Src Oncogene Research Articles

Src is activated by the nuclear receptor peroxisome proliferator‐activated receptor β/δ in ultraviolet radiation‐induced skin cancer

Although non-melanoma skin cancer (NMSC) is the most common human cancer and its incidence continues to rise worldwide, the mechanisms underlying its development remain incompletely understood. Here, we unveil a cascade of events involving peroxisome proliferator-activated receptor (PPAR) β/δ and the oncogene Src, which promotes the development of ultraviolet (UV)-induced skin cancer in mice. UV-induced PPARβ/δ activity, which directly stimulated Src expression, increased Src kinase activity and enhanced the EGFR/Erk1/2 signalling pathway, resulting in increased epithelial-to-mesenchymal transition (EMT) marker expression. Consistent with these observations, PPARβ/δ-null mice developed fewer and smaller skin tumours, and a PPARβ/δ antagonist prevented UV-dependent Src stimulation. Furthermore, the expression of PPARβ/δ positively correlated with the expression of SRC and EMT markers in human skin squamous cell carcinoma (SCC), and critically, linear models applied to several human epithelial cancers revealed an interaction between PPARβ/δ and SRC and TGFβ1 transcriptional levels. Taken together, these observations motivate the future evaluation of PPARβ/δ modulators to attenuate the development of several epithelial cancers.

P120catenin alteration in cancer and its role in tumour invasion

Since its discovery in 1989 as a substrate of the Src oncogene, p120catenin has been revealed as an important player in cancer initiation and tumour dissemination. p120catenin regulates a wide range of cellular processes such as cell-cell adhesion, cell polarity and cell proliferation and plays a pivotal role in morphogenesis, inflammation and innate immunity. The pleiotropic effects of p120catenin rely on its interactions with numerous partners such as classical cadherins at the plasma membrane, Rho-GTPases and microtubules in the cytosol and transcriptional modulators in the nucleus. Alterations of p120catenin in cancer not only concern its expression level but also its intracellular localization and can lead to both pro-invasive and anti-invasive effects. This review focuses on the p120catenin-mediated pathways involved in cell migration and invasion and discusses the potential consequences of major cancer-related p120catenin alterations with respect to tumour spread.

SRChing for the substrates of Src

By the mid 1980's, it was clear that the transforming activity of oncogenic Src was linked to the activity of its tyrosine kinase domain and attention turned to identifying substrates, the putative next level of control in the pathway to transformation. Among the first to recognize the potential of phosphotyrosine-specific antibodies, Parsons and colleagues launched a risky shotgun-based approach that led ultimately to the cDNA cloning and functional characterization of many of today's best-known Src substrates (for example, p85-Cortactin, p110-AFAP1, p130Cas, p125FAK and p120-catenin). Two decades and over 6000 citations later, the original goals of the project may be seen as secondary to the enormous impact of these protein substrates in many areas of biology. At the request of the editors, this review is not restricted to the current status of the substrates, but reflects also on the anatomy of the project itself and some of the challenges and decisions encountered along the way.

Chemopreventive activity of plant flavonoid isorhamnetin in colorectal cancer is mediated by oncogenic Src and β-catenin.

Analysis of the Polyp Prevention Trial showed an association between an isorhamnetin-rich diet and a reduced risk of advanced adenoma recurrence; however, the mechanism behind the chemoprotective effects of isorhamnetin remains unclear. Here, we show that isorhamnetin prevents colorectal tumorigenesis of FVB/N mice treated with the chemical carcinogen azoxymethane and subsequently exposed to colonic irritant dextran sodium sulfate (DSS). Dietary isorhamnetin decreased mortality, tumor number, and tumor burden by 62%, 35%, and 59%, respectively. MRI, histopathology, and immunohistochemical analysis revealed that dietary isorhamnetin resolved the DSS-induced inflammatory response faster than the control diet. Isorhamnetin inhibited AOM/DSS-induced oncogenic c-Src activation and β-catenin nuclear translocation, while promoting the expression of C-terminal Src kinase (CSK), a negative regulator of Src family of tyrosine kinases. Similarly, in HT-29 colon cancer cells, isorhamnetin inhibited oncogenic Src activity and β-catenin nuclear translocation by inducing expression of csk, as verified by RNA interference knockdown of csk. Our observations suggest the chemoprotective effects of isorhamnetin in colon cancer are linked to its anti-inflammatory activities and its inhibition of oncogenic Src activity and consequential loss of nuclear β-catenin, activities that are dependent on CSK expression.

