Strategy For Renal Cell Carcinoma Research Articles

The selective MEK1 inhibitor Selumetinib enhances the antitumor activity of everolimus against renal cell carcinoma in vitro and in vivo.

Renal cell carcinoma (RCC) is a urologic malignant cancer and often diagnosed at an advanced stage, which results in high mortality. Targeted therapy may improve the quality of life and survival of patients who are not suitable for nephrectomy. Everolimus, an mTOR inhibitor, is currently used as sequential or second-line therapy for RCC refractory to Sunitinib or sorafenib. However, its efficiency is palliative. In this study, we evaluated whether the antitumor activity of everolimus against RCC is enhanced by selumetinib, a selective MEK1 inhibitor. We discovered that everolimus in combination with selumetinib synergistically inhibited the proliferation of Caki-1, 786-O and 769-P cells in vitro. Mechanistically, this combination decreased p-RPS6 and p-4E-BP1 dramatically, which causes G1 cell cycle arrest and prevents reactivation of AKT and ERK. In vivo, the antitumor efficacy and pharmacodynamic biomarkers of the combination therapy were recapitulated in Caki-1 xenograft model. In addition, this combination treatment potently inhibited angiogenesis in xenograft models by impairing VEGF secretion from tumor cells. Our findings provide a sound evidence that combination of everolimus and selumetinib is a potential dual-targeted strategy for renal cell carcinoma.

Repurposing Sunitinib with Oncolytic Reovirus as a Novel Immunotherapeutic Strategy for Renal Cell Carcinoma.

In addition to their direct cytopathic effects, oncolytic viruses are capable of priming antitumor immune responses. However, strategies to enhance the immunotherapeutic potential of these agents are lacking. Here, we investigated the ability of the multi-tyrosine kinase inhibitor and first-line metastatic renal cell carcinoma (RCC) agent, sunitinib, to augment the antitumor immune response generated by oncolytic reovirus. In vitro, oncolysis and chemokine production were assessed in a panel of human and murine RCC cell lines after exposure to reovirus, sunitinib, or their combination. In vivo, the RENCA syngeneic murine model of RCC was employed to determine therapeutic and tumor-specific immune responses after treatment with reovirus (intratumoral), sunitinib, or their combination. Parallel investigations employing the KLN205 syngeneic murine model of lung squamous cell carcinoma (NSCLC) were conducted for further validation. Reovirus-mediated oncolysis and chemokine production was observed following RCC infection. Reovirus monotherapy reduced tumor burden and was capable of generating a systemic adaptive antitumor immune response evidenced by increased numbers of tumor-specific CD8+ IFNγ-producing cells. Coadministration of sunitinib with reovirus further reduced tumor burden resulting in improved survival, decreased accumulation of immune suppressor cells, and the establishment of protective immunity upon tumor rechallenge. Similar results were observed for KLN205 tumor-bearing mice, highlighting the potential broad applicability of this approach. The ability to repurpose sunitinib for augmentation of reovirus' immunotherapeutic efficacy positions this novel combination therapy as an attractive strategy ready for clinical testing against a range of histologies, including RCC and NSCLC. Clin Cancer Res; 22(23); 5839-50. ©2016 AACR.

Tyrosine Kinase Signaling in Clear Cell and Papillary Renal Cell Carcinoma Revealed by Mass Spectrometry-Based Phosphotyrosine Proteomics.

