TRPM8 ion channel is aberrantly expressed and required for preventing replicative senescence in pancreatic adenocarcinoma

Abstract

Pancreatic adenocarcinoma is mostly fatal and generally resistant to conventional treatments, such that effective therapies with tolerable side effects are desperately needed. Ion channels including the transient receptor potential (TRP) channels have been implicated in human malignancies, but their roles in pancreatic cancer were mostly unknown. Recent identification of the melastatin-subfamily members of the TRP family of ion channels, and their functions in pancreatic epithelia and adenocarcinoma, is expected to provide a new perspective to understanding the mechanism underlying pancreatic tumorigenesis. In this report, we present the clinical and pathological features of a mini-series of patients with pancreatic adenocarcinoma, which aberrantly exhibits immunoreactivity against the TRPM8 channel. We have recently demonstrated the proliferative role of TRPM8 channel in pancreatic cancer cells. Here, we present evidence that RNA interference-mediated silencing of TRPM8 induces replicative senescence in pancreatic adenocarcinoma cells. This suggests that the aberrantly expressed TRPM8 channel may contribute to pancreatic tumorigenesis by preventing oncogene-induced senescence, and targeted inhibition of TRPM8 may enhance tumor sensitivity to therapeutics. Based on these observations, we hypothesize that the TRPM8 ion channel plays a crucial role in the growth and progression of pancreatic neoplasia during tumorigenesis. We propose that TRPM8 can be exploited as a clinical biomarker and as a therapeutic target for developing personalized therapy in pancreatic adenocarcinoma.

Keywords: :

• biomarker
• calcium
• cellular senescence
• ion channel
• pancreatic adenocarcinoma
• target
• transient receptor potential
• TRPM7
• TRPM8

Introduction

Pancreatic adenocarcinoma is a highly lethal disease; its incidence is rising, and afflicted individuals generally have poor prognosis.¹ Most of the patients die from metastatic disease that is typically resistant to conventionally used chemotherapeutic agents. Novel approaches targeting the genetic alterations involved in pancreatic carcinogenesis are expected to facilitate design of effective and patient-tailored therapy. Novel biomarkers and molecular targets are emerging, and this area of research continues to be a focus of active investigation with the goal of improving treatment response in patients with pancreatic adenocarcinoma.

Molecular studies have revealed the genetics and biology of multi-step pancreatic carcinogenesis. Development of pancreatic neoplasia involves pre-senescence of pancreatic epithelia and deregulation of developmental pathways, oncogenes, and tumor suppressors. Pancreatic intra-epithelial neoplasia (PanINs) are pre-malignant lesions, and their progression to invasive adenocarcinoma is associated with aberrant activation of hedgehog signaling, overexpression of epidermal growth factor receptor (EGFR), oncogenic mutations in K-RAS, and inactivation or loss of the tumor suppressors WRN, p16^INK4A, SMAD4, LKB1, and p53.^2-6 Besides, accumulating evidence has revealed important roles of ion channels in human cancers, but very little is known about their identity and functional roles in pancreatic cancer.

The transient receptor potential (TRP) family of ion channels play diverse physiological roles by sensing physical stimuli in the cellular environment and responding with modulation of intracellular ionic currents and signaling pathways.^7,8 The TRP members possess common features, including a trans-plasma membrane channel that exhibits relative ionic selectivity, and they exert regulatory effects on cellular proliferation, survival, differentiation, and migration. Consistent with these cellular functions, aberrantly expressed TRP channels play important roles in the pathogenesis of many human diseases, including cancer. We have recently shown that the TRP melastatin-subfamily members, TRPM7 and TRPM8, are required for proliferation of pancreatic adenocarcinoma cells.^9,10 Here, we present the clinical and pathologic features of a mini-series of patients with pancreatic adenocarcinoma, which exhibits aberrant immunoreactivity against TRPM8. Moreover, we show that targeted silencing of TRPM8 with small interfering RNA (siRNA) induces replicative senescence in pancreatic adenocarcinoma cells. A working model is proposed for TRPM8-mediated signaling that modulates EGF-induced cellular proliferation and survival in pancreatic adenocarcinoma. We hypothesize that TRPM8 contributes to development and progression of pancreatic neoplasia, and we suggest that TRPM8 can be developed as a clinical biomarker and a therapeutic target in pancreatic adenocarcinoma.

