YES1 as a Therapeutic Target for HER2-Positive Breast Cancer after Trastuzumab and Trastuzumab-Emtansine (T-DM1) Resistance Development.

Trastuzumab-emtansine (T-DM1) is a therapeutic agent molecularly targeting human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer (MBC), and it is especially effective for MBC with resistance to trastuzumab. Although several reports have described T-DM1 resistance, few have examined the mechanism underlying T-DM1 resistance after the development of acquired resistance to trastuzumab. We previously reported that YES1, a member of the Src family, plays an important role in acquired resistance to trastuzumab in HER2-amplified breast cancer cells. We newly established a trastuzumab/T-DM1-dual-resistant cell line and analyzed the resistance mechanisms in this cell line. At first, the T-DM1 effectively inhibited the YES1-amplified trastuzumab-resistant cell line, but resistance to T-DM1 gradually developed. YES1 amplification was further enhanced after acquired resistance to T-DM1 became apparent, and the knockdown of the YES1 or the administration of the Src inhibitor dasatinib restored sensitivity to T-DM1. Our results indicate that YES1 is also strongly associated with T-DM1 resistance after the development of acquired resistance to trastuzumab, and the continuous inhibition of YES1 is important for overcoming resistance to T-DM1.

Preventing Engrailed-1 activation in fibroblasts yields wound regeneration without scarring.

Skin scarring, the end result of adult wound healing, is detrimental to tissue form and function. Engrailed-1 lineage-positive fibroblasts (EPFs) are known to function in scarring, but Engrailed-1 lineage-negative fibroblasts (ENFs) remain poorly characterized. Using cell transplantation and transgenic mouse models, we identified a dermal ENF subpopulation that gives rise to postnatally derived EPFs by activating Engrailed-1 expression during adult wound healing. By studying ENF responses to substrate mechanics, we found that mechanical tension drives Engrailed-1 activation via canonical mechanotransduction signaling. Finally, we showed that blocking mechanotransduction signaling with either verteporfin, an inhibitor of Yes-associated protein (YAP), or fibroblast-specific transgenic YAP knockout prevents Engrailed-1 activation and promotes wound regeneration by ENFs, with recovery of skin appendages, ultrastructure, and mechanical strength. This finding suggests that there are two possible outcomes to postnatal wound healing: a fibrotic response (EPF-mediated) and a regenerative response (ENF-mediated).

SRC family kinase (SFK) inhibitor dasatinib improves the antitumor activity of anti-PD-1 in NSCLC models by inhibiting Treg cell conversion and proliferation.

INTRODUCTION: The use of immune-checkpoint inhibitors has drastically improved the management of patients with non-small cell lung cancer (NSCLC), but innate and acquired resistances are hurdles needed to be solved. Immunomodulatory drugs that can reinvigorate the immune cytotoxic activity, in combination with antiprogrammed cell death 1 (PD-1) antibody, are a great promise to overcome resistance. We evaluated the impact of the SRC family kinases (SFKs) on NSCLC prognosis, and the immunomodulatory effect of the SFK inhibitor dasatinib, in combination with anti-PD-1, in clinically relevant mouse models of NSCLC. METHODS: A cohort of patients from University Clinic of Navarra (n=116) was used to study immune infiltrates by multiplex immunofluorescence (mIF) and YES1 protein expression in tumor samples. Publicly available resources (TCGA, Km Plotter, and CIBERSORT) were used to study patient's survival based on expression of SFKs and tumor infiltrates. Syngeneic NSCLC mouse models 393P and UNSCC680AJ were used for in vivo drug testing. RESULTS: Among the SFK members, YES1 expression showed the highest association with poor prognosis. Patients with high YES1 tumor levels also showed high infiltration of CD4+/FOXP3+ cells (regulatory T cells (Tregs)), suggesting an immunosuppressive phenotype. After testing for YES1 expression in a panel of murine cell lines, 393P and UNSCC680AJ were selected for in vivo studies. In the 393P model, dasatinib+anti-PD-1 treatment resulted in synergistic activity, with 87% tumor regressions and development of immunological memory that impeded tumor growth when mice were rechallenged. In vivo depletion experiments further showed that CD8+ and CD4+ cells are necessary for the therapeutic effect of the combination. The antitumor activity was accompanied by a very significant decrease in the number of Tregs, which was validated by mIF in tumor sections. In the UNSCC680AJ model, the antitumor effects of dasatinib+anti-PD-1 were milder but similar to the 393P model. In in vitro assays, we demonstrated that dasatinib blocks proliferation and transforming growth factor beta-driven conversion of effector CD4+ cells into Tregs through targeting of phospholymphocyte-specific protein tyrosine kinase and downstream effectors pSTAT5 and pSMAD3. CONCLUSIONS: YES1 protein expression is associated with increased numbers of Tregs in patients with NSCLC. Dasatinib synergizes with anti-PD-1 to impair tumor growth in NSCLC experimental models. This study provides the preclinical rationale for the combined use of dasatinib and PD-1/programmed death-ligand 1 blockade to improve outcomes of patients with NSCLC.

