Down-regulation of the tumour suppressor κ-opioid receptor predicts poor prognosis in hepatocellular carcinoma patients

Hepatocellular carcinoma (HCC), as a common malignancy, has an increasing incidence and mortality globally, especially in Asia [ 1]. Worldwide, there are more than 50% of the cases in China alone, according to the epidemiologic report [ 2]. This cancer has a very poor survival rate even with advanced diagnostic strategies and improved therapies. The prognosis for HCC after resection is still discouraging due to the potential for residual tumor and the high rate of tumor recurrence, which exceeds 60% [ 3]. Therefore, investing a valuable biomarker to better evaluate the diagnosis and prognosis of HCC patients will be beneficial in the guidance of treatment and inhibition of metastasis.

Opioids are wildly used in pain management of cancer patients, no doubt that interest in the possibility of opioids may influence the course of cancer development is not recent. Opioids have been shown to accelerate the growth of tumour cells and induce metastasis [ 4, 5], whereas other studies have reported that opioids can induce apoptosis in several cancer cells, such as lung cancer, colon cancer and breast cancer [ 6– 9]. Opioid receptors, with opioids as ligands, belong to a group of G protein-coupled receptors [ 10]. In general, opioid receptors contain three subtypes, μ, κ, and δ (MOR, KOR and DOR, respectively), which modulate a variety of physiological functions such as pain regulation, emotional tone, and cognitive functions [ 11]. Opioid receptors were discovered both in neural tissues (brain and spinal cord) and a wide spectrum of peripheral extraneural tissues (spleen, stomach, lung, pancreas, liver, heart, blood, and blood vessels) [ 12]. The expression profile of opioid receptors in different cancer cells has also been reported [ 13] and experimental studies in investing the effects of opioid receptor agonists and antagonists on the proliferation and metastasis of cancer both in vivo and in vitro study have received lots of attention. Morphine, a MOR agonist, were shown to possess antitumor effects [ 6]. In contrast, other reports described tumor-promoting effects of morphine by immunosuppression [ 14] or inducing angiogenesis [ 15].

Although the reports in the literature are inconsistent, opioid receptors expressed in tumour cells may have an implication in tumour progression [ 16, 17]. KOR expression has been reported in various cancer cells, such as small lung cancer cell and oesophageal cancer cell [ 18, 19]. Furthermore, KOR expression is up-regulated in esophageal squamous cell carcinoma (ESCC) tissues and patients with an elevated nuclear KOR expression in ESCC have a worse prognosis [ 19]. In contrast, an in vivo assay revealed that xenograft tumors in KOR knock-out mice demonstrated increased tumour growth and promoted angiogenesis [ 20]. Therefore, the effect of KOR expression in different cancers is variable. The potential role of KOR in HCC progression, including recurrence and metastasis, is unknown. In this research, we aimed to detect the clinical significance of the expression of KOR in HCC patients and investigate the potential effects of KOR expression on patient prognosis.

Patients with HCC, which is the most prevalent malignant carcinoma, exhibit a poor outcome, even after curative resection [ 1, 22]. As previous reports have shown, the 5-year recurrence rate of HCC patients ranges from 50 to 70% [ 2]. In our cohort study, the 5-year recurrence rate of HCC patients was 61%. Therefore, dependable tumour biomarkers are urgently needed to help identify patients who are at a high risk of poor survival and to establish individualized treatment programmes.

The reports showed that opioids administration is one of the factors that could influence tumour progression [ 23– 25]. Opioid receptors (MOR, KOR, and DOR) are the targets of opioids and are classified into the superfamily of G-protein-coupled receptors, which modulate pain and emotional regulation [ 12]. Opioid receptors are expressed in many tumours and are implicated in cell proliferation and metastasis. DOR and MOR are overexpressed in lung cancer [ 26, 27], and the overexpression of MOR enhances cancer progression by regulating Epidermal Growth Factor (EGF)-induced signalling events [ 28] and epithelial mesenchymal transition (EMT) events [ 17]. In addition, previous studies have demonstrated that KOR takes part in the tumourigenesis and progression of ESCC [ 19] but that the activation of KOR inhibits the growth of lung cancer cells [ 18]. These findings indicate that KOR expression plays different roles in various carcinomas. Nevertheless, the expression status of KOR in HCC remains unknown.

