RESUMO
Cadmium is a xenobiotic involved in neoplastic transformation. Cadmium enters the cells through divalent cation transporters including the Transient Receptor Potential Melastatin-related 7 (TRPM7) which is known to be involved in cancer cell fate. This work aimed to study the role of TRPM7 in neoplastic transformation induced by cadmium exposure in non-cancer epithelial cells. Non-cancer epithelial cells were chronically exposed to low-dose of cadmium. TRPM7 expression and function were studied by Western-Blot, Patch-Clamp and calcium and magnesium imaging. Finally, cell migration and invasion were studied by Boyden chamber assays. Chronic cadmium exposure induced TRPM7 overexpression and increased the membrane currents (P < 0.001). Cells exposed to cadmium had higher intracellular calcium and magnesium levels (P < 0.05). TRPM7 silencing restored calcium levels but strongly decreased intracellular magnesium concentration (P < 0.001). Moreover, cadmium exposure enhanced both cell migration and invasion, but TRPM7 silencing strongly decreased these features (P < 0.001). Furthermore, mammary epithelial cells exposed to cadmium became rounded and had less cell-to-cell junctions. Cadmium exposure decreased epithelial markers while the mesenchymal ones were increased. Importantly, TRPM7 silencing was able to reverse these phenotypic modifications (P < 0.05). To summarize, our data show that chronic cadmium exposure enhanced TRPM7 expression and activity in non-cancer epithelial cells. TRPM7 overexpression induced intracellular magnesium increase and stimulated cell migration and invasion. These neoplastic properties could be linked to a TRPM7-dependent epithelial-to-mesenchymal transition reprogramming in cell exposed to cadmium. These findings provide new insights into the regulation of cell fates by cadmium exposure.
Assuntos
Cádmio/toxicidade , Movimento Celular/efeitos dos fármacos , Substâncias Perigosas/toxicidade , Canais de Cátion TRPM/metabolismo , Transição Epitelial-Mesenquimal , HumanosRESUMO
STAT5a and -5b (signal transducers and activators of transcription 5a and 5b) proteins play an essential role in hematopoietic cell proliferation and survival and are frequently constitutively active in hematologic neoplasms and solid tumors. Because STAT5a and STAT5b differ mainly in the carboxyl-terminal transactivation domain, we sought to identify new proteins that bind specifically to this domain by using a bacterial two-hybrid screening. We isolated hTid1, a human DnaJ protein that acts as a tumor suppressor in various solid tumors. hTid1 interacts specifically with STAT5b but not with STAT5a in hematopoietic cell lines. This interaction involves the cysteine-rich region of the hTid1 DnaJ domain. We also demonstrated that hTid1 negatively regulates the expression and transcriptional activity of STAT5b and suppresses the growth of hematopoietic cells transformed by an oncogenic form of STAT5b. Our findings define hTid1 as a novel partner and negative regulator of STAT5b.
Assuntos
Proteínas de Choque Térmico HSP40/metabolismo , Fator de Transcrição STAT5/metabolismo , Animais , Células COS , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Chlorocebus aethiops , Proteínas de Choque Térmico HSP40/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células Jurkat , Camundongos , Estrutura Terciária de Proteína , Fator de Transcrição STAT5/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
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 Ca(2+) entry and MDA-MB-231 cell migration without affecting cell proliferation. Recent studies have reported that Ca(2+) entry through Orai1 channels is required for MDA-MB-231 cell migration. Down-regulation of hEag1 or Orai1 reduced Ca(2+) influx and cell migration with similar efficiency. Interestingly, no additive effects on Ca(2+) 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 Ca(2+) entry through Orai1 channels. hEag1 may therefore represent a potential target for the suppression of BC cell migration, and thus prevention of metastasis development.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Movimento Celular/fisiologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Canais de Cálcio/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Canais de Potássio Éter-A-Go-Go/genética , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica , Linfonodos/patologia , Manganês , Invasividade Neoplásica , Proteína ORAI1 , Técnicas de Patch-Clamp , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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 Ca(2+) and Mg(2+) 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-Mg(2+) solution activated a nonselective current with a strong outward rectification. This cation current is inhibited by intracellular Mg(2+) and by TRPM7 silencing. The downregulation of TRPM7 by small interference RNA dramatically inhibited intracellular Mg(2+) fluorescence and cell migration without affecting cell proliferation, suggesting that TRPM7 contributes to Mg(2+) entry and cell migration. Moreover, external Mg(2+) 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 Mg(2+)-dependent mechanism and may be a potential biomarker of poor prognosis of PDAC.
