Structure-Based Virtual Screening, Synthesis and Biological Evaluation of Potential FAK-FAT Domain Inhibitors for Treatment of Metastatic Cancer.

Focal adhesion kinase (FAK) is a tyrosine kinase that is overexpressed and activated in several advanced-stage solid cancers. In cancer cells, FAK promotes the progression and metastasis of tumours. In this study, we used structure-based virtual screening to filter a library of more than 210K compounds against the focal adhesion targeting FAK-focal adhesion targeting (FAT) domain to identify 25 virtual hit compounds which were screened in the invasive breast cancer line (MDA-MB-231). Most notably, compound I showed low micromolar antiproliferative activity, as well as antimigratory activity. Moreover, examination in a model of triple negative breast cancer (TNBC), revealed that, despite not effecting FAK phosphorylation, compound I significantly impairs proliferation whilst impairing focal adhesion growth and turnover leading to reduced migration. Further optimisation and synthesis of analogues of the lead compound I using a four-step synthetic procedure was performed, and analogues were assessed for their antiproliferative activity against three breast cancer (MDA-MB-231, T47D, BT474) cell lines and one pancreatic cancer (MIAPaCa2) cell line. Compound 5f was identified as a promising lead compound with IC50 values in the range of 4.59-5.28 µM in MDA-MB-231, T47D, BT474, and MIAPaCa2. Molecular modelling and pharmacokinetic studies provided more insight into the therapeutic features of this new series.

3D culture of Her2+ breast cancer cells promotes AKT to MAPK switching and a loss of therapeutic response.

BACKGROUND: The Her2 receptor is overexpressed in up to 25 % of breast cancers and is associated with a poor prognosis. Around half of Her2+ breast cancers also express the estrogen receptor and treatment for such tumours can involve both endocrine and Her2-targeted therapies. However, despite preclinical data supporting the effectiveness of these agents, responses can vary widely in the clinical setting. In light of the increasing evidence pointing to the interplay between the tumour and its extracellular microenvironment as a significant determinant of therapeutic sensitivity and response here we investigated the impact of 3D matrix culture of breast cancer cells on their therapeutic sensitivity. METHODS: A 3D Matrigel-based culture system was established and optimized for the growth of ER+/Her2+ breast cancer cell models. Growth of cells in response to trastuzumab and endocrine agents in 3D culture versus routine monolayer culture were assessed using cell counting and Ki67 staining. Endogenous and trastuzumab-modulated signalling pathway activity in 2D and 3D cultures were assessed using Western blotting. RESULTS: Breast cancer cells in 3D culture displayed an attenuated response to both endocrine agents and trastuzumab compared with cells cultured in traditional 2D monolayers. Underlying this phenomenon was an apparent matrix-induced shift from AKT to MAPK signalling; consequently, suppression of MAPK in 3D cultures restores therapeutic response. CONCLUSION: These data suggest that breast cancer cells in 3D culture display a reduced sensitivity to therapeutic agents which may be mediated by internal MAPK-mediated signalling. Targeting of adaptive pathways that maintain growth in 3D culture may represent an effective strategy to improve therapeutic response clinically.

Computer-aided identification of novel anticancer compounds with a possible dual HER1/HER2 inhibition mechanism.

HER1 and HER2 are frequently overexpressed in human tumors where they drive cellular proliferation. For this reason they are considered important targets in anticancer therapy with dual HER1/HER2 inhibitors being recently approved and marketed. In this paper we report the identification of a series of compounds with anticancer activity by a combined virtual screening approach on the kinase domains of HER1 and HER2. 6 hit compounds that present a sub- or low-micromolar activity in two cell-based assays, were initially identified and a subsequent design cycle led to the synthesis of a compound with nanomolar activity in the cell-based assays.

A mechanism for epithelial-mesenchymal transition and anoikis resistance in breast cancer triggered by zinc channel ZIP6 and STAT3 (signal transducer and activator of transcription 3).

