Simultaneous inhibition of key growth pathways in melanoma cells and tumor regression by a designed bidentate constrained helical peptide.

Protein-protein interactions are part of a large number of signaling networks and potential targets for drug development. However, discovering molecules that can specifically inhibit such interactions is a major challenge. S100B, a calcium-regulated protein, plays a crucial role in the proliferation of melanoma cells through protein-protein interactions. In this article, we report the design and development of a bidentate conformationally constrained peptide against dimeric S100B based on a natural tight-binding peptide, TRTK-12. The helical conformation of the peptide was constrained by the substitution of α-amino isobutyric acid--an amino acid having high helical propensity--in positions which do not interact with S100B. A branched bidentate version of the peptide was bound to S100B tightly with a dissociation constant of 8 nM. When conjugated to a cell-penetrating peptide, it caused growth inhibition and rapid apoptosis in melanoma cells. The molecule exerts antiproliferative action through simultaneous inhibition of key growth pathways, including reactivation of wild-type p53 and inhibition of Akt and STAT3 phosphorylation. The apoptosis induced by the bidentate constrained helix is caused by direct migration of p53 to mitochondria. At moderate intravenous dose, the peptide completely inhibits melanoma growth in a mouse model without any significant observable toxicity. The specificity was shown by lack of ability of a double mutant peptide to cause tumor regression at the same dose level. The methodology described here for direct protein-protein interaction inhibition may be effective for rapid development of inhibitors against relatively weak protein-protein interactions for de novo drug development.

Reduced phosphorylation of Stat3 at Ser-727 mediated by casein kinase 2 - protein phosphatase 2A enhances Stat3 Tyr-705 induced tumorigenic potential of glioma cells.

Signal transducer and activator of transcription 3 (Stat3) is a transcription factor that is involved in cell survival and proliferation and has been found to be persistently activated in most human cancers mainly through its phosphorylation at Tyr-705. However, the role and regulation of Stat3 Ser-727 phosphorylation in cancer cells have not been clearly evaluated. In our findings, correlation studies on the expression of CK2 and Stat3 Ser-727 phosphorylation levels in human glioma patient samples as well as rat orthotopic tumor model show a degree of negative correlation. Moreover, brain tumor cell lines were treated with various pharmacological inhibitors to inactivate the CK2 pathway. Here, increased Stat3 Ser-727 phosphorylation upon CK2 inhibition was observed. Overexpression of CK2 (α, α' or ß subunits) by transient transfection resulted in decreased Stat3 Ser-727 phosphorylation. Stat3 Tyr-705 residue was conversely phosphorylated in similar situations. Interestingly, we found PP2A, a protein phosphatase, to be a mediator in the negative regulation of Stat3 Ser-727 phosphorylation by CK2. In vitro assays prove that Ser-727 phosphorylation of Stat3 affects the transcriptional activity of its downstream targets like SOCS3, bcl-xl and Cyclin D1. Stable cell lines constitutively expressing Stat3 S727A mutant showed increased survival, proliferation and invasion which are characteristics of a cancer cell. Rat tumor models generated with the Stat3 S727A mutant cell line formed more aggressive tumors when compared to the Stat3 WT expressing stable cell line. Thus, in glioma, reduced Stat3 Ser-727 phosphorylation enhances tumorigenicity which may be regulated in part by CK2-PP2A pathway.

Mechanism of ß-catenin-mediated transcriptional regulation of epidermal growth factor receptor expression in glycogen synthase kinase 3 ß-inactivated prostate cancer cells.

Wnt/ß-catenin and EGFR pathways are important in cancer development and often aberrantly activated in human cancer. However, it is very important to understand the mechanism responsible for this activation and the relation between them. Here, we report the mechanism of EGFR expression by transcriptionally active ß-catenin in GSK3ß-inactivated prostate cancer cells that eventually leads to its enhanced proliferation and survival. Expressions of ß-catenin and EGFR are elevated in various cancers specifically in prostate cancer cells, DU145. When GSK3ß is inactivated in these cells, ß-catenin gets stabilized, phosphorylated at Ser-552 by protein kinase A, accumulates in the nucleus, and regulates the expression of its target genes that include EGFR. Chromatin immunoprecipitation (ChIP) and promoter analysis revealed that the EGFR promoter gets occupied by transcriptionally active ß-catenin when elevated in GSK3ß-inactivated cells. This phenomenon not only leads to increased expression of EGFR but also initiates the activation of its downstream molecules such as ERK1/2 and Stat3, ultimately resulting in up-regulation of multiple genes involved in cell proliferation and survival.

The chaperone-assisted E3 ligase C terminus of Hsc70-interacting protein (CHIP) targets PTEN for proteasomal degradation.

The tumor suppressor, PTEN is key to the regulation of diverse cellular processes, making it a prime candidate to be tightly regulated. The PTEN level is controlled in a major way by E3 ligase-mediated degradation through the Ubiquitin-Proteasome System (UPS). Nedd 4-1, XIAP, and WWP2 have been shown to maintain PTEN turnover. Here, we report that CHIP, the chaperone-associated E3 ligase, induces ubiquitination and regulates the proteasomal turnover of PTEN. It was apparent from our findings that PTEN transiently associates with the molecular chaperones and thereby gets diverted to the degradation pathway through its interaction with CHIP. The TPR domain of CHIP and parts of the N-terminal domain of PTEN are required for their interaction. Overexpression of CHIP leads to elevated ubiquitination and a shortened half-life of endogenous PTEN. On the other hand, depletion of endogenous CHIP stabilizes PTEN. CHIP is also shown to regulate PTEN-dependent transcription presumably through its down-regulation. PTEN shared an inverse correlation with CHIP in human prostate cancer patient samples, thereby triggering the prospects of a more complex mode of PTEN regulation in cancer.