Targeted therapies in renal cell carcinoma (RCC) are limited by acquired resistance. Novel therapeutic targets are needed to combat resistance and, ideally, target the unique biology of RCC subtypes. Tyrosine kinases provide critical oncogenic signaling and their inhibition has significantly impacted cancer care. To describe a landscape of tyrosine kinase activity in RCC that could inform novel therapeutic strategies, we performed a mass spectrometry-based system-wide survey of tyrosine phosphorylation in 10 RCC cell lines as well as 15 clear cell and 15 papillary RCC human tumors. To prioritize identified tyrosine kinases for further analysis, a 63 tyrosine kinase inhibitor (TKI) drug screen was performed. Among the cell lines, 28 unique tyrosine phosphosites were identified across 19 kinases and phosphatases including EGFR, MET, JAK2, and FAK in nearly all samples. Multiple FAK TKIs decreased cell viability by at least 50% and inhibited RCC cell line adhesion, invasion, and proliferation. Among the tumors, 49 unique tyrosine phosphosites were identified across 44 kinases and phosphatases. FAK pY576/7 was found in all tumors and many cell lines, whereas DDR1 pY792/6 was preferentially enriched in the papillary RCC tumors. Both tyrosine kinases are capable of transmitting signals from the extracellular matrix and emerged as novel RCC therapeutic targets. Tyrosine kinase profiling informs novel therapeutic strategies in RCC and highlights the unique biology among kidney cancer subtypes. Clin Cancer Res; 22(22); 5605-16. ©2016 AACR.

Abstract 5056: PIM-1 kinase regulates vitamin D receptor signaling in human renal carcinoma cells

Abstract Dysregulation of vitamin D signaling and metabolism enzymes is a frequent change in many cancers and may confound approaches to vitamin -D based therapies. The mechanisms by which aberrant vitamin D signaling and metabolism lead to resistance to vitamin D action are poorly understood. The kidney plays a critical role in vitamin D synthesis and metabolism; in renal cell carcinoma (RCC) cells vitamin D metabolism is dysregulated. Recently, we discovered that an oncogenic protein kinase PIM-1 is involved in vitamin D-induced expression of 24-hydroxylase, a key catalytic enzyme of vitamin D encoded by CYP24A1 gene, in bladder and prostate cancer. To study the impact of PIM1 on vitamin D signaling and vitamin D-mediated anti-tumor activity in RCC, we analyzed 4 human renal cell carcinoma cell lines (Caki-1, ACHN, A498 and 786-O) and 25 samples of human RCC tumor tissue and 13 benign kidney tissues (including 10 matched tumor and benign tissue samples) by qRT-PCR, immunohistochemistry (IHC), Western blot analysis, MTT and clonogenic assays. VDR mRNA and protein were detected in all RCC cell lines. IHC showed that VDR was strongly expressed in the normal kidney tissues, but decreased in RCC tissues. As expected, VDR expression was significantly increased by treatment with 1,25-dihydroxyvitamin D3 (1,25D3) in RCC cell lines. MTT and clonogenic assays showed that 1,25D3 inhibited RCC cell growth (p<0.05). We also noted a significant increase of CYP24A1 expression induced by vitamin D3 (p<0.0001); and effect which would be expected to promote the catabolism of 1,25D3. qRT-PCR and Western blot analysis demonstrated that PIM-1 is expressed in RCC cells. PIM-1 inhibition by siRNA or PIM-1 inhibitor JP_11646 significantly reduced 1,25D3-induced CYP24A1 expression (p<0.05). Therefore, we hypothesized that targeting PIM-1 kinase activity with a PIM-1 inhibitor may be an effective strategy to reduce 1,25D3 metabolism, thereby enhancing vitamin D-mediated anti-tumor activity. Inhibition of PIM1 kinase activity by JP_11646 reduced CYP24A1 expression at the transcriptional level in RCC cells. We further evaluated the efficacy of the PIM1 inhibitor alone or in combination with 1,25D3. Inhibition of PIM1 kinase activity by JP_11646 reduced cell growth in RCC cell lines in a dose-dependent manner as measured by MTT and clonogenic assays. Furthermore, inhibition of PIM1 kinase activity enhances 1,25D3-mediated inhibitory effect on cell growth in RCC cells (p<0.05). This study indicates that PIM-1 is involved in the regulation of vitamin D signaling in RCC. Inhibition of PIM kinase activity combined with vitamin D may be an attractive new therapeutic strategy for RCC. Citation Format: Wei Luo, Yingyu Ma, Carmen Baldino, Justin Caserta, Swathi Ramakrishnan, Pili Roberto, Candace Johnson, Donald Trump. PIM-1 kinase regulates vitamin D receptor signaling 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 5056. doi:10.1158/1538-7445.AM2015-5056

