Isabelle Dhennin-Duthille

Evidence that TRPM7 is required for breast cancer cell proliferation

Because transient receptor potential (TRP) channels have been implicated in tumor progression, we have investigated the potential role of TRPM7 channel in breast cancer cell proliferation. Under whole cell patch clamp, a Mg(2+)-inhibited cationic (MIC) current was observed in MCF-7 cells. This current was characterized by an inward current and a strong outward rectifying current that were both inhibited in a concentration-dependent manner by the presence of intracellular Mg(2+) or Mg(2+)-ATP. The inward current was reduced by La(3+), and the outward current was sensitive to 2-aminoethoxydiphenyl borate (2-APB), spermine, La(3+), and flufenamic acid. Importantly, a similar MIC current was also recorded in the primary culture of human breast cancerous epithelial cells (hBCE). Moreover, TRPM7 transcripts were found in both hBCE and MCF-7 cells. In MCF-7 cells, the MIC current was inhibited by TRPM7 small interfering RNA. Interestingly, we found that cell proliferation and intracellular Ca(2+) concentration were also reduced by TRPM7 silencing in MCF-7 cells. TRPM7 channels were also found in both human breast cancer and healthy tissues. Importantly, TRPM7 channel was overexpressed in grade III breast cancer samples associated with important Ki67 or tumor size. Our findings strongly suggest that TRPM7 is involved in the proliferative potentiality of breast cancer cells, probably by regulating Ca(2+) influx.

High Expression of Transient Receptor Potential Channels in Human Breast Cancer Epithelial Cells and Tissues: Correlation with Pathological Parameters

Background: Transient Receptor Potential (TRP) channels are expressed in many solid tumors. However, their expression in breast cancer remains largely unknown. Here, we investigated the profile expression of 13 TRP channels in human breast ductal adenocarcinoma (hBDA) and performed a correlation between their overexpression and pathological parameters. Methods: The TRP channels expression was determined by RT-PCR in hBDA tissue, in human breast cancer epithelial (hBCE) primary culture and in MCF-7 cell line. The TRP protein level was evaluated by immunohistochemistry in hBDA tissue samples of 59 patients. Results: TRPC1, TRPC6, TRPM7, TRPM8, and TRPV6 channels were overexpressed in hBDA compared to the adjacent non-tumoral tissue. Most interestingly, TRPC1, TRPM7 and TRPM8 expression strongly correlated with proliferative parameters (SBR grade, Ki67 proliferation index, and tumor size), and TRPV6 was mainly overexpressed in the invasive breast cancer cells. Using laser capture microdissection, we found that TRPV6 expression was higher in invasive areas, compared to the corresponding non-invasive ones. Moreover, TRPV6 silencing inhibited MDA-MB-231 migration and invasion, and MCF-7 migration. Conclusion: TRP channels are aberrantly expressed in hBDA, hBCE primary cultures, and cell lines, and associated with pathological parameters. The high expression of TRP channels in tumors suggests the potential of these channels for diagnostic, prognosis and/or therapeutic approaches in human breast ductal adenocarcinoma.

Transient receptor potential melastatin-related 7 channel is overexpressed in human pancreatic ductal adenocarcinomas and regulates human pancreatic cancer cell migration

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive forms of cancer with a tendency to invade surrounding healthy tissues, leading to a largely incurable disease. Despite many advances in modern medicine, there is still a lack of early biomarkers as well as efficient therapeutical strategies. The melastatin‐related transient receptor potential 7 channel (TRPM7) is a nonselective cation channel that is involved in maintaining Ca2+ and Mg2+ homeostasis. It has been recently reported to regulate cell differentiation, proliferation and migration. However, the role of TRPM7 in PDAC progression is far to be understood. In our study, we show that TRPM7 is 13‐fold overexpressed in cancer tissues compared to the healthy ones. Furthermore, TRPM7 staining is stronger in tumors with high grade, suggesting a correlation between TRPM7 expression and PDAC progression. Importantly, TRPM7 expression is inversely related to patient survival. In BxPC‐3 cell line, dialyzing the cytoplasm during the patch‐clamp whole‐cell recording with a 0‐Mg2+ solution activated a nonselective current with a strong outward rectification. This cation current is inhibited by intracellular Mg2+ and by TRPM7 silencing. The downregulation of TRPM7 by small interference RNA dramatically inhibited intracellular Mg2+ fluorescence and cell migration without affecting cell proliferation, suggesting that TRPM7 contributes to Mg2+ entry and cell migration. Moreover, external Mg2+ following TRPM7 silencing fully restored the cell migration. In summary, our results indicate that TRPM7 is involved in the BxPC‐3 cell migration via a Mg2+‐dependent mechanism and may be a potential biomarker of poor prognosis of PDAC.

