c-Src Inhibitor Research Articles

Decreased E‐Cadherin in MCF7 Human Breast Cancer Cells Forming Multicellular Spheroids Exposed to Simulated Microgravity

MCF7 human breast cancer cells were cultured under normal gravity (1 g) and on a random positioning machine (RPM) preventing sedimentation. After 2 weeks, adherent 1 g-control and adherent RPM cells (AD) as well as multicellular spheroids (MCS) were harvested. AD and MCS had been exposed to the RPM in the same culture flask. In a subsequent proteome analysis, the majority of the proteins detected showed similar label-free quantification (LFQ) scores in each of the respective subpopulations, but in both AD or MCS cultures, proteins were also found whose LFQs deviated at least twofold from their counterparts in the 1 g-control cells. They included the cell junction protein E-cadherin, which was diminished in MCS cells, where proteins of the E-cadherin autodegradation pathway were enhanced and c-Src (proto-oncogene tyrosine-protein kinase c-Src) was detected. Spheroid formation was prevented by inhibition of c-Src but promoted by antibodies blocking E-cadherin activity. An interaction analysis of the detected proteins that are involved in forming and regulating junctions or adhesion complexes and in E-cadherin autodegradation indicated connections between the two protein groups. This suggests that the balance of proteins that up- or downregulate E-cadherin mediates the tendency of MCF7 cells to form MCS during RPM exposure.

PO-438 Internalisation of cell-penetrating peptides based on CONNEXIN43 in glioma stem cells and neural cells

Introduction Connexin43 (Cx43), the main gap junction channel-forming protein in astrocytes, is downregulated in malignant gliomas. These tumours are composed of a heterogeneous population of cells that include many with stem cell-like properties, called glioma stem cells (GSCs). Interestingly, restoring Cx43 in these cells reverses GSC phenotype and reduces their tumorigenicity. This effect is based on the interaction between the carboxy tail of Cx43 and the tyrosine kinase c-Src. A cell-penetrating peptide (CPP) containing residues 266–283 of Cx43 (TAT-Cx43266–283) is able to recruit c-Src inhibitors CSK and PTEN and to inhibit c-Src activity. The aim of this work was to study the internalisation of this CPP into GSCs and other cells of the brain parenchyma. Material and methods Glioma cell lines and primary rat neurons and astrocytes were expanded in adherent culture and incubated with a CPP containing amino acids 266–283 of Cx43 fused to TAT at the N-terminus and to biotin at the C terminus (TAT-Cx43266–283-B) for 5 min at 37°C. The internalisation of the peptide was studied by confocal microscopy. GSCs were microinjected in organotypic brain slice cultures, allowed to engraft into the tissue and incubated with TAT-Cx43266–283-B to study the uptake of the peptide in GSCs exposed to brain tissue interactions. The internalisation of the peptide by the surrounding cells was examined by immunofluorescence. Results and discussions We observed a higher uptake of TAT-Cx43266–283-B in glioma cells and GSCs compared to astrocytes, especially at low concentrations of the peptide (6 µM). We did not find TAT-Cx43266–283-B internalisation by neurons under these experimental conditions. We noted a specific localization of TAT-Cx43266–283-B at the plasma membrane that was not shown by CPPs containing other regions of Cx43. Colocalization studies revealed TAT-Cx43266–283-B colocalization with c-Src, which is myristoilated in its active form, near the plasma membrane. We explored the uptake of TAT-Cx43266–283-B in organotypic slice cultures and found high levels of TAT-Cx43266–283-B internalisation by GSCs engrafted in the tissue. Interestingly, TAT-Cx43266–283-B was also found within the cells of the tumour microenvironment. Conclusion The specific subcellular localization of TAT-Cx43266–283-B at the plasma membrane helps to understand the mechanism by which TAT-Cx43266–283 inhibits c-Src and its consequent antitumorigenic effect. Additionally, the efficient internalisation of the peptide in GSCs increases its therapeutic possibilities.

