Purpose Hypoxia-inducible factor-1 alpha (HIF-1 α) expression has been linked with increased mortality and treatment failure in various cancers. The purpose of this study was to test the hypothesis that transcatheter arterial embolization (TAE) induces expression of HIF-1 α within the same rabbit VX2 liver tumor. Materials and Methods Seven VX2 tumors were grown in the livers of 5 New Zealand white rabbits. Ultrasound guided biopsies were taken before and 10 min after TAE from all tumors. Pre- and post- TAE tumor biopsy specimens along with post- TAE whole liver tumor sections were stained with HIF-1 α antibody and analyzed for percentage of HIF-1 α positive nuclei using a spectral unmixing system mounted on a high powered microscope. Statistical data comparisons were performed using Wilcoxon signed-rank test (alpha=0.05). Results TAE of liver tumors resulted in a statistically significant increase in the mean percentage of HIF-1 α expression. The mean percentage of HIF-1 α positive stained nuclei increased from 23 % ± 3.5% in pre- TAE biopsy specimens to 41% ± 8.7% (p< 0.02) in post- TAE biopsy specimens. The increase was even more significant when mean percentage of HIF-1 α positive stained nuclei from the same pre- TAE biopsy specimens were compared to sections from post- TAE whole tumor specimens [60% ± 8.9% (p<0.02)]. Conclusions The study revealed that hypoxia caused by TAE of VX2 liver tumors activates HIF-1 α, a transcription factor that in turn regulates other pro-angiogenic factors.

The authors recommend (a) the use of fresh VX2 cell suspension for percutaneous injection in the hind limbs of rabbits to maintain the VX2 cell strain and (b) the surgical implantation of freshly harvested VX2 tumor fragment into the liver parenchyma to establish liver tumors.

The E3 ubiquitin ligase Casitas B lymphoma protein (Cbl) controls the ubiquitin-dependent degradation of EGF receptor (EGFR), but its role in regulating downstream signaling elements with which it associates and its impact on biological outcomes of EGFR signaling are less clear. Here, we demonstrate that stimulation of EGFR on human mammary epithelial cells disrupts adherens junctions (AJs) through Vav2 and Rac1/Cdc42 activation. In EGF-stimulated cells, Cbl regulates the levels of phosphorylated Vav2 thereby attenuating Rac1/Cdc42 activity. Knockdown of Cbl and Cbl-b enhanced the EGF-induced disruption of AJs and cell motility. Overexpression of constitutively active Vav2 activated Rac1/Cdc42 and reorganized junctional actin cytoskeleton; these effects were suppressed by WT Cbl and enhanced by a ubiquitin ligase-deficient Cbl mutant. Cbl forms a complex with phospho-EGFR and phospho-Vav2 and facilitates phospho-Vav2 ubiquitinylation. Cbl can also interact with Vav2 directly in a Cbl Tyr-700-dependent manner. A ubiquitin ligase-deficient Cbl mutant enhanced the morphological transformation of mammary epithelial cells induced by constitutively active Vav2; this effect requires an intact Cbl Tyr-700. These results indicate that Cbl ubiquitin ligase plays a critical role in the maintenance of AJs and suppression of cell migration through down-regulation of EGFR-Vav2 signaling.

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