Inhibition of Hsp90 Leads to Cell Cycle Arrest and Apoptosis in Human Malignant Pleural Mesothelioma

Thoracic Oncology Laboratory, Department of Surgery, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, California, USA.
Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer (Impact Factor: 5.28). 11/2008; 3(10):1089-95. DOI: 10.1097/JTO.0b013e3181839693
Source: PubMed


Heat shock protein 90 (Hsp90) is an abundant molecular chaperone that mediates the maturation and stability of a variety of proteins associated with the promotion of cell growth and survival. Inhibition of Hsp90 function leads to proteasomal degradation of its mis-folded client proteins. Recently, Hsp90 has emerged as being of prime importance to the growth and survival of cancer cells and its inhibitors have already been used in phase I and II clinical trials.
We investigated how 17-allylamino-17-demethoxygeldanamycin (17-AAG), a small molecule inhibitor of Hsp90, is implicated in human malignant pleural mesothelioma (MM).
We found that 17-AAG led to significant G1 or G2/M cell cycle arrest, inhibition of cell proliferation, and decrease of AKT, AKT1, and survivin expression in all human malignant pleural mesothelioma cell lines examined. We also observed significant apoptosis induction in all MM cell lines treated with 17-AAG. Furthermore, 17-AAG induced apoptosis in freshly cultured primary MM cells and caused signaling changes identical to those in 17-AAG treated MM cell lines.
These results suggest that Hsp90 is strongly associated with the growth and survival of MM and that inhibition of Hsp90 may have therapeutic potential in the treatment of MM.