Abstract 1123: Glutaminase inhibitors suppress pyrimidine synthesis and promote DNA replication stress in VHL-deficient human renal cancer cells

Abstract Hypoxic and VHL-deficient cells use glutamine to generate citrate and lipids through reductive carboxylation (RC) of a-ketoglutarate. To gain insights into the role of HIF and the molecular mechanisms underlying RC, we took advantage of a panel of disease associated Von Hippel-Lindau (VHL) mutants and showed that HIF2a expression is necessary and sufficient for the induction of RC in human renal cell carcinoma (RCC) cells. Reductive carboxylation can be detected in vivo. HIF2a-mediated glutamine metabolism is induced by low intracellular citrate levels, which promote the reductive flux by mass action to maintain lipogenesis. In addition to being a source of lipogenic acetyl-CoA, glutamine can be used in the Krebs cycle to generate aspartate - the carbon source for pyrimidine biosynthesis. We showed that VHL-deficient RCC cells rely on RC-derived aspartate to maintain de novo pyrimidine biosynthesis. Pharmacological inhibition of glutaminase (GLS) by the commercially available glutaminase inhibitor BPTES and a glutaminase inhibitor which is in Phase I clinical study (CB-839, Calithera Biosciences Inc) depleted the levels of pyrimidines in VHL-deficient cells but not in VHL-reconstituted cells, which utilized glucose oxidation to supply the aspartate pool. Nucleoside depletion activates an intra-S-phase checkpoint corresponding to DNA replication stress and suppressed S-phase transition in VHL-deficient RCC cells. DNA replication stress was rescued by administration of glutamate or dimethyl-alpha-ketoglutarate. Addition of nucleobases partially rescued the effect of glutaminase inhibitors on cell growth. Our data suggest that glutaminase inhibitors suppress de novo pyrimidine synthesis, leading to DNA replication stress in VHL-deficient RCC cells. We currently test the hypothesis that this mechanistic insight may allow the development of novel therapeutic strategies for RCC by combining glutaminase inhibitors with drugs promoting DNA replication stress. Citation Format: Arimichi Okazaki, Paulo Gameiro, Gregory Stephanopoulos, Othon Iliopoulos. Glutaminase inhibitors suppress pyrimidine synthesis and promote DNA replication stress in VHL-deficient human renal cancer 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 1123. doi:10.1158/1538-7445.AM2015-1123

Abstract 3823: Dysregulation of bone morphogenetic protein 2 serves as a candidate molecular marker in human renal cell carcinoma

Abstract Introduction and Objective Bone morphogenetic protein-2 (BMP-2) has been shown to function as a tumor suppressor with several different cancers, while its expression level is down-regulated by CpG hypermethylation. We hypothesized that impaired regulation of BMP-2 through epigenetic pathways is associated with the pathogenesis of renal cell carcinoma (RCC). Methods To test our hypothesis, CpG methylation of the BMP-2 gene was analyzed using 2 RCC cell lines, and 96 matched RCC and normal renal tissues. We also performed functional analysis using BMP-2 restored RCC cells. Results BMP-2 mRNA expression was significantly enhanced in the RCC cells by 5-aza-2′-deoxycitidie treatment. The prevalence of BMP-2 promoter methylation was significantly higher, while BMP-2 mRNA expression was significantly lower in RCC samples as compared to normal kidney samples. Furthermore, a significant correlation was found between BMP-2 promoter methylation and mRNA transcription in the tumors. There was also a significant association of BMP-2 mRNA expression with tumor stage and lymph node involvement, while lower BMP-2 mRNA expression was significantly associated with poor overall survival after a radical nephrectomy. In RCC cells, BMP-2 restoration significantly inhibited cell proliferation, migration, invasion, and colony formation. In addition, BMP-2 overexpression effectively induced p21WAF1/CIP1 and p27KIP1 expression, and cellular apoptosis in RCC cells. Conclusion This is the first study to show inactivation of the BMP-2 gene via epigenetic pathways in RCC. Aberrant BMP-2 methylation and the resultant loss of BMP-2 expression may be useful as a molecular marker for designing improved diagnostic and therapeutic strategies for RCC. Citation Format: Yozo Mitsui, Hiroshi Hirata, Naoko Arichi, Miho Hiraki, Hiroaki Yasumono, Inik Chang, Shinichiro Fukuhara, Soichiro Yamamura, Varahram Shahryari, Guoren Deng, Sharanjot Saini, Shahana Majid, Yuichiro Tanaka, Rajvir Dahiya, Hiroaki Shiina. Dysregulation of bone morphogenetic protein 2 serves as a candidate molecular marker in human renal cell carcinoma. [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 3823. doi:10.1158/1538-7445.AM2015-3823