Assuntos
Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/patologia , Movimento Celular , Neoplasias Pancreáticas/patologia , Canais de Cátion TRPM/fisiologia , Adenocarcinoma/química , Adenocarcinoma/metabolismo , Cálcio/metabolismo , Carcinoma Ductal Pancreático/química , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Humanos , Magnésio/metabolismo , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinases , Canais de Cátion TRPM/análiseRESUMO
Pancreatic diseases, such as pancreatitis or pancreatic ductal adenocarcinoma, are characterized by the presence of activated pancreatic stellate cells (PSCs). These cells represent key actors in the tumor stroma, as they actively participate in disease development and progression: reprograming these PSCs into a quiescent phenotype has even been proposed as a promising strategy for restoring the hallmarks of a healthy pancreas. Since TRPM7 channels have been shown to regulate hepatic stellate cells proliferation and survival, we aimed to study the role of these magnesium channels in PSC activation and proliferation. PS-1 cells (isolated from a healthy pancreas) were used as a model of healthy PSCs: quiescence or activation were induced using all-trans retinoic acid or conditioned media of pancreatic cancer cells, respectively. The role of TRPM7 was studied by RNA silencing or by pharmacological inhibition. TRPM7 expression was found to be correlated with the activation status of PS-1 cells. TRPM7 expression was able to regulate proliferation through modulation of cell cycle regulators and most importantly p53, via the PI3K/Akt pathway, in a magnesium-dependent manner. Finally, the analysis of TCGA database showed the overexpression of TRPM7 in cancer-associated fibroblasts. Taken together, we provide strong evidences that TRPM7 can be considered as a marker of activated PSCs.
Assuntos
Neoplasias Pancreáticas , Canais de Cátion TRPM , Humanos , Magnésio/metabolismo , Neoplasias Pancreáticas/patologia , Células Estreladas do Pâncreas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Neoplasias PancreáticasRESUMO
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.
Assuntos
Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica , Canais de Potencial de Receptor Transitório/metabolismo , Neoplasias da Mama/metabolismo , Células Cultivadas , Feminino , Humanos , Imuno-Histoquímica , Estadiamento de Neoplasias , Proteínas Serina-Treonina Quinases , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Canais de Cátion TRPC/metabolismo , Canal de Cátion TRPC6 , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/genéticaRESUMO
Activated pancreatic stellate cells (aPSCs), the crucial mediator of pancreatic desmoplasia, are characterized, among others, by high proliferative potential and abundant transforming growth factor ß1 (TGFß1) secretion. Over the past years, the involvement of Ca2+ channels in PSC pathophysiology has attracted great interest in pancreatic cancer research. We, thus, aimed to investigate the role of the Orai1 Ca2+ channel in these two PSC activation processes. Using the siRNA approach, we invalided Orai1 expression and assessed the channel functionality by Ca2+ imaging, the effect on aPSC proliferation, and TGFß1 secretion. We demonstrated the functional expression of the Orai1 channel in human aPSCs and its implication in the store-operated Ca2+ entry (SOCE). Orai1 silencing led to a decrease in aPSC proliferation, TGFß1 secretion, and AKT activation. Interestingly, TGFß1 induced a higher SOCE response by increasing Orai1 mRNAs and proteins and promoted both AKT phosphorylation and cell proliferation, abolished by Orai1 silencing. Together, our results highlight the role of Orai1-mediated Ca2+ entry in human aPSC pathophysiology by controlling cell proliferation and TGFß1 secretion through the AKT signaling pathway. Moreover, we showed a TGFß1-induced autocrine positive feedback loop by promoting the Orai1/AKT-dependent proliferation via the stimulation of Orai1 expression and function.
