Activation Of c-Src Research Articles

C-Src Is Responsible for Mitochondria-Mediated Arrhythmic Risk in Ischemic Cardiomyopathy.

Increased mitochondrial Ca2+ uptake has been implicated in the QT prolongation and lethal arrhythmias associated with nonischemic cardiomyopathy. We attempted to define the role of mitochondria in ischemic arrhythmic risk and to identify upstream regulators. Myocardial infarction (MI) was induced in wild-type FVB/NJ mice by ligation of the left anterior descending coronary artery. Western blot, immunoprecipitation, ECG telemetry, and patch-clamp techniques were used. After MI, c-Src (proto-oncogene tyrosine-protein kinase Src) and its active form (p-Src Y416) were increased. The activation of c-Src was associated with increased diastolic Ca2+ sparks, action potential duration prolongation, and arrhythmia in MI mice. c-Src upregulation and arrhythmia could be reversed by treatment of mice with the Src inhibitor PP1 but not with the inactive analogue PP3. Tyrosine phosphorylated mitochondrial Ca2+ uniporter (MCU) was upregulated in the heart tissues of MI mice and patients with ischemic cardiomyopathy. In a heterologous expression system, c-Src could bind MCU and phosphorylate MCU tyrosines. Overexpression of wild-type c-Src significantly increased the mitochondrial Ca2+ transient while overexpression of dominant-negative c-Src significantly decreased the mitochondrial Ca2+ transient. c-Src inhibition by PP1, MCU inhibition by Ru360, or MCU knockdown could reduce the action potential duration, Ca2+ sparks, and arrhythmia after MI. The human heart tissue showed that patients with ischemic cardiomyopathy had significantly increased c-Src active form associated with increased MCU tyrosine phosphorylation and ventricular arrhythmia. MI leads to increased c-Src active form that results in MCU tyrosine phosphorylation, increased mitochondrial Ca2+ uptake, QT prolongation, and arrhythmia, suggesting c-Src or MCU may represent novel antiarrhythmic targets.

Deciphering Mutational Impacts on c-Src-HK2 Interaction in Colorectal Cancer Progression, and Identification of Potential Phytocompounds Inhibitors: A Molecular Simulation and Free Energy Calculation Approach.

Colorectal cancer (CRC) stands as the third most widespread cancer worldwide in both men and women, witnessing a concerning rise, especially in younger demographics. Abnormal activation of the Non-Receptor Tyrosine Kinase c-Src has been linked to the advancement of several human cancers, including colorectal, breast, lung, and pancreatic ones. The interaction between c-Src and Hexokinase 2 (HK2) triggers enzyme phosphorylation, significantly boosting glycolysis, and ultimately contributing to the development of CRC. The objectives of this study are to examine the influence of newly identified mutations on the interaction between c-Src and the HK2 enzyme and to discover potent phytocompounds capable of disrupting this interaction. In this study, we utilized molecular docking to check the effect of the identified mutation on the binding of c-Src with HK2. Virtual drug screening, MD simulation, and binding free energy were employed to identify potent drugs against the binding interface of c-Src and HK2. Among these mutations, six (W151C, L272P, A296S, A330D, R391H, and P434A) were observed to significantly disrupt the stability of the c-Src structure. Additionally, through molecular docking analysis, we demonstrated that the mutant forms of c-Src exhibited high binding affinities with HK2. The wildtype showed a docking score of -271.80 kcal/mol, while the mutants displayed scores of -280.77 kcal/mol, -369.01 kcal/mol, -324.41 kcal/mol, -362.18 kcal/mol, 266.77 kcal/mol, and -243.28 kcal/mol for W151C, L272P, A296S, A330D, R391H, and P434A respectively. Furthermore, we identified five lead phytocompounds showing strong potential to impede the binding of c-Src with HK2 enzyme, essential for colon cancer progression. These compounds exhibit robust bonding with c-Src with docking scores of -7.37 kcal/mol, -7.26 kcal/mol, -6.88 kcal/mol, -6.81 kcal/mol, and -6.73 kcal/mol. Moreover, these compounds demonstrate dynamic stability, structural compactness, and the lowest residual fluctuation during MD simulation. The binding free energies for the top five hits (-42.44±0.28 kcal/mol, -28.31±0.25 kcal/mol, -34.95±0.44 kcal/mol, -38.92±0.25 kcal/mol, and -30.34±0.27 kcal/mol), further affirm the strong interaction of these drugs with c-Src which might impede the cascade of events that drive the progression of colon cancer. Our findings serve as a promising foundation, paving the way for future discoveries in the fight against colorectal cancer.

C-Src-induced vascular malformations require localised matrix degradation at focal adhesions.

