Targeting heat-shock protein 90 with ganetespib for molecularly targeted therapy of gastric cancer

Gastric cancer (GC) remains the fifth most common cancer worldwide. Heat-shock protein 90 (HSP90) has become an attractive therapeutic target in treating cancers, because of its abnormally high expression in cancers. Several successful cases of HSP90 inhibitors capable of inhibiting GC inspired us to try ganetespib, a clinically promising and actively investigated second-generation HSP90 inhibitor in GC treatment. In our study, we show that ganetespib markedly reduced the growth of MGC-803 and also significantly inhibited the growth of SGC-7901 and MKN-28 in a dose-dependent manner. It induced G2/M cell-cycle arrest and apoptosis in all three cell lines, together with the related markers affected significantly. Mechanistically, ganetespib caused pronounced decrease of expression of classic HSP90 client proteins. Specifically, it greatly affected epidermal growth factor receptor (EGFR) signaling cascades by markedly decreasing the levels of total EGFR and EGFR on cell membranes. EGFR knockdown also induced cell-cycle arrest and apoptosis accompanied with a decrease of several EGFR downstream proteins. These results strongly support that EGFR signaling greatly contributes to the ganetespib inhibitory effects. Besides, we found that the responses of GC cell lines to ganetespib correlated well with their EGFR expression levels: MGC-803, as well as AGS and BGC-803, with higher EGFR expression responded to ganetespib better, whereas SGC-7901 and MKN-28 with lower EGFR levels were much less sensitive to ganetespib. Although SGC-7901 and MKN-28 were not very sensitive to ganetespib, ganetespib worked synergistically with radiation and cisplatin in killing them. Importantly, ganetespib significantly inhibited the growth of xenograft tumors in vivo as a single agent or in combination with cisplatin. Results of hematoxylin/eosin staining, TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assays, and immunohistochemistry staining of phosphorylated cyclin-dependent kinase 1 (pCDK1), EGFR and Ki-67 revealed significant differences in ganetespib-treated tumors. Collectively, our data suggest that ganetespib, as a new potent treatment option, can be used for the molecularly targeted therapy of GC patients according to their expression profiles of EGFR.Gastric cancer (GC) remains the fifth most common cancer worldwide, with an estimated 9 52 000 new cases (7% of total cancer incidence) and 7 23 000 deaths (9% of total cancer mortality) in 2012.¹ As a highly aggressive and lethal malignancy, the aggressive nature of GC is linked to mutations in tumor suppressor genes, oncogenes, growth factors and their receptors, and so on.² Till now, there are few effective treatment options for advanced patients with distant metastasis or recurrence.³ The detailed mechanisms that regulate GC are not yet fully understood; therefore, such situations underscore the persistent unmet need to identify therapeutics that target pathways involved in GC progression.Consequently, identification of key regulatory molecules in GC is of high priority for understanding the mechanism for tumor dissemination as well as the development of novel interventions. Aberrant expression and kinase activity of Src have been found in many different tumors, including GC.^{4, 5} Previous studies have shown that phosphorylated mammalian target of rapamycin (p-mTOR) was significantly overexpressed in advanced GC patients'' tumors and suggested that the PI3K/AKT/mTOR (phosphoinositide 3-kinase/AKT/mTOR) pathway is activated in GC with potential prognostic and predictive significance.^{6, 7} Aurora A overexpression has recently been reported in GC, and it was suggested to be associated with cancer progression and poor prognosis.^{8, 9, 10}In our previous work, we conducted data mining meta-analyses integrating results from multiple small interfering RNA (siRNA) screens to identify gene targets, which are necessary for the growth of different cancer cells. Among those genes, we found that heat-shock protein 90 (HSP90) was one of the most vital proteins for cancer cell survival.¹¹ As we know, HSP90 is involved in the regulation of numerous proteins important for GC pathogenesis, such as proteins important for cell adhesion (e.g., focal adhesion kinase), cell motility (e.g., epidermal growth factor receptor (EGFR), c-Src, phosphoinositide-dependent protein kinase 1 (PDK1)), and angiogenesis (e.g., hypoxia-inducible factor-1 (HIF-1), vascular endothelial growth factor receptor (VEGFR)).^{12, 13, 14, 15} For these reasons, HSP90 has been of considerable interest as a therapeutic target in GC.As an ATP-dependent molecular chaperone protein, HSP90 conducts the proper folding of myriad proteins.^{12, 14} Abnormally high expression of HSP90 has been found in GC and been greatly considered as an independent prognostic marker of GC progression.^{16, 17, 18} HSP90 remains an attractive therapeutic target in a variety of cancers,^{19, 20, 21, 22} and inhibition of HSP90 showed potent growth inhibitory effects on GC in cell cultures and in mouse models.^{23, 24, 25} Ganetespib is a particularly promising second-generation HSP90 inhibitor that does not suffer from the toxicity issues associated with earlier-generation HSP90 inhibitors and exhibits increased potency compared with first- and other second-generation agents.^{11, 26, 27, 28, 29}In this current study, using cell culture and xenograft mouse models, we sought to evaluate the effects of ganetespib treatments on GC cells, individually or in combination with other treatments. In addition, we searched for the possible mechanisms underlying the antitumor activities of ganetespib. And, our results suggested that, as a promising drug candidate, ganetespib has potent antitumor activities on GC, and it is worth being investigated further clinically for the molecularly targeted therapy of GC patients.