Ras and macropinocytosis: trick and treat

Oncogene-driven adaptation of metabolism during tumorigenesis includes steps that stimulate the uptake of nutrients, especially glucose and glutamine, to sustain cell growth and proliferation. Macropinocytosis, a clathrin- and caveolin-independent endocytotic process that had previously been linked to the action of oncogenic Ras and Src, is now shown to contribute to amino acid uptake via enhanced delivery of extracellular proteins to lysosomes.

Modulation of natural killer cell effector functions through lenalidomide/dasatinib and their combined effects against multiple myeloma cells

The multikinase inhibitor dasatinib blocks the constitutive activation of oncogenic Src kinases in multiple myeloma (MM) cells and potentially enhances natural killer (NK) cell activity. Therefore, we tested combination effects of dasatinib and lenalidomide regarding MM cell viability and NK cell effector functions. The drug combination mostly had little influence on the viability of MM cell lines, and produced mixed results on primary MM cells. Prolonged lenalidomide treatment enhanced NK cell effector functions, and dasatinib addition at late stages of NK cell expansion increased levels of CD107a/b and interferon-γ (IFNγ), but not of tumor necrosis factor-α (TNFα). Additive effects were observed for the enhancement of cytokine production and degranulation, but only lenalidomide increased NK cell cytotoxicity against MM cells. This effect correlated with increased TNF-related apoptosis-inducing ligand (TRAIL) expression and was attenuated by dasatinib, or suppressors of TRAIL or TNFα. Our data thus indicate a functional role for the TRAIL/TRAIL-R system in lenalidomide-mediated NK-cell activity against MM cells, but also show that dasatinib is unsuitable to support or boost this effect.

Differential anti-tumor activities of curcumin against Ras- and Src-activated human adenocarcinoma cells

Although curcumin has been studied as a potential anticancer drug targeting multiple signaling molecules, the role of oncogenic Src and Ras in curcumin sensitivity remains unknown. Using HAG-1 human adenocarcinoma cells transfected with either activated Src or Ras, we investigated here the functional role of these oncogenes in curcumin sensitivity. Activation of either Src or Ras did not confer resistance to curcumin, compared to vehicle-transfected cells. Curcumin enhanced Erk1/2 predominantly in Ras-activated cells, but inhibited Akt and its downstream molecules (mTOR and S6K1) regardless of these oncogene activations. The sub-G0/G1 apoptotic populations were substantially increased with demonstrable cleavage of PARP, but this increase was most prominent in Src-activated cells. Suppression of Bcl-xL level and enhanced expression of Bax were demonstrated in Src-activated, but not Ras-activated cells. By contrast, drastic increases of G2/M cell populations were seen in Ras-activated cells rather than Src-activated cells, suggesting a potential role of Ras/Erk1/2 activation in curcumin-induced G2/M arrest. These data indicate that curcumin-induced growth inhibition would be mediated mainly by G2/M arrest in Ras-driven cells but by apoptosis induction in Src-driven cells, providing a mechanistic rationale for the potential use of curcumin in the treatment of human cancers with activated Src or Ras.

A randomized phase II study of cediranib (CED) alone versus CED plus dasatinib (DAS) in patients (pts) with castration-resistant prostate cancer (CRPC).