C-Cbl reverses HER2-mediated tamoxifen resistance in human breast cancer cells

BackgroundTamoxifen is a frontline therapy for estrogen receptor (ER)-positive breast cancer in premenopausal women. However, many patients develop resistance to tamoxifen, and the mechanism underlying tamoxifen resistance is not well understood. Here we examined whether ER-c-Src-HER2 complex formation is involved in tamoxifen resistance.MethodsMTT and colony formation assays were used to measure cell viability and proliferation. Western blot was used to detect protein expression and protein complex formations were detected by immunoprecipitation and immunofluorescence. SiRNA was used to examine the function of HER2 in of BT474 cells. An in vivo xenograft animal model was established to examine the role of c-Cbl in tumor growth.ResultsMTT and colony formation assay showed that BT474 cells are resistant to tamoxifen and T47D cells are sensitive to tamoxifen. Immunoprecipitation experiments revealed ER-c-Src-HER2 complex formation in BT474 cells but not in T47D cells. However, ER-c-Src-HER2 complex formation was detected after overexpressing HER2 in T47D cells and these cells were more resistant to tamoxifen. HER2 knockdown by siRNA in BT474 cells reduced ER-c-Src-HER2 complex formation and reversed tamoxifen resistance. ER-c-Src-HER2 complex formation was also disrupted and tamoxifen resistance was reversed in BT474 cells by the c-Src inhibitor PP2 and HER2 antibody trastuzumab. Nystatin, a lipid raft inhibitor, reduced ER-c-Src-HER2 complex formation and partially reversed tamoxifen resistance. ER-c-Src-HER2 complex formation was disrupted by overexpression of c-Cbl but not by the c-Cbl ubiquitin ligase mutant. In addition, c-Cbl could reverse tamoxifen resistance in BT474 cells, but the ubiquitin ligase mutant had no effect. The effect of c-Cbl was validated in BT474 tumor-bearing nude mice in vivo. Immunofluorescence also revealed ER-c-Src-HER2 complex formation was reduced in tumor tissues of nude mice with c-Cbl overexpression.ConclusionsOur results suggested that c-Cbl can reverse tamoxifen resistance in HER2-overexpressing breast cancer cells by inhibiting the formation of the ER-c-Src-HER2 complex.

Acid Stimulation of the Citrate Transporter NaDC-1 Requires Pyk2 and ERK1/2 Signaling Pathways.

Background Urine citrate is reabsorbed exclusively along the renal proximal tubule via the apical Na+-dicarboxylate cotransporter NaDC-1. We previously showed that an acid load in vivo and media acidification in vitro increase NaDC-1 activity through endothelin-1 (ET-1)/endothelin B (ETB) signaling. Here, we further examined the signaling pathway mediating acid-induced NaDC-1 activity.Methods We transiently transfected cultured opossum kidney cells, a model of the proximal tubule, with NaDC-1 and ETB and measured [14C]-citrate uptake after media acidification under various experimental conditions, including inactivation of Pyk2 and c-Src, which were previously shown to be activated by media acidification. Wild-type (Pyk2+/+) and Pyk2-null (Pyk2-/-) mice were exposed to NH4Cl loading and euthanized after various end points, at which time we harvested the kidneys for immunoblotting and brush border membrane NaDC-1 activity studies.Results Inhibition of Pyk2 or c-Src prevented acid stimulation but not ET-1 stimulation of NaDC-1 in vitro Consistent with these results, NH4Cl loading stimulated NaDC-1 activity in kidneys of wild-type but not Pyk2-/- mice. In cultured cells and in mice, ERK1/2 was rapidly phosphorylated by acid loading, even after Pyk2 knockdown, and it was required for acid but not ET-1/ETB stimulation of NaDC-1 in vitro Media acidification also induced the phosphorylation of Raf1 and p90RSK, components of the ERK1/2 pathway, and inhibition of these proteins blocked acid stimulation of NaDC-1 activity.Conclusions Acid stimulation of NaDC-1 activity involves Pyk2/c-Src and Raf1-ERK1/2-p90RSK signaling pathways, but these pathways are not downstream of ET-1/ETB in this process.

C-Src inhibitor selectively inhibits triple-negative breast cancer overexpressed Vimentin invitro and invivo.

