Expression of myeloid Src-family kinases is associated with poor prognosis in AML and influences Flt3-ITD kinase inhibitor acquired resistance.

Unregulated protein-tyrosine kinase signaling is a common feature of AML, often involving mutations in Flt3 and overexpression of myeloid Src-family kinases (Hck, Fgr, Lyn). Here we show that high-level expression of these Src kinases predicts poor survival in a large cohort of AML patients. To test the therapeutic benefit of Flt3 and Src-family kinase inhibition, we used the pyrrolopyrimidine kinase inhibitor A-419259. This compound potently inhibits Hck, Fgr, and Lyn as well as Flt3 bearing an activating internal tandem duplication (ITD). Flt3-ITD expression sensitized human TF-1 myeloid cells to growth arrest by A-419259, supporting direct action on the Flt3-ITD kinase domain. Cells transformed with the Flt3-ITD mutants D835Y and F691L were resistant to A-419259, while co-expression of Hck or Fgr restored inhibitor sensitivity to Flt3-ITD D835Y. Conversely, Hck and Fgr mutants with engineered A-419259 resistance mutations decreased sensitivity of TF-1/Flt3-ITD cells. To investigate de novo resistance mechanisms, A-419259-resistant Flt3-ITD+ AML cell populations were derived via long-term dose escalation. Whole exome sequencing identified a distinct Flt3-ITD kinase domain mutation (N676S/T) among all A-419259 target kinases in each of six independent resistant cell populations. These studies show that Hck and Fgr expression influences inhibitor sensitivity and the pathway to acquired resistance in Flt3-ITD+ AML.

Lyn regulates epithelial-mesenchymal transition in CS-exposed model through Smad2/3 signaling.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by airflow limitation that is progressive and not fully reversible. Cigarette smoking is one of the most commonly and important risk factors for COPD, which contributes to airway remodeling, the outstanding pathological changes in COPD. One potential mechanism which might be important for airway remodeling is the process called epithelial-mesenchymal transition (EMT). However, the underlying molecular mechanisms of EMT in CS-induced COPD are still poorly understood. METHODS: Two Gene Expression Omnibus (GEO) datasets (GSE108134 and GSE5058) were combined to identify the key genes involved in COPD. Then, single-gene analysis of Lyn was performed. Lyn expression was confirmed in patients with COPD. 16HBE cells were treated with cigarette smoking extracts (CSE). Wild type (WT) C57BL/6 J mice and Lyn+/+ transgenic mice were exposed to CSE to establish CS-exposed model. Pathological changes were observed by hematoxylin-eosin staining. The expression levels of EMT markers were examined by using western blot and immunofluorescence. The expression and phosphorylation levels of Lyn and Smad2/3 were detected as well. RESULTS: The gain of mesenchymal markers vimentin and α-SMA with a concomitant loss of E-cadherin was observed in both in vivo and in vitro studies. Meanwhile, cigarette smoking extracts (CSE) induced EMT in 16HBE cells in a time- and dose- dependent manner. Furthermore, by analyzing GEO datasets and using molecular methods, we explored a kinase, Lyn, its expression correlated with the expression of E-cadherin, vimentin and α-SMA in CS-exposed model. Moreover, we found that EMT induced by CSE was regulated by activated Lyn through phosphorylation of Smad2/3. CONCLUSIONS: In summary, we found that Lyn regulates epithelial-mesenchymal transition in CS-exposed model through Smad2/3 signaling. As a kinase, Lyn is "druggable", and might provide a therapeutic opportunity for targeting EMT. Therefore, our research might provide a new method to treat COPD by targeting Lyn kinase specifically.

A novel SRC-2-dependent regulation of epithelial-mesenchymal transition in breast cancer cells.