In this study, we first measured the KOR mRNA expression levels and found that KOR was significantly down-regulated in HCC tumour tissues. Furthermore, we examined the correlation between KOR expression and clinicopathological parameters. Interestingly, the results exhibited that no matter in mRNA or protein level, low KOR expression was significantly associated with aggressive parameters, such as large tumour size, vascular invasion, poor differentiation and advanced TNM stage. A recent study showed that KOR-regulated lung carcinoma or melanoma invasiveness and metastasis were accompanied by changes in vascular endothelial growth factor (VEGF) [ 20]. The Kaplan-Meier survival analysis displayed that HCC patients in the KOR-positive group had a better OS and RFS than did those in the KOR-negative group. Unlike the principle of classification described above, we also grouped 174 patients into 3 novel groups. Consistent with our previous results, patients in the KOR gain group (KOR ^T > N) had the best outcomes among all patients, although HCC showed down-regulation of KOR expression. A multivariate analysis demonstrated that down-regulation of KOR in HCC was an independent and significant risk factor for both OS and RFS after surgery. A different report demonstrated that KOR expression in ESCC was associated with poor prognosis [ 19]. However, our data suggested that KOR might act as a tumour suppressor and could be a potential prognostic factor for HCC.

Numerous biomarkers of hepatocarcinogenesis have been identified in recent researches, and AFP has been recognized as the standard HCC tumour biomarker for a long time [ 29]. Elevated AFP levels are closely associated with HCC carcinogenesis and a high recurrence and mortality rate after hepatectomy [ 30]. In the current study, we classified 174 patients into 2 groups based on AFP levels with a cut-off point of 20 ng/ml. Interestingly, we found that patients in the KOR-positive group with a high AFP level exhibited better outcomes than did those in the KOR-negative group; the OS and RFS were 78 and 53% versus 35 and 25%, respectively. These results revealed that KOR overexpression in tumour tissues, even in tissues with a high AFP level, were more effective at predicting patient prognosis and supported the assumption that KOR could function as a tumour suppressor in HCC.

Clinical stage is the predominant determining factor of prognosis in patients with HCC, and the TNM stage system is one of the commonly used systems [ 31]. According to the TNM stage system, patients in stage I are believed to be in an early stage of HCC and to experience better outcomes after surgical resection [ 32]. However, patients in the same TNM stage often display various clinical outcomes, and a few patients will still have a poor prognosis. Our research demonstrated that in patients with TNM stage I, the OS and RFS rates at 5 years for KOR- positive patients were 77 and 56%, respectively, whereas the OS and RFS rates 5 years for KOR- negative patients were 44 and 25%, respectively. These results suggest that the KOR down-regulation in tumour tissues predicted poorer outcomes in patients in an early stage. Moreover, the identical correlation existed in the differentiation I/II group. In conclusion, these findings indicted that the low expression of KOR in tumour tissues could indicate a worse prognosis in early stage HCC patients, which would influence treatment decisions regarding individual clinical therapy.

Kuzumaki demonstrated that U50,488H (KOR agonist) reduced the growth of gefitinib-resistant lung cancer cells through the activation of phosphorylated-glycogen synthase kinase 3β [ 18]. Kohei Yamamizu reported that KOR agonists inhibited tumour angiogenesis and tumour growth by suppressing VEGF signalling in both in vivo and in vitro assays [ 20]. These researches suggested that KOR agonists could inhibit the growth of cancer cells through the stimulation of KOR. A recent review demonstrated that the activation of KOR could be useful for inhibiting vascular formation in cancers, and suggested that KOR could be a therapeutic target [ 33]. These findings have implications for the decision to use opioids of activated MOR or KOR type in cancer patients during surgery or treatment of chronic pain. Growing evidence indicates that analgesics of the MOR agonist type stimulate angiogenesis and tumour progression [ 34]. In contrast, the analgesics of the KOR agonist type could offer therapeutic benefits by suppressing tumour angiogenesis. Our results showed that KOR up-regulation in HCC was associated with better prognosis. Low KOR expression was associated with vascular invasion in HCC patients, which indicated that activated KOR might induce the inhibition of angiogenesis and metastasis. In addition, KOR down-regulation was related with poor tumor differentiation and advanced TNM stage, which suggested that KOR might take an important part in the HCC development and progression. We therefore inferred that KOR might be a novel potential target for therapy. Nevertheless, additional studies are required to illustrate the mechanisms that underlie the antitumour effects of KOR on HCC progression.

In conclusion, our research demonstrated that KOR, which was frequently down-regulated in HCC, was significantly associated with large tumour size, increased vascular invasion, poor differentiation and advanced TNM stage. Moreover, we revealed the relevance between KOR expression in tumour recurrence and patient prognosis and suggested that KOR was an independent and significant risk factor in HCC. Taken together, KOR might be a potential tumour suppressor in HCC progression and could provide a therapeutic target for HCC treatment.