Cancer Letters

Cancer Letters

Volume 453, 1 July 2019, Pages 1-9
Cancer Letters

Original Articles
The μ-opioid receptor (MOR) promotes tumor initiation in hepatocellular carcinoma

https://doi.org/10.1016/j.canlet.2019.03.038Get rights and content

Highlights

  • MOR is positively correlated to hepatocarcinoma progression, cancer cell proliferation and tumor formation.

  • MOR + hepatocellular carcinoma cancer cells, which have cancer stem cell properties, demonstrates the potential of a new therapeutic target.

  • MOR affects CSC tumorigenesis by regulating the NFAT signal pathway, which provides a promising approach to HCC treatment.

Abstract

Hepatocellular carcinoma (HCC) is the most prevalent subtype of liver cancer. Anesthetic regimens possibly influence cancer development. Exploration of novel, effective targets for liver cancer is the current hotspot in cancer treatment. A previous study conducted by us has demonstrated that enhanced expression of the μ-opioid receptor (MOR) promotes cell proliferation, adhesion, migration, and tumorigenesis. The current study investigates whether MOR regulates self-renewal of hepatocellular carcinoma stem cells (HCSCs). We utilize cell function assays, siRNA, shRNA, flow cytometry sorting, and other molecular biology techniques for this purpose. The results indicate that MOR expression is positively related to hepatocarcinoma progression. Silencing MOR greatly reduce HCC-related tumorigenesis both in vitro and in vivo and significantly extend the survival of tumor-bearing mice. Moreover, MOR silencing will greatly reduce colony formation by HCC cells, indicating down-regulation of cancer initiation. In conclusion, these results establish that MOR can be a novel and reliable HCSC marker and a potential therapeutic target against HCC via MOR-NFAT signaling.

Introduction

Hepatocellular carcinoma (HCC) is the fifth common malignant tumor and the third leading cause of death among all cancer victims [1]. Owing to recent progress in early diagnosis methods and surgical techniques, the incidence and death rates associated with HCC have declined slightly, but issues related to postoperative recurrence and metastasis continue to be problematic.

Opioids are typically used in cancer pain management, including pain associated with tumor resection and preoperative analgesia. Opioids work on receptors, blocking pain conduction in the nervous system. Generally, opioid receptors are categorized into 3 subtypes; μ, κ, and δ (MOR, KOR, DOR), which can modulate pain relief, cognition, and emotion, respectively [2]. Many studies have indicated that opioid receptors also exist in cancer cells of humans and other animals [[3], [4], [5], [6]]. The Μu opioid receptor, MOR, a G protein-coupled receptor found in squamous cells of lung, breast, colon, and prostate cancer cells, is among the most widely used opioid receptors [[7], [8], [9]]. A previous study of ours has demonstrated that MOR is specifically expressed in HCC tissues, whereas it shows no or very low expression in paracancerous tissues. We tested the hypothesis that MOR is associated with HCC, with a view to elucidating the mechanism underlying MOR-facilitated cancer development.

Recent studies have demonstrated the critical role played by cancer stem cells (CSCs) or cancer-initiating cells in tumorigenesis of many cancers [10,11], including hepatocellular carcinoma [12,13]. CSCs display the capacity to self-renew, differentiate, and give rise to new tumors [14]. In HCC, EpCAM, CD133, CD13, CD90 and CD44 have been defined as CSC surface markers [[15], [16], [17], [18]]. However, molecular therapeutic targets for CSC of HCC remain unestablished. The complicated nature of HCC treatment requires an interdisciplinary approach to ensure optimal outcomes. In a previous study, we sorted CD133+ and CD133- populations in Huh7 and HepG2 cells and evaluated MOR expression using flow cytometry, revealing that CD133+ subsets expressed higher levels of MOR than the CD133- subsets in both HCC cell lines, which predicates that MOR is associated with CSC.

We reported for the first time that MOR + cells have CSC properties in HCC and investigated whether MOR + cells offered a new therapeutic target. Furthermore, we explored possible signaling pathways of MOR in HCC. We found that downregulation of MOR inhibits the proliferation of hepatocarcinoma cells and can significantly retard tumor growth. Therapy targeting MOR can provide a new and promising approach to HCC treatment.

Section snippets

Animal models

Nude mice used in this study were purchased from the Animal Center of the Chinese Academy of Medical Science (Beijing, China). Six-week-old female nude mice, weighing approximately 15 g, were used in the current study. The mice were fed under specific-pathogen-free (SPF) conditions. Xenografts of tumor cells were generated via subcutaneous injection of tumor cells (1 × 106 cells suspended in 200 μl PBS) into the dorsal skin of nude mice. Tumor sizes were monitored twice a week. Animal studies

MOR is generally expressed in the hepatocellular carcinoma cell lines, human tumor tissues and MOR + hepatocellular carcinoma cancer cells resembled cancer stem cells

MOR transcription was measured via qPCR in a normal liver cell line (L02) and several HCC cell lines (PLC, HLE, Huh7 and HepG2). MOR expression was the lowest in L02, higher in HCC cell lines at different levels. But the highest expression of MOR was observed in Huh7 and HepG2 (Fig. 1A).

CD133 is a surface marker of CSC. In order to analyze whether MOR is a potential marker for HCC CSCs, we sorted both CD133+ and CD133- populations in Huh7 and HepG2 cells, and determined their MOR expression

Discussion

All opioid receptor family members are G protein-coupled receptors. The μ-opioid receptor (MOR) is the main member of the opioid receptor super family, which plays an important role in relieving acute postoperative pain and chronic pain. MOR agonists are commonly used not only as analgesics during cancer surgery but also to relieve cancer pain in patients with late malignant tumors. Besides, opioid receptors have been reported to affect cardiovascular and immune systems significantly [20]. MOR

Conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (81472413, 81672956, 81602644), Beijing Science and Technology Project (Z181100003818003) and Fund for Fostering Young Scholars of Peking University Health Science Center (BMU2018PY020).

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    These authors contributed equally and are joint first authors for this work.

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