Genes involved in normal developmental processes attract attention as mediators of tumour progression as they facilitate migration of tumour cells. EMT (epithelial-mesenchymal transition), an essential part of embryonic development, tissue remodelling and wound repair, is crucial for tumour metastasis. Previously, zinc transporter ZIP6 [SLC39A6; solute carrier family 39 (zinc transporter), member 6; also known as LIV-1) was linked to EMT in zebrafish gastrulation through a STAT3 (signal transducer and activator of transcription 3) mechanism, resulting in nuclear localization of transcription factor Snail. In the present study, we show that zinc transporter ZIP6 is transcriptionally induced by STAT3 and unprecedented among zinc transporters, and is activated by N-terminal cleavage which triggers ZIP6 plasma membrane location and zinc influx. This zinc influx inactivates GSK-3ß (glycogen synthase kinase 3ß), either indirectly or directly via Akt or GSK-3ß respectively, resulting in activation of Snail, which remains in the nucleus and acts as a transcriptional repressor of E-cadherin (epithelial cadherin), CDH1, causing cell rounding and detachment. This was mirrored by ZIP6-transfected cells which underwent EMT, detached from monolayers and exhibited resistance to anoikis by their ability to continue proliferating even after detachment. Our results indicate a causative role for ZIP6 in cell motility and migration, providing ZIP6 as a new target for prediction of clinical cancer spread and also suggesting a ZIP6-dependent mechanism of tumour metastasis.

Critical research gaps and translational priorities for the successful prevention and treatment of breast cancer.

INTRODUCTION: Breast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice. METHODS: More than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer 'stem' cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account. RESULTS: The 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working. CONCLUSIONS: With resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years.

Overexpression of CD44 accompanies acquired tamoxifen resistance in MCF7 cells and augments their sensitivity to the stromal factors, heregulin and hyaluronan.

BACKGROUND: Acquired resistance to endocrine therapy in breast cancer is a significant problem with relapse being associated with local and/or regional recurrence and frequent distant metastases. Breast cancer cell models reveal that endocrine resistance is accompanied by a gain in aggressive behaviour driven in part through altered growth factor receptor signalling, particularly involving erbB family receptors. Recently we identified that CD44, a transmembrane cell adhesion receptor known to interact with growth factor receptors, is upregulated in tamoxifen-resistant (TamR) MCF7 breast cancer cells. The purpose of this study was to explore the consequences of CD44 upregulation in an MCF7 cell model of acquired tamoxifen resistance, specifically with respect to the hypothesis that CD44 may influence erbB activity to promote an adverse phenotype. METHODS: CD44 expression in MCF7 and TamR cells was assessed by RT-PCR, Western blotting and immunocytochemistry. Immunofluorescence and immunoprecipitation studies revealed CD44-erbB associations. TamR cells (± siRNA-mediated CD44 suppression) or MCF7 cells (± transfection with the CD44 gene) were treated with the CD44 ligand, hyaluronon (HA), or heregulin and their in vitro growth (MTT), migration (Boyden chamber and wound healing) and invasion (Matrigel transwell migration) determined. erbB signalling was assessed using Western blotting. The effect of HA on erbB family dimerisation in TamR cells was determined by immunoprecipitation in the presence or absence of CD44 siRNA. RESULTS: TamR cells overexpressed CD44 where it was seen to associate with erbB2 at the cell surface. siRNA-mediated suppression of CD44 in TamR cells significantly attenuated their response to heregulin, inhibiting heregulin-induced cell migration and invasion. Furthermore, TamR cells exhibited enhanced sensitivity to HA, with HA treatment resulting in modulation of erbB dimerisation, ligand-independent activation of erbB2 and EGFR and induction of cell migration. Overexpression of CD44 in MCF7 cells, which lack endogenous CD44, generated an HA-sensitive phenotype, with HA-stimulation promoting erbB/EGFR activation and migration. CONCLUSIONS: These data suggest an important role for CD44 in the context of tamoxifen-resistance where it may augment cellular response to erbB ligands and HA, factors that are reported to be present within the tumour microenvironment in vivo. Thus CD44 may present an important determinant of breast cancer progression in the setting of endocrine resistance.

Pro-metastatic tumor-stroma interactions in breast cancer.

The vast majority of breast cancer-related deaths are due to metastatic disease. Reciprocal and complex interactions between epithelial tumor cells and the various components of the tumor microenvironment influence tumor progression and metastases although the molecular mechanisms underlying these metastasis-promoting effects are not fully characterized. Identifying and understanding pathways of tumor-stroma cross-talk are likely to lead to the development of novel prognostic biomarkers for metastasis and strategies to prevent metastasis at its earliest stages, resulting in improved patient outcomes.

