It is widely accepted that Kv channels participate in cancer development and progression and their expression has shown to be aberrant in several types of tumor tissue, also in PDAC (Serrano-Novillo et al., 2019; Teisseyre et al., 2019). It has been shown that Kv1.3 is expressed in different human PDAC cell lines, harboring mutation in p53 (Zaccagnino et al., 2017). The authors demonstrate that the inhibition of Kv1.3 by clofazimine, induces apoptosis in-vitro and reduces tumor weight in-vivo (Zaccagnino et al., 2017). Another study has reported a remodeling of Kv1.3 and Kv1.5 on a large cohort of human tissue samples (Bielanska et al., 2009). In fact, they showed that protein expression of Kv1.3 was lower in PDAC tissue, while Kv1.5 had a higher protein expression in PDAC tissue compared to healthy tissue (Bielanska et al., 2009; Comes et al., 2013). The low expression of Kv1.3 in PDAC can be explained by a hypermethylation of the KCNA3 gene promoter (Brevet et al., 2009). Similar to Kv1.3 the expression of Kv7.1 has recently been shown to be down-regulated in PDAC (Zaccagnino et al., 2016; Tawfik et al., 2020). KCNQ1 (gene coding for Kv7.1) was downregulated in PDAC tissue, compared to normal tissue. In addition, downregulation of KCNQ1 was found in a system comparing PDAC A818–6 cells grown as a highly malignant undifferentiated monolayer (ML) or as three-dimensional (3D) single layer hollow spheres (HS). Database analysis showed that KCNQ1 was involved in the enrichment of pancreatic secretion in normal pancreatic epithelium and HS, suggesting that a downregulation of KCNQ1 might impair fluid secretion in PDAC and ML cells, while being maintained in normal pancreas and HS cells (Tawfik et al., 2020). Another comprehensive study has been investigating the gene-expression levels of the transportome in PDAC and normal specimens (Zaccagnino et al., 2016). The authors showed the downregulation of five different K⁺ channels, including the K⁺ voltage-gated channels; KCNQ1 and KCNE1. Moreover, their results showed a downregulation of genes coding for the Kir4.2 (KCNJ15), Kir5.1 (KCNJ16), and the K₂P channel TWIK-3 (KCNK7). In addition, the expression of KCNJ15 and KCNK7 was associated with the expression of EMT transcription factors (Zaccagnino et al., 2016). The authors also suggested that the higher expression of K⁺ channels in normal pancreatic epithelium takes part in setting the resting membrane potential, which generates the driving force of fluid and ion secretion in the pancreatic ducts (Zaccagnino et al., 2016).

Kv10.1 is another Kv channel that has been reported in pancreatic cancer. The expression of Kv10.1 in peripheral tissues is very restricted (Hemmerlein et al., 2006), including pancreatic tissue (Pardo et al., 1999). A xenograft mouse model of pancreatic cancer showed that monoclonal antibodies blocking the Kv10.1 current exerts antitumor activity (Gomez-Varela et al., 2007). Because Kv10.1 is nearly absent in normal tissue, there is a certain tumor selectivity for Kv10.1 expression, which gives rise to the possibility that Kv10.1 can be used as a targeting channel for the delivery of cytotoxic compounds (Pardo and Stuhmer, 2014). However, the expression and function of Kv10.1 in PDAC must be further investigated.

Interestingly, another Kv channel, Kv11.1 has been implicated as an oncogene in various cancers, including PDAC (Arcangeli et al., 2014; Lastraioli et al., 2015a). In contrast, to the Kv10.1 expression, Kv11.1 is ubiquitously expressed in normal human tissues including heart where it is mainly expressed (Sanguinetti et al., 1995; McDonald et al., 1997; Pond et al., 2000; Camacho, 2006; Comes et al., 2015). KCNH2 (gene coding for Kv11.1) was identified as a gene with somatic mutations that could drive the metastatic process of PDAC (Zhou et al., 2012). Here, exome sequencing analysis showed that KCNH2 clustered into a single network related to cancer development. To investigate the involvement of KCNH2 in PDAC progression, the authors showed that knockdown of Kv11.1 reduced proliferation, colony formation and migration in PDAC cell lines. Immunohistochemical analysis of Kv11.1 expression showed expression in 8 out of 38 (21%) PDAC tissues, versus one out of 37 (2.7%) in normal tissues (Zhou et al., 2012). Another study further investigated the expression and role of Kv11.1 in PDAC (Feng et al., 2014). Here, immunohistochemical analysis confirmed a strong expression in PDAC tissues, with highest expression in the cytoplasm and membrane. In contrary, normal tissue showed only weak expression. The expression was confirmed in PDAC cell lines (Feng et al., 2014). Knockdown of Kv11.1 showed a significant decreased proliferation rate, higher number of cells undergoing apoptosis, cell cycle arrest in G1 phase and a reduction of migration and invasion, suggesting that Kv11.1 has a role in different aspects of PDAC progression (Feng et al., 2014). This was confirmed in a xenograft mouse model, were a knockdown of Kv11.1 in CFPAC-1 cells showed reduced tumor growth and fewer metastatic nodules, compared to tumors in mice injected with control cells (Feng et al., 2014). Furthermore, it was found that miR-96 was downregulated in tumor tissue and PDAC cells. The overexpression of miR-96 reduced cell proliferation, migration, and invasion in-vitro and reduced the Kv11.1 expression, tumor growth, and formation of metastasis in-vivo (Feng et al., 2014). This indicates that Kv11.1 could function as an oncogene in PDAC and be a potential target of miR-96 (Feng et al., 2014). Further investigations showed that Kv11.1 promotes pancreatic cancer cell migration, by modulation of F-actin organization and dynamics (Lastraioli et al., 2015b) suggesting its involvement in cancer metastasis (Arcangeli et al., 2014; Manoli et al., 2019).