Clinicopathological Features of Pancreatic Adenocarcinoma that Aberrantly Expresses TRPM8

Among the patients included in this clinical case series, one of them is presented in detail with the medical history, clinical course, radiological images, and histopathology. The clinicopathological features and outcomes of all of these five patients are summarized in Table 1. The first patient is a 67 y-old Caucasian man, who initially presented with painless jaundice, dark urine, acholic stools, and weight loss of 60 lb in one year. Computed tomographic scan of the abdomen revealed a mass in the head and neck region of the pancreas, with compression of the bile duct (Fig. 1A). Endoscopic retrograde cholangiopancreatography was performed with placement of a biliary stent, and the signs and symptoms associated with biliary obstruction resolved. He was further evaluated using endoscopic ultrasonography (Fig. 1B) with fine needle aspiration of the pancreatic mass; the cytology was consistent with adenocarcinoma.

Table 1. Clinicopathological features and anti-TRPM8 immunoreactivity of pancreatic adenocarcinoma in a mini-series of five patients

Age	Gender	Smoking	Signs and symptoms	Pathological features	Serum CA19–9	TRPM8 Immuno-reactivity	Clinical outcomes	Fig.
67	Male	Denies	Jaundice, dark urine, acholic stool, weight loss	Stage IIb (T3 N1 M0)	Pre-op. 449 U/ml Post-op. Not available	Positive	Pancreaticoduodenectomy followed by adjuvant chemotherapy and radiation therapy. Developed pulmonary emboli. Treated with anti-coagulant. Complicated by hematochezia. Died.	1, 2
53	Male	2 p.p.d. for 5 y	Dark urine, acholic stool, jaundice, pruritis	Stage IIb (T3 N1 M0)	Pre-op. 140 U/ml Post-op. 100 U/ml	Positive	Pancreaticoduodenectomy followed by adjuvant chemotherapy and radiation therapy. Developed abdominal polymicrobial abscesses. Died.	3A
56	Female	1 p.p.d. for 34 y	Abdominal pain, jaundice, acholic stool, dark urine	Stage IIa (T2 N0 M0)	Not available	Positive	Neoadjuvant chemoradiation therapy. Pancreaticoduodenectomy with tumor at retroperitoneal resection margin and 8 negative lymph nodes. Complicated by leak of a hepaticojejunostomy anastomosis causing bacterial peritonitis, sepsis, and death.	3B
58	Female	Denies	Jaundice, anorexia, weight loss, dark urine, acholic stool	Stage IIa (T3 N0 M0)	Pre-op. 253 U/ml Post-op. Sixteen U/ml	Positive	Pancreaticoduodenectomy with negative surgical margin and 15 negative lymph nodes, followed by adjuvant chemotherapy and radiation therapy. No tumor recurrence for 4 y.	3C
49	Female	Denies	Dark urine, jaundice, epigastric pain radiating to back	Stage IIb (T2 N1 M0)	Pre-op. 43 U/ml, Post-op. 34 U/ml	Positive	Pancreaticoduodenectomy with tumor present at distal pancreatic duct margin and in 4 of 10 peri-pancreatic lymph nodes. Received adjuvant chemotherapy and radiation therapy. Local Recurrence in body of remnant pancreas with elevation of serum CA19–9 to 95 U/ml 3 y later. Undergone total pancreatectomy followed by adjuvant gemcitabine.	3D

Age, age in years-old at diagnosis of pancreatic adenocarcinoma; p.p.d., pack of cigarettes per day; Pre-op., pre-operative; Post-op., post-operative. 5-fluorouracil was used as part of the neoadjuvant and adjuvant therapy unless indicated otherwise

