Basal breast cancer cells feature high expression of the Src family kinase Lyn that has been implicated in the pathogenicity of this disease. In this study, we identified novel Lyn kinase substrates, the most prominent of which was the atypical kinase SgK269 (PEAK1). In breast cancer cells, SgK269 expression associated with the basal phenotype. In primary breast tumors, SgK269 overexpression was detected in a subset of basal, HER2-positive, and luminal cancers. In immortalized MCF-10A mammary epithelial cells, SgK269 promoted transition to a mesenchymal phenotype and increased cell motility and invasion. Growth of MCF-10A acini in three-dimensional (3D) culture was enhanced upon SgK269 overexpression, which induced an abnormal, multilobular acinar morphology and promoted extracellular signal-regulated kinase (Erk) and Stat3 activation. SgK269 Y635F, mutated at a major Lyn phosphorylation site, did not enhance acinar size or cellular invasion. We show that Y635 represents a Grb2-binding site that promotes both Stat3 and Erk activation in 3D culture. RNA interferencemediated attenuation of SgK269 in basal breast cancer cells promoted acquisition of epithelial characteristics and decreased anchorage-independent growth. Together, our results define a novel signaling pathway in basal breast cancer involving Lyn and SgK269 that offers clinical opportunities for therapeutic intervention.

BackgroundCharacterization of molecular mechanisms underpinning development of pancreatic ductal adenocarcinoma (PDAC) may lead to the identification of novel therapeutic targets and biomarkers. SgK223, also known as Pragmin, is a pseudokinase and scaffolding protein closely related to SgK269/PEAK1. Both proteins are implicated in oncogenic tyrosine kinase signaling, but their mechanisms and function remain poorly characterized.MethodsExpression of SgK223 in PDAC and PDAC cell lines was characterized using gene expression microarrays, mass spectrometry (MS)-based phosphoproteomics and Western blotting. SgK223 was overexpressed in human pancreatic ductal epithelial (HPDE) cells via retroviral transduction, and knocked down in PDAC cells using siRNA. Cell proliferation was determined using a colorimetric cell viability assay, and cell migration and invasion using transwells. Expression of markers of epithelial-mesenchyme transition (EMT) was assayed by quantitative PCR. SgK223 and Stat3 signaling was interrogated by immunoprecipitation, Western blot and gene reporter assays. The functional role of specific kinases and Stat3 was determined using selective small molecule inhibitors.ResultsElevated site-selective tyrosine phosphorylation of SgK223 was identified in subsets of PDAC cell lines, and increased expression of SgK223 detected in several PDAC cell lines compared to human pancreatic ductal epithelial (HPDE) cells and in PDACs compared to normal pancreas. Expression of SgK223 in HPDE cells at levels comparable to those in PDAC did not alter cell proliferation but led to a more elongated morphology, enhanced migration and invasion and induced gene expression changes characteristic of a partial EMT. While SgK223 overexpression did not affect activation of Erk or Akt, it led to increased Stat3 Tyr705 phosphorylation and Stat3 transcriptional activity, and SgK223 and Stat3 associated in vivo. SgK223-overexpressing cells exhibited increased JAK1 activation, and use of selective inhibitors determined that the increased Stat3 signaling driven by SgK223 was JAK-dependent. Pharmacological inhibition of Stat3 revealed that Stat3 activation was required for the enhanced motility and invasion of SgK223-overexpressing cells.ConclusionsIncreased expression of SgK223 occurs in PDAC, and overexpression of SgK223 in pancreatic ductal epithelial cells promotes acquisition of a migratory and invasive phenotype through enhanced JAK1/Stat3 signaling. This represents the first association of SgK223 with a particular human cancer, and links SgK223 with a major signaling pathway strongly implicated in PDAC progression.

