Akt and Src mediate the photocrosslinked fibroin-induced neural differentiation.

Neural transplantation is a promising modality for treatment of neurodegenerative diseases, traumatic brain injury and stroke. Biocompatible scaffolds with optimized properties improve the survival of transplanted neural cells and differentiation of progenitor cells into the desired types of neurons. Silk fibroin is a biocompatible material for tissue engineering. Here, we describe thin-film scaffolds based on photocrosslinked methacrylated silk fibroin (FBMA). These scaffolds exhibit an increased mechanical stiffness and improved water stability. Photocrosslinking of fibroin increased its rigidity from 25 to 480 kPa and the contact angle from 59.7 to 70.8, the properties important for differentiation of neural cells. Differentiation of SH-SY5Y neuroblastoma cells on FBMA increased the length of neurites as well as the levels of neural differentiation markers MAP2 and ßIII-tubulin. Growth of SH-SY5Y cells on the unmodified fibroin and FBMA substrates led to a spontaneous phosphorylation of Src and Akt protein kinases critical for neuronal differentiation; this effect was paralleled by neural cell adhesion molecule elevation. Thus, FBMA is an easily manufactured, cytocompatible material with improved and sustainable properties applicable for neural tissue engineering.

Crosstalk with lung epithelial cells regulates Sfrp2-mediated latency in breast cancer dissemination.

The process of metastasis is complex1. In breast cancer, there are frequently long time intervals between cells leaving the primary tumour and growth of overt metastases2,3. Reasons for disease indolence and subsequent transition back to aggressive growth include interactions with myeloid and fibroblastic cells in the tumour microenvironment and ongoing immune surveillance4-6. However, the signals that cause actively growing cells to enter an indolent state, thereby enabling them to survive for extended periods of time, are not well understood. Here we reveal how the behaviour of indolent breast cancer cells in the lung is determined by their interactions with alveolar epithelial cells, in particular alveolar type 1 cells. This promotes the formation of fibronectin fibrils by indolent cells that drive integrin-dependent pro-survival signals. Combined in vivo RNA sequencing and drop-out screening identified secreted frizzled-related protein 2 (SFRP2) as a key mediator of this interaction. Sfrp2 is induced in breast cancer cells by signals from lung epithelial cells and promotes fibronectin fibril formation and survival, whereas blockade of Sfrp2 expression reduces the burden of indolent disease.

EGFR-c-Src-Mediated HDAC3 Phosphorylation Exacerbates Invasion of Breast Cancer Cells.

Breast cancer is one of the leading causes of morbidity and mortality among women. Epidermal growth factor receptor (EGFR) and proto-oncogene tyrosine-protein kinase Src (c-Src) are critical components of the signaling pathways that are associated with breast cancer. However, the regulatory mechanism of histone deacetylase 3 (HDAC3) in these pathways remains unclear. Using the Net Phos 3.1 program for the analysis of kinase consensus motifs, we found two c-Src-mediated putative phosphorylation sites, tyrosine (Tyr, Y)-328 and Y331 on HDAC3, and generated a phospho-specific HDAC3 antibody against these sites. c-Src-mediated phosphorylation was observed in the cells expressing wild-type HDAC3 (HDAC3WT), but not in cells overexpressing phosphorylation-defective HDAC3 (HDAC3Y328/331A). Phosphorylated HDAC3 showed relatively higher deacetylase activity, and PP2, which is a c-Src inhibitor, blocked HDAC3 phosphorylation and reduced its enzymatic activity. EGF treatment resulted in HDAC3 phosphorylation in both MDA-MB-231 and EGFR-overexpressing MCF7 (MCF7-EGFR) cells, but not in MCF7 cells. Total internal reflection fluorescence analysis showed that HDAC3 was recruited to the plasma membrane following EGF stimulation. HDAC3 inhibition with either c-Src knockdown or PP2 treatment significantly ameliorated the invasiveness of breast cancer cells. Altogether, our findings reveal an EGF signaling cascade involving EGFR, c-Src, and HDAC3 in breast cancer cells.

Autophagy-Src Regulates Connexin43-Mediated Gap Junction Intercellular Communication in Irradiated HepG2 Cells.

