Sorafenib (BAY 43-9006) inhibits tumor growth and vascularization and induces tumor apoptosis and hypoxia in RCC xenograft models.

PURPOSE: New research findings have revealed a key role for vascular endothelial growth factor (VEGF) in the stimulation of angiogenesis in clear cell renal carcinoma (RCC) which is a highly vascularized and treatment-resistant tumor. Sorafenib (BAY 43-9006, Nexavar) is a multi-kinase inhibitor which targets receptor tyrosine and serine/threonine kinases involved in tumor progression and tumor angiogenesis. The effect of sorafenib on tumor growth and tumor histology was assessed in both ectopic and orthotopic mouse models of RCC. METHODS: Sorafenib was administered orally to mice bearing subcutaneous (SC, ectopic) or sub-renal capsule (SRC, orthotopic) tumors of murine (Renca) or human (786-O) RCC. Treatment efficacy was determined by measurements of tumor volume and tumor growth delay. In mechanism of action studies, using the 786-O and Renca RCC tumor models, the effect of sorafenib was assessed after dosing for 3 or 5 days in the SC models and 21 days in the SRC models. Inhibition of tumor angiogenesis was assessed by measuring level of CD31 and alpha-smooth muscle actin (alphaSMA) staining by immunohistochemistry (IHC). The effect of sorafenib on MAPK signaling, cell cycle progression and cell proliferation was also assessed by IHC by measuring levels of phospho-ERK, phospho-histone H3 and Ki-67 staining, respectively. The extent of tumor apoptosis was measured by terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assays. Finally, the effects of sorafenib on tumor hypoxia was assessed in 786-O SC model by injecting mice intravenously with pimonidazole hydrochloride 1 h before tumor collection and tumor sections were stained with a FITC-conjugated Hypoxyprobe antibody. RESULTS: Sorafenib produced significant tumor growth inhibition (TGI) and a reduction in tumor vasculature of both ectopic and orthotopic Renca and 786-O tumors, at a dose as low as 15 mg/kg when administered daily. Inhibition of tumor vasculature was observed as early as 3 days post-treatment, and this inhibition of angiogenesis correlated with increased level of tumor apoptosis (TUNEL-positive) and central necrosis. Consistent with these results, a significant increase in tumor hypoxia was also observed 3 days post-treatment in 786-O SC model. However, no significant effect of sorafenib on phospho-ERK, phospho-histone H3 or Ki-67 levels in either RCC tumor model was observed. CONCLUSION: Our results show the ability of sorafenib to potently inhibit the growth of both ectopically- and orthotopically-implanted Renca and 786-O tumors. The observed tumor growth inhibition and tumor stasis or stabilization correlated strongly with decreased tumor angiogenesis, which was due, at least in part, to inhibition of VEGF and PDGF-mediated endothelial cell and pericyte survival. Finally, sorafenib-mediated inhibition of tumor growth and angiogenesis occurred at concentrations equivalent to those achieved in patients in the clinic.

Loss of the cell cycle inhibitors p21(Cip1) and p27(Kip1) enhances tumorigenesis in knockout mouse models.

Events that contribute to tumor formation include mutations in the ras gene and loss or inactivation of cell cycle inhibitors such as p21(Cip1) and p27(Kip1). In our previous publication, we showed that mice expressing the MMTV/v-Ha-ras transgene developed tumors earlier and at higher multiplicities in the absence than in the presence of p21(Cip1). To further evaluate the combinatorial role of genetic alterations and loss of cell cycle inhibitors in tumorigenesis, we performed two companion studies. In the first study, wild type and p21(Cip1)-null mice were exposed to the chemical carcinogen, urethane. Similar to its effects in v-Ha-ras mice, loss of p21(Cip1) accelerated tumor onset and increased tumor multiplicity in urethane-treated mice. Lung tumors were the predominant tumor type in urethane-treated mice regardless of p21(Cip1) status. In the second study, tumor formation was monitored in v-Ha-ras mice expressing or lacking p27(Kip1). Unlike p21(Cip1), the absence of p27(Kip1) had no effect on the timing or multiplicity of tumor formation, which was largely restricted to mammary and salivary glands. However, once tumors appeared, they grew faster in p27(Kip1)-null mice than in p27(Kip1)-wild type mice. Increases in growth rate were particularly striking for salivary tumors in ras/p27(-/-) mice. Loss of p21(Cip1), on the other hand, had no effect on tumor growth rate in v-Ha-ras mice. Collectively, our data suggest that p21(Cip1) suppresses tumor formation elicited by multiple agents and that p21(Cip1) and p27(Kip1) suppress tumor formation in different ways.

