| Background:Breast cancer is the highest morbidity of cancer of the female population. On a global scale, the morbidity showed an increasing trend in recent years. "Global cancer statistics,2012 " that were published online in CA journal in 2015 show that about 1.2 million women suffering from breast cancer, which killed about 500,000 patients, and the incidence rate of 7% to 10% of the annual of various malignant tumors. According to "2015 China Annual Cancer Registration", Chinese morbidity of breast cancer in women was 35 to 45 people/10 million that showed an increasing trend, and was already the highest incidence of cancer of the female. Additionlly, the Chinese women suffering from breast cancer show a 10 years earlier average age of onset than that in Western countries and there is a tendency to younger development. Visible, prevention and treatment of breast cancer in China is facing severe form. As the disease screening and diagnosis and treatment progress, early diagnosis and treatment of breast cancer has made significant progress, leading to survival and quality of life of patients significantly improved. But there are a considerable number of deaths due to breast cancer patients, and the most important factor is the metastasis of breast cancer. The current treatments are difficult to suppress that process and the treatments for metastasis patients are also unsatisfactory. Therefore, to further elucidate the mechanisms of breast cancer metastasis and find new ways to treat breast cancer are the focus of prevention and treatment.Renin-angiotensin system (RAS) system is an important system that regulating body fluid equilibrium and blood pressure. Highlights of previous studies concern the role of angiotensin-converting enzyme (ACE)/Angiotensin II (AngⅡ)/ AT1R axis. In recent years, a new component of RAS that Angiotensin-converting enzyme2 (ACE2)/Angiotensin-(1-7) (Ang-(1-7))/Mas receptor axis becoming ever more attention. ACE2 that discovered in the year 2000 is similar ACE. ACE is a peptide phthalocyanine dipeptidase that has two enzyme catalytic domain. ACE2 is a carboxypeptidase that only has one enzyme catalytic domain. ACE can hydrolyses AngI to generate AngⅡ, ACE2 can hydrolyses the C-terminus of the peptide chain of AngI and AngⅡ to generate Ang (1-9) and Ang-(1-7) respectively, and Ang-(1-9) is hydrolyses to further generate Ang-(1-7) by ACE. The effects of Ang-(1-9) are currently rarely reported. Because ACE hydrolysis Ang-(1-9) to generate the amount of Ang-(1-7) is very small, Ang-(1-7) is mainly composed of ACE2 hydrolysis AngⅡ generated, ACE2 is Ang-(1-7) generation a key enzyme. Mas is the receptor of Ang-(1-7) and belongs to the rhodopsin-like class A G protein coupled receptor (GPCR) subfamily that containing G protein coupled receptor shared very conservative tertiary structure, namely seven hydrophobic transmembrane domains. Ang-(1-7) could regulate body fluid equilibrium and blood pressure, such as vasodilatory, diuretic, lowering blood pressure and so on, by Mas receptor。Recently reported, in addition to the systemic RAS system, there are also RAS system within each tissue and organ, namely the local RAS system. Increase local AngⅡ promotes cell proliferation, inflammation, metabolism and cell transfer capability and so on in tissue and organs. On the contrary, Ang-(1-7) could inhibit cardiac hypertrophy, proliferation of vascular smooth muscle, inhibition of inflammation. Further research also found that Ang-(1-7) could inhibit lung cancer A549 cells and prostate cancer PC3 cells proliferation by restraining the activiaty of mitogen-activated protein kinase/MAP kinase and cyclooxygenase-2 (COX-2), the expression of VEGF (vascular endothelial growth factor) or reducing thymidine uptake to inhibited DNA replication. Ang-(1-7) also can inhibit PC3 cells and A549 cells metastasis. These founds indicated that ACE2/Ang-(1-7)/Mas receptor axis with anti-tumor effect. Recent reports in the international authoritative journal---Cancer research, Ang-(1-7) can inhibit breast cancer growth by reducing the adjacent fibrosis, indicating that ACE2/Ang-(1-7)/Mas receptor axis may also inhibite breast cancer occurrence and development. So, whether has ACE2/Ang-(l-7)/Mas receptor axis an inhibiting effect on breast cancer metastasis? There are few reports currently. Qingyun Li et al have reported that ACE2 protein could upregulated E-cadherin