Connexin molecules are an important component of the gap junction, with connexin43 (Cx43) being the most abundantly expressed type. Src is a nonreceptor tyrosine-protein kinase that affects Cx43 activity by multiple mechanisms. However, it is not clear how Src regulates Cx43 to influence radiation-induced bystander effects (RIBEs). In this study, we demonstrated that Cx43 on Tyr265 was phosphorylated by activated Src in α-irradiated HepG2 cells, with the total expression of Cx43 unchanged. After inhibition of Cx43 phosphorylation in irradiated cells, the frequency of γ-H2AX foci formation in adjacent nonirradiated bystander cells was significantly enhanced. Furthermore, this study showed that autophagy regulated the activity of Src and phosphorylation of Cx43, and the level of autophagy was correlated with the radiation-induced reactive oxygen species (ROS). These results suggest that ROS and autophagy play an important role in regulating the Src-Cx43 axis to affect the RIBEs. Our findings provide new insights into the Cx43-mediated gap junction intercellular communication, as well as the underlying mechanism of RIBEs.

Src Cooperates with Oncogenic Ras in Tumourigenesis via the JNK and PI3K Pathways in Drosophila epithelial Tissue.

The Ras oncogene (Rat Sarcoma oncogene, a small GTPase) is a key driver of human cancer, however alone it is insufficient to produce malignancy, due to the induction of cell cycle arrest or senescence. In a Drosophila melanogaster genetic screen for genes that cooperate with oncogenic Ras (bearing the RasV12 mutation, or RasACT), we identified the Drosophila Src (Sarcoma virus oncogene) family non-receptor tyrosine protein kinase genes, Src42A and Src64B, as promoting increased hyperplasia in a whole epithelial tissue context in the Drosophila eye. Moreover, overexpression of Src cooperated with RasACT in epithelial cell clones to drive neoplastic tumourigenesis. We found that Src overexpression alone activated the Jun N-terminal Kinase (JNK) signalling pathway to promote actin cytoskeletal and cell polarity defects and drive apoptosis, whereas, in cooperation with RasACT, JNK led to a loss of differentiation and an invasive phenotype. Src + RasACT cooperative tumourigenesis was dependent on JNK as well as Phosphoinositide 3-Kinase (PI3K) signalling, suggesting that targeting these pathways might provide novel therapeutic opportunities in cancers dependent on Src and Ras signalling.

WNT5A promotes migration and invasion of human osteosarcoma cells via SRC/ERK/MMP-14 pathway.

WNT5A, a representative ligand of activating several non-canonical WNT signal pathways, plays significant roles in oncogenesis and tumor inhibition. It has been shown that the non-receptor tyrosine kinase SRC is required for WNT5A-induced invasion of osteosarcoma cells. However, the precise molecular mechanism underlying WNT5A/SRC-mediated osteosarcoma cells invasion remains poorly defined. The study was designed to explore the role of ERK1/2 in WNT5A/SRC-induced osteosarcoma cells invasion and the downstream target of the SRC/ERK1/2 signalings. We found that WNT5A (100 ng/mL) remarkably stimulated migration and invasion of human osteosarcoma MG-63 cells, whereas inhibiting either SRC kinase activity by siRNA-mediated SRC silence or ERK1/2 phosphorylation by PD98059 treatment suppressed these effects, which suggested that the activation of SRC and ERK1/2 is essential for WNT5A-induced MG-63 cells migration and invasion. Furthermore, ERK1/2 phosphorylation induced by WNT5A was dramatically blocked by SRC siRNA. Additionally, our study further demonstrated that MMP-14 was upregulated after exposure to WNT5A in MG-63 cells, and the increased expression was blocked by SRC siRNA or PD98059. Collectively, these results indicate that WNT5A activates SRC/ERK1/2 signal pathway, leading to the upregulation of MMP-14 expression and MG-63 cells migration and invasion.

c-Src activity is differentially required by cancer cell motility modes.

Cancer cell migration requires that cells respond and adapt to their surroundings. In the absence of extracellular matrix cues, cancer cells will undergo a mesenchymal to ameboid transition, whereas a highly confining space will trigger a switch to "leader bleb-based" migration. To identify oncogenic signaling pathways mediating these transitions, we undertook a targeted screen using clinically useful inhibitors. Elevated Src activity was found to change actin and focal adhesion dynamics, whereas inhibiting Src triggered focal adhesion disassembly and blebbing. On non-adherent substrates and in collagen matrices, amoeboid-like, blebbing cells having high Src activity formed protrusions of the plasma membrane. To evaluate the role of Src in confined cells, we use a novel approach that places cells under a slab of polydimethylsiloxane (PDMS), which is held at a defined height. Using this method, we find that leader bleb-based migration is resistant to Src inhibition. High Src activity was found to markedly change the architecture of cortical actomyosin, reduce cell mechanical properties, and the percentage of cells that undergo leader bleb-based migration. Thus, Src is a signal transducer that can potently influence transitions between migration modes with implications for the rational development of metastasis inhibitors.