Grade-Dependent Metabolic Reprogramming in Kidney Cancer Revealed by Combined Proteomics and Metabolomics Analysis.

Kidney cancer [or renal cell carcinoma (RCC)] is known as "the internist's tumor" because it has protean systemic manifestations, suggesting that it utilizes complex, nonphysiologic metabolic pathways. Given the increasing incidence of this cancer and its lack of effective therapeutic targets, we undertook an extensive analysis of human RCC tissue employing combined grade-dependent proteomics and metabolomics analysis to determine how metabolic reprogramming occurring in this disease allows it to escape available therapeutic approaches. After validation experiments in RCC cell lines that were wild-type or mutant for the Von Hippel-Lindau tumor suppressor, in characterizing higher-grade tumors, we found that the Warburg effect is relatively more prominent at the expense of the tricarboxylic acid cycle and oxidative metabolism in general. Further, we found that the glutamine metabolism pathway acts to inhibit reactive oxygen species, as evidenced by an upregulated glutathione pathway, whereas the β-oxidation pathway is inhibited, leading to increased fatty acylcarnitines. In support of findings from previous urine metabolomics analyses, we also documented tryptophan catabolism associated with immune suppression, which was highly represented in RCC compared with other metabolic pathways. Together, our results offer a rationale to evaluate novel antimetabolic treatment strategies being developed in other disease settings as therapeutic strategies in RCC.

Tumor suppressive microRNA-200a inhibits renal cell carcinoma development by directly targeting TGFB2.

A large body of evidence indicates that microRNAs play a critical role in tumor initiation and progression by negatively regulating oncogenes or tumor suppressor genes. Here, we report that the expression of miR-200a was notably downregulated in 45 renal cell carcinoma (RCC) samples. Restoration of miR-200a suppressed cell proliferation, migration, and invasion in two RCC cell lines. Furthermore, we used an epithelial-to-mesenchymal transition PCR array to explore the putative target genes of miR-200a. By performing quantitative real-time PCR, ELISA, and luciferase reporter assays, transforming growth factor beta2 (TGFB2) was validated as a direct target gene of miR-200a. Moreover, siRNA-mediated knockdown of TGFB2 partially phenocopied the effect of miR-200a overexpression. These results suggest that miR-200a suppresses RCC development via directly targeting TGFB2, indicating that miR-200a may present a novel target for diagnostic and therapeutic strategies in RCC.

New strategies in renal cell carcinoma: targeting the genetic and metabolic basis of disease.

The development of new forms of treatment of advanced renal cell carcinoma over the past two decades has been primarily focused on targeting the VHL/HIF pathway. The recent identification of mutations of chromatin-remodeling genes in clear-cell renal carcinoma (ccRCC), of genomic heterogeneity, and of a Warburg-like metabolic phenotype in advanced disease has had a profound effect on our understanding of the evolution of ccRCC and on potential approaches to personalized therapy. Early approaches to therapy for patients with advanced type I papillary RCC that have centered around the MET/HGF pathway will expand as more genomic information becomes available. Sporadic and familial type II papillary renal cell carcinoma are characterized by enhanced aerobic glycolysis and share an antioxidant response phenotype. In fumarate hydratase-deficient RCC, fumarate-induced succination of KEAP1 activates Nrf2 signaling. CUL3 and Nrf2 mutations as well as an Nrf2 activation phenotype are found in sporadic type II papillary RCC. Therapeutic approaches designed to target the Nrf2 pathway as well as to impair blood flow and glucose delivery in these cancers that are highly dependent on a robust tumor vasculature and on ready availability of glucose for energy production and glycolysis are in development.

