The signal transducer and activator of transcription 3 (STAT3) signaling pathway has been implicated in cell apoptosis and inflammatory processes. Ischemic preconditioning (IPC) and ischemic postconditioning (IPTC) inhibit both of these processes. In the present study, we investigated the role of phosphorylated STAT3 (p-STAT3)-mediated apoptosis and inflammation following non-invasive remote limb IPTC (NRIPoC) using a classic rat model of focal cerebral ischemia. Forty-five adult male Sprague-Dawley rats were divided randomly into 3 groups (n=15 per group): the sham-operated, ischemia/reperfusion (I/R) and NRIPoC groups. NRIPoC was implemented at the beginning of reperfusion. At 24 h after cerebral reperfusion, we evaluated the neurological deficit score (NDS), assessed the cerebral infarct size and tissue morphology, and evaluated neuronal apoptosis. The protein expression levels of Bcl-2, Bax, nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNF-α) and p-STAT3 in the penumbra region were assessed by western blot analysis. The cerebral infarct volume, the number of apoptotic cells and the protein expression levels of Bcl-2, Bax, NF-κB and TNF-α were all found to be increased in the I/R group compared with the sham-operated group. However, these levels were decreased in the NRIPoC group compared with the I/R group. The number of apoptotic cells in the penumbra in the I/R group was increased compared with that in the NRIPoC and sham-operated groups. The protein expression of p-STAT3 was increased in the NRIPoC group compared with the sham-operated and I/R groups. These results indicate that the protective effects of NRIPoC against cerebral I/R injury may be related to the attenuation of neuronal apoptosis and inflammation through the activation of STAT3.

Background: Breast cancer is a heterogeneous disease. Hence, stratification of patients based on the subtype of breast cancer is key to its successful treatment. Among all the breast cancer subtypes, basal-like breast cancer is the most aggressive subtype with limited treatment options. Interestingly, we found focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase, is highly overexpressed and activated in basal-like breast cancer. Methods: To understand the role of FAK in this subtype, we generated mice with conditional deletion of FAK and a knock-in mutation in its kinase domain in MMTV-Wnt1-driven basal-like mammary tumors. Tumor initiation, growth, and metastasis were characterized for these mice cohorts. Immunohistochemical and transcriptomic analysis of Wnt1-driven tumors were also performed to elucidate the mechanisms underlying FAK-dependent phenotypes. Pharmacological inhibition of FAK and mTOR in human basal-like breast cancer cell lines was also tested. Results: We found that in the absence of FAK or its kinase function, growth and metastasis of the tumors were significantly suppressed. Furthermore, immunohistochemical analyses of cleaved caspase 3 revealed that loss of FAK results in increased tumor cell apoptosis. To further investigate the mechanism by which FAK regulates survival of the Wnt1-driven tumor cells, we prepared an isogenic pair of mammary tumor cells with and without FAK and found that FAK ablation increased their sensitivity to ER stress-induced cell death, as well as reduced tumor cell migration and tumor sphere formation. Comparative transcriptomic profiling of the pair of tumor cells and gene set enrichment analysis suggested mTOR pathway to be downregulated upon loss of FAK. Immunoblot analyses further confirmed that absence of FAK results in reduction of AKT and downstream mTOR pathways. We also found that inhibition of FAK and mTOR pathways both induces apoptosis, indicating the importance of these pathways in regulating cell survival. Conclusions: In summary, our studies show that in a basal-like tumor model, FAK is required for survival of the tumor cells and can serve as a potential therapeutic target.

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