Figure 1. Radiographic and pathological features of pancreatic adenocarcinoma with metastasis to regional lymph nodes. (A) computed tomograph showing heterogeneous attenuation in the head and neck region of the pancreas (green arrow) with abrupt termination of common bile duct and pancreatic duct consistent with pancreatic cancer. (B) endoscopic ultrasonograph showing a large, hypoechoic mass (indicated diagonally by yellow and blue dotted lines) in the head of the pancreas with irregular outer margins. (C–F) histology of resected pancreatic adenocarcinoma stained with hematoxylin and eosin. (C) pancreatic tumor that contains atypical glands infiltrating a markedly desmoplastic stroma consistent with well-differentiated adenocarcinoma (20x magnification). (D) pancreatic adeno-carcinoma, in which atypical glandular epithelia contain large, pleomorphic nuclei with prominent nucleoli (200x magnification). (E and F) peri-pancreatic lymph nodes containing metastatic adenocarcinoma (20x magnification).

His past medical history was significant for previously treated prostate cancer and non-insulin-dependent diabetes mellitus. He denied any family history of cancer and any tobacco use. The patient appeared cachectic, and physical examination was otherwise unremarkable. Laboratory tests showed elevated serum levels of the tumor marker CA 19–9 (449 U/ml, normal range 0–37 U/ml), glucose 197 mg/dl (normal range 65–99 mg/dl), and alkaline phosphatase 128 U/l (normal range 35–104 U/l). Hepatic transaminases and total bilirubin were within the normal limits.

The patient underwent pancreaticoduodenectomy (Whipple’s procedure) for resection of the localized pancreatic tumor. Pathological evaluation revealed a 3.5-cm pancreatic adenocarcinoma extending beyond the pancreas, but showed no involvement of celiac axis and superior mesenteric artery, and no tumor was detected in the surgical margins (Fig. 1C and D). However, metastatic tumor cells were present in four peri-pancreatic lymph nodes (Fig. 1E and F). Therefore, the pathological stage of his pancreatic adenocarcinoma is IIb (T3 N1 M0). Following surgical resection of the pancreatic tumor, he received adjuvant chemotherapy (5-fluorouracil) and radiation therapy. He tolerated the adjuvant therapy fairly well, except for febrile neutropenia. Subsequently, he was diagnosed with bilateral pulmonary emboli and treated with enoxaparin for anti-coagulation. He then developed hematochezia, after which his condition began to deteriorate, and he eventually died. Autopsy was offered for investigation of his death but declined by his family.

The other four patients in this mini-series were also diagnosed with stage II pancreatic adenocarcinoma, and they were treated by surgical resection. One of them received adjuvant chemoradiation therapy and subsequently died from sepsis. The other died from sepsis following the operation. One of the patients received adjuvant chemoradiation therapy, and tumor recurrence has not been detected so far. The last patient on the list was treated with adjuvant chemoradiation therapy; she then developed tumor recurrence in the remaining pancreas, and she underwent total pancreatectomy, and received further adjuvant chemotherapy (Table 1).

The clinicopathological features of these patients are somewhat representative of the majority of cases of pancreatic adenocarcinoma.^1,2,11 Consistent with the previous studies showing that cigarette smoking accounts for one third of the cases of pancreatic cancer, 60% of the patients described here did not have a history of smoking. The clinical presentations of these patients resulted from biliary obstruction caused by the tumors located in the pancreatic head, which is a common location for the majority of this tumor. CA19-9 is a commonly used clinical biomarker of pancreatic adenocarcinoma, and its lack of good correlation with tumor status can be observed in this mini-case series. In general, the median survival of patients with stage II pancreatic adenocarcinoma is 12–24 mo, and adjuvant therapy has been shown to prolong survival by approximately 8 mo. In the patients discussed above, post-operative complications and possibly recurrence of pancreatic cancer contribute to mortality. These data further support the urgent need of identifying and validating clinically useful biomarkers, therapeutic targets, and agents to facilitate early detection and improve treatment response of this malignant disease.

