Effects of TGF-ß signaling in clear cell renal cell carcinoma cells.

Clear cell renal cell carcinoma (ccRCC) is by far the most common type of kidney cancer and is characterized by loss of the tumor suppressor gene von Hippel-Lindau (VHL). ccRCC patients with metastatic disease has poor prognosis and today's therapy is insufficient. The cytokine Transforming Growth Factor-ß (TGF-ß) has been extensively studied in tumor biology and is believed to serve a variety of functions in tumor progression. We have previously shown that inhibition of NOTCH signaling causes a reduced migratory and invasive capacity of ccRCC cells, at least partly by a cross-talk with the TGF-ß pathway. In the present study we aimed to further clarify the role of TGF-ß signaling in ccRCC. We investigated the effects of TGF-ß pathway modulation and showed that TGF-ß inhibition attenuates the invasive capacity of ccRCC cells. By performing expression profiling we obtained a gene signature of the TGF-ß induced response in ccRCC cells. The expression analyses revealed an extensive overlap between the TGF-ß response and genes regulated by the hypoxia inducible factor (HIF). The link between the hypoxic and the TGF-ß pathways was further corroborated by functional experiments, which demonstrated that TGF-ß pathway activity was attenuated upon reintroduction of functional VHL in ccRCC.

Sarcomatoid conversion of clear cell renal cell carcinoma in relation to epithelial-to-mesenchymal transition.

Approximately 8% of clear cell renal cell carcinoma cases contain regions of radically different morphology, demonstrating a mesenchymal appearance histologically resembling sarcomas. These biphasic neoplasms are called sarcomatoid clear cell renal cell carcinoma. Patients diagnosed with sarcomatoid clear cell renal cell carcinoma face a considerably worse prognosis due to an increased propensity for metastasis. In the present study we investigate whether the sarcomatoid conversion of clear cell renal cell carcinoma could be interpreted as linked to the process of epithelial-mesenchymal transition. Using 6 biphasic clear cell renal cell carcinoma cases we show that sarcomatoid clear cell renal cell carcinoma shares characteristic markers associated with loss of von Hippel-Lindau tumor suppressor with conventional clear cell renal cell carcinoma and also exhibits a markedly higher proliferative index. Furthermore the sarcomatoid elements demonstrate an enhanced expression of epithelial-mesenchymal transition related mesenchymal markers as compared with the clear cell renal cell carcinoma counterparts. We further selected a representative case, clinically demonstrating direct overgrowth of the sarcomatoid component into the liver and colon, for extended immunohistochemical characterization, resulting in a further set of positive and negative epithelial-mesenchymal transition markers as well as pronounced transforming growth factor ß positivity, indicating that sarcomatoid clear cell renal cell carcinoma may be associated to epithelial-mesenchymal transition. Transforming growth factor ß1 exposure of in vitro cultured primary clear cell renal cell carcinoma cells resulted in cells adopting a mesenchymal morphology similar to sarcomatoid clear cell renal cell carcinoma. Corresponding changes in RNA levels for key epithelial-mesenchymal transition markers were also seen. We therefore suggest that sarcomatoid clear cell renal cell carcinoma morphologically and immunohistochemically may represent a completed epithelial-mesenchymal transition and that transforming growth factor ß1 could be an important driving force during the sarcomatoid transdifferentiation of clear cell renal cell carcinoma.

The notch and TGF-ß signaling pathways contribute to the aggressiveness of clear cell renal cell carcinoma.

BACKGROUND: Despite recent progress, therapy for metastatic clear cell renal cell carcinoma (CCRCC) is still inadequate. Dysregulated Notch signaling in CCRCC contributes to tumor growth, but the full spectrum of downstream processes regulated by Notch in this tumor form is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We show that inhibition of endogenous Notch signaling modulates TGF-ß dependent gene regulation in CCRCC cells. Analysis of gene expression data representing 176 CCRCCs showed that elevated TGF-ß pathway activity correlated significantly with shortened disease specific survival (log-rank test, p = 0.006) and patients with metastatic disease showed a significantly elevated TGF-ß signaling activity (two-sided Student's t-test, p = 0.044). Inhibition of Notch signaling led to attenuation of both basal and TGF-ß1 induced TGF-ß signaling in CCRCC cells, including an extensive set of genes known to be involved in migration and invasion. Functional analyses revealed that Notch inhibition decreased the migratory and invasive capacity of CCRCC cells. CONCLUSION: An extensive cross-talk between the Notch and TGF-ß signaling cascades is present in CCRCC and the functional properties of these two pathways are associated with the aggressiveness of this disease.