Abstract 3529: KIM-1 is a novel therapeutic target in renal cell carcinoma

Abstract Background. Renal cell carcinoma (RCC) is characterized by lack of early warning signs, diverse clinical manifestations, absence of a reliable diagnostic and predictive biomarker, and resistance to targeted therapy. Given the high incidence and worse prognosis, novel approaches for diagnosis, management and treatment of RCC are urgently needed. Kidney Injury Molecule-1 (KIM-1) is not expressed in normal kidney tissues but markedly up-regulated in dedifferentiated proximal tubular epithelial cells following renal injury. We demonstrated KIM-1 is expressed ubiquitoulsy by both primary and metastatic lesions of clear cell and papillary renal cell carcinoma, while barely detectable in other subtypes of RCC. The ectodomain of KIM-1 molecule cleaves by matrix metaloproteinases and sheds into surround milieu. In the present study, we investigated the role of KIM-1 in the pathogenesis of RCC. Methods. KIM-1 is stably transfected in immortalized tubular epithelial cells (LLC-PK1, gain-of-function) and KIM-1 was knockdown in RCC cell lines (loss-of-function) using lentiviral apporach and interoggated KIM-1 function in carcinogenesis. Results. Heterologous expression of KIM-1 increased cell proliferation, induced anchorage indepdent growth, increased migration and invasion potential, and increased the secreation of tumor promoting fractors, including TGF beta and IL-6 in immortalized tubular epithelial cells (PK1-KIM-1); while depletion of KIM-1 in RCC cells leads to cell cycle arrest at G1 phase and senescence. Recently, we and others have demonstrated that KIM-1 is a phosphatildyl serine receptor and the expression of KIM-1 converts a normal epithelial cell into a “semi professional phagocyte” and facilitates the removal of apoptotic and necrotic cells by recognizing phosphotidyl serine (PS) on their cell surface. Our data suggests that during this process, tumor epithelial cells undergo reprogramming and secrete and activate cytokines involved in cell survival and angiogenesis programs. Conclusions. The present study demonstrates that KIM-1-expression promotes tumorigenesis primarily by two distinct mechanisms: i) by directly conferring oncogenic properties to renal epithelial cells; ii) by resulting in production of a variety of chemokines and cytokines that support tumor progression and angiogenesis. We believe that our findings can significantly contribute to the development of novel therapeutic apporaches and in the advancement of management strategies in renal cell carcinoma and help reduce associated mortality and morbidity. Citation Format: Venkata S. Sabbisetti. KIM-1 is a novel therapeutic target in renal cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3529. doi:10.1158/1538-7445.AM2014-3529

Molecular Biomarkers in Advanced Renal Cell Carcinoma

The availability of agents directly targeting tumorigenic and angiogenic pathways has significantly improved the outcomes of patients with advanced renal cell carcinoma (RCC) in recent years. However, all patients eventually become resistant and a substantial percentage experience immediate disease progression with first-line targeted therapy. In addition, patients have variable clinical benefit and/or tolerance to different agents, including drugs within the same class. Thus, the choice of therapy for an individual patient remains empiric at present. Upon this landscape, several molecular biomarkers have been investigated with the purpose of guiding therapy. This review discusses prognostic biomarkers correlating with the outcome of patients independent of therapy, and predictive biomarkers of treatment response, including circulating biomarkers (such as VEGF and VEGF-related proteins, cytokine and angiogenic factors, and lactate dehydrogenase), and tissue-based biomarkers (such as single-nucleotide polymorphisms). Many potential prognostic and predictive molecular biomarkers have now been identified in RCC, although none has yet entered into clinical practice, and all require prospective validation in appropriately designed randomized studies. In the near future, however, validated biomarkers may become integral to management strategies in RCC, enabling tailored treatment for individual patients to improve clinical outcomes.