Adrenomedullin Promotes the Proliferation and Inhibits Apoptosis of Dental Pulp Stem Cells Involved in Divergence Pathways.

INTRODUCTION: Adrenomedullin (ADM) is highly expressed in dental tissues at the critical developmental time points during tooth development. However, its role in pulp repair and pulp injury is still unknown. The study aimed to investigate the mechanisms by which ADM affects the proliferation and apoptosis of human dental pulp stem cells (DPSCs). METHODS: Fifty-four freshly extracted wisdom teeth were obtained from 27 patients with caries and 27 without. Enzyme-linked immunosorbent assay and immunohistochemistry were used to examine the expression levels of ADM in caries and normal pulp tissues. Human primary DPSCs from the third passage were incubated with ADM (10(-8) mol/L) for 24 hours; the cell cycle and apoptosis were investigated by flow cytomery, and protein expression was investigated using Western blot analysis. RESULTS: The levels of ADM in caries pulp tissues were significantly higher than those in normal pulp tissues. Incubation with ADM enhanced the percentage of G2/S/M phase DPSCs (P < .01) by the addition of a JNK/c-Jun inhibitor. Incubation with ADM reduced DPSC apoptosis by the addition of the Src/glycogen synthase kinase-3 inhibitor. ADM also ameliorated CoCl2-induced apoptosis and caspase-3 expression. CONCLUSIONS: ADM enhances the proliferation of DPSCs through activation of the JNK/c-Jun signaling pathway and inhibits apoptosis of DPSCs through activation of the Src/glycogen synthase kinase-3 signaling pathway.

Integrin αvß3 expression in tongue squamous carcinoma cells Cal27 confers anticancer drug resistance through loss of pSrc(Y418).

Integrins play key roles in the regulation of tumor cell adhesion, migration, invasion and sensitivity to anticancer drugs. In the present study we investigate the mechanism of resistance of tongue squamous carcinoma cells Cal27 with de novo integrin αvß3 expression to anticancer drugs. Cal27-derived cell clones, obtained by transfection of plasmid containing integrin subunit ß3 cDNA, as compared to control cells demonstrate: expression of integrin αvß3; increased expression of integrin αvß5; increased adhesion to fibronectin and vitronectin; resistance to cisplatin, mitomycin C, doxorubicin and 5-fluorouracil; increased migration and invasion, increased amount of integrin-linked kinase (ILK) and decreased amounts of non-receptor tyrosine kinase (Src) and pSrc(Y418). Knockdown of ILK and integrin ß5 in cells expressing integrin αvß3 ruled out their involvement in drug resistance. Opposite, Src knockdown in Cal27 cells which led to a reduction in pSrc(Y418), as well as treatment with the pSrc(Y418) inhibitors dasatinib and PP2, conferred resistance to all four anticancer drugs, indicating that the loss of pSrc(Y418) is responsible for the observed effect. We identified differential integrin signaling between Cal27 and integrin αvß3-expressing cells. In Cal27 cells integrin αv heterodimers signal through pSrc(Y418) while this is not the case in integrin αvß3-expressing cells. Finally, we show that dasatinib counteracts the effect of cisplatin in two additional head and neck squamous cell carcinoma (HNSCC) cell lines Cal33 and Detroit562. Our results suggest that pSrc(Y418) inhibitors, potential drugs for cancer therapy, may reduce therapeutic efficacy if combined with chemotherapeutics, and might not be recommended for HNSCC treatment.

E-cadherin and Src associate with extradesmosomal Dsg3 and modulate desmosome assembly and adhesion.