5039 Background: Activation of the Vascular Endothelial Growth Factor Receptor (VEGFR) and the oncogenic Src pathway has been implicated in the development of CRPC in preclinical models. CED and DAS are multi-kinase inhibitors targeting VEGFR and Src respectively. Phase II studies of CED (Karakunnel et al ASCO 2009) and DAS (Yu et al Urology 2011) in CRPC have shown single agent activity. Methods: Docetaxel-preteated CRPC pts were randomized to arm A: CED alone (20 mg/day) vs arm B: CED (20 mg/day) plus DAS (100 mg/day) given orally on 4-week cycles. Primary endpoint was 12 week progression-free survival (PFS) as per the Prostate Cancer Clinical Trials Working Group (PCWG2). Patient reported outcomes were evaluated using Functional Assessment of Cancer Therapy-Prostate (FACT-P) and Present Pain Intensity (PPI) scales. Correlative studies of bone turnover markers (BTM), including bone alkaline phosphate (BAP) and serum beta-C telopeptide (B-CTx) were serially assayed. Results: 22 patients, 11 per arm, were enrolled. Baseline demographics were similar in both arms. Median number of cycles = 4 in arm A (range 1-12) and 2 in arm B (range 1-9). Twelve-week PFS was 73 % in arm A vs 18 % in arm B (p = 0.03). Median PFS in months (arm A vs B) was: 5.2 vs 2.6 (95% CI: 1.9-6.5 vs 1.4-not reached). Most common grade 3 toxicities were hypertension, anemia and thrombocytopenia in arm A and hypertension, diarrhea and fatigue in arm B. One treatment-related death (retroperitoneal hemorrhage) was seen in arm A. FACT-P and PPI scores did not significantly change in either arm. No correlation between BTM and PFS was seen in both arms A and B. Conclusions: Although limited by small numbers, this randomized study showed that the combination of VEGFR and Src targeted therapy did not result in improved efficacy and may be associated with a worse outcome than VEGFR targeted therapy alone in pts with CRPC. Clinical trial information: NCT01260688.

FGFR4 Role in Epithelial-Mesenchymal Transition and Its Therapeutic Value in Colorectal Cancer

Fibroblast growth factor receptor 4 (FGFR4) is vital in early development and tissue repair. FGFR4 expression levels are very restricted in adult tissues, except in several solid tumors including colorectal cancer, which showed overexpression of FGFR4. Here, FGFR4 mutation analysis discarded the presence of activating mutations, other than Arg388, in different colorectal cancer cell lines and tumoral samples. Stable shRNA FGFR4-silencing in SW480 and SW48 cell lines resulted in a significant decrease in cell proliferation, adhesion, cell migration and invasion. This decrease in the tumorigenic and invasive capabilities of colorectal cancer cells was accompanied by a decrease of Snail, Twist and TGFβ gene expression levels and an increase of E-cadherin, causing a reversion to a more epithelial phenotype, in three different cell lines. In addition, FGFR4-signaling activated the oncogenic SRC, ERK1/2 and AKT pathways in colon cancer cells and promoted an increase in cell survival. The relevance of FGFR4 in tumor growth was supported by two different strategies. Kinase inhibitors abrogated FGFR4-related cell growth and signaling pathways at the same extent than FGFR4-silenced cells. Specific FGFR4-targeting using antibodies provoked a similar reduction in cell growth. Moreover, FGFR4 knock-down cells displayed a reduced capacity for in vivo tumor formation and angiogenesis in nude mice. Collectively, our data support a crucial role for FGFR4 in tumorigenesis, invasion and survival in colorectal cancer. In addition, FGFR4 targeting demonstrated its applicability for colorectal cancer therapy.

Abstract 4284: Cadherin-11 function is required for full neoplastic transformation of mouse fibroblasts by v-Src.