Inhibition of focal adhesion kinase suppresses the adverse phenotype of endocrine-resistant breast cancer cells and improves endocrine response in endocrine-sensitive cells.

Acquired resistance to endocrine therapy in breast cancer is a major clinical problem. Previous reports have demonstrated that cell models of acquired endocrine resistance have altered cell-matrix adhesion and a highly migratory phenotype, features which may impact on tumour spread in vivo. Focal adhesion kinase (FAK) is an intracellular kinase that regulates signalling pathways central to cell adhesion, migration and survival and its expression is frequently deregulated in breast cancer. In this study, we have used the novel FAK inhibitor PF573228 to address the role of FAK in the development of endocrine resistance. Whilst total-FAK expression was similar between endocrine-sensitive and endocrine-resistant MCF7 cells, FAK phosphorylation status (Y397 or Y861) was altered in resistance. PF573228 promoted a dose-dependent inhibition of FAK phosphorylation at Y397 but did not affect other FAK activation sites (pY407, pY576 and pY861). Endocrine-resistant cells were more sensitive to these inhibitory effects versus MCF7 (mean IC(50) for FAK pY397 inhibition: 0.43 µM, 0.05 µM and 0.13 µM for MCF7, TamR and FasR cells, respectively). Inhibition of FAK pY397 was associated with a reduction in TamR and FasR adhesion to, and migration over, matrix components. PF573228 as a single agent (0-1 µM) did not affect the growth of MCF7 cells or their endocrine-resistant counterparts. However, treatment of endocrine-sensitive cells with PF573228 and tamoxifen combined resulted in greater suppression of proliferation versus single agent treatment. Together these data suggest the importance of FAK in the process of endocrine resistance, particularly in the development of an aggressive, migratory cell phenotype and demonstrate the potential to improve endocrine response through combination treatment.

Reduced kinase D­interacting substrate of 220 kDa (Kidins220) in pancreatic cancer promotes EGFR/ERK signalling and disease progression.

Kidins220 is a transmembrane scaffold protein involved in several types of cancer. The aim of the present study was to examine the role of Kidins220 in tumorigenesis and disease progression of pancreatic cancer. The relevant signalling pathways including EGFR, EMT, and MMP were also investigated. The expression of Kidins220 was examined at the transcript and protein level. The Kidins220 knockdown cell model was established and its influence on cellular functions was determined. Involvement of Kidins220 in tumorigenesis and metastasis was examined in CD1 mice, respectively. The results showed that, reduced Kidin220 expression was associated with tumorigenesis, metastasis, and overall survival of pancreatic cancer. Knockdown of Kidins220 promoted proliferation, colony formation and tumorigenic capacity of pancreatic cancer cells in vitro and in vivo, respectively. Kidins220 regulated pancreatic cancer cell migration through the EGFR/AKT/ERK signalling pathway. Furthermore, enhanced EMT was observed in the pancreatic cancer cell lines with the knockdown of Kidins220, underlying EGFR regulation. Kidins220 also affected cell invasion via MMP1. A reduced expression of Kidins220 was observed in pancreatic cancer, which is associated with disease progression, distant metastasis and poor prognosis. The loss of Kidins220 in pancreatic cancer may contribute to disease progression through the upregulation of EGFR and downstream signalling.

ZIP7-mediated intracellular zinc transport contributes to aberrant growth factor signaling in antihormone-resistant breast cancer Cells.

Antiestrogens such as tamoxifen are the mainstay of treatment for estrogen receptor-positive breast cancer. However, their effectiveness is limited by the development of endocrine resistance, allowing tumor regrowth and progression. Importantly, in vitro MCF7 cell models of acquired tamoxifen resistance (TamR cells) display an aggressive, invasive phenotype in which activation of epithelial growth factor receptor/IGF-I receptor/Src signaling plays a critical role. In this study, we report that TamR cells have increased levels of zinc and zinc transporter, ZIP7 [solute carrier family 39 (zinc transporter) member 7, also known as SLC39A7], resulting in an enhanced response to exogenous zinc, which is manifested as a greatly increased growth factor receptor activation, leading to increased growth and invasion. Removal of ZIP7, using small interfering RNA, destroys this activation of epithelial growth factor receptor/IGF-I receptor/Src signaling by reducing intracellular zinc levels. Similarly, it also blocks the activation of HER2, -3, and -4. These data suggest that intracellular zinc levels may be a critical factor in determining growth factor responses and that the targeting of zinc transporters may have novel therapeutic implications. We show that ZIP7 is a critical component in the redistribution of zinc from intracellular stores to the cytoplasm and, as such, is essential for the zinc-induced inhibition of phosphatases, which leads to activation of growth factor receptors. Removal of ZIP7 therefore offers a means through which zinc-induced activation of growth factor receptors may be effectively suppressed and provides a mechanism of targeting multiple growth factor pathways, increasing tumor kill, and preventing further development of resistance in breast cancer.

