Anti-FAK (phospho-Y397) Anticorpo (A27599)

INTRODUCTION:

Acquired tamoxifen resistance remains the major obstacle to breast cancer endocrine therapy. β1-integrin was identified as one of the target genes of G protein-coupled estrogen receptor (GPER), a novel estrogen receptor recognized as an initiator of tamoxifen resistance. Here, we investigated the role of β1-integrin in GPER-mediated tamoxifen resistance in breast cancer.

METHODS:

The expression of β1-integrin and biomarkers of epithelial-mesenchymal transition were evaluated immunohistochemically in 53 specimens of metastases and paired primary tumors. The function of β1-integrin was investigated in tamoxifen-resistant (MCF-7R) subclones, derived from parental MCF-7 cells, and MCF-7R β1-integrin-silenced subclones in MTT and Transwell assays. Involved signaling pathways were identified using specific inhibitors and Western blotting analysis.

RESULTS:

GPER, β1-integrin and mesenchymal biomarkers (vimentin and fibronectin) expression in metastases increased compared to the corresponding primary tumors; a close expression pattern of β1-integrin and GPER were in metastases. Increased β1-integrin expression was also confirmed in MCF-7R cells compared with MCF-7 cells. This upregulation of β1-integrin was induced by agonists of GPER and blocked by both antagonist and knockdown of it in MCF-7R cells. Moreover, the epidermal growth factor receptor/extracellular regulated protein kinase (EGFR/ERK) signaling pathway was involved in this transcriptional regulation since specific inhibitors of these kinases also reduced the GPER-induced upregulation of β1-integrin. Interestingly, silencing of β1-integrin partially rescued the sensitivity of MCF-7R cells to tamoxifen and the α5β1-integrin subunit is probably responsible for this phenomenon. Importantly, the cell migration and epithelial-mesenchymal transition induced by cancer-associated fibroblasts, or the product of cancer-associated fibroblasts, fibronectin, were reduced by knockdown of β1-integrin in MCF-7R cells. In addition, the downstream kinases of β1-integrin including focal adhesion kinase, Src and AKT were activated in MCF-7R cells and may be involved in the interaction between cancer cells and cancer-associated fibroblasts.

CONCLUSIONS:

GPER/EGFR/ERK signaling upregulates β1-integrin expression and activates downstream kinases, which contributes to cancer-associated fibroblast-induced cell migration and epithelial-mesenchymal transition, in MCF-7R cells. GPER probably contributes to tamoxifen resistance via interaction with the tumor microenvironment in a β1-integrin-dependent pattern. Thus, β1-integrin may be a potential target to improve anti-hormone therapy responses in breast cancer patients.

The receptor for advanced-glycation end products (RAGE) is upregulated in various cancers and has been associated with tumor progression, but little is known about its expression and regulation by microRNAs (miRNAs) in esophageal squamous cell carcinoma (ESCC). Here, we describe miR-185, which represses RAGE expression, and investigate the biological role of miR-185 in ESCC. In this study, we found that the high level of RAGE expression in 29 pairs of paraffin-embedded ESCC tissues was correlated positively with the depth of invasion by immunohistochemistry, suggesting that RAGE was involved in ESCC. We used bioinformatics searches and luciferase reporter assays to investigate the prediction that RAGE was regulated directly by miR-185. Besides, overexpression of miR-185 in ESCC cells was accompanied by 27% (TE-11) and 49% (Eca-109) reduced RAGE expression. The effect was further confirmed in RAGE protein by immunofluorescence in both cell lines. The effects were reversed following cotransfection with miR-185 and high-level expression of the RAGE vector. Furthermore, the biological role of miR-185 in ESCC cell lines was investigated using assays of cell viability, Ki-67 staining, and cell migration and invasion, as well as in a xenograft model. We found that overexpression of miR-185 inhibited migration and invasion by ESCC cells in vitro and reduced their capacity to develop distal pulmonary metastases in vivo partly through the RAGE/heat shock protein 27 pathway. Interestingly, in clinical specimens, the level of plasma miR-185 expression was decreased significantly (P = 0.002) in patients with ESCC [0.500; 95% confidence interval (CI) 0.248-1.676] compared with healthy controls (2.410; 95% CI 0.612-5.671). The value of the area under the receiver-operating characteristic curve was 0.73 (95% CI 0.604-0.855). In conclusion, our findings shed novel light on the role of miR-185/RAGE in ESCC metastasis, and plasma miR-185 has potential as a novel diagnostic biomarker in ESCC.

