Gumz, Michelle L.

The importance of normal circadian rhythms is underscored by the known relationship between circadian disruption and increased cancer risk. The goal of the present study was to investigate the role of the circadian clock in the regulation of genes encoding products that function in cellular transformation. Microarray analysis was used to evaluate changes in gene expression following siRNA‐mediated knockdown of the circadian clock protein Per 1 in a murine renal collecting duct cell line (mpkCCD c14 ). Several hundred genes underwent significant changes in expression following Per1 knockdown, including 21 genes involved in cellular transformation. Cdkn1a and Cyclin E1, both cell cycle regulators, claudin 1 and 12, involved in tight junction formation, and sFRP1, a regulator of Wnt signaling and a tumor suppressor in renal cell carcinoma, were all confirmed as novel Per1 target genes. In an independent sample set, Cdkn1a, Cyclin E1, Claudin 1 and 12, and sFRP1 were all up‐regulated following Per1 knockdown in mpkCCD c14 cells. The chromophobe subtype of renal cell carcinoma is thought to arise from the collecting duct. Interestingly, dysregulation of the circadian clock genes Per2 and Cry2 was observed in patient chromophobe tumor samples compared to normal tissues. These observations support a role for the circadian clock in the control of cellular transformation in the kidney.

Abstract Anaplastic thyroid carcinoma (ATC) is a highly aggressive form of the disease for which new therapeutic options are desperately needed. Previously, we showed that the high-affinity peroxisome proliferator–activated receptor γ (PPARγ) agonist, RS5444, inhibits cell proliferation of ATC cells via induction of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21). We show here that up-regulation of RhoB is a critical step in PPARγ-mediated activation of p21-induced cell stasis. Using multiple independently derived ATC cell lines, we found that treatment with RS5444 leads to the up-regulation of RhoB and subsequent activation of p21, and that silencing of RhoB by RNAi blocks the ability of RS5444 to induce p21 and to inhibit cell proliferation. Our results show that transcriptional regulation of RhoB by the nuclear transcription factor PPARγ is responsible for the induction of p21 mRNA and protein. We further implicate RhoB as a key signaling effector for the growth inhibition of ATC, as treatment with a histone deacetylase inhibitor shown to increase RhoB expression in lung cancer cells caused the up-regulation of RhoB in ATC cells accompanied by increased expression of p21 and inhibition of cell proliferation; this effect occurred even in ATC cells that were unresponsive to RS5444 due to a lack of expression of PPARγ. Our results implicate RhoB as a novel intermediate in critical signaling pathways and as an additional target for therapeutic intervention in ATC. [Cancer Res 2009;69(4):1536–44]

Abstract Purpose: Incidence and mortality rates for renal cell carcinoma (RCC) have been rising for decades. Unfortunately, the molecular events that support RCC carcinogenesis remain poorly understood. In an effort to gain a better understanding of signaling events in clear cell RCC (cRCC), we investigated the antitumor activity of secreted frizzled-related protein 1 (sFRP1), a negative regulator of Wnt signaling. Experimental Design: Genomic profiling of cRCC tumors and patient-matched normal tissues was done and confirmed using quantitative PCR and immunohistochemistry. Methylation-specific PCR was done on patient samples to evaluate the mechanism responsible for sFRP1 loss. sFRP1 expression was restored in cRCC cells and the effects on tumor phenotype were characterized. Results: Genomic profiling, quantitative PCR, and immunohistochemistry indicated that loss of sFRP1 occurred in cRCC and papillary RCC patient tissues. Twelve Wnt-regulated genes were up-regulated in cRCC tissues, including c-myc and cyclin D1, potentiators of cell proliferation and survival. Methylation of the sFRP1 gene was one mechanism identified for attenuation of sFRP1 mRNA. Stable reexpression of sFRP1 in cRCC cells resulted in decreased expression of Wnt target genes, decreased growth in cell culture, inhibition of anchorage-independent growth, and decreased tumor growth in athymic nude mice. Conclusions: To our knowledge, this is the first report to show that stable restoration of sFRP1 expression in cRCC cells attenuates the cRCC tumor phenotype. Our data support a role for sFRP1 as a tumor suppressor in cRCC and that perhaps loss of sFRP1 is an early, aberrant molecular event in renal cell carcinogenesis.