IK (KCa3.1) channels are the K⁺ channels most frequently studied among this family in PDAC. Even though, transcripts of KCa4.1 and KCa4.2 also have been shown in some cell lines (Hayashi et al., 2012). Investigation of the KCa3.1 mRNA expression in primary pancreatic cancer tumors show that 8 of 9 tumors (89%) contain a 6- to 66-fold higher expression, compared to normal pancreatic tissue (Jager et al., 2004). KCa3.1 is also found overexpressed in several PDAC cell lines (Jager et al., 2004). The over-expression of KCa3.1 was associated with an increased Ca²⁺-activated K⁺-current. Pharmacological inhibition (by TRAM-34, Clotrimazole) of KCa3.1 completely suppressed cell proliferation of MiaPaCa-2 and BxPC-3 cells but not PANC-1 cells (Jager et al., 2004). Moreover, application of [Ca²⁺]_o while inhibiting with TRAM-34 or Clotrimazole rescued the MiaPaCa-2 and BxPC-3 cell proliferation but did not affect this of PANC-1 suggesting that PANC-1 cell line grows independently of functional KCa3.1 channels (Jager et al., 2004). Bonito and co-workers have also reported the role of KCa3.1 in PDAC cell proliferation and migration (Bonito et al., 2016). They showed a significant mRNA upregulation of KCa3.1 in MiaPaCa-2 and BxPC-3, but not in Capan-1 and PANC-1 cells. In addition, Patch clamp measurements revealed a Ca²⁺-activated K⁺ current, which was reduced by TRAM-34 and clotrimazole. Interestingly, a transient gene silencing of KCa3.1 in MiaPaCa-2 cells completely abolished the Ca²⁺ current (Bonito et al., 2016). MiaPaca-2 cell proliferation was inhibited with TRAM-34 and 1% FBS, whereas no effect was found by application of TRAM-34 and 10% FBS in the culture media, as shown before (Jager et al., 2004). Silencing of KCa3.1 removed the ability of MiaPaCa-2 cells to proliferate, and attenuated their cell invasion and migration. Surprisingly treatment upon TRAM-34 or clotrimazole increased cell migration. It was hypothesized that this could be due to Ca²⁺ homeostasis, which was investigated by Ca²⁺ imaging that confirmed that TRAM-34 evoked an increase of [Ca²⁺]_i (Bonito et al., 2016) possibly leading to promotion of cell migration (Lotz et al., 2004). This indicates that KCa3.1 expression and function are important for cell proliferation, migration and invasion (Bonito et al., 2016). Another study has identified KCa3.1 as a regulator of oxidative phosphorylation in MiaPaCa-2 cells as silencing and inhibition of KCa3.1 determined the effect of channel dependent-oxidative phosphorylation in proliferation and ATP generation (Kovalenko et al., 2016). In addition, MiaPaCa-2 cells showed mRNA and protein levels in mitochondria, suggesting that KCa3.1 is involved in proliferation through metabolic processes (Kovalenko et al., 2016). Three other studies have identified KCNN4 (gene coding for KCa3.1) as a gene related to PDAC as its transcripts and gene-level were upregulated compared to normal pancreatic tissue (Zaccagnino et al., 2016; Jiang et al., 2017; Shen et al., 2017). The upregulation of KCNN4 was associated with the gene expression of different EMT transcription factors (Zaccagnino et al., 2016).

The outward conducting, pH and membrane potential activated K₂P channels have an impact on physiological processes. They can regulate the cell volume, the membrane potential in form of being pH sensitive, modulate ion transport and Ca²⁺ homeostasis. They are involved in cancer progression due to their impact on cell growth survival and migration, as it has been shown in different types of cancer (Mu et al., 2003; Kim et al., 2004; Voloshyna et al., 2008; Alvarez-Baron et al., 2011; Lee G. W. et al., 2012; Nagy et al., 2014; Sauter et al., 2016). A broad data base analysis of K₂P expression in different cancers revealed an aberrant expression of different K₂P in PDAC (Williams et al., 2013). mRNA expression of KCNK1 (gene coding for TWIK-1) was upregulated in PDAC compared to normal tissue, and KCNK3 (gene coding for TASK-1) were downregulated (Williams et al., 2013). One study has found the functional mRNA and protein expression of KCNK5 (gene coding for TASK-2) in PDAC cell lines HPAF, but the role in cancer progression was not further studied (Fong et al., 2003). In another study a pH sensitive K⁺ current was identified in BxPC-3 cells and was probably mediated by TREK-1 (Sauter et al., 2016). TREK-1 protein expression was shown in PDAC cell lines where it was shown that TREK-1 was involved in proliferation (Sauter et al., 2016). A similar pattern was shown in a scratch wound healing assay were activation of TREK-1 lead to decreased migration. These results indicate that TREK-1 has a potential inhibiting role in PDAC proliferation and migration (Sauter et al., 2016). Very few studies have been done on the role of K₂P channels in PDAC. However, it can be suggested from other types of cancer that these channels can be related to cancer progression (Comes et al., 2015).