RESUMO
Despite magnesium (Mg2+) representing the second most abundant cation in the cell, its role in cellular physiology and pathology is far from being elucidated. Mg2+ homeostasis is regulated by Mg2+ transporters including Mitochondrial RNA Splicing Protein 2 (MRS2), Transient Receptor Potential Cation Channel Subfamily M, Member 6/7 (TRPM6/7), Magnesium Transporter 1 (MAGT1), Solute Carrier Family 41 Member 1 (SCL41A1), and Cyclin and CBS Domain Divalent Metal Cation Transport Mediator (CNNM) proteins. Recent data show that Mg2+ transporters may regulate several cancer cell hallmarks. In this review, we describe the expression of Mg2+ transporters in digestive cancers, the most common and deadliest malignancies worldwide. Moreover, Mg2+ transporters' expression, correlation and impact on patient overall and disease-free survival is analyzed using Genotype Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) datasets. Finally, we discuss the role of these Mg2+ transporters in the regulation of cancer cell fates and oncogenic signaling pathways.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Neoplasias Gastrointestinais/metabolismo , Magnésio/metabolismo , Animais , Biomarcadores , Proteínas de Transporte de Cátions/genética , Suscetibilidade a Doenças , Neoplasias Gastrointestinais/etiologia , Neoplasias Gastrointestinais/mortalidade , Neoplasias Gastrointestinais/patologia , Regulação Neoplásica da Expressão Gênica , Homeostase , Humanos , Transporte de Íons , Ligação Proteica , Transdução de SinaisRESUMO
BACKGROUND: The 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. METHODS: RT-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. RESULTS: TRPM8 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 microM) 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 ERalpha 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. CONCLUSION: Taken together, these results show that TRPM8 channels are expressed and functional in breast cancer and that their expression is regulated by ER alpha.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias da Mama/metabolismo , Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Canais de Cátion TRPM/metabolismo , Adenocarcinoma/genética , Western Blotting , Neoplasias da Mama/genética , Cálcio/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Potenciais da Membrana , Técnicas de Patch-Clamp , Pirimidinonas/farmacologia , Interferência de RNA , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPM/agonistas , Canais de Cátion TRPM/genética , Fatores de TempoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer-related deaths in United States and Europe. It is predicted that PDAC will become the second leading cause of cancer-related deaths during the next decades. The development of PDAC is not well understood, however, studies have shown that dysregulated exocrine pancreatic fluid secretion can contribute to pathologies of exocrine pancreas, including PDAC. The major roles of healthy exocrine pancreatic tissue are secretion of enzymes and bicarbonate rich fluid, where ion channels participate to fine-tune these biological processes. It is well known that ion channels located in the plasma membrane regulate multiple cellular functions and are involved in the communication between extracellular events and intracellular signaling pathways and can function as signal transducers themselves. Hereby, they contribute to maintain resting membrane potential, electrical signaling in excitable cells, and ion homeostasis. Despite their contribution to basic cellular processes, ion channels are also involved in the malignant transformation from a normal to a malignant phenotype. Aberrant expression and activity of ion channels have an impact on essentially all hallmarks of cancer defined as; uncontrolled proliferation, evasion of apoptosis, sustained angiogenesis and promotion of invasion and migration. Research indicates that certain ion channels are involved in the aberrant tumor growth and metastatic processes of PDAC. The purpose of this review is to summarize the important expression, localization, and function of ion channels in normal exocrine pancreatic tissue and how they are involved in PDAC progression and development. As ion channels are suggested to be potential targets of treatment they are furthermore suggested to be biomarkers of different cancers. Therefore, we describe the importance of ion channels in PDAC as markers of diagnosis and clinical factors.
RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a malignancy with a very poor prognosis due to highly metastatic profile. Cell migration is an essential step of the metastatic cascade allowing cancer cells to spread toward target tissues. Recent studies strongly suggest that bioactive elastin peptides, also named elastokines or elastin-derived peptides (EDPs), are released in the extracellular microenvironment during tumoral remodeling of the stroma. EDPs stimulate cancer cell migration by interacting with their membrane receptor, ribosomal protein SA (RPSA). Others membrane proteins like ion channels are also involved in cancer cell migration. It has been recently shown that the transient receptor potential melastatin-related 7 (TRPM7) channel regulates PDAC cell migration and invasion. The objective of this work was to study the effect of EDPs on TRPM7 channel in human pancreatic cancer cells. We showed that EDPs promote MIA PaCa-2 cell migration using Boyden chamber assay. Cells transfected with a siRNA targeting TRPM7 were not able to migrate in response to EDPs indicating that TRPM7 regulated cell migration induced by these peptides. Moreover, EDPs were able to stimulate TRPM7 currents recorded by Patch-Clamp. Finally, we showed that TRPM7 channels and RPSA receptors are colocalized at the plasma membrane of human pancreatic cancer cells. Taken together, our data suggest that TRPM7/RPSA complex regulated human pancreatic cancer cell migration. This complex may be a promising therapeutic target in PDAC.
RESUMO
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.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Canais de Cátion TRPM/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Magnésio/metabolismo , Manganês , Potenciais da Membrana/fisiologia , Proteínas Serina-Treonina Quinases , Canais de Cátion TRPM/genética , Técnicas de Cultura de TecidosRESUMO
In the battle against cancer cells, therapeutic modalities are drastically limited by intrinsic or acquired drug resistance. Resistance to therapy is not only common, but expected: if systemic agents used for cancer treatment are usually active at the beginning of therapy (i.e., 90% of primary breast cancers and 50% of metastases), about 30% of patients with early-stage breast cancer will have recurrent disease. Altered expression of ion channels is now considered as one of the hallmarks of cancer, and several ion channels have been linked to cancer cell resistance. While ion channels have been associated with cell death, apoptosis and even chemoresistance since the late 80s, the molecular mechanisms linking ion channel expression and/or function with chemotherapy have mostly emerged in the last ten years. In this review, we will highlight the relationships between ion channels and resistance to chemotherapy, with a special emphasis on the underlying molecular mechanisms.
