Abstract Background: The eukaryotic heat shock protein 90 (Hsp90) is a ubiquitous chaperone protein that regulates many client proteins involved in signal transduction, cell cycle control and transcriptional regulation. These include the kinases implicated in tumorigenesis such as EGFR, Raf-1, ErbB-2, v-Src family kinases and Cdk4. Our previous data indicated that the interaction of Hsp90 with EGFR is critical for head and neck cancer cell survival upon treatment with cisplatin. We have also identified a potential region in EGFR that is involved in its interaction with Hsp90. We hypothesized that a small peptide analogous to this binding region of EGFR will competitively inhibit Hsp90 binding to EGFR, and therefore EGFR will not be protected from cisplatin induced degradation leading to cell death. Methods and Results: Using Immunoblotting and clonogenic survival assays in eight head and neck squamous cancer cell lines we found that cisplatin-induced EGFR degradation significantly correlates with cytotoxicity. Immunoprecipitation of Hsp90 and/or EGFR showed that cisplatin induced EGFR degradation followed with its dissociation from Hsp90 in cisplatin sensitive cells. In contrary, cisplatin resistant cells showed a stabilized Hsp90-EGFR interaction and therefore EGFR was protected from cisplatin induced degradation. We also found that the Hsp90-EGFR interaction is reduced between EGFR mutants for the binding region of Hsp90 when compared to wild-type EGFR in Chinese hamster ovary cells (EGFR negative). Therefore, we design peptides targeting Hsp90-EGFR binding region. Importantly, we found that transfection of a novel peptide (8 amino acids) abolished the Hsp90-EGFR interaction in cisplatin resistant UMSCC-1 cells, promoted EGFR degradation and sensitized these cells to cisplatin in both culture and xenograft models. We further confirmed by using HIV-TAT-biotin derivative of this peptide that the peptide is getting internalized in cells and tumors. We also found the interaction of specific peptide with Hsp90. Conclusions: Our results indicated that enhanced Hsp90-EGFR interaction is an adaptive response that can protect EGFR degradation induced by cisplatin treatment. We have identified a region in EGFR which is required for its interaction with Hsp90. Our data also indicated that using a novel peptide this interaction can be disrupted which promotes EGFR degradation and increases cytotoxicity in both culture and xenograft models. We are now investigating the role of this peptide in radiosensitization in head and neck carcinoma cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5451.
Transcriptional dysregulation is a hallmark of prostate cancer (PCa). We mapped the RNA polymerase II-associated (RNA Pol II-associated) chromatin interactions in normal prostate cells and PCa cells. We discovered thousands of enhancer-promoter, enhancer-enhancer, as well as promoter-promoter chromatin interactions. These transcriptional hubs operate within the framework set by structural proteins - CTCF and cohesins - and are regulated by the cooperative action of master transcription factors, such as the androgen receptor (AR) and FOXA1. By combining analyses from metastatic castration-resistant PCa (mCRPC) specimens, we show that AR locus amplification contributes to the transcriptional upregulation of the AR gene by increasing the total number of chromatin interaction modules comprising the AR gene and its distal enhancer. We deconvoluted the transcription control modules of several PCa genes, notably the biomarker KLK3, lineage-restricted genes (KRT8, KRT18, HOXB13, FOXA1, ZBTB16), the drug target EZH2, and the oncogene MYC. By integrating clinical PCa data, we defined a germline-somatic interplay between the PCa risk allele rs684232 and the somatically acquired TMPRSS2-ERG gene fusion in the transcriptional regulation of multiple target genes - VPS53, FAM57A, and GEMIN4. Our studies implicate changes in genome organization as a critical determinant of aberrant transcriptional regulation in PCa.
The epidermal growth factor receptor (EGFR) has been targeted for inhibition using tyrosine kinase inhibitors and monoclonal antibodies, with improvement in outcome in subsets of patients with head and neck, lung, and colorectal carcinomas. We have previously found that EGFR stability plays a key role in cell survival after chemotherapy and radiotherapy. Heat shock protein 90 (HSP90) is known to stabilize mutant EGFR and ErbB2, but its role in cancers with wild-type (WT) WT-EGFR is unclear. In this report, we demonstrate that fully mature, membrane-bound WT-EGFR interacts with HSP90 independent of ErbB2. Further, the HSP90 inhibitors geldanamycin (GA) and AT13387 cause a decrease in WT-EGFR in cultured head and neck cancer cells. This decrease results from a significantly reduced half-life of WT-EGFR. WT-EGFR was also lost in head and neck xenograft specimens after treatment with AT13387 under conditions that inhibited tumor growth and prolonged survival of the mice. Our findings demonstrate that WT-EGFR is a client protein of HSP90 and that their interaction is critical for maintaining both the stability of the receptor as well as the growth of EGFR-dependent cancers. Furthermore, these findings support the search for specific agents that disrupt HSP90's ability to act as an EGFR chaperone.