It is well known that physiological Ca²⁺ signaling has many effects in the exocrine pancreas, and takes part in stimulating secretion of HCO₃^- and other ions (Hegyi and Petersen, 2013; Maleth and Hegyi, 2014). In non-excitable cells, such as cancer cells, Ca²⁺ entry occurs mainly through SOCs (Mo and Yang, 2018) but also through transient receptor potential channels (TRP), which are selective for both Ca²⁺ and Na⁺ (Worley et al., 2007).

There is increasing evidence of dysregulated Ca²⁺ signaling in cancer. This evidence is based on the implication of SOCs and TRP in key hallmarks of cancer progression and as prognostic markers in several types of cancers (Prevarskaya et al., 2007; Shapovalov et al., 2016; Chen et al., 2019). Some members of SOCs and TRP have been studied in PDAC, even though knowledge is less pronounced compared to other types of cancer, such as breast-, cervical-, and colorectal cancer (Chen et al., 2019).

The complex of ORAI1 and STIM1 has been shown to play a role in carcinogenesis and to be involved in regulation of proliferation, migration, invasion and apoptosis in different types of cancer (Chen et al., 2019). Only two studies have been performed on PDAC showing that ORAI1 and STIM1 mediate SOC entry and that they are involved in proliferation, survival and apoptosis (Kondratska et al., 2014; Khan et al., 2020). It has been shown that both ORAI1 and STIM1 were expressed in several PDAC cell lines at mRNA and protein levels, with PANC-1 showing the highest levels of both. Knockdown of ORAI1 and STIM1 with siRNA showed a significant reduction of Ca²⁺ entry. This was confirmed in PANC-1, AsPC-1, MiaPaCa-2, and Capan-1 cells, indicating that SOC entry is mediated by ORAI1 and STIM1 in different PDAC cell lines. A recent study has revealed the involvement of Calcium Release-Activated calcium (CRAC) channel (ORAI1) in proliferation of PDAC (Khan et al., 2020). An inhibition with CRAC channel inhibitor, RP4010, showed a significant reduction of cell proliferation and colony formation in MiaPaCa-2 cells and in L3.6pl (a pancreatic adenosquamous carcinoma derivated cell line). The influx of calcium was also inhibited upon treatment with RP4010, suggesting that cell proliferation is mediated by regulation of Ca²⁺ entry through CRAC channel (Khan et al., 2020). It was proposed that cell proliferation was calcium-regulated through the AKT/mTOR signaling pathway as RP4010 inhibition decreased the mRNA levels and protein expression of phosphorylated AKT, modulated the expression of proteins important for downstream AKT/mTOR signaling. Furthermore, RP4010 or ORAI1 knockdown showed a decrease in mRNA levels and in nuclear translocation of NFAT1, suggesting that CRAC channel takes part in modulating calcium signaling associated with NFAT translocation and that PDAC proliferation is regulated through the calcium-activated AKT/mTOR/NFAT signaling (Khan et al., 2020). To test if RP4010 could enhance anticancer activity of standard used treatments gemcitabine and Nab-Paclitaxel, a combination of the three drugs were used to treat PDAC cell lines. The results showed a decrease in proliferation. A synergistic effect of certain dose combinations of RP4010 with gemcitabine/Nab-Paclitaxel was found to inhibit cell growth. In addition, this synergistic treatment downregulated the expression of NFATC1 and mTOR mRNA and NFAT1, NF-κB, and phosphorylated S6K proteins, suggesting that inhibition of cell proliferation through CRAC channel are mediated by a downregulation of mTOR, NFAT and NF-κB signaling (Khan et al., 2020). The anticancer activity and the synergistic effect of RP4010/Gemcitabine/Nab-Paclitaxel were tested in-vivo. In a patient-derived xenograft mouse model, it was shown that Ki-67 expression decreased with the treatment of RP4010 or by the triple combination treatment (Khan et al., 2020). The overexpression found by Kondratska and co-workers can explain increased [Ca²⁺]_i levels in PDAC cell lines, and that this is a mechanism for survival (Kondratska et al., 2014). In contrast, another study has found decreased gene expression levels of ORAI1 (Zaccagnino et al., 2016).