In an attempt to gain insight into the clinical significance of TRPM8 in pancreatic cancer, we analyzed its expression in the surgical specimens of pancreatic adenocarcinoma by immuno-histochemistry. In contrast to normal pancreatic ducts and acini,¹⁰ aberrant expression of TRPM8 is consistently observed in the pancreatic adenocarcinoma (Figs. 2 and 3; Table 1). In agreement with the proliferative role of TRPM8 in pancreatic cancer cells,¹⁰ these findings further support our hypothesis for a contributory role of TRPM8 in pancreatic tumorigenesis. Although the mechanisms that mediate the functional role of TRPM8 in pancreatic cancer cells are unknown, biochemical and molecular studies of TRPM8 in other types of cells have begun to elucidate the regulation of its ionic channel activity and its cellular functions.

Figure 2. TRPM8 is aberrantly expressed in pancreatic adenocarcinoma. The paraffin-embedded tumor sections from the surgically resected specimen were analyzed by immunohistochemistry using rabbit anti-human TRPM8 antibodies (Lifespan Biosciences) at 1:50 dilution, followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG (EnVision+ System, Dako). The signals were detected by color reaction using 3,3′-diaminobenzidine (Dako), counterstained with hematoxylin (Richard-Allan Scientific), and mounted using Permount (Sigma). The brown color indicates the presence of TRPM8 protein. Strong immunoreactivity against TRPM8 can be observed in the ductal epithelia of the pancreatic adenocarcinoma, with staining in both plasma membrane and cytoplasm. There is heterogeneity for expression of TRPM8 in the tumor. (A) 40x magnification. (B) 200x magnification. (C) 400x magnification. Tissue sections incubated in the absence of anti-TRPM8 antibodies were processed in parallel as control, and they did not exhibit any detectable immunoreactivity against TRPM8 (data not shown).

Figure 3. Expression of TRPM8 in pancreatic adenocarcinoma tissues. Surgically resected specimens were embedded in paraffin and processed for immunohistochemistry using rabbit anti-human TRPM8 antibodies (Lifespan Biosciences) followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG (EnVision+ System, Dako). The signals were detected by color reaction using 3,3′-diaminobenzidine (Dako), counterstained with hematoxylin (Richard-Allan Scientific), and mounted using Permount (Sigma). The brown color indicates the presence of TRPM8 protein. Note the heterogeneity for expression of TRPM8 in the tumors. Controls using the consecutive tissue sections for each paraffin block were processed in the absence of anti-TRPM8 antibodies, and no immunoreactivity against TRPM8 was detected (data not shown). The panels A though D correspond to the patients as described in Table 1.

Biochemical Functions and Molecular Genetics of TRPM8

Structure and channel activity of TRPM8

TPRM8 is a Ca²⁺ permeable, non-selective, and voltage-gated ion channel. It consists of 1,104 amino acid residues, with a relative molecular mass of 128,000.^12,13 TRPM8 contains six transmembrane domains located between amino acids 695–1,024.¹² The S1-S4 transmembrane domains demonstrate weak voltage-sensing properties, and the channel pore is located between the S5 and S6 domains.^12,13 The ion channel of TRPM8 can be activated by cold temperature (between 25°C and 15°C) and cooling compounds (such as menthol, eucalyptol, and icilin), resulting in increased intracellular Ca²⁺ levels.^12,13 Indeed, TRPM8 is permeable to multiple cations—monovalent ions such as Na⁺ and K⁺ as well as divalent ions including Ca²⁺ and Ba²⁺. Opening of the channel is voltage-dependent and the probability of opening increases with membrane depolarization.¹⁴ Furthermore, experiments in Chinese hamster oocytes and trigeminal neurons showed that TRPM8 had a large outward rectifying current with poor cationic selectivity.^12,13 However, in the prostatic cancer cell line LNCaP (lymph node carcinoma of the prostate), TRPM8 shows a strong inward rectifying current with high Ca²⁺ selectivity. Such difference in channel characteristics of TRPM8 may be related to its localization to both endoplasmic reticulum (ER) membrane and plasma membrane of LNCaP cells.¹⁵ It has been demonstrated that, in LNCaP cells, TRPM8 mediates Ca²⁺ release from the ER as induced by cold temperature or menthol and also from the plasma membrane store-operated channels.¹⁶ Thus, TRPM8 functions as a non-selective cation channel that is active at depolarized membrane potentials, and its channel activity appears to depend on the cellular context.