Oncogene c‐Src has been found to be a potential target for the treatment of triple‐negative breast cancer (TNBC). However, the therapeutic effects of the c‐Src inhibitor on TNBC patients are controversial compared to those on cell lines. The molecular mechanisms of the inhibitory effects of the c‐Src inhibitor on TNBC remain unclear. Herein, we showed that a specific c‐Src inhibitor, PP2, was effective in inhibiting phosphorylation of c‐Src in 4 cell lines: T‐47D, SK‐BR‐3, SUM1315MO2, and MDA‐MB‐231, regardless of hormone receptors and human epidermal growth factor receptor 2 (HER2) expression levels. Giving PP2 preferentially reduced the S phase of cell cycles and inhibited colony formation in SUM1315MO2 and MDA‐MB‐231, but not in SK‐BR‐3 and T‐47D cells. Furthermore, PP2 effectively blocked cell migration/invasion and epithelial‐mesenchymal transition (EMT) in TNBC cell lines, SUM1315MO2 and MDA‐MB‐231. An EMT biomarker, vimentin, was highly expressed in 2 TNBC cell lines when they were compared with SK‐BR‐3 and T‐47D cells. Further depletion of vimentin by shRNA remarkably attenuated the inhibitory effects of the c‐Src inhibitor on TNBC cells in vitro and in vivo, indicating a crucial action of vimentin to affect the function of c‐Src in TNBC. This study provides an important rationale for the clinic to precisely select TNBC patients who would benefit from c‐Src inhibitor treatment. This finding suggests that traditional markers for TNBC are not sufficient to precisely define this aggressive type of cancer. Vimentin is identified as an important biomarker to enable categorization of TNBC.

Vascular endothelial growth factor C promotes cervical cancer cell invasiveness via regulation of microRNA-326/cortactin expression

Vascular endothelial growth factor C (VEGF-C) accelerates cervical cancer metastasis, while the detailed mechanism remains largely unknown. Recent evidence indicates that microRNA play a crucial role in controlling cancer cell invasiveness. In the present study, we investigated the role of miR-326 in VEGF-C-induced cervical cancer cell invasion. VEGF-C expression was higher and miR-326 was much lower in primary cervical cancer specimens than that in non-cancerous specimens, and a negative correlation between VEGF-C and miR-326 was found. On cervical carcinoma cell line SiHa cells, treatment with VEGF-C downregulated miR-326 level and increased cortactin protein expression. Transfection with miR-326 mimic reversed cortactin expression induced by VEGF-C, suggesting that VEGF-C increased cortactin via downregulation of miR-326. VEGF-C activated c-Src and c-Src inhibitor PP2 abolished VEGF-C effect on miR-326 and cortactin expression, implying that VEGF-C regulated miR-326/cortactin via c-Src signaling. VEGF-C promoted SiHa cell invasion index, which was largely inhibited by transfection with miR-326 antagonist or by siRNA against cortactin. In conclusion, our findings implied that VEGF-C reduced miR-326 expression and increased cortactin expression through c-Src signaling, leading to enhanced cervical cancer invasiveness. This may shed light on potential therapeutic strategies for cervical cancer therapy.

C-Src-Dependent and -Independent Functions of Matk in Osteoclasts and Osteoblasts.

The non-receptor tyrosine kinase c-Src participates in bone metabolism by regulating the activities of both the bone-resorbing osteoclasts and bone-forming osteoblasts. In this study, we investigated whether megakaryocyte-associated tyrosine kinase (Matk), a potent inhibitor of c-Src, affects the functions of murine osteoclasts and osteoblasts. Results revealed that the formation of osteoclasts with actin rings was attenuated by Matk overexpression in osteoclast precursor cells but was enhanced by Matk knockdown. The inhibitory effect of Matk on osteoclasts was closely related with the inhibition of c-Src activity. Intriguingly, Matk overexpression in osteoblasts reduced bone nodule formation. Conversely, Matk knockdown increased osteoblast function. Most importantly, binding of Matk to Runx2 resulted in the inhibition of Runx2 translocation into the nucleus and downregulation of Runx2 target genes. Taken together, our findings demonstrated that Matk plays a critical role in bone metabolism by impairing the functions of osteoclasts and osteoblasts via distinct mechanisms involving inhibition of c-Src-dependent and -independent signaling pathways.

