New insights into the phosphorylation of the threonine motif of the β1 integrin cytoplasmic domain

Integrins require an activation step before ligand binding and signaling that is mediated by talin and kindlin binding to the β integrin cytosolic domain (β-tail). Conflicting reports exist about the contribution of phosphorylation of a conserved threonine motif in the β1-tail (β1-pT788/pT789) to integrin activation. We show that widely used and commercially available antibodies against β1-pT788/pT789 integrin do not detect specific β1-pT788/pT789 integrin signals in immunoblots of several human and mouse cell lysates but bind bi-phosphorylated threonine residues in numerous proteins, which were identified by mass spectrometry experiments. Furthermore, we found that fibroblasts and epithelial cells expressing the phospho-mimicking β1-TT788/789DD integrin failed to activate β1 integrins and displayed reduced integrin ligand binding, adhesion initiation and cell spreading. These cellular defects are specifically caused by the inability of kindlin to bind β1-tail polypeptides carrying a phosphorylated threonine motif or phospho-mimicking TT788/789DD substitutions. Our findings indicate that the double-threonine motif in β1-class integrins is not a major phosphorylation site but if phosphorylated would curb integrin function.

Correlation between the features of β1 and β3 integrin expression and lymphangitic metastatic spread in nonspecific invasive breast carcinoma based on tumor morphological heterogeneity

Objective. To study β1 and β3 integrin expression in nonspecific invasive breast carcinoma and to find the associations with parameters of tumor morphological heterogeneity and lymphatic dissemination.Material and Methods. Study group comprised 107 patients with breast cancer. Histological type of tumor corresponded to invasive carcinoma of a nonspecific type (invasive ductal carcinoma) in 100% of cases. Patients did not receive any preoperative treatment. In each case, we performed morphological examination of samples of primary tumor and axillary lymph nodes obtained at the surgical stage of treatment (radical mastectomy or sectoral resection of mammary gland with axillary lymphadenectomy). The parameters of β1 and β3 integrin expression in primary tumor tissue were assessed by immunohistochemistry.Results. The study demonstrated that an increase in the degree of malignancy of breast carcinoma was associated with a decrease in the incidence of positive expression of β1 integrin as well as with an increase in the incidence of positive expression of β3 integrin. Metastases in lymph nodes were significantly less frequently detected in the presence of positive expression of β1 integrin in the alveolar and solid structures compared with the cases of absent expression of the marker in similar structures (48%; χ2 = 3.5; p = 0.05 and 48%; χ2 = 4.8; p = 0.02, respectively). Lymphogenic metastasis were detected significantly more often in cases with positive expression of β3 integrin in discrete groups of cells compared with the cases where the expression of study marker in the described structures was absent (47 and 23%, respectively; χ2 = 5.1; p = 0.02).Conclusion. The results of work showed the presence of relationships between the morphological heterogeneity of the tumor and the parameters of β1 and β3 integrin expression in the parenchymal structures of the neoplasm. The study showed the association of described parameters with the frequency of lymphatic dissemination in patients with breast cancer. Obtained data expand and support previously known evidence and suggest the possibility of assessing the markers as potential prognostic factors predicting the course of cancer.

Bacterial Outer Membrane Protein OmpX Regulates β1 Integrin and Epidermal Growth Factor Receptor (EGFR) Involved in Invasion of M-HeLa Cells by Serratia proteamaculans

Opportunistic pathogen Serratia proteamaculans are able to penetrate the eukaryotic cells. The penetration rate can be regulated by bacterial surface protein OmpX. OmpX family proteins are able to bind to host cell surface to the epidermal growth factor receptor (EGFR) and the extracellular matrix protein fibronectin, whose receptors are in return the α5 β1 integrins. Here we elucidated the involvement of these host cell proteins in S. proteamaculans invasion. We have shown that, despite the absence of fibronectin contribution to S. proteamaculans invasion, β1 integrin was directly involved in invasion of M-HeLa cells. Herewith β1 integrin was not the only receptor that determines sensitivity of host cells to bacterial invasion. Signal transfer from EGFR was also involved in the penetration of these bacteria into M-HeLa cells. However, M-HeLa cells have not been characterized by large number of these receptors. It turned out that S. proteamaculans attachment to the host cell surface resulted in an increment of EGFR and β1 integrin genes expression. Such gene expression increment also caused Escherichia coli attachment, transformed with a plasmid encoding OmpX from S. proteamaculans. Thus, an OmpX binding to the host cell surface caused an increase in the EGFR and β1 integrin expression involved in S. proteamaculans invasion.

Emergence of β1 integrin-deficient breast tumours from dormancy involves both inactivation of p53 and generation of a permissive tumour microenvironment

The molecular and cellular mechanisms underlying mammary tumour dormancy and cancer recurrence are unclear and remain to be elucidated. Here, we report that mammary epithelial-specific disruption of β1 integrin in a murine model of Luminal B human breast cancer drastically impairs tumour growth with proliferation block, apoptosis induction and cellular senescence. β1 integrin-deficient dormant lesions show activation of the tumour suppressor p53, and tumours that circumvent dormancy possess p53 mutation analogous to those in human disease. We further demonstrate that mammary epithelial deletion of p53 in β1 integrin-deficient mice fully rescues tumour dormancy and bypasses cellular senescence. Additionally, recurrent β1 integrin-deficient tumours exhibit fibrosis with increased cancer-associated fibroblast infiltration and extracellular matrix deposition, absent in fast-growing β1 integrin/p53-deficient lesions. Taken together, these observations argue that β1 integrin modulates p53-dependent cellular senescence resulting in tumour dormancy and that pro-tumourigenic stromal cues and intrinsic genetic mutation are required for dormancy exit.

Mutant collagen COL11A1 enhances cancerous invasion

Collagens are the most abundant proteins in the body and comprise the basement membranes and stroma through which cancerous invasion occurs; however, a pro-neoplastic function for mutant collagens is undefined. Here we identify COL11A1 mutations in 66 of 100 cutaneous squamous cell carcinomas (cSCCs), the second most common U.S. cancer, concentrated in a triple helical region known to produce trans-dominant collagens. Analysis of COL11A1 and other collagen genes found that they are mutated across common epithelial malignancies. Knockout of mutant COL11A1 impairs cSCC tumorigenesis in vivo. Compared to otherwise genetically identical COL11A1 wild-type tissue, gene-edited mutant COL11A1 skin is characterized by induction of β1 integrin targets and accelerated neoplastic invasion. In mosaic tissue, mutant COL11A1 cells enhanced invasion by neighboring wild-type cells. These results suggest that specific collagens are commonly mutated in cancer and that mutant collagens may accelerate this process.

The solute carrier MFSD1 decreases β1 integrins activation status and thus tumor metastasis

Solute carriers are increasingly recognized as participating in a plethora of pathologies, including cancer. We describe here the involvement of the orphan solute carrier MFSD1 in the regulation of tumor cell migration. Loss of MFSD1 enabled higher levels of metastasis in a mouse model. We identified an increased migratory potential in MFSD1-/- tumor cells which was mediated by increased focal adhesion turn-over, reduced stability of mature inactive β1 integrin, and the resulting increased integrin activation index. We show that MFSD1 promoted recycling to the cell surface of endocytosed inactive β1 integrin and thereby protected β1 integrin from proteolytic degradation; this led to dampening of the integrin activation index. Furthermore, down-regulation of MFSD1 expression was observed during early steps of tumorigenesis and higher MFSD1 expression levels correlate with a better cancer patient prognosis. In sum, we describe a requirement for endolysosomal MFSD1 in efficient β1 integrin recycling to suppress tumor spread.