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 sFRP1loss. 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 sFRP1occurred 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 sFRP1in 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 sFRP1as a tumor suppressor in cRCC and that perhaps loss of sFRP1is an early, aberrant molecular event in renal cell carcinogenesis.
Renal cell carcinoma (RCC) is a major health issue. Whereas localized disease can be cured surgically, there is no effective therapy for metastatic disease. The development of an effective therapy will require an understanding of the pathways that are important in RCC carcinogenesis and progression. Using genomic profiling of patientmatched tissue, we have identified aberrations in the transforming growth factor b (TGFb) signaling pathway in RCC. We observed loss of type III TGFb receptor (TBR3) expression in all RCC samples. This suggests that TBR3 loss is an early event in RCC carcinogenesis and plays a sentinel role in the acquisition of a tumorigenic phenotype. We also observed subsequent loss of type II TGFb receptor (TBR2) expression in metastatic RCCs. We propose that loss of TBR3 is necessary for RCC carcinogenesis, and that loss of TBR2 leads to acquisition of a metastatic phenotype. To this end, we have identified a human renal cell carcinoma line (UMRC6) that is representative of localized, nonmetastatic RCC, reflecting a loss of TBR3, but not TBR2 expression. Another cell line, UMRC3, is highly metastatic, having lost TBR3 and TBR2 expression. We demonstrate functional loss of TGFb responsiveness in these cell lines as observed through phenotypic and transcriptional responsiveness to exogenous TGFb. Restoring TBR2 and TBR3 expression in UMRC3 cells attenuates cell proliferation, completely restores TGFb-mediated transcriptional responses, and completely blocks anchorage independent-growth: while restoration of TBR2 partially restores TGFb-mediated signaling. Based on these data, we propose that dysregulation in TGFb signaling, through stepwise loss in receptor expression, plays a prominent role in RCC carcinogenesis and progression. In addition, these studies unequivocably demonstrate a link between loss of TBR3 and a human disease.
Double-stranded DNA cleavage of light-activated lysine conjugates is strongly enhanced at the slightly acidic pH (<7) suitable for selective targeting of cancer cells. This enhancement stems from the presence of two amino groups of different basicities. The first amino group plays an auxiliary role by enhancing solubility and affinity to DNA whereas the second amino group which is positioned next to the light-activated DNA-cleaver undergoes protonation at the desired pH threshold. This protonation results in two synergetic effects which account for the increased DNA-cleaving ability at the lower pH. First, lysine conjugates show tighter binding to DNA at the lower pH, which is consistent with the anticipated higher degree of interaction between two positively charged ammonium groups with the negatively charged phosphate backbone of DNA. Second, the unproductive pathway which quenches the excited state of the photocleaver through intramolecular electron transfer is eliminated once the donor amino group next to the chromophore is protonated. Experiments in the presence of traps for diffusing radicals show that reactive oxygen species do not contribute significantly to the mechanism of DNA cleavage at the lower pH, which is indicative of tighter binding to DNA under these conditions. This feature is valuable not only because many solid tumors are hypoxic but also because cleavage which does not depend on diffusing species is more localized and efficient. Sequence-selectivity experiments suggest combination of PET and base alkylation as the chemical basis for the observed DNA-damage. The utility of these molecules for phototherapy of cancer is confirmed by the drastic increase in toxicity of five conjugates against cancer cell lines upon photoactivation.
Loss of Transforming Growth Factor β Receptor III (TβRIII) correlates with loss of Transforming Growth Factor β (TGF-β) responsiveness and suggests a role for dysregulated TGF-β signaling in clear cell renal cell carcinoma (ccRCC) progression and metastasis. Here we identify that for all stages of ccRCC TβRIII expression is down-regulated in patient-matched tissue samples and cell lines. We find that this loss of expression is not due to methylation of the gene and we define GATA3 as the first transcriptional factor to positively regulate TβRIII expression in human cells. We localize GATA3s binding to a 10bp region of the TβRIII proximal promoter. We demonstrate that GATA3 mRNA is down-regulated in all stages of ccRCC, mechanistically show that GATA3 is methylated in ccRCC patient tumor tissues as well as cell lines and that inhibiting GATA3 expression in normal renal epithelial cells down-regulates TβRIII mRNA and protein expression. These data support a sequential model whereby loss of GATA3 expression via epigenetic silencing decreases TβRIII expression during ccRCC progression.
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