Suppression of rho B expression in invasive carcinoma from head and neck cancer patients.

PURPOSE: In contrast to Ras small GTPases, which contribute to human malignancy when overexpressed or constitutively activated, convincing evidence for the involvement of Ras homologous (Rho) GTPases in human cancer is still missing. In cell culture and animal models, RhoB antagonizes malignant transformation, but no data are available regarding the expression of RhoB in human tumors. In this study, we have analyzed the status of the RhoB protein and the closely related family member RhoA in human head and neck squamous cell carcinomas. EXPERIMENTAL DESIGN: Protein immunoexpression was quantitated by image analysis in the context of tumor invasion and differentiation. To account for possible individual variations, expression levels of RhoB and RhoA were evaluated in the tumor and its adjacent nonneoplastic tissue. Potential gene deletions or mutations were assessed by PCR and RT-PCR. RESULTS: RhoB expression is readily detected in normal epithelium, carcinomas in situ, and well-differentiated tumors, but it becomes weak to undetectable as tumors become deeply invasive and poorly differentiated. In contrast, Ki67 (proliferation marker) and RhoA protein levels increase with tumor progression. Furthermore, whereas in nonneoplastic keratinocytes RhoB is localized mainly in the nucleus, in carcinomas RhoB is predominantly located in the cytoplasm. RhoB gene deletions or mutations were not found. CONCLUSIONS: These results give additional support to the notion that RhoB may play a tumor suppressive role in squamous cell carcinomas of the head and neck. The lack of RhoB expression in deeply invasive carcinoma argues against inhibition of RhoB farnesylation as a mediator of farnesyltransferase inhibitors' antitumor activity.

RhoB, not RhoA, represses the transcription of the transforming growth factor beta type II receptor by a mechanism involving activator protein 1.

The transforming growth factor-beta (TGF-beta) type I (T beta R-I) and type II (T beta R-II) receptors are responsible for transducing TGF-beta signals. We have previously shown that inhibition of farnesyltransferase activity results in an increase in T beta R-II expression, leading to enhanced TGF-beta binding, signaling, and inhibition of tumor cell growth, suggesting that a farnesylated protein(s) exerts a repressive effect on T beta R-II expression. Likely candidates are farnesylated proteins such as Ras and RhoB, which are both farnesylated and involved in cell growth control. Neither a dominant negative Ha-Ras, constitutively activated Ha-Ras, or a pharmacological inhibitor of MEK1 affected T beta R-II transcription. However, ectopic expression of RhoB, but not the closely related family member RhoA, resulted in a 5-fold decrease of T beta R-II promoter activity. Furthermore, ectopic expression of RhoB, but not RhoA, resulted in a significant decrease of T beta R-II protein expression and resistance of tumor cells to TGF-beta-mediated cell growth inhibition. Deletion analysis of the T beta R-II promoter identified a RhoB-responsive region, and mutational analysis of this region revealed that a site for the transcription factor activator protein 1 (AP1) is critical for RhoB-mediated repression of T beta R-II transcription. Electrophoretic mobility shift assays clearly showed that the binding of AP1 to its DNA-binding site is strongly inhibited by RhoB. Consequently, transcription assays using an AP1 reporter showed that AP1-mediated transcription is down-regulated by RhoB. Altogether, these results identify a mechanism by which RhoB antagonizes TGF-beta action through transcriptional down-regulation of AP1 in T beta R-II promoter.