C‐Src mediates intestinal stem cell response to high fat diet

c‐Src is a commonly upregulated oncogene in colorectal cancer. High fat diet (HFD) augments the numbers and function of Lgr5+ intestinal stem cells (ISCs), increasing the capacity of these stems cells to initiate tumors. Discoveries in triple negative breast cancer models and patient specimens suggest that mitochondrial reprogramming to fatty acid oxidation (FAO) activates c‐Src. Here, we determined the role of FAO induced by HFD on c‐Src activation and ISC self‐renewal. Lgr5‐EGFP‐IRES‐creERT2 (Lrg5‐EGFP) knock‐in mice were fed a dietary chow consisting of 45% kcal fat or a low fat matching diet ad libitum from 8 to 20 weeks of age. Beginning at age 10 weeks, mice were i.p. injected with 20 mg/kg Etomoxir (ETX), an inhibitor of FAO, twice per week. Intestinal enteroids derived from Lgr5‐EGFP mice were treated with constituents of HFD (30 μM palmitic acid and 30 μM oleic acid) every 3 days for 2 weeks ± 20 μM PP2, a Src inhibitor. Numbers of intestinal Lgr5‐GFP+ cells were quantitated and isolated by flow cytometry and expression of c‐Src target gene Cyclin D1 and ISC self‐renewal gene Olfm4 were measured by qPCR. c‐Src activation was measured in human colon cancer cell lines HCT‐116 and Caco2 treated with 200 μM ETX or 10 mM L‐carnitine (FAO activator) for 16 hr. HFD increased the number of Lgr5+ ISCs and expression of Cyclin D1 and Olfm4, which were prevented by ETX. PP2 inhibition of c‐Src in enteroids prevented the palmitic acid/oleic acid‐induced increase in Lgr5+ ISC numbers. ETX decreased and L‐carnitine induced c‐Src activity in HCT‐116 and Caco2 cells. Our data provide novel evidence that the Src pathway plays an important role in ISC response to constituents of HFD.Support or Funding InformationCollaborative Faculty Research Investment Program (CFRIP), Baylor Scott & White Health and Baylor College of MedicineThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Smooth muscle Ca2+ sensitization causes hypercontractility of middle cerebral arteries in mice bearing the familial hemiplegic migraine type 2 associated mutation

Familial hemiplegic migraine type 2 (FHM2) is associated with inherited point-mutations in the Na,K-ATPase α2 isoform, including G301R mutation. We hypothesized that this mutation affects specific aspects of vascular function, and thus compared cerebral and systemic arteries from heterozygote mice bearing the G301R mutation (Atp1a2+/−G301R) with wild type (WT). Middle cerebral (MCA) and mesenteric small artery (MSA) function was compared in an isometric myograph. Cerebral blood flow was assessed with Laser speckle analysis. Intracellular Ca2+ and membrane potential were measured simultaneously. Protein expression was semi-quantified by immunohistochemistry. Protein phosphorylation was analysed by Western blot. MSA from Atp1a2+/−G301R and WT showed similar contractile responses. The Atp1a2+/−G301R MCA constricted stronger to U46619, endothelin and potassium compared to WT. This was associated with an increased depolarization, although the Ca2+ change was smaller than in WT. The enhanced constriction of Atp1a2+/−G301R MCA was associated with increased cSrc activation, stronger sensitization to [Ca2+]i and increased MYPT1 phosphorylation. These differences were abolished by cSrc inhibition. Atp1a2+/−G301R mice had reduced resting blood flow through MCA in comparison with WT mice. FHM2-associated mutation leads to elevated contractility of MCA due to sensitization of the contractile machinery to Ca2+, which is mediated via Na,K-ATPase/Src-kinase/MYPT1 signalling.

SMAD4-independent activation of TGF-β signaling by MUC1 in a human pancreatic cancer cell line.

Pancreatic Ductal Adenocarcinoma (PDA) has a mortality rate that nearly matches its incidence rate. Transforming Growth Factor Beta (TGF-β) is a cytokine with a dual role in tumor development switching from a tumor suppressor to a tumor promoter. There is limited knowledge of how TGF-β function switches during tumorigenesis. Mucin 1 (MUC1) is an aberrantly glycosylated, membrane-bound, glycoprotein that is overexpressed in >80% of PDA cases and is associated with poor prognosis. In PDA, MUC1 promotes tumor progression and metastasis via signaling through its cytoplasmic tail (MUC1-CT) and interacting with other oncogenic signaling molecules. We hypothesize that high levels of MUC1 in PDA may be partly responsible for the TGF-β functional switch during oncogenesis. We report that overexpression of MUC1 in BxPC3 human PDA cells (BxPC3.MUC1) enhances the induction of epithelial to mesenchymal transition leading to increased invasiveness in response to exogenous TGF-β1. Simultaneously, these cells resist TGF-β induced apoptosis by downregulating levels of cleaved caspases. We show that mutating the tyrosines in MUC1-CT to phenylalanine reverses the TGF-β induced invasiveness. This suggests that the tyrosine residues in MUC1-CT are required for TGF-β induced invasion. Some of these tyrosines are phosphorylated by the tyrosine kinase c-Src. Thus, treatment of BxPC3.MUC1 cells with a c-Src inhibitor (PP2) significantly reduces TGF-β induced invasiveness. Similar observations were confirmed in the Chinese hamster ovarian (CHO) cell line. Data strongly suggests that MUC1 may regulate TGF-β function in PDA cells and thus have potential clinical relevance in the use of TGF-β inhibitors in clinical trials.

