Abstract IA28: Targeting stress-induced molecular chaperones CLU and Hsp27 to inhibit EMT and metastases
Authors: Gleave Martin , Zoubeidi Amina , Chi Kim 
Published: August 2014

Stress-activated adaptive pathways help drive cancer cell survival, metastases, and acquired treatment resistance. Heat shock and other molecular chaperones play central roles in these stress responses, regulating protein homeostasis (proteostasis) as well as many signaling and transcriptional survival networks to facilitate cell survival and treatment resistance. Clusterin (CLU) and Hsp27 are stress-activated chaperones that treatment-induced and associated with metastases and treatment resistance in many cancers, functioning to protect cells from many varied therapeutic stressors that induce apoptosis. Both CLU and HSP27 levels increase after androgen deprivation and become highly expressed in metastatic CRPC.

While studies have defined CLU and Hsp27 as key mediators of adaptive survival pathways and treatment resistance, they also regulate epithelial plasticity and epithelial-to-mesenchymal transition (EMT). Defining mechanistic linkages between treatment stress and effectors of EMT will yield new insights into metastasis and treatment strategies. EMT endows malignant cells with enhanced migratory and survival attributes that facilitate cancer metastases. EMT can be initiated by a wide variety of signals in the tumor environment that activate the transcription factors Twist and Snail, leading to repression of epithelial cell markers like E-cadherin and cytokeratins, and gain of mesenchymal markers like N-cadherin and vimentin. We recently defined roles for both CLU and Hsp27, downstream of TGF-β and IL-6, respectively, as key mediators of EMT and metastases. We found that factors such as TGF-β and IL-6 known to induce EMT, up-regulate molecular chaperones CLU and Hsp27. We then set out to define molecular mechanisms by which TGF-β and IL-6 regulate CLU and Hsp27 expression, respectively, and whether CLU and Hsp27 mediate TGF-β and IL-6 regulated EMT and metastasis.

CLU is a mediator of TGF-β-induced EMT. Our data show that the transcription factor Twist1 mediates TGF-β-induced CLU expression, binding to E-boxes in the distal promoter region of CLU gene to regulate basal and TGF-β-induced CLU transcription. TGF-β induced stimulation of Twist1 as well as CLU transcript expression was potently suppressed by Twist1 silencing. CLU silencing reduced TGF-β induction of the mesenchymal markers N-cadherin and fibronectin, and inhibited migratory and invasive properties induced by TGF-β. Targeted inhibition of CLU also suppressed metastasis in an in vivo model. Taken together, these findings identify CLU as a mediator of TGF-β-induced EMT, and suggest that CLU suppression may represent a promising therapeutic option for suppressing prostate cancer metastatic progression.

Hsp27 facilitates IL-6 mediated EMT via modulation of STAT3/Twist. In addition to a role for CLU in EMT, we found that Hsp27 overexpression drives, while its knockdown reverses, EMT, with congruent effects on cell migration, invasion and matrix metalloprotease activity. Mechanistically, silencing Hsp27 decreased IL-6 dependent STAT3 phosphorylation, nuclear translocation, and abrogated STAT3 binding to the Twist promoter, suggesting that Hsp27 is required for IL-6 mediated EMT via modulation of STAT3/Twist signaling. Elevated expression of Hsp27 and Twist were also found in high grade PCa tumors. Overall, these data define a role of Hsp27 as a critical regulator of IL-6 dependent and independent EMT and highlights Hsp27 as an attractive anti-cancer target for metastatic CRPC.

CLU and Hsp27 as Anti-Cancer Targets. Since Hsp27 and CLU are ATP-independent chaperones and not amenable to inhibition by small molecules, antisense strategies have been used to therapeutically target this chaperones, with evidence of anti-cancer activity in clinical trials. We developed OGX-011 as a CLU inhibitor and demonstrated pre-clinical activity and dose-dependent target suppression in Phase I studies. Moreover, we led a randomized Phase II trial that reported a 7-month-survival benefit when the CLU inhibitor, OGX-011, was combined with docetaxel in patients with CRPC; OGX-011 is now in phase III trials in metastatic CRPC and NSCLC.

We also developed OGX-427 as an Hsp27 inhibitor and demonstrated pre-clinical activity and tolerability in Phase I studies. A randomized phase II study of OGX-427 plus Prednisone (P) vs. P alone enrolled 64 mCRPC patients. At 12 weeks, the proportion of patients progression free was 71% in OGX-427+P treated patients and 40% in patients on P. A ≥50% PSA decline occurred in 50% of patients on OGX-427+P and 20% patients treated with P. Measurable disease response occurred in 44% on OGX-427+P and 0 patients on P. CTC conversion from ≥5 to &lt;5/7.5 ml occurred in 55% evaluable patients on OGX-427+P and 41% treated with P. These results confirm, for the first time, single agent activity for an Hsp27 inhibitor in cancer, and Phase II trials in metastatic bladder and CRPC are ongoing.

Citation Format: Martin Gleave, Amina Zoubeidi, Kim Chi. Targeting stress-induced molecular chaperones CLU and Hsp27 to inhibit EMT and metastases. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr IA28.