Cell Death and Survival Involvement of Extracellular Vesicle Long Noncoding RNA (linc-VLDLR) in Tumor Cell Responses to Chemotherapy

Hepatocellular cancer (HCC) is a highly treatment-refractory cancer and is also highly resistant to adverse cellular stress. Although cell behavior can be modulated by noncoding RNAs (ncRNA) within extracellular vesicles (EV), the contributions of long noncoding RNAs (lncRNAs) are largely unknown. To this end, the involvement and functional roles of lncRNAs contained within EVs during chemotherapeutic stress in human HCC were determined. Expression profiling identified a subset of lncRNAs that were enriched in tumor cell– derived vesicles released from two different cell lines. Of these, lincRNA-VLDLR (linc-VLDLR) was significantly upregulated in malignant hepatocytes. Exposure of HCC cells to diverse anticancer agents such as sorafenib, camptothecin, and doxorubicin increased linc-VLDLR expression in cells as well as within EVs released from these cells. Incubation with EVs reduced chemotherapy-induced cell death and also increased linc-VLDLR expression in recipient cells. RNAi-mediated knockdown of linc-VLDLR decreased cell viability and abrogated cell-cycle progression. Moreover, knockdown of VLDLR reduced expression of ABCG2 (ATPbinding cassette, subfamily G member 2), whereas overexpression of this protein reduced the effects of VLDLR knockdown on sorafenib-induced cell death. Here, linc-VLDLR is identified as an EV-enriched lncRNA that contributes to cellular stress responses. Implications: These findings provide new insight into the role of EVs and demonstrate the capacity of lncRNAs to mediate chemotherapeutic stress response in HCC. Mol Cancer Res; 1–11. 2014 AACR. Introduction Hepatocellular cancer (HCC) is one of the most prevalent cancers worldwide, with an annual incidence of around 750,000 new cases (1). This tumor is characterized by the alteration of multiple signaling pathways that modulate tumor behavior, local spread, and a tendency for multifocal tumor development (2). HCC is highly resistant to conventional therapies. Tumor progression is increased by the ability of HCC cells to resist adverse environmental stress such as hypoxia, radiation, and chemotherapy. Acquired resistance to adverse environmental conditions enhances tumor propagation, malignant progression, and resistance to therapy, and is a central issue in both the pathophysiology and the therapy of HCC (3, 4). Understanding the primary mechanisms or acquiring resistance to cellular stress will enable us to develop more effective treatment for HCC. The major focus of attention in genetic regulation of HCC development, progression, and behavior has been on protein-coding genes, and more recently microRNAs (miRNA). In contrast, the contribution of long noncoding RNA (lncRNA) to hepatocarcinogenesis has only recently become appreciated. LncRNAs are defined as noncoding RNAs more than 200 nucleotides in length (5–8). Like miRNA, these lncRNA can affect regulation of gene expression and have an impact on many different cellular processes. In contrast to miRNA, however, they have complex RNA structures and can function through a diverse and broad range of mechanisms. Our previous studies suggested that TUC338, a lncRNA that contains an ultra-conserved element, is significantly increased in human cirrhosis and HCC and can promote cell growth of HCC cells (9). On the other hand, maternally expressed gene 3 is strikingly downregulated in HCC relative to expression in nonmalignant hepatocytes and plays a crucial role as a tumor suppressor (10). Although these and other lncRNAs such as MALAT-1, HULC, and H19 have been implicated in human HCC, the functional contribution of these and other lncRNA genes is mostly unknown. Department of Internal Medicine, Department of Transplantation, and Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida. Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). Corresponding Author: Tushar Patel, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL 32224. Phone: 904-956-3257; Fax: 904-956-3359; E-mail: [email protected] doi: 10.1158/1541-7786.MCR-13-0636 2014 American Association for Cancer Research. Molecular Cancer Research www.aacrjournals.org OF1 on June 19, 2017. © 2014 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from Published OnlineFirst May 29, 2014; DOI: 10.1158/1541-7786.MCR-13-0636