Anti-oestrogens but not oestrogen deprivation promote cellular invasion in intercellular adhesion-deficient breast cancer cells.

INTRODUCTION: Anti-oestrogens have been the mainstay of therapy in patients with oestrogen-receptor (ER) positive breast cancer and have provided significant improvements in survival. However, their benefits are limited by tumour recurrence in a significant proportion of initially drug-responsive breast cancer patients because of acquired anti-oestrogen resistance. Relapse on such therapies clinically presents as local and/or regional recurrences, frequently with distant metastases, and the prognosis for these patients is poor. The selective ER modulator, tamoxifen, classically exerts gene inhibitory effects during the drug-responsive phase in ER-positive breast cancer cells. Paradoxically, this drug is also able to induce the expression of genes, which in the appropriate cell context may contribute to an adverse cell phenotype. Here we have investigated the effects of tamoxifen and fulvestrant treatment on invasive signalling and compared this with the direct effects of oestrogen withdrawal to mimic the action of aromatase inhibitors. METHODS: The effect of oestrogen and 4-hydroxy-tamoxifen on the invasive capacity of endocrine-sensitive MCF-7 cells, in the presence or absence of functional E-cadherin, was determined by Matrigel invasion assays. Studies also monitored the impact of oestrogen withdrawal or treatment with fulvestrant on cell invasion. Western blotting using phospho-specific antibodies was performed to ascertain changes in invasive signalling in response to the two anti-oestrogens versus both oestradiol treatment and withdrawal. RESULTS: To the best of our knowledge, we report for the first time that tamoxifen can promote an invasive phenotype in ER-positive breast cancer cells under conditions of poor cell-cell contact and suggest a role for Src kinase and associated pro-invasive genes in this process. Our studies revealed that although this adverse effect is also apparent for further classes of anti-oestrogens, exemplified by the steroidal agent fulvestrant, it is absent during oestrogen withdrawal. CONCLUSIONS: These data highlight a previously unreported effect of tamoxifen (and potentially further anti-oestrogens), that such agents appear able to induce breast cancer cell invasion in a specific context (absence of good cell-cell contacts), where these findings may have major clinical implications for those patients with tumours that have inherently poor intercellular adhesion. In such patients oestrogen deprivation with aromatase inhibitors may be more appropriate.

The discovery of new and more potent chloropyramine (C4) analogues for the potential treatment of invasive breast cancer.

Breast cancer is the second most common cancer worldwide, accounting for 25% of all female cancers. Although the survival rate has increased significantly in the past few decades, patients who develop secondary site metastasis as well as those diagnosed with triple negative breast cancer still represent a real unmet medical challenge. Previous studies have shown that chloropyramine (C4) inhibits FAK-VEGFR3 signalling. More recently, C4 is reported to have SASH1 inducing properties. However, C4 exerts its antitumour and antiangiogenic effects at high micromolar concentrations (>100 µm) that would not be compatible with further drug development against invasive breast cancer driven by FAK signalling. In this study, molecular modelling guided structural modifications have been introduced to the chloropyramine C4 scaffold to improve its activity in breast cancer cell lines. Seventeen compounds were designed and synthesized, and their antiproliferative activity was evaluated against three human breast cancer lines (MDA-MB-231, BT474 and T47D). Compound 5c was identified to display an average activity of IC50  = 23.5-31.3 µm, which represents a significant improvement of C4 activity in the same assay model. Molecular modelling and pharmacokinetic studies provided more promising insights into the mechanistic features of this new series.

Co-Administration of Fish Oil With Signal Transduction Inhibitors Has Anti-Migration Effects in Breast Cancer Cell Lines, in vitro.