A recent study has been investigating the role of STIM1 in PDAC progression (Wang et al., 2019). shRNA knockdown of STIM1 showed decreased proliferation, invasion, and upregulation of E-cadherin protein levels and downregulation of vimentin levels, suggesting that STIM1 is involved in carcinogenesis of PDAC and in some way involved in Epithelial-Mesenchymal transition (EMT). Even though, E-cadherin levels have shown to be upregulated, in contrary to what is usually seen in cells undergoing EMT where E-cadherin decrease in favor of N-cadherin (Gheldof and Berx, 2013). Furthermore, tissue microarray analysis showed that the STIM1 expression positively correlated with HIF-1α (Wang et al., 2019). It was further shown that similar protein expression levels of STIM1 and HIF-1α were expressed in different PDAC cell lines. STIM1 and HIF-1α protein levels were also upregulated in some PDAC tumor samples compared to non-tumor samples. Knockdown of HIF-1α in PANC-1 cells revealed a significantly lower mRNA and protein expression of STIM1. The co-upregulation of both proteins and the downregulation of STIM1 upon knockdown of HIF-1α indicate that STIM1 is regulated by HIF-1α on the transcriptional level. STIM1 promoter activity was tested in PANC-1 cells upon normoxia or hypoxia, where HIF-1α binding sites, under hypoxic conditions reduced STIM1 promoter activity (Wang et al., 2019). These results indicate that HIF-1α probably regulates STIM1 transcription and that STIM1 overexpression, in a hypoxic environment, can promote PDAC progression and invasion (Wang et al., 2019).

The EMT process is stimulated upon loss of cell-cell contact and occurs in migrating cancer cells (Gheldof and Berx, 2013). It has been shown in disconnected individual PANC-1 cells that ER/Plasma membrane junctions containing STIM1, together with the IP₃Rs, redistribute to the leading edge of focal adhesions (Okeke et al., 2016). An inhibition of IP₃Rs and SOC entry reduced the migrating capacity of PANC-1 cells. This mechanism indicates the importance of Ca²⁺ signaling in migration through SOC entry and intracellular calcium channels (Okeke et al., 2016).

TRP form an adaptable family of ion channel proteins where the majority are calcium permeable and show regulatory patterns that are sensitive to different environmental factors (Shapovalov et al., 2016). The role of TRP has been reported in different types of cancer (Prevarskaya et al., 2007). It has been proposed that TRPC1 can regulate PDAC cell proliferation through TGF-β signaling, as TGF-β has been shown to be one of the key modulators of EMT in mammary epithelial cells (Radisky and LaBarge, 2008). In PDAC cell line BxPC-3, TGF-β has shown to induce [Ca²⁺]_i increase leading to activation of the Ca²⁺-dependent protein kinase Cα (PKC-α) and its translocation to the plasma membrane. PKC-α activation by TGF-β initiates the motility and migration by inhibiting tumor suppressor PTEN (Chow et al., 2008). Further on, it has been shown that there is a high expression of TRPC1, TRPC4 and TRPC6 in BxPC-3 cells (Dong et al., 2010). Here, it was confirmed that TGF-β induces cytosolic Ca²⁺ concentrations through TRPC1, followed by a PKC-α activation, thus initiating motility and migration. This was shown by a pharmacological inhibition of SOC entry pathways with 2-APB and La³⁺, which abolishes the TGF-β induced cytosolic Ca²⁺ increase. Furthermore, blocking of PKC-α with selective PKC-α inhibitors inhibited the TGF-β mediated Ca²⁺ entry. In addition, knockdown of TRPC1 with siRNA reversed the effect of TGF-β on cell motility, although, knockdown of TRPC4 and TRPC6 did not have an effect on motility of TGF-β mediated BxPC-3 cell motility (Dong et al., 2010). These observations suggest that dysregulated Ca²⁺ entry through TRPC1 could be involved in EMT, and thereby invasion and metastasis of PDAC.

TRPV channels function as sensors in the central and peripheral nervous system where the majority is sensitive to voltage and temperature (Premkumar and Abooj, 2013). TRPV1 has shown to be related to oncogenesis and is expressed in different types of cancer (Domotor et al., 2005; Lazzeri et al., 2005; Sanchez et al., 2005; Miao et al., 2008; Morelli et al., 2014; Vercelli et al., 2014). TRPV1 can be activated by multiple pathways, which can promote pancreatic inflammation and pain, but also pancreatic cancer (Hartel et al., 2006; Huang et al., 2020). TRPV1 was shown to be upregulated at the mRNA and protein level in PDAC tissue compared to normal pancreatic tissue (Hartel et al., 2006). TRPV1 staining has been shown in both normal acini and ducts but with highest intensity in nerves of inflamed tissue surrounding the cancer. The elevated TRPV1 expression in infiltrating nerves was associated with pain in patients with PDAC. The same authors showed that inhibition of TRPV1 with resiniferatoxin induces apoptosis by targeting mitochondrial respiration and decreases cell growth in some PDAC cell lines (Hartel et al., 2006).

Recently, it has been shown that TRPV1 regulates the Epidermal Growth Factor Receptor (EGFR) in PANC-1 cell line (Huang et al., 2020). In this study, an overexpression of TRPV1 has been associated with a decrease in protein expression of EGFR in PANC-1. Vice versa, the downregulation and inhibition of TRPV1 increases the protein expression of EGFR. In addition, an overexpression of TRPV1 showed increased levels of ubiquitinated EGFR. The membranous fractions of EGFR were reduced, while the cytoplasmic were increased compared to the control (Huang et al., 2020). This indicates that TRPV1 promotes EGFR ubiquitination and thereby a downregulation of EGFR activity, resulting in EGFR cytoplasmic translocation and degradation, which was found to be mainly through the lysosomal pathway (Huang et al., 2020). Furthermore, it was shown that TRPV1 overexpression inhibited proliferation, probably through the MAPK signaling pathway. Overexpression of TRPV1 resulted in decreased mRNA levels of KRAS and AKT2 and a treatment with EGF reduced the protein expression of ERK, JNK, and CREB, suggesting that a TRPV1 overexpression decreases EGFR/MAPK dependent proliferation in PANC-1 cells (Huang et al., 2020). The two above mentioned studies show contrary results in form of how the expression of TRPV1 is related to proliferation. Hartel et al., demonstrated that inhibition of TRPV1 terminate cell growth and induced apoptosis, where Huang et al., found that an overexpression of TRPV1 leads to a reduced proliferation rate (Hartel et al., 2006; Huang et al., 2020).