Heat-Induced Epithelial Barrier Dysfunction Occurs via C-Src Kinase and P120ctn Expression Regulation in the Lungs

Background/Aims: Thermal injury causes pulmonary edema and can lead to death. Intercellular junctions are composed of adhesive (p120ctn, E-cadherin, α-catenin and β-catenin) and compact (occludin and ZO-1) junctions. Heat deteriorates intercellular junctions and increases cell gaps to ultimately induce pulmonary edema, but the underlying mechanism remains elusive. Methods: Mouse lung epithelial (MLE-12) cells pre-treated with the c-Src inhibitor PP2, p120ctn catenin (p120ctn) small interfering RNA and p120ctn catenin (p120ctn) complementary DNA were subjected to heat treatment. Western blotting and real-time polymerase chain reaction assays were used to evaluate junction protein expression changes after heat treatment, and co-immunoprecipitation was used to test the binding state of junction proteins. In addition, hematoxylin and eosin staining and immunohistochemistry were used to evaluate changes in junction protein expression and lung injury in a Wistar rat model of thermal inhalation injury. Results: Heat increased cell permeability; induced ZO-1, occludin, α-catenin and β-catenin degradation; and decreased E-cadherin distribution in cell membranes. Heat also activated c-Src and decreased both p120ctn expression levels and occludin and ZO-1 association. c-Src inhibitor (PP2) treatment and p120ctn overexpression reversed these effects and attenuated lung injury in vivo. Conclusion: Heat induces junction protein degradation and dissociation to increase membrane permeability and cause lung edema via c-Src kinase activation and p120ctn expression downregulation.

A c-Src Inhibitor Peptide Based on Connexin43 Exerts Neuroprotective Effects through the Inhibition of Glial Hemichannel Activity.

The non-receptor tyrosine kinase c-Src is an important mediator in several signaling pathways related to neuroinflammation. Our previous study showed that cortical injection of kainic acid (KA) promoted a transient increase in c-Src activity in reactive astrocytes surrounding the neuronal lesion. As a cell-penetrating peptide based on connexin43 (Cx43), specifically TAT-Cx43266–283, inhibits Src activity, we investigated the effect of TAT-Cx43266–283 on neuronal death promoted by cortical KA injections in adult mice. As expected, KA promoted neuronal death, estimated by the reduction in NeuN-positive cells and reactive gliosis, characterized by the increase in glial fibrillary acidic protein (GFAP) expression. Interestingly, TAT-Cx43266–283 injected with KA diminished neuronal death and reactive gliosis compared to KA or KA+TAT injections. In order to gain insight into the neuroprotective mechanism, we used in vitro models. In primary cultured neurons, TAT-Cx43266–283 did not prevent neuronal death promoted by KA, but when neurons were grown on top of astrocytes, TAT-Cx43266–283 prevented neuronal death promoted by KA. These observations demonstrate the participation of astrocytes in the neuroprotective effect of TAT-Cx43266–283. Furthermore, the neuroprotective effect was also present in non-contact co-cultures, suggesting the contribution of soluble factors released by astrocytes. As glial hemichannel activity is associated with the release of several factors, such as ATP and glutamate, that cause neuronal death, we explored the participation of these channels on the neuroprotective effect of TAT-Cx43266–283. Our results confirmed that inhibitors of ATP and NMDA receptors prevented neuronal death in co-cultures treated with KA, suggesting the participation of astrocyte hemichannels in neurotoxicity. Furthermore, TAT-Cx43266–283 reduced hemichannel activity promoted by KA in neuron-astrocyte co-cultures as assessed by ethidium bromide (EtBr) uptake assay. In fact, TAT-Cx43266–283 and dasatinib, a potent c-Src inhibitor, strongly reduced the activation of astrocyte hemichannels. In conclusion, our results suggest that TAT-Cx43266–283 exerts a neuroprotective effect through the reduction of hemichannel activity likely mediated by c-Src in astrocytes. These data unveil a new role of c-Src in the regulation of Cx43-hemichannel activity that could be part of the mechanism by which astroglial c-Src participates in neuroinflammation.

Src Kinase Inhibition Attenuates Morphine Tolerance without Affecting Reinforcement or Psychomotor Stimulation.