protein levels by inhibiting transcription factors (ZEB1、TWIST1 and Snaill), resulting in metastatic suppression of non-small cell lung cancer. Ang-(1-7) also could increase E-cadherin protein levels in renal tubule epithelia. These results suggest that, ACE2/Ang-(1-7)/Mas axis receptor may inhibit the metastasis of breast cancer by up-regulating E-cadherin protein.Store-operated calcium entry (SOCE) widely exists in the non-excitatory and excitatory cells and is the predominant Ca2+entry mechanism. Stromal interaction moleculel (Stiml) and Orail are responsible for SOCE. Stiml is endoplasmic reticulum (ER)-localized Ca2+ sensor protein and Orail is plasma membrane (PM)-localized Ca2+ channel. Ca2+ depletion in the ER is sensed by the luminal EF-hand of Stim proteins, causing their multimerization, and subsequent translocation within close proximity of the PM. The interaction of the cytoplasmic C termini of Stim with PM components, and association with PM-localized Orail channels, cause their activation in the PM, and mediates Ca2+ influx. That plays a vital role to sustain Ca2+ homeostasis and also ensures the accuracy of intracellular Ca2+ delivery. SOCE mediated Ca2+ influx promotes cell metastasis by regulating energy metabolism, cell skeleton movement, focal adhesion turnover and so on. Knockdown Stiml and Orail protein can inhibit breast cancer MDA-MB-231 cells metastatisis. On the contrary, the metastatisis of normal breast epithelial cells MCF-10A that were low metastatic ability and overexpressed with Stiml and Orail protein would been increased. More interestingly, SOCE also mediated tumor cell EMT by activating Snaill to inhibit the expression levels of E-cadherin. These indicate that SOCE mediated Ca2+ influx promotes cell metastasis by regulating cells motility and metastasis-associated protein expression. Previous studies have reported that Ca2+ activated NF-kB pathway can promote Snaill expression and p21 activated kinase 1 (PAK1) can activated the nuclear translocation of Snaill. These suggested that SOCE decreased the expression levels of E-cadherin by activating NF-kB/PAK1/Snaill signal pathway.The studies of cardiovascular research found that AngⅡ accelerated smooth muscle proliferation by activating SOCE. But the role of Ang-(1-7) that an endogenous inhibitor of AngⅡ on SOCE is not clear. Whether the effect of ACE2/Ang-(1-7)/Mas receptor axis on upregulating E-cadherin expression is associated with SOCE? That’s all not clear.Objective1. To explore that whether ACE2 protein expression levels is associated with breast cancer metastasis and further define the effects of ACE2/Ang-(1-7)/Mas receptor axis on breast cancer metastasis.2. To explore the effect and mechanism of SOCE on the expression of E-cadherin.3. To define that whether the effects of ACE2/Ang-(1-7)/Mas receptor axis on SOCE in breast cancer cells.Methods1. The total protein of three highly metastatic ability of breast cancer cell lines (BT549, ZR-75-30 and MDA-MB-231) and three low metastatic ability of breast cancer cell lines (BT474, T-470 and MCF-7) were extracted and the expression of ACE2 protein were determined by western blot. Breast cancer cell lines that are low ACE2 protein expression were transfected with lentivirus to overpress ACE2 protein. On the contrary, breast cancer cell lines that are high ACE2 protein expression were transfected with shRNA-vector to knowdown ACE2 protein or were treated with Mas receptor inhibitor, A-779. Wound healing assay and Transwell assay were preform to detect the migration and invation of Breast cancer cell lines for confirming the relation between the expression levels of ACE2 protein and metastasis of breast cancer cells.2. Wound healing assay and Transwell assay were preform to respectively detect the migration and invation of MDA-MB-231 cells that were treated with Ang-(1-7) for confirming the effects of Ang-(1-7) on metastasis of MDA-MB-231 cells. Meantime, MDA-MB-231 cells were pretreated with A-779 for observing whether A-779 could inhibite the effects of A-779.3. The total protein of breast cancer cells that were overexpressed or knockdown for ACE2 pretein, or treatment with Ang-(1-7) were extracted for the detection of change of E-cadherin protein and transcription factors (ZEB1, TWIST1 and Snaill). In addition, nucleoprotein NF-kB and Snaill, and cytosolic proteins phosphorylated PAK1 were detected by western blot. The nuclear