The Screening Of Novel HSP90 Inhibitors And Clarifying Its Activities And Mechanism Of Anti-proliferation Against Pancreatic Cancer The HSP90 Inhibitors Induced IGF-1β Degradation Through Autophagy Lysosomal Pathway In Pancreatic Cancer Cells

Pancreatic cancer (PC) is one of the most common malignant tumors of digestive system, which is characterized by difficult diagnosis in early stage, high invasion and metastasis, acquired resistance to chemotherapy, resulting in poor prognosis. Generally, the resection rate of pancreatic cancer patients after surgical treatment reached to 20-25%. Gemcitabine single-agent or combination with 5-Fu are recommended to treat with advanced pancreatic cancer patients. These chemotherapeutic regimens have various potential limitations, such as systemic toxicity and drug resistance, leading to a marginal survival benefit over no therapy. The overall five-year survival rate is less than 5%. Given the complexity of PC with its multiple genetic abnormalities, targeting a single pathway is unlikely to be effective. Thus, identification of new targets that modulate multiple signaling pathways would be desired for PC therapy.Heat shock protein 90 (HSP90) is known as an essential molecular chaperone, which regulates the folding, assembly and maturation of a wide range of client proteins involved in cell growth and surviva. The majority of identified HSP90 clients are oncogenic proteins, including transmembrane tyrosine kinases (HER-2, EGFR and IGF-1R), signaling proteins (AKT, c-RAF, P53, V-src), cell cycle regulators (CDK4, CDK6) and transcription factor (STAT3, HIF-la), etc. Many of these oncoproteins directly participate in the growth and angiogenesis of PC. HSP90 itself was also reported to be 2 to 10 folds over-expressed in various human cancers, including pancreatic carcinomals,which exerts its chaperone function to remain the activity of excessive activated or mutated onco-proteins and avoid the degradation of them, in order to maintain the malignant phenotype and accelerate tumor progression. Targeting of HSP90 chaperone exhibits combined antitumor effect by simultaneous destabilization and inactivation of various oncogenic proteins. Excitingly, HSP90 is maintained in an activated, super-chaperone complex form in tumor cells, which has a much higher affinity for HSP90 inhibitors versus normal cells.Therefore, targeting HSP90 became an appealing, selective and validated target for cancer therapy.The crystal structure analysis showed that HSP90 is a constitutive dimer that contains three domains permonomer:NH2-terminal ATPase domain (P1-E245), the middle domain (K246-D528) and the C-terminal dimerization domain (G529-D723). The ATP binding and ADP/ATP exchange act as a conformational switch to regulation the chaperone function of HSP90. Currently, most of the known HSP90 inhibitors all bind to the ATP-binding pocket located in the N-domain, and cause un-functionality of HSP90. Some of them have been developed and tested in preclinical and clinical models for their anticancer activity. Geldanamycins (GAs) represent the first-in-class HSP90 inhibitors.17-AAG is one of the well-known GAs class of HSP90 inhibitors, which has potent antitumor activity (IC50,10-～-9M). Whereas, GAs have various potential limitations, including unacceptable hepatotoxicity, dependency on polymorphic enzymes metabolism, and multidrug resistance, these cause to preclude their clinical application .With the rapid development of chemical structure and high-throughput screening technology, several research groups also focus on purine scaffold and resorcinol derivatives to develop various potent HSP90 inhibitors, such as, PU3, CCT018159 and VER-52296. Crystallographic and structure-activity studies reveal that resorcinol ring and the adjacent isoxazole amide VER-52296 can bind deeply into the HSP90 ATP binding site, and dramatically deplete the client proteins including c-RAF and HER-2, leading to potent inhibition of the proliferation in a wide range of human cancer cell lines .However, VER-52296 is also active against nontumorigenic human epithelial cells as well as human cancer cells. Latent toxicity problems are likely to be revealed after using it in a phase I study on the patients with advanced solid tumors, such as nausea, vomiting and night blindness.Therefore, in our study, we firstly