nutlin-3a and Kidney-Neoplasms

Sunitinib continues to be administered as a first-line therapeutic agent in metastatic renal cell carcinoma (mRCC). This study examined the potential role of p53 in sunitinib resistance and as a predictive marker in mRCC.. We analysed the effects of p53 knockout on sunitinib resistance. p53 expression in 53 mRCC patients receiving first-line sunitinib was determined immunohistochemically. We performed in silico analysis to examine the predictive value of p53 in mRCC.. WST-1 assays showed that p53 knockout decreased sensitivity to sunitinib. Sunitinib and nutlin-3 together suppressed cell growth. Immunohistochemistry revealed 11 p53-positive cases among 53 patients with mRCC. Kaplan-Meier analysis showed that p53-positive cases tended to be associated with poor progression-free survival (PFS) after first-line sunitinib treatment. In the JAVELIN 101 study, TP53 mutation was significantly associated with poor PFS after sunitinib treatment.. p53 may be involved in sunitinib resistance and represent a valuable marker for sunitinib treatment in mRCC.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Renal Cell; Cell Proliferation; Computer Simulation; Drug Resistance, Neoplasm; Drug Synergism; Female; Gene Knockout Techniques; Humans; Imidazoles; Kaplan-Meier Estimate; Kidney Neoplasms; Male; Middle Aged; Piperazines; Progression-Free Survival; Retrospective Studies; Sunitinib; Treatment Outcome; Tumor Suppressor Protein p53; Up-Regulation

Although the role of p53 as a tumour suppressor in renal cell carcinoma (RCC) is unclear, our recent analysis suggests that increased wild-type p53 protein expression is associated with poor outcome. A growing body of evidence also suggests that p53 expression and increased co-expression of MDM2 are linked with poor prognosis in RCC. We have therefore examined whether an MDM2 antagonist; Nutlin-3, might rescue/increase p53 expression and induce growth inhibition or apoptosis in RCC cells that retain wild-type p53. We show that inhibition of p53 suppression by MDM2 in RCC cells promotes growth arrest and p53-dependent senescence - phenotypes known to mediate p53 tumour suppression in vivo. We propose that future investigations of therapeutic strategies for RCC should incorporate MDM2 antagonism as part of strategies aimed at rescuing/augmenting p53 tumour suppressor function.

Topics: Antineoplastic Agents; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Kidney Neoplasms; Piperazines; Proto-Oncogene Proteins c-mdm2; RNA, Messenger; Tumor Suppressor Protein p53

The renal cell carcinoma (RCC) is one of the top 10 cancers in USA. The renal tumors are highly angiogenic and are resistant to conventional interventions, particularly radiotherapy. The advent of multi-specific tyrosine kinase inhibitor sorafenib has improved the progression-free survival in RCC, but overall survival in recurrent and metastatic RCC is still a concern that has lead to characterization of combinatorial regimens. Hence, we studied the effect of combination of nutlin-3, an MDM2 inhibitor, which increases p53 levels, and sorafenib in RCC. Sorafenib along with nutlin-3 synergistically inhibited the cell survival and enhanced caspase-3 cleavage leading to apoptosis in RCC. Nutlin-3 and sorafenib were more effective in reducing the migration of RCC, in combination than as single agents. Sorafenib and nutlin-3 decreased the phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) and ERK along with inducing p53 activity. The sorafenib and nutlin-3 co-treatment lead to enhanced levels of p53, p-p53, and increase in the levels of p53 pro-apoptotic effector PUMA, Bax, and decrease in the anti-apoptotic Bcl-2 levels. Importantly, our studies revealed that sorafenib alone can activate p53 in a concentration dependent manner. Thus, co-treatment of nutlin-3 with sorafenib leads to increased half-life of p53, which in turn can be activated by sorafenib, to induce downstream pro-apoptotic and anti-proliferative effects. This is the first report showing the synergistic effect of sorafenib and nutlin-3 while providing a strong clinical-translational rationale for further testing of sorafenib and nutlin-3 combinatorial regimen in human RCC.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Benzenesulfonates; Carcinoma, Renal Cell; Caspase 3; Cell Line, Tumor; Cell Movement; Humans; Imidazoles; Kidney Neoplasms; MAP Kinase Signaling System; Niacinamide; Phenylurea Compounds; Phosphorylation; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; Pyridines; Sorafenib; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor Receptor-2