Prolonged opioid administration leads to tolerance characterized by reduced analgesic potency. Pain management is additionally compromised by the hedonic effects of opioids, the cause of their misuse. The multifunctional protein β-arrestin2 regulates the hedonic effects of morphine and participates in tolerance. These actions might reflect µ opioid receptor up-regulation through reduced endocytosis. β-Arrestin2 also recruits kinases to µ receptors. We explored the role of Src kinase in morphine analgesic tolerance, locomotor stimulation, and reinforcement in C57BL/6 mice. Analgesic (tail withdrawal latency; percentage of maximum possible effect, n = 8 to 16), locomotor (distance traveled, n = 7 to 8), and reinforcing (conditioned place preference, n = 7 to 8) effects of morphine were compared in wild-type, µ, µ, and β-arrestin2 mice. The influence of c-Src inhibitors dasatinib (n = 8) and PP2 (n = 12) was examined. Analgesia in morphine-treated wild-type mice exhibited tolerance, declining by day 10 to a median of 62% maximum possible effect (interquartile range, 29 to 92%). Tolerance was absent from mice receiving dasatinib. Tolerance was enhanced in µ mice (34% maximum possible effect; interquartile range, 5 to 52% on day 5); dasatinib attenuated tolerance (100% maximum possible effect; interquartile range, 68 to 100%), as did PP2 (91% maximum possible effect; interquartile range, 78 to 100%). By contrast, c-Src inhibition affected neither morphine-evoked locomotor stimulation nor reinforcement. Remarkably, dasatinib not only attenuated tolerance but also reversed established tolerance in µ mice. The ability of c-Src inhibitors to inhibit tolerance, thereby restoring analgesia, without altering the hedonic effect of morphine, makes c-Src inhibitors promising candidates as adjuncts to opioid analgesics.

Dual Src Kinase/Pretubulin Inhibitor KX-01, Sensitizes ERα-negative Breast Cancers to Tamoxifen through ERα Reexpression.

Unlike breast cancer that is positive for estrogen receptor-α (ERα), there are no targeted therapies for triple-negative breast cancer (TNBC). ERα is silenced in TNBC through epigenetic changes including DNA methylation and histone acetylation. Restoring ERα expression in TNBC may sensitize patients to endocrine therapy. Expression of c-Src and ERα are inversely correlated in breast cancer suggesting that c-Src inhibition may lead to reexpression of ERα in TNBC. KX-01 is a peptide substrate-targeted Src/pretubulin inhibitor in clinical trials for solid tumors. KX-01 (1 mg/kg body weight-twice daily) inhibited growth of tamoxifen-resistant MDA-MB-231 and MDA-MB-157 TNBC xenografts in nude mice that was correlated with Src kinase inhibition. KX-01 also increased ERα mRNA and protein, as well as increased the ERα targets progesterone receptor (PR), pS2 (TFF1), cyclin D1 (CCND1), and c-myc (MYC) in MDA-MB-231 and MDA-MB-468, but not MDA-MB-157 xenografts. MDA-MB-231 and MDA-MB-468 tumors exhibited reduction in mesenchymal markers (vimentin, β-catenin) and increase in epithelial marker (E-cadherin) suggesting mesenchymal-to-epithelial transition (MET). KX-01 sensitized MDA-MB-231 and MDA-MB-468 tumors to tamoxifen growth inhibition and tamoxifen repression of the ERα targets pS2, cyclin D1, and c-myc. Chromatin immunoprecipitation (ChIP) of the ERα promoter in KX-01-treated tumors demonstrated enrichment of active transcription marks (acetyl-H3, acetyl-H3Lys9), dissociation of HDAC1, and recruitment of RNA polymerase II. Methylation-specific PCR and bisulfite sequencing demonstrated no alteration in ERα promoter methylation by KX-01. These data demonstrate that in addition to Src kinase inhibition, peptidomimetic KX-01 restores ERα expression in TNBC through changes in histone acetylation that sensitize tumors to tamoxifen.Implications: Src kinase/pretubulin inhibitor KX-01 restores functional ERα expression in ERα- breast tumors, a novel treatment strategy to treat triple-negative breast cancer. Mol Cancer Res; 15(11); 1491-502. ©2017 AACR.

Tyrosine Kinase Inhibitors Protect the Salivary Gland from Radiation Damage by Inhibiting Activation of Protein Kinase C-δ.