BACKGROUND: There is an urgent need for new therapies to treat cancer metastasis. Fish oil, with high omega 3 fatty acid content, has shown anticancer activity and signal transduction inhibitors have shown anti-metastatic properties. OBJECTIVE: To provide preliminary in vitro data on the anti-migration potential of signal transduction inhibitors and co-administered fish oil. METHODS: MCF-7, TamR and FasR breast cancer cell lines were used to determine the effects of combinations of PD98059, LY294002 and fish oil in growth assays. Modulations of p-Src and COX-2, both mediators of motility and invasion, were then determined by Western blotting and IHC to ascertain effects on migration potential. RESULTS: Migration rates for the three cell lines examined were ranked: FasR>TamR>MCF-7 (p <0.05). Addition of fish oil reduced the number of TamR cells migrating after 48h (p <0.05), while the addition of PD98059 and LY294002 also decreased migratory potential of TamR cells (p <0.05). Addition of PD98059 and LY294002 to TamR cells did not result in a significant decrease in p-Src levels; as was the case when PD98059, LY294002 and 4-hydroxytamoxifen were added to MCF-7 cells. However, the co-administration of fish oil markedly reduced p-Src and COX-2 expression in both cell lines. CONCLUSION: Co-administration of a commercial fish oil with signal transduction inhibitors results in decreased cell migration via an unknown co-operative mechanism and could constitute a novel approach for the treatment of breast cancer metastasis.

Src kinase promotes adhesion-independent activation of FAK and enhances cellular migration in tamoxifen-resistant breast cancer cells.

Src kinase is intimately involved in the control of matrix adhesion and cell migration through its ability to modulate the activity of focal adhesion kinase (FAK). In light of our previous observations that acquisition of tamoxifen resistance in breast cancer cells is accompanied by elevated Src kinase activity, we wish to investigate whether FAK function is also altered in these cells and if this leads to an enhanced migratory phenotype. In in vitro adhesion assays, tamoxifen-resistant (TamR) MCF7 cells had a greater affinity for the matrix proteins fibronectin, laminin, vitronectin and collagen and subsequently demonstrated a much greater migratory capacity across these substrates compared to their weakly-migratory, endocrine-sensitive counterparts. Additionally, elevated levels of activated Src in TamR cells promoted an increase in FAK phosphorylation at Y861 and Y925 and uncoupled FAK activation from an adhesion-dependent process. Inhibition of Src activity using the Src/Abl inhibitor AZD0530 reduced FAK activity, suppressed cell spreading on matrix-coated surfaces and significantly inhibited cell migration. Our data thus suggest that Src kinase plays a central role in the enhanced migratory phenotype that accompanies endocrine resistance through its modulation of FAK signalling and demonstrates the potential use of Src inhibitors as potent suppressors of tumour cell migration.

Src as a therapeutic target in anti-hormone/anti-growth factor-resistant breast cancer.

Endocrine therapy is the treatment of choice in hormone receptor-positive breast cancer. However, the effectiveness of anti-hormone drugs, such as tamoxifen, is limited because of the development of resistance, ultimately leading to disease progression and patient mortality. Using in vitro cell models of anti-hormone resistance, we have previously demonstrated that altered growth factor signalling contributes to an endocrine insensitive phenotype. Significantly, our recent studies have revealed that the acquisition of endocrine resistance in breast cancer is accompanied by a greatly enhanced migratory and invasive phenotype. Furthermore, therapeutic intervention using anti-growth factor monotherapies, despite an initial growth suppressive phase, again results in the development of a resistant state and a further augmentation of their invasive phenotype. Using the dual specific Src/Abl kinase inhibitor, AZD0530, we have highlighted a central role for Src kinase in promoting the invasive phenotype that accompanies both anti-hormone and anti-growth factor resistance. Importantly, the use of Src inhibitors in combination with anti-growth factor therapies appears to be additive, producing a marked inhibitory effect on cell growth, migration and invasion and ultimately prevents the emergence of a resistant phenotype. These observations suggest that the inhibition of Src activity may present a novel therapeutic intervention strategy, particularly when used as an adjuvant in endocrine-resistant breast disease, with the potential to delay or prevent the acquisition of subsequent resistance to anti-growth factor therapies.

Chronic exposure to fulvestrant promotes overexpression of the c-Met receptor in breast cancer cells: implications for tumour-stroma interactions.