Steroid receptor coactivator 2 (SRC-2) is a nuclear receptor coactivator, important for the regulation of estrogen receptor alpha (ERα)-mediated transcriptional activity in breast cancer cells. However, the transcriptional role of SRC-2 in breast cancer is still ambiguous. Here we aimed to unravel a more precise transcriptional role of SRC-2 and uncover unique target genes in MCF-7 breast cancer cells, as opposed to the known oncogene SRC-3. Gene expression analyses of cells depleted of either SRC-2 or SRC-3 showed that they transcriptionally regulate mostly separate gene sets. However, individual unique gene sets were implicated in some of the same major gene ontology biological processes, such as cellular structure and development. This finding was supported by three-dimensional cell cultures, demonstrating that depletion of SRC-2 and SRC-3 changed the morphology of the cells into epithelial-like hollow acinar structures, indicating that both SRC proteins are involved in maintaining the hybrid E/M phenotype. In clinical ER-positive, HER2-negative breast cancer samples the expression of SRC-2 was negatively correlated with the expression of MCF-7-related luminal, cell cycle and cellular morphogenesis genes. Finally, elucidating SRC-2 unique transcriptional effects, we identified Lyn kinase (an EMT biomarker) to be upregulated exclusively after SRC-2 depletion. In conclusion, we show that both SRC-2 and SRC-3 are essential for the EMT in breast cancer cells, controlling different transcriptional niches.

The suppressive effect of arsenic trioxide on TET2-FOXP3-Lyn-Akt axis-modulated MCL1 expression induces apoptosis in human leukemia cells.

Arsenic trioxide (ATO) has been reported to inhibit the activity of Ten-eleven translocation methylcytosine dioxygenase (TET). TET modulates FOXP3 expression, while dysregulation of FOXP3 expression promotes the malignant progression of leukemia cells. We examined the role of TET-FOXP3 axis in the cytotoxic effects of ATO on the human acute myeloid leukemia cell line, U937. ATO-induced apoptosis in U937 cells was characterized by activation of caspase-3/-9, mitochondrial depolarization, and MCL1 downregulation. In addition, ATO-treated U937 cells showed ROS-mediated inhibition of TET2 transcription, leading to downregulation of FOXP3 expression and in turn, suppression of FOXP3-mediated activation of Lyn and Akt. Overexpression of FOXP3 or Lyn minimized the suppressive effect of ATO on Akt activation and MCL1 expression. Promoter luciferase activity and chromatin immunoprecipitation assays revealed the crucial role of Akt-mediated CREB phosphorylation in MCL1 transcription. Further, ATO-induced Akt inactivation promoted GSK3ß-mediated degradation of MCL1. Transfection of constitutively active Akt expression abrogated ATO-induced MCL1 downregulation. MCL1 overexpression lessened the ATO-induced depolarization of mitochondrial membrane and increased the viability of ATO-treated cells. Thus, our data suggest that ATO induces mitochondria-mediated apoptosis in U937 cells through its suppressive effect on TET2-FOXP3-Lyn-Akt axis-modulated MCL1 transcription and protein stabilization. Our findings also indicate that the same pathway underlies ATO-induced death in human leukemia HL-60 cells.

Estrogen-regulated feedback loop limits the efficacy of estrogen receptor-targeted breast cancer therapy.

Endocrine therapy resistance invariably develops in advanced estrogen receptor-positive (ER+) breast cancer, but the underlying mechanisms are largely unknown. We have identified C-terminal SRC kinase (CSK) as a critical node in a previously unappreciated negative feedback loop that limits the efficacy of current ER-targeted therapies. Estrogen directly drives CSK expression in ER+ breast cancer. At low CSK levels, as is the case in patients with ER+ breast cancer resistant to endocrine therapy and with the poorest outcomes, the p21 protein-activated kinase 2 (PAK2) becomes activated and drives estrogen-independent growth. PAK2 overexpression is also associated with endocrine therapy resistance and worse clinical outcome, and the combination of a PAK2 inhibitor with an ER antagonist synergistically suppressed breast tumor growth. Clinical approaches to endocrine therapy-resistant breast cancer must overcome the loss of this estrogen-induced negative feedback loop that normally constrains the growth of ER+ tumors.

Tyrosine Kinase Inhibition in HPV-related Squamous Cell Carcinoma Reveals Beneficial Expression of cKIT and Src.