Histone Deacetylase Inhibitors and Epigenetic Modifications as a Novel Strategy in Renal Cell Carcinoma

Recent investigations of renal cell carcinoma (RCC) have revealed several epigenetic modifications, as well as alterations in the genes and enzymes that regulate these changes. Preclinical models have revealed that histone gene modifiers and epigenetic alterations may play a critical role in RCC tumorigenesis. Specific changes in DNA methylation and mutations of histone modifiers have been identified and may be associated with an aggressive phenotype. In addition, the potential of reversing the effects of these enzymes and hence reversing the cellular epigenetic landscape to a "normal phenotype" have led to an increasing interest in developing targeted chromatin remodeling agents. However, the translation of the understanding of these changes to the clinic for the treatment of RCC has posed significant challenges, partly due to tumor heterogeneity. This review describes the aberrant histone and DNA alterations recently reported in RCC and highlights the potential targeted chromatin remodeling therapies in the management of this disease.

Development of a novel interferon-α2b gene construct with a repetitive hypoxia-inducible factor binding site and its suppressive effects on human renal cell carcinoma cell lines in vitro

Despite the advent of targeted therapies, interferon-alpha (IFN-α) remains a therapeutic option for advanced renal cell carcinoma (RCC), especially in Japan, with a treatment response rate of 15-20%. To improve the efficacy of IFN-α-based therapies, we evaluated a novel treatment strategy for RCC using an IFN-α2b gene construct with a repetitive hypoxia-inducible factor binding site. We constructed an expression plasmid designated 5HREp-IFN-α2b containing the coding region of the IFN-α2b gene. Five copies of the hypoxia-response element (HRE) sequences were inserted upstream of the IFN-α2b gene, and the construct was transfected into human RCC cell lines ACHN, 786-O and KU19-20. The concentrations of IFN-α2b in the conditioned media were measured by enzyme-linked immunosorbent assay. Cell viabilities were determined by MTS assays. Construct-induced IFN-α secretion was confirmed in all three cell lines. IFN-α production was significantly enhanced by the hypoxia-mimicking agent deferoxamine mesylate in cell lines expressing the wild-type von Hippel-Lindau (VHL) gene (KU19-20 and ACHN) compared with cells expressing the mutant VHL gene (786-O). The construct exerted significant suppressive effects on the viabilities of all RCC cell lines. This is the first study to report on the construction of a cytokine gene with a repetitive hypoxia-inducible factor binding site and its application in the suppression of human cancer cells. Gene therapy using this IFN-α2b gene construct with HREs may represent a novel treatment modality for advanced RCC.

Abstract 5195: Kidney injury molecule-1: a novel therapeutic target in renal cell carcinoma.