Another member of the TRPV family, TRPV6, was also found to be overexpressed in the primary pancreatic cancer tissues at both protein and mRNA levels. Moreover, by immunohistochemical analysis, it was found that TRPV6 is mainly localized in the cytoplasm in both tumor and normal tissue (Song et al., 2018). In-vitro, the highest level of TRPV6 was found in two pancreatic cell lines, Capan-2 and SW1990. The knockdown of TRPV6, by siRNA, resulted in reduced proliferation, cell cycle arrest in G0/G1 phase, promotion of apoptosis, and suppression of cell migration and invasion (Song et al., 2018). Furthermore, the silencing of TRPV6 resulted in a significant increase of sensitivity to the chemotherapeutic reagent oxaliplatin (Song et al., 2018). In contrast to this finding, Zaccagnino et al., 2016 showed a downregulation of TRPV6 in PDAC tissue, compared to normal pancreatic epithelium. Moreover, Tawfik et al., 2020 found also a downregulation of TRPV6 expression in a PDAC cell line A818–6 grown in a highly malignant undifferentiated monolayer. The authors suggest that a lower expression of TRPV6 could contribute to an inhibited epithelial fluid secretion in PDAC (Tawfik et al., 2020).

The TRPM family is also constituted with several members, which have been found to be implicated in carcinogenesis. One of the most studied in PDAC is TRPM7, which is a particular channel having an intrinsic kinase, together with its closest homolog TRPM6 (Yee et al., 2012a). TRMP7 is ubiquitously expressed and controls cellular homeostasis of ions, especially Mg²⁺ and Ca²⁺. Interpreting that the developmental role of TRPM7 in zebrafish could be the same in humans, the role of TRPM7 has been studied in the development and progression of PDAC. Here, it has been shown that there was an overexpression of TRPM7 protein in PDAC tissue compared to normal tissue, and that TRPM7 is required for Mg²⁺-regulated proliferation. Knockdown of TRPM7 with siRNA showed that this channel is necessary to prevent cell cycle arrest in G0/G1 phases. Furthermore, the proliferation of TRPM7-deficient PDAC cells was rescued by adding Mg²⁺ to the cell culture medium (Yee et al., 2011). Another study confirmed the overexpression of TRPM7 both at mRNA and protein levels in PDAC tissue (Rybarczyk et al., 2012). Furthermore, it was shown that TRPM7 silenced BxPC-3 cells decreased [Mg²⁺]_i, suggesting that TRPM7 takes part in regulating Mg²⁺ uptake in PDAC cells. In contrary to previous findings, these authors demonstrated that the silencing of TRPM7 had no effect on cell viability or proliferation, but a significant decrease of BxPC-3 cell migration (Rybarczyk et al., 2012). TRPM7 has also been found to be involved in cell invasion in both MiaPaCa2- and PANC-1 cells. In the two last cell lines, TRPM7 regulates constitutive cation currents, the influx and homeostasis of Mg²⁺, and cell invasion through the Hsp90α/uPA/MMP-2 proteolytic pathway (Rybarczyk et al., 2017).

Besides TRPM7, also other TRPM channels are found to be expressed in pancreatic cancer (Yee et al., 2010; Yee et al., 2014). TRPM8 is expressed in different types of adult human tissue and has also been found to be expressed in PDAC. In a panel of PDAC cell lines, mRNA TRPM8 was consistently overexpressed compared to the control cell line (H6c7) (Yee et al., 2010). This pattern has further been confirmed by immunohistochemistry in human PDAC tumors, compared to normal pancreatic tissue (Yee et al., 2010; Yee et al., 2014). Here, it was found that TRPM8 has a role in carcinogenesis in form of proliferation, migration and senescence. TRPM8 is required for proliferation by promoting cell cycle progression in PANC-1 and BxPC-3 cells, as a knockdown of TRPM8 showed a significant decrease in proliferation rate and a cell cycle arrest in G0/G1 phase (Yee et al., 2010). In another study, the knockdown of TRPM8 showed the opposite effect on proliferation. Here, the proliferation increased by 30% in PANC-1 cells and in contrary the proliferation was suppressed in HEK/M8 cells. It was found that TRPM8 is expressed in a non-glycosylated form in different PDAC cell lines, and that the channel in this form might have a protective role in PDAC (Ulareanu et al., 2017). Concerning the involvement of TRPM8 in migration and invasion, two studies show opposite results. One study demonstrated that TRPM8 also is required for cell migration, as a knockdown of the channel impaired migration of BxPC-3 and MiaPaCa-2 by 60% and 45%, respectively (Yee et al., 2014). Where another study found that it enhanced the motility of PANC-1 cells (Cucu et al., 2014).

