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Treatment of neuropathic pain (NP) continues to be a clinical challenge and the underlying mechanisms of NP remain elusive. More evidence suggests that glial cell line-derived neurotrophic factor (GDNF) has potent anti-nociceptive effects on NP, but the underlying mechanisms are still largely unknown. Recent data have shown that integrin β1 plays an important part in NP induction, and that the activity of integrin β1 signaling is associated with the phosphorylation of the conserved threonines in the cytoplasmic domain and recruitment of focal adhesion kinase (FAK) to the integrin β1 tail and phosphorylation. We assessed the effect of GDNF on integrinβ1/FAK signaling in NP states. Immunostaining results showed that integrin β1 was mainly observed in the superficial dorsal horn in the spinal cord of rats, and was mostly expressed in intrinsic neurons. Expression of p-integrin β1 and the phosphorylation of integrin β1-associated FAK, but not integrin β1 itself, was up-regulated after chronic constriction injury (CCI), which could be reversed by GDNF, and the effect of GDNF on integrin β1/FAK signaling was inhibited by pre-treatment with RET function-blocking antibody (RET Ab). Moreover, pre-treatment with RET Ab could antagonize the effect of GDNF on inhibiting the NP induced by CCI. These data suggest that GDNF can regulate integrin β1 activity via a RET-related mechanism., (Copyright © 2017. Published by Elsevier Inc.)

Specific cleavage of the transmembrane molecule, CUB domain-containing protein-1 (CDCP1), by plasmin-like serine proteases induces outside-in signal transduction that facilitates early stages of spontaneous metastasis leading to tumor cell intravasation, namely cell escape from the primary tumor, stromal invasion and transendothelial migration. We identified active β1 integrin as a biochemical and functional partner of the membrane-retained 70-kDa CDCP1 fragment, newly generated from its full-length 135-kDa precursor though proteolytic cleavage by serine proteases. Both in cell cultures and in live animals, active β1 integrin complexed preferentially with functionally activated, phosphorylated 70-kDa CDCP1. Complexing of β1 integrin the 70-kDa with CDCP1 fragment induced intracellular phosphorylation signaling, involving focal adhesion kinase-1 (FAK) and PI3 kinase (PI3K)-dependent Akt activation. Thus, inhibition of FAK/PI3K activities by specific inhibitors as well as short-hairpin RNA downregulation of β1 integrin significantly reduced FAK/Akt phosphorylation under conditions where CDCP1 was processed by serine proteases, indicating that FAK/PI3K/Akt pathway operates downstream of cleaved CDCP1 complexed with β1 integrin. Furthermore, this complex-dependent signaling correlated positively with high levels of tumor cell intravasation and dissemination. Correspondingly, abrogation in vivo of CDCP1 cleavage either by unique cleavage-blocking monoclonal antibody 10-D7 or by inhibition of proteolytic activity of plasmin-like serine proteases with aprotinin prevented β1 integrin/CDCP1 complexing and downstream FAK/Akt signaling concomitant with significant reduction of stromal invasion and spontaneous metastasis. Therefore, β1 integrin appears to serve as a motility-regulating partner mediating cross-talk between proteolytically cleaved, membrane-retained CDCP1 and members of FAK/PI3K/Akt pathway. This CDCP1 cleavage-induced signaling cascade constitutes a unique mechanism, independent of extracellular matrix remodeling, whereby a proteolytically cleaved CDCP1 regulates in vivo locomotion and metastasis of tumor cells through β1 integrin partnering. Our findings indicate that CDCP1 cleavage, occurring at the apex of a β1 integrin/FAK/PI3K/Akt signaling cascade, may represent a therapeutic target for CDCP1-positive cancers.

Integrin signaling critically contributes to the progression, growth, and therapy resistance of malignant tumors. Here, we show that targeting of β₁ integrins with inhibitory antibodies enhances the sensitivity to ionizing radiation and delays the growth of human head and neck squamous cell carcinoma cell lines in 3D cell culture and in xenografted mice. Mechanistically, dephosphorylation of focal adhesion kinase (FAK) upon inhibition of β₁ integrin resulted in dissociation of a FAK/cortactin protein complex. This, in turn, downregulated JNK signaling and induced cell rounding, leading to radiosensitization. Thus, these findings suggest that robust and selective pharmacological targeting of β₁ integrins may provide therapeutic benefit to overcome tumor cell resistance to radiotherapy.

beta1 integrin and collagen matrix interactions regulate the survival of cells by associating with focal adhesion kinase (FAK) and initiating MAPK/ERK signalling, but little is known about these signalling pathways during human fetal islet ontogeny. The purpose of this study was to investigate whether beta1 integrin/FAK activation of the MAPK/ERK pathway regulates human fetal islet cell expression of endocrine cell markers and survival. Isolated human (18-21 weeks fetal age) islet-epithelial cell clusters, cultured on collagen I, were examined using beta1 integrin blocking antibody, beta1 integrin siRNA and FAK expression vector. Perturbing beta1 integrin function in the human fetal islet-epithelial cell clusters resulted in a marked decrease in cell adhesion, in parallel with a reduction in the number of cells expressing PDX-1, insulin and glucagon (p < 0.05). beta1 integrin blockade disorganized focal adhesion contacts in the PDX-1(+) cells and decreased activation of FAK and ERK1/2 signalling in parallel with an increase in expression of cleaved caspases 9 and 3 (p < 0.01). Similar results were obtained following an siRNA knock-down of beta1 integrin expression. In contrast, over-expression of FAK not only increased phospho-ERK and the expression of PDX-1, insulin and glucagon (p < 0.05) but also abrogated the decreases in phospho-ERK and PDX-1 by beta1 integrin blockade. This study demonstrates that activation of the FAK/ERK signalling cascade by beta1 integrin is involved in the differentiation and survival of human fetal pancreatic islet cells., (2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

The molecular determinants which dictate survival and apoptosis/anoikis in human intestinal crypt cells remain to be fully understood. To this effect, the roles of beta1 integrin/Fak/Src signaling to the PI3-K/Akt-1, MEK/Erk, and p38 pathways, were investigated. The regulation of six Bcl-2 homologs (Bcl-2, Mcl-1, Bcl-X(L), Bax, Bak, Bad) was likewise analyzed. We report that: (1) Anoikis causes a down-activation of Fak, Src, Akt-1 and Erk1/2, a loss of Fak-Src association, and a sustained/enhanced activation of p38beta, which is required as apoptosis/anoikis driver; (2) PI3-K/Akt-1 up-regulates the expression of Bcl-X(L) and Mcl-1, down-regulates Bax and Bak, drives Bad phosphorylation (both serine112/136 residues) and antagonizes p38beta activation; (3) MEK/Erk up-regulates Bcl-2, drives Bad phosphorylation (serine112 residue), but does not antagonize p38bactivation; (4) PI3-K/Akt-1 is required for survival, whereas MEK/Erk is not; (5) Src acts as a cornerstone in the engagement of both pathways by beta1 integrins/Fak, and is crucial for survival; and (6) beta1 integrins/Fak and/or Src regulate Bcl-2 homologs as both PI3-K/Atk-1 and MEK/Erk combined. Hence, beta1 integrin/Fak/Src signaling translates into integrated mediating functions of p38beta activation and regulation of Bcl-2 homologs by PI3-K/Akt-1 and MEK/Erk, consequently determining their requirement (or not) for survival.