AKT2S128/CCTαS315/319/323-positive cancer-associated fibroblasts (CAFs) mediate focal adhesion kinase (FAK) inhibitors resistance via secreting phosphatidylcholines (PCs).

Abnormal metabolism is regarded as an oncogenic hallmark related to tumor progression and therapeutic resistance. Present study employed multi-omics, including phosphoproteomics, untargeted metabolomics and lipidomics, to demonstrate that the pAKT2 Ser128 and pCCTα Ser315/319/323-positive cancer-associated fibroblasts (CAFs) substantially release phosphatidylcholines (PCs), contributing to the resistance of focal adhesion kinase (FAK) inhibitors in esophageal squamous cell carcinoma (ESCC) treatment. Additionally, we observed extremely low levels of FAK Tyr397 expression in CAFs, potentially offering no available target for FAK inhibitors playing their anti-growth role in CAFs. Consequently, FAK inhibitor increased the intracellular concentration of Ca2+ in CAFs, promoting the formation of AKT2/CCTα complex, leading to phosphorylation of CCTα Ser315/319/323 sites and eventually enhancing stromal PC production. This activation could stimulate the intratumoral Janus kinase 2 (JAK2)/Signal transducer and activator of transcription 3 (STAT3) pathway, triggering resistance to FAK inhibition. Analysis of clinical samples demonstrated that stromal pAKT2 Ser128 and pCCTα Ser315/319/323 are related to the tumor malignancy and reduced patient survival. Pseudo-targeted lipidomics and further validation cohort quantitatively showed that plasma PCs enable to distinguish the malignant extent of ESCC patients. In conclusion, inhibition of stroma-derived PCs and related pathway could be possible therapeutic strategies for tumor therapy.

Co-targeting FAK and Gli1 inhibits the tumor-associated macrophages-released CCL22-mediated esophageal squamous cell carcinoma malignancy.

Esophageal squamous cell carcinoma (ESCC) is a frequently seen esophageal tumor type in China. Activation of signaling proteins and relevant molecular mechanisms in ESCC are partially explored, impairing the antitumor efficiency of targeted therapy in ESCC treatment. Tumor-associated macrophages (TAMs)-released C-C motif chemokine 22 (CCL22) can activate intratumoral focal adhesion kinase (FAK), thus promoting the progression of ESCC. Here, we demonstrated that highly secreted CCL22 by TAMs (CCL22-positive TAMs) induced ESCC cell stemness and invasion through facilitating transcriptional activity of intratumoral glioma-associated oncogene 1 (Gli1), a downstream effector for Hedgehog (HH) pathway. Mechanistically, FAK-activated protein kinase B (AKT) mediated Gli1 phosphorylation at its Ser112/Thr115/Ser116 sites and released Gli1 from suppressor of fused homolog, the endogenous inhibitor of Gli1 to activate downstream stemness-associated factors, such as SRY-box transcription factor 2 (SOX2), Nanog homeobox (Nanog), or POU class 5 homeobox (OCT4). Furthermore, inhibition of FAK activity by VS-4718, the FAK inhibitor, enhanced antitumor effect of GDC-0449, the HH inhibitor, both in xenografted models and in vitro assays. Clinically, CCL22/Gli1 axis is used to evaluate ESCC prognosis. Overall, our study establishes the communication of FAK with HH pathway and offers the novel mechanism related to Gli1 activation independent of Smoothened as well as the rationale for the anti-ESCC combination treatment.

GDF11 promotes wound healing in diabetic mice via stimulating HIF-1ɑ-VEGF/SDF-1ɑ-mediated endothelial progenitor cell mobilization and neovascularization.

