TNK2 preserves epidermal growth factor receptor expression on the cell surface and enhances migration and invasion of human breast cancer cells

Introduction Amplification of the TNK2 gene in primary tumours correlates with poor prognosis. In accordance, TNK2 overexpression was shown to promote invasion of cancer cells - but the mechanism by which TNK2 mediates these effects is unresolved. TNK2 was suggested to regulate Cdc42-driven migration by activation of breast cancer antioestrogen resistance 1 (BCAR1); however, distinct from this effect is evidence for a role of TNK2 in the regulation of epidermal growth factor receptor (EGFR) endocytosis and degradation. In the present study we sought to investigate whether negative targeting of TNK2 by siRNA could be used to inhibit cancer cell invasion, to establish the contribution of its effect on the EGFR and to consequently attempt to resolve the issue of TNK2's mechanism of action. Methods We used siRNA to knockdown expression of TNK2 and its proposed effector BCAR1 in order to analyse the effect of this knockdown on cancer cell behaviour in vitro. We examined morphological changes using phase-contrast microscopy and immunohistochemistry. Functional parameters examined included apoptosis, proliferation, migration and invasion. We also performed flow cytometry analysis to examine EGFR cell surface expression and carried out western blot to examine the total EGFR levels. Results We observed that targeting of TNK2 by siRNA in breast cancer cells resulted in distinct morphological changes characterised by a stellate appearance and an absence of protrusions at membrane edges. These changes were not recapitulated upon siRNA targeting of BCAR1. We thus hypothesised that a component of the effects induced by TNK2 may be independent of BCAR1. Consistent with the idea of an alternative mechanism for TNK2, we observed that TNK2 associates with activated EGFR in breast cancer cells in a TNK2-kinase-independent manner. Furthermore, we demonstrated that TNK2 functions to maintain EGFRs on the cell surface. We could demonstrate that the main functional effect of activating these surface EGFRs in breast cancer cells is stimulation of migration. In accordance, TNK2 silencing by siRNA led to a significant reduction in cell surface EGFR and to a concomitant decrease in the migratory and invasive capacity of breast cancer cells. Conclusion Our data suggest that TNK2 can enhance migration and invasion of breast cancer cells via preservation of EGFR expression, notwithstanding its previously reported signalling via BCAR1, explaining its oncogenic behaviour in vitro and correlation with metastatic human breast cancer in vivo

Pubertal mammary gland development : Elucidation of in vivo morphogenesis using Murine models

During the past 25 years, the combination of increasingly sophisticated gene targeting technology with transplantation techniques has allowed researchers to address a wide array of questions about postnatal mammary gland development. These in turn have significantly contributed to our knowledge of other branched epithelial structures. This review chapter highlights a selection of the mouse models exhibiting a pubertal mammary gland phenotype with a focus on how they have contributed to our overall understanding of in vivo mammary morphogenesis. We discuss mouse models that have enabled us to assign functions to particular genes and proteins and, more importantly, have determined when and where these factors are required for completion of ductal outgrowth and branch patterning. The reason for the success of the mouse mammary gland model is undoubtedly the suitability of the postnatal mammary gland to experimental manipulation. The gland itself is very amenable to investigation and the combination of genetic modification with accessibility to the tissue has allowed an impressive number of studies to inform biology. Excision of the rudimentary epithelial structure postnatally allows genetically modified tissue to be readily transplanted into wild type stroma or vice versa, and has thus defined the contribution of each compartment to particular phenotypes. Similarly, whole gland transplantation has been used to definitively discern local effects from indirect systemic effects of various growth factors and hormones. While appreciative of the power of these tools and techniques, we are also cognizant of some of their limitations, and we discuss some shortcomings and future strategies that can overcome them

Molecular profiling of breast cancer: transcriptomic studies and beyond

Utilisation of 'omics' technologies, in particular gene expression profiling, has increased dramatically in recent years. In basic research, high-throughput profiling applications are increasingly used and may now even be considered standard research tools. In the clinic, there is a need for better and more accurate diagnosis, prognosis and treatment response indicators. As such, clinicians have looked to omics technologies for potential biomarkers. These prediction profiling studies have in turn attracted the attention of basic researchers eager to uncover biological mechanisms underlying clinically useful signatures. Here we highlight some of the seminal work establishing the arrival of the omics, in particular transcriptomics, in breast cancer research and discuss a sample of the most current applications. We also discuss the challenges of data analysis and integrated data analysis with emphasis on utilising the current publicly available gene expression datasets

