Triple knockdown of CDC37, HSP90-alpha and HSP90-beta diminishes extracellular vesicles-driven malignancy events and macrophage M2 polarization in oral cancer

ABSTRACT

Evidence has been accumulating to indicate that extracellular vesicles (EVs), including exosomes, released by cancer cells can foster tumour progression. The molecular chaperones – CDC37, HSP90α and HSP90β play key roles in cancer progression including epithelial-mesenchymal transition (EMT), although their contribution to EVs-mediated cell–cell communication in tumour microenvironment has not been thoroughly examined. Here we show that triple depletion of the chaperone trio attenuates numerous cancer malignancy events exerted through EV release. Metastatic oral cancer-derived EVs (MEV) were enriched with HSP90α HSP90β and cancer-initiating cell marker CD326/EpCAM. Depletion of these chaperones individually induced compensatory increases in the other chaperones, whereas triple siRNA targeting of these molecules markedly diminished the levels of the chaperone trio and attenuated EMT. MEV were potent agents in initiating EMT in normal epithelial cells, a process that was attenuated by the triple chaperone depletion. The migration, invasion, and in vitro tumour initiation of oral cancer cells were significantly promoted by MEV, while triple depletion of CDC37/HSP90α/β reversed these MEV-driven malignancy events. In metastatic oral cancer patient-derived tumours, HSP90β was significantly accumulated in infiltrating tumour-associated macrophages (TAM) as compared to lower grade oral cancer cases. HSP90-enriched MEV-induced TAM polarization to an M2 phenotype, a transition known to support cancer progression, whereas the triple chaperone depletion attenuated this effect. Mechanistically, the triple chaperone depletion in metastatic oral cancer cells effectively reduced MEV transmission into macrophages. Hence, siRNA-mediated knockdown of the chaperone trio (CDC37/HSP90α/HSP90β) could potentially be a novel therapeutic strategy to attenuate several EV-driven malignancy events in the tumour microenvironment.

Abbreviations

CDC37: cell division control 37; EMT: epithelial-mesenchymal transmission; EV: extracellular vesicles; HNSCC: head and neck squamous cell carcinoma; HSP90: heat shock protein 90; TAM: tumour-associated macrophage

KEYWORDS:

• Extracellular vesicles
• epithelial-mesenchymal transition
• tumour-associated macrophage
• CDC37
• HSP90
• tetraspanin
• oral cancer

Acknowledgments

This paper is dedicated to the memory of one of our mentors, Professor Ken-ichi Kozaki, who passed away on 29 May 2016. The authors thank Haruo Urata for the operation of TEM, Tomonari Kasai and Masaharu Seno for particle size analysis using ELS-8000, Kazuko Kobayashi for the operation of Zetasizer, Eriko Aoyama for maintenance of HCS analyzer. The authors thank Manabu Itoh and Kazuya Arai at JSR for technical advice in NCP. The authors thank Keisuke Nakano for illuminating discussion and encouraging support.

Author contributions

TE, KOn and SKC conceptualized and administrated the study. TE acquired funding. HN, AS, KK, MT, KOk, HO, SI and TE prepared resources. TE, CS, MTT, HK, KOn, KK, HO and KK devised methodology. KOn, HK, MTT, EAT, YL, YO, CS and MWO performed the experimentation. KOn, HK, TE, MTT and CS curated, validated and visualized data. TE, CS, KOk, HO and KK supervised. TE, KOn and HK wrote the original draft. TE and SKC reviewed and edited, and revised the manuscript. All authors reviewed the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Supplementry material

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Additional information

Funding

This work was supported by JSPS KAKENHI, grant numbers [17K11642 (T.E.), 17K11643 (C.S., T.E.), 17K11669 (K.Oh., T.E.), 19K24072 (K.On.), 16K11722-JM (T.E., H.N.), 19K10288-JM (H.N.), 18K09789-KN (H.N., T.E), JP16K11441 (H.N.), JP20H03888 (H.N., T.E.), 19H03817 (M.T., T.E.), 19H04051 (H.O., T.E.) and 20K09904 (C.S., T.E., K.Ok.)], Ryobi Teien Memorial Foundation Grant (K.Ok., C.S., T.E.), Suzuken Memorial Foundation Grant (T.E.) and NIH Grant CA176326-05 (SKC).