Abstract The signal transducer and activator of transcription-3 (Stat3) is an important signal transducer with a role in cancer etiology. Following activation of a number of growth factor or cytokine receptors or expression of oncogenes such as vSrc, Stat3 is phosphorylated at tyr705, dimerizes and migrates to the nucleus where it activates transcription from a number of genes involved in cell division and survival. We recently discovered a novel pathway of activation of Stat3: Engagement of cadherin-11 (cad11), a cell to cell adhesion protein, known to be involved in metastasis, induces a dramatic increase in the levels of the Rac and Cdc42 small GTPases, and this leads to Stat3 activation. Stat3 in turn, downregulates the p53 antioncogene and upregulates anti-apoptotic genes, thus offering a potent survival signal. A large number of oncogenes such as vSrc activate the E2F transcription factor family, which induce the expression of genes involved in cell division. However, at the same time E2F is a potent apoptosis inducer, hence the high demand of transformed cells for anti-apoptotic signals. Since cad11 activates Stat3, a potent survival factor, we examined the effect of downregulation of cad11 upon transformation of mouse Balb/c3T3 fibroblasts by vSrc. Forced expression of high levels of activated Src caused a dramatic reduction in cad11 levels. However, intermediate Src levels, which are physiologically relevant, did allow cad11 expression, albeit at lower levels. Cad11 knockdown in these cells through shRNA expression with a retroviral vector caused a dramatic reduction in Stat3 activity and induced apoptosis. That is, the residual amounts of cad11 present are sufficient to activate Stat3 and prevent apoptosis induced by the Src/E2F axis. Taken together, these findings indicate that cad11, despite its presumed structural role, and in addition to its known role in promoting metastasis, is actually also necessary for full neoplastic transformation by the Src oncogene. Citation Format: Stephanie Guy, Mulu Geletu, Rozanne Arulanandam, Leda Raptis. Cadherin-11 function is required for full neoplastic transformation of mouse fibroblasts by v-Src. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4284. doi:10.1158/1538-7445.AM2013-4284

Abstract 4934: The role of Aurora A kinase-dependent phosphorylation of cortactin in invadopodia formation.

Abstract Aurora A kinase (AURKA) is a serine/threonine kinase known for its critical role in regulation of cell cycle progression. Overexpression and activation of AURKA is implicated in development of multiple types of human cancer. AURKA upregulation is considered as independent prognostic marker for breast cancer patients and associated with poor survival outcome. Recent work by our laboratory and others suggests a new role for AURKA in promotion of tumor cells migration and invasion through regulation of actin dynamics. However, the specific manner in which AURKA regulates actin-based machinery of migrating cancer cells is unknown. In our current work we show that phosphorylation of cortactin (CTTN) by AURKA directly regulates actin dynamics in breast cancer cells. CTTN is known to be regulated by oncogenic Src kinase. This phosphorylation is pivotal for formation of free barbed ends and further actin polymerization. Dynamic polymerization/depolimerization is required for invasion of cancer cells. Thus inhibition of Src-dependant phosphorylation of CTTN is critical for invadopodia maturation. In our current work we identify the phosphorylation site on CTTN targeted by AURKA and show that phosphorylation of CTTN by AURKA could potentially inhibit further phosphorylation of CTTN by Src leading to stabilization of invadopodia. Inhibition of AURKA activity or depletion of AURKA in cancer cells led to decrease in invasion and invadopodia maturation. In addition, we showed that adaptor protein NEDD9 which is known to bind both AURKA and Src also binds to CTTN and therefore potentially can mediate Src-CTTN-AURKA complex formation that regulates the sequential phosphorylation events necessary for invadopodia genesis. It is our expectation that AURKA-phosphorylated CTTN will inhibit Src-driven tyrosine-phosphorylation of CTTN, regulating a mechanistic switch between complementary actin polymerization and actin stabilization programs. Citation Format: Varvara K. Kozyreva, Laura Kelley, Sarah L. McLaughlin, Elena N. Pugacheva. The role of Aurora A kinase-dependent phosphorylation of cortactin in invadopodia formation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4934. doi:10.1158/1538-7445.AM2013-4934

Involvement of Src and the actin cytoskeleton in the antitumorigenic action of adenosine dialdehyde

