Tumour cell invasiveness is crucial for cancer metastasis and is not yet understood. Here we describe two functional screens for proteins required for the invasion of fibrosarcoma cells that identified the molecular chaperone heat shock protein 90 (hsp90). The hsp90 alpha isoform, but not hsp90 beta, is expressed extracellularly where it interacts with the matrix metalloproteinase 2 (MMP2). Inhibition of extracellular hsp90 alpha decreases both MMP2 activity and invasiveness. This role for extracellular hsp90 alpha in MMP2 activation indicates that cell-impermeant anti-hsp90 drugs might decrease invasiveness without the concerns inherent in inhibiting intracellular hsp90.
Heat-shock protein 90 (Hsp90) chaperones a key subset of signaling proteins and is necessary for malignant transformation. Hsp90 is subject to an array of posttranslational modifications that affect its function, including acetylation. Histone deacetylase (HDAC) inhibitors and knockdown of HDAC6 induce Hsp90 acetylation and inhibit its activity. However, direct determination of the functional consequences of Hsp90 acetylation has awaited mapping of specific sites. We now demonstrate that Hsp90 K294 is acetylated. Mutational analysis of K294 shows that its acetylation status is a strong determinant of client protein and cochaperone binding. In yeast, Hsp90 mutants that cannot be acetylated at K294 have reduced viability and chaperone function compared to WT or to mutants that mimic constitutive acetylation. These data suggest that acetylation/deacetylation of K294 plays an important role in regulating the Hsp90 chaperone cycle.
Although therapeutically targeting a single signaling pathway that drives tumor development and/or progression has been effective for a number of cancers, in many cases this approach has not been successful. Targeting networks of signaling pathways, instead of isolated pathways, may overcome this problem, which is probably due to the extreme heterogeneity of human tumors. However, the possibility that such networks may be spatially arranged in specialized subcellular compartments is not often considered in pathway-oriented drug discovery and may influence the design of new agents. Hsp90 is a chaperone protein that controls the folding of proteins in multiple signaling networks that drive tumor development and progression. Here, we report the synthesis and properties of Gamitrinibs, a class of small molecules designed to selectively target Hsp90 in human tumor mitochondria. Gamitrinibs were shown to accumulate in the mitochondria of human tumor cell lines and to inhibit Hsp90 activity by acting as ATPase antagonists. Unlike Hsp90 antagonists not targeted to mitochondria, Gamitrinibs exhibited a "mitochondriotoxic" mechanism of action, causing rapid tumor cell death and inhibiting the growth of xenografted human tumor cell lines in mice. Importantly, Gamitrinibs were not toxic to normal cells or tissues and did not affect Hsp90 homeostasis in cellular compartments other than mitochondria. Therefore, combinatorial drug design, whereby inhibitors of signaling networks are targeted to specific subcellular compartments, may generate effective anticancer drugs with novel mechanisms of action.
Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an inherited cancer syndrome linked to biallelic inactivation of the gene encoding the tricarboxylic acid cycle enzyme fumarate hydratase (FH).Individuals with HLRCC are at risk to develop cutaneous and uterine leiomyomas and an aggressive form of kidney cancer. Pseudohypoxic drive-the aberrant activation of cellular hypoxia response pathways despite normal oxygen tension-is considered to be a likely mechanism underlying the etiology of this tumor. Pseudohypoxia requires the oxygen-independent stabilization of the ␣ subunit of the hypoxia-inducible transcription factor (HIF-1␣). Under normoxic conditions, proline hydroxylation of HIF-1␣ permits VHL recognition and subsequent targeting for proteasomal degradation. Here, we demonstrate that inactivating mutations of FH in an HLRCC-derived cell line result in glucose-mediated generation of cellular reactive oxygen species (ROS) and ROS-dependent HIF-1␣ stabilization. Additionally, we demonstrate that stable knockdown of FH in immortalized renal epithelial cells results in ROS-dependent HIF-1␣ stabilization. These data reveal that the obligate glycolytic switch present in HLRCC is critical to HIF stabilization via ROS generation.Patients with hereditary leiomyomatosis and renal cell cancer (HLRCC) harbor germ line mutations of the FH gene, which encodes the tricarboxylic acid cycle enzyme fumarate hydratase, and affected individuals are at risk for the development of leiomyomas of the skin and uterus (fibroids) as well as kidney cancer (11,25,37). Genetic analysis of tumor samples indicates that FH acts as a tumor suppressor gene (37). The renal tumors that develop in HLRCC patients are notable for their aggressiveness, and effective systemic therapies are lacking at this time. Hence, identification of the molecular mechanisms that underlie the pathogenesis of this disease is needed to facilitate the development of targeted therapeutic strategies. Moreover, such studies may provide further insight into the role of mitochondrial metabolism in both normal and aberrant cellular physiology.FH catalyzes the enzymatic step of the tricarboxylic (TCA) cycle that hydrates fumarate to form malate. Proposed mechanisms for HLRCC tumor formation include apoptotic resistance, oxidative stress, and pseudohypoxic drive (10). Of these, most reports to date support a role for pseudohypoxic drive, based specifically on studies of hypoxia-inducible transcription factor 1␣ (HIF-1␣) expression. Pseudohypoxia is defined as the aberrant activation of hypoxia response pathways under normal oxygen conditions. HIF-1␣ expression is elevated both in HLRCC tumor specimens and in normoxic cells in which FH expression has been transiently suppressed with small interfering RNA (siRNA) (16). HIF-2␣ expression is also elevated in HLRCC tumor samples, although to a lesser extent than is HIF-1␣. In addition, there is clear evidence of upregulated transcription of HIF target genes in HLRCC tumor samples and in FH siRNA-treated cells (16,30). Fur...
Summary Swe1 (Saccharomyces WEE1), the only “true” tyrosine kinase in budding yeast, is an Hsp90 client protein. Here we show that Swe1Wee1 phosphorylates a conserved tyrosine residue (Y24 in yeast Hsp90 and Y38 in human Hsp90α) in the N-domain of Hsp90. Phosphorylation is cell cycle-associated and modulates the ability of Hsp90 to chaperone a selected clientele, including v-Src and several other kinases. Non-phosphorylatable mutants have normal ATPase activity, support yeast viability, and productively chaperone the Hsp90 client glucocorticoid receptor. Deletion of SWE1 in yeast increases Hsp90 binding to its inhibitor geldanamycin, and pharmacologic inhibition/silencing of Wee1 sensitizes cancer cells to Hsp90 inhibitor-induced apoptosis. These findings demonstrate that Hsp90 chaperoning of distinct client proteins is differentially regulated by specific post-translational modification of a unique subcellular pool of the chaperone, and they provide a novel strategy to increase the cellular potency of Hsp90 inhibitors.
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