Recently, it has been shown that a third member of the TRPM family, also plays a role in PDAC progression (Lin et al., 2018). An overexpression of TRPM2 enhanced the proliferative, migrative and invasive abilities of PANC-1 cells, compared to the control cells and the results were inversed when TRPM2 was silenced in PANC-1 cells. It should be noted, that the study does not mention the application of a proliferation inhibitor during the Scratch wound-healing assay, which investigates the migratory role of TRPM2. Therefore, one can speculate if the wound-healing could be caused by proliferation, and not migration. Nevertheless, these results suggest that TRPM2 is involved at least in cell growth and invasion (Lin et al., 2018).

The expression of two voltage dependent Ca²⁺ channels have been found to be dysregulated in PDAC, namely, CaV2.1 (CACNA1A) and CaV3.1 (CACNA1G) are upregulated and downregulated, respectively (Zaccagnino et al., 2016). These sparse data indicate that voltage-dependent Ca²⁺ channels might have a role in PDAC progression.

Aberrant expression and dysregulated function of Cl^- channels have shown to be involved in carcinogenesis, especially their role in cell volume regulation has shown to be important for cancer cell migration and infiltration (Duran et al., 2010; Prevarskaya et al., 2010; Anderson et al., 2019). In Capan-1 cells, CaCC are expressed at the apical membrane, as shown for normal pancreatic acinar and ductal cells (Park et al., 2001; Wang et al., 2013; Wang and Novak, 2013).

The functional role of TMEM16A has been found to vary between different types of cancer (Ayoub et al., 2010; Liu et al., 2012; Ruiz et al., 2012; Britschgi et al., 2013). While a pro-proliferative role was found in breast and prostate cancer, the role in pancreatic cancer has been found to be contradictory. An anti-proliferative effect was found by a knockdown and overexpression strategy in CFPAC-1 cells (Ruiz et al., 2012), where another study found that inhibition with the TMEM16A specific inhibitor T16A_inh-A01 decreased the proliferation rate in CFPAC-1 cells (Mazzone et al., 2012). Both studies lack the comparison of PDAC cell lines with a normal pancreatic epithelial control cell line. This was considered in a recent study, where the role of TMEM16A was investigated in PDAC cell lines and compared to a normal pancreatic epithelial control cell line (Sauter et al., 2015). The mRNA expression of TMEM16A was upregulated, with a 1,450-fold, in AsPC-1, BxPC-3, and especially in Capan-1 cells (Sauter et al., 2015). The upregulation was confirmed by an increase in TMEM16A protein expression for all three cell lines. Furthermore, it was shown that TMEM16A carries the major component of CaCC current in these cell lines (Sauter et al., 2015). Moreover, the authors found that knockdown of TMEM16A had no effect on proliferation. Inhibition, by T16A_inh-A01 or other CaCC inhibitors, failed to affect PDAC cell lines proliferation, while T16A_inh-A01 had a significant effect on the control cell line cell proliferation, which almost completely lack the expression of TMEM16A. These results suggest that the inhibition by T16A_inh-A01 is unspecific for TMEM16A, and that this channel has no implication in proliferation, at least in these three PDAC cell lines (Sauter et al., 2015). According to the role of TMEM16A in migration, gene silencing reduced the migratory capability of AsPC-1 and BxPC-3 cells, where the inhibition with T16A_inh-A01 was ineffective (Sauter et al., 2015). Other CaCC inhibitors caused a decrease in migration of BxPC-3 cells. Nevertheless, Capan-1 cells showed the highest expression of TMEM16A, the migration was very slow, suggesting that TMEM16A is not implicated in the role of migration in Capan-1 cells and supporting that TMEM16A has different roles in carcinogenesis of PDAC cells (Sauter et al., 2015).

Another recent study has performed a database investigation on the expression of TMEM16A and found that mRNA TMEM16A expression is upregulated in pancreatic cancer (Crottes et al., 2019). The authors found that extracellular application of EGF increased [Ca²⁺]_i and the outward-rectifying Cl^- current, which were both inhibited by different TMEM16A inhibitors. The regulation of Cl^- currents and the Ca²⁺ response were probably due to SOC entry. Furthermore, silencing of TMEM16A in AsPC-1 cells reduced migration even under EGF treatments, while EGF induced migration in the control cell line. This indicates that TMEM16A is involved in EGF-induced PDAC migration and progression, probably through Ca²⁺ signaling. In addition, this study investigated the possible role of TMEM16A to classify PDAC patients (Crottes et al., 2019). They found 10 genes involved in EGF-induced TMEM16A-dependent Ca²⁺ signaling, which could distinguish neuro-endocrine tumors from other pancreatic cancers. In PDAC, these genes formed three clusters with different genetic profiles that could reflect different molecular characterizations (Crottes et al., 2019).