RESUMO
BACKGROUND: TRP 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. METHODS: Molecular (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). RESULTS: A 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 TRPM7, 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. CONCLUSION: Our 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.
Assuntos
Neoplasias da Mama/metabolismo , Glândulas Mamárias Humanas/metabolismo , Neoplasias Epiteliais e Glandulares/metabolismo , Canais de Cátion TRPC/biossíntese , Western Blotting , Neoplasias da Mama/patologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Glândulas Mamárias Humanas/citologia , Estadiamento de Neoplasias , Neoplasias Epiteliais e Glandulares/patologia , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPC/fisiologia , Canal de Cátion TRPC6RESUMO
Intestinal magnesium (Mg) uptake is essential for systemic Mg homeostasis. Colon cells express the two highly homologous transient receptor potential melastatin type (TRPM) 6 and 7 Mg2+ channels, but their precise function and the consequences of their mutual interaction are not clear. To explore the functional role of TRPM6 and TRPM7 in the colon, we used human colon cell lines that innately express both channels and analyzed the functional consequences of genetic knocking-down, by RNA interference, or pharmacological inhibition, by NS8593, of either channel. TRPM7 silencing caused an increase in Mg2+ influx, and correspondingly enhanced cell proliferation and migration, while downregulation of TRPM6 did not affect significantly either Mg2+ influx or cell proliferation. Exposure to the specific TRPM6/7 inhibitor NS8593 reduced Mg2+ influx, and consequently cell proliferation and migration, but Mg supplementation rescued the inhibition. We propose a model whereby in colon cells the functional Mg2+ channel at the plasma membrane may consist of both TRPM7 homomers and TRPM6/7 heteromers. A different expression ratio between the two proteins may result in different functional properties. Altogether, our findings confirm that TRPM6 cannot be replaced by TRPM7, and that TRPM6/7 complexes and TRPM6/7-mediated Mg2+ influx are indispensable in human epithelial colon cells.
Assuntos
Colo/citologia , Colo/metabolismo , Células Epiteliais/fisiologia , Magnésio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Canais de Cátion TRPM/metabolismo , Linhagem Celular Tumoral , Humanos , Mucosa Intestinal/citologia , Proteínas Serina-Treonina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPM/genéticaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a very poor prognosis. There is an urgent need to better understand the molecular mechanisms that regulate PDAC cell aggressiveness. The transient receptor potential melastatin 7 (TRPM7) is a nonselective cationic channel that mainly conducts Ca2+ and Mg2+. TRPM7 is overexpressed in numerous malignancies including PDAC. In the present study, we used the PANC-1 and MIA PaCa-2 cell lines to specifically assess the role of TRPM7 in cell invasion and matrix metalloproteinase secretion. We show that TRPM7 regulates Mg2+ homeostasis and constitutive cation entry in both PDAC cell lines. Moreover, cell invasion is strongly reduced by TRPM7 silencing without affecting the cell viability. Conditioned media were further studied, by gel zymography, to detect matrix metalloproteinase (MMP) secretion in PDAC cells. Our results show that MMP-2, urokinase plasminogen activator (uPA), and heat-shock protein 90α (Hsp90α) secretions are significantly decreased in TRPM7-deficient PDAC cells. Moreover, TRPM7 expression in human PDAC lymph node metastasis is correlated to the channel expression in primary tumor. Taken together, our results show that TRPM7 is involved in PDAC cell invasion through regulation of Hsp90α/uPA/MMP-2 proteolytic axis, confirming that this channel could be a promising biomarker and possibly a target for PDAC metastasis therapy.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Canais de Cátion TRPM/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Expressão Gênica , Inativação Gênica , Humanos , Metástase Linfática , Magnésio/metabolismo , Modelos Biológicos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteólise , Canais de Cátion TRPM/genéticaRESUMO