Non-healing diabetic wounds (DW) are a serious clinical problem that remained poorly understood. We recently found that topical application of growth differentiation factor 11 (GDF11) accelerated skin wound healing in both Type 1 DM (T1DM) and genetically engineered Type 2 diabetic db/db (T2DM) mice. In the present study, we elucidated the cellular and molecular mechanisms underlying the action of GDF11 on healing of small skin wound. Single round-shape full-thickness wound of 5-mm diameter with muscle and bone exposed was made on mouse dorsum using a sterile punch biopsy 7 days following the onset of DM. Recombinant human GDF11 (rGDF11, 50 ng/mL, 10 µL) was topically applied onto the wound area twice a day until epidermal closure (maximum 14 days). Digital images of wound were obtained once a day from D0 to D14 post-wounding. We showed that topical application of GDF11 accelerated the healing of full-thickness skin wounds in both type 1 and type 2 diabetic mice, even after GDF8 (a muscle growth factor) had been silenced. At the cellular level, GDF11 significantly facilitated neovascularization to enhance regeneration of skin tissues by stimulating mobilization, migration and homing of endothelial progenitor cells (EPCs) to the wounded area. At the molecular level, GDF11 greatly increased HIF-1ɑ expression to enhance the activities of VEGF and SDF-1ɑ, thereby neovascularization. We found that endogenous GDF11 level was robustly decreased in skin tissue of diabetic wounds. The specific antibody against GDF11 or silence of GDF11 by siRNA in healthy mice mimicked the non-healing property of diabetic wound. Thus, we demonstrate that GDF11 promotes diabetic wound healing via stimulating endothelial progenitor cells mobilization and neovascularization mediated by HIF-1ɑ-VEGF/SDF-1ɑ pathway. Our results support the potential of GDF11 as a therapeutic agent for non-healing DW.

LncRNA ROR modulates myocardial ischemia-reperfusion injury mediated by the miR-185-5p/CDK6 axis.

LncRNAs and miRNAs are correlated with the pathogenesis of myocardial ischemia-reperfusion injury (MIRI). Whether lncRNA ROR or miR-185-5p plays a crucial role in MIRI is still unclear. In in-vitro, human cardiac myocytes (HCMs) were treated with hypoxia/reoxygenation (H/R). Wistar rats were used to set up an in-vitro I/R model by means of recanalization after ligation. Evaluation of the myocardial injury marker lactate dehydrogenase (LDH) in HCMs cells was performed. The expression of miR-185-5p and ROR, IL-1ß, and IL-18 were detected by qRT-PCR. ELISA was also performed to evaluate the secretion of IL-1ß and IL-18. Western blotting was carried out to determine CDK6, NLRP3, GSDMD-N, ASC, and cleaved-caspase1 protein expression. The relationship between miR-185-5p and CDK6 or ROR was confirmed by a dual-luciferase reporter assay. Our findings revealed that H/R treated HCMs showed a significantly decreased miR-185-5p expression and increased expression of CDK6 and ROR. ROR knockdown reduced H/R induced pyroptosis and inflammation, while knockdown of miR-185-5p accelerated the effect. Furthermore, miR-185-5p was negatively regulated and absorbed by ROR in HCMs. Overexpression of miR-185-5p reversed the H/R-induced cell pyroptosis and upregulation of LDH, IL-1ß, and IL-18. In HCMs, miR-185-5p was also negatively regulated and related to CDK6 expression. Moreover, overexpression of CDK6 significantly inhibited the effects of miR-185-5p mimics on the inflammatory response and pyroptosis of HCMs. Knockdown of ROR alleviated H/R-induced myocardial injury by elevating miR-185-5p and inhibiting CDK6 expression. Taken together, our results show that the ROR/miR-185-5p/CDK6 axis modulates cell pyroptosis induced by H/R and the inflammatory response of HCMs.

Up-regulation of miR-195 contributes to cardiac hypertrophy-induced arrhythmia by targeting calcium and potassium channels.

Previous studies have confirmed that miR-195 expression is increased in cardiac hypertrophy, and the bioinformatics website predicted by Targetscan software shows that miR-195 can directly target CACNB1, KCNJ2 and KCND3 to regulate Cavß1, Kir2.1 and Kv4.3 proteins expression. The purpose of this study is to confirm the role of miR-195 in arrhythmia caused by cardiac hypertrophy. The protein levels of Cavß1, Kir2.1 and Kv4.3 in myocardium of HF mice were decreased. After miR-195 was overexpressed in neonatal mice cardiomyocytes, the expression of ANP, BNP and ß-MHC was up-regulated, and miR-195 inhibitor reversed this phenomenon. Overexpression of miR-195 reduced the estimated cardiac function of EF% and FS% in wild-type (WT) mice. Transmission electron microscopy showed that the ultrastructure of cardiac tissues was damaged after miR-195 overexpression by lentivirus in mice. miR-195 overexpression increased the likelihood of arrhythmia induction and duration of arrhythmia in WT mice. Lenti-miR-195 inhibitor carried by lentivirus can reverse the decreased EF% and FS%, the increased incidence of arrhythmia and prolonged duration of arrhythmia induced by TAC in mice. After miR-195 treatment, the protein expressions of Cavß1, Kir2.1 and Kv4.3 were decreased in mice. The results were consistent at animal and cellular levels, respectively. Luciferase assay results showed that miR-195 may directly target CACNB1, KCNJ2 and KCND3 to regulate the expression of Cavß1, Kir2.1 and Kv4.3 proteins. MiR-195 is involved in arrhythmia caused by cardiac hypertrophy by inhibiting Cavß1, Kir2.1 and Kv4.3.