Desmosomes provide strong intercellular cohesion essential for the integrity of cells and tissues exposed to continuous mechanical stress. For desmosome assembly, constitutively synthesized desmosomal cadherins translocate to the cell-cell border, cluster and mature in the presence of Ca(2+) to stable cell contacts. As adherens junctions precede the formation of desmosomes, we investigated in this study the relationship between the classical cadherin E-cadherin and the desmosomal cadherin Desmoglein 3 (Dsg3), the latter of which is indispensable for cell-cell adhesion in keratinocytes. By using autoantibodies from patients with the blistering skin disease pemphigus vulgaris (PV), we showed in loss of function studies that E-cadherin compensates for effects of desmosomal disassembly. Overexpression of E-cadherin reduced the loss of cell cohesion induced by PV autoantibodies and attenuated activation of p38 MAPK. Silencing of E-cadherin abolished the localization of Dsg3 at the membrane and resulted in a shift of Dsg3 from the cytoskeletal to the non-cytoskeletal protein pool which conforms to the notion that E-cadherin regulates desmosome assembly. Mechanistically, we identified a complex consisting of extradesmosomal Dsg3, E-cadherin, ß-catenin and Src and that the stability of this complex is regulated by Src. Moreover, Dsg3 and E-cadherin are phosphorylated on tyrosine residues in a Src-dependent manner and Src activity is required for recruiting Dsg3 to the cytoskeletal pool as well as for desmosome maturation towards a Ca(2+)-insensitive state. Our data provide new insights into the role of E-cadherin and the contribution of Src signaling for formation and maintenance of desmosomal junctions.

The proto-oncogene c-Src and its downstream signaling pathways are inhibited by the metastasis suppressor, NDRG1.

N-myc downstream regulated gene-1 (NDRG1) is a potent metastasis suppressor that plays a key role in regulating signaling pathways involved in mediating cancer cell invasion and migration, including those derived from prostate, colon, etc. However, the mechanisms and molecular targets through which NDRG1 reduces cancer cell invasion and migration, leading to inhibition of cancer metastasis, are not fully elucidated. In this investigation, using NDRG1 over-expression models in three tumor cell-types (namely, DU145, PC3MM and HT29) and also NDRG1 silencing in DU145 and HT29 cells, we reveal that NDRG1 decreases phosphorylation of a key proto-oncogene, cellular Src (c-Src), at a well-characterized activating site (Tyr416). NDRG1-mediated down-regulation of EGFR expression and activation were responsible for the decreased phosphorylation of c-Src (Tyr416). Indeed, NDRG1 prevented recruitment of c-Src to EGFR and c-Src activation. Moreover, NDRG1 suppressed Rac1 activity by modulating phosphorylation of a c-Src downstream effector, p130Cas, and its association with CrkII, which acts as a "molecular switch" to activate Rac1. NDRG1 also affected another signaling molecule involved in modulating Rac1 signaling, c-Abl, which then inhibited CrkII phosphorylation. Silencing NDRG1 increased cell migration relative to the control and inhibition of c-Src signaling using siRNA, or a pharmacological inhibitor (SU6656), prevented this increase. Hence, the role of NDRG1 in decreasing cell migration is, in part, due to its inhibition of c-Src activation. In addition, novel pharmacological agents, which induce NDRG1 expression and are currently under development as anti-metastatic agents, markedly increase NDRG1 and decrease c-Src activation. This study leads to important insights into the mechanism involved in inhibiting metastasis by NDRG1 and how to target these pathways with novel therapeutics.

Src mediates extracellular signal-regulated kinase 1/2 activation and autophagic cell death induced by cardiac glycosides in human non-small cell lung cancer cell lines.

Aberrant Na(+) /K(+) -ATPases (NKA) expression is closely related to the incidence and development of cancer, making NKA targeted cancer therapy more intriguing. Cardiac glycosides (CGs) belong to NKA inhibitors and possess potent anti-cancer properties in many cancers. Our previous work demonstrates that CGs family member digoxin or ouabain induces autophagic cell death in human non-small cell lung cancer (NSCLC) cell lines through regulation of both mammalian target of rapamycin and extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. However, what acts as an upstream regulator of ERK1/2 activation during autophagy induction remains obscure. In the present study, the role of Src in the ERK1/2 signaling pathway as well as autophagic cell death induced by either digoxin or ouabain was examined in A549 and H460 cells. Src is significantly activated simultaneously with mitogen-activated protein kinase kinase 1/2 (MEK1/2) and ERK1/2 activation upon the drug treatment. Moreover, Src inhibitor PP2 could block either drug induced MEK1/2 and ERK1/2 phosphorylation, together with autophagic phenotypes in the cells. Knockdown of Src with siRNA causes the similar effect as PP2, both of which markedly alleviate the drugs' cytotoxicity. In addition, increased levels of intracellular reactive oxygen species (ROS) are found to be involved in Src mediated autophagy. Together, this work provides evidences showing that Src mediates MEK1/2 and ERK1/2 pathway as well as ROS generation, and regulates autophagic cell death induced by the cardiac glycosides. These observations may further help understand the molecular mechanisms of autophagy induced by NKA inhibitors in NSCLC cells.