Endothelial cells lining the blood vessel wall communicate intricately with the surrounding extracellular matrix, translating mechanical cues into biochemical signals. Moreover, vessels require the capability to enzymatically degrade the matrix surrounding them, to facilitate vascular expansion. c-Src plays a key role in blood vessel growth, with its loss in the endothelium reducing vessel sprouting and focal adhesion signalling. Here, we show that constitutive activation of c-Src in endothelial cells results in rapid vascular expansion, operating independently of growth factor stimulation or fluid shear stress forces. This is driven by an increase in focal adhesion signalling and size, with enhancement of localised secretion of matrix metalloproteinases responsible for extracellular matrix remodelling. Inhibition of matrix metalloproteinase activity results in a robust rescue of the vascular expansion elicited by heightened c-Src activity. This supports the premise that moderating focal adhesion-related events and matrix degradation can counteract abnormal vascular expansion, with implications for pathologies driven by unusual vascular morphologies.

C-Src Facilitates ER-to-Mitochondria Ca2+ Transport and Activates Cardiac Fibroblasts under Pulmonary Arterial Hypertension

Introduction: Similar to left ventricular (LV) fibrosis, growing evidence suggests a potential link between right ventricular (RV) fibrosis, poor function of the pressure-overloaded RV, and mortality in patients with pulmonary arterial hypertension (PAH). However, no therapies are currently available that specifically target RV fibrosis. Although the pathological roles of an oxidative stress-sensitive tyrosine kinase c-Src in LV hypertrophy and failure are well characterized, it remains unclear whether c-Src is involved in RV pathology under PAH. Using heterologous expression systems, we previously showed that mitofusin 2 (Mfn2), a key component of the tethering structures between the endoplasmic reticulum (ER) and mitochondria (Mito) is a novel c-Src substrate in mitochondria; and c-Src-dependent tyrosine phosphorylation (P-Tyr) of Mfn2 at the outer mitochondrial membrane (OMM) decreases the ER-Mito distance, possibly through conformational changes of Mfn2, which facilitates ER-to-Mito Ca2+ transfer and increases reactive oxygen species (ROS). Hypothesis: c-Src signaling alters the size of ER-Mito microdomains and enhances oxidative stress in the RV during PAH. Methods: Primary adult cardiac fibroblasts (CFs) from human ventricles and a preclinical rat PAH model induced by Su gen 5416 injection and 3-week h ypo x ia followed by 4-week normoxia (SuHx) were used for in vitro and in vivo assays, respectively. RV tissues from pulmonary hypertension (PH) patients and sex-/aged-matched controls were used for biochemical and cell biological assays. Results: c-Src activation occurred specifically in CFs in the RV, but not in RV myocytes in a rat SuHx PAH model and human PH-RVs as assessed by immunohistochemistry, which is notably different from the reports on LV hypertrophy and failure. P-Tyr of Mfn2 was detectable from human PH-RVs by Western Blots. RV-CFs from PAH rats exhibited increased c-Src activity, decreased ER-Mito distance, increased ROS, and proliferative signaling activation compared to RV-CFs from control rats as assessed by live cell imaging, immunocytochemistry, and transmission electron microscopy. All of these changes in rat CFs from PAH-RVs were recapitulated by overexpressing c-Src in human CFs in vitro. In addition, c-Src significantly increased the speed and amount of mitochondrial Ca2+ uptake without changing cytosolic Ca2+ dynamics in human CFs, suggesting a potential link between c-Src activation, the size of ER-Mito microdomains, and RV fibrosis under PAH. To further dissect the role of c-Src activity in CFs especially in the mitochondria, we specifically inhibited c-Src activity at the OMM, where Mfn2 is located, using an OMM-targeted dominant-negative c-Src (termed mt-c-Src-DN) that was generated by the addition of an OMM-targeting sequence (amino acid 1-33 from human TOM20) to the N-terminus of kinase-dead c-Src-K295R/Y527F. The introduction of mt-c-Src-DN increased the ER-Mito distance and reduced the speed of ER-to-Mito Ca2+ transport in human CFs. Conclusion: c-Src-dependent P-Tyr of Mfn2 alters the ER-Mito tethering structure, facilitates ER-to-Mito Ca2+ transport, and increases mitochondrial ROS generation, which promotes CF activation in PAH. Therefore, CF- and mitochondria-specific inhibition of c-Src may provide an additional therapeutic strategy to attenuate RV fibrosis and failure in response to PAH. NIH R01HL136757 (to J.O.-U.), R01HL160699 (to B.S.J.), P30GM1114750 (Targeted project to J.O.-U.), P30GM110759 (Targeted project to J.O.-U.), U54GM115677 (Targeted project to B.S.J.), and American Heart Association (AHA) 18CDA34110091 (to B.S.J.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Icariin regulates RANKL-induced osteoclast differentiation via the ERα/c-Src/RANK signaling

