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Background: Invadopodia facilitate cancer cell extravasation, but the molecular mechanism whereby invadopodia-specific proteases such as MT1-MMP are called to invadopodia is unclear., Methods: Mass spectrometry and immunoprecipitation were used to identify interactors of MT1-MMP in metastatic breast cancer cells. After identification, siRNA and small molecule inhibitors were used to assess the effect these interactors had on cellular invasiveness. The chicken embryo chorioallantoic membrane (CAM) model was used to assess extravasation and invadopodia formation in vivo., Results: In metastatic breast cancer cells, MT1-MMP was found to associate with plectin, a cytolinker and scaffolding protein. Complex formation between plectin and MT1-MMP launches invadopodia formation, a subtype we termed i plectin ( i  = invadopodial). i Plectin delivers MT1-MMP to invadopodia and is indispensable for regulating cell surface levels of the enzyme. Genetic depletion of plectin with siRNA reduced invadopodia formation and cell invasion in vitro. In vivo extravasation efficiency assays and intravital imaging revealed i plectin to be a key contributor to invadopodia ultrastructure and essential for extravasation. Pharmacologic inhibition of plectin using the small molecule Plecstatin-1 (PST-1) abrogated MT1-MMP delivery to invadopodia and extravasation efficiency., Conclusions: Anti-metastasis therapeutic approaches that target invadopodia are possible by disrupting interactions between MT1-MMP and i plectin., Clinical Trial Registration Number: NCT04608357., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The loss of intercellular adhesion molecule E-cadherin is a hallmark of the epithelial-mesenchymal transition (EMT), during which tumor cells transition into an invasive phenotype. Accordingly, E-cadherin has long been considered a tumor suppressor gene; however, E-cadherin expression is paradoxically correlated with breast cancer survival rates. Using novel multi-compartment organoids and multiple in vivo models, we show that E-cadherin promotes a hyper-proliferative phenotype in breast cancer cells via interaction with the transmembrane receptor EGFR. The E-cad and EGFR interaction results in activation of the MEK/ERK signaling pathway, leading to a significant increase in proliferation via activation of transcription factors, including c-Fos. Pharmacological inhibition of MEK activity in E-cadherin positive breast cancer significantly decreases both tumor growth and macro-metastasis in vivo. This work provides evidence for a novel role of E-cadherin in breast tumor progression and identifies a new target to treat hyper-proliferative E-cadherin-positive breast tumors, thus providing the foundation to utilize E-cadherin as a biomarker for specific therapeutic success., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The acquisition of cellular invasiveness by breast epithelial cells and subsequent transition from ductal carcinoma in situ (DCIS) to invasive breast cancer is a critical step in breast cancer progression. Little is known about the molecular dynamics governing this transition. We have previously shown that overexpression of the transcriptional regulator TBX3 in DCIS-like cells increases survival, growth, and invasiveness. To explore this mechanism further and assess direct transcriptional targets of TBX3 in a high-resolution, isoform-specific context, we conducted genome-wide chromatin-immunoprecipitation (ChIP) arrays coupled with transcriptomic analysis. We show that TBX3 regulates several epithelial-mesenchymal transition (EMT)-related genes, including SLUG and TWIST1. Importantly, we demonstrate that TBX3 is a direct regulator of SLUG expression, and SLUG expression is required for TBX3-induced migration and invasion. Assessing TBX3 by immunohistochemistry in early-stage (stage 0 and stage I) breast cancers revealed high expression in low-grade lesions. Within a second independent early-stage non-high-grade cohort, we observed an association between TBX3 level in the DCIS and size of the invasive focus. Additionally, there was a positive correlation between TBX3 and SLUG, and TBX3 and TWIST1 in the invasive carcinoma. Pathway analysis revealed altered expression of several proteases and their inhibitors, consistent with the ability to degrade basement membrane in vivo. These findings strongly suggest the involvement of TBX3 in the promotion of invasiveness and progression of early-stage pre-invasive breast cancer to invasive carcinoma through the low-grade molecular pathway. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Invadopodia are cell protrusions that mediate cancer cell extravasation but the microenvironmental cues and signaling factors that induce invadopodia formation during extravasation remain unclear. Using intravital imaging and loss of function experiments, we determined invadopodia contain receptors involved in chemotaxis, namely GABA receptor and EGFR. These chemotaxis capabilities are mediated in part by PAK1 which controls invadopodia responsiveness to ligands such as GABA and EGF via assembly, stability, and turnover of invadopodia in vivo. PAK1 knockdown rendered cells unresponsive to chemotactic stimuli present in the stroma, resulting in dramatically lower rates of cancer cell extravasation and metastatic colony formation compared to stimulated cancer cells. In an experimental mouse model of brain metastasis, inhibition of PAK1 significantly reduced overall tumor burden and reduced the average size of brain metastases. In summary, invadopodia contain chemotaxis receptors that can respond to microenvironmental cues to guide cancer cell extravasation, and when PAK1 is depleted, brain tropism of metastatic breast cancer cells is significantly reduced, blocking secondary colony growth at sites otherwise permissive for metastatic outgrowth.