Differential effects of c-Src and c-Yes on the endocytic vesicle-mediated trafficking events at the Sertoli cell blood-testis barrier: an in vitro study.

The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in the mammalian body. However, it undergoes cyclic restructuring during the epithelial cycle of spermatogenesis in which the "old" BTB located above the preleptotene spermatocytes being transported across the immunological barrier is "disassembled," whereas the "new" BTB found behind these germ cells is rapidly "reassembled," i.e., mediated by endocytic vesicle-mediated protein trafficking events. Thus, the immunological barrier is maintained when preleptotene spermatocytes connected in clones via intercellular bridges are transported across the BTB. Yet the underlying mechanism(s) in particular the involving regulatory molecules that coordinate these events remains unknown. We hypothesized that c-Src and c-Yes might work in contrasting roles in endocytic vesicle-mediated trafficking, serving as molecular switches, to effectively disassemble and reassemble the old and the new BTB, respectively, to facilitate preleptotene spermatocyte transport across the BTB. Following siRNA-mediated specific knockdown of c-Src or c-Yes in Sertoli cells, we utilized biochemical assays to assess the changes in protein endocytosis, recycling, degradation and phagocytosis. c-Yes was found to promote endocytosed integral membrane BTB proteins to the pathway of transcytosis and recycling so that internalized proteins could be effectively used to assemble new BTB from the disassembling old BTB, whereas c-Src promotes endocytosed Sertoli cell BTB proteins to endosome-mediated protein degradation for the degeneration of the old BTB. By using fluorescence beads mimicking apoptotic germ cells, Sertoli cells were found to engulf beads via c-Src-mediated phagocytosis. A hypothetical model that serves as the framework for future investigation is thus proposed.

Src kinases and ERK activate distinct responses to Stitcher receptor tyrosine kinase signaling during wound healing in Drosophila.

Metazoans have evolved efficient mechanisms for epidermal repair and survival following injury. Several cellular responses and key signaling molecules that are involved in wound healing have been identified in Drosophila, but the coordination of cytoskeletal rearrangements and the activation of gene expression during barrier repair are poorly understood. The Ret-like receptor tyrosine kinase (RTK) Stitcher (Stit, also known as Cad96Ca) regulates both re-epithelialization and transcriptional activation by Grainy head (Grh) to induce restoration of the extracellular barrier. Here, we describe the immediate downstream effectors of Stit signaling in vivo. Drk (Downstream of receptor kinase) and Src family tyrosine kinases bind to the same docking site in the Stit intracellular domain. Drk is required for the full activation of transcriptional responses but is dispensable for re-epithelialization. By contrast, Src family kinases (SFKs) control both the assembly of a contractile actin ring at the wound periphery and Grh-dependent activation of barrier-repair genes. Our analysis identifies distinct pathways mediating injury responses and reveals an RTK-dependent activation mode for Src kinases and their central functions during epidermal wound healing in vivo.

RIG-I modulates Src-mediated AKT activation to restrain leukemic stemness.

Retinoic acid (RA)-inducible gene I (RIG-I) is highly upregulated and functionally implicated in the RA-induced maturation of acute myeloid leukemia (AML) blasts. However, the underlying mechanism and the biological relevance of RIG-I expression to the maintenance of leukemogenic potential are poorly understood. Here, we show that RIG-I, without priming by foreign RNA, inhibits the Src-facilitated activation of AKT-mTOR in AML cells. Moreover, in a group of primary human AML blasts, RIG-I reduction renders the Src family kinases hyperactive in promoting AKT activation. Mechanistically, a PxxP motif in RIG-I, upon the N-terminal CARDs' association with the Src SH1 domain, competes with the AKT PxxP motif for recognizing the Src SH3 domain. In accordance, mutating PxxP motif prevents Rig-I from inhibiting AKT activation, cytokine-stimulated myeloid progenitor proliferation, and in vivo repopulating capacity of leukemia cells. Collectively, our data suggest an antileukemia activity of RIG-I via competitively inhibiting Src/AKT association.

SLAP displays tumour suppressor functions in colorectal cancer via destabilization of the SRC substrate EPHA2.