The cortactin oncoprotein is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC), often due to amplification of the encoding gene (CTTN). While cortactin overexpression enhances invasive potential, recent research indicates that it also promotes cell proliferation, but how cortactin regulates the cell cycle machinery is unclear. In this article we report that stable short hairpin RNA-mediated cortactin knockdown in the 11q13-amplified cell line Cortactin is an F-actin binding protein involved in a variety of cellular processes, including endocytosis, vesicle trafficking, and the formation of cellular protrusions such as lamellipodia (14). In order to mediate these functions, cortactin interacts with a variety of proteins depending on the cell type and subcellular compartment, and this is achieved via distinct binding domains located within the cortactin molecule. The N terminus of cortactin harbors an acidic region that represents the interaction site for the actin-polymerizing Arp2/Arp3 complex, and this is followed by a repeat region containing the F-actin binding site. A Src homology 3 (SH3) domain located at the C terminus of cortactin recruits diverse proteins, including components of the endocytosis machinery (e.g., CD2AP and dynamin) and regulators of Rho family GTPases (e.g., BPGAP1 and Fgd1) and actin polymerization (e.g., N-WASP), while an adjacent proline-rich region contains phosphorylation sites for Src family kinases (14).The cortactin gene (CTTN) is located at chromosome band 11q13, a region that is commonly amplified in many human malignancies, particularly breast, ovarian, and bladder cancers and head and neck squamous cell carcinoma (HNSCC) (43).Several genes lie within this chromosomal region and are overexpressed upon its amplification. However, of these genes, the amplification of cyclin D1 (CCND1) and CTTN is most frequently associated with poor clinical outcomes such as decreased patient survival and increased metastasis (34,43). Chromosomal mapping of the 11q13 locus has revealed four distinct regions that can be individually or coordinately amplified (12,20,34). Within this locus, CCND1 and CTTN are located on different amplicons, and independent amplification of these genes has been demonstrated (34). In HNSCC, a tumor type in which 11q13 amplification occurs at the relatively high frequency of ϳ30% (43), CTTN amplification has been identified as an independent predictor of reduced disease-specific survival while CCND1 amplification is not prognostic in this tumor type (16,18,38,39). This strongly suggests that cortactin overexpression can act independently to promote tumor progression in cases of HNSCC.Due to the ability of cortactin to promote actin polymerization, many previous studies on cancer cells have focused on the role of cortactin in promoting cell motility and invasion (35,40,54), effects mediated by increased lamellipodial persistence (5), invadopodia formation (4), and protease secretion (10, 11). In agreement with this, cortactin overexpression has b...

Yeast cells begin to bud and enter the S phase when growth conditions are favorable during the G 1 phase. When subjected to some oxidative stresses, cells delay entry at G 1 , allowing repair of cellular damage. Hence, oxidative stress sensing is coordinated with the regulation of cell cycle. We identified a novel function of the cell cycle regulator of Saccharomyces cerevisiae, Swi6p, as a redox sensor through its cysteine residue at position 404. When alanine was substituted at this position, the resultant mutant, C404A, was sensitive to several reactive oxygen species and oxidants including linoleic acid hydroperoxide, the superoxide anion, and diamide. This mutant lost the ability to arrest in G 1 phase upon treatment with lipid hydroperoxide. The Cys-404 residue of Swi6p in wild-type cells was oxidized to a sulfenic acid when cells were subjected to linoleic acid hydroperoxide. Mutation of Cys-404 to Ala abolished the down-regulation of expression of the G 1 cyclin genes CLN1, CLN2, PCL1, and PCL2 that occurred when cells of the wild type were exposed to the lipid hydroperoxide. In conclusion, oxidative stress signaling for cell cycle regulation occurs through oxidation of the G 1 /S-speicific transcription factor Swi6p and consequently leads to suppression of the expression of G 1 cyclins and a delay in cells entering the cell cycle.The cell cycle consists of a series of coordinated events that ensure duplication of genetic material, chromosome segregation, cell growth, and cytokinesis, producing two daughter cells. In eukaryotes, cell cycle events are governed by phasespecific cyclins that complex and activate cyclin-dependent kinase (CDK) 2 for activation of phase-specific events. Cell division is regulated in part by the periodic expression of genes specific to each of the four phases (G 1 , S, G 2 , and M) (1, 2). In Saccharomyces cerevisiae, the single CDK, Cdc28, can complex with nine cyclins that are transcribed distinctly in the four stages of the cell cycle. Cells enter the cell cycle to undergo division when both intrinsic and extrinsic requirements are met. In S. cerevisiae, when cells attain a critical size in the presence of sufficient nutrients in the late G 1 phase, they reach an interval called "Start," at which the bud begins to emerge, DNA replication is initiated, and cells duplicate their spindle pole body preparing for mitosis and cytokinesis (3).Cell cycle initiation and progression are tightly regulated to ensure that cell division does not take place under unfavorable conditions (4, 5). In proliferating cells, oxidative stress can arise from reactive oxygen species that are generated from incomplete reduction of oxygen from the electron transport chain. Environmental factors, including heat and other stresses, ionizing radiation, metal ions, herbicides such as paraquat, and the vitamin K precursor menadione, can also induce reactive oxygen species production in cells (6). Reactive oxygen species are highly toxic because of their ability to directly damage nucleic acids, proteins, an...

BackgroundThe receptor tyrosine kinase RON exhibits increased expression during pancreatic cancer progression and promotes migration, invasion and gemcitabine resistance of pancreatic cancer cells in experimental models. However, the prognostic significance of RON expression in pancreatic cancer is unknown.MethodsRON expression was characterized in several large cohorts, including a prospective study, totaling 492 pancreatic cancer patients and relationships with patient outcome and clinico-pathologic variables were assessed.ResultsRON expression was associated with outcome in a training set, but this was not recapitulated in the validation set, nor was there any association with therapeutic responsiveness in the validation set or the prospective study.ConclusionsAlthough RON is implicated in pancreatic cancer progression in experimental models, and may constitute a therapeutic target, RON expression is not associated with prognosis or therapeutic responsiveness in resected pancreatic cancer.