Amphiregulin: Role in mammary gland development and breast cancer

Extensive epithelial cell proliferation underlies the ductal morphogenesis of puberty that generates the mammary tree that will eventually fill the fat pad. This estrogen-dependent process is believed to be essentially dependent on locally produced growth factors that act in a paracrine fashion. EGF-like growth factor ligands, acting through EGF receptors are some of the principal promoters of pubertal ductal morphogenesis. Amphiregulin is the most abundant EGF-like growth factor in the pubertal mammary gland. Its gene is transcriptionally regulated by ER alpha, and recent evidence identifies it as a key mediator of the estrogen-driven epithelial cell proliferation of puberty: The pubertal deficiency in mammary gland ductal morphogenesis in ER alpha, amphiregulin, and EGFR knockout mice phenocopy each other. As a prognostic indicator in human breast cancer, amphiregulin indicates an outcome identical to that predicted by ER alpha presence. Despite this, a range of studies both on preneoplastic human breast tissue and on cell culture based models of breast cancer, suggest a possibly significant role for amphiregulin in driving human breast cancer progression. Here we summarise our current understanding of amphiregulin's contribution to mammary gland development and breast cancer progression

TNK2 preserves epidermal growth factor receptor expression on the cell surface and enhances migration and invasion of human breast cancer cells-1

NK2 are shown relative to β-actin protein levels in the following cell lines: HB2, normal human breast epithelial cells; MCF-7, MDA-MB-468, and T47D human breast cancer cells; HC11 murine mammary epithelial cells; and human breast cancer cell line, MDA-MB-231. TNK2 is recruited to the EGFR following activation of the receptor in MDA-MB-231 cells, as indicated by coimmunoprecipitation in the presence and absence of epidermal growth factor (EGF) stimulation at 100 ng/ml. The association of EGFR and TNK2 in MDA-MB-231 cells is not dependent on the phosphorylation status of TNK2, as indicated by the ability of wildtype (Wt), mutant kinase-deficient (Kd) and constitutively active (Ca) TNK2 to immunoprecipitate activated EGFR. The percentage cell surface expression of EGFR is shown in serum-starved MDA-MB-231 cells treated with EGF (100 ng/ml) for the indicated times. Reduced numbers of basal cell surface EGFRs are seen in cells treated with targeting SiRNA (S1) relative to the nontargeting SiRNA control (N), as measured by a fluorescein isothiocyanate-conjugated anti-EGFR antibody by fluorescence-activated cell sorting analysis. On average, a 27% reduction of cell surface receptors can be seen at timepoint 0 (= 0.0135). A representative western blot illustrating the downregulation of TNK2 achieved by SiRNA treatment relative to β-actin is shown.<p><b>Copyright information:</b></p><p>Taken from "TNK2 preserves epidermal growth factor receptor expression on the cell surface and enhances migration and invasion of human breast cancer cells"</p><p>http://breast-cancer-research.com/content/10/2/R36</p><p>Breast Cancer Research : BCR 2008;10(2):R36-R36.</p><p>Published online 24 Apr 2008</p><p>PMCID:PMC2397538.</p><p></p

TNK2 preserves epidermal growth factor receptor expression on the cell surface and enhances migration and invasion of human breast cancer cells-0

Ontargeting SiRNA control (N). These changes consisted of an increased elongated appearance and a reduction in the number of protrusions at the membrane edges. A representative western blot illustrating the downregulation of TNK2 achieved by siRNA (S1, S3) treatment relative to β-actin is shown. The morphology changes observed in TNK2 SiRNA-treated cells were not observed in MDA-MB-231 cells treated with SiRNA directed against breast cancer antioestrogen resistance 1 (BCAR1). (i) Cells transfected with nontargeting (N), TNK2-targeting (S1) and BCAR1-targeting (#3) siRNA. (ii) The morphological changes induced can also be seen as an alteration in the actin fibre network. Staining for F-actin and costaining for the nuclear compartment with 4',6-diamidino-2-phenylindole is shown for cells treated with nontargeting (N), TNK2-targeting (S3) and BCAR1-targeting siRNA (#3). A representative western blot illustrating the downregulation of TNK2 (S1) and BCAR1 (#3) achieved by SiRNA treatment relative to β-actin is shown.<p><b>Copyright information:</b></p><p>Taken from "TNK2 preserves epidermal growth factor receptor expression on the cell surface and enhances migration and invasion of human breast cancer cells"</p><p>http://breast-cancer-research.com/content/10/2/R36</p><p>Breast Cancer Research : BCR 2008;10(2):R36-R36.</p><p>Published online 24 Apr 2008</p><p>PMCID:PMC2397538.</p><p></p