Transmethylation is an important reaction that transfers a methyl group in S-adenosylmethionine (SAM) to substrates such as DNA, RNA, and proteins. It is known that transmethylation plays critical roles in various cellular responses. In this study, we examined the effects of transmethylation on tumorigenic responses and its regulatory mechanism using an upregulation strategy of adenosylhomocysteine (SAH) acting as a negative feedback inhibitor. Treatment with adenosine dialdehyde (AdOx), an inhibitor of transmethylation-suppressive adenosylhomocysteine (SAH) hydrolase (SAHH), enhanced the level of SAH and effectively blocked the proliferation, migration, and invasion of cancer cells; the treatment also induced the differentiation of C6 glioma cells and suppressed the neovascular genesis of eggs in a dose-dependent manner. Through immunoblotting analysis, it was found that AdOx was capable of indirectly diminishing the phosphorylation of oncogenic Src and its kinase activity. Interestingly, AdOx disrupted actin cytoskeleton structures, leading to morphological changes, and suppressed the formation of a signaling complex composed of Src and p85/PI3K, which is linked to various tumorigenic responses. In agreement with these data, the exogenous treatment of SAH or inhibition of SAHH by specific siRNA or another type of inhibitor, 3-deazaadenosine (DAZA), similarly resulted in antitumorigenic responses, suppressive activity on Src, the alteration of actin cytoskeleton, and a change of the colocalization pattern between actin and Src. Taken together, these results suggest that SAH/SAHH-mediated transmethylation could be linked to the tumorigenic processes through cross-regulation between the actin cytoskeleton and Src kinase activity.

Chronic nicotine inhibits the therapeutic effects of gemcitabine on pancreatic cancer in vitro and in mouse xenografts

Aim of studySmoking is an established risk factor for pancreatic cancer and nicotine replacement therapy (NRT) often accompanies chemotherapy. The current study has tested the hypothesis that chronic exposure to low dose nicotine reduces the responsiveness of pancreatic cancer to the leading therapeutic for this cancer, gemcitabine. MethodsThe effects of chronic nicotine (1μm/L) on two pancreatic cancer cell lines in vitro and in a xenograft model were assessed by immunoassays, Western blots and cell proliferation assays. ResultsExposure in vitro to nicotine for 7days inhibited the gemcitabine-induced reduction in viable cells, gemcitabine-induced apoptosis as indicated by reduced expression of cleaved caspase-3 while inducing the phosphorylation of signalling proteins extracellular signal-regulated kinase (ERK), v-akt thymoma viral oncogene homolog (protein kinase B, AKT) and Src. Nicotine (1μm/L) in the drinking water for 4weeks significantly reduced the therapeutic response of mouse xenografts to gemcitabine while reducing the induction of cleaved caspase-3 and the inhibition of phosphorylated forms of multiple signalling proteins by gemcitabine in xenograft tissues. ConclusionsOur experimental data suggest that continued moderate smoking and NRT may negatively impact therapeutic outcomes of gemcitabine on pancreatic cancer and that clinical studies in cancer patients are now warranted.

Involvement of Src in the Adaptation of Cancer Cells under Microenvironmental Stresses

Protein-tyrosine phosphorylation, which is catalyzed by protein-tyrosine kinase (PTK), plays a pivotal role in a variety of cellular functions related to health and disease. The discovery of the viral oncogene Src (v-Src) and its cellular nontransforming counterpart (c-Src), as the first example of PTK, has opened a window to study the relationship between protein-tyrosine phosphorylation and the biology and medicine of cancer. In this paper, we focus on the roles played by Src and other PTKs in cancer cell-specific behavior, that is, evasion of apoptosis or cell death under stressful extracellular and/or intracellular microenvironments (i.e., hypoxia, anoikis, hypoglycemia, and serum deprivation).

Adhesion-mediated cytoskeletal remodeling is controlled by the direct scaffolding of Src from FAK complexes to lipid rafts by SSeCKS/AKAP12