BACKGROUND/AIM: Therapeutic options of locally advanced or metastatic head and neck squamous cell carcinoma (HNSCC) are limited. Src and cKIT are key protein regulators for local tumor progression. The aim of the study was to investigate the therapeutic potential of targeted therapies in human squamous cell carcinoma (HNSCC) in vitro. Therefore, the influence of the selective tyrosine kinase inhibitors niotinib, dasatinib, erlotinib, gefitinib and afatinib on Src and cKIT expression in Human papilloma virus (HPV)-positive and HPV-negative squamous cancer cells (SCC) was analyzed in vitro. MATERIALS AND METHODS: ELISA was performed to evaluate the expression of Src and cKIT under the influence of nilotinib, dasatinib, erlotinib, gefitinib and afatinib (10 µmol/l) in HPV-negative and HPV-positive SCC (24-96 h of incubation). RESULTS: Gefitinib significantly increased cKIT expression in HPV-positive and HPV-negative cells whereas nilotinib and afatinib decreased cKIT expression in HPV-positive SCC. The influence of tyrosine kinase inhibitors in HPV-negative SCC was marginal. Surprisingly, Src expression was significantly increased by all tested tyrosine kinase inhibitors in HPV-positive SCC. CONCLUSION: The results revealed beneficial and unexpected information concerning the interaction of selective tyrosine kinase inhibitors and the tumor biology of HNSCC.

Hepatitis B virus core protein promotes hepatocarcinogenesis by enhancing Src expression and activating the Src/PI3K/Akt pathway.

Hepatitis B virus core protein (HBc) is expressed preferentially in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC). HBc can function as an oncogene arising from its gene regulatory properties, but how it contributes functionally to hepatocarcinogenesis remains unclear. In this study, we determined the molecular and functional roles of HBc during HBV-associated hepatocellular tumorigenesis. HBc increased tumor formation of hepatoma cells. Moreover, expression of HBc specifically promoted proliferation of hepatoma cells in vitro. Mechanistic investigations revealed that these effects were caused by activation of the Src/PI3K/Akt pathway through proximal switch from inactive Src to the active form of the kinase by HBc. HBc-mediated sarcoma (Src) kinase activation was associated with down-regulation of C-terminal Src kinase (Csk). In addition, HBc enhances Src expression by activation of alternative Src 1A promoter in an Sp1 transcription factor-dependent manner. Proliferation induced by stable HBc expression was associated with increased G1-S cell cycle progression mediated by Src kinase activation. HBc-induced cellular proliferation and tumor formation were reversed by administration of the Src inhibitor saracatinib. Together, our findings suggest that HBc promotes tumorigenesis of hepatoma cells by enhancing the expression of total Src and the active form of the kinase and subsequently activates Src/PI3K/Akt signaling pathway, revealing novel insights into the underlying mechanisms of HBV-associated hepatocarcinogenesis.-Liu, W., Guo, T.-F., Jing, Z.-T., Yang, Z., Liu, L., Yang, Y.-P., Lin, X., Tong, Q.-Y. Hepatitis B virus core protein promotes hepatocarcinogenesis by enhancing Src expression and activating the Src/PI3K/Akt pathway.

A novel BLK-induced tumor model.

B-lymphoid tyrosine kinase (BLK) is a non-receptor tyrosine kinase belonging to the SRC family kinases. BLK is known to be functionally involved in B-cell receptor signaling and B-cell development. New evidence suggests that B-lymphoid tyrosine kinase is ectopically expressed and is a putative oncogene in cutaneous T-cell lymphoma and other T-cell malignancies. However, little is known about the role of BLK in lymphomagenesis, and the oncogenic function seems to depend on the cellular context. Importantly, BLK is also ectopically expressed in other hematological and multiple non-hematological malignancies including breast, kidney, and lung cancers, suggesting that BLK could be a new potential target for therapy. Here, we studied the oncogenic potential of human BLK. We found that engrafted Ba/F3 cells stably expressing constitutive active human BLK formed tumors in mice, whereas neither Ba/F3 cells expressing wild type BLK nor non-transfected Ba/F3 cells did. Inhibition of BLK with the clinical grade and broadly reacting SRC family kinase inhibitor dasatinib inhibited growth of BLK-induced tumors. In conclusion, our study provides evidence that human BLK is a true proto-oncogene capable of inducing tumors, and we demonstrate a novel BLK activity-dependent tumor model suitable for studies of BLK-driven lymphomagenesis and screening of novel BLK inhibitors in vivo.

Photocontrol of the Src Kinase in Mammalian Cells with a Photocaged Intein.

Recently developed methods that can photochemically control protein activities and functions in live cells have opened up new opportunities for studying signaling networks at the cellular and subcellular levels. Our laboratory has reported a genetically encoded photoactivatable intein, which allows the direct photocontrol of primary sequences of proteins, and consequently, their activities and functions in live mammalian cells. Herein, we provide details on experimental design and the utilization of this photocaged intein to photoactivate the Src tyrosine kinase in human embryonic kidney (HEK) 293T cells. The described procedures may be adopted to photocontrol other proteins in other types of mammalian cells.

