Hepatocellular carcinoma (HCC) is currently is the fifth most common malignancy worldwide and the poor because of frequent metastasis and resistance to chemotherapy. Circulating tumor cells (CTCs) in blood have attracted attention as potential seeds for metastasis and an important indicator of treatment outcome. However, the biological properties of CTCs are largely unknown due to rarity and lack of CTC-specific surface markers. Major vault protein (MVP) is upregulated during malignant progression and drug resistance development in various cancer cells, although it is ubiquitously expressed in many normal tissues. Here, we found for the first time that MVP was expressed on the surface of various cancer cells including HCC cell lines, although MVP has been known as a cytoplasmic and nuclear protein. To investigate the role of cell-surface MVP (csMVP) on HCC cell lines, MVP expression was knocked down in Huh7 cells by small interfering RNA. MVP knockdown decreased cell growth and increased apoptotic cell death. When Huh7 cells were treated with a polyclonal anti-MVP antibody (α-MVP) recognizing csMVP, Huh7 cell proliferation was decreased without apoptotic cell death. Cell sorting revealed that csMVP-positive Huh7 cells showed a higher proliferation and survival rate than csMVP-negative Huh7 cells under the stress of low density seeding. Huh7 cells treated with α-MVP also inhibited cell invasion and migration in vitro. Thus, csMVP is positively associated with HCC cell proliferation, survival, invasion and migration. Analysis of signaling molecules showed that MVP knockdown caused a significant decrease in the levels of phosphorylation of FAK, ERK, AKT and S6K. MVP knockdown also increased E-cadherin but decreased vimentin, indicating that MVP promotes the process of epithelial-to-mesenchymal transition. To further analyze the role of csMVP during HCC metastasis, blood samples from 62 HCC patients and 10 healthy volunteers were stained with csMVP, EpCAM, HSA, EGFR, CK, and/or vimentin antibodies after the depletion of red blood cells and CD45-positive cells. csMVP-positive CTCs were detected in approximately 85.5% (> 0.46CTCs/ml) of patients, and the cell count measured in ml of blood ranged between 0.48 and 37.8. Double staining showed that almost all csMVP-positive cells were panCK-negative while approximately one fifth of csMVP-positive CTCs was HSA-positive. Triple staining further showed that among csMVP-positive cells, EpCAM+Vimentin-, EpCAM+Vimentin+, EpCAM-Vimentin+ and EpCAM-Vimentin- cells were approximately 0, 0, 49 and 51%, respectively, suggesting that a half of csMVP-positive cells are EMT-phenotypic cells and the rest of them are both EpCAM- and vimentin-negative cells. The results suggest that csMVP is a novel marker on CTCs in patients with HCCs, where it is expressed predominantly on EMT phenotypic and EpCAM-vimentin- intermediate CTCs.

Citation Format: Hyun Min Lee, Jae Won Joh, Won Tae Kim, Min Kyu Kim, Se Ri Seo, Hong Seo Choi, Hee Jin Chang, Young Joo Jang, Dae Shick Kim, Chun Jeih Ryu. Cell-surface major vault protein is a novel marker for circulating tumor cells with nonepithelial phenotypes in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1966. doi:10.1158/1538-7445.AM2017-1966