Targeting heat shock factor 1 with a triazole nucleoside analog to elicit potent anticancer activity on drug-resistant pancreatic cancer
Introduction
When subjected to insults such as heat shock or the exposure to heavy metal ions or cytotoxic agents, cells respond by over-expressing heat shock proteins (HSPs), which protect the cells and enable them to survive under otherwise stressful and lethal conditions [1], [2]. The whole process and related events is denoted the heat shock response (HSR). Regulation of the HSR is complicated and is linked to multiple cellular signaling pathways. While many questions remain concerning the HSR, increasing evidence suggests that heat shock factors (HSFs) mediate the inducible heat shock gene expression at the transcriptional level and hence regulate the HSR [3]. The most highly expressed HSFs in mammalian cells include HSF1, HSF2 and HSF4. Though all of them have been identified to modulate HSP expression, HSF1 is the most important regulator of the HSR in response to various stimuli [3], [4], [5]. Indeed HSF1 can regulate a multitude of signaling and metabolic pathways to promote the survival of cells. In addition, cancer cells are more dependent on HSF1 than normal cells for proliferation and survival due to the constant exposure of cancer cells to stresses both from internal oncogenic processes (such as mutated proteins and deregulated signal pathways) and external environmental factors (such as hypoxia and nutrient deprivation) [3]. Consequently, targeting HSF1 has attracted considerable attention as an appealing anticancer approach [3], [5], [6].
As the dominant upstream transcriptional regulator for stress inducible HSPs, HSF1 activates their transcription and regulates their expression in response to stress. Among the superfamily of HSPs, HSP90, HSP70 and HSP27 chaperones are particularly attractive as anticancer targets due to their function as master regulators of multiple oncoproteins and their tumorigenic properties [7], [8], [9], [10], [11], [12], [13], [14]. Different inhibitors of HSP90, mainly derived from the natural product geldanamycin, have led to encouraging results in clinical trials on multiple myeloma and gastrointestinal stromal tumors [15]. Antisense oligonucleotides developed to down-regulate HSP27 have also been demonstrated to show effective anticancer activity [16], [17], [18] and are at advanced stages in clinical trials for use in the treatment of several human cancers including lung, breast, prostate, bladder and ovarian [19]. As for HSP70, different types of inhibitors have been reported including small molecular chemical entities, antisenses and siRNAs, peptides, aptamers, etc. [14], although no candidate has yet entered into clinical trials for cancer treatment.
While current anticancer strategies focus mainly on the inhibition of individual HSPs, we believe that targeting multiple HSPs at once or their upstream controlling genes may be more effective. Given that HSF1 and HSPs form the HSR network and are up-regulated in cancer to promote cell survival and development, we postulated that targeting the heat shock response (HSR) pathways via the down-regulation of HSF1 might allow the simultaneous down-regulation of several HSPs, cause depletion of multiple oncoproteins and attack several pathways necessary for cancer development. This would ultimately lead to a more efficacious anticancer activity, while at the same time, reducing the likelihood of the tumor acquiring resistance to any single therapeutic pathway [14], [15], [20], [21].
In the present work, we disclose our finding of a triazole nucleoside analog which targets HSF1-mediated heat shock response pathways leading to the simultaneous down-regulation of the HSPs: HSP27, HSP70 and HSP90, and consequent depletion of their corresponding oncogenic client proteins. This triazole nucleoside analog yields effective caspase-dependent apoptosis with a potent anticancer effect in the drug-resistant pancreatic cancer model in vitro and in vivo. It is important to mention here that pancreatic cancer is one of the most lethal and devastating human cancers, constituting a major unsolved health problem worldwide [22], [23]. Conventional treatments have little impact on this disease due to its poor diagnosis, aggressive metastasis and rapid development of drug-resistance. The current standard chemotherapy for patients with advanced pancreatic cancer is the nucleoside drug gemcitabine [24], [25], though it is only moderately effective and results in a mere 12% response and 3% overall survival rate with a median survival of less than 6 months. Consequently, there is a pressing need to explore more efficacious drug candidates with novel modes of action to combat pancreatic cancer, particularly its drug-resistant form. Recently HSF1 was reported to play a pro-survival role in the pathogenesis of pancreatobiliary tumors [26]. Inhibition of HSF1 expression by the HSF1 siRNA sequences led to time-dependent death in pancreatic cancer cell lines in which a caspase-dependent apoptotic pathway was activated, whereas did not significantly decrease the viability of immortalized pancreatic ductal cells [26]. This hence suggested that down-regulation of HSF1 could emerge as cancer-specific therapy for pancreatic cancer. Targeting HSF1 using the triazole nucleoside disclosed here may therefore constitute a promising novel strategy.
Section snippets
Cell culture and reagents
Human pancreatic cancer MiaPaCa-2 and Panc-1 cells were purchased from American Type Culture Collection. The cells were grown in Dulbecco’s modified eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS). Gemcitabine (Gemzar) was purchased from Lilly France, SAS (Suresnes, France). (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide, MTT) and Actinomycin D was purchased from Sigma–Aldrich. PE Annexin V and 7-aminoactinomycin D (7-AAD) was purchased from BD
Lead optimization results in more efficacious candidates
We have previously identified a triazole nucleoside analog 1 (Fig. 1A) which effectively down-regulated HSP27 and resulted in apoptosis-associated anticancer activity against drug-resistant pancreatic cancer [27]. During our optimization process based on 1, we found that by varying the alkyl chain length we could critically modulate the anticancer activity: the analogs 2 and 3 bearing shorter alkyl chains were almost inactive or showed little activity, whereas compounds 4 and 5 (Fig. 1A)
Discussion
The targeting of individual HSPs constitutes an emerging trend in the search for efficacious anticancer candidates with novel mechanisms of action, however these inhibitors sometimes cause counterproductive effects on anticancer efficiency via their induction of other HSPs. An example of this is shown with one of the earliest HSP90 inhibitors, 17-AAG, which was found to up-regulate the expression of HSP70, a HSP with strong cytoprotective properties [15]. As HSF1 is the master transcription
Conclusion
In conclusion, we identified a novel triazole nucleoside which can effectively target HSF1 leading to the simultaneous down-regulation of HSP27, HSP70 and HSP90. Following the suppression of these HSPs, their client oncoproteins such as STAT3 and AKT were also significantly depleted. The final global consequence was a caspase-dependent induction of apoptosis and a potent anticancer effect on drug-resistant pancreatic cancer in vitro and in vivo. To our knowledge, this is the first triazole
Acknowledgments
This project is supported by the National Mega Project on Major Drug Development (2009ZX09301-014), La Ligue Contre le Cancer (BL-8670), Wuhan University, CNRS, INSERM and INSERM Transfert. Y.X. is supported by la Fondation pour la Recherche Médicale, M.W. le program de bourses d’excellene d’Eiffel, and Y.F. the China Scholarship Council. We thank Joël Tardivel-Lacombe for his assistance with cell culture, and Sandrine Henri for the help with radioactivity experiments and Drs. Emily Witty and
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