Metastatic cell migration and invasion are regulated by altered adhesion-mediated signaling to the actin-based cytoskeleton via activated Src-FAK complexes. SSeCKS (the rodent orthologue of human Gravin/AKAP12), whose expression is downregulated by oncogenic Src and in many human cancers, antagonizes oncogenic Src pathways including those driving neovascularization at metastatic sites, metastatic cell motility and invasiveness. This is likely manifested through its function as a scaffolder of F-actin and signaling proteins such as cyclins, calmodulin, protein kinase (PK) C and PKA. Here, we show that in contrast to its ability to inhibit haptotaxis, SSeCKS increased prostate cancer cell adhesion to fibronectin (FN) and type I collagen in a FAK-dependent manner, correlating with a relative increase in FAKpoY397 levels. In contrast, SSeCKS suppressed adhesion-induced Src activation (SrcpoY416) and phosphorylation of FAK at Y925, a known Src substrate site. SSeCKS also induced increased cell spreading, cell flattening, integrin β1 clustering and formation of mature focal adhesion plaques. An in silico analysis identified a Src-binding domain on SSeCKS (a.a.153–166) that is homologous to the Src binding domain of Caveolin-1, and this region is required for SSeCKS-Src interaction, for SSeCKS-enhanced Src activity and sequestration to lipid rafts, and for SSeCKS-enhanced adhesion of MAT-LyLu and CWR22Rv1 prostate cancer cells. Our data suggest a model in which SSeCKS suppresses oncogenic motility by sequestering Src to caveolin-rich lipid rafts, thereby disengaging Src from FAK-associated adhesion and signaling complexes.

High Expression of IL-13 Receptor α2 in Colorectal Cancer Is Associated with Invasion, Liver Metastasis, and Poor Prognosis

Autocrine secretion of cytokines by metastatic colorectal cancer cells and their role during invasion and liver homing has been poorly characterized. In this study, we used cytokine arrays to analyze the secretomes of poorly and highly metastatic colorectal cancer cells. Compared with poorly metastatic cancer cells, highly metastatic cells expressed increased levels of the immunosuppressive cytokines interleukin (IL)-4 and IL-13 in addition to increased surface expression of the high affinity IL-13 receptor IL-13Rα2, suggesting that IL-13Rα2 mediates IL-13 effects in colorectal cancer cells. Silencing of IL-13Rα2 in highly metastatic cells led to a decrease in adhesion capacity in vitro and a reduction in liver homing and increased survival in vivo, revealing a role for this receptor in cell adhesion, migration, invasion, and metastatic colonization. In support of this, IL-13 signaling activated the oncogenic signaling molecules phosphoinositide 3-kinase, AKT, and SRC in highly metastatic cells. Clinically, high expression of IL-13Rα2 was associated with later stages of disease progression and poor outcome in patients with colorectal cancer. Our findings therefore support a critical role for IL-13Rα2 expression in colon cancer invasion and metastasis.

Abstract 1236: Ephrin signaling in medulloblastoma

Abstract Eph receptor and ephrin ligand expression profiles have been associated with poor outcome in several malignancies, but to date, there have been no comprehensive studies characterizing Eph/ephrin expression and signaling function in medulloblastoma (MB), the most common malignant brain tumor of childhood. Determining if Eph receptors and ephrin ligands have a functional role in MB tumorigenesis would be important to our understanding of the molecular regulation of MB and for identifying more effective and less toxic targeted therapies for MB. A few studies have shown that the level of specific Eph receptor mRNA expression and Eph tyrosine kinase activity appears to be increased in MB, particularly in those subtypes with a propensity to metastasize. However, to our knowledge, this is the first study to comprehensively characterize the ephrin receptor and ligand expression profile (mRNA and protein) of MB tumors and to demonstrate the functional effects of ephrin signaling in medulloblastoma cells in vitro. We hypothesize that interactions between specific Eph receptor and ligands results in bidirectional signaling that is critical for MB cell proliferation and migration. We have generated profiles characterizing Eph receptor and cognate ligand expression at the mRNA and protein level in two medulloblastoma cell lines and in 29 (mRNA) and 60 (protein) human medulloblastoma tissues. Based on our data, the candidates of interest for functional characterization are Eph receptors B1 and B2, expressed in over 98% of tumors, and the cognate ephrin ligands b1 and b2. Of these targets, only ephrin b2 was detected in developing fetal cerebellum, with positive expression by IHC restricted to the purkinje cells, suggesting that these ephrin family members are tumor-specific. To assess the functional role of these molecules in MB cells, we knocked down ephrin b2 and ephrin b1 using siRNA. Cells with altered Eph/ephrin expression were then compared to control transfectants for the ability to signal downstream through Src and for changes in cell function using bioassays of proliferation (MTT) and migration (scratch assay). We demonstrate that ephrin b2 is diffusely expressed in 100% of medulloblastoma tumors analyzed, signals through src oncogene, and promotes MB cell proliferation. In contrast, ephrin b1 is heterogeneously expressed in 20% of tumors analyzed, localizes to cells with high MIB proliferative index within the tumor that are associated with an aggressive phenotype of medulloblastoma, and promotes MB cell migration. Together, these data indicate that ephrin b1 and b2 have distinct and differential functional roles in the regulation of Eph signaling and cellular behavior in MB cells. Further preclinical investigation of these specific targets in MB in vivo is warranted to validate these Eph family members as potential novel therapeutic targets in MB. The complete tumor expression profiles and functional data of altered Eph/ephrin expression in MB cells will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1236. doi:1538-7445.AM2012-1236

