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Elevated Transient Receptor Potential Melastatin 8 (TRPM8) Expression Is Correlated with Poor Prognosis in Pancreatic Cancer

Background The transient receptor potential melastatin 8 (TRPM8) was found to be expressed abnormally in a variety of tumors and is associated with unfavorable prognosis in human cancers. However, its clinical significance in pancreatic cancer (PC) is mostly unknown. Material/Methods qRT-PCR was performed to measure the expression of TRPM8 in 110 pairs of PC tissues and the adjacent non-cancerous tissues. The association of TRPM8 expression with the clinical characters of PC patients was analyzed using the chi-square test. Furthermore, the prognostic value of TRPM8 was determined with Kaplan-Meier survival curve and Cox regression analysis. Results We found that the expression level of TRPM8 was significantly elevated in PC tissues compared to the non-cancerous controls (P<0.001). In addition, a close relationship was observed between elevated TRPM8 expression with large tumor size (P=0.001), advanced TNM (P=0.013), and distant metastasis (P=0.034). Survival analysis suggested that patients with high TRPM8 expression has worse OS (P=0.001) and DFS (P<0.001) than those with low TRPM8 expression. Moreover, TRPM8 was confirmed as a valuable prognostic biomarker for OS (HR=1.913; 95% CI: 1.020–3.589; P=0.043) or DFS (HR=2.374; 95% CI: 1.269–4.443; P=0.007) of PC patients. Conclusions This study shows that TRPM8 expression is significantly up-regulated in PC and it might be a useful prognostic factor for patients with PC.


Background
Pancreatic cancer (PC) remains one of most lethal malignant tumors; it is responsible for approximately 227 000 deaths worldwide every year due to its high recurrence and mortality rates [1][2][3]. Recently, the mortality rate of PC has been increasing and the average age of patients has decreased [4]. Surgical resection remains the only potentially curative treatment for PC [5,6]. However, due to the lack of specific clinical symptoms at early stages, most cases are diagnosed in advanced stages and are ineligible for potentially curative resection; therefore, the prognosis is poor and the 5-year survival rate is only 3-6% [7,8]. Several clinicopathologic parameters have been identified as prognostic factors of PC, including neural invasion, lymph node metastasis, CA19-9 level, and TNM [9][10][11][12]. Moreover, a variety of biological prognostic factors have been reported in the development and progression of PC [13], but there currently is no satisfactory way to effectively improve the survival of PC patients.
The family of transient receptor potential (TRP) channels are essential mediators of signaling pathways involved in cellular functions and associated with progression and treatment of various cancers [14]. As a member of the TRP family, transient receptor potential melastatin 8 (TRPM8) is a Ca 2+ -permeable cation channel [15] and is required for the transduction of moderately cold temperatures [16,17]. Initially, human TRPM8 was known as trp-p8 and has been identified to have a high expression level in prostate cancer [18]. Moreover, increased expression of TRPM8 was found in prostate cancer cells and it emerged as a promising prognostic marker in prostate cancer [19]. In fact, more and more reports have suggested that TRPM8 plays vital functions in carcinogenesis of many cancers, including melanoma [20], bladder cancer [21], oral cancer [22], lung cancer, colon cancer, and breast cancer [18]. More importantly, studies on the relationship between TRPM8 and tumors reveal that high expression of TRPM8 appears to be associated with poor prognosis [23,24]. Hence, TRPM8 had been reported to be a valuable prognostic biomarker and might be a potential treatment target [25]. It has reported that TRPM8 expression is elevated in PC, and the aberrantly over-expressed TRPM8 is required for cellular proliferation and invasion [26,27]. However, its clinical significance in prognosis for PC has not been previously reported. The present study investigated the potential of TRPM8 as a prognostic biomarker in PC patients.

Material and Methods
The collection of patients and samples The present study was approved by the Ethics Committees of the General Hospital of the PLA. A total of 110 fresh PC tissues and corresponding adjacent non-cancerous tissues were obtained from patients who were pathologically diagnosed with PC in the General Hospital of the PLA. All patients provided written informed consent and none of them had received any related anti-tumor therapies (e.g., chemotherapy, radiotherapy, or hormone therapy) before surgery. The TNM stage was classified according to the International Association of Cancer. For the expression of CA19-9 and CEA in serum, blood samples were randomly obtained before surgery, and serum was obtained by routine centrifuging. The expressions of CA19-9 and CEA were determined by double-antibody sandwich ELISA (electrochemiluminescence immunoassay). All patients had complete 3-year follow-up. Clinicopathological data of all patients are summarized in Table 1.

RNA extraction and quantitative real-time PCR (qRT-PCR)
All the RNA was exacted from the collected PC tissues and adjacent normal specimens with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocols. The purity of RNA was determined by NanoDrop ND-1000 (Wilmington, DE, USA). First-strand complementary DNA (cDNA) was transcribed using the PrimeScript RT reagent kit (TaKaRa, Dalian, China). The qRT-PCR was performed using SYBR Premix Ex Taq (Takara, Dalian, China). The relative expression of TRPM8 was normalized to GAPDH and quantified by 2 -DDCT method. All experiments were performed in triplicate.
Statistical analysis SPSS 18.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA) were used to perform statistical analyses. The TRPM8 expression differences of PC tissues with adjacent normal specimens were measured by t test. The chi-square test was used to analyze the relationship between TRPM8 expression and the clinical features of PC patients. Kaplan-Meier analysis with log-rank test was used for survival analysis. The prognostic value of TRPM8 mRNA in pancreatic cancer was determined using multivariate Cox regression analysis after adjusting the baseline characteristics, including age, sex, tumor size, metastasis, differentiation, tumor location, CA19-9, and CEA. P<0.05 was considered to be statistically significant.