Expression of TRPC6 channels in human epithelial breast cancer cells

BackgroundTRP channels have been shown to be involved in tumour generation and malignant growth. However, the expression of these channels in breast cancer remains unclear. Here we studied the expression and function of endogenous TRPC6 channels in a breast cancer cell line (MCF-7), a human breast cancer epithelial primary culture (hBCE) and in normal and tumour breast tissues.MethodsMolecular (Western blot and RT-PCR), and immunohistochemical techniques were used to investigate TRPC6 expression. To investigate the channel activity in both MCF-7 cells and hBCE we used electrophysiological technique (whole cell patch clamp configuration).ResultsA non selective cationic current was activated by the oleoyl-2-acetyl-sn-glycerol (OAG) in both hBCE and MCF-7 cells. OAG-inward current was inhibited by 2-APB, SK&F 96365 and La3+. TRPC6, but not TRPC7, was expressed both in hBCE and in MCF-7 cells. TRPC3 was only expressed in hBCE. Clinically, TRPC6 mRNA and protein were elevated in breast carcinoma specimens in comparison to normal breast tissue. Furthermore, we found that the overexpression of TRPC6 protein levels were not correlated with tumour grades, estrogen receptor expression or lymph node positive tumours.ConclusionOur results indicate that TRPC6 channels are strongly expressed and functional in breast cancer epithelial cells. Moreover, the overexpression of these channels appears without any correlation with tumour grade, ER expression and lymph node metastasis. Our findings support the idea that TRPC6 may have a role in breast carcinogenesis.

Estrogen regulation of TRPM8 expression in breast cancer cells

BackgroundThe calcium-permeable cation channel TRPM8 (melastatin-related transient receptor potential member 8) is over-expressed in several cancers. The present study aimed at investigating the expression, function and potential regulation of TRPM8 channels by ER alpha (estrogen receptor alpha) in breast cancer.MethodsRT-PCR, Western blot, immuno-histochemical, and siRNA techniques were used to investigate TRPM8 expression, its regulation by estrogen receptors, and its expression in breast tissue. To investigate the channel activity in MCF-7 cells, we used the whole cell patch clamp and the calcium imaging techniques.ResultsTRPM8 channels are expressed at both mRNA and protein levels in the breast cancer cell line MCF-7. Bath application of the potent TRPM8 agonist Icilin (20 μM) induced a strong outwardly rectifying current at depolarizing potentials, which is associated with an elevation of cytosolic calcium concentration, consistent with established TRPM8 channel properties. RT-PCR experiments revealed a decrease in TRPM8 mRNA expression following steroid deprivation for 48 and 72 hours. In steroid deprived medium, addition of 17-beta-estradiol (E2, 10 nM) increased both TRPM8 mRNA expression and the number of cells which respond to Icilin, but failed to affect the Ca2+ entry amplitude. Moreover, silencing ERα mRNA expression with small interfering RNA reduced the expression of TRPM8. Immuno-histochemical examination of the expression of TRPM8 channels in human breast tissues revealed an over-expression of TRPM8 in breast adenocarcinomas, which is correlated with estrogen receptor positive (ER+) status of the tumours.ConclusionTaken together, these results show that TRPM8 channels are expressed and functional in breast cancer and that their expression is regulated by ER alpha.

Human ether à‐gogo K+ channel 1 (hEag1) regulates MDA‐MB‐231 breast cancer cell migration through Orai1‐dependent calcium entry

Breast cancer (BC) has a poor prognosis due to its strong metastatic ability. Accumulating data present ether à go‐go (hEag1) K+ channels as relevant player in controlling cell cycle and proliferation of non‐invasive BC cells. However, the role of hEag1 in invasive BC cells migration is still unknown. In this study, we studied both the functional expression and the involvement in cell migration of hEag1 in the highly metastatic MDA‐MB‐231 human BC cells. We showed that hEag1 mRNA and proteins were expressed in human invasive ductal carcinoma tissues and BC cell lines. Functional activity of hEag1 channels in MDA‐MB‐231 cells was confirmed using astemizole, a hEag1 blocker, or siRNA. Blocking or silencing hEag1 depolarized the membrane potential and reduced both Ca2+ entry and MDA‐MB‐231 cell migration without affecting cell proliferation. Recent studies have reported that Ca2+ entry through Orai1 channels is required for MDA‐MB‐231 cell migration. Down‐regulation of hEag1 or Orai1 reduced Ca2+ influx and cell migration with similar efficiency. Interestingly, no additive effects on Ca2+ influx or cell migration were observed in cells co‐transfected with sihEag1 and siOrai1. Finally, both Orai1 and hEag1 are expressed in invasive breast adenocarcinoma tissues and invaded metastatic lymph node samples (LNM+). In conclusion, this study is the first to demonstrate that hEag1 channels are involved in the serum‐induced migration of BC cells by controlling the Ca2+ entry through Orai1 channels. hEag1 may therefore represent a potential target for the suppression of BC cell migration, and thus prevention of metastasis development. J. Cell. Physiol. 227: 3837–3846, 2012.

TRP channels: diagnostic markers and therapeutic targets for breast cancer?

Breast cancer is the most frequently occurring cancer in women and has the highest rate of mortality. Ion channels such as the transient receptor potential (TRP) channels could play a critical role in the development and progression of cancer. Although these channels are frequently and abundantly expressed in many tumors, their expression, activity, and roles in the context of breast cancer remain poorly understood. This review summarizes our current knowledge regarding TRP channels expressed in human breast tissue, primary human breast epithelial cells, and cell lines, the functional role of TRP channels during breast cancer cell growth and migration, as well as their relationship with clinical and pathological features.