Isolation and characterization of progenitor-like cells from human renal proximal tubules.

The tubules of the kidney display a remarkable capacity for self-renewal on damage. Whether this regeneration is mediated by dedifferentiating surviving cells or, as recently suggested, by stem cells has not been unequivocally settled. Herein, we demonstrate that aldehyde dehydrogenase (ALDH) activity may be used for isolation of cells with progenitor characteristics from adult human renal cortical tissue. Gene expression profiling of the isolated ALDH(high) and ALDH(low) cell fractions followed by immunohistochemical interrogation of renal tissues enabled us to delineate a tentative progenitor cell population scattered through the proximal tubules (PTs). These cells expressed CD24 and CD133, previously described markers for renal progenitors of Bowman's capsule. Furthermore, we show that the PT cells, and the glomerular progenitors, are positive for KRT7, KRT19, BCL2, and vimentin. In addition, tubular epithelium regenerating on acute tubular necrosis displayed long stretches of CD133(+)/VIM(+) cells, further substantiating that these cells may represent a progenitor cell population. Furthermore, a potential association of these progenitor cells with papillary renal cell carcinoma was discovered. Taken together, our data demonstrate the presence of a previously unappreciated subset of the PT cells that may be endowed with a more robust phenotype, allowing increased resistance to acute renal injury, enabling rapid repopulation of the tubules.

The miR-17-92 microRNA cluster regulates multiple components of the TGF-ß pathway in neuroblastoma.

The miR-17-92 microRNA cluster is often activated in cancer cells, but the identity of its targets remains elusive. Using SILAC and quantitative mass spectrometry, we examined the effects of activation of the miR-17-92 cluster on global protein expression in neuroblastoma (NB) cells. Our results reveal cooperation between individual miR-17-92 miRNAs and implicate miR-17-92 in multiple hallmarks of cancer, including proliferation and cell adhesion. Most importantly, we show that miR-17-92 is a potent inhibitor of TGF-ß signaling. By functioning both upstream and downstream of pSMAD2, miR-17-92 activation triggers downregulation of multiple key effectors along the TGF-ß signaling cascade as well as direct inhibition of TGF-ß-responsive genes.

Gas6 and the receptor tyrosine kinase Axl in clear cell renal cell carcinoma.

BACKGROUND: The molecular biology of renal cell carcinoma (RCC) is complex and not fully understood. We have recently found that the expression of the receptor tyrosine kinase Axl in the RCC tumors independently correlates with survival of the patients. PRINCIPAL FINDINGS: Here, we have investigated the role of Axl and its ligand Gas6, the vitamin-K dependent protein product of the growth arrest-specific gene 6, in clear cell RCC (ccRCC) derived cells. The Axl protein was highly expressed in ccRCC cells deficient in functional von Hippel-Lindau (VHL) protein, a tumor suppressor gene often inactivated in ccRCC. VHL reconstituted cells expressed decreased levels of Axl protein, but not Axl mRNA, suggesting VHL to regulate Axl expression. Gas6-mediated activation of Axl in ccRCC cells resulted in Axl phosphorylation, receptor down-regulation, decreased cell-viability and migratory capacity. No effects of the Gas6/Axl system could be detected on invasion. Moreover, in ccRCC tumor tissues, Axl was phosphorylated and Gas6 gamma-carboxylated, suggesting these molecules to be active in vivo. SIGNIFICANCE: These results provide novel information regarding the complex function of the Gas6/Axl system in ccRCC.