Nutlin-3 is a novel small-molecule antagonist of the human homolog of mouse double minute (MDM2) that binds MDM2 in the p53-binding pocket and activates the p53 signaling pathway. In this study, we show that nutlin-3 sensitizes Caki human renal cancer cells, but not normal human skin fibroblast (HSF) cells or human mesangial cells, to TRAIL-mediated apoptosis. Combined treatment with nutlin-3 and TRAIL markedly induces apoptosis in HCT116 cells (p53 wild type), but not in HCT116 p53-/- cells, suggesting that p53 is critical for the sensitizing effect of nutlin-3 on TRAIL-induced apoptosis. Pretreatment with N-acetylcysteine (NAC) significantly inhibited nutlin-3-induced DR5 upregulation and cell death induced by the combined treatment with nutlin-3 and TRAIL, suggesting that reactive oxygen species (ROS) mediate nutlin-3-induced DR5 upregulation, which contributes toward TRAIL-mediated apoptosis. However, the upregulation of the p53-mediated protein p53 upregulated modulator of apoptosis (PUMA) by nutlin-3 is likely to be ROS independent because antioxidants failed to block PUMA upregulation. Interestingly, a combined treatment with NAC and PUMA small interfering RNAs significantly blocks nutlin-3-induced and TRAIL-induced apoptosis. Therefore, the present study shows that nutlin-3 enhances TRAIL-induced apoptosis in human renal cancer cells by ROS-mediated or p53-mediated DR5 upregulation and p53-induced PUMA upregulation. These results may offer a novel therapeutic approach to TRAIL-based cancer therapy.

Topics: Acetylcysteine; Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Fibroblasts; Genes, p53; HCT116 Cells; Humans; Imidazoles; Kidney Neoplasms; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Reactive Oxygen Species; Receptors, TNF-Related Apoptosis-Inducing Ligand; TNF-Related Apoptosis-Inducing Ligand; Up-Regulation

The interplay among hypoxia-inducible factor 1-alpha (HIF-1alpha), p53 and human orthologue of murine double minute 2 (Hdm2) has been introduced as a key event in tumor promotion and angiogenesis. Recently, nutlin-3, a small-molecule antagonist of Hdm2, was demonstrated to inhibit the HIF-1-mediated vascular endothelial growth factor production and tumor angiogenesis. Yet, the mechanism by which nutlin-3 inhibits HIF-1 is an open question. We here addressed the mode-of-action of nutlin-3 with respect to the HIF-1alpha-p53-Hdm2 interplay. The effect of nutlin-3 on HIF-1alpha function was examined by reporter analyses, immunoprecipitation and immunoblotting. Nutlin-3 downregulated HIF-1alpha, which occurred p53-dependently but von Hippel-Lindau-independently. On the contrary, nutlin-3 blunted the hypoxic induction of vascular endothelial growth factor by inactivating HIF-1 even in p53-null cells. The C-terminal transactivation domain (CAD) of HIF-1alpha was inactivated by nutlin-3, and furthermore, the factor-inhibiting hypoxia-inducible factor (FIH) hydroxylation of Asn803 was required for the nutlin-3 action. In terms of protein interactions, Hdm2 competed with FIH in CAD binding and inhibited the Asn803 hydroxylation both in vivo and in vitro, which facilitated p300 recruitment. Moreover, nutlin-3 reinforced the FIH binding and Ans803 hydroxylation by inhibiting Hdm2. In conclusion, Hdm2 functionally activates HIF-1 by inhibiting the FIH interaction with CAD, and the Hdm2 inhibition by nutlin-3 results in HIF-1 inactivation and vascular endothelial growth factor suppression. The interplays among HIF-1alpha, Hdm2, FIH and p300 could be potential targets for treating tumors overexpressing HIF-1alpha.

Topics: Aryl Hydrocarbon Receptor Nuclear Translocator; Carcinoma, Hepatocellular; Cell Line, Tumor; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Genes, Reporter; HCT116 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Imidazoles; Kidney Neoplasms; Liver Neoplasms; Mixed Function Oxygenases; Piperazines; Plasmids; Proto-Oncogene Proteins c-mdm2; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Vascular Endothelial Growth Factor A