Elaine A. McSherry

Altered Cytoplasmic-to-Nuclear Ratio of Survivin Is a Prognostic Indicator in Breast Cancer

Purpose: Survivin (BIRC5) is a promising tumor biomarker. Conflicting data exist on its prognostic effect in breast cancer. These data may at least be partly due to the manual interpretation of immunohistochemical staining, especially as survivin can be located in both the nucleus and cytoplasm. Quantitative determination of survivin expression using image analysis offers the opportunity to develop alternative scoring models for survivin immunohistochemistry. Here, we present such a model. Experimental Design: A breast cancer tissue microarray containing 102 tumors was stained with an anti-survivin antibody. Whole-slide scanning was used to capture high-resolution images. These images were analyzed using automated algorithms to quantify the staining. Results: Increased nuclear, but not cytoplasmic, survivin was associated with a reduced overall survival (OS; P = 0.038) and disease-specific survival (P = 0.0015). A high cytoplasmic-to-nuclear ratio (CNR) of survivin was associated with improved OS (P = 0.005) and disease-specific survival (P = 0.05). Multivariate analysis revealed that the survivin CNR was an independent predictor of OS (hazard ratio, 0.09; 95% confidence interval, 0.01-0.76; P = 0.027). A survivin CNR of >5 correlated positively with estrogen receptor (P = 0.019) and progesterone receptor (P = 0.033) levels, whereas it was negatively associated with Ki-67 expression (P = 0.04), p53 status (P = 0.005), and c-myc amplification (P = 0.016). Conclusion: Different prognostic information is supplied by nuclear and cytoplasmic survivin in breast cancer. Nuclear survivin is a poor prognostic marker in breast cancer. Moreover, CNR of survivin, as determined by image analysis, is an independent prognostic factor.

Breast cancer cell migration is regulated through junctional adhesion molecule-A-mediated activation of Rap1 GTPase

IntroductionThe adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells.MethodsMCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and β1-integrin, we examined activation of the β1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and β1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation.ResultsJAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the β1-integrin substrate fibronectin. This was accompanied by reduced protein expression of β1-integrin and its binding partners αV- and α5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and β1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between JAM-A, AF-6 and the Rap1 activator PDZ-GEF2 in MCF7 cells and in primary cultures from breast cancer patients.ConclusionsOur findings provide compelling evidence of a novel role for JAM-A in driving breast cancer cell migration via activation of Rap1 GTPase and β1-integrin. We speculate that JAM-A over-expression in some breast cancer patients may represent a novel therapeutic target to reduce the likelihood of metastasis.

Tight junctions: a barrier to the initiation and progression of breast cancer?

Breast cancer is a complex and heterogeneous disease that arises from epithelial cells lining the breast ducts and lobules. Correct adhesion between adjacent epithelial cells is important in determining the normal structure and function of epithelial tissues, and there is accumulating evidence that dysregulated cell-cell adhesion is associated with many cancers. This review will focus on one cell-cell adhesion complex, the tight junction (TJ), and summarize recent evidence that TJs may participate in breast cancer development or progression. We will first outline the protein composition of TJs and discuss the functions of the TJ complex. Secondly we will examine how alterations in these functions might facilitate breast cancer initiation or progression; by focussing on the regulatory influence of TJs on cell polarity, cell fate and cell migration. Finally we will outline how pharmacological targeting of TJ proteins may be useful in limiting breast cancer progression. Overall we hope to illustrate that the relationship between TJ alterations and breast cancer is a complex one; but that this area offers promise in uncovering fundamental mechanisms linked to breast cancer progression.

A novel mechanism of regulating breast cancer cell migration via palmitoylation-dependent alterations in the lipid raft affiliation of CD44

IntroductionMost breast cancer-related deaths result from metastasis, a process involving dynamic regulation of tumour cell adhesion and migration. The adhesion protein CD44, a key regulator of cell migration, is enriched in cholesterol-enriched membrane microdomains termed lipid rafts. We recently reported that raft affiliation of CD44 negatively regulates interactions with its migratory binding partner ezrin. Since raft affiliation is regulated by post-translational modifications including palmitoylation, we sought to establish the contribution of CD44 palmitoylation and lipid raft affiliation to cell migration.MethodsRecovery of CD44 and its binding partners from raft versus non-raft membrane microdomains was profiled in non-migrating and migrating breast cancer cell lines. Site-directed mutagenesis was used to introduce single or double point mutations into both CD44 palmitoylation sites (Cys286 and Cys295), whereupon the implications for lipid raft recovery, phenotype, ezrin co-precipitation and migratory behaviour was assessed. Finally CD44 palmitoylation status and lipid raft affiliation was assessed in primary cultures from a small panel of breast cancer patients.ResultsCD44 raft affiliation was increased during migration of non-invasive breast cell lines, but decreased during migration of highly-invasive breast cells. The latter was paralleled by increased CD44 recovery in non-raft fractions, and exclusive non-raft recovery of its binding partners. Point mutation of CD44 palmitoylation sites reduced CD44 raft affiliation in invasive MDA-MB-231 cells, increased CD44-ezrin co-precipitation and accordingly enhanced cell migration. Expression of palmitoylation-impaired (raft-excluded) CD44 mutants in non-invasive MCF-10a cells was sufficient to reversibly induce the phenotypic appearance of epithelial-to-mesenchymal transition and to increase cell motility. Interestingly, cell migration was associated with temporal reductions in CD44 palmitoylation in wild-type breast cells. Finally, the relevance of these findings is underscored by the fact that levels of palmitoylated CD44 were lower in primary cultures from invasive ductal carcinomas relative to non-tumour tissue, while CD44 co-localisation with a lipid raft marker was less in invasive ductal carcinoma relative to ductal carcinoma in situ cultures.ConclusionOur results support a novel mechanism whereby CD44 palmitoylation and consequent lipid raft affiliation inversely regulate breast cancer cell migration, and may act as a new therapeutic target in breast cancer metastasis.