Osteoporosis (OP) is a common metabolic bone disease. Excessive osteoclastic activity significantly contributes to the development of OP. Icariin (ICA) is a flavonol glycoside derived from herbal plants and possesses curative effects on postmenopausal OP and bone fracture. This study aimed to investigate the effects of ICA on osteoclast differentiation induced by receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) and the involvement of estrogen receptor α (ERα) and RANK signaling cascade in this process. RANKL was used to induce the differentiation of RAW264.7 cells to into osteoclasts. Small interfering RNA technique was used to knockdown ERα in cells. Cell counting kit-8 assay was performed to determine the cytotoxicity of ICA. The number of tartrate-resistant acid phosphatase (TRAP)-positive cells was quantified by TRAP staining. RANKL induced the differentiation of RAW264.7 cells into osteoclasts, while ICA abolished the pro-osteoporotic effect of RANKL. Moreover, ERα knockdown abolished the effects of ICA on RANKL-induced osteoclastogenesis. Further exploration revealed that ICA inhibited the phosphorylation of c-Src in osteoclasts via regulating ERα, while inactivation of c-Src reversed ERα knockdown-promoted osteoclastogenesis. Lastly, ICA inhibited the activation of the mitogen-activated protein kinase signaling pathway and downregulated the expressions of target osteoclastogenic proteins in RANKL-treated RAW 264.7 cells, while ERα knockdown almost completely diminished the effects of ICA. ICA inhibited RANKL-induced osteoclast differentiation via regulating the ERα/c-Src/RANK signaling. These findings elucidated a novel mechanism by which ICA exerts an anti-osteoporotic effect.

Thrombin-Induced COX-2 Expression and PGE2 Synthesis in Human Tracheal Smooth Muscle Cells: Role of PKCδ/Pyk2-Dependent AP-1 Pathway Modulation.

In this study, we confirmed that thrombin significantly increases the production of COX-2 and PGE2 in human tracheal smooth muscle cells (HTSMCs), leading to inflammation in the airways and lungs. These molecules are well-known contributors to various inflammatory diseases. Here, we investigated in detail the involved signaling pathways using specific inhibitors and small interfering RNAs (siRNAs). Our results demonstrated that inhibitors targeting proteins such as protein kinase C (PKC)δ, proline-rich tyrosine kinase 2 (Pyk2), c-Src, epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), or activator protein-1 (AP-1) effectively reduced thrombin-induced COX-2 and PGE2 production. Additionally, transfection with siRNAs against PKCδ, Pyk2, c-Src, EGFR, protein kinase B (Akt), or c-Jun mitigated these responses. Furthermore, our observations revealed that thrombin stimulated the phosphorylation of key components of the signaling cascade, including PKCδ, Pyk2, c-Src, EGFR, Akt, and c-Jun. Thrombin activated COX-2 promoter activity through AP-1 activation, a process that was disrupted by a point-mutated AP-1 site within the COX-2 promoter. Finally, resveratrol (one of the most researched natural polyphenols) was found to effectively inhibit thrombin-induced COX-2 expression and PGE2 release in HTSMCs through blocking the activation of Pyk2, c-Src, EGFR, Akt, and c-Jun. In summary, our findings demonstrate that thrombin-induced COX-2 and PGE2 generation involves a PKCδ/Pyk2/c-Src/EGFR/PI3K/Akt-dependent AP-1 activation pathway. This study also suggests the potential use of resveratrol as an intervention for managing airway inflammation.

Magnetic photoactive Fe3O4@Sb2S3 nanospheres assisted homogeneous photoelectrochemical–electrochemical biosensing platform integrating with MOFs-derived bifunctional NPC@Thi nanopolyhedra-mediated dual “signal-on” strategy for protein tyrosine kinase activity