The channel activity of TRPM8 can be modulated by chemical and physical stimuli in the cellular environment as well as intracellular signaling molecules. Thermal cooling decreases the depolarization potential required to activate the channel.¹⁷ Menthol and cold temperature can directly activate TRPM8, whereas icilin activates TRPM8 only in the presence of extracellular Ca²⁺ ions.^13,18 Acidic pH produces an inhibitory effect whereas alkaline pH exerts a potentiating effect on the channel activity.¹⁹ Besides, the lipid signaling molecule phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) also plays a regulatory role in the channel activity of TRPM8. PtdIns(4,5)P₂ is necessary for the activation of TRPM8 by external stimuli including cold temperatures and cooling agents.^20,21 Addition of exogenous PtdIns(4,5)P₂ to cells with desensitized TRPM8 partially restores the response of TRPM8 to cold stimuli and cooling agents as well as directly activates the channel. Phosphoinositide (PI) 4-kinase is involved in PtdIns(4,5)P₂ synthesis, and addition of PI 4-kinase inhibitors to whole cells reduced TRPM8 activity confirming the importance of PtdIns(4,5)P₂ in its channel function.²⁰ Evidence indicates that PtdIns(4,5)P₂ interacts with the positive carboxy terminus residues in TRPM8, and the affinity of PtdIns(4,5)P₂ for TRPM8 is increased by cold or cooling agents. On the other hand, the influx of Ca²⁺ activates a Ca²⁺-sensitive phospholipase C that deplete PtdIns(4,5)P₂, causing desensitization of TRPM8.²¹ Taken together, a variety of environmental cues contribute to the regulation of TRPM8 channel activity, whereas PtdIns(4,5)P₂ can act as a messenger regulating the function of TRPM8 in response to environmental stimuli, including cold temperature and alkaline pH.

Expression and cellular functions of TRPM8

In adult humans, TRPM8 is expressed in a tissue-selective pattern, and hormones play a regulatory role. The prostate gland expresses relatively high level of TRPM8 mRNA.²² It is also expressed at a discernable level in the liver. In dorsal root ganglion (DRG) and trigeminal ganglion (TG) neurons, TRPM8 is expressed in the C- and Ad- fibers that sense cold temperature and pain.^12,23 In prostatic carcinoma, expression of TRPM8 is upregulated in apical epithelial cells and androgen-dependent. Putative androgen response elements have been found upstream and downstream of the coding sequence of TRPM8 in prostate carcinoma cell lines. As the epithelia become de-differentiated and androgen-independent, TRPM8 expression decreases.^15,24,25 TRPM8 has been shown to localize in both plasma membrane and ER of prostate cancer cells.¹⁵ Androgen appears to play a role in the localization and function of TPRM8 on the plasma membrane, whereas localization to the ER is androgen-independent.²⁶ In breast cancer cell lines, expression of TRPM8 also seems to be hormone-dependent, since estrogen upregulates TRPM8 levels in estrogen receptor-positive cells.²⁷

TRPM8 plays a role in cellular proliferation and survival as well as detecting and mediating thermosensation,^15,23 yet how these cellular functions are inter-related and mediated remain to be determined. PtdIns(4,5)P₂ is necessary for the activation of TRPM8 by cold temperature, and it may be important in regulating TRPM8-mediated proliferation.^20,21 Geraniol is an intermediate in the biosynthesis of cholesterol; it enhances cell proliferation in prostatic epithelia, possibly by acting as an intracellular messenger that interacts with TRPM8.^15,28 It has been demonstrated that TRPM8 mediates depletion of intracellular Ca²⁺ store in prostatic cancer cells.¹⁶ Hypothetically, external stimuli that influence phosphatidylinositol metabolism and cholesterol homeostasis regulate the production of intra-cellular messengers such as PtdIns(4,5)P₂ and geraniol, which in turn modulate the function of TRPM8 and intracellular calcium levels, and thus cellular proliferation and survival.