Successful embryonic implantation requires an effective maternal-embryonic molecular dialogue. However, the detailed mechanisms of epithelial-embryo adhesion remain poorly understood. Here, we report that matrix metalloproteinase-26 (MMP-26) is a novel downstream target gene of homeobox a 10 (HOXA10) in human endometrial cells. HOXA10 binds directly to a conserved TTAT unit (-442 to -439) located within the 5' regulatory region of the MMP-26 gene and regulates the expression and secretion of MMP-26 in a concentration-dependent manner. Moreover, the adenovirus-mediated overexpression of MMP-26 in Ishikawa cells markedly increased BeWo spheroid adhesion. An antibody blocking assay further demonstrated that the promotion of BeWo spheroid adhesion by HOXA10 and MMP-26 was significantly inhibited by pre-treatment with a specific antibody against MMP-26. These results demonstrate that the HOXA10-mediated expression of MMP-26 promotes embryo adhesion during the process of embryonic implantation.

Glucose regulated protein 78 (GRP78) is predominantly located in the endoplasmic reticulum as a molecular chaperone. It has also been found on the membranes of some cancer cells, acting as a receptor for a wide variety of ligands. However, its presence on colorectal cancer (CRC) cell surface and its role in CRC metastatic progression remain elusive. Here we reported that GRP78 was predominantly present in the form of clustering aggregates on CRC cell surfaces, and its surface abundance was strongly correlated with CRC differentiation stage. Interestingly, we observed that cell-surface GRP78 had an interaction with the ECM adhesion molecule β1-integrin and was involved in cell-matrix adhesion through regulation of focal adhesion kinase (FAK). Moreover, the present data also implicated that surface GRP78 promoted the cell invasion process, and this effect was at least partly mediated through its association with uPA-uPAR protease system. Together, our data suggests that surface GRP78 promotes CRC cell migration and invasion by regulating cell-matrix adhesion and ECM degradation, which is independent of its signaling receptor function.

Pemphigus vulgaris (PV) is an autoimmune blistering skin disease characterized by suprabasal acantholysis produced as a consequence of desmoglein (Dsg) and non-Dsg autoantibodies binding to several targeting molecules localized on the membrane of keratinocytes. Nitric oxide (NO) may exert a pathogenic function in several immunological processes. We have previously demonstrated that neural nitric oxide synthase (nNOS) plays part in PV acantholysis. Also, our group has described a relevant role for HER [human epidermal growth factor receptor (EGFR) related] isoforms and several kinases such as Src (Rous sarcoma), mammalian target of rapamycin (mTOR) and focal adhesion kinase (FAK), as well as caspases in PV development. Using a passive transfer mouse model of PV, we aimed to investigate the relationship between the increase in nNOS and EGFR, Src, mTOR and FAK kinase upregulation observed in PV lesions. Our results revealed a new function for nNOS, which contributes to EGFR-mediated PV acantholysis through the upregulation of Src, mTOR and FAK. In addition, we found that nNOS participates actively in PV at least in part by increasing caspase-9 and caspase-3 activities. These findings underline the important issue that in PV acantholysis, caspase activation is a nNOS-linked process downstream of Src, mTOR and FAK kinase upregulation.

Angiogenesis has become an attractive target for the treatment of certain diseases such as cancer and rheumatoid arthritis. Our previous studies demonstrated that the saponin fraction from Gleditsia sinensis fruits had anti-angiogenic potential, and Gleditsiosides B (GB) was probably the main active constituent. In the present study, we assessed the effect of GB on endothelial cell migration, a crucial event in angiogenesis, and explored the underlying mechanisms. The migration of endothelial cells was assessed by transwell. The expressions of MMP-2/-9 and TIMP-1/-2 were analyzed by Western blotting, and the activities of MMP-2/-9 were detected by gelatin zymography assay. Moreover, migration-related proteins and signaling pathways, including FAK, MAPKs and PI3K/AKT, were analyzed by Western blotting. It was shown that GB, at a concentration of 10 μM without significant cytotoxicity, could effectively abrogate the migration of human umbilical vein endothelial cells (HUVECs) induced by bFGF. GB also inhibited the expression and activity of MMP-2, elevated the expression of TIMP-1, and restrained the phosphorylations of FAK, ERK, PI3K and AKT in a concentration-dependent manner. The findings suggest that GB was able to abrogate the migration of endothelial cells through down-regulating the activation of MMP-2 and FAK via preventing ERK and PI3K/AKT signaling pathways.