YES1 amplification confers trastuzumab-emtansine (T-DM1) resistance in HER2-positive cancer.

BACKGROUND: Trastuzumab-emtansine (T-DM1), one of the most potent HER2-targeted drugs, shows impressive efficacy in patients with HER2-positive breast cancers. However, resistance inevitably occurs and becomes a critical clinical problem. METHODS: We modelled the development of acquired resistance by exposing HER2-positive cells to escalating concentrations of T-DM1. Signalling pathways activation was detected by western blotting, gene expression was analysed by qRT-PCR and gene copy numbers were determined by qPCR. The role of Yes on resistance was confirmed by siRNA-mediated knockdown and stable transfection-mediated overexpression. The in vivo effects were tested in xenograft model. RESULTS: We found that Yes is overexpressed in T-DM1-resistant cells owing to amplification of chromosome region 18p11.32, where the YES1 gene resides. Yes activated multiple proliferation-related signalling pathways, including EGFR, PI3K and MAPK, and led to cross-resistance to all types of HER2-targeted drugs, including antibody-drug conjugate, antibody and small molecule inhibitor. The outcome of this cross-resistance may be a clinically incurable condition. Importantly, we found that inhibiting Yes with dasatinib sensitised resistant cells in vitro and in vivo. CONCLUSIONS: Our study revealed that YES1 amplification conferred resistance to HER2-targeted drugs and suggested the potential application of the strategy of combining HER2 and Yes inhibition in the clinic.

Long non-coding TUG1 accelerates prostate cancer progression through regulating miR-128-3p/YES1 axis.

OBJECTIVE: Dysregulation of long non-coding RNAs (lncRNAs) is being found to have relevance to human cancers, including prostate cancer (PCa). Taurine-upregulated gene 1 (TUG1) has been demonstrated to have a potential oncogenic role in PCa. Then the aim of this study was to investigate the molecular mechanisms of TUG1 on PCa progression. PATIENTS AND METHODS: The expression levels of TUG1, YES proto-oncogene 1 (YES1) mRNA and miR-128-3p were assessed using quantitative real-time polymerase chain reaction. Cell proliferation ability, apoptosis, and migration and invasion capacities were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, flow cytometry and transwell assay, respectively. Western blot analysis was employed to evaluate the indicated proteins levels. The interaction between miR-128-3p and TUG1 or YES1 was determined using the Dual-Luciferase reporter assay. In vivo assay was used to observe the effect of TUG1 on tumor growth in vivo. RESULTS: Our data indicated that TUG1 was upregulated in PCa tissues and cells and predicted poor prognosis. TUG1 knockdown weakened PCa cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and accelerated cell apoptosis in vitro. Mechanistically, TUG1 directly interacted with miR-128-3p and miR-128-3p mediated the regulatory effects of TUG1 depletion on PCa cell progression. YES1 was a direct target of miR-128-3p and TUG1 modulated YES1 expression by sponging miR-128-3p. Moreover, TUG1 silencing repressed PCa cell progression in vitro through YES1. Additionally, TUG1 silencing mitigated tumor growth in vivo. CONCLUSIONS: Our study suggested that TUG1 silencing retarded PCa cell progression in vitro and tumor growth in vivo through miR-128-3p/YES1 axis, showing that targeting TUG1 might be a novel therapeutic strategy for PCa management.