Inhibition of SRC family kinases reduces myeloid-derived suppressor cells in head and neck cancer.

SRC family kinases (SFKs), a group of nonreceptor tyrosine kinases, modulate multiple cellular functions, such as cell proliferation, differentiation and metabolism. SFKs display aberrant activity in progressive stages of human cancers. However, the precise role of SFKs in the head and neck squamous cell carcinoma (HNSCC) signaling network is far from clear. In this study, we found that the inhibition of SFKs activity by dasatinib effectively reduced the tumor size and population of MDSCs in the HNSCC mouse model. Molecular analysis indicates that phosphorylation of LYN, rather than SRC, was inhibited by dasatinib treatment. Next, we analyzed LYN expression by immunostaining and found that it was overexpressed in the human HNSCC specimens. Moreover, LYN expression in stromal cells positively correlated with myeloid-derived suppressor cells (MDSCs) makers CD11b and CD33 in human HNSCC. The dual positive expression of LYN in epithelial and stromal cells (EPI+ SRT+ ) was associated with unfavorable overall survival of HNSCC patients. These findings indicate that SFKs may be a potential target for an effective immunotherapy of HNSCC by decreasing MDSCs and moreover, LYN will have an impact on such therapeutic strategy.

Src Family Kinases Modulate the Loss of Endothelial Barrier Function in Response to TNF-α: Crosstalk with p38 Signaling.

Activation of Src Family Kinase (SFK) signaling is required for the increase in endothelial permeability induced by a variety of cytokines and growth factors. However, we previously demonstrated that activation of endogenous SFKs by expression of dominant negative C-terminal Src Kinase (DN-Csk) is not sufficient to decrease endothelial adherens junction integrity. Basal SFK activity has been observed in normal venular endothelia and was not associated with increased basal permeability. The basal SFK activity however was found to contribute to increased sensitivity of the venular endothelium to inflammatory mediator-induced leakage. How SFK activation achieves this is still not well understood. Here, we show that SFK activation renders human dermal microvascular endothelial cells susceptible to low doses of TNF-α. Treatment of DN-Csk-expressing cells with 50 pg/ml TNF-α induced a loss of TEER as well as drastic changes in the actin cytoskeleton and focal adhesion proteins. This synergistic effect was independent of ROCK or NF-κB activity. TNF-α-induced p38 signaling was required for the synergistic effect on barrier function, and activation of the p38 MAPK alone was also able to induce changes in permeability only in monolayers with active SFKs. These results suggest that the activation of endogenous levels of SFK renders the endothelial barrier more susceptible to low, physiologic doses of TNF-α through activation of p38 which leads to a loss of endothelial tight junctions.

Role of RANKL in the regulation of NFATc1 and c­Src mRNA expression in osteoclast­like cells.

This study was designed to determine the effects of receptor activator of nuclear factor κB ligand (RANKL) on the mRNA expression of nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) and c­Src in rat osteoclast­like cells. The marrow cells were exposed to macrophage colony-stimulating factor (M­CSF; 25 ng/ml) and different concentrations of RANKL (0, 50, 75 and 100 ng/ml) for 9 days. The mRNA expression of NFATc1 and c­Src was determined by polymerase chain reaction. Compared with the M­CSF (25 ng/ml)+RANKL (0 ng/ml) group, the levels of NFATc1 and c­Src mRNA expression were significantly increased in the M­CSF (25 ng/ml)+RANKL (75 and 100 ng/ml) groups (P<0.01, P<0.01, P<0.01 and P<0.01, respectively). Compared with the M­CSF (25 ng/ml)+RANKL (50 ng/ml) group, the levels of NFATc1 and c­Src mRNA expression was significantly increased in the M­CSF (25 ng/ml)+RANKL (75 and 100 ng/ml) groups (P<0.05, P<0.01, P<0.01 and P<0.01, respectively). Compared with M­CSF (25 ng/ml)+RANKL (75 ng/ml) group, the levels of NFATc1 and c­Src mRNA expression was significantly increased in the M­CSF (25 ng/ml)+RANKL (100 ng/ml) group, (P<0.01 and P<0.01, respectively). These data suggest that RANKL could regulate the expression of NFATc1 and c­Src mRNA in the marrow culture system.