Role of TRPM8 in Pancreatic Cancer

Aberrant expression of TRPM8 in pancreatic adenocarcinoma

Until recently, the role of TRP ion channels in pancreatic adenocarcinoma had been under-investigated. Of the eight mammalian TRPM channels, only the expression of TRPM8 is consistently elevated in the seven human pancreatic adenocarcinoma cell lines being examined.¹⁰ This is confirmed by analysis of TRPM8 immunoreactivity in surgically resected specimens of pancreatic adenocarcinoma (reference 10 and this study). The mechanism underlying the role of TRPM8 in cellular proliferation and survival of pancreatic epithelia and neoplasia is unclear, and it has become an area of active investigation in our laboratory.

In comparison with the minimal, if any, expression in normal pancreatic ducts,¹⁰ TPRM8 is aberrantly expressed in pancreatic adenocarcinoma (Figs. 2 and 3). The mechanism underlying the elevated expression remains to be determined. Hypothetically, amplification of the TRPM8 gene, or epigenetic mechanisms such as histone modifications or DNA hypomethylation of the TRPM8 promoter region, can lead to elevated expression of the TRPM8 gene. Androgen receptor is variably expressed in human pancreatic adenocarcinoma cell lines,²⁹ and it is possible that androgen may upregulate expression of TRPM8 in pancreatic adenocarcinoma in a similar manner as in prostatic carcinoma. We are currently examining a large number of malignant pancreatic tissues for expression of TRPM8 and correlating it with the clinicopathological features and molecular alterations. These studies are expected to help shed light on the clinical significance of TRPM8 in pancreatic cancer. Furthermore, we plan to test our hypothesis that TRPM8 is functionally expressed in pancreatic cancer stem cells or tumor-initiating cells using flow cytometry with stem cell-specific markers.

Proliferative role of TRPM8 in pancreatic adenocarcinoma

Expression of TRPM8 is essential for maintaining cellular proliferation in pancreatic adenocarcinoma. This is supported by our recent evidence that RNA interference-mediated silencing of TRPM8 in pancreatic adenocarcinoma cell lines impaired cellular proliferation and impeded cell cycle progression.¹⁰ As examined by phase contrast microscopy and DAPI staining of nuclei, the TRPM8-deficient cells are large and flattened, and they contain multiple nuclei and cytoplasmic vacuoles.¹⁰ These features are suggestive of non-apoptotic cell death through mitotic catastrophe and cellular senescence.³⁰ In agreement with these findings, flow cytometric analysis indicated that targeted knockdown of TRPM8 expression did not increase the proportion of apoptotic cells, as compared with the control siRNA-transfected cells (Fig. 4).

Figure 4. Anti-TRPM8 siRNA did not induce apoptotic cell death. The BxPC-3 and PANC-1 cells were transfected with anti-TRPM8 siRNA or non-targeting control siRNA by using Nucleofector^® (Amaxa/Lonza) as described.¹⁰ Following transfection, the cells were seeded at 2 x 10⁵ cells / 3 ml in each well of a 6-well cell culture cluster (Corning Inc., costar) and incubated at 37°C for 72 h. The cells were then washed with phosphate buffered saline (PBS, pH 7.4), incubated with fluorescein isothiocyanate (FITC)-conjugated Annexin V (Invitrogen) and propidium iodide (PI, Invitrogen), and analyzed for apoptosis by flow cytometry as described.⁴ The proportion of cells undergoing early apoptosis (lower right quadrant) and late apoptosis (upper right quadrant) is indicated. The baseline level of apoptotic cells in the control siRNA-transfected group may be attributed to electrical pulsation during Nucleofection^®, detachment of cells by trypsinization, and disaggregation of cells by filtration prior to flow cytometric analysis.