Abstract Background. Renal cell carcinoma (RCC) is characterized by lack of early warning signs, diverse clinical manifestations, absence of a reliable diagnostic and predictive biomarker, and resistance to targeted therapy. Thus, novel approaches for diagnosis, management and treatment of RCC are urgently needed. Kidney Injury Molecule-1 (KIM-1) is not expressed in normal kidney tissues but markedly up-regulated in dedifferentiated proximal tubular epithelial cells following renal injury. We demonstrated that KIM-1 is expressed ubiquitoulsy by both primary and metastatic lesions of clear cell and papillary renal cell carcinoma, while barely detectable in other subtypes of RCC. Additionally, the ectodomain of KIM-1 is cleaved by matrix metalloproteinases and sheds into the surrounding milieu. Methods. KIM-1 was stably transfected in immortalized renal tubular epithelial cells (LLC-PK1, gain-of-function) and KIM-1 was downmodulated in RCC cell lines (loss-of-function) using a siRNA lentiviral approach. KIM-1 function was assessed using these two systems. Results. Stable expression of KIM-1 in non-malignant renal epithelial cells increased cell proliferation, migration and invasion potential, induced anchorage independent growth, and increased the secretion of tumor promoting and angiogenic factors including VEGF, Ang2, TGF beta and IL-6 (PK1-KIM-1); while depletion of KIM-1 in RCC cells led to G1 cell cycle arrest phase and senescence. Recently, we and others have demonstrated that KIM-1 is a phosphatildyl serine receptor and the expression of KIM-1 converts a normal epithelial cell into a “semi professional phagocyte” and facilitates the removal of apoptotic and necrotic cells by recognizing phosphotidyl serine (PS) on their cell surface. Our data suggests that during this process, tumor epithelial cells undergo reprogramming and secrete and activate cytokines involved in cell survival and angiogenesis programs. Conclusions. The present study shows that KIM-1-expression promotes tumorigenesis primarily by two distinct mechanisms: i) by directly conferring oncogenic properties to renal epithelial cells; ii) by resulting in production of a variety of chemokines and cytokines that support tumor progression and angiogenesis. We believe that our findings can significantly contribute to the development of novel therapeutic apporaches and in the advancement of management strategies in RCC and help reduce associated mortality and morbidity. Citation Format: Venkata Sabbisetti, Rupal Bhatt, Swetha Ramadesikan, Joseph Bonventre. Kidney injury molecule-1: a novel therapeutic target in renal cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5195. doi:10.1158/1538-7445.AM2013-5195 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Conservative treatment of renal cell carcinoma in kidney transplantation

PurposeTo evaluate the new treatment strategies in renal cell carcinoma (RCC) that affects the graft in renal recipients. MethodsA literature review is made, analyzing all the published cases of conservative surgery in renal graft RCC. Synthesis of evidenceA total of 51 partial nephrectomies in renal graft patients have been described, with a graft survival rate of 88% and a recurrence rate of 6%. Most of the patients (75%) were asymptomatic at the time of diagnosis, and the mean lesion size was 2.8cm. Enucleation was the most frequent technique employed. 77% of all immunosuppressor regimens included cyclosporine A. Six patients with graft RCC were subjected to radiofrequency ablation and two patients underwent percutaneous cryoablation, with a single case of relapse and a graft survival rate of 100%. ConclusionsNephron-sparing surgery is a good management option in renal graft RCC, affording good oncological control and graft survival. Modification of immunosuppression with the withdrawal of cyclosporine A and the introduction of mTOR inhibitors is an adequate measure in such patients.

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant. In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma as well as other solid tumors. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis. Protein based analysis of RCC is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

Treatment of Renal Cell Carcinoma with 2-Stage Totalen blocSpondylectomy after Marked Response to Molecular Target Drugs

Metastatic renal cell carcinoma of the bone occurs at a high rate, and the prognosis is poor. In general, total en bloc spondylectomy is considered when there is only one vertebral metastasis and the primary disease is treated. However, palliative surgery is selected when the primary disease is not being treated or metastasis occurs to an important organ. We encountered a patient in whom lung and vertebra metastases were already present at the time of the first examination at our department and the prognosis was considered poor. However, molecular targeted therapy was markedly effective and enabled 2-stage total en bloc spondylectomy. As of one year after total en bloc spondylectomy, the condition has improved to cane gait, and surgery for lung metastasis is planned. Molecular target drugs might markedly change the current therapeutic strategy for renal cell carcinoma.

