A Novel Role of the TRPM4 Ion Channel in Exocytosis.

Under physiological conditions, the widely expressed calcium-activated TRPM4 channel conducts sodium into cells. This sodium influx depolarizes the plasma membrane and reduces the driving force for calcium entry. The aberrant expression or function of TRPM4 has been reported in various diseases, including different types of cancer. TRPM4 is mainly localized in the plasma membrane, but it is also found in intracellular vesicles, which can undergo exocytosis. In this study, we show that calcium-induced exocytosis in the colorectal cancer cell line HCT116 is dependent on TRPM4. In addition, the findings from some studies of prostate cancer cell lines suggest a more general role of TRPM4 in calcium-induced exocytosis in cancer cells. Furthermore, calcium-induced exocytosis depends on TRPM4 ion conductivity. Additionally, an increase in intracellular calcium results in the delivery of TRPM4 to the plasma membrane. This process also depends on TRPM4 ion conductivity. TRPM4-dependent exocytosis and the delivery of TRPM4 to the plasma membrane are mediated by SNARE proteins. Finally, we provide evidence that calcium-induced exocytosis depends on TRPM4 ion conductivity, not within the plasma membrane, but rather in TRPM4-containing vesicles.

Investigation of Novel Small Molecular TRPM4 Inhibitors in Colorectal Cancer Cells.

(1) Background: Transient receptor potential melastatin (TRPM4) ion channel aberrant expression or malfunction contributes to different types of cancer, including colorectal cancer (CRC). However, TRPM4 still needs to be validated as a potential target in anti-cancer therapy. Currently, the lack of potent and selective TRPM4 inhibitors limits further studies on TRPM4 in cancer disease models. In this study, we validated novel TRPM4 inhibitors, CBA, NBA, and LBA, in CRC cells. (2) Methods: The potency to inhibit TRPM4 conductivity in CRC cells was assessed with the whole-cell patch clamp technique. Furthermore, the impact of TRPM4 inhibitors on cellular functions, such as viability, proliferation, and cell cycle, were assessed in cellular assays. (3) Results: We show that in CRC cells, novel TRPM4 inhibitors irreversibly block TRPM4 currents in a low micromolar range. NBA decreases proliferation and alters the cell cycle in HCT116 cells. Furthermore, NBA reduces the viability of the Colo205 cell line, which highly expresses TRPM4. (4) Conclusions: NBA is a promising new TRPM4 inhibitor candidate, which could be used to study the role of TRPM4 in cancer disease models and other diseases.

TRPM4 in Cancer-A New Potential Drug Target.

Transient receptor potential melastatin 4 (TRPM4) is widely expressed in various organs and associated with cardiovascular and immune diseases. Lately, the interest in studies on TRPM4 in cancer has increased. Thus far, TRPM4 has been investigated in diffuse large B-cell lymphoma, prostate, colorectal, liver, breast, urinary bladder, cervical, and endometrial cancer. In several types of cancer TRPM4 is overexpressed and contributes to cancer hallmark functions such as increased proliferation and migration and cell cycle shift. Hence, TRPM4 is a potential prognostic cancer marker and a promising anticancer drug target candidate. Currently, the underlying mechanism by which TRPM4 contributes to cancer hallmark functions is under investigation. TRPM4 is a Ca2+-activated monovalent cation channel, and its ion conductivity can decrease intracellular Ca2+ signaling. Furthermore, TRPM4 can interact with different partner proteins. However, the lack of potent and specific TRPM4 inhibitors has delayed the investigations of TRPM4. In this review, we summarize the potential mechanisms of action and discuss new small molecule TRPM4 inhibitors, as well as the TRPM4 antibody, M4P. Additionally, we provide an overview of TRPM4 in human cancer and discuss TRPM4 as a diagnostic marker and anticancer drug target.

Small Molecular Inhibitors Block TRPM4 Currents in Prostate Cancer Cells, with Limited Impact on Cancer Hallmark Functions.