Nuclear expression of Lyn, a Src family kinase member, is associated with poor prognosis in renal cancer patients.

BACKGROUND: 8000 cases of renal cancer are diagnosed each year in the UK, with a five-year survival rate of 50%. Treatment options are limited; a potential therapeutic target is the Src family kinases (SFKs). SFKs have roles in multiple oncogenic processes and promote metastases in solid tumours. The aim of this study was to investigate SFKs as potential therapeutic targets for clear cell renal cell carcinoma (ccRCC). METHODS: SFKs expression was assessed in a tissue microarray consisting of 192 ccRCC patients with full clinical follow-up. SFK inhibitors, dasatinib and saracatinib, were assessed in early ccRCC cell lines, 786-O and 769-P and a metastatic ccRCC cell line, ACHN (± Src) for effects on protein expression, apoptosis, proliferation and wound healing. RESULTS: High nuclear expression of Lyn and the downstream marker of activation, paxillin, were associated with decreased patient survival. Conversely, high cytoplasmic expression of other SFK members and downstream marker of activation, focal adhesion kinase (FAK) were associated with increased patient survival. Treatment of non-metastatic 786-O and 769-P cells with dasatinib, dose dependently reduced SFK activation, shown via SFK (Y(419)) and FAK (Y(861)) phosphorylation, with no effect in metastatic ACHN cells. Dasatinib also increased apoptosis, while decreasing proliferation and migration in 786-O and 769-P cell lines, both in the presence and absence of Src protein. CONCLUSIONS: Our data suggests that nuclear Lyn is a potential therapeutic target for ccRCC and dasatinib affects cellular functions associated with cancer progression via a Src kinase independent mechanism.

Inhibitory effect of fermented Arctium lappa fruit extract on the IgE-mediated allergic response in RBL­2H3 cells.

Arctium lappa fruit has been used in traditional medicine, and it is known to exert beneficial effects, such as antioxidant, anti-inflammatory and anticancer effects. However, the effects of the Arctium lappa fruit on the allergic response remain unknown. In this study, we evaluated the anti-allergic effects of Arctium lappa fruit extract (AFE) and its fermented form (F-AFE) using immunoglobulin E (IgE)-activated RBL­2H3 cells. To investigate the anti-allergic effects of AFE or F-AFE, we examined the release of ß-hexosaminidase, a key biomarker of degranulation during an allergic reaction, and the production of pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) in the cells treated with or without the above-mentioned extracts. AFE weakly inhibited the release of ß-hexosaminidase, whereas F-AFE significantly suppressed the release of ß-hexosaminidase in a dose-dependent manner. Consistently, F-AFE suppressed the production of TNF-α and PGE2 in a dose-dependent manner. F-AFE exerted an inhibitory effect on the production of ß-hexosaminidase, TNF-α and PGE2 with an IC50 value of 30.73, 46.96 and 36.27 µg/ml, respectively. Furthermore, F-AFE inhibited the phosphorylation of Lyn, Fyn and Syk, which are involved in the FcεRI signaling pathway, that of phosphoinositide phospholipase C (PLC)γ1/2 and protein kinase C (PKC)δ, which are associated with the degranulation process, as well as that of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK), p38 and Akt, which are associated with cytokine expression. In the late phase, F-AFE partially suppressed the phosphorylation of cytosolic phospholipase A2 (cPLA2), but not the expression of cyclooxygenase (COX)-2. To compare and identify the major components of the two extracts, we used high-performance liquid chromatography. The levels of arctigenin, one of the major compounds, were elevated 6-fold in F-AFE compared with AFE, whereas the levels of arctiin, an arctigenin glycoside, were decreased in F-AFE by approximately 57.40%. These results suggest that arctigenin plays an important role in the anti-allergic effects of F-AFE. Taken together, F-AFE containing anti-allergic phytochemicals, including arctigenin, inhibited the activation of the FcεRI receptor induced by the antigen­IgE complex. Such effects may provide further information for the development of a phytomedicine for allergic diseases.

miR-124 and Androgen Receptor Signaling Inhibitors Repress Prostate Cancer Growth by Downregulating Androgen Receptor Splice Variants, EZH2, and Src.

