Heat-shock factor 1 both positively and negatively affects cellular clonogenic growth depending on p53 status.

HSF1 (heat-shock factor 1) is the master regulator of the heat-shock response; however, it is also activated by cancer-associated stresses and supports cellular transformation and cancer progression. We examined the role of HSF1 in relation to cancer cell clonogenicity, an important attribute of cancer cells. Ectopic expression or HSF1 knockdown demonstrated that HSF1 positively regulated cancer cell clonogenic growth. Furthermore, knockdown of mutant p53 indicated that HSF1 actions were mediated via a mutant p53-dependent mechanism. To examine this relationship more specifically, we ectopically co-expressed mutant p53(R273H) and HSF1 in the human mammary epithelial cell line MCF10A. Surprisingly, within this cellular context, HSF1 inhibited clonogenicity. However, upon specific knockdown of endogenous wild-type p53, leaving mutant p53(R273H) expression intact, HSF1 was observed to greatly enhance clonogenic growth of the cells, indicating that HSF1 suppressed clonogenicity via wild-type p53. To confirm this we ectopically expressed HSF1 in non-transformed and H-Ras(V12)-transformed MCF10A cells. As expected, HSF1 significantly reduced clonogenicity, altering wild-type p53 target gene expression levels consistent with a role of HSF1 increasing wild-type p53 activity. In support of this finding, knockdown of wild-type p53 negated the inhibitory effects of HSF1 expression. We thus show that HSF1 can affect clonogenic growth in a p53 context-dependent manner, and can act via both mutant and wild-type p53 to bring about divergent effects upon clonogenicity. These findings have important implications for our understanding of HSF1's divergent roles in cancer cell growth and survival as well as its disparate effect on mutant and wild-type p53.

HSP90 inhibitors enhance differentiation and MITF (microphthalmia transcription factor) activity in osteoclast progenitors.

The HSP90 (heat-shock protein 90) inhibitor 17-AAG (17-allylamino-demethoxygeldanamycin) increases osteoclast formation both in vitro and in vivo, an action that can enhance cancer invasion and growth in the bone microenvironment. The cellular mechanisms through which 17-AAG exerts this action are not understood. Thus we sought to clarify the actions of 17-AAG on osteoclasts and determine whether other HSP90 inhibitors had similar properties. We determined that 17-AAG and the structurally unrelated HSP90 inhibitors CCT018159 and NVP-AUY922 dose-dependently increased RANKL [receptor activator of NF-κB (nuclear factor κB) ligand]-stimulated osteoclastogenesis in mouse bone marrow and pre-osteoclastic RAW264.7 cell cultures. Moreover, 17-AAG also enhanced RANKL- and TNF (tumour necrosis factor)-elicited osteoclastogenesis, but did not affect RANKL-induced osteoclast survival, suggesting that only differentiation mechanisms are targeted. 17-AAG affected the later stages of progenitor maturation (after 3 days of incubation), whereas the osteoclast formation enhancer TGFß (transforming growth factor ß) acted prior to this, suggesting different mechanisms of action. In studies of RANKL-elicited intracellular signalling, 17-AAG treatment did not increase c-Fos or NFAT (nuclear factor of activated T-cells) c1 protein levels nor did 17-AAG increase activity in luciferase-based NF-κB- and NFAT-response assays. In contrast, 17-AAG treatment (and RANKL treatment) increased both MITF (microphthalmia-associated transcription factor) protein levels and MITF-dependent vATPase-d2 (V-type proton ATPase subunit d2) gene promoter activity. These results indicate that HSP90 inhibitors enhance osteoclast differentiation in an NFATc1-independent manner that involves elevated MITF levels and activity.