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Available from: Yu-Ching Lin
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    • "However, despite the evidence for tyrosine kinase activation in mesothelioma pathogenesis, targeted antikinase therapies have not shown dramatic clinical activity in mesothelioma patients. However, the observed activation of multiple RTKs in mesothelioma suggests that transforming activity is dependent on coordinated activity of multiple tyrosine kinases (Kawaguchi et al, 2009; Menges et al, 2010; Perrone et al, 2010; Brevet et al, 2011; Ou et al, 2011b), and simultaneous inhibition of multiple kinases by cocktails of small-molecule kinase inhibitors or single-agent HSP90 inhibitors elicits compelling pro-apoptotic and anti-proliferative responses in mesothelioma preclinical models (Okamoto et al, 2008; Kawaguchi et al, 2009; Ou et al, 2011b). In addition to the evidence for PI3K/AKT and RAF/MEK/MAPK activation in mesothelioma initiation, there is likewise substantial evidence that these key signalling pathways are crucial in maintaining the transformed state, and in mesothelioma metastasis (Altomare et al, 2005; Cole et al, 2006; Patel et al, 2007; Jacobson et al, 2009; Suzuki et al, 2009; Shukla et al, 2011; Carbone and Yang, 2012; Cedres et al, 2012; Fischer et al, 2012; Menges et al, 2012; Miyoshi et al, 2012; Pinton et al, 2012). "
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    ABSTRACT: Background: Mesothelioma is a notoriously chemotherapy-resistant neoplasm, as is evident in the dismal overall survival for patients with those of asbestos-associated disease. We previously demonstrated co-activation of multiple receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), MET, and AXL in mesothelioma cell lines, suggesting that these kinases could serve as novel therapeutic targets. Although clinical trials have not shown activity for EGFR inhibitors in mesothelioma, concurrent inhibition of various activated RTKs has pro-apoptotic and anti-proliferative effects in mesothelioma cell lines. Thus, we hypothesised that a coordinated network of multi-RTK activation contributes to mesothelioma tumorigenesis. Methods: Activation of PI3K/AKT/mTOR, Raf/MAPK, and co-activation of RTKs were evaluated in mesotheliomas. Effects of RTK and downstream inhibitors/shRNAs were assessed by measuring mesothelioma cell viability/growth, apoptosis, activation of signalling intermediates, expression of cell-cycle checkpoints, and cell-cycle alterations. Results: We demonstrate activation of the PI3K/AKT/p70S6K and RAF/MEK/MAPK pathways in mesothelioma, but not in non-neoplastic mesothelial cells. The AKT activation, but not MAPK activation, was dependent on coordinated activation of RTKs EGFR, MET, and AXL. In addition, PI3K/AKT/mTOR pathway inhibition recapitulated the anti-proliferative effects of concurrent inhibition of EGFR, MET, and AXL. Dual targeting of PI3K/mTOR by BEZ235 or a combination of RAD001 and AKT knockdown had a greater effect on mesothelioma proliferation and viability than inhibition of individual activated RTKs or downstream signalling intermediates. Inhibition of PI3K/AKT was also associated with MDM2-p53 cell-cycle regulation. Conclusions: These findings show that PI3K/AKT/mTOR is a crucial survival pathway downstream of multiple activated RTKs in mesothelioma, underscoring that PI3K/mTOR is a compelling target for therapeutic intervention.
    Full-text · Article · Apr 2014 · British Journal of Cancer
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    • "When active, mTOR stimulates via S6K1 the translation of many transcription factors which regulate the expression of a large number of genes involved in a multitude of biological processes, including, among the others, cell cycle progression, ribosome biogenesis , transcription, translation and unfolded protein response, which all appear to be significantly altered at 2 – 48 h. The interplay between these processes is a topic which has been largely investigated: the inhibition of cellular transcription negatively affects cell cycle progression (Adolph et al., 1993) and similarly specific phases of translation, such as elongation, both finely regulate and respond to cell cycling (White-Gilbertson et al., 2009); furthermore, the inhibition of the folding of newly produced peptides by HSP90, leads to cell cycle arrest at the G2/S transition (Karkoulis et al., 2013; Okamoto et al., 2008). Other regulatory loops downstream of mTOR contribute to the maintenance of its signaling, the most relevant of which involves ER-␣, the target of two drugs whose effect on transcription was highly connected to fullerene 2, fulvestrant and clomifene. "
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    ABSTRACT: The interest on functionalized fullerenes in the field of nanomedicine has seen a significant increase in the past decade. However, the different methods employed to increase C60 solubility profoundly influence the physicochemical properties and the toxicological effects of these compounds, thus complicating the evaluation of their toxicity and potential therapeutic use. Here we report a whole-transcriptome RNA-seq analysis assessing the effect of two fullerenes (1 and 2) on gene expression in the human MCF7 cell line. Although these two compounds had previously been characterized by in vitro studies as having a cytotoxic and null effect respectively, to date the mechanisms at the basis of this different behavior and, more in general, at the basis of the effect of most fullerene derivatives in living cells are still completely unknown. Our data evidence that: a) fullerene 2 caused a significant, time-dependent alteration of gene expression, whereas 1 only had a negligible effect; b) the biological processes mostly influenced over the 48hours experimental time course were transcription, protein synthesis, cell cycle progression and cell adhesion; c) the gene expression signature of 2-treated cells was strikingly similar to those induced by selective inhibitors of mTOR signaling, thus suggesting an effect on this pathway for fullerene 2. Our work represents the first approach towards the application of RNA-seq to the study of the molecular mechanisms underlying the interaction of fullerenes with cellular systems and provides an objective view of the feasibility and the safety of these nanomaterials for a medical application.
    Full-text · Article · Oct 2013 · Toxicology
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    • "Furthermore, E2F1 protein expression levels displayed a prominent downregulation in both drug-treated cell lines, rendering the transcription factor almost undetectable at the higher dose of 10 μΜ and therefore suggesting its critical implication in the geldanamycin-induced G1-block described here. Hsp90 inhibition is known to facilitate cell cycle arrest in all checkpoints of the cell cycle, depending on malignancy grade and cellular context [21]. In the bladder cancer cell lines examined in the present study, geldanamycin administration primarily leads to a dose-dependent G1-checkpoint cell cycle arrest, while analysis of expression and activation profiles of several determinants of the cell cycle (Cdk4, pRb, Cyclin D1 and E2F1) correlate well with the observed block in cycle progression. "
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    ABSTRACT: Background Geldanamycin (GA) can be considered a relatively new component with a promising mode of action against human malignancies. It specifically targets heat shock protein 90 (Hsp90) and interferes with its function as a molecular chaperone. Methods In this study, we have investigated the effects of geldanamycin on the regulation of Hsp90-dependent oncogenic signaling pathways directly implicated in cell cycle progression, survival and motility of human urinary bladder cancer cells. In order to assess the biological outcome of Hsp90 inhibition on RT4 (grade I) and T24 (grade III) human urinary bladder cancer cell lines, we applied MTT assay, FACS analysis, Western blotting, semi-quantitative (sq) RT-PCR, electrophoretic mobility shift assay (EMSA), immunofluorescence and scratch-wound assay. Results We have herein demonstrated that, upon geldanamycin treatment, bladder cancer cells are prominently arrested in the G1 phase of cell cycle and eventually undergo programmed cell death via combined activation of apoptosis and autophagy. Furthermore, geldanamycin administration proved to induce prominent downregulation of several Hsp90 protein clients and downstream effectors, such as membrane receptors (IGF-IR and c-Met), protein kinases (Akt, IKKα, IKKβ and Erk1/2) and transcription factors (FOXOs and NF-κΒ), therefore resulting in the impairment of proliferative -oncogenic- signaling and reduction of cell motility. Conclusions In toto, we have evinced the dose-dependent and cell line-specific actions of geldanamycin on cell cycle progression, survival and motility of human bladder cancer cells, due to downregulation of critical Hsp90 clients and subsequent disruption of signaling -oncogenic- integrity.
    Full-text · Article · Feb 2013 · Cancer Cell International
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