Transient receptor potential melastatin 4 (TRPM4) is a broadly expressed Ca2+ activated monovalent cation channel that contributes to the pathophysiology of several diseases. For this study, we generated stable CRISPR/Cas9 TRPM4 knockout (K.O.) cells from the human prostate cancer cell line DU145 and analyzed the cells for changes in cancer hallmark functions. Both TRPM4-K.O. clones demonstrated lower proliferation and viability compared to the parental cells. Migration was also impaired in the TRPM4-K.O. cells. Additionally, analysis of 210 prostate cancer patient tissues demonstrates a positive association between TRPM4 protein expression and local/metastatic progression. Moreover, a decreased adhesion rate was detected in the two K.O. clones compared to DU145 cells. Next, we tested three novel TRPM4 inhibitors with whole-cell patch clamp technique for their potential to block TRPM4 currents. CBA, NBA and LBA partially inhibited TRPM4 currents in DU145 cells. However, none of these inhibitors demonstrated any TRPM4-specific effect in the cellular assays. To evaluate if the observed effect of TRPM4 K.O. on migration, viability, and cell cycle is linked to TRPM4 ion conductivity, we transfected TRPM4-K.O. cells with either TRPM4 wild-type or a dominant-negative mutant, non-permeable to Na+. Our data showed a partial rescue of the viability of cells expressing functional TRPM4, while the pore mutant was not able to rescue this phenotype. For cell cycle distribution, TRPM4 ion conductivity was not essential since TRPM4 wild-type and the pore mutant rescued the phenotype. In conclusion, TRPM4 contributes to viability, migration, cell cycle shift, and adhesion; however, blocking TRPM4 ion conductivity is insufficient to prevent its role in cancer hallmark functions in prostate cancer cells.

The name tells the story: Two-pore channels.

TPC2-A1-N and TPC2-A1-P, two novel small molecules, differentially activate two-pore channel 2 (TPC2) and mimic the activation of TPC2 with NAADP and PIP2, resulting in distinct ion channel selectivities. These two different modes of TPC2 activity have physiological, and possibly pathophysiological, implications as they can modulate vesicle trafficking and lysosomal exocytosis.

TRP Channels in Digestive Tract Cancers.

Cancers of the digestive tract are among the most prevalent types of cancer. These types of cancers are often diagnosed at a late stage, which results in a poor prognosis. Currently, many biomedical studies focus on the role of ion channels, in particular transient receptor potential (TRP) channels, in cancer pathophysiology. TRP channels show mostly non-selective permeability to monovalent and divalent cations. TRP channels are often dysregulated in digestive tract cancers, which can result in alterations of cancer hallmark functions, such as enhanced proliferation, migration, invasion and the inability to induce apoptosis. Therefore, TRP channels could serve as potential diagnostic biomarkers. Moreover, TRP channels are mostly expressed on the cell surface and ion channel targeting drugs do not need to enter the cell, making them attractive candidate drug targets. In this review, we summarize the current knowledge about TRP channels in connection to digestive tract cancers (oral cancer, esophageal cancer, liver cancer, pancreatic cancer, gastric cancer and colorectal cancer) and give an outlook on the potential of TRP channels as cancer biomarkers or therapeutic targets.

TRPM4 is highly expressed in human colorectal tumor buds and contributes to proliferation, cell cycle, and invasion of colorectal cancer cells.

Transient receptor potential melastatin-4 channel (TRPM4) dysregulation contributes to heart conditions, immune diseases, and cervical and prostate cancer. Up to now, the involvement of TRPM4 in colorectal cancer (CRC) pathophysiology remains unknown. Here, we investigated tumor tissue microarrays from 379 CRC patients and analyzed TRPM4 protein expression, tumor characteristics, and clinical outcome. High TRPM4 protein expression was associated with unfavorable tumor features characteristic for epithelial-mesenchymal transition and infiltrative growth patterns, that is, a high number of tumor buds and a low percentage in tumor border configuration. Compared to CRC cells representing early cancer stages, TRPM4 protein expression was the highest in cells representing late-stage metastatic cancer. Investigation of CRC cell line HCT116 and five CRISPR/cas9 TRPM4 knockout clones demonstrated that TRPM4 exhibited large Na+ current densities (~ 60 pA/pF). In addition, CRISPR/cas9 TRPM4 knockout clones showed a tendency toward decreased migration and invasion, cell viability, and proliferation and exhibited a shift in cell cycle when compared to HCT116. Stable overexpression of TRPM4 (TRPM4 wild-type) in two CRISPR/cas9 TRPM4 knockout clones rescued the decrease in cell viability and cell cycle shift. Stable overexpression of a nonconducting, dominant-negative TRPM4 mutant (TRPM4 D894A) did not rescue the decrease in viability or cell cycle shift. Taken together, these findings pointed to TRPM4 ion channel conductivity as the underlying mechanism for decreased viability and cell cycle shift in the TRPM4 knockout clones. Together with previous findings, our present data suggest that TRPM4 plays a versatile role in cancer cell proliferation, cell cycle, and invasion.