Abnormal protein tyrosine kinase (PTK) expression is associated with various diseases and thus it is of great significance in analysis of PTK activity via a reliable method. Herein, magnetic photoactive Fe3O4@Sb2S3 nanospheres and bifunctional thionine-embedded metal organic frameworks (MOFs)-derived nitrogen-doped porous carbon (NPC@Thi) nanopolyhedra were successfully synthesized for constructing a highly sensitive magnetic-assisted homogeneous photoelectrochemical (PEC)–electrochemical (EC) dual-mode biosensing platform for the detection of PTK (c-Src) activity. The Fe3O4@Sb2S3 nanospheres with good magnetic and photoactive properties assisted adequate biological recognition in a homogeneous system, simplified sample separation and facilitated sensor construction. After c-Src-mediated specific recognition, bifunctional NPC@Thi nanopolyhedra were connected to Fe3O4@Sb2S3 nanospheres. NPC@Thi not only enhanced the photocurrent of Fe3O4@Sb2S3 but also acted as the electrochemical probe. Thus, highly sensitive and reliable dual “signal-on” assay of c-Src activity with wide linear response ranges and low detection limits was achieved by the successful cooperation of PEC (10 fM to 10 nM, 7.2 fM) and EC (5 fM to 10 nM, 3.8 fM) biosensing strategies. The proposed homogeneous PEC–EC dual-mode biosensing platform is prospective to be extended to the analysis of other PTK activity and the discovery of PTK-targeted drug.

Abstract 133: Urine Derived Human Renal Proximal Tubule Cells Isolated From Inverse Salt Sensitive Participants Have Higher C-src Kinase Activity And Aminopeptidase N Expression

We have previously published that 11% of individuals in a salt sensitivity clinical trial are determined to be Inverse Salt Sensitive (ISS), and are associated with SNPs in the Dopamine D2 Receptor (D 2 R) that are shown to have decrease D 2 R expression in urine derived human renal proximal tubule cells (uRPTC) compared to salt resistant (SR) and salt sensitive (SS) individuals. We also previously have shown that ISS uRPTCs have increased expression of aminopeptidase N (APN), the enzyme capable of converting AngIII to AngIV. Inhibition of this activity in ISS may be a therapeutic strategy for ISS individuals. We previously reported that a nanobody designed to inhibit human APN activity added to cells reverses a number of adverse cell physiologic responses to low sodium conditions in ISS derived uRPTCs. Here we show evidence that the increased expression of APN in ISS uRPTCs is due to increased constitutive C-SRC activity. Three stable ISS uRPTCs, three SR uRPTCs and three SS uRPTCs were grown and lysed for analysis by Western blotting. The human homolog of the V-Src Avian Sarcoma viral oncogene tyrosine kinase (C-Src) activation state was measure using a Y-416 C-Src tyrosine phosphorylation specific antibody. There is a 266.1±21.7% increase in ISS Y416 p-Src vs SR (p<0.01 ISS vs SR, N=3) and no difference in SS vs SR. Plasma membrane localized APN was measured by In-cell Western and the increase in APN expression between ISS and SR was completely blocked by incubating cells for 4 hours with the C-Src inhibitor PP2 (ISS 49.1±8.8% increase over SR vs -14.6±11.9% with PP2, p<0.01, N=4 per group). Similarly lithium chloride (10 mM 4hrs), and D 2 R overexpression normalized the differential expression of D 2 R in ISS and completely blocked the increased APN expression found in ISS. In summary, low D 2 R expression in ISS leads to constitutively active C-Src and increased APN expression, and re-expression of D 2 R or inhibition of C-Src normalizes expression of APN. Future studies will determine if the tyrosine phosphorylation site in the intracellular domain of APN is a direct target of C-Src.

EGFR-Induced and c-Src-Mediated CD47 Phosphorylation Inhibits TRIM21-Dependent Polyubiquitylation and Degradation of CD47 to Promote Tumor Immune Evasion.

Tumor cells often overexpress immune checkpoint proteins, including CD47, for immune evasion. However, whether or how oncogenic activation of receptor tyrosine kinases, which are crucial drivers in tumor development, regulates CD47 expression is unknown. Here, it is demonstrated that epidermal growth factor receptor (EGFR) activation induces CD47 expression by increasing the binding of c-Src to CD47, leading to c-Src-mediated CD47 Y288 phosphorylation. This phosphorylation inhibits the interaction between the ubiquitin E3 ligase TRIM21 and CD47, thereby abrogating TRIM21-mediated CD47 K99/102 polyubiquitylation and CD47 degradation. Knock-in expression of CD47 Y288F reduces CD47 expression, increases macrophage phagocytosis of tumor cells, and inhibits brain tumor growth in mice. In contrast, knock-in expression of CD47 K99/102R elicits the opposite effects compared to CD47 Y288F expression. Importantly, CD47-SIRPα blockade with an anti-CD47 antibody treatment significantly enhances EGFR-targeted cancer therapy. In addition, CD47 expression levels in human glioblastoma (GBM) specimens correlate with EGFR and c-Src activation and aggravation of human GBM. These findings elucidate a novel mechanism underlying CD47 upregulation in EGFR-activated tumor cells and underscore the role of the EGFR-c-Src-TRIM21-CD47 signaling axis in tumor evasion and the potential to improve the current cancer therapy with a combination of CD47 blockade with EGFR-targeted remedy.