The active forms of STAT5A (signal transducer and activator of transcription 5A) and STAT5B are able to relieve the cytokine dependence of haematopoietic cells and to induce leukaemia in mice. We have demonstrated previously that activation of the PI3K (phosphoinositide 3-kinase) signalling cascade plays a major role in cell growth and survival induced by these proteins. Interaction between STAT5 and p85, the regulatory subunit of the PI3K, has been suggested to be required for this activation. We show in the present study that the scaffolding protein Gab2 [Grb2 (growth-factor-receptor-bound protein 2)-associated binder-2] is an essential component of this interaction. Gab2 is persistently tyrosine-phosphorylated in Ba/F3 cells expressing caSTAT5 (constitutively activated STAT5), independent of JAK2 (Janus kinase 2) activation where it interacts with STAT5, p85 and Grb2, but not with Shp2 [SH2 (Src homology 2)-domain-containing tyrosine phosphatase] proteins. Interaction of STAT5 with Gab2 was also observed in Ba/F3 cells stimulated with interleukin-3 or expressing the oncogenic fusion protein Tel-JAK2. The MAPKs (mitogen-activated protein kinases) ERK1 (extracellular-signal-regulated kinase 1) and ERK2 were constitutively activated in the caSTAT5-expressing cells and were found to be required for caSTAT5-induced cell proliferation. Overexpression of Gab2-3YF, a mutant of Gab2 incapable of binding PI3K, inhibited the proliferation and survival of caSTAT5-expressing cells as well as ERK1/2 and Akt/protein kinase B phosphorylation. Taken together, our results indicate that Gab2 is required for caSTAT5-induced cell proliferation by regulating both the PI3K/Akt and the Ras/MAPK pathways.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas do Leite/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Proteínas ras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , Proliferação de Células , Proteína Adaptadora GRB2 , Mutação , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt , Fator de Transcrição STAT5 , Transdução de SinaisRESUMO
Transient Receptor Potential Melastatin-related 7 (TRPM7) is a non-selective cation channel fused with a functional kinase domain. Physiologically, TRPM7 channel is involved in magnesium homeostasis, cell survival and gastrulation. The channel part is responsible for calcium, magnesium, and metal trace entries. Cation current through TRPM7 channel is inhibited by both intracellular magnesium and magnesium complexed with nucleotides. In parallel, the kinase is able to phosphorylate cytoskeleton proteins like myosin chain regulating cell tension and motility. Moreover, TRPM7 kinase domain can be cleaved by caspase and participates to apoptosis signaling. Importantly, TRPM7 channel expression is aberrant in numerous cancers including breast, glioblastoma, nasopharynx, ovarian, and pancreatic. Moreover, TRPM7 high expression is an independent biomarker of poor outcome in breast cancer. Pharmacological modulation or silencing of TRPM7 strongly affects proliferation, adhesion, migration or invasion in cancer cell lines. Nevertheless, it is still not clear by which mechanism TRPM7 channels may disturb cancer cell hallmarks. In the present review, we will discuss the role of TRPM7 channels in malignancies. In particular, we will distinguish the role of cation signaling from kinase function in order to better understand how TRPM7 channels may play a central role in cancer progression. We will also discuss the recent advances in pharmacological blockers of TRPM7 and their potential use for cancer therapy.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Canais de Cátion TRPM/metabolismo , Acetatos/toxicidade , Produtos Biológicos/toxicidade , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Diterpenos/toxicidade , Humanos , Magnésio/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPM/química , Canais de Cátion TRPM/genéticaRESUMO
Calcium (Ca(2+)) and magnesium (Mg(2+)) are important metal elements that regulate a variety of cellular processes such as proliferation, migration, and apoptosis, in cancer cells. Among the ionic channels mediating intracellular entry, the transient receptor potential melastatin type 7 (TRPM7) channel is of particular interest, it being a non-selective, cationic channel mediating both Ca(2+) and Mg(2+) influx. TRPM7 is highly expressed in a number of human cancer tissues and cell lines. In this review, we summarise current knowledge on the physiological role of the dual function TRPM7 chanzyme, the potential application of TRPM7 as a diagnostic and prognostic marker of cancer progression with respect to clinical and pathological characteristics, and the molecular mechanisms implicated in cancerogenesis that specifically involve Ca(2+) and Mg(2+) influx through TRPM7 or kinase activity and interaction with cytoskeletal proteins.
Assuntos
Magnésio/metabolismo , Proteínas de Neoplasias/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Canais de Cátion TRPM/fisiologia , Animais , Apoptose/fisiologia , Biomarcadores Tumorais , Cálcio/metabolismo , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Movimento Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Homeostase , Humanos , Transporte de Íons , Mamíferos/metabolismo , Proteínas de Neoplasias/química , Neoplasias/química , Neoplasias/mortalidade , Prognóstico , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , Transdução de Sinais/fisiologia , Canais de Cátion TRPM/químicaRESUMO
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.