translocation of NF-kB and Snaill was analyze by immunofluorescence.4. Cells were incubated with Fura-4/AM in Hanks’buffered salt solution. The Ca2+ influx images were recorded by FV10-ASW 1.7 viewer software under FV1000-IX71 laser scanning confocal microscopes. Observation and comparison of SOCE activity between the ability of highly metastatic MDA-MB-231 cells and the ability of lowly metastatic MCF-7 cells. Complex levels of Stimland Orail were detected by Immunofluorescence staining and Co-IP to evaluate the interaction of Orail and Stiml in MDA-MB-231 cells and MCF-7 cells.5. MDA-MB-231 cells were treatment with SOCE inhibitors (2-APB and SKF96365) and calcium chelator (EGTA) and were evaluated as follow:the changes of migration and invation were detected by Wound healing assay and Transwell assay. Cells were incubated with Fura-4/AM in Hanks’buffered salt solution. The Ca2+influx images were recorded by FV10-ASW 1.7 viewer software under FV1000-IX71 laser scanning confocal microscopes. Complex levels of Stimland Orail were detected by Immunofluorescence staining and Co-IP to evaluate the interaction of Orail and Stiml. Western blotting was used to analyze cytoplasmic E-cadherin, Snaill, p-PAKl/PAK1 and nuclear NF-kB and snaill. Nuclear translocation of NF-kB and snaill were analyzed by Immunofluorescence staining.6. Cells were incubated with Fura-4/AM in Hanks’ buffered salt solution. The Ca2+ influx images were recorded by FV10-ASW 1.7 viewer software under FV1000-1X71 laser scanning confocal microscopes. Observation and comparison of SOCE activity in breast cancer cells that were overexpressed or knockdown for ACE2 protein, or treatment with Ang-(1-7). Complex levels of Stimland Orail were detected by Immunofluorescence staining and Co-IP to evaluate the interaction of Orail and Stiml in breast cancer cells that were overexpressed or knockdown for ACE2 pretein, or treatment with Ang-(1-7).7. A complete set of clinical data including age, tumor stage, stage grouping and so on, and Surgical biopsy specimens including adjacent cancer tissue, ductal cancer tissue, invasive cancer tissue and lymph node of breast cancer patient were collected. Immunohistochemistry staining for ACE2 was performed to analysis the correlation between the expression levels ACE2 in tissues and tumour clinical stages.8. Mice tumor model:Mice were divided randomly into four groups and injected via the tail vein with 1×106 overexpressed or knockdown for ACE2 protein cells. Lungs were collected for H&E staining and analyzed for macro and micro metastatic lesions.9. Statistical analysis:All statistical analysis was done with SPSS for Windows version 13.0. Chi-square analysis and Fisher exact probability were applied to analyze the relationship between ACE2 protein expression and clinicopathological status. Normality test was performed for data from experiment in vitro and in vivo. Results were expressed as mean ± SD. Differences between multiple groups were assessed by one-way analysis of variance, and Student’s unpaired t test was used to compare the value of the test and control group. A value of P< 0.05 was considered as significant difference.Results1. The expression levels of ACE2 protein in different metastatic breast cancer cell lines.The expression levels of ACE2 protein in highly metastatic ability of breast cancer cell lines (BT549, ZR-75-30 and MDA-MB-231) were significant high than that in low metastatic ability of breast cancer cell lines (BT474, T-470 and MCF-7). Differences between the two groups was statistically significant (P<0.05).2. Build a stable ACE2 overexpression of MDA-MB-231 cells and stable ACE2 protein knockdown MCF-7 cells.The expression levels of ACE2 protein of MDA-MB-231 cells in lenti-ACE2 group were significant high than that in lenti-NC group, the difference was statistically significant (P<0.05). MCF-7 cells were transfected with shNC vector and four shACE2 vector, and the expression levels of ACE2 protein in shACE2 group were significant low than that in shNC group, the difference was statistically significant (P<0.05). The effect of ACE2 knockdown of shACE2#2 was the most obvious, so the following experiments were done in MCF-7 cells that were transfected shACE2#2.3. The roles of ACE2/Ang-(1-7)/Mas receptor axis in migration and invation of breast cancer cells.The migration and invation were inbihited in ACE2 overexpression and Ang-(1-7) treated MAD-MB-231 