YES1 Drives Lung Cancer Growth and Progression and Predicts Sensitivity to Dasatinib.

Rationale: The characterization of new genetic alterations is essential to assign effective personalized therapies in non-small cell lung cancer (NSCLC). Furthermore, finding stratification biomarkers is essential for successful personalized therapies. Molecular alterations of YES1, a member of the SRC (proto-oncogene tyrosine-protein kinase Src) family kinases (SFKs), can be found in a significant subset of patients with lung cancer.Objectives: To evaluate YES1 (v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1) genetic alteration as a therapeutic target and predictive biomarker of response to dasatinib in NSCLC.Methods: Functional significance was evaluated by in vivo models of NSCLC and metastasis and patient-derived xenografts. The efficacy of pharmacological and genetic (CRISPR [clustered regularly interspaced short palindromic repeats]/Cas9 [CRISPR-associated protein 9]) YES1 abrogation was also evaluated. In vitro functional assays for signaling, survival, and invasion were also performed. The association between YES1 alterations and prognosis was evaluated in clinical samples.Measurements and Main Results: We demonstrated that YES1 is essential for NSCLC carcinogenesis. Furthermore, YES1 overexpression induced metastatic spread in preclinical in vivo models. YES1 genetic depletion by CRISPR/Cas9 technology significantly reduced tumor growth and metastasis. YES1 effects were mainly driven by mTOR (mammalian target of rapamycin) signaling. Interestingly, cell lines and patient-derived xenograft models with YES1 gene amplifications presented a high sensitivity to dasatinib, an SFK inhibitor, pointing out YES1 status as a stratification biomarker for dasatinib response. Moreover, high YES1 protein expression was an independent predictor for poor prognosis in patients with lung cancer.Conclusions: YES1 is a promising therapeutic target in lung cancer. Our results provide support for the clinical evaluation of dasatinib treatment in a selected subset of patients using YES1 status as predictive biomarker for therapy.

Identification of type I and type II inhibitors of c-Yes kinase using in silico and experimental techniques.

c-Yes kinase is considered as one of the attractive targets for anti-cancer drug design. The DFG (Asp-Phe-Gly) motif present in most of the kinases will adopt active and inactive conformations, known as DFG-in and DFG-out and their inhibitors are classified into type I and type II, respectively. In the present study, two screening protocols were followed for identification of c-Yes kinase inhibitors. (i) Structure-based virtual screening (SBVS) and (ii) Structure-based (SB) and Pharmacophore-based (PB) tandem screening. In SBVS, the c-Yes kinase structure was obtained from homology modeling and seven ensembles with different active site scaffolds through molecular dynamics (MD) simulations. For SB-PB tandem screening, we modeled ligand bound active and inactive conformations. Physicochemical properties of inhibitors of Src kinase family and c-Yes kinase were used to prepare target focused libraries for screenings. Our screening procedure along with docking showed 520 probable hits in SBVS and tandem screening (120 and 400, respectively). Out of 5000 compounds identified from different computational methods, 2410 were examined using kinase inhibition assays. It includes 266 compounds (5.32%) identified from our method. We observed that 14 compounds (12%) are identified by the present method out of 168 that showed > 30% inhibition. Among them, three compounds are novel, unique, and showed good inhibition. Further, we have studied the binding of these compounds at the DFG-in and DFG-out conformations and reported the probable class (type I or type II). Hence, we suggest that these compounds could be novel drug leads for regulation of colorectal cancer.