Next, we analyzed TRPM8-deficient pancreatic cancer cells for cellular senescence by determining the activity of senescence-associated β-galactosidase (SA β-gal) as a marker. Results of the SA β-gal assay indicate that RNA interference-mediated silencing of TRPM8 induced senescence in the pancreatic cancer cell lines BxPC-3 and PANC-1 (Fig. 5). These data suggest that TRPM8 is required to prevent non-apoptotic cell death that involves replicative senescence. In addition, molecular analysis revealed that the impaired cell cycle progression in the TRPM8-deficient cells is associated with increased mRNA levels of the cyclin-dependent kinase inhibitors, p21^CDKN1A and p27^CDKN1B.¹⁰ On the basis of these data, we hypothesize that aberrantly upregulated expression of TRPM8 in pancreatic adenocarcinoma contributes to the uncontrolled proliferation of the cancer cells by facilitating cell cycle progression and by preventing non-apoptotic cell death through replicative senescence. We propose a working model that will form the basis for testing our hypotheses regarding the signaling mechanisms that mediate the proliferative and pro-survival effects of TRPM8 in pancreatic adenocarcinoma cells (Fig. 6).

Figure 5. RNA interference-mediated silencing of TRPM8 induced replicative senescence. The BxPC-3 and PANC-1 cells were transfected with either anti-TRPM8 siRNA or non-targeting control siRNA and then seeded at 4 x 10⁴ cells / 2 ml medium in each well of a 12-well cell culture cluster (Corning Inc., costar). The transfected cells were incubated at 37°C for 72 h, and then analyzed for activity of SA β-gal using the Senescence Detection Kit (Biovision Medical Products) according to the manufacturer’s instructions. Images were acquired under an inverted light microscope with phase contrast (Nikon TE-300) and processed using Adobe Photoshop CS3 extended. Appearance of blue color indicates SA β-gal activity (arrows), which is detected in the anti-TRPM8 siRNA-transfected cells but not in those transfected with control siRNA.

Figure 6. A working model for the proliferative and pro-survival roles of TRPM8 in pancreatic adenocarcinoma cells. Cold temperature, menthol, alkaline pH, or phosphatidylinositol-4,5-bis-phosphate (PtdIns(4,5)P₂) activates the ion channel of TRPM8. Conversely, acidic pH inhibits the channel activity of TRPM8. Once activated, TRPM8 allows inflow of Ca²⁺ from the extracellular medium into the cytosol, leading to activation of Ca²⁺-sensitive phospholipase C (PLC) and hydrolysis of PtdIns(4,5)P₂, thus providing negative feedback inhibition of the TRPM8 channel activity. Hydrolysis of PtdIns(4,5)P₂ produces inositol 1,4,5-triphosphate (Ins(1,4,5)P₃), which causes release of Ca²⁺ from intracellular stores and generates diacylglycerol (DAG). The increased Ca²⁺ or DAG activates protein kinase C (PKC). PKC in turn activates RAF in the RAS/ERK pathway,³⁴ leading to transcription of a variety of genes and resulting in cellular proliferation and survival.³⁵

Potential of TRPM8 as a diagnostic biomarker and a therapeutic target

Considering the functional expression of TRPM8 in pancreatic adenocarcinoma, we intend to explore the potential utility of TRPM8 as a biomarker for early detection and monitoring therapeutic response as well as a target for therapy. Assay for the expression level of TRPM8 along with other biomarkers in pancreatic tissues and patients’ blood with clinicopathological correlation may help establish TRPM8 as part of the molecular phenotype of pancreatic adenocarcinoma. The molecular signature thus established may be developed into a clinical tool for improving diagnosis and early detection of this disease, predicting and monitoring response to therapy, and determining prognosis.

