Thrombospondin 4/integrin α2/HSF1 axis promotes proliferation and cancer stem-like traits of gallbladder cancer by enhancing reciprocal crosstalk between cancer-associated fibroblasts and tumor cells

Abstract

Background Cancer-associated fibroblasts (CAFs), the primary component of tumor stroma in tumor microenvironments, are well-known contributors to the malignant progression of gallbladder cancer (GBC). Thrombospondins (THBSs or TSPs) comprise a family of five adhesive glycoproteins that are overexpressed in many types of cancers. However, the expression and potential roles of TSPs in the crosstalk between CAFs and GBC cells has remained unclear. Methods Peritumoral fibroblasts (PTFs) and CAFs were extracted from GBC tissues. Thrombospondin expression in GBC was screened by RT-qPCR. MTT viability assay, colony formation, EdU incorporation assay, flow cytometry analysis, Transwell assay, tumorsphere formation and western blot assays were performed to investigate the effects of CAF-derived TSP-4 on GBC cell proliferation, EMT and cancer stem-like features. Subcutaneous tumor formation models were established by co-implanting CAFs and GBC cells or GBC cells overexpressing heat shock factor 1 (HSF1) to evaluate the roles of TSP-4 and HSF1 in vivo. To characterize the mechanism by which TSP-4 is involved in the crosstalk between CAFs and GBC cells, the levels of a variety of signaling molecules were detected by coimmunoprecipitation, immunofluorescence staining, and ELISA assays. Results In the present study, we showed that TSP-4, as the stromal glycoprotein, is highly expressed in CAFs from GBC and that CAF-derived TSP-4 induces the proliferation, EMT and cancer stem-like features of GBC cells. Mechanistically, CAF-secreted TSP-4 binds to the transmembrane receptor integrin α2 on GBC cells to induce the phosphorylation of HSF1 at S326 and maintain the malignant phenotypes of GBC cells. Moreover, the TSP-4/integrin α2 axis-induced phosphorylation of HSF1 at S326 is mediated by Akt activation (p-Akt at S473) in GBC cells. In addition, activated HSF1 signaling increased the expression and paracrine signaling of TGF-β1 to induce the transdifferentiation of PTFs into CAFs, leading to their recruitment into GBC and increased TSP-4 expression in CAFs, thereby forming a positive feedback loop to drive the malignant progression of GBC. Conclusions Our data indicate that a complex TSP-4/integrin α2/HSF1/TGF-β cascade mediates reciprocal interactions between GBC cells and CAFs, providing a promising therapeutic target for gallbladder cancer patients.

Pathological Significance and Prognostic Roles of Thrombospondin-3, 4 and 5 in Bladder Cancer

“…However, contrary to our expectations, positively stained cells for TSP-3 to 5 were rare in the cancer stromal area. This finding was similar to that in other types of cancer (34,37,53). Another limitation of the study is that the detailed pathological roles of TPS-4 are unclear.…”

“…Like TSP-5, TSP-4 is reported to be associated with malignant potential, tumor growth, and progression in several cancers including gastric cancer, hepatocellular carcinoma, and gallbladder cancer (34)(35)(36)(37). However, our results showed no significant pathological role of TSP-4 expression in BC tissues.…”

“…In short, although over 40% of patients were judged as positive for TSP-4 expression despite its lack of expression in normal urothelial cells, no significant role of TSP-4 was determined. Based on previous reports in other malignancies (34)(35)(36)(37), we speculate that TSP-4 might play significant roles in BC, except in cell survival and angiogenesis. In addition, TSP-4 expressed in stromal cells might play important roles in malignant aggressiveness, though the methods used in this study cannot detect such expression.…”

“…Unfortunately, the reasons underlying this discrepancy between BC and other cancers are unknown because there are no in vivo and in vitro studies on the pathological significance and prognostic role of TSP-4 in BC. However, TSP-4 immunoreactivity was mainly detected in the "cancer cells" of BC in the present study, whereas in previous studies, TSP-4 expression was evaluated in "stromal cells" of tumor tissues or without distinguishing between "cancer cells" and "stromal cells" (34,(36)(37)(38). Therefore, we first speculated that the pathological activities of TSP-4 might originate from stromal cell expression, but not from cancer cells.…”

“…Overall, these studies suggest that TSP-4 expression in cancer cells plays a significant role in tumor growth and cancer cell progression. However, there are contrasting opinions regarding whether TSP-4 acts as a tumor-promoter or suppressor, based on different types of malignancies (34)(35)(36)(37)(39)(40)(41). Finally, we emphasize that further detailed studies using cancer cell lines, animal models, and clinicopathological data are necessary to discuss and conclude the clinical significance and pathological roles of TSP-4 in patients with BC.…”