An iterative compound screening contest method for identifying target protein inhibitors using the tyrosine-protein kinase Yes.

We propose a new iterative screening contest method to identify target protein inhibitors. After conducting a compound screening contest in 2014, we report results acquired from a contest held in 2015 in this study. Our aims were to identify target enzyme inhibitors and to benchmark a variety of computer-aided drug discovery methods under identical experimental conditions. In both contests, we employed the tyrosine-protein kinase Yes as an example target protein. Participating groups virtually screened possible inhibitors from a library containing 2.4 million compounds. Compounds were ranked based on functional scores obtained using their respective methods, and the top 181 compounds from each group were selected. Our results from the 2015 contest show an improved hit rate when compared to results from the 2014 contest. In addition, we have successfully identified a statistically-warranted method for identifying target inhibitors. Quantitative analysis of the most successful method gave additional insights into important characteristics of the method used.

Identification of potential inhibitors based on compound proposal contest: Tyrosine-protein kinase Yes as a target.

A search of broader range of chemical space is important for drug discovery. Different methods of computer-aided drug discovery (CADD) are known to propose compounds in different chemical spaces as hit molecules for the same target protein. This study aimed at using multiple CADD methods through open innovation to achieve a level of hit molecule diversity that is not achievable with any particular single method. We held a compound proposal contest, in which multiple research groups participated and predicted inhibitors of tyrosine-protein kinase Yes. This showed whether collective knowledge based on individual approaches helped to obtain hit compounds from a broad range of chemical space and whether the contest-based approach was effective.

The inhibition of SRC family kinase suppresses pancreatic cancer cell proliferation, migration, and invasion.

OBJECTIVES: Src is considered a rising therapeutic target for the treatment of solid tumors, and Src family kinases (SFKs) participate in cancer cell proliferation and survival. The role of SFK suppression was investigated in the proliferation, migration, and invasion of pancreatic cancer cells. METHODS: Knockdown of the SFKs in pancreatic cancer cells was achieved by transfecting small interfering RNAs, and its effects were investigated using proliferation, wound, and invasion assays. RESULTS: The SFK inhibitors suppressed proliferation and induced cell cycle arrest in pancreatic cancer cells. The SFK messenger RNA profiles showed that Yes1, Lyn, Fyn, Frk, Hck, and Src were expressed. Specific small interfering RNA transfection suppressed the messenger RNA expressions of Yes1, Lyn, Fyn, Frk, and Src, and the knockdown suppressed cell proliferation by 16.7% to 47.3% in PANC-1 cells. Knockdown of any of these 5 SFKs suppressed proliferation in other pancreatic cancer cell lines by 3.0% to 40.5%. The knockdowns significantly reduced pancreatic cancer cell migration by 24.9% to 66.7% and completely inhibited invasion. CONCLUSIONS: These results suggest that the knockdown of Yes1, Lyn, Fyn, Frk, or Src reduce human pancreatic cancer cell proliferation, migration, and invasion, and that SFKs should be viewed as critical therapeutic targets of pancreatic cancer.

Identification of potent Yes1 kinase inhibitors using a library screening approach.

Yes1 kinase has been implicated as a potential therapeutic target in a number of cancers including melanomas, breast cancers, and rhabdomyosarcomas. Described here is the development of a robust and miniaturized biochemical assay for Yes1 kinase that was applied in a high throughput screen (HTS) of kinase-focused small molecule libraries. The HTS provided 144 (17% hit rate) small molecule compounds with IC50 values in the sub-micromolar range. Three of the most potent Yes1 inhibitors were then examined in a cell-based assay for inhibition of cell survival in rhabdomyosarcoma cell lines. Homology models of Yes1 were generated in active and inactive conformations, and docking of inhibitors supports binding to the active conformation (DFG-in) of Yes1. This is the first report of a large high throughput enzymatic activity screen for identification of Yes1 kinase inhibitors, thereby elucidating the polypharmacology of a variety of small molecules and clinical candidates.