In patients undergoing irradiation (IR) therapy, injury to nontumor tissues can result in debilitating, and sometimes permanent, side effects. We have defined protein kinase C-δ (PKCδ) as a regulator of DNA damage-induced apoptosis and have shown that phosphorylation of PKCδ by c-Abl and c-Src activates its proapoptotic function. Here, we have explored the use of tyrosine kinase inhibitors (TKI) of c-Src and c-Abl to block activation of PKCδ for radioprotection of the salivary gland. Dasatinib, imatinib, and bosutinib all suppressed tyrosine phosphorylation of PKCδ and inhibited IR-induced apoptosis in vitro To determine whether TKIs can provide radioprotection of salivary gland function in vivo, mice were treated with TKIs and a single or fractionated doses of irradiation. Delivery of dasatinib or imatinib within 3 hours of a single or fractionated dose of irradiation resulted in >75% protection of salivary gland function at 60 days. Continuous dosing with dasatinib extended protection to at least 5 months and correlated with histologic evidence of salivary gland acinar cell regeneration. Pretreatment with TKIs had no impact on clonogenic survival of head and neck squamous cell carcinoma (HNSCC) cells, and in mice harboring HNSCC cell-derived xenografts, combining dasatinib or imatinib with fractionated irradiation did not enhance tumor growth. Our studies indicate that TKIs may be useful clinically to protect nontumor tissue in HNC patients undergoing radiotherapy, without negatively impacting cancer treatment. Mol Cancer Ther; 16(9); 1989-98. ©2017 AACR.

Morphine activation of mu opioid receptors causes disinhibition of neurons in the ventral tegmental area mediated by \u03b2-arrestin2 and c-Src

The tyrosine kinase, c-Src, participates in mu opioid receptor (MOP) mediated inhibition in sensory neurons in which β-arrestin2 (β-arr2) is implicated in its recruitment. Mice lacking β-arr2 exhibit increased sensitivity to morphine reinforcement; however, whether β-arr2 and/or c-Src participate in the actions of opioids in neurons within the reward pathway is unknown. It is also unclear whether morphine acts exclusively through MOPs, or involves delta opioid receptors (DOPs). We examined the involvement of MOPs, DOPs, β-arr2 and c-Src in the inhibition by morphine of GABAergic inhibitory postsynaptic currents (IPSCs) recorded from neurons in the mouse ventral tegmental area. Morphine inhibited spontaneous IPSC frequency, mainly through MOPs, with only a negligible effect remaining in MOP−/− neurons. However, a reduction in the inhibition by morphine for DOP−/− c.f. WT neurons and a DPDPE-induced decrease of IPSC frequency revealed a role for DOPs. The application of the c-Src inhibitor, PP2, to WT neurons also reduced inhibition by morphine, while the inactive PP3, and the MEK inhibitor, SL327, had no effect. Inhibition of IPSC frequency by morphine was also reduced in β-arr2−/− neurons in which PP2 caused no further reduction. These data suggest that inhibition of IPSCs by morphine involves a β-arr2/c-Src mediated mechanism.

Glucocorticoids promote Von Hippel Lindau degradation and Hif-1α stabilization

Glucocorticoid (GC) and hypoxic transcriptional responses play a central role in tissue homeostasis and regulate the cellular response to stress and inflammation, highlighting the potential for cross-talk between these two signaling pathways. We present results from an unbiased in vivo chemical screen in zebrafish that identifies GCs as activators of hypoxia-inducible factors (HIFs) in the liver. GCs activated consensus hypoxia response element (HRE) reporters in a glucocorticoid receptor (GR)-dependent manner. Importantly, GCs activated HIF transcriptional responses in a zebrafish mutant line harboring a point mutation in the GR DNA-binding domain, suggesting a nontranscriptional route for GR to activate HIF signaling. We noted that GCs increase the transcription of several key regulators of glucose metabolism that contain HREs, suggesting a role for GC/HIF cross-talk in regulating glucose homeostasis. Importantly, we show that GCs stabilize HIF protein in intact human liver tissue and isolated hepatocytes. We find that GCs limit the expression of Von Hippel Lindau protein (pVHL), a negative regulator of HIF, and that treatment with the c-src inhibitor PP2 rescued this effect, suggesting a role for GCs in promoting c-src-mediated proteosomal degradation of pVHL. Our data support a model for GCs to stabilize HIF through activation of c-src and subsequent destabilization of pVHL.

