Isoform-specific function of calpains in cell adhesion disruption: studies in postlactational mammary gland and breast cancer

2016 ◽  
Vol 473 (18) ◽  
pp. 2893-2909 ◽  
Author(s):  
Lucía Rodríguez-Fernández ◽  
Iván Ferrer-Vicens ◽  
Concha García ◽  
Sara S. Oltra ◽  
Rosa Zaragozá ◽  
...  

Cleavage of adhesion proteins is the first step for physiological clearance of undesired cells during postlactational regression of the mammary gland, but also for cell migration in pathological states such as breast cancer. The intracellular Ca2+-dependent proteases, calpains (CAPNs), are known to cleave adhesion proteins. The isoform-specific function of CAPN1 and CAPN2 was explored and compared in two models of cell adhesion disruption: mice mammary gland during weaning-induced involution and breast cancer cell lines according to tumor subtype classification. In both models, E-cadherin, β-catenin, p-120, and talin-1 were cleaved as assessed by western blot analysis. Both CAPNs were able to cleave adhesion proteins from lactating mammary gland in vitro. Nevertheless, CAPN2 was the only isoform found to co-localize with E-cadherin in cell junctions at the peak of lactation. CAPN2/E-cadherin in vivo interaction, analyzed by proximity ligation assay, was dramatically increased during involution. Calpain inhibitor administration prevented the cytosolic accumulation of truncated E-cadherin cleaved by CAPN2. Conversely, in breast cancer cells, CAPN2 was restricted to the nuclear compartment. The isoform-specific expression of CAPNs and CAPN activity was dependent on the breast cancer subtype. However, CAPN1 and CAPN2 knockdown cells showed that cleavage of adhesion proteins and cell migration was mediated by CAPN1, independently of the breast cancer cell line used. Data presented here suggest that the subcellular distribution of CAPN1 and CAPN2 is a major issue in target-substrate recognition; therefore, it determines the isoform-specific role of CAPNs during disruption of cell adhesion in either a physiological or a pathological context.

Author(s):  
Chen-Long Wang ◽  
Jing-Chi Li ◽  
Ci-Xiang Zhou ◽  
Cheng-Ning Ma ◽  
Di-Fei Wang ◽  
...  

Abstract Purpose Tumor metastasis is the main cause of death from breast cancer patients and cell migration plays a critical role in cancer metastasis. Recent studies have shown long non-coding RNAs (lncRNAs) play an essential role in the initiation and progression of cancer. In the present study, the role of an LncRNA, Rho GTPase Activating Protein 5- Antisense 1 (ARHGAP5-AS1) in breast cancer was investigated. Methods RNA sequencing was performed to find out dysregulated LncRNAs in MDA-MB-231-LM2 cells. Transwell migration assays and F-actin staining were utilized to estimate cell migration ability. RNA pulldown assays and RNA immunoprecipitation were used to prove the interaction between ARHGAP5-AS1 and SMAD7. Western blot and immunofluorescence imaging were used to examine the protein levels. Dual luciferase reporter assays were performed to evaluate the activation of TGF-β signaling. Results We analyzed the RNA-seq data of MDA-MB-231 and its highly metastatic derivative MDA-MB-231-LM2 cell lines (referred to as LM2) and identified a novel lncRNA (NR_027263) named as ARHGAP5-AS1, which expression was significantly downregulated in LM2 cells. Further functional investigation showed ARHGAP5-AS1 could inhibit cell migration via suppression of stress fibers in breast cancer cell lines. Afterwards, SMAD7 was further identified to interact with ARHGAP5-AS1 by its PY motif and thus its ubiquitination and degradation was blocked due to reduced interaction with E3 ligase SMURF1 and SMURF2. Moreover, ARHGAP5-AS1 could inhibit TGF-β signaling pathway due to its inhibitory role on SMAD7. Conclusion ARHGAP5-AS1 inhibits breast cancer cell migration via stabilization of SMAD7 protein and could serve as a novel biomarker and a potential target for breast cancer in the future.


2012 ◽  
Vol 214 (2) ◽  
pp. 165-175 ◽  
Author(s):  
Jorge Diaz ◽  
Evelyn Aranda ◽  
Soledad Henriquez ◽  
Marisol Quezada ◽  
Estefanía Espinoza ◽  
...  

Progesterone and progestins have been demonstrated to enhance breast cancer cell migration, although the mechanisms are still not fully understood. The protease-activated receptors (PARs) are a family of membrane receptors that are activated by serine proteases in the blood coagulation cascade. PAR1 (F2R) has been reported to be involved in cancer cell migration and overexpressed in breast cancer. We herein demonstrate that PAR1 mRNA and protein are upregulated by progesterone treatment of the breast cancer cell lines ZR-75 and T47D. This regulation is dependent on the progesterone receptor (PR) but does not require PR phosphorylation at serine 294 or the PR proline-rich region mPRO. The increase in PAR1 mRNA was transient, being present at 3 h and returning to basal levels at 18 h. The addition of a PAR1-activating peptide (aPAR1) to cells treated with progesterone resulted in an increase in focal adhesion (FA) formation as measured by the cellular levels of phosphorylated FA kinase. The combined but not individual treatment of progesterone and aPAR1 also markedly increased stress fiber formation and the migratory capacity of breast cancer cells. In agreement with in vitro findings, data mining from the Oncomine platform revealed that PAR1 expression was significantly upregulated in PR-positive breast tumors. Our observation that PAR1 expression and signal transduction are modulated by progesterone provides new insight into how the progestin component in hormone therapies increases the risk of breast cancer in postmenopausal women.


2021 ◽  
pp. 002215542110552
Author(s):  
Chidalu A. Edechi ◽  
Maryam Amini ◽  
Mohammad K. Hamedani ◽  
Lucas E.L. Terceiro ◽  
Barbara E. Nickel ◽  
...  

The tight junction membrane protein claudin 1 and the adherens junction protein E-cadherin play critical roles in cell–cell communication and in cell signaling. As a result, their protein levels and distribution in cancer have been a focus of cancer researchers in recent years. The loss of sensitivity to contact inhibition and the establishment of invasive properties in cancer are thought to be a result of the mislocalization of these membrane proteins to the cytoplasm. However, reports on their distribution and levels have been inconsistent. It is therefore critical that the techniques used to determine the cellular localization of these proteins be both consistent and reliable. This study was undertaken to determine the optimal fixation method, methanol or formalin, for the detection of claudin 1 and E-cadherin by immunofluorescence in five different human breast cancer cell lines. Both methods exhibited staining of the cell membrane and cytoplasm, but the strongest and most distinct signals were obtained using methanol fixation. Interestingly, cell-specific differences were also observed that appeared to be associated with levels of claudin 1 and E-cadherin as seen by Western blotting. Therefore, when evaluating cellular localization of the junction proteins claudin 1 and E-cadherin, expression level and cell type differences must be considered:


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