Targeting Renal Cell Carcinoma with Gambogic Acid in Combination with Sunitinib in Vitro and in Vivo

To evaluated the effect of the gambogic acid (GA), one of the effective components of Garcinia, in combination with a new multi-targeted oral medication, sunitinib (SU) on renal cancer cell proliferation in vitro and on tumor growth in vivo. After treatment with GA or SU, either alone or in combination, MTT and FACS analysis were used to examine cell viability and cycle distribution of the renal carcinoma cell lines 786-0 and Caki-1. Western blotting was employed to examine the expression of proteins related to the cell cycle and vascular formation. Furthermore, a xenograft model was applied to study the antitumor efficacy of SU or GA alone or in combination, with immunohistochemistry to detect expression of proteins related to xenograft growth and angiogenesis. Western blotting was used to examine NF-?B signaling pathway elements in xenografts. Treatment of 786-0 and Caki-1 cells with GA or SU resulted in decreased tumor cell proliferation, especially with joint use. Cells accumulated more strongly in the sub-G1 phase after joint treatment with GA and SU than treatment of GA and SU alone. Western blotting arrays showed 1 protein significantly upregulated, 2 proteins downregulated, and 2 proteins unchanged. Moreover, combined use of GA and SU inhibited the growth and angiogenesis of xenografts generated from Caki-1 significantly. Immunohistochemistry arrays showed downregulation of the expression of proteins promoting xenograft growth and angiogenesis, and Western blotting showed inhibition of the NF-?B signaling pathway after treatment by GA alone and in combination with SU in xenografts. Our results show that the joint use of GA and SU can provide greater antitumor efficacy compared to either drug alone and thus may offer a new treatment strategy for renal cell carcinoma.

Tratamiento conservador del carcinoma de células renales en el injerto renal

PurposeTo evaluate the new treatment strategies in renal cell carcinoma (RCC) that affects the graft in renal recipients. Acquisition of evidenceA literature review is made, analyzing all the published cases of conservative surgery in renal graft RCC. Synthesis of evidenceA total of 51 partial nephrectomies in renal graft patients have been described, with a graft survival rate of 88% and a recurrence rate of 6%. Most of the patients (75%) were asymptomatic at the time of diagnosis, and the mean lesion size was 2.8cm. Enucleation was the most frequent technique employed. 77% of all immunosuppressor regimens included cyclosporine A. Six patients with graft RCC were subjected to radiofrequency ablation and two patients underwent percutaneous cryoablation, with a single case of relapse and a graft survival rate of 100%. ConclusionsNephron-sparing surgery is a good management option in renal graft RCC, affording good oncological control and graft survival. Modification of immunosuppression with the withdrawal of cyclosporine A and the introduction of mTOR inhibitors is an adequate measure in such patients.

MiR-21 Downregulated TCF21 to Inhibit KISS1 in Renal Cancer

To investigate whether the microRNA-21 gene (miR-21) could regulate renal cancer cells invasion by downregulation of TFC21 and KISS1. Quantitative real-time polymerase chain reaction was applied to evaluate the expression level of miRNA-21 in renal cancer and normal renal cell samples. The regulated effects of miR-21 to TCF21 were detected by Western blot after pre/anti-miR-21 was transfected to Caki-1 cells. The luciferase activity assay was used to reveal the predicted target gene of miR-21 was direct and specific. Small interfering RNA-TCF21 was transfected to Caki-1 cells to inhibit the expression of the TCF21 gene. Next, the expression of the KISS1 gene was detected by Western blot in Caki-1 cells with TCF21 gene silencing. The expression vector, pcDNA3.1-KISS1, was transfected to Caki-1 cells to upregulate the expression of the KISS1 gene. The invasion ability of Caki-1 cells with KISS1 overexpression was analyzed using the Transwell assay. Our study showed that miR-21 was upregulated in human renal cell carcinoma specimens compared with its expression in normal renal cell specimens. Pre-miR-21 could upregulate the expression of miR-21 and downregulate the expression of TCF21, and anti-miR-21 showed the opposite effects. siRNA-TCF21 decreased the expression of the TCF21 protein, and the expression of KISS1 was downregulated in Caki-1 cells with TCF21 gene silencing. pcDNA3.1-KISS1 transfection upregulated the expression of the KISS1 protein, and the invasion ability of Caki-1 cells with KISS1 overexpression decreased markedly. Aberrantly expressed miR-21 might regulate the TCF21-KISS1-associated renal cell carcinoma cell invasion pathway, and this miRNA signature could offer a novel potential therapeutic strategy for renal cell carcinoma.