established a fluorescence polarization system to screen N-terminal HSP90 inhibitors and examined the binding affinity towards HSP90 of 80 new compounds derived from resorcinylic scaffold. Secondly, the effects of these compounds on anti-proliferation were evaluated in many of tumor cells. Thirdly, the anti-proliferative activities and of candidate compound named Y306zh were evaluated in vitro and in vivo. Finally, we further investigated the mode of actions of Y306zh against pancreatic cancer in order to support the further medicinal chemistry development of this kind of compounds.In the first part, the recombinant plasmid pET24a(+)-HSP90a was transformed into E. coli strain BL21-DE3, and induced with 1mM IPTG. The target fusion protein was purified by Ni-NTA column. The fluorescence polarization assay for screening HSP90 inhibitors was established and optimized using varying concentrations of recombinant HSP90 protein and probe VER00051001. In addition, NVP-AUY922 or GA was added into FP buffer to verify the reliability of the screening model.In the second part, the cytotoxic effects of 80 screened compounds with the structure of resorcinol and the adjacent triazolopiperazine or isoxazole were examined at seven kinds of commonly used human cancer cells. The HSP90 binding affinity of these compounds which exhibited potent anti-proliferation was examined by fluorescence polarization assay in order to obtain candidate compounds. One of them named Y306zh exhibited strong HSP90 binding affinity and effective cell growth inhibition. So, we further investigated the efficacy and mechanism of Y306zh against cancer.In the third part, we performed MTT assay in fifteen kinds of human cancer cell lines to determine the anti-proliferation activity of Y306zh. The PC cell lines were chosen as they exhibited sensitive to Y306zh. Then, the cell growth inhibition of Y306zh was examined on five kinds of PC cells and normal cells, the former have different levels of HSP90a. Flow cytometric analysis was performed to detect the cell cycle distribution and apoptosis after treatment with Y306zh in PC cells. In vivo, the body weight, tumor volumes, tumor mass and Ki-67 positive cells of tumor sections in the Mia-paca2 xenograft mice was detected to evaluate its efficacy.In the last part, the binding affinity of Y306zh towards N-terminal HSP90 was examined by the fluorescence polarization assay. The effect of Y306zh on ATP-bound active HSP90 and HSP90-cochaperone complex were investigated using ATP-sepharose binding assay and co-immunoprecipitation assay in Mia-paca2 cells. Western blot analysis was used to detect the expression of EGFR and IGF-1Rβ, the well-known HSP90 client proteins associated with tumorgenesis, chemoresistance and poor prognosis of PC, and the key regulators of their downstream signaling pathways PI3K/AKT and MAPK. A well-known proteasome inhibitor MG132 was used to block the proteasome function to investigate the degradation of clients (EGFR, IGF-1R(3, AKT, C-RAF and CDK4) induced by Y306zh. Co-immunoprecipitation assay was used to detect the relationship between the degradation of these client proteins via ubiquitin-proteasomal pathway induced by Y306zh and the formation of the CHIP-HSP70-HSP90-client complex.In summary, the major advancements are as follows:1) Two kinds of model were established:the pET24α (+)-HSP90a expression system to express and purify the activity recombinant full-length HSP90 protein, the fluorescence polarization assay for screening HSP90 inhibitors.2) From 80 screened compounds with the structure of resorcinol and the adjacent triazolopiperazine or isoxazole, we identified 24 compounds which effectively inhibited the tumor cell viability with the IC50 of 10-6～10-7M, and most of them (about 20) compounds decreased the binding between the probe VER00051001 and HSP90 by more than 1-fold at 10-7～10-8M.3) As a novel and potent HSP90 inhibitor, Y306zh effectively inhibited the proliferation of pancreatic cancer cells with more selective and better therapeutic window in vitro and in vivo.4) Y306zh attenuated the HSP90 activity by interrupting ATP binding to HSP90 and HSP90-p23 interaction, leading to destabilization of EGFR and IGF-1Rβ, hence simultaneous suppression of pro-proliferative/survival signaling pathways PI3K/AKT and MAPK to carry out its antitumor activity.