Transient receptor potential melastatin 7 is involved in oestrogen receptor-negative metastatic breast cancer cells migration through its kinase domain

Oestrogen receptor negative (ER(-)) invasive ductal carcinoma (IDC) represents a significant clinical challenge and therefore prompts the discovery of novel biomarkers. Transient receptor potential melastatin 7 (TRPM7), a channel protein that also contains a regulatory kinase domain, is overexpressed in IDC and regulates migration. However, the molecular mechanism remains poorly defined. Here, we examined whether TRPM7 regulates migration by its channel function or by its kinase domain. A Magnesium Inhibited Cation current was recorded in two ER(-) highly metastatic breast cancer cell lines. Down-regulation of TRPM7 neither affected Ca(2+)-, nor Mg(2+)-homoeostasis but significantly reduced cell migration via a Ca(2+)-independent pathway. Notably, the overexpression of the truncated kinase domain form of TRPM7 decreased cell migration, while the overexpression of the wild-type form strongly increased it. Concomitantly, TRPM7 silencing reduced the myosin IIA heavy chain phosphorylation. Furthermore, we found higher TRPM7 expression in ER(-) IDC tissues and lymph nodes than in the non-invasive tumoural samples. In conclusion, TRPM7 plays a critical role in breast cancer cell migration through its kinase domain, and our data support the consideration of using TRPM7 as a novel biomarker and a potential therapeutic target in the treatment of human ER(-) IDC.

New insights into pharmacological tools to TR(i)P cancer up

The aim of this review is to address the recent advances regarding the use of pharmacological agents to target transient receptor potential (TRP) channels in cancer and their potential application in therapeutics. Physiologically, TRP channels are responsible for cation entry (Ca2+, Na+, Mg2+) in many mammalian cells and regulate a large number of cellular functions. However, dysfunction in channel expression and/or activity can be linked to human diseases like cancer. Indeed, there is growing evidence that TRP channel expression is altered in cancer tissues in comparison with normal ones. Moreover, these proteins are involved in many cancerous processes, including cell proliferation, apoptosis, migration and invasion, as well as resistance to chemotherapy. Among the TRP superfamily, TRPC, TRPV, TRPM and TRPA1 have been shown to play a role in many cancer types, including breast, digestive, gliomal, head and neck, lung and prostate cancers. Pharmacological modulators are used to characterize the functional implications of TRP channels in whole‐cell membrane currents, resting membrane potential regulation and intracellular Ca2+ signalling. Moreover, pharmacological modulation of TRP activity in cancer cells is systematically linked to the effect on cancerous processes (proliferation, survival, migration, invasion, sensitivity to chemotherapeutic drugs). Here we describe the effects of such TRP modulators on TRP activity and cancer cell phenotype. Furthermore, the potency and specificity of these agents will be discussed, as well as the development of new strategies for targeting TRP channels in cancer.

Canaux cationiques TRP dans le cancer du sein : expression, rôle et corrélation avec des paramètres cliniques

Breast cancer (BC) has the highest incidence rate in women in industrialized countries. Statistically, it is estimated that one out of 10 women will develop BC during her life. Evidence is accumulating for the role of ion channels in the development of cancer. Most studied ion channels in BC are K(+) channels, which are involved in cell proliferation, cell cycle progression and cell migration, and Na(+) channels, which correlate with invasiveness. Emerging studies demonstrated the role of Ca(2+) signaling in cancer cell proliferation, survival and migration. Recent findings demonstrated that the expression and/or activity of the transient receptor potential (TRP) channels are altered in several cancers. Among the TRP families, TRPC (canonical or classical), TRPM (melastatin) and TRPV (vanilloid) are related to malignant growth and cancer progression. Although these channels are frequently and abundantly expressed in many tumors, their specific expression, activity and roles in BC are still poorly understood. The expression of TRP channels has also been proposed as a tool for diagnosis, prognosis and/or therapeutic issues of several diseases. In cancer, TRPV6 and TRPM8 have been proposed as tumor progression markers of prostate cancer outcome and TRPC6 as a novel therapeutic target for esophageal carcinoma. Interestingly high levels of TRPC3 expression correlate with a favorable prognosis in patients with lung adenocarcinoma. Our team has recently reported the expression and role of TRPC1, TRPC6, TRPM7, TRPM8 and TRPV6 in BC cell lines and primary cultures. We have also investigated TRP expression and their clinical significance in human breast adenocarcinoma and we suggest that TRP channels are new potential BC markers. Indeed TRPC1 and TRPM8 may be considered as good prognosis markers of well-differentiated tumors, TRPM7 as a proliferative marker of poorly differentiated tumors and TRPV6 as a prognosis marker of aggressive cancers. In this review, we summarize the data reported to date regarding the changes in TRP expression associated with BC. We also discuss the importance of TRP channels in BC cells proliferation and migration and their interest as new BC markers.