Previous studies indicate that breast cancer cells with high aldehyde dehydrogenase (ALDH) activity and CD44 expression (ALDH hi CD44⁺) contribute to metastasis and therapy resistance, and that ALDH1 correlates with poor outcome in breast cancer patients. The current study hypothesized that ALDH1 functionally contributes to breast cancer metastatic behavior and therapy resistance. Expression of ALDH1A1 or ALDH1A3 was knocked down in MDA-MB-468 and SUM159 human breast cancer cells using siRNA. Resulting impacts on ALDH activity (Aldefluor ® assay); metastatic behavior and therapy response in vitro (proliferation/adhesion/migration/colony formation/chemotherapy and radiation) and extravasation/metastasis in vivo (chick choroiallantoic membrane assay) was assessed. Knockdown of ALDH1A3 but not ALDH1A1 in breast cancer cells decreased ALDH activity, and knockdown of ALDH1A1 reduced breast cancer cell metastatic behavior and therapy resistance relative to control ( p < 0.05). In contrast, knockdown of ALDH1A3 did not alter proliferation, extravasation, or therapy resistance, but increased adhesion/migration and decreased colony formation/metastasis relative to control ( p < 0.05). This is the first study to systematically examine the function of ALDH1 isozymes in individual breast cancer cell behaviors that contribute to metastasis. Our novel results indicate that ALDH1 mediates breast cancer metastatic behavior and therapy resistance, and that different enzyme isoforms within the ALDH1 family differentially impact these cell behaviors., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Background: MicroRNAs (miRs) are involved in the regulation of many processes that contribute to malignancy, including cell proliferation, radiation resistance, invasion and metastasis. The role of miR-330-3p, an miR upregulated in breast cancer, remains unclear., Methods: We examine the association of miR-330-3p with distant relapse-free survival in the Oxford cohort of breast cancer patients. We also study miR-330-3p function using in vitro invasion and ex ovo metastasis assays. Using in vitro luciferase assays, we validate a novel target gene for miR-330-3p, Collagen And Calcium Binding EGF Domains 1 (CCBE1). We assess functional consequences of CCBE1 loss by using siRNA-mediated knockdown followed by in vitro invasion assays. Lastly, we examine the expression profile of CCBE1 in breast carcinomas in the Curtis and TCGA Breast Cancer data sets using Oncomine Platform as well as distant relapse-free and overall survival of patients in the Helsinki University breast cancer data set according to CCBE1 expression status., Results: miR-330-3p is enriched in breast cancer, and higher levels of miR-330-3p expression are associated with lower distant relapse-free survival in a cohort of breast cancer patients. Consistent with these observations, overexpression of miR-330-3p in breast cancer cell lines results in greater invasiveness in vitro, and miR-330-3p-overexpressing cells also metastasise more aggressively ex ovo. We identify CCBE1 as a direct target of miR-330-3p, and show that knockdown of CCBE1 results in a greater invasive capacity. Accordingly, in breast cancer patients CCBE1 is frequently downregulated, and its loss is associated with reduced distant relapse-free and overall survival., Conclusions: We show for the first time that miR-330-3p targets CCBE1 to promote invasion and metastasis. miR-330-3p and CCBE1 may represent promising biomarkers in breast cancer.

Human breast cancers that exhibit high proportions of immune cells and elevated levels of pro-inflammatory cytokines predict poor prognosis. Here, we demonstrate that treatment of human MCF-7 breast cancer cells with pro-inflammatory cytokines results in ERα-dependent activation of gene expression and proliferation, in the absence of ligand or presence of 4OH-tamoxifen (TOT). Cytokine activation of ERα and endocrine resistance is dependent on phosphorylation of ERα at S305 in the hinge domain. Phosphorylation of S305 by IKKβ establishes an ERα cistrome that substantially overlaps with the estradiol (E2)-dependent ERα cistrome. Structural analyses suggest that S305-P forms a charge-linked bridge with the C-terminal F domain of ERα that enables inter-domain communication and constitutive activity from the N-terminal coactivator-binding site, revealing the structural basis of endocrine resistance. ERα therefore functions as a transcriptional effector of cytokine-induced IKKβ signaling, suggesting a mechanism through which the tumor microenvironment controls tumor progression and endocrine resistance., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Membrane-type-1 Matrix Metalloproteinase (MT1-MMP) is a multifunctional protease that regulates ECM degradation, proMMP-2 activation, and varied cellular processes including migration and viability. MT1-MMP is believed to be a central mediator of tumourigenesis whose role is dictated by its functionally distinct protein domains. Both the localization and signal transduction capabilities of MT1-MMP are dependent on its cytoplasmic domain, exemplifying diverse regulatory functions. To further our understanding of the multifunctional contributions of MT1-MMP to cellular processes, we overexpressed cytoplasmic domain altered constructs in MCF-7 breast cancer cells and analyzed migration and viability in 2D culture conditions, morphology in 3D Matrigel culture, and tumorigenic ability in vivo. We found that the cytoplasmic domain was not needed for MT1-MMP mediated migration