Abstract 4790: Mapping the oncogenic kinome using a kinase activity screening array

Abstract The oncogenic activity of kinases harnesses myriad signalling cascades driving tumorigenesis. Improper control of phosphorylation networks represents one of the most relevant regulator and biomarker of cell dys-functions leading to malignancies. Surprisingly however, measuring the phosphorylation activity of kinases, and potentially monitoring the functionality of the entire kinome at once to identify molecular signatures causing cancers and guide therapeutic interventions, remains largely unexplored. Specifically, a highly convenient tool for clinical applications or research purposes would be the advent of a microarray-like chip to map phosphorylation networks. We undertook to develop such a kinase-sensing platform, and present here unique strategies, technical advances and data -including the detection of oncogenic Src and HER kinases activity in biological samples- supporting such potential breakthrough. The devised kinase activity-sensing system relies on peptide probes that act as beacons to evaluate enzymes functionality, and emit distinctive optical signals before and after (de-)phosphorylation. The following steps have been achieved. First, in order to use baits relevant to human cancer etiology, we created a protein and peptide repertoire cataloguing 6173 distinctive kinase-substrate active nodes and compiling 2702 unique biological kinase peptide targets that all represent latent nano-sensors usable to comprehensively track kinase signaling networks. Second, we established that our label-free detection approach (namely Raman scattering of probes monitored via surface-enhanced Raman spectroscopy (SERS)) is a functional system that reliably and robustly identifies the phosphorylation state of peptide probes. Third, real-time kinetics and kinase inhibitor experiments using biochemical samples and biological extracts from mammalian cells demonstrated that our approach could directly assess the functionality of kinases. Fourth, in an attempt to identify hyperactive Src kinase in tumor cells, our assay discerned normal from oncogenic Src-mutated cells. Finally, using breast cancer cell models of tumor progression and tissue samples from patients, we were able to measure EGFR kinases (hyper-)activity in vivo. Together, our strategy allows for highly sensitive, specific, rapid, one-step, label-free, multiplex capture and measurement of phosphorylation events. Our efforts will now focus on expending this approach into a fully functional kinomic-screening platform to explore oncogenic kinase networks in human cancers. We will translate such device into the clinic, and map phosphorylation signatures that cause breast malignancies to eventually guide therapeutic interventions that best match breast cancer patients’ disease characteristics and predicted responses to (chemo-)therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4790. doi:1538-7445.AM2012-4790

Abstract 2303: MicroRNA-23b is a tumor suppressor and a useful prognostic biomarker in prostate cancer

