Imprinted genes that regulate early mammalian growth are coexpressed in somatic stem cellsImprinted genes that regulate early mammalian growth are coexpressed in somatic stem cells

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Involvement of Lyn and the Atypical Kinase SgK269/PEAK1 in a Basal Breast Cancer Signaling Pathway

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 interference–mediated 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.National Health and Medical Research Council of Australia, Cancer Council New South Wales (NSW) and Science Foundation Ireland (Grant No. 06/CE/B1129). DRC and DS were supported by Fellowships from Cancer Institute (CI) NSW. FH is supported by The Ministry of Education and Research, Bundesministerium für Bildung und Forschung (03Z1CN21). CMT is the recipient of a Research Scholarship from CINSW, and LZ an Australian Postgraduate Award. DGO is supported by a National Breast Cancer Foundation (NBCF) and Cure Cancer Australia Foundation Postdoctoral Fellowship. AS is supported by an Early Career Fellowship from the NBCF.Deposited by bulk impor

Loss of osteoclasts contributes to development of osteosarcoma pulmonary metastases

We conducted a transcriptomic screen of osteosarcoma (OS) biopsies and found that expression of osteoclast-specific tartrate-resistant acid phosphatase 5 (ACP5/TRAP) is significantly downregulated in OS compared with nonmalignant bone (P < 0.0001). Moreover, lesions from OS patients with pulmonary metastases had 2-fold less ACP5/TRAP expression (P < 0.018) than lesions from patients without metastases. In addition, we found a direct correlation (P = 0.0166) between ACP5/TRAP expression and time to metastasis. Therefore, we examined whether metastasis-competent (MC) OS cells could induce loss of ACP5(+) osteoclasts and contribute to metastasis. We found that MC OS cell lines can inhibit osteoclastogenesis in vitro and in vivo. In addition, osteoclasts can inhibit the migration of MC OS cells in vitro. Finally, ablation of osteoclasts with zoledronic acid increases the number of metastatic lung lesions in an orthotopic OS model, whereas fulvestrant treatment increases osteoclast numbers and reduces metastatic lesions. These data indicate that the metastatic potential of OS is determined early in tumor development and that loss of osteoclasts in the primary lesion enhances OS metastasis.Liliana Endo-Munoz, Andrew Cumming, Danny Rickwood, Danielle Wilson, Claudia Cueva, Charlotte Ng, Geoffrey Strutton, A. Ian Cassady, Andreas Evdokiou, Scott Sommerville, Ian Dickinson, Alexander Guminski, and Nicholas A. Saunder

Tyrosine phosphorylation profiling reveals the signaling network characteristics of basal breast cancer cells

To identify therapeutic targets and prognostic markers for basal breast cancers, breast cancer cell lines were subjected to mass spectrometry-based profiling of protein tyrosine phosphorylation events. This revealed that luminal and basal breast cancer cells exhibit distinct tyrosine phosphorylation signatures that depend on pathway activation as well as protein expression. Basal breast cancer cells are characterized by elevated tyrosine phosphorylation of Met, Lyn, EphA2, epidermal growth factor receptor (EGFR), and FAK, and Src family kinase (SFK) substrates such as p130Cas. SFKs exert a prominent role in these cells, phosphorylating key regulators of adhesion and migration and promoting tyrosine phosphorylation of the receptor tyrosine kinases EGFR and Met. Consistent with these observations, SFK inhibition attenuated cellular proliferation, survival, and motility. Basal breast cancer cell lines exhibited differential responsiveness to small molecule inhibitors of EGFR and Met that correlated with the degree of target phosphorylation, and reflecting kinase coactivation, inhibiting two types of activated network kinase (e. g., EGFR and SFKs) was more effective than single agent approaches. FAK signaling enhanced both proliferation and invasion, and Lyn was identified as a proinvasive component of the network that is associated with a basal phenotype and poor prognosis in patients with breast cancer. These studies highlight multiple kinases and substrates for further evaluation as therapeutic targets and biomarkers. However, they also indicate that patient stratification based on expression/activation of drug targets, coupled with use of multi-kinase inhibitors or combination therapies, may be required for effective treatment of this breast cancer subgroup. Cancer Res; 70(22); 9391-401. (C) 2010 AACR.</p