miR-124 targets the androgen receptor (AR) transcript, acting as a tumor suppressor to broadly limit the growth of prostate cancer. In this study, we unraveled the mechanisms through which miR-124 acts in this setting. miR-124 inhibited proliferation of prostate cancer cells in vitro and sensitized them to inhibitors of androgen receptor signaling. Notably, miR-124 could restore the apoptotic response of cells resistant to enzalutamide, a drug approved for the treatment of castration-resistant prostate cancer. We used xenograft models to examine the effects of miR-124 in vivo when complexed with polyethylenimine-derived nanoparticles. Intravenous delivery of miR-124 was sufficient to inhibit tumor growth and to increase tumor cell apoptosis in combination with enzalutamide. Mechanistic investigations revealed that miR-124 directly downregulated AR splice variants AR-V4 and V7 along with EZH2 and Src, oncogenic targets that have been reported to contribute to prostate cancer progression and treatment resistance. Taken together, our results offer a preclinical rationale to evaluate miR-124 for cancer treatment.

Tumor necrosis factor-alpha induces VCAM-1-mediated inflammation via c-Src-dependent transactivation of EGF receptors in human cardiac fibroblasts.

BACKGROUND: Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine and elevated in the regions of tissue injury and inflammatory diseases. The deleterious effects of TNF-α on fibroblasts may aggravate heart inflammation mediated through the up-regulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1). However, the mechanisms underlying TNF-α-induced VCAM-1 expression in cardiac fibroblasts remain unknown. This study aimed to investigate the roles of TNF-α in VCAM-1 expression and its effects on human cardiac fibroblasts (HCFs). RESULTS: The primary culture HCFs were used in this study. The results obtained with Western blotting, real time-quantitative PCR, and promoter activity analyses showed that TNF-α-induced VCAM-1 expression was mediated through TNF receptor (TNFR) 1-dependent gene up-regulation. Activation of TNFR1 by TNF-α transactivated c-Src-dependent EGF receptor (EGFR) linking to PI3K/Akt cascade, and then led to transcriptional activity of NF-κB. Moreover, the results of promoter reporter assay demonstrated that the phosphorylated p65 NF-κB turned on VCAM-1 gene expression. Subsequently, up-regulation of VCAM-1 promoted monocytes adhesion to HCFs challenged with TNF-α determined by cell adhesion assay. CONCLUSIONS: Taken together, these results indicate that in HCFs, activation of NF-κB by c-Src-mediated transactivation of EGFR/PI3K/Akt cascade is required for TNF-α-induced VCAM-1 expression. Finally, increased VCAM-1 enhances monocytes adhering to HCFs challenged with TNF-α. Understanding the mechanisms of VCAM-1 up-regulated by TNF-α on HCFs may provide rationally therapeutic interventions for heart injury or inflammatory diseases.

CD44 regulates cell proliferation, migration, and invasion via modulation of c-Src transcription in human breast cancer cells.

CD44 was recently identified as a cancer initiation marker on the cell membrane. The cytoplasmic tail of CD44 is known to bind ERM (ezrin, radixin, moesin) proteins, cytoskeletal proteins like ankyrin, and the non-receptor tyrosine kinase c-Src. CD44 transmits its oncogenic signaling via c-Src and its downstream effectors. To investigate the role of CD44 in breast cancer cells, we generated CD44 knock-down cells via retroviral delivery of shRNA against CD44. We found that silencing of CD44 decreased the proliferation, migration, and invasion of breast cancer cells. The expression and activity of cell migration-related proteins, including c-Src, paxillin, and FAK were decreased by CD44 silencing. We also found that the c-Jun protein level was negatively regulated via induction of a GSK-3ß-dependent degradation pathway in CD44 knock-down cells. The expression level of Sp1, a target gene product of c-Jun, was also decreased in these cells. Finally, CD44 knock-down suppressed both mRNA and protein levels of c-Src and its downstream MAPK pathway as a result of down-regulation of Sp1 as a transcription factor for c-Src. Collectively, these results indicate that biological changes induced by CD44 silencing are mediated by cumulative down-regulation of c-Jun, Sp1, and c-Src in human breast cancer cells.

Aflatoxin B1 induces Src phosphorylation and stimulates lung cancer cell migration.