Loss-of-function screen in rhabdomyosarcoma identifies CRKL-YES as a critical signal for tumor growth.

To identify novel signaling pathways necessary for rhabdomyosarcoma (RMS) survival, we performed a loss-of-function screen using an inducible small hairpin RNA (shRNA) library in an alveolar and an embryonal RMS cell line. This screen identified CRKL expression as necessary for growth of alveolar RMS and embryonal RMS both in vitro and in vivo. We also found that CRKL was uniformly highly expressed in both RMS cell lines and tumor tissue. As CRKL is a member of the CRK adapter protein family that contains an SH2 and two SH3 domains and is involved in signal transduction from multiple tyrosine kinase receptors, we evaluated CRKL interaction with multiple tyrosine kinase receptor signaling pathways in RMS cells. While we saw no interaction of CRKL with IGFIR, MET or PI3KAKT/mTOR pathways, we determined that CRKL signaling was associated with SRC family kinase (SFK) signaling, specifically with YES kinase. Inhibition of SFK signaling with dasatinib or another SFK inhibitor, sarcatinib, suppressed RMS cell growth in vitro and in vivo. These data identify CRKL as a novel critical component of RMS growth. This study also demonstrates the use of functional screening to identify a potentially novel therapeutic target and treatment approach for these highly aggressive pediatric cancers.

ß-Catenin-driven cancers require a YAP1 transcriptional complex for survival and tumorigenesis.

Wnt/ß-catenin signaling plays a key role in the pathogenesis of colon and other cancers; emerging evidence indicates that oncogenic ß-catenin regulates several biological processes essential for cancer initiation and progression. To decipher the role of ß-catenin in transformation, we classified ß-catenin activity in 85 cancer cell lines in which we performed genome-scale loss-of-function screens and found that ß-catenin active cancers are dependent on a signaling pathway involving the transcriptional regulator YAP1. Specifically, we found that YAP1 and the transcription factor TBX5 form a complex with ß-catenin. Phosphorylation of YAP1 by the tyrosine kinase YES1 leads to localization of this complex to the promoters of antiapoptotic genes, including BCL2L1 and BIRC5. A small-molecule inhibitor of YES1 impeded the proliferation of ß-catenin-dependent cancers in both cell lines and animal models. These observations define a ß-catenin-YAP1-TBX5 complex essential to the transformation and survival of ß-catenin-driven cancers.

Differential effects on cell motility, embryonic stem cell self-renewal and senescence by diverse Src kinase family inhibitors.

The Src family of non-receptor tyrosine kinases (SFKs) has been shown to play an intricate role in embryonic stem (ES) cell maintenance. In the present study we have focused on the underlying molecular mechanisms responsible for the vastly different effects induced by various commonly used SFK inhibitors. We show that several diverse cell types, including fibroblasts completely lacking SFKs, cannot undergo mitosis in response to SU6656 and that this is caused by an unselective inhibition of Aurora kinases. In contrast, PP2 and PD173952 block motility immediately upon exposure and forces cells to grow in dense colonies. The subsequent halt in proliferation of fibroblast and epithelial cells in the center of the colonies approximately 24 h post-treatment appears to be caused by cell-to-cell contact inhibition rather than a direct effect of SFK kinase inhibition. Interestingly, in addition to generating more homogenous and dense ES cell cultures, without any diverse effect on proliferation, PP2 and PD173652 also promote ES cell self-renewal by reducing the small amount of spontaneous differentiation typically observed under standard ES cell culture conditions. These effects could not be mirrored by the use of Gleevec, a potent inhibitor of c-Abl and PDGFR kinases that are also inhibited by PP2.