An eight-amino acid segment is known to be responsible for the marked difference in the rates of degradation of the EGF receptor (ErbB1) and ErbB2 upon treatment of cells with the Hsp90 inhibitor geldanamycin. We have scrambled the first six amino acids of this segment of the EGF receptor (EGFR), which lies in close association with the ATP binding cleft and the dimerization face. Scrambling these six amino acids markedly reduces EGFR stability, EGF-stimulated receptor dimerization, and autophosphorylation activity. Two peptides were synthesized as follows: one containing the wild-type sequence of the eight-amino acid segment, which we call Disruptin; and one with the scrambled sequence. Disruptin inhibits Hsp90 binding to the EGFR and causes slow degradation of the EGFR in two EGFR-dependent cancer cell lines, whereas the scrambled peptide is inactive. This effect is specific for EGFR versus other Hsp90 client proteins. In the presence of EGF, Disruptin, but not the scrambled peptide, inhibits EGFR dimerization and causes rapid degradation of the EGFR. In contrast to the Hsp90 inhibitor geldanamycin, Disruptin inhibits cancer cell growth by a nonapoptotic mechanism. Disruptin provides proof of concept for the development of a new class of anti-tumor drugs that specifically cause EGFR degradation.Background: An eight-amino acid segment lying within the αC-β4 loop region of many protein kinases determines sensitivity to Hsp90 inhibitors.Results: A peptide comprised of this segment of the EGFR inhibits both Hsp90 binding and EGF-dependent EGFR dimerization.Conclusion: The peptide selectively degrades EGFR versus other Hsp90 clients.Significance: This peptide represents a unique approach to the therapy of EGFR-driven tumors. An eight-amino acid segment is known to be responsible for the marked difference in the rates of degradation of the EGF receptor (ErbB1) and ErbB2 upon treatment of cells with the Hsp90 inhibitor geldanamycin. We have scrambled the first six amino acids of this segment of the EGF receptor (EGFR), which lies in close association with the ATP binding cleft and the dimerization face. Scrambling these six amino acids markedly reduces EGFR stability, EGF-stimulated receptor dimerization, and autophosphorylation activity. Two peptides were synthesized as follows: one containing the wild-type sequence of the eight-amino acid segment, which we call Disruptin; and one with the scrambled sequence. Disruptin inhibits Hsp90 binding to the EGFR and causes slow degradation of the EGFR in two EGFR-dependent cancer cell lines, whereas the scrambled peptide is inactive. This effect is specific for EGFR versus other Hsp90 client proteins. In the presence of EGF, Disruptin, but not the scrambled peptide, inhibits EGFR dimerization and causes rapid degradation of the EGFR. In contrast to the Hsp90 inhibitor geldanamycin, Disruptin inhibits cancer cell growth by a nonapoptotic mechanism. Disruptin provides proof of concept for the development of a new class of anti-tumor drugs that specifically cause EGFR degradation. Background: An eight-amino acid segment lying within the αC-β4 loop region of many protein kinases determines sensitivity to Hsp90 inhibitors. Results: A peptide comprised of this segment of the EGFR inhibits both Hsp90 binding and EGF-dependent EGFR dimerization. Conclusion: The peptide selectively degrades EGFR versus other Hsp90 clients. Significance: This peptide represents a unique approach to the therapy of EGFR-driven tumors. A New Way to Target Degradation of the Epidermal Growth Factor Receptor in Cancer Cells♦: Destabilization of the Epidermal Growth Factor Receptor (EGFR) by a Peptide That Inhibits EGFR Binding to Heat Shock Protein 90 and Receptor DimerizationJournal of Biological ChemistryVol. 288Issue 37Preview♦ See referenced article, J. Biol. Chem. 2013, 288, 26879–26886 Full-Text PDF Open Access
'/%3e%3c/defs%3e%3cpath fill='%23012169' d='M0 0h10080v5040H0z'/%3e%3cpath stroke='%23fff' stroke-width='.6' d='m0 0 6 3m0-3L0 3' clip-path='url(%23b)' transform='scale(840)'/%3e%3cpath stroke='%23e4002b' stroke-width='.4' d='m0 0 6 3m0-3L0 3' clip-path='url(%23c)' transform='scale(840)'/%3e%3cpath stroke='%23fff' stroke-width='840' d='M2520 0v2520M0 1260h5040'/%3e%3cpath stroke='%23e4002b' stroke-width='504' d='M2520 0v2520M0 1260h5040'/%3e%3cg fill='%23fff'%3e%3cuse xlink:href='%23d' x='2520' y='3780'/%3e%3cuse xlink:href='%23a' x='7560' y='4200'/%3e%3cuse xlink:href='%23a' x='6300' y='2205'/%3e%3cuse xlink:href='%23a' x='7560' y='840'/%3e%3cuse xlink:href='%23a' x='8680' y='1869'/%3e%3cuse xlink:href='%23e' x='8064' y='2730'/%3e%3c/g%3e%3c/svg%3e)