Store-operated calcium entry in disease: Beyond STIM/Orai expression levels.

Precise intracellular calcium signaling is crucial to numerous cellular functions. In non-excitable cells, store-operated calcium entry (SOCE) is a key step in the generation of intracellular calcium signals. Tight regulation of SOCE is important, and dysregulation is involved in several pathophysiological cellular malfunctions. The current underlying SOCE, calcium release-activated calcium current (ICRAC), was first discovered almost three decades ago. Since its discovery, the molecular components of ICRAC, Orai1 and stromal interaction molecule 1 (STIM1), have been extensively investigated. Several regulatory mechanisms and proteins contribute to alterations in SOCE and cellular malfunctions in cancer, immune and neurodegenerative diseases, inflammation, and neuronal disorders. This review summarizes these regulatory mechanisms, including glycosylation, pH sensing, and the regulatory proteins golli, α-SNAP, SARAF, ORMDL3, CRACR2A, and TRPM4 channels.

Correction to: Store-Operated Ca2+ Entry as a Prostate Cancer Biomarker - a Riddle with Perspectives.

[This corrects the article DOI: 10.1007/s40610-017-0072-8.].

Store-Operated Ca2+ Entry as a Prostate Cancer Biomarker - a Riddle with Perspectives.

PURPOSE OF REVIEW: Store-operated calcium entry (SOCE) is dysregulated in prostate cancer, contributing to increased cellular migration and proliferation and preventing cancer cell apoptosis. We here summarize findings on gene expression levels and functions of SOCE components, stromal interaction molecules (STIM1 and STIM2), and members of the Orai protein family (Orai1, 2, and 3) in prostate cancer. Moreover, we introduce new research models that promise to provide insights into whether dysregulated SOCE signaling has clinically relevant implications in terms of increasing the migration and invasion of prostate cancer cells. RECENT FINDINGS: Recent reports on Orai1 and Orai3 expression levels and function were in part controversial probably due to the heterogeneous nature of prostate cancer. Lately, in prostate cancer cells, transient receptor melastatin 4 channel was shown to alter SOCE and play a role in migration and proliferation. We specifically highlight new cancer research models: a subpopulation of cells that show tumor initiation and metastatic potential in mice and zebrafish models. SUMMARY: This review focuses on SOCE component dysregulation in prostate cancer and analyzes several preclinical, cellular, and animal cancer research models.

ADAM17 silencing in mouse colon carcinoma cells: the effect on tumoricidal cytokines and angiogenesis.

ADAM17 (a disintegrin and metalloprotease 17) is a major sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules and is often overexpressed in malignant cells. It is generally accepted that ADAM17 promotes tumor development via activating growth factors from the EGF family, thus facilitating autocrine stimulation of tumor cell proliferation and migration. Here we show, using MC38CEA murine colon carcinoma model, that ADAM17 also regulates tumor angiogenesis and cytokine profile. When ADAM17 was silenced in MC38CEA cells, in vivo tumor growth and in vitro cell motility were significantly diminished, but no effect was seen on in vitro cell proliferation. ADAM17-silencing was accompanied by decreased in vitro expression of vascular endothelial growth factor-A and matrix metalloprotease-9, which was consistent with the limited angiogenesis and slower growth seen in ADAM17-silenced tumors. Among the growth factors susceptible to shedding by ADAM17, neuregulin-1 was the only candidate to mediate the effects of ADAM17 on MC38CEA motility and tumor angiogenesis. Concentrations of TNF and IFNγ, cytokines that synergistically induced proapoptotic effects on MC38CEA cells, were significantly elevated in the lysates of ADAM17-silenced tumors compared to mock transfected controls, suggesting a possible role for ADAM17 in host immune suppression. These results introduce new, complex roles of ADAM17 in tumor progression, including its impact on the anti-tumor immune response.