The checkpoint inhibitor PD-1H/VISTA controls osteoclast-mediated multiple myeloma bone disease

Multiple myeloma bone disease is characterized by the development of osteolytic bone lesions. Recent work identified matrix metalloproteinase 13 as a myeloma-derived fusogen that induces osteoclast activation independent of its proteolytic activity. We now identify programmed death-1 homolog, PD-1H, as the bona fide MMP-13 receptor on osteoclasts. Silencing PD-1H or using Pd-1h-/- bone marrow cells abrogates the MMP-13-enhanced osteoclast fusion and bone-resorptive activity. Further, PD-1H interacts with the actin cytoskeleton and plays a necessary role in supporting c-Src activation and sealing zone formation. The critical role of PD-1H in myeloma lytic bone lesions was confirmed using a Pd-1h-/- myeloma bone disease mouse model wherein myeloma cells injected into Pd-1h-/-Rag2-/- results in attenuated bone destruction. Our findings identify a role of PD-1H in bone biology independent of its known immunoregulatory functions and suggest that targeting the MMP-13/PD-1H axis may represent a potential approach for the treatment of myeloma associated osteolysis.

Small artery remodeling and stiffening in deoxycorticosterone acetate-salt hypertensive rats involves the interaction between endogenous ouabain/Na + K + -ATPase/cSrc signaling.

Endogenous ouabain (EO) increases in some patients with hypertension and in rats with volume-dependent hypertension. When ouabain binds to Na + K + -ATPase, cSrc is activated, which leads to multieffector signaling activation and high blood pressure (BP). In mesenteric resistance arteries (MRA) from deoxycorticosterone acetate (DOCA)-salt rats, we have demonstrated that the EO antagonist rostafuroxin blocks downstream cSrc activation, enhancing endothelial function and lowering oxidative stress and BP. Here, we examined the possibility that EO is involved in the structural and mechanical alterations that occur in MRA from DOCA-salt rats. MRA were taken from control, vehicle-treated DOCA-salt or rostafuroxin (1 mg/kg per day, for 3 weeks)-treated DOCA-salt rats. Pressure myography and histology were used to evaluate the mechanics and structure of the MRA, and western blotting to assess protein expression. DOCA-salt MRA exhibited signs of inward hypertrophic remodeling and increased stiffness, with a higher wall:lumen ratio, which were reduced by rostafuroxin treatment. The enhanced type I collagen, TGFβ1, pSmad2/3 Ser465/457 /Smad2/3 ratio, CTGF, p-Src Tyr418 , EGFR, c-Raf, ERK1/2 and p38MAPK protein expression in DOCA-salt MRA were all recovered by rostafuroxin. A process combining Na + K + -ATPase/cSrc/EGFR/Raf/ERK1/2/p38MAPK activation and a Na + K + -ATPase/cSrc/TGF-1/Smad2/3/CTGF-dependent mechanism explains how EO contributes to small artery inward hypertrophic remodeling and stiffening in DOCA-salt rats. This result supports the significance of EO as a key mediator for end-organ damage in volume-dependent hypertension and the efficacy of rostafuroxin in avoiding remodeling and stiffening of small arteries.

Repurposing of rilpivirine for preventing platelet β3 integrin-dependent thrombosis by targeting c-Src active autophosphorylation

BackgroundHIV-infected individuals are known to be at higher risk for thrombotic cardiovascular disease (CVD), which may also be differentially affected by components of anti-HIV drugs. To identify the effects of a series of FDA-approved anti-HIV drugs on platelet aggregation in humans, focusing on the novel pharmacological effects of rilpivirine (RPV), a reverse transcriptase inhibitor, on platelet function both in vitro and in vivo and the mechanisms involved. Methods and resultsIn vitro studies showed that RPV was the only anti-HIV reagent that consistently and efficiently inhibited aggregation elicited by different agonists, exocytosis, morphological extension on fibrinogen, and clot retraction. Treatment of mice with RPV significantly prevented thrombus formation in FeCl3-injured mesenteric vessels, postcava with stenosis surgery, and ADP -induced pulmonary embolism models without defects in platelet viability, tail bleeding, and coagulation activities. RPV also improved cardiac performance in mice with post-ischemic reperfusion. A mechanistic study revealed that RPV preferentially attenuated fibrinogen-stimulated Tyr773 phosphorylation of β3-integrin by inhibiting Tyr419 autophosphorylation of c-Src. Molecular docking and surface plasmon resonance analyses showed that RPV can bind directly to c-Src. Further mutational analysis showed that the Phe427 residue of c-Src is critical for RPV interaction, suggesting a novel interaction site for targeting c-Src to block β3-integrin outside-in signaling. ConclusionThese results demonstrated that RPV was able to prevent the progression of thrombotic CVDs by interrupting β3-integrin-mediated outside-in signaling via inhibiting c-Src activation without hemorrhagic side effects, highlighting RPV as a promising reagent for the prevention and therapy of thrombotic CVDs.