Pemphigus vulgaris (PV) is an autoimmune blistering skin disease characterized by suprabasal acantholysis and by autoantibodies against desmoglein 3 localized on desmosomes. In addition, caspases also seem to participate in this blistering disease. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase involved in cytoskeleton remodelling and formation and disassembly of cell adhesion structures. We have previously demonstrated that HER (human epidermal growth factor receptor related) isoforms, Src (Rous sarcoma) and mammalian target of rapamycin (mTOR), three molecules implicated in signalling processes, take part in suprabasal acantholysis and apoptosis induced by PV-IgG in a mouse model. Our aim was to investigate whether upregulation of FAK is implicated in the development of PV lesions. Herein, using a mouse model, PV-IgG administration showed an increased level of FAK phosphorylated on 397 and 925 tyrosine residues in the basal layer of epidermis. When mice were pretreated with a FAK inhibitor (FI), the acantholysis of the basal layer of epidermis was absent. More interestingly, we observed that phosphorylated FAK (Y397/925) decreased when HER isoforms, Src, mTOR and pan-caspases inhibitors were employed before PV-IgG administration. In addition, pretreatment with the FI before PV-IgG injection prevented the changes in both Bax and Bcl-2 expression and caspase-9 and caspase-3 activities induced by PV-IgG. Finally, FI reduced the expression of phosphorylated Src and mTOR in the basal cells of epidermis. In conclusion, our data reveal a novel role of phosphorylated FAK (Y397/925) in PV development involving HER isoforms, Src and mTOR kinases.

Because cells are sensitive to mechanical forces,microgravity might act on stress-dependent cell changes. Regulation of focal adhesions (FAs) and cytoskeletal activity plays a role in cell maintenance, cell movement,and migration. Human MCF-7 cells were exposed to modeled microgravity (MMG) to test the hypothesis that migration responsiveness to microgravity is associated with cytoskeleton and FA anomalies. MMG acts on MCF-7 cells by disorganizing cytoskeleton filaments (microfilaments and microtubules). Microfilaments in MMG did not display their typical radial array. Likewise, microtubules were disrupted in MCF-7 cells within 4 h of initiation of MMG and were partly reestablished by 48 h. FAs generated inmicrogravity were less mature than those established in controls, shown by reduced FAs number and clustering. In parallel, MMG decreased kinases activity (such as FAK,PYK2, and ILK) of FAs in MCF-7 cells. The expression of both integrinbeta1 and integrinbeta4 were downregulated by MMG. We conclude that cytoskeletal alterations and FAs changes in MMG are concomitant with cell invasion and migration retardation. We suggest that reduced migration response in MCF-7 cells following MMG is linked to changes of cytoskeleton and FAs.

BACKGROUND:

To investigate the role of focal adhesion kinase (FAK)-mediated signaling in hypoxia-induced cardiac fibroblasts (CFs) differentiation and cardiac fibrosis post-myocardial infarction (MI) on a mice model.

METHODS:

CFs of neonatal C57BL/6 mice were treated under normoxic, hypoxic, or hypoxic+PP2 (known as a Src kinase family inhibitor) conditions. Gene expressions of FAK, alpha-smooth muscle actin (α-SMA) and collagen type I alpha 1 (Col1α1), or α-SMA and vimentin levels were performed by RT-PCR and immunofluorescence staining, respectively. Thirty mice were surgically treated into Sham (n=7) and MI (n=23) groups; and FAK inhibitor PF-562271 was given to six survivor MI mice (as PF group, from 15 survivors). Heart function and collagenous tissues were examined by echocardiography, as well as by Masson‘s trichrome and Sirius red staining, respectively. Type I collagen, FAK protein, mTOR, ERK1/2, AKT, P70S6K and phospho-FAK levels were also analyzed.

RESULTS:

FAK inhibition with PP2 significantly decreased CFs differentiation and collagen synthesis under hypoxia treatment. In vivo, PF-562271 treatment resulted in fibrosis attenuation; however, deteriorated heart function of MI mice could not be significantly improved. PF-562271 may affect phospho-mTOR (p<0.05), phospho-ERK1/2 (p<0.01), phospho-AKT (p<0.001) and phospho-P70S6K (p<0.05) to exert its benefits. FAK can be activated either under hypoxia in CFs or MI in a mouse model to promote fibrosis. However, pharmacological inhibition of FAK can attenuate fibrosis response.

CONCLUSION:

This study provides novel evidence that FAK inhibition may become a promising pharmaceutical strategy to attenuate fibrosis post-MI.