gender. Multivariate Cox regression analysis demonstrated that the level of AR expression is an independent predictor of cancer specific survival (HR, 15.546; 95% CI, 1.320–183.131; P = 0.029). In tissue culture, treatment with DHT caused proliferation in AR-positive cell lines while enzalutamide resulted in a reduction in cell viability in a dose-dependent manner. CONCLUSIONS In patients with localized RCC, AR mRNA expression level is associated with shorter progression-free and cancer-specific survival, independent of gender. In addition, cell culture data suggest that enzalutamide may have an effect in limiting the growth of AR-positive RCC. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e553-e554 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Parth Modi More articles by this author Yun-Sok Ha More articles by this author Geuntaek Lee More articles by this author Han-Jong Ahn More articles by this author Wun-Jae Kim More articles by this author Isaac Kim More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...