Loss of E2F7 expression is an early event in squamous differentiation and causes derepression of the key differentiation activator Sp1

Squamous differentiation is controlled by key transcription factors such as Sp1 and E2F. We have previously shown that E2F1 can suppress transcription of the differentiation-specific gene, transglutaminase type 1 (TG1), by an indirect mechanism mediated by Sp1. Transient transfection of E2F1-E2F6 indicated that E2F-mediated reduction of Sp1 transcription was not responsible for E2F-mediated suppression of squamous differentiation. However, we found that E2F4 and E2F7, but not E2Fs 1, 2, 3, 5, or 6, could suppress the activation of the Sp1 promoter in differentiated keratinocytes (KCs). E2F4-mediated suppression could not be antagonized by E2Fs 1, 2, 3, 5, or 6 and was localized to a region of the human Sp1 promoter spanning-139 to +35 bp. Chromatin immunoprecipitation analysis, as well as transient overexpression and short hairpin RNA knockdown experiments indicate that E2F7 binds to a unique binding site located between-139 and-119 bp of the Sp1 promoter, and knockdown of E2F7 in proliferating KCs leads to a derepression of Sp1 expression and the induction of TG1. In contrast, E2F4 knockdown in proliferating KCs did not alter Sp1 expression. These data indicate that loss of E2F7 during the initiation of differentiation leads to the derepression of Sp1 and subsequent transcription of differentiation-specific genes such as TG1

Valproic acid as a therapeutic agent for head and neck squamous cell carcinomas

Here we investigate if valproic acid (VA) can enhance the efficacy of commonly used therapies for head and neck squamous cell carcinomas (HNSCC) and the molecular mechanisms that may be related to its anticancer effects. Proliferation and viability of distinct cell types subjected to VA treatment alone or in combination regimens were measured through BrdU incorporation and LDH release, respectively. Molecular markers compatible with histone deacetylase inhibitory activity of VA were assessed through western blots assays in lysates obtained from cultured cells and tumour biopsies. Treatment of all cell types with VA resulted in a dose-dependent increase in histone H3 acetylation and p21 expression, as well as dose-dependent cytostasis. In contrast, the cytotoxic response to VA was variable and did not correlate with cytostasis, histone acetylation or p21 induction. The variability in response to VA was also observed in tumour biopsy samples collected from patients prior to and following a 1 week oral course of VA. In addition, we found that a combination of a clinically achievable concentration of VA plus cisplatin caused a threefold to sevenfold increase in cisplatin cytotoxicity in vitro. VA acts as a histone deacetylase inhibitor (HDI) in SCC cells and normal human keratinocytes (HKs), potentiates the cytotoxic effect of cisplatin in SCC cell lines and decreases the viability of SCC cells as opposed to HKs. Taken together, the results provide initial evidence that VA might be a valuable drug in the development of better therapeutic regimens for HNSCC

Tumor-initiating activity and tumor morphology of HNSCC is modulated by interactions between clonal variants within the tumor

Tumor initiation (TI) in xenotransplantation models of head and neck squamous cell carcinoma (HNSCC) is an inefficient process. Poor TI could be due to (1) posttransplant cell loss, (2) a rare sub-population of cancer stem cells or (3) a requirement for specific cellular interactions, which rely on cell number. By tracking GFP-expressing HNSCC cells, we conclude that the posttransplant loss of cancer cells is minimal in the xenotransplant model. Furthermore, an examination of putative cancer stem cell markers (such as CD133, CD44, SP and label retention) in HNSCC cell lines revealed no correlation between marker expression and tumorigenicity. In addition, single-cell clones randomly isolated from HNSCC cell lines and then transplanted into mice were all capable of initiating tumors with efficiencies varying almost 34-fold. As the observed variation in the clones was both more and less tumorigenic than the parental cells, a combination of two clones, at suboptimal cell numbers for TI, was implanted into mice and was found to modulate the tumor-initiating activity, thus indicating that TI is dependent on a critical number of cells and, for the first time, that interactions between clonal variants within tumors can modulate the overall tumor-initiating activity. Put in context with previous literature on tumorigenic activity, we believe that interactions between clonal variants within a tumor as well as (1) stromal interactions, (2) angiogenic activity, (3) immunocompetence and (4) cancer stem cells may all contribute to tumorigenic potential and the propensity for tumor growth and recurrence