Specific oncogenic activity of the Src-family tyrosine kinase c-Yes in colon carcinoma cells.

c-Yes, a member of the Src tyrosine kinase family, is found highly activated in colon carcinoma but its importance relative to c-Src has remained unclear. Here we show that, in HT29 colon carcinoma cells, silencing of c-Yes, but not of c-Src, selectively leads to an increase of cell clustering associated with a localisation of ß-catenin at cell membranes and a reduction of expression of ß-catenin target genes. c-Yes silencing induced an increase in apoptosis, inhibition of growth in soft-agar and in mouse xenografts, inhibition of cell migration and loss of the capacity to generate liver metastases in mice. Re-introduction of c-Yes, but not c -Src, restores transforming properties of c-Yes depleted cells. Moreover, we found that c-Yes kinase activity is required for its role in ß-catenin localisation and growth in soft agar, whereas kinase activity is dispensable for its role in cell migration. We conclude that c-Yes regulates specific oncogenic signalling pathways important for colon cancer progression that is not shared with c-Src.

Comprehensive screening of human genes with inhibitory effects on yeast growth and validation of a yeast cell-based system for screening chemicals.

To evaluate yeast as a high-throughput cell-based system for screening chemicals that may lead to drug development, 10,302 full-length human cDNAs (~50% of the total cDNAs) were introduced into yeast. Approximately 5.6% (583 clones) of the cDNAs repressed the growth of yeast. Notably, ~25% of the repressive cDNAs encoded uncharacterized proteins. Small chemicals can be readily surveyed by monitoring their restorative effects on the growth of yeast. The authors focused on protein kinases because protein kinases are involved in various diseases. Among 263 protein kinase cDNAs (~50% of the total) expressed in yeast, 60 cDNAs (~23%), including c-Yes, a member of the Src tyrosine kinase family, inhibited the growth of yeast. Known inhibitors for protein kinases were examined for whether they reversed the c-Yes-induced inhibition of the yeast growth. Among 85 inhibitors tested, 6 compounds (PP2, PP1, SU6656, purvalanol, radicicol, and geldanamycin) reversed the inhibition, indicating a high specificity sufficient for validating this screening system. Human c-Yes was found to interact with Hsc82, one of the yeast chaperones. Radicicol and geldanamycin probably exerted their actions through interactions with Hsc82. These results indicate that when human proteins requiring molecular chaperones for their activities are subjected to the yeast screening system, 2 groups of chemicals may be found. The actions of one group are exerted through direct interactions with the human proteins, whereas those of the other group are mediated through interactions with chaperones.

Activity of the multikinase inhibitor dasatinib against ovarian cancer cells.

BACKGROUND: Here, we explore the therapeutic potential of dasatinib, a small-molecule inhibitor that targets multiple cytosolic and membrane-bound tyrosine kinases, including members of the Src kinase family, EphA2, and focal adhesion kinase for the treatment of ovarian cancer. METHODS: We examined the effects of dasatinib on proliferation, invasion, apoptosis, cell-cycle arrest, and kinase activity using a panel of 34 established human ovarian cancer cell lines. Molecular markers for response prediction were studied using gene expression profiling. Multiple drug effect/combination index (CI) isobologram analysis was used to study the interactions with chemotherapeutic drugs. RESULTS: Concentration-dependent anti-proliferative effects of dasatinib were seen in all ovarian cancer cell lines tested, but varied significantly between individual cell lines with up to a 3 log-fold difference in the IC(50) values (IC(50) range: 0.001-11.3 micromol l(-1)). Dasatinib significantly inhibited invasion, and induced cell apoptosis, but less cell-cycle arrest. At a wide range of clinically achievable drug concentrations, additive and synergistic interactions were observed for dasatinib plus carboplatin (mean CI values, range: 0.73-1.11) or paclitaxel (mean CI values, range: 0.76-1.05). In this study, 24 out of 34 (71%) representative ovarian cancer cell lines were highly sensitive to dasatinib, compared with only 8 out of 39 (21%) representative breast cancer cell lines previously reported. Cell lines with high expression of Yes, Lyn, Eph2A, caveolin-1 and 2, moesin, annexin-1, and uPA were particularly sensitive to dasatinib. CONCLUSIONS: These data provide a clear biological rationale to test dasatinib as a single agent or in combination with chemotherapy in patients with ovarian cancer.

