Inhibition of the CRAF/prohibitin interaction reverses CRAF-dependent resistance to vemurafenib.

Activating BRAF mutations promote constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway and are common in a variety of human malignancies, including melanoma and colon cancer. Several small molecule BRAF inhibitors such as vemurafenib have been developed and demonstrate remarkable clinical efficacy. However, resistance typically emerges in most melanoma patients. Studies have demonstrated that reactivation of MAPK signaling via CRAF overexpression and dysregulation is a mechanism for vemurafenib resistance in melanoma. Prohibitins (PHBs) are highly conserved proteins that are thought to control the cell cycle, senescence and tumor suppression. PHB1 is essential for CRAF-mediated ERK1/2 activation through direct binding to CRAF. We developed a CRAF-mediated model of vemurafenib resistance in melanoma cells to assess the importance of the interaction between CRAF and PHB1 in resistance to BRAF-targeting agents. We demonstrate that CRAF overexpression renders melanoma cells resistant to BRAF-targeting agents. Moreover, treatment with the natural compound rocaglamide A disrupts the interaction between PHB and CRAF in melanoma cells, thus reducing MEK1/2 and ERK1/2 signaling, inhibiting melanoma cell growth and inducing apoptosis. The efficacy of these compounds was also demonstrated in a human melanoma xenograft model. Taken together, these data suggest that PHB1 may serve as a novel, druggable target in CRAF-mediated vemurafenib resistance.

Cloning and characterization of murine p53 upstream sequences reveals additional positive transcriptional regulatory elements.

Transcriptional regulation of the p53 gene plays an important role leading to elevated expression of mutant p53 alleles in tumor cells. In addition, alterations in p53 transcription levels occur in response to changes in the cell cycle. Previous work had identified a number of regulatory sites at the 5'-end of the murine p53 promoter. During the characterization of the 5'-end of the cloned murine p53 promoter, we identified a 28 bp positive regulatory element that participates in three distinct DNA-protein complexes. The binding by nuclear factors to each one of these sites contributes to the overall activity of the p53 promoter. One site is a potential recognition sequence for members of the ETS family of transcription factors, which are known regulators of the human p53 promoter. Since six nucleotides in the middle of this required element were not present in the previously published sequence of the murine promoter, we recloned this region from C57/BL6 cells and confirmed their presence in the genome. The removal of this regulatory element completely abolishes p53 promoter activity.