Our previous investigations using cell models of tamoxifen resistance have shown that the acquisition of an endocrine-insensitive state is accompanied by an invasive in vitro phenotype. In this study, we wished to determine whether this was specifically related to partial oestrogen receptor agonists or whether similar phenomena arise with the newer 'pure' anti-oestrogens, exemplified by fulvestrant. Our data demonstrate that the development of fulvestrant resistance in two breast cancer cell lines, MCF7 and T47D, is accompanied by an augmented migratory and invasive phenotype in vitro and overexpression of the HGF/SF receptor, c-Met. Importantly, upregulated c-Met expression in these cells facilitates their stimulation by HGF/SF-secreting stromal fibroblasts, leading to the activation of Src, Akt and ERK1/2 and a profound enhancement of their aggressive phenotype in vitro. These effects could be specifically attributable to activation of the c-Met receptor since the inclusion of neutralising antibodies to c-Met, or siRNA-mediated knockdown of c-Met expression, suppressed both invasion and migration stimulated by either exogenous HGF/SF, fibroblast-conditioned medium or following co-culture with fibroblast cells. Together, these in vitro data suggest that the development of fulvestrant resistance in vivo may confer a metastatic advantage to the cells by allowing their migratory and invasive behaviour to be augmented by surrounding stromal cells.

The Role of BCA2 in the Endocytic Trafficking of EGFR and Significance as a Prognostic Biomarker in Cancer.

Breast Cancer Associated gene 2 (BCA2) is an E3 ubiquitin ligase that is over-expressed in >50% of primary breast cancers, and has been shown to increase in vitro cell proliferation and invasion. The protein has been linked to alterations in EGFR degradation; however there is some dispute as to its role and influence on the biology of this receptor. Our work aimed to ascertain the role of BCA2 in EGFR endocytosis and down-regulation and to examine its links with breast cancer outcome. Data generated with the online expression analysis tool KM-Plotter showed that high BCA2 levels are associated with poor prognosis in ovarian, gastric and breast cancer, particularly HER2 over-expressing breast cancers. Experimentally, we demonstrate that over-expression of BCA2 induced a reduction in total EGFR levels. BCA2 over-expressing cells stimulated with EGF exhibited reduced lysosomal degradation of both this ligand and its receptor. Signalling downstream of EGFR in BCA2 over-expressing cells was characterized by a lower magnitude but increased duration. Our findings support a role for BCA2 in receptor endocytosis. Consistent with this we show that BCA2 over-expression reduces the level of vesicle-associated Rab7, a regulator of late endocytosis and documented interaction partner of BCA2. Levels of transferrin receptor and the uptake of transferrin were unaltered by over-expression of BCA2 indicating that trafficking changes may be limited to late endocytic sorting events. This report offers a thorough exploration of BCA2 biology and suggests a context-dependent role for the protein in the endocytic regulation of EGFR and as a prognostic biomarker in cancer.

Overexpression of Specific CD44 Isoforms Is Associated with Aggressive Cell Features in Acquired Endocrine Resistance.

While endocrine therapy is the mainstay of ER+ breast cancer, the clinical effectiveness of these agents is limited by the phenomenon of acquired resistance that is associated with disease relapse and poor prognosis. Our previous studies revealed that acquired resistance is accompanied by a gain in cellular invasion and migration and also that CD44 family proteins are overexpressed in the resistant phenotype. Given the association of CD44 with tumor progression, we hypothesized that its overexpression may act to promote the aggressive behavior of endocrine-resistant breast cancers. Here, we have investigated further the role of two specific CD44 isoforms, CD44v3 and CD44v6, in the endocrine-resistant phenotype. Our data revealed that overexpression of CD44v6, but not CD44v3, in endocrine-sensitive MCF-7 cells resulted in a gain in EGFR signaling, enhanced their endogenous invasive capacity, and attenuated their response to endocrine treatment. Suppression of CD44v6 in endocrine-resistant cell models was associated with a reduction in their invasive capacity. Our data suggest that upregulation of CD44v6 in acquired resistant breast cancer may contribute to a gain in the aggressive phenotype of these cells and loss of endocrine response through transactivation of the EGFR pathway. Future therapeutic targeting of CD44v6 may prove to be an effective strategy alongside EGFR-targeted agents in delaying/preventing acquired resistance in breast cancer.