Tyrphostin A9 attenuates glioblastoma growth by suppressing PYK2/EGFR-ERK signaling pathway.

Glioblastoma (GBM) is a fatal primary brain tumor with extremely poor clinical outcomes. The anticancer efficiency of tyrosine kinase inhibitors (TKIs) has been shown in GBM and other cancer, with limited therapeutic outcomes. In the current study, we aimed to investigate the clinical impact of active proline-rich tyrosine kinase-2 (PYK2) and epidermal growth factor receptor (EGFR) in GBM and evaluate its druggability by a synthetic TKI-Tyrphostin A9 (TYR A9). The expression profile of PYK2 and EGFR in astrocytoma biopsies (n = 48) and GBM cell lines were evaluated through quantitative PCR, western blots, and immunohistochemistry. The clinical association of phospho-PYK2 and EGFR was analyzed with various clinicopathological features and the Kaplan-Meier survival curve. The phospho-PYK2 and EGFR druggability and subsequent anticancer efficacy of TYR A9 was evaluated in GBM cell lines and intracranial C6 glioma model. Our expression data revealed an increased phospho-PYK2, and EGFR expression aggravates astrocytoma malignancy and is associated with patients' poor survival. The mRNA and protein correlation analysis showed a positive association between phospho-PYK2 and EGFR in GBM tissues. The in-vitro studies demonstrated that TYR A9 reduced GBM cell growth, cell migration, and induced apoptosis by attenuating PYK2/EGFR-ERK signaling. The in-vivo data showed TYR A9 treatment dramatically reduced glioma growth with augmented animal survival by repressing PYK2/EGFR-ERK signaling. Altogether, this study report that increased phospho-PYK2 and EGFR expression in astrocytoma was associated with poor prognosis. The in-vitro and in-vivo evidence underlined translational implication of TYR A9 by suppressing PYK2/EGFR-ERK modulated signaling pathway. The schematic diagram displayed proof of concept of the current study indicating activated PYK2 either through the Ca2+/Calmodulin-dependent protein kinase II (CAMKII) signaling pathway or autophosphorylation at Tyr402 induces association to the SH2 domain of c-Src that leads to c-Src activation. Activated c-Src in turn activates PYK2 at other tyrosine residues that recruit Grb2/SOS complex and trigger ERK½ activation. Besides, PYK2 interaction with c-Src acts as an upstream of EGFR transactivator that can activate the ERK½ signaling pathway, which induces cell proliferation and cell survival by increasing anti-apoptotic proteins or inhibiting pro-apoptotic proteins. TYR A9 treatment attenuate GBM cell proliferation and migration; and induce GBM cell death by inhibiting PYK2 and EGFR-induced ERK activation.

The c-Src/LIST Positive Feedback Loop Sustains Tumor Progression and Chemoresistance.

Chemotherapy resistance and treatment failure hinder clinical cancer treatment. Src, the first mammalian proto-oncogene to be discovered, is a valuable anti-cancer therapeutic target. Although several c-Src inhibitors have reached the clinical stage, drug resistance remains a challenge during treatment. Herein, a positive feedback loop between a previously uncharacterized long non-coding RNA (lncRNA), which the authors renamed lncRNA-inducing c-Src tumor-promoting function (LIST), and c-Src is uncovered. LIST directly binds to and regulates the Y530 phosphorylation activity of c-Src. As a c-Src agonist, LIST promotes tumor chemoresistance and progression in vitro and in vivo in multiple cancer types. c-Src can positively regulate LIST transcription by activating the NF-κB signaling pathway and then recruiting the P65 transcription factor to the LIST promoter. Interestingly, the LIST/c-Src interaction is associated with evolutionary new variations of c-Src. It is proposed that the human-specific LIST/c-Src axis renders an extra layer of control over c-Src activity. Additionally, the LIST/c-Src axis is of high physiological relevance in cancer and may be a valuable prognostic biomarker and potential therapeutic target.