Abstract Introduction and Objective: MicroRNAs are small noncoding RNAs that regulate the expression of >90% of all human genes, either inhibiting target mRNA translation or inducing its degradation. These genes are abnormally expressed in human malignancies, making their biological importance increasingly apparent. The main objective of this study was to investigate the role of miRNA-23b in prostate carcinogenesis and assess whether miR-23b has any prognostic implication in prostate cancer (PC). Methods: The methods employed in this study include quantitative-real time PCR; methylation specific quantitative-real time PCR; western blot; in-situ hybridization; fluorescence-activated cell sorting assays for cell cycle distribution and apoptosis; assays for cell viability, clonability, migration and invasiveness of prostate cancer cells. Luciferase reporter and mutational assays and in-vivo studies in nude mice were also performed. Results: The expression of miR-23b was significantly suppressed or silenced in human PC tissue samples and cell lines when compared with normal tissues and a non-malignant cell line. The mechanism of miR-23b silencing in PC was found to be due to hypermethylation that was reversed by 5Aza-deoxycitidine (5Aza) treatments. Lower expression of miR-23b also correlated with higher pathological stage and higher Gleason grade making it a useful prognostic biomarker in prostate cancer. Ectopic expression of miR-23b induced G0/G1 cell cycle arrest and apoptosis, suppressed cell proliferation, colony formation, migration, and invasion in prostate cancer cells. miR-23b exerted these functional effects by directly targeting the proto-oncogene Src kinase. Over-expression of miR-23b significantly suppressed the luciferase activity of reporter plasmid containing the 3′UTR sequences complementary to Src kinase, which was abolished by mutations in these 3′UTR regions. Genistein treatment also induced cell cycle arrest and apoptosis, and suppressed Src kinase expression at the protein level in prostate cancer cells. Genistein and miR-23b reduced the Src-regulated downstream ERK/AKT pathway molecules and also inhibited Src kinase and tumor cell growth in vivo. Conclusion: This is the first study demonstrating that i) miRNA-23b is silenced by CpG hypermethylation in PC; ii) miR-23b acts as a tumor suppressor and is a prognostic biomarker in PC; iii) genistein, a nontoxic chemopreventive agent induces the expression of miR-23b and iv) both genistein and miR-23b inhibited oncogenic Src kinase in-vitro and in-vivo. The use of RNAi is currently being implemented as a gene-specific approach for molecular medicine. By the same principle, it is possible that reexpression of silenced miR-23b by nontoxic chemopreventive agents may cause down-regulation of target oncogenes thereby leading to the development of novel therapeutic approaches for the treatment of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2303. doi:1538-7445.AM2012-2303

Abstract 4957: Mixed Lineage Kinase 3 mediates vanadium-induced cell death in cancer cells

Abstract Vanadium is a transition metal and occurs naturally in a variety of minerals. In mammals, vanadium has been shown to promote cancer cell death and can also mimic insulin action, most probably through the inhibition of cellular tyrosine phosphatase and subsequent activation of signaling pathways. The mechanism by which vanadium causes cancer cell death is still not known. Interestingly, it has been shown that various mitogen activated protein kinase (MAPK) members are activated upon vanadium treatment. However, the contribution of any specific MAPK member that mediates vanadium-induced cell death has not been clearly delineated. It has been reported that JNK is activated by vanadium and therefore we investigated the role of Mixed Lineage Kinase 3 (MLK3) on vanadium-induced cell death. MLK3 is a MAP3K member that is known to specifically activate JNK pathway. We have earlier reported that MLK3 activation promotes cell death in cancer and neuronal cells. Our results demonstrated that endogenous MLK3 was activated by vanadium and MLK3 was highly phosphorylated on tyrosine residues. We also identified Src as a tyrosine kinase that phosphorylated MLK3 on specific tyrosine residues and phosphorylation of these tyrosine residues by Src was essential for MLK3 activation. Vanadium-induced cancer cell death was mediated by MLK3 or MLKs, because CEP-1347, a pan MLK inhibitor, prevented this cell death. Furthermore, the tyrosine phospho-deficient mutants of MLK3 also prevented vanadium-induced cell death. Interestingly, the activity of Src was seen to be in parallel with MLK3 kinase activity in human primary breast tumors. Taken together, our data demonstrate that tyrosine phosphorylation of MLK3 by oncogenic Src leads to MLK3 activation, which subsequently promotes vanadium-induced cell death in cancer cells. Our results also implicate a paradoxical pro-apoptotic role of Src in cancer cells, and thus it is conceivable that in tumors where both Src and MLK3/MLKs activities are higher, targeting of this axis might promote cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4957. doi:1538-7445.AM2012-4957