AflatoxinB1 (AFB1) is well known as a potent carcinogen. Epidemiological studies have shown an association between AFB1 exposure and lung cancer in humans. AFB1 can induce the mutations of genes such as tumor suppressor p53 through its metabolite AFB1-8,9-exo-epoxide, which acts as a mutagen to react with DNA. In addition, recent study demonstrates AFB1 positively regulates type I insulin-like growth factor receptor (IGF-IR) signaling in hepatoma cells. The current study aims to determine the effects of AFB1 on Src kinase and insulin receptor substrate (IRS) in lung cancer cells and the effects of AFB1 on lung cancer cell migration. To this end, the effects of AFB1 on IRS expression, Src, Akt, and ERK phosphorylation were measured by Western blot analysis. The migration of lung cancer cells was detected by wound-healing assay. AFB1 downregulates IRS1 but paradoxically upregulates IRS2 through positive regulation of the stability of IRS2 and the proteasomal degradation of IRS1 in lung cancer cell lines A549 and SPCA-1. In addition, AFB1 induces Src, Akt, and ERK1/2 phosphorylation. Treatment of lung cancer cells with Src inhibitor saracatinib abrogates AFB1-induced IRS2 accumulation. Moreover, AFB1 stimulates lung cancer cell migration, which can be inhibited by saracatinib. We conclude that AFB1 may upregulate IRS2 and stimulate lung cancer cell migration through Src.

Sphingosine-1-phosphate mediates COX-2 expression and PGE2 /IL-6 secretion via c-Src-dependent AP-1 activation.

Sphingosine-1-phosphate (S1P) has been shown to regulate cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2 ) expression and IL-6 secretion in various respiratory diseases. However, the mechanisms underlying S1P-induced COX-2 expression and PGE2 production in human tracheal smooth muscle cells (HTSMCs) remain unclear. Here we demonstrated that S1P markedly induced COX-2 expression. S1P also induced PGE2 and IL-6 secretion which were reduced by the inhibitors of COX-2 (NS-398 and celecoxib). Pretreatment with the inhibitor of S1PR1 (W123), S1PR3 (CAY10444), c-Src (PP1), PYK2 (PF431396), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of S1PR1, S1PR3, c-Src, PYK2, p38, p42, JNK2, c-Jun, or c-Fos reduced S1P-induced COX-2 expression and PGE2 /IL-6 secretion. Moreover, S1P induced c-Src, PYK2, p42/p44 MAPK, JNK1/2, p38 MAPK, and c-Jun phosphorylation. We observed that S1P-induced p42/p44 MAPK and JNK1/2, but not p38 MAPK activation was mediated via a c-Src/PYK2-dependent pathway. S1P also enhanced c-Fos, but not c-Jun mRNA and protein expression and the AP-1 promoter activity. S1P-induced c-Fos mRNA and protein expression, c-Jun phosphorylation, and AP-1 promoter activity was reduced by W123, CAY10444, PP1, PF431396, U0126, SP600125, or SB202190. These results demonstrated that S1P-induced COX-2 expression and PGE2 /IL-6 generation was mediated through S1PR1/3/c-Src/PYK2/p42/p44 MAPK- or JNK1/2- and S1PR1/3/c-Src/p38 MAPK-dependent AP-1 activation.

EGF regulates claudin-2 and -4 expression through Src and STAT3 in MDCK cells.

Epidermal Growth Factor (EGF) is a key regulator of epithelial paracellular permeability, a property that depends on tight junctions (TJ) and can be evaluated through the measurement of the transepithelial electrical resistance (TER). EGF increases the TER of MDCK monolayers by inducing ERK1/2-dependent downregulation of claudin-2 (CLDN-2) and upregulation of claudin-4 (CLDN-4). Because either increments or decrements in TER often involve Src activation and epithelial cell differentiation occasionally depends on STAT3, here we investigated whether EGF might control CLDN-2 downregulation and CLDN-4 upregulation through those proteins. We found that EGF induces Src activation necessary for the reduction of CLDN-2 at the TJ, the degradation of this CLDN, the reduction of the cellular levels of its mRNA and the resulting increase of TER. EGF-induced changes on CLDN-2 protein and mRNA also depend on STAT3 activity. This growth factor increases the levels of STAT3 phosphorylated at Y705 in the nucleus, a process that depends on Src activation. Interestingly, Src and STAT3 activation do not exclusively mediate the EGF-induced downregulation of CLDN-2, but they are also implicated in the EGF-induced CLDN-4 transcription, translation, and exocytic fusion into TJ. Our results indicate that EGF controls the levels of CLDN-2 and -4 proteins and mRNAs through Src and STAT3 activity.