MicroRNA-132 regulates total protein of Nav1.1 and Nav1.2 in the hippocampus and cortex of rat with chronic cerebral hypoperfusion.

Nav1.1 and Nav1.2 are the voltage-gated sodium channel alpha subunit1 and 2, encoded by the genes of SCN1A and SCN2A. Previous studies have shown that chronic cerebral hypoperfusion (CCH) could induce neuropathological and cognitive impairment and increased total Nav1.1 and Nav1.2protein levels, yet the detailed mechanisms are not fully understood. MicroRNAs (miRNAs) are a class of small, non-coding RNAs that are involved in the regulation of dementia. miR-132 is known to play a key role in neurodegenerative disease. Here, we determined that miR-132 regulates Nav1.1 and Nav1.2 under CCH state. In this study, the expression of miR-132 was decreased in both the hippocampus and cortex of ratsfollowing CCH generated by bilateral common carotid artery occlusion (2VO). Lentiviral-mediated overexpression of miR-132 ameliorated dementia vulnerability induced by 2VO. At the molecular level, miR-132 repressed the increased protein expression of Nav1.1 and Nav1.2 in both the hippocampus and cortex induced by 2VO. MiR-132 suppressed, while AMO-miR-132 enhanced, the level of Nav1.1 and Nav1.2 in primary cultured neonatal rat neurons (NRNs) detected by both western blot analysis and immunofluorescence analysis. Results obtained by dual luciferase assay showed that overexpression of miR-132 inhibited the expression of Nav1.1 and Nav1.2 in human embryonic kidney 293 (HEK293T) cells. Additionally, binding-site mutation failed to influence Nav1.1 and Nav1.2, indicating that Nav1.1 and Nav1.2 are potential targets for miR-132. Taken together, our findings demonstrated that miR-132 protects against CCH-induced learning and memory impairments by down-regulating the expression of Nav1.1 and Nav1.2, and SCN1A and SCN2A are the target genes of miR-132.

Activation of cardiac TrkB receptor by its small molecular agonist 7,8-dihydroxyflavone inhibits doxorubicin-induced cardiotoxicity via enhancing mitochondrial oxidative phosphorylation.

Brain-derived neurotrophic factor (BDNF)/tropomyosin-related receptor kinase B (TrkB) pathway has been revealed as a novel therapeutic target for several neurological diseases. Recently, small-molecule TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) has received considerable attention as a novel potential candidate for the treatment of various BDNF-implicated human disorders. However, its roles in cardiac diseases are not fully understood. Here, the present study aimed to clarify the effects and mechanisms of 7,8-DHF on doxorubicin (Dox)-induced cardiotoxicity. Kunming mice and H9c2 cells were employed to investigate the functional role of 7,8-DHF both in vivo and in vitro. 7,8-DHF markedly increased cell viability and reduced cell death of Dox-treated cells. Meanwhile, 7,8-DHF significantly increased mitochondrial respiration, membrane potential, and optic atrophy 1 (OPA1) protein expression. 7,8-DHF improved cardiac function and attenuated cardiac injury in Dox mice model. Expression of AMP-activated protein kinase (AMPK) and signal transducers and activators of transcription 3 (STAT3) was restored by 7,8-DHF. Furthermore, the protective role of 7,8-DHF was abolished by ANA-12 (a specific antagonist of TrkB). In elucidating the molecular mechanism, the phosphorylation of Akt was significantly increased while extracellular regulated protein kinase (ERK) was decreased after 7,8-DHF treatment. The regulatory effects of 7,8-DHF on STAT3 and AMPK was reversed by Akt inhibitor. In summary, 7,8-DHF attenuated Dox-induced cardiotoxicity by activating Akt and increasing mitochondrial oxidative phosphorylation and thereby regulating STAT3, AMPK, and ERK signals. The present study enhanced current understanding of TrkB receptor in the cardiovascular system and provided a novel target for prevention and treatment of heart diseases.