The adaptor SLAP is a negative regulator of receptor signalling in immune cells but its role in human cancer is ill defined. Here we report that SLAP is abundantly expressed in healthy epithelial intestine but strongly downregulated in 50% of colorectal cancer. SLAP overexpression suppresses cell tumorigenicity and invasiveness while SLAP silencing enhances these transforming properties. Mechanistically, SLAP controls SRC/EPHA2/AKT signalling via destabilization of the SRC substrate and receptor tyrosine kinase EPHA2. This activity is independent from CBL but requires SLAP SH3 interaction with the ubiquitination factor UBE4A and SLAP SH2 interaction with pTyr594-EPHA2. SRC phosphorylates EPHA2 on Tyr594, thus creating a feedback loop that promotes EPHA2 destruction and thereby self-regulates its transforming potential. SLAP silencing enhances SRC oncogenicity and sensitizes colorectal tumour cells to SRC inhibitors. Collectively, these data establish a tumour-suppressive role for SLAP in colorectal cancer and a mechanism of SRC oncogenic induction through stabilization of its cognate substrates.

Src mediates ERK reactivation in gefitinib resistance in non-small cell lung cancer.

To study epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) resistance mechanisms, we established a novel gefitinib-resistant lung cancer cell line derived from an EGFR-mutant non-small cell lung cancer cell line (PC-9) pretreated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (designated PC9-GR). We found that gefitinib substantially suppressed the EGFR signaling pathway, whereas ERK was reactivated after several hours in PC9-GR but not in PC-9. The combination of gefitinib with ERK inhibition (by U0126) restored gefitinib susceptibility in PC9-GR, but PI3K-Akt inhibition with LY294002 did not. Although the levels of phosphorylated Src were up-regulated simultaneously with ERK reactivation, neither ERK suppression using U0126 nor an ERK-specific siRNA induced Src phosphorylation. Furthermore, dual inhibition of EGFR and Src restored gefitinib sensitivity in PC9-GR in vitro and in vivo. In conclusion, our results indicate that Src-mediated ERK reactivation may play a role in a novel gefitinib resistance mechanism, and that the combined use of gefitinib with a Src inhibitor may be a potent strategy to overcome this resistance.

Fibroblast growth factor 2 causes G2/M cell cycle arrest in ras-driven tumor cells through a Src-dependent pathway.

We recently reported that paracrine Fibroblast Growth Factor 2 (FGF2) triggers senescence in Ras-driven Y1 and 3T3(Ras) mouse malignant cell lines. Here, we show that although FGF2 activates mitogenic pathways in these Ras-dependent malignant cells, it can block cell proliferation and cause a G2/M arrest. These cytostatic effects of FGF2 are inhibited by PD173074, an FGF receptor (FGFR) inhibitor. To determine which downstream pathways are induced by FGF2, we tested specific inhibitors targeting mitogen-activated protein kinase (MEK), phosphatidylinositol 3 kinase (PI3K) and protein kinase C (PKC). We show that these classical mitogenic pathways do not mediate the cytostatic activity of FGF2. On the other hand, the inhibition of Src family kinases rescued Ras-dependent malignant cells from the G2/M irreversible arrest induced by FGF2. Taken together, these data indicate a growth factor-sensitive point in G2/M that likely involves FGFR/Ras/Src pathway activation in a MEK, PI3K and PKC independent manner.

c-Src regulates cell cycle proteins expression through protein kinase B/glycogen synthase kinase 3 beta and extracellular signal-regulated kinases 1/2 pathways in MCF-7 cells.

We have demonstrated that c-Src suppression inhibited the epithelial to mesenchymal transition in human breast cancer cells. Here, we investigated the role of c-Src on the cell cycle progression using siRNAs and small molecule inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Western blot analysis demonstrated the down-regulation of cyclin D1 and cyclin E and up-regulation of p27 Kip1 after c-Src suppression by PP2. Incubation of cells in the presence of PP2 significantly blocked the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), protein kinase B (AKT), and glycogen synthase kinase 3 beta (GSK3ß). Specific pharmacological inhibitors of MEK1/2/ERK1/2 and phosphatidylinositide 3-kinase/AKT pathways were used to demonstrate the relationship between the signal cascade and cell cycle proteins expression. The expression of cyclin D1 and cyclin E were decreased after inhibition of ERK1/2 or AKT activity, whereas the p27 Kip1 expression was increased. In addition, knockdown of c-Src by siRNAs reduced cell proliferation and phosphorylation of ERK1/2, AKT, and GSK3ß. After c-Src depletion by siRNAs, we observed significant down-regulation of cyclin D1 and cyclin E, and up-regulation of p27 Kip1. These results suggest that c-Src suppression by PP2 or siRNAs may regulate the progression of cell cycle through AKT/GSK3ß and ERK1/2 pathways.