Mechanotransduction in an extracted cell model: Fyn drives stretch- and flow-elicited PECAM-1 phosphorylation.

Mechanosensing followed by mechanoresponses by cells is well established, but the mechanisms by which mechanical force is converted into biochemical events are poorly understood. Vascular endothelial cells (ECs) exhibit flow- and stretch-dependent responses and are widely used as a model for studying mechanotransduction in mammalian cells. Platelet EC adhesion molecule 1 (PECAM-1) is tyrosine phosphorylated when ECs are exposed to flow or when PECAM-1 is directly pulled, suggesting that it is a mechanochemical converter. We show that PECAM-1 phosphorylation occurs when detergent-extracted EC monolayers are stretched, indicating that this phosphorylation is mechanically triggered and does not require the intact plasma membrane and soluble cytoplasmic components. Using kinase inhibitors and small interfering RNAs, we identify Fyn as the PECAM-1 kinase associated with the model. We further show that stretch- and flow-induced PECAM-1 phosphorylation in intact ECs is abolished when Fyn expression is down-regulated. We suggest that PECAM-1 and Fyn are essential components of a PECAM-1-based mechanosensory complex in ECs.

Abl collaborates with Src family kinases to stimulate actin-based motility of vaccinia virus.

Local activation of Src at the plasma membrane by extracellular vaccinia virus results in a signalling cascade that acts to stimulate actin polymerization beneath the virus to enhance its cell-to-cell spread. Initiation of this signalling cascade involves Src-mediated phosphorylation of tyrosine 112 and 132 of the viral membrane protein A36R. Here we show that recruitment of Src is dependent on its myristoylation and an interaction with A36R upstream of tyrosine 112 and 132. We further show that Src, Fyn and Yes have unique specificities towards these tyrosine residues. Using cell lines deficient in Src, Fyn and Yes, we demonstrate that multiple Src family members can stimulate vaccinia-induced actin polymerization and also uncover a role for Abl family kinases. Additionally, Abl and Arg are able to phosphorylate A36R in vitro and are recruited to vaccinia-induced actin tails. The ability of multiple families of tyrosine kinases to directly phosphorylate A36R ensures robust cell-to-cell spread of vaccinia virus will occur under a variety of cellular conditions.

Src family kinase members have a common response to histone deacetylase inhibitors in human colon cancer cells.

Histone deacetylase inhibitors (HDIs) induce cell cycle arrest, differentiation and/or apoptosis in numerous cancer cell types and have shown promise in clinical trials. These agents are particularly novel, given their ability to selectively influence gene expression. Previously, we demonstrated that the HDIs butyrate and trichostatin A (TSA) directly repress c-Src proto-oncogene expression in many cancer cell lines. Activation and/or overexpression of c-Src have been frequently observed in numerous malignancies, especially of the colon. Therefore, our observation was particularly interesting since butyrate is a naturally abundant component of the large intestine and has been suggested to be a cancer-preventive agent. However, c-Src is not the only Src family kinase (SFK) member to be implicated in the development of human cancers, including those of the colon. Therefore, the relative expression levels of known SFKs were examined in a panel of human colon cancer cell lines. We found a surprisingly diverse expression pattern but noted that most cell lines expressed relatively high levels of at least 2 SFKs. When the effects of butyrate and TSA were examined in representative cell lines, the expression of all SFKs was repressed in a dose- and time-dependent manner. Further, detailed examination of Lck, Yes and Lyn demonstrated that this repression had a direct effect on transcription and was independent of new protein synthesis. These results mirror our earlier data obtained with c-Src and suggest that SFKs are a major target of HDIs and likely account in part for the anticancer effects of these promising new drugs.