cells, the difference was statistically significant (P<0.05). The effects of ACE2 and Ang-(1-7) could be reversed by A-779. On the contrary, knockdown ACE2 or A-779 could enhance the migration and invation of MCF-7 cells, the difference was statistically significant (P<0.05).4. The effects of ACE2/Ang-(1-7)/Mas receptor axis on the expression levels of transcription factors (ZEB1、TWIST1 and Snaill).The expression levels of transcription factors (ZEB1、TWISTland Snaill) were downexpressed in ACE2 overexpression and Ang-(1-7) treated MDA-MB-231 cells. But knockdown ACE2 or A-779 could increase The expression levels of transcription factors (ZEB1、TWIST1 and Snaill). The expression changes of Snaill was the most obvious.5. The effects of ACE2/Ang-(1-7)/Mas receptor axis on NF-kB/PAK1/Snaill signaling pathway.ACE2 and Ang-(1-7) upregulated the E-cadherin expression via Mas receptor in MAD-MB-231 cells. In contrary, high E-cadherin expression was decreased in ACE2-knockdown MCF-7, whereas Ang-(1-7) restored E-cadherin expression. Significant decrease of NF-kB level in MDA-MB-231 cells that overexpress ACE2 or with Ang-(1-7) treatment, and an opposite increase of NF-kB in ACE2-knockdown MCF-7 cells. The phosphorylation levels of cytoplasmic PAKl and nuclear Snaill were dramatically reduced in ACE2-overexpressing or Ang-(1-7) treatedMDA-MB-231 cells, whereas A-779 treatment abolished the effects of ACE2 and Ang-(1-7). The knockdown of ACE2 and treatment with A-779 increased p-PAK1 and nuclear NF-kB levels in MCF-7 cells. On the other hand, Ang-(1-7) repressed the increase of the p-PAK1 and nuclear NF-kB p65 after knockdown of ACE2 in MCF-7 cells, while A-779 treatment also abolished the effects of Ang-(1-7).6. Participation of SOCE in regulation of NF-kB/PAK1/Snail1/signal pathwaySOCE activity was significantly greater high-metastatic MDA-MB-231 cells than in low-metastatic MCF-7 cells. Consistent with this result, the expression of Stiml and Orail, which regulate SOCE, was increased as demonstrated by dual IHC staining and Co-IP. The phosphorylation level of PAK1 and the nuclear translocation of NF-kB and Snaill were increased in MDA-MB-231 cells than in MCF-7 cells, similarly correlated with the SOCE activity. To further investigate the role of SOCE activity in PAK1/NF-kB/Snaill signal pathway, we used SOCE inhibitors (2-APB and SFK96365) to block SOCE activity and EGTA to chelate extracellular Ca2+in MDA-MB-231 cells. Meanwhile, the levels for Snaill protein, p-PAKl and nuclear NF-kB and Snaill were significantly decreased, while E-cadherin expression was obviously induced in MDA-MB-231 cells with 2-APB, SFK96365 or EGTA treatment.7. ACE2/Ang-(1-7)/Mas axis inhibits the activity of SOCEThe SOCE activity was repressed in ACE2-overexpression and Ang-(1-7) treated MDA-MB-231 cells. In addition, Ang-(1-7) also reduced the SOCE activity that was elevated with ACE2 knockdown in MCF-7. Further, A-779 treatment abolished the effects of ACE2 expression and Ang-(1-7) treatment.8. The expression levels of ACE2 protein in human breast cancer tumors from 40 patients with different clinical and pathological characteristics.ACE2 was highly expressed in adjacent and ductal cancer samples. On the contrary, low ACE2 expression was observed in breast cancer samples with distant metastasis or samples that had spread to lymph node, the difference was statistically significant (P<0.05).9. The effects of ACE2 protein on breast cancer metastasis in vivo.The animals with stable expression of ACE2 in MDA-MB-231 cells showed significantly less metastatic foci in the lung, the difference was statistically significant (P<0.05). Consistent with this finding, increased metastatic lesions were found in the lungs of ACE2-knockdown MCF-7 cell-injected mice at day 42, the difference was statistically significant (P<0.05).Conclution1. ACE2/Ang-(1-7)/Mas receptor axis could inhibit breast cancer cell metastasis through inactivation of NF-KB/PAK1/Snaill signal pathway, leading to reduce the inhibitory effect of Snaill to the expression of E-cadherin.2. SOCE increases extracellular Ca2+influx, activating NF-κB/PAKl/Snaill signal pathway and reducing E-cadherin expression, leading to enhance breast cancer cell migration and invasion.3. ACE2/Ang-(1-7)/Mas receptor axis inactivates NF-kB/PAK1/Snaill signal pathway by reducing the activation of SOCE. |
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