Neurite outgrowth induced by stimulation of angiotensin II AT2 receptors in SH-SY5Y neuroblastoma cells involves c-Src activation

Neuroblastoma, the most common extracranial solid tumor occurring in childhood, originates from the aberrant proliferation of neural crest cells. Accordingly, the mechanism underling neuronal differentiation could provide new strategies for neuroblastoma treatment. It is well known that neurite outgrowth could be induced by Angiotensin II (Ang II) AT2 receptors; however, the signaling mechanism and its possible interaction with NGF (neural growth factor) receptors remain unclear. Here, we show that Ang II and CGP42112A (AT2 receptor agonist) promote neuronal differentiation by inducing neurite outgrowth and βIII-tubulin expression in SH-SY5Y neuroblastoma cells. In addition, we demonstrate that treatment with PD123319 (AT2 receptor antagonist) reverts Ang II or CGP42112A-induced differentiation. By using specific pharmacological inhibitors we established that neurite outgrowth induced by CGP42112A requires the activation of MEK (mitogen-activated protein kinase kinase), SphK (sphingosine kinase) and c-Src but not PI3K (phosphatidylinositol 3-kinase). Certainly, CGP42112A stimulated a rapid and transient (30 s, 1 min) phosphorylation of c-Src at residue Y416 (indicative of activation), following by a Src deactivation as indicated by phosphorylation of Y527. Moreover, inhibition of the NGF receptor tyrosine kinase A (TrkA) reduced neurite outgrowth induced by Ang II and CGP42112A. In summary, we demonstrated that AT2 receptor-stimulated neurite outgrowth in SH-SY5Y cells involves the induction of MEK, SphK and c-Src and suggests a possible transactivation of TrkA. In that regard, AT2 signaling pathway is a key player in neuronal differentiation and might be a potential target for therapeutic treatments.

Progesterone and its metabolite allopregnanolone promote invasion of human glioblastoma cells through metalloproteinase‑9 and cSrc kinase.

Glioblastomas are the most aggressive and common primary brain tumors in adults. Glioblastoma cells have a great capacity to migrate and invade the brain parenchyma, often reaching the contralateral hemisphere. Progesterone (P4) and its metabolite, allopregnanolone (3α-THP), promote the migration and invasion of human glioblastoma-derived cells. P4 induces migration in glioblastoma cells by the activation of the proto-oncogene tyrosine-protein kinase Src (cSrc) and focal adhesion kinase (Fak). In breast cancer cells, cSrc and Fak promote invasion by increasing the expression and activation of extracellular matrix metalloproteinases (MMPs). However, the mechanism of action by which P4 and 3a-THP promote invasion in glioblastoma cells remains unclear. The effects of P4 and 3α-THP on the protein expression levels of MMP-2 and -9 and the participation of cSrc in progestin effects in U251 and U87 human glioblastoma-derived cells were evaluated. It was determined by western blotting that the P4 increased the protein expression level of MMP-9 in U251 and U87 cells, and 3α-THP increased the protein expression level of MMP-9 in U87 cells. None of these progestins modified MMP-2 protein expression levels. The increase in MMP-9 expression was reduced when the intracellular progesterone receptor and cSrc expression were blocked with small interfering RNAs. Cell invasion induced by P4 and 3α-THP was also blocked by inhibiting cSrc activity with PP2 or by cSrc gene silencing. These results suggest that P4 and its metabolite 3α-THP induce the invasion of glioblastoma cells by increasing MMP-9 expression through the cSrc kinase family. The results of this study provide information of interest in the context of targeted therapies against molecular pathways involved in glioblastoma invasion.

Coordinated activation of c-Src and FOXM1 drives tumor cell proliferation and breast cancer progression.

Activation of the tyrosine kinase c-Src promotes breast cancer progression and poor outcomes, yet the underlying mechanisms are incompletely understood. Here, we have shown that deletion of c-Src in a genetically engineered model mimicking the luminal B molecular subtype of breast cancer abrogated the activity of forkhead box M1 (FOXM1), a master transcriptional regulator of the cell cycle. We determined that c-Src phosphorylated FOXM1 on 2 tyrosine residues to stimulate its nuclear localization and target gene expression. These included key regulators of G2/M cell-cycle progression as well as c-Src itself, forming a positive feedback loop that drove proliferation in genetically engineered and patient-derived models of luminal B-like breast cancer. Using genetic approaches and small molecules that destabilize the FOXM1 protein, we found that targeting this mechanism induced G2/M cell-cycle arrest and apoptosis, blocked tumor progression, and impaired metastasis. We identified a positive correlation between FOXM1 and c-Src expression in human breast cancer and show that the expression of FOXM1 target genes predicts poor outcomes and associates with the luminal B subtype, which responds poorly to currently approved therapies. These findings revealed a regulatory network centered on c-Src and FOXM1 that is a targetable vulnerability in aggressive luminal breast cancers.