Background: Despite intensive treatment, patients with high-risk neuroblastoma (HR-NB) have a five-year survival rate of only 50%, largely due to intrinsic or acquired drug resistance. The multidrug transporter P-glycoprotein (P-gp; ABCB1) effluxes conventional agents used in HR-NB induction therapy, including vincristine, etoposide, and doxorubicin, and has been reported as a resistance mechanism to the ALK inhibitors crizotinib and ceritinib. We assessed the prevalence of P-gp expression and its role in resistance to standard-of-care chemotherapies and molecularly targeted therapies. Methods and Results: Analysis of ABCB1 levels in large patient tumor datasets demonstrates that high expression is more common in neuroblastoma than in other cancers and may be attributable to the sympathoadrenal lineage of the disease. Moreover, high relative expression of ABCB1 in HR-NB tumors is associated with poorer outcome, consistent with its multidrug transporter function. Frequent high ABCB1/P-gp expression in HR-NB was confirmed using RNA-sequencing, immunohistochemistry, and Western blot on panels of neuroblastoma tumor samples, patient-derived xenograft models, and cell lines. We demonstrated that the P-gp inhibitor tariquidar and P-gp knockdown (shRNA) both strongly sensitize high P-gp expressing neuroblastoma cells to vincristine, doxorubicin, and etoposide but not to ALK inhibitors or to relapse therapy regimens. Further, P-gp knockdown sensitized human neuroblastoma xenografts to vincristine, substantially extending animal survival. We have generated ABCB1-null HR-NB cell lines by CRISPR/Cas9 gene editing to further assess the role of P-gp in HR-NB drug resistance, have established flow-based assays to measure P-gp activity in tumor samples, and are investigating the benefits of tariquidar as adjuncts to conventional chemotherapy in PDX models. Conclusions: Elevated P-gp expression is common in HR-NB. In laboratory models, P-gp can confer resistance to standard-of-care chemotherapies but not to ALK inhibitors. Our findings suggest that tumor P-gp activity might be used to guide treatment options for individual patients in order to avoid ineffective treatments. The potential of P-gp inhibitors as adjuncts to conventional chemotherapy for HR-NB should be further investigated. Citation Format: Caroline Atkinson, Carole Tactacan, Alvin Kamili, Federica Saletta, Christine Gana, Georgina Eden, Chelsea Mayoh, Richard Lock, Murray Norris, Michelle Haber, Andrew Gifford, Toby Trahair, Jamie Fletcher. P-Glycoprotein is a resistance mechanism for conventional induction chemotherapy but not ALK inhibitors in high-risk neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A35.

Background: Neuroblastoma is the most common extracranial solid malignancy in children, comprising 15% of cancer related deaths. Despite intensive treatment, patients with high-risk neuroblastoma (HR-NB) have a survival rate of ~50%, largely due to intrinsic or acquired drug resistance. The multidrug transporter P-glycoprotein (P-gp; ABCB1) effluxes several conventional agents used in HR-NB induction therapy, including doxorubicin, vincristine and etoposide, as well as the ALK inhibitor crizotinib. We observed high P-gp expression in HR-NB cell lines and patient-derived xenograft (PDX) models, so sought to assess the prevalence of P-gp expression and its role in resistance to standard-of-care chemotherapies and relevant targeted agents. Methods and Results: Using RNA-sequencing, immunohistochemistry and western blot on panels of neuroblastoma tumour samples, PDX models and cell lines, we demonstrated that high ABCB1/P-gp expression is frequent in HR-NB. Analysis of ABCB1 levels in large patient tumor datasets suggests that high expression is attributable to the sympathoadrenal lineage of the disease, that high expression is more common in HR-NB than in most other cancers, and that high relative expression of ABCB1 in HR-NB tumours is associated with poorer outcome, consistent with its multidrug transporter function. We demonstrated that the P-gp inhibitor tariquidar and P-gp knockdown (shRNA) both strongly sensitize cultured high P-gp expressing neuroblastoma cells to vincristine, doxorubicin and etoposide but not to ALK inhibitors. Further, P-gp knockdown sensitized human neuroblastoma xenografts to vincristine, substantially extending survival. Conclusions: Elevated P-gp expression is common in HR-NB and can be sufficient to confer resistance to standard-of-care chemotherapies in model systems. Our findings suggest that tumour P-gp levels might be used to guide treatment options for individual patients, and to avoid ineffective treatments. The potential of P-gp inhibitors as adjuncts to conventional chemotherapy for HR-NB should be further investigated. Citation Format: Caroline Atkinson, Carole M. Tactacan, Alvin Kamili, Federica Saletta, Christine Gana, Georgina L. Eden, Chelsea Mayoh, Richard B. Lock, Murray D. Norris, Michelle Haber, Andrew J. Gifford, Toby N. Trahair, Jamie I. Fletcher. Re-evaluating the role of P-glycoprotein in the resistance of high-risk neuroblastoma to standard-of-care chemotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4741.

No abstract