Resveratrol prevents angiotensin II-induced hypertrophy of vascular smooth muscle cells through the transactivation of growth factor receptors

We previously showed that augmented levels of endogenous angiotensin II (AngII) contribute to vascular smooth muscle cell (VSMC) hypertrophy through the transactivation of growth factor receptors in spontaneously hypertensive rats. Resveratrol (RV), a polyphenolic component of red wine, has also been shown to attenuate AngII-evoked VSMC hypertrophy; however, the molecular mechanism mediating this response is obscure. The present study was therefore undertaken to examine whether RV could prevent AngII-induced VSMC hypertrophy through the transactivation of growth factor receptor and associated signaling pathways. AngII treatment of VSMC enhanced the protein synthesis that was attenuated towards control levels by RV pretreatment as well as by the inhibitors of NADPH oxidase, c-Src, and growth factor receptors. Furthermore, RV pretreatment also inhibited enhanced levels of superoxide anion, NADPH oxidase activity, increased expression of NADPH oxidase subunits, and phosphorylation of c-Src, EGF-R, PDGE-R, ERK1/2, and AKT1/2. In conclusion, these results indicate that RV attenuates AngII-induced VSMC hypertrophy through the inhibition of enhanced oxidative stress and activation of c-Src, growth factor receptors, and MAPK/AKT signaling. We suggest that RV could be used as a therapeutic agent in the treatment of vascular complications associated with hypertension and hypertrophy.

Binding of CIB1 to the αIIb tail of αIIbβ3 is required for FAK recruitment and activation in platelets.

BackgroundIt is believed that activation of c-Src bound to the integrin β3 subunit initiates outside-in signaling. The involvement of αIIb in outside-in signaling is poorly understood.ObjectivesWe have previously shown that CIB1 specifically interacts with the cytoplasmic domain of αIIb and is required for αIIbβ3 outside-in signaling. Here we evaluated the role of CIB1 in regulating outside-in signaling in the absence of inside-out signaling.MethodsWe used αIIb cytoplasmic domain peptide and CIB1-function blocking antibody to inhibit interaction of CIB1 with αIIb subunit as well as Cib1-/- platelets to evaluate the consequence of CIB1 interaction with αIIb on outside-in signaling.ResultsFibrinogen binding to αIIbβ3 results in calcium-dependent interaction of CIB1 with αIIb, which is not required for filopodia formation. Dynamic rearrangement of cytoskeleton results in CIB1-dependent recruitment of FAK to the αIIb complex and its activation. Disruption of the association of CIB1 and αIIb by incorporation of αIIb peptide or anti-CIB1 inhibited both FAK association and activation. Furthermore, FAK recruitment to the integrin complex was required for c-Src activation. Inhibition of c-Src had no effect on CIB1 accumulation with the integrin at the filopodia, suggesting that c-Src activity is not required for the formation of CIB1-αIIb-FAK complex.ConclusionOur results suggest that interaction of CIB1 with αIIb is one of the early events occurring during outside-in signaling. Furthermore, CIB1 recruits FAK to the αIIbβ3 complex at the filopodia where FAK is activated, which in turn activates c-Src, resulting in propagation of outside-in signaling leading to platelet spreading.

RACK1 to modulate expression of MMP10 via Src/NF-κB pathway in gastric cancer.

e15529 Background: Receptor of activated C kinase 1 (RACK1) is regarded as a scaffolding protein in multiple intracellular signal transduction pathways. Our previous works had demonstrated that RACK1 might involve in progression of gastric cancer, and could be a criterion to predict the good prognosis for the patients after surgical resection. The aim of this study is to explore the regulatory role of RACK1 in matrix metalloproteinase 10 (MMP10) autocrine, and which may be involved in invasion of gastric cancer. Methods: RACK1 knockdown gastric cell lines were established by shRNA. Secretion of cytokines secretion, as well as MMP10 was measured by antibody arrays and enzyme-linked immunosorbent assay. Cell migration and invasion was compared between normal and RACK1 knockdown cells. Pharmacological inhibitors were employed in searching specific signaling pathway. The correlation of RACK1 and MMP10 expression in gastric cancer samples was analyzed by Spearman’s ρ test. Results: Expression of MMP10 was substantially enhanced after knockdown of RACK1 in vitro, which contributed to enhanced migration and invasiveness of gastric cancer cells in autocrine manner. Specific inhibitors of NF-κB and c-Src, as well as overexpression of dominant-negative c-Src mutant, could suppress the expression of MMP10. Finally, an inverse correlation of RACK1 and IL-8 expression was confirmed in gastric cancer samples. Conclusions: Down-regulation of RACK1 resulted in the up-regulation of MMP10 in gastric cancer, which might enhance invasiveness via Src/NF-κB pathway partially.