TRPM8 expression was up-regulated in PC tissues
To illustrate the abnormal expression of TRPM8 in PC, we investigated its mRNA expression level in PC tissues and adjacent non-cancerous tissues by use of qRT-PCR. We observed that the expression of TRPM8 was obviously higher in PC tissues than that in adjacent non-cancerous tissues (mean ±SD: 1.45±0.55 versus 0.85±0.44, P<0.001; Figure 1).

Relationship of TRPM8 expression with clinical parameters of PC patients
To further determine the clinical significance of TRPM8 expression in PC, the relationship of TRPM8 expression with clinical parameters was investigated. As shown in Table 1, based on the median expression level of TRPM8 mRNA in PC tissues (1.23), the participants were divided into 2 groups: the high-expression group (n=57), and the low-expression group (n=53). The results showed that the high expression of TRPM8 was significantly associated with tumor size (P=0.001), TNM (P=0.013), and distant metastasis (P=0.034). However, no close relationship was found between TRPM8 expression and age, sex, tumor differentiation, tumor location, CA19-9, or CEA (all, P>0.05).

The prognostic value of TRPM8 expression in PC
The results of Kaplan-Meier curves showed that the survival time of patients in the high-TRPM8 expression group was  (Table 3).

Discussion
In the present study, our data further verified the findings of previous studies showing that TRPM8 expression is elevated in PC tissues compared to that in adjacent non-cancerous tissues. In addition, our data demonstrate for the first time that high TRPM8 expression is obviously associated with poor OS and DFS in PC patients, and it could be a valuable prognostic biomarker for patients with PC.
As a Ca2+-permeable cation channel, TRPM8 has been increasingly reported in recent years. TRPM8 was initially thought to be over-expressed in prostate cancer [18]; subsequently, an increasing number of studies demonstrated the consistent profile of TRPM8 expression in other human tumors such as urothelial carcinoma of the bladder [24], osteosarcoma [28], and breast cancer [29]. In PC, Yee et al. indicated that the expression of TRPM8 is increased in pancreatic adenocarcinoma and it plays important role in cellular proliferation [26]. Consistent with the previous study, our data also revealed that TRPM8 mRNA levels are obviously elevated in PC tissues compared to the adjacent non-cancerous tissues. All these data show that TRPM8 may be a tumor promoter in the development of PC. Therefore, to assess the clinical significance of TRPM8 in PC progression, we evaluated its association with the clinicopathological characteristics. The results showed significant correlations between TRPM8 expression and tumor size, TNM stage, and distant metastasis.

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A growing body of evidence supports the idea that TRPM8 mRNA expression can be used as a prognostic marker and as a tool for the design of novel cancer therapies [23,30,31]. For example, Zhao et al. showed that both the mRNA and protein expression levels of TRPM8 were high in osteosarcoma patients and the over-expression of TRPM8 was a valuable prognostic biomarker for patient survival; therefore, it may be a novel potential molecular target for the treatment of osteosarcoma patients [23]. Although the previous study has supported exploration of TRPM8 as a biomarker and target of PC [32], its role in prognosis of PC is still unclear. In the present study we found that patients in the high-TRPM8 expression group had worse outcomes than those with low expression, and TRPM8 might be a potential valuable prognosis biomarker for PC patients.
Previous reports have indicated that TRPM8 affects the development of human tumors by regulating cell proliferation, invasiveness, and adhesion [16,33]. Moreover, the expression of TRPM8 is involved in a variety of signal channels of cells, such as the activation of phospholipase C and phosphoinositides [34]. However, the mechanism underlying the over-expression of TRPM8 in PC cells remains unclear. The expression profiles of TRPM8 mRNA were reported to be significantly associated with Ca 2+ channel activity and the Ca 2+ signaling pathway is involved in both cell survival and death through 3724 the transfection of Ca 2+ from the endoplasmic reticulum (ER) to the mitochondria [35]. The transfection process may result in alterations in multiple biological processes, such as energy production, and reactive oxygen species (ROS) production, thus significantly influencing cell survival status [36,37]. We found that the alterations in Ca(2+)-permeable channel induced by over-expression of TRPM8 played contributory roles in cellular proliferation, thus contributing to pancreatic tumor growth and metastasis. TRPM8 might be a potential biomarker for the treatment and therapy of pancreatic adenocarcinoma [26]. Therefore, in the future, more attention should be focused on the role of TRPM8 expression in the generation of Ca 2+ release channels in PC cells.

Conclusions
In conclusion, the present study demonstrates that TRPM8 levels are significantly increased in PC tissues. Moreover, high TRPM8 expression level correlates with worse OS and DFS of PC; therefore, it could be an effective prognosis factor for PC patients.