The purported role of TRPM8 as a therapeutic target of pancreatic adenocarcinoma may be facilitated by the studies using small molecule inhibitors to probe the biophysical functions of TRPM8. For instance, SKF96365, 4-(3-chloro-pyridin-2-yl)-piperazine-1-carboxylic acid (4-tert-butyl-phenyl)-amide (BCTC), and 1,10-phenanthroline have been shown to be negative allosteric modulators of TRPM8 channel function.³³ Each acts to shift the voltage for activation of TRPM8 to more depolarized membrane potentials. The utility of these small molecules suggests the possibility of developing novel therapy through chemical modulation of TRPM8 channel activity. Moreover, targeted inhibition of TRPM8 expression and/or activity may enhance tumor sensitivity to the cytotoxic effects of chemotherapeutics or radiation therapy through induction of replication senescence. Thus, application of high-throughput screening of small molecule modulators of TRPM8 channel activity in combination with automated patch-clamp assays of ionic currents may help expedite discovery of anti-tumor therapies in pancreatic cancer. To prevent any effect on non-tumor target organs such as the prostate gland and sensory neurons, nanoparticles mediated delivery of the small molecule modulators of TRPM8 or siRNA directed against TRPM8 are expected to provide specific targeting of pancreatic cancer. Besides, TRPM8 may be utilized as a target for ligand-directed nanoparticles for pancreatic cancer-specific delivery of anti-tumor agents.

Conclusion and Prospective

Ion channels are functionally expressed in various types of cancer and they have the potential for development into clinical biomarkers and therapeutic targets. Pancreatic adenocarcinoma aberrantly expresses the TRPM8 ion channel, which is required for proliferation of the cancer cells by promoting cell cycle progression and by suppressing non-apoptotic cell death through replicative senescence. These data suggest that TRPM8 contributes to the uncontrolled growth of pancreatic neoplasia during the multi-step carcinogenesis, and provide a novel link between pancreatic cancer and cellular sensation. In ongoing studies, we aim to determine the mechanisms that mediate the proliferative and pro-survival role of TRPM8 by investigating TRPM8-mediated signal transduction in pancreatic adenocarcinoma cells and potentially in pancreatic cancer stem cells. By determining the expression levels of TRPM8 and other molecular markers in pancreatic adenocarcinoma and in patients’ blood, we aim to evaluate TRPM8 as a clinical biomarker for early detection, predicting and monitoring therapeutic response, and prognostic indication. Using small molecule modulators of TRPM8 along with ligand-directed nanoparticles, we will test and validate its role as a molecular target for development of cancer-specific therapy in this devastating disease.

Abbreviations:
BCTC	=	4-(3-chloro-pyridin-2-yl)-piperazine-1-carboxylic acid (4-tert-butyl-phenyl)-amide
CBD	=	common bile duct
DAG	=	diacylglycerol
DAPI	=	4’6 diamidino-2-phenylindole
DRG	=	dorsal root ganglion
EGFR	=	epidermal growth factor receptor
ER	=	endoplasmic reticulum
IHC	=	immunohistochemistry
IP3	=	inositol 1,4,5-triphosphate
PanIN	=	pancreatic intra-epithelial neoplasia
PCR	=	polymerase chain reaction
PI	=	phosphoinositide
PIP2	=	phosphatidylinositol 4,5-bisphosphate
PKC	=	protein kinase C
PLC	=	phospholipase C
siRNA	=	small interfering RNA
TG	=	trigeminal ganglion
TRP	=	transient receptor potential
TRPM7 and TRPM8	=	melastatin-subfamily member 7 and 8 of the TRP family

Acknowledgments

N.S.Y. is supported by the Physician Scientist Stimulus Package from The Pennsylvania State University College of Medicine. R.D.B. is a recipient of the Medical Student Research Fellowship from The University of Iowa Carver College of Medicine. Funding support of this work was obtained from Penn State Hershey Cancer Institute (N.S.Y.) and Cancer Center Support Grant (P30 CA 086862 by National Cancer Institute to Holden Comprehensive Cancer Center at The University of Iowa (N.S.Y.).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.