Geldanamycins Trigger a Novel Ron Degradative Pathway, Hampering Oncogenic Signaling

Abstract

Ron, the tyrosine kinase receptor for macrophage-stimulating protein is responsible for proliferation and migration of cells from different tissues. Ron can acquire oncogenic potential by single point mutations in the kinase domain, and dysregulated Ron signaling has been involved in the development of different human cancers. We have previously shown that ligand-activated Ron recruits the negative regulator c-Cbl, which mediates its ubiquitylation and degradation. Here we report that Ron is ubiquitylated also by the U-box E3 ligase C-terminal Hsc70-interacting protein (CHIP), recruited via chaperone intermediates Hsp90 and Hsc70. Gene silencing shows that CHIP activity is necessary to mediate Ron degradation upon cell treatment with Hsp90 inhibitors geldanamycins. The oncogenic Ron M1254T receptor escapes from c-Cbl negative regulation but retains a strong association with CHIP. This constitutively active mutant of Ron displays increased sensitivity to geldanamycins, enhanced physical interaction with Hsp90, and more rapid degradation rate. Cell growth and migration, as well as the transforming potential evoked by Ron M1254T , are abrogated upon Hsp90 inhibition. These data highlight a novel mechanism for Ron degradation and propose Hsp90 antagonists like geldanamycins as suitable pharmacological agents for therapy of cancers where altered Ron signaling is involved. Dysregulated activation of receptor tyrosine kinases (RTKs)3 has been extensively documented in different types of human tumors (1) and frequently correlates with poor responsiveness to conventional therapies, pointing at RTKs as potential targets for molecule-based cancer therapy (2, 3). Among the different therapeutic approaches a promising strategy relies on forcing RTKs toward degradative pathways (4).The Ron tyrosine kinase, receptor for macrophage-stimulating protein (MSP), is a member of the hepatocyte growth factor (HGF) receptor subfamily (5, 6). Ron is expressed in a variety of human tissues, and the engagement by its cognate ligand activates multiple intracellular signaling pathways controlling normal cell proliferation, migration, and adhesion-dependent survival (7-9). A single point mutation (M1254T), targeted to a conserved residue of the tyrosine kinase domain is oncogenic (Ron M1254T ), by increasing kinase efficiency and subverting substrate specificity (10, 11). Growing evidence indicates that Ron can be involved in cancer development and progression in humans (12, 13) and in murine models (14, 15). Therefore, targeting Ron expression by forcing its down-regulation may help to elucidate its role in tumor development and progression.It has been reported that many kinases that are deregulated in human cancers are dependent on the chaperone activity of the Heat shock protein 90 (Hsp90) for their conformational maturation and stability (16). Hsp90 is a ubiquitous chaperone protein abundantly expressed in mammalian cells, where it performs housekeeping functions assisting in the folding, activation, and assembly of a variety of proteins...

Degradation of HER2 by Cbl-Based Chimeric Ubiquitin Ligases

“…Therefore, Hsp90 inhibitors, by causing inactivation, destabilization, and eventual degradation of the client proteins, exhibit potent antitumor activity (46). Growing evidences showed that such effects are mostly mediated by chaperone-dependent ubiquitin ligase CHIP, which is independent of Cbl ubiquitin ligase and does not require the kinase activity of oncoproteins (32,35,(47)(48)(49). More importantly, nascent kinases and some mutant forms of RTKs, which either escape from Cbl-negative regulation or resist to their inhibitors, still remain sensitive to CHIP overexpressionor geldanamycin/17-AAG-induced degradation (32,(48)(49)(50).…”

Smad4-dependent TGF-β Signaling Suppresses RON Receptor Tyrosine Kinase-dependent Motility and Invasion of Pancreatic Cancer Cells

“…These studies imply that Smads act as transcriptional repressors of RON, perhaps in a manner similar to that seen for c-Myc. Studies by others (35) show that RON protein turnover is mediated through ubiquitylation/proteasome pathway. Our studies indicate that in the absence of exogenous RON ligand RON protein is relatively long lived in both Smad4-intact and Smad4-deficient cells.…”

“…This finding suggests that RON protein has a relatively long half-life in both Smad4-intact and Smad4-deficient cells. A recent study (35) showed that degradation of RON protein is through an ubiquitin-proteasome pathway and that heat shock protein 90 (Hsp90), a chaperone protein, stabilizes the RON from proteasome degradation. To examine whether Smad4 might accelerate RON degradation by this mechanism, BxPC3 and Bx/Smad4 cells were treated with an Hsp90 inhibitor, 17-allylamino-17-demethoxy-geldanamycin (17-AAG), and a proteasome inhibitor, MG132.…”

Ron-Receptor Tyrosine Kinase In Tumorigenesis and Metastasis

“…This molecule is able to inhibit HGFL-induced phosphorylation of Ron and also has inhibitory effects on the growth of colon cancer cells [54]. Another possible treatment modality for negatively regulating receptor tyrosine kinase activity in human cancers include a class of anti-tumor drugs called geldanamycins, which have recently been shown to inhibit tumor cell growth by preventing proper folding and degradation of oncogenic proteins, including the Ron receptor [55]. These small molecules that negatively modulate Ron activity provide an alternative approach to targeting this receptor in vivo.…”