Role of c-Src and reactive oxygen species in cardiovascular diseases.

Oxidative stress, caused by the over production of oxidants or inactivity of antioxidants, can modulate the redox state of several target proteins such as tyrosine kinases, mitogen-activated protein kinases and tyrosine phosphatases. c-Src is one such non-receptor tyrosine kinase which activates NADPH oxidases (Noxs) in response to various growth factors and shear stress. Interaction between c-Src and Noxs is influenced by cell type and primary messengers such as angiotensin II, which binds to G-protein coupled receptor and activates the intracellular signaling cascade. c-Src stimulated activation of Noxs results in elevated release of intracellular and extracellular reactive oxygen species (ROS). These ROS species disturb vascular homeostasis and cause cardiac hypertrophy, coronary artery disease, atherosclerosis and hypertension. Interaction between c-Src and ROS in the pathobiology of cardiac fibrosis is hypothesized to be influenced by cell type and stimuli. c-Src and ROS have a bidirectional relationship, thus increased ROS levels due to c-Src mediated activation of Noxs can further activate c-Src by promoting the oxidation and sulfenylation of critical cysteine residues. This review highlights the role of c-Src and ROS in mediating downstream signaling pathways underlying cardiovascular diseases. Furthermore, due to the central role of c-Src in activation of various signaling proteins involved in differentiation, migration, proliferation, and cytoskeletal reorganization of vascular cells, it is presented as therapeutic target for treating cardiovascular diseases except cardiac fibrosis.

SHP-1 tyrosine phosphatase binding to c-Src kinase phosphor-dependent conformations: A comparative structural framework.

SHP-1 is a cytosolic tyrosine phosphatase that is primarily expressed in hematopoietic cells. It acts as a negative regulator of numerous signaling pathways and controls multiple cellular functions involved in cancer pathogenesis. This study describes the binding preferences of SHP-1 (pY536) to c-Srcopen (pY416) and c-Srcclose (pY527) through in silico approaches. Molecular dynamics simulation analysis revealed more conformational changes in c-Srcclose upon binding to SHP-1, as compared to its active/open conformation that is stabilized by the cooperative binding of the C-SH2 domain and C-terminal tail of SHP-1 to c-Src SH2 and KD. In contrast, c-Srcclose and SHP-1 interaction is mediated by PTP domain-specific WPD-loop (WPDXGXP) and Q-loop (QTXXQYXF) binding to c-Srcclose C-terminal tail residues. The dynamic correlation analysis demonstrated a positive correlation for SHP-1 PTP with KD, SH3, and the C-terminal tail of c-Srcclose. In the case of the c-Srcopen-SHP-1 complex, SH3 and SH2 domains of c-Srcopen were correlated to C-SH2 and the C-terminal tail of SHP-1. Our findings reveal that SHP1-dependent c-Src activation through dephosphorylation relies on the conformational shift in the inhibitory C-terminal tail that may ease the recruitment of the N-SH2 domain to phosphotyrosine residue, resulting in the relieving of the PTP domain. Collectively, this study delineates the intermolecular interaction paradigm and underlying conformational readjustments in SHP-1 due to binding with the c-Src active and inactive state. This study will largely help in devising novel therapeutic strategies for targeting cancer development.

Estradiol-Induced Epithelial to Mesenchymal Transition and Migration Are Inhibited by Blocking c-Src Kinase in Breast Cancer Cell Lines.

The epithelial-to-mesenchymal transition (EMT) is the main event that favors cell migration and metastasis in breast cancer. Previously, we demonstrated that 1 nM estradiol (E2) promotes EMT, induced by c-Src kinase, causing changes in the localization of proteins that compose the tight junction (TJ) and adherens junction (AJ). The present work highlights the central role of c-Src in the initiation of metastasis, induced by E2, through increasing the ability of MCF-7 and T47-D cells, which express estrogen receptor alpha (ERα), to migrate and invade before they become metastatic. Treatment with E2 can activate two signaling pathways, the first one by the phosphorylated c-Src (p-Src) which forms the p-Src/E-cadherin complex. This phenomenon was completely prevented by incubation with a selective inhibitor of c-Src (5 µM PP2). p-Src then promotes the downregulation of E-cadherin and occludin, which are epithelial phenotype marker proteins of the AJ and TJ, respectively. In the second pathway, E2 binds to ERα, creating a complex that translocates to the nucleus, inducing the synthesis of SNAIL1 and N-cadherin proteins, markers of the mesenchymal phenotype. Both processes increased the migratory and invasive capacities of both cell lines. The present study demonstrate that E2 enhance EMT and migration, through c-Src activation, in human breast cancer cells that express ERα and become potential therapeutic targets.