TNK2 preserves epidermal growth factor receptor expression on the cell surface and enhances migration and invasion of human breast cancer cells-2

period of 72 hours. Epidermal growth factor (EGF) treatment (100 ng/ml) enhanced MDA-MB-231 cell and MCF-7 cell migration as measured by a scratch-wound migration assay. Healing due to cell migration was measured over a period of 48 hours following wounding. The difference in migratory ability induced by EGF was significant in both cell types (= 0.0483 and = 0.013 at 48 hours for MDA-MB-231 cells and MCF-7 cells, respectively). Treatment of both EGF-treated MDA-MB-231 and EGF-treated MCF-7 cells with the epidermal growth factor receptor kinase inhibitor PD153035 (PD) significantly inhibited wound healing in serum-containing medium (= 0.0004 and = 0.0006 at the end points, respectively). Treatment of both MDA-MB-231 cells and MCF-7 cells with PD significantly inhibited wound healing in serum-containing medium (= 0.028 at 24 hours and = 0.0264 at 48 hours, respectively).<p><b>Copyright information:</b></p><p>Taken from "TNK2 preserves epidermal growth factor receptor expression on the cell surface and enhances migration and invasion of human breast cancer cells"</p><p>http://breast-cancer-research.com/content/10/2/R36</p><p>Breast Cancer Research : BCR 2008;10(2):R36-R36.</p><p>Published online 24 Apr 2008</p><p>PMCID:PMC2397538.</p><p></p

TNK2 preserves epidermal growth factor receptor expression on the cell surface and enhances migration and invasion of human breast cancer cells-5

Ontargeting SiRNA control (N). These changes consisted of an increased elongated appearance and a reduction in the number of protrusions at the membrane edges. A representative western blot illustrating the downregulation of TNK2 achieved by siRNA (S1, S3) treatment relative to β-actin is shown. The morphology changes observed in TNK2 SiRNA-treated cells were not observed in MDA-MB-231 cells treated with SiRNA directed against breast cancer antioestrogen resistance 1 (BCAR1). (i) Cells transfected with nontargeting (N), TNK2-targeting (S1) and BCAR1-targeting (#3) siRNA. (ii) The morphological changes induced can also be seen as an alteration in the actin fibre network. Staining for F-actin and costaining for the nuclear compartment with 4',6-diamidino-2-phenylindole is shown for cells treated with nontargeting (N), TNK2-targeting (S3) and BCAR1-targeting siRNA (#3). A representative western blot illustrating the downregulation of TNK2 (S1) and BCAR1 (#3) achieved by SiRNA treatment relative to β-actin is shown.<p><b>Copyright information:</b></p><p>Taken from "TNK2 preserves epidermal growth factor receptor expression on the cell surface and enhances migration and invasion of human breast cancer cells"</p><p>http://breast-cancer-research.com/content/10/2/R36</p><p>Breast Cancer Research : BCR 2008;10(2):R36-R36.</p><p>Published online 24 Apr 2008</p><p>PMCID:PMC2397538.</p><p></p

ERalpha-CITED1 co-regulated genes expressed during pubertal mammary gland development: implications for breast cancer prognosis

Expression microarray analysis identified over 930 genes regulated during puberty in the mouse mammary gland. Most prominent were genes whose expression increased in parallel with pubertal development and remained high thereafter. Members of the Wnt, transforming growth factor-beta and oestrogen-signalling pathways were significantly overrepresented. Comparison to expression data from CITED1 knockout mice identified a subset of oestrogen-responsive genes displaying altered expression in the absence of CITED1. Included in this subset are stanniocalcin2 (Stc2) and amphiregulin (Areg). Chromatin immunoprecipitation revealed that ERalpha binds to oestrogen response elements in both the Stc2 and Areg genes in the mammary gland during puberty. Additionally, CITED1 and ERalpha localize to the same epithelial cells of the pubertal mammary gland, supporting a role for interaction of these two proteins during normal development. In a human breast cancer data set, expression of Stc2, Areg and CITED1 parallel that of ERalpha. Similar to ERalpha, CITED1 expression correlates with good outcome in breast cancer, implying that potential maintenance of the ERalpha-CITED1 co-regulated signalling pathway in breast tumours can indicate good prognosis