Junctional adhesion molecule-A is co-expressed with HER2 in breast tumors and acts as a novel regulator of HER2 protein degradation and signaling.

Junctional adhesion molecule-A (JAM-A) is a membranous cell–cell adhesion protein involved in tight-junction formation in epithelial and endothelial cells. Its overexpression in breast tumors has recently been linked with increased risk of metastasis. We sought to identify if JAM-A overexpression was associated with specific subtypes of breast cancer as defined by the expression of human epidermal growth factor receptor-2 (HER2), estrogen receptor (ER) and progesterone receptor. To this end, JAM-A immunohistochemistry was performed in two breast cancer tissue microarrays. In parallel, cross-talk between JAM-A, HER2 and ER was examined in several breast cell lines, using complementary genetic and pharmacological approaches. High JAM-A expression correlated significantly with HER2 protein expression, ER negativity, lower patient age, high-grade breast cancers, and aggressive luminal B, HER2 and basal subtypes of breast cancer. JAM-A and HER2 were co-expressed at high levels in vitro in SKBR3, UACC-812, UACC-893 and MCF7-HER2 cells. Knockdown or functional antagonism of HER2 did not alter JAM-A expression in any cell line tested. Interestingly, however, JAM-A knockdown decreased HER2 and ER-α expression, resulting in reduced levels of phospho-(active) AKT without an effect on the extracellular signal-related kinase phosphorylation. The downstream effects of JAM-A knockdown on HER2 and phospho-AKT were partially reversed upon treatment with the proteasomal inhibitor MG132. We conclude that JAM-A is co-expressed with HER2 and associates with aggressive breast cancer phenotypes. Furthermore, we speculate that JAM-A may regulate HER2 proteasomal degradation and activity, potentially offering a promise as a therapeutic target in HER2-positive breast cancers.

Abstract 3328: Novel regulation of HER2 degradation and breast cancer cell proliferation by junctional adhesion molecule-a (JAM-A)

Background: We have previously shown that the adhesion protein Junctional Adhesion Molecule-A (JAM-A) is over-expressed in a population of aggressive and metastatic breast tumours, and that it regulates the migration of breast cells. JAM-dependent regulation of migration could contribute to the likelihood of metastatic disease, since migration is an early event in metastasis. Our objective was to further interrogate the links between JAM-A signalling and markers of disease progression in breast cancer. Approach and Results: Immunohistochemical analysis of JAM-A in two breast cancer tissue microarrays (TMA) revealed that high JAM-A expression correlated significantly with HER2 protein expression, ERnegativity, high grade breast cancers and aggressive luminal B, HER2 and basal subtypes of breast cancer. Using LCC1 and MCF7-HER2 cells which co-express high levels of JAM-A and HER2, we silenced the gene expression of each one in order to examine potential consequences for protein expression of the other. Knockdown of HER2 had no effect on JAM-A protein levels, but knockdown of JAM-A significantly reduced the protein expression of HER2 and its downstream pro-survival effector pAkt. Accordingly, JAM-A knockdown increased apoptosis via a pathway involving Akt activation, as evidenced by apoptosis normalisation upon pharmacological inhibition of Akt. The expressional regulation of HER2 and pAkt by JAM-A was sensitive to inhibition by the proteasomal inhibitor MG132, suggesting that JAM-A regulates the proteasomal degradation of HER2. Finally, over-expression of wild-type JAM-A was sufficient to significantly enhance proliferative and migratory behaviour in breast cell lines. Conclusions: Our results are consistent with a model whereby JAM-A expression inhibits the proteasomal degradation of HER2, facilitating a pro-survival phenotype through increased Akt activity. Therefore JAM-A antagonists may have future therapeutic value in the treatment of breast tumours overexpressing HER2. Citation Format: Kieran Brennan, Elaine A. McSherry, Lance Hudson, Elaine W. Kay, Leonie S. Young, Arnold DK Hill, Ann M. Hopkins. Novel regulation of HER2 degradation and breast cancer cell proliferation by junctional adhesion molecule-a (JAM-A). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3328. doi:10.1158/1538-7445.AM2014-3328

The Molecular Aspects of Tight Junctions

Tight junctions (TJs) are multi-protein complexes whose principal function is to mediate cell-cell adhesion between epithelial or endothelial cells. While once thought to participate solely as passive effectors of adhesion, it is increasingly being recognised that TJs are dynamic structures which regulate many aspects of cellular function and physiology. Accordingly, dysregulation of TJ-based adhesion or signalling is emerging as an intriguing contributor to several pathophysiologies including cancer. This review will attempt to summarise the current state of knowledge about molecular aspects which regulate, and are regulated by, TJs. The first section will outline selected physiological processes known to influence TJ structure or function, under the headings of cell adhesion/polarity, cell-matrix signalling, ion transport, hormone effects, pro-inflammatory cytokines and hypoxia. The second section will describe selected functional behaviours within the pathophysiology of cancer which TJs have been demonstrated to influence, encompassing cell proliferation and apoptosis, migration and invasion, cell fate and differentiation, metastasis across the blood brain barrier and finally angiogenesis. Collectively, these sections illustrate that a wealth of mechanistic information can be gained from interrogating the contribution of TJs to normal physiology. In turn they highlight how TJ-based disturbances can promote some of the functional behaviours associated with cancer, and thereby offer insight into new TJ-based targets that may offer pharmacological promise in halting tumour progression.