HSF1 associates with metastasis associated protein 1 (MTA1) as well as other NURD complex members (HDAC1, HDAC2) in a manner that is enhanced by either heregulin exposure or heat shock

A. KHALEQUE; A. BHARTI; J. GONG; D.R. CIOCCA; A. STATI; M. FANELLI,; F.D. CUELLO-CARRIÓN; S.K. CALDERWOOD

Universidad de Concepción, Concepción, Chile

Workshop; 5th International Workshop on the Molecular Biology of Sress Responses; 2006

Cell Stress Society International

The mechanisms underlying elevated Hsp expression in tumor cells are not known, but may reflect the genetic alterations accompanying malignancy or the disordered state of the tumor microenvironment which would be expected to lead to cellular stress. These changes may reflect the altered activity of HSF1 which is elevated in concentration in some carcinomas. Our recent studies have suggested for the first time a mechanism that may underlie the elevation of Hsp seen in malignant cells involving the secreted survival factor heregulin. Heregulin is a very effective inducer of HSF1 activity and of Hsp synthesis in breast cancer cells as well as other cells expressing receptors for heregulin. Heregulin is known to increase the expression of a class of pro-metastatic proteins (metastatic associated proteins, MTA). These include MTA1, a protein found elevated in breast carcinomas and other cancers. MTA1 functions (at least partially) as a component of the NURD complex, a co-repressor complex with both ATP-dependent chromatin remodeling and histone deacetylase (HDAC) activity. Kumar and co-workers have recently shown that MTA1 can repress the estrogen receptor in a heregulin dependent manner, evidently by recruitment of the NURD complex to estrogen responsive elements (ERE) containing promoters. We have shown previously that HSF1 is a potent gene repressor and have defined two independent repression domains on HSF1 as well as a complex mechanism of target gene repression involving the quenching of activating factors on the promoter as well as the recruitment of co-repressor molecules. In the present study we have explored the association of HSF1 with MTA1 and with other components of the NURD complex, observing the effects of heregulin and heat shock. MCF7, HeLa, MEF and MEF hsf1-/- cells were used for the studies. Cells were treated with 1 nM HRG-b1 for 24 h or 48 h. In some cases cells were treated with inhibitors [AG-825 (50 mM), PD98059 (50 mM) or LY-294, 002 (30 mM)] for 2 hr prior to HRG-b1 treatment. Heat shock was carried out at 430C for 30 minutes. To induce apoptosis cells were either treated with cis-platinum II (cis-diamminedichloroplatinum, 100 mM) or heat shocked at 470C for 1 h after the HRGb1 treatment. The proteins were detected by immunoblot with and without immunoprecipitation. Luciferase and b-galactosidase activity assays were performed. Colony formation was scored for anchorage independent growth. Finally, the tissue co-localization of HSF1 and MTA1 was studied in human breast cancer biopsy samples by immunohistochemistry. Western analysis of the GST pulldown indicated efficient association of HSF1 with MTA1 particularly under conditions of HSF1 overexpression. We have confirmed these experiments using immunoprecipitation from the HeLa lysates. In cells expressing Myc-MTA1, HSF1 was co-immunoprecipitated and the degree of HSF1 recovery was increased by HSF1 overexpression. We also carried out HSF1 immunoprecipitation using cells overexpressing either Flag-HSF1 or control Flag tag and observed the co-immunoprecipitation of MTA1 with Flag-HSF1 but not Flag control. As most HSF1 exists in innate complexes in untreated cells we examined the effects of HSF1 activation by stress on the association of HSF1 with MTA1. Heat shock caused an increase in MTA1 association with Flag-HSF1 and this effect was amplified in cells overexpressing Myc-MTA1. We next examined the potential association of HSF1 with other components of the NURD complex, showing that histone deacetylase 1 (HDAC1) is co-immunoprecipitated with HSF1 in cells overexpressing Flag-HSF1 and probed by anti-HDAC1. In addition association of HDAC1 with HSF1 was increased by heat shock activation. We found that heregulin induces MTA1 expression in a HSF1 dependent manner and that HSF1 and MTA1 cooperate in gene repression. The tissue co-localization of HSF1 and MTA1 was confirmed by immunohistochemistry in human breast cancer samples. Our experiments indicate that HSF1 binds MTA1 in a heregulin dependent manner and represses target promoters, both proteins co-localize in human breast tumors. Our experiments show that HSF1 associates with MTA1 as well as other NURD complex members (HDAC1, HDAC2) in a manner that is enhanced by either heregulin exposure or heat shock