promotion, but was necessary to maintain viability during serum depravation in 2D culture. Similarly, during 3D Matrigel culture the cytoplasmic domain of MT1-MMP was not needed to initiate a protrusive phenotype, but was necessary to prevent colony blebbing when cells were serum deprived. We also tested in vivo tumorigenic potential to show that cells expressing cytoplasmic domain altered constructs demonstrated a reduced ability to vascularize tumours. These results suggest that the cytoplasmic domain regulates MT1-MMP function in a manner required for cell survival, but is dispensable for cell migration., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) is a multifunctional protease implicated in metastatic progression ostensibly due to its ability to degrade extracellular matrix (ECM) components and allow migration of cells through the basement membrane. Despite in vitro studies demonstrating this principle, this knowledge has not translated into the use of MMP inhibitors (MMPi) as effective cancer therapeutics, or been corroborated by evidence of in vivo ECM degradation mediated by MT1-MMP, suggesting that our understanding of the role of MT1-MMP in cancer progression is incomplete., Methods: MCF-7 and MDA-MB 231 breast cancer cell lines were created that stably overexpress different levels of MT1-MMP. Using 2D culture, we analyzed proMMP-2 activation (gelatin zymography), ECM degradation (fluorescent gelatin), ERK signaling (immunoblot), cell migration (transwell/scratch closure/time-lapse imaging), and viability (colorimetric substrate) to assess how different MT1-MMP levels affect these cellular parameters. We also utilized Matrigel 3D cell culture and avian embryos to examine how different levels of MT1-MMP expression affect morphological changes in 3D culture, and tumourigenecity and extravasation efficiency in vivo., Results: In 2D culture, breast cancer cells expressing high levels of MT1-MMP were capable of widespread ECM degradation and TIMP-2-mediated proMMP-2 activation, but were not the most migratory. Instead, cells expressing low levels of MT1-MMP were the most migratory, and demonstrated increased viability and ERK activation. In 3D culture, MCF-7 breast cancer cells expressing low levels of MT1-MMP demonstrated an invasive protrusive phenotype, whereas cells expressing high levels of MT1-MMP demonstrated loss of colony structure and cell fragment release. Similarly, in vivo analysis demonstrated increased tumourigenecity and metastatic capability for cells expressing low levels of MT1-MMP, whereas cells expressing high levels were devoid of these qualities despite the production of functional MT1-MMP protein., Conclusions: This study demonstrates that excessive ECM degradation mediated by high levels of MT1-MMP is not associated with cell migration and tumourigenesis, while low levels of MT1-MMP promote invasion and vascularization in vivo.

Background: TBX3 is a T-box transcription factor repressor that is elevated in metastatic breast cancer and is believed to promote malignancy of tumor cells, possibly by promoting cell survival and epithelial-mesenchymal transition., Methods: The relative expression of TBX3 was assessed in the 21T cell lines which were derived from an individual patient and represent three distinct stages of breast cancer progression: 21PT, atypical ductal hyperplasia; 21NT, ductal carcinoma in situ; and 21MT-1, invasive mammary carcinoma. Two different isoforms of TBX3 (TBX3iso1 and TBX3iso2) were overexpressed to evaluate cell survival/colony forming ability, growth, and invasion in the ductal carcinoma in situ-like 21NT cell line using an in vitro Matrigel model of cancer progression. In addition, TBX3 expression was knocked down to evaluate the effects of downregulating TBX3 on the invasive mammary carcinoma-like 21MT-1 cell line. Finally, PCR array profiling was used to assess alterations in gene expression due to TBX3 overexpression in the 21NT cells., Results: TBX3 is abundant in the invasive 21MT-1 cell line, while being minimally expressed in the non-invasive 21NT and 21PT cell lines. Overexpression of either TBX3iso1 or TBX3iso2 in 21NT cells resulted in increased cell survival/colony forming ability, growth vs. apoptosis and invasion in Matrigel. In contrast, short hairpin RNA-mediated knockdown of TBX3 in the 21MT-1 cells resulted in smaller colonies, with a more regular, less dispersed (less infiltrative) morphology. Array profiling of the 21NT TBX3 iso1 and iso2 transfectants showed that there are common alterations in expression of several genes involved in signal transduction, cell cycle control/cell survival, epithelial-mesenchymal transition and invasiveness., Conclusions: Overall, these results indicate that TBX3 (isoform 1 or 2) expression can promote progression in a model of early breast cancer by altering cell properties involved in cell survival/colony formation and invasiveness, as well as key regulatory and EMT/invasiveness-related gene expressions.

Planar cell polarity (PCP) signaling has been shown in different studies to either promote or inhibit the malignancy of breast cancer. Using the 21T cell lines, which were derived from an individual patient and represent distinct stages of progression, we show that the prototypical PCP ligand, WNT5A, is expressed highest in 21MT-1 cells (invasive mammary carcinoma) and lowest in 21PT (atypical ductal hyperplasia) and 21NT (ductal carcinoma in situ) cells. Overexpression of WNT5A decreased spherical colony formation and increased invasion and