Another TMEM16 protein expressed in pancreatic cancer is the TMEM16J protein, which also has been found to be overexpressed (Jun et al., 2017). TMEM16J is not a well characterized protein, but it is proposed that it might function as a cation channel activated by the cAMP/PKA signaling pathway (Falzone et al., 2018; Kim et al., 2018). An upregulation of TMEM16J gene-, mRNA-, and protein overexpression were found in AsPC-1, BxPC-3, and Capan-2 cell lines and a small overexpression in PANC-1 cells. An overexpression of TMEM16J in PANC-1 cells resulted in phosphorylated ERK1/2 levels, but not total ERK1/2 levels. Furthermore, both EGFR and phosphorylated EGFR levels were upregulated in PANC-1 cells overexpressing TMEM16J and an immunoprecipitation assay revealed that both TMEM16A and TMEM16J formed protein complexes with EGFR, but the binding affinity for TMEM16J was 132% higher, than for the one of TMEM16A (Jun et al., 2017), suggesting that TMEM16J are involved in upregulation and activation of EGFR. In contrary, a knockdown of TMEM16J in AsPC-1 cells resulted in inhibition of phosphorylated ERK1/2, EGFR and phosphorylated EGFR and a decreased proliferation rate. These results were confirmed in-vivo, were a xenograft mouse model was made by implanting PANC-1 cells stably overexpressing TMEM16J. It was shown that tumor growth was significantly increased and immunohistochemistry of these tumors confirmed the TMEM16J overexpression (Jun et al., 2017). These results indicate that TMEM16J is implicated in cell proliferation and tumor growth. Another member of the TMEM16 family, TMEM16E, has been shown to be implicated in PDAC. It is not yet clear whether the TMEM16E protein function as an ion channel or scramblase (Falzone et al., 2018). It has been shown, by immunohistochemical analysis, that TMEM16E is entirely expressed in PDAC but not in normal pancreatic tissue (Song et al., 2019). The highest expression of both mRNA and protein of TMEM16E was found in PANC-1 cells. The impact of TMEM16E on migration was investigated by a wound-scratch assay and a siRNA knockdown of TMEM16E showed a significant decrease in PANC-1 cell migration (Song et al., 2019). Even though, it should be mentioned that the authors do not account for the possible effect of proliferation in this assay. The migration was in some ways confirmed by the downregulation of vimentin protein expression, compared to the control, which showed a higher expression of vimentin, suggesting that TMEM16E is implicated in migration of PANC-1 cells. In addition, the proliferation of PANC-1 cells was significantly decreased upon knockdown of TMEM16E suggesting its role in proliferation (Song et al., 2019). This assay supports the speculation on the TMEM16E role in migration.

Besides being activated by Ca²⁺, CaCC can also be activated and regulated by specific proteins, namely Calcium-activated Chloride channel regulators (CLCAs) also called Calcium Chloride channel accessory proteins. CLCAs are expressed in different types of cancer and have been implicated in regulation of proliferation, migration and metastasis (Yurtsever et al., 2012; Lang and Stournaras, 2014; Stock and Schwab, 2015). CLCA1 has been shown to be overexpressed in pancreatic cancer (Hu et al., 2018a; Hu et al., 2018b). However, the expression pattern and underlying molecular mechanism of its role in PDAC is less known. Finally, low gene expression of Chloride Channel Kb (CLCNKB) and Chloride Voltage-Gated Channel 1 (CLCN1) have been reported in human PDAC tissue compared to normal pancreatic epithelium (Zaccagnino et al., 2016).

It has been shown that CFTR is expressed in some PDAC cell lines. An early study showed that CFTR only was expressed in Capan-1 cells among nine different pancreatic cell lines and that the expression varied as a function of confluence (Chambers and Harris, 1993). Singh et al., confirmed the almost non-existent expression of CFTR in PDAC cell lines. Indeed, mRNA levels were detectable in normal pancreatic tissue and three (Capan-1, Suit2 and SW1990) out of 16 pancreatic cell lines (Singh et al., 2007). Furthermore, Zaccagnino et al., reported the downregulation of CFTR, at gene-level, in human PDAC tissue compared to normal pancreatic epithelium. This downregulation was associated with gene expression of EMT transcription factors (Zaccagnino et al., 2016). Furthermore, Singh et al., showed that wild type CFTR negatively regulated MUC4 expression while silencing of CFTR upregulated MUC4 expression. As MUC4 is a protein involved in tumor migration and metastasis, the negative regulation by CFTR indicates a protective role and a tumor suppressing function by inhibiting MUC4 and hence pancreatic cancer progression (Singh et al., 2007).

A recent study has investigated CFTR expression in patient derivated PDAC organoids, in order to enable routine organoid subtyping for personalized treatment (Hennig et al., 2019). It has been suggested that subtyping could be based on the expression of cytokeratin 81 (KRT81) and hepatocyte nuclear factor 1A (HNF1A). As the antibody for HNF1A was no longer available, the authors permitted CFTR to replace it as a potential marker instead of HNF1A (Hennig et al., 2019). Organoids can be categorized into the established quasi-mesenchymal, exocrine-like, and classical subtypes. Immunofluorescence staining showed a mutual expression pattern where exocrine-like organoids were CFTR⁺/KRT81^- and quasi-mesenchymal CFTR^-/KRT81⁺. The protein expression revealed by IF was compared to mRNA levels of CFTR, which matched in 8 out of 10 cases (Hennig et al., 2019). In addition, it was confirmed, by immunohistochemical analysis, that both CFTR and KRT81 were preserved in 6 out of 7 tumors, indicating that the organoids had the same subtype as their primary tumor (Hennig et al., 2019). These results suggest that CFTR could be a supplement marker for HFN1A and that CFTR/KRT81 together might be a suitable way to evaluate subtype organoids for personal treatments (Hennig et al., 2019).

CLICs are ubiquitously expressed and have been identified in several types of cancer, where they are either overexpressed or downregulated compared to the normal tissue (Peretti et al., 2015). In PDAC, CLICs are mostly found upregulated, even though their specific role in PDAC progression and development is not yet understood. CLIC2, CLIC3, and CLIC5 have been shown to be expressed at mRNA and protein levels in a HPAF cell line. By an electrophysiological study, the authors revealed that there was no single channel/conductance for apical Cl^- secretion, but that these CLICs rather contributed to provide a constant net conductance across the plasma membrane (Fong et al., 2003). Another study has shown the importance of CLIC3 in PDAC, as immunohistochemical analysis and mRNA levels showed an overexpression of CLIC3 in PDAC tissue compared to normal pancreatic tissue (Dozynkiewicz et al., 2012). It was also found that CLIC3 in collaboration with Rab25 promoted cancer cell invasion and migration by integrin recycling from late endosomes/lysosomes (Dozynkiewicz et al., 2012). CLIC1 was overexpressed in primary tumors compared to normal pancreatic tissue, and strongly expressed in MiaPaCa-2 and PANC-1 cells (Lu et al., 2015). Silencing of CLIC1 showed a significant decrease in the proliferation rate, colony formation and the invasive abilities of both MiaPaCa-2 and PANC-1 cells, suggesting that CLIC1 contributes to the aggressive role of these PDAC cells (Lu et al., 2015). In addition, gene expression levels of CLIC5 has been found to be downregulated in PDAC tissue, compared to normal pancreatic epithelium and to be associated with gene expression of transcription factors related to cell differentiation (Zaccagnino et al., 2016).

It has recently been shown that acid-sensing ion channels (ASICs), an H⁺-gated subgroup of ENaC, are expressed in PDAC cell lines and tissue (Zhu et al., 2017). ASIC1 and ASIC3 were found functionally expressed and mRNA and protein expression were also found in PDAC cell lines. In all cases, the expression was upregulated compared to the normal control cell line. These results were confirmed in PDAC tissue where immunohistochemical analysis and qPCR revealed the overexpression compared to non-cancerous pancreatic tissue, suggesting that ASIC1 and ASIC3 have a pathophysiological role in PDAC (Zhu et al., 2017). Separate inhibition or knockdown of ASIC1 and ASIC3 decreased the acidity-promoted invasion and migration capacity of PDAC cell lines, but did not decrease the proliferation rate, suggesting that ASIC1 and ASIC3 are involved in the metastatic process of PDAC, but not tumor cell growth (Zhu et al., 2017). Furthermore, it was shown that ASIC1 and ASIC3 are involved in acidity-promoted EMT, as silencing or inhibition of ASIC1 or ASIC3 in PDAC cells showed decreased protein expression of mesenchymal markers Vimentin, N-cadherin, Snail, and ZEB1, while the epithelial marker E-cadherin showed increased protein expression. In contrary, PDAC cells overexpressing ASIC1 and ASIC3 showed an increase in mesenchymal markers and a decrease in epithelial markers, under acidic conditions. This was confirmed in human PDAC tissue samples by IF analysis. It was further investigated whether this mechanism was regulated by [Ca²⁺]_i, where it was found that inhibition of ASIC1 or ASIC3 resulted in a decrease of [Ca²⁺]_i upon acidification. In addition, the removal of [Ca²⁺]_i upon acidic conditions decreased mesenchymal markers and increased the epithelial ones. It was determined that the RhoA pathway, which is involved in cytoskeleton re-arrangement and cell migration, was a major effector of EMT induced by ASIC1/3-[Ca²⁺]_i activation in acidic conditions (Zhu et al., 2017). The role of ASIC1 and ASIC3 was further confirmed in-vivo, where a xenograft mouse model injected with BxPC-3 cells with a stable knockdown of ASIC1 and ASIC3 showed a significant decrease in lung and liver metastasis, but no obvious effect on tumor growth (Zhu et al., 2017).

Another subfamily of Na⁺ channels, namely voltage gated sodium channels (VGSCs), has shown to be implicated in cancer progression (Angus and Ruben, 2019). An early study has shown that Ca²⁺ blockers Phenytoin and Verapamil inhibited the growth of pancreatic cancer cell lines MiaPaCa-2 and CAV, both in-vitro and in-vivo (Sato et al., 1994). Phenytoin and Verapamil were chosen because they appeared to be blocking different Ca²⁺ channels; T-type and L-type voltage dependent Ca²⁺ channels, respectively (Sato et al., 1994). It has been suggested that this growth-inhibition of pancreatic cancer cell was rather due to the block of VGSC than the block of Ca²⁺ channels, as both Phenytoin and Verapamil show high affinity for VGSC in the inactivated state of the channel (Ragsdale et al., 1991; Koltai, 2015). In addition, the expression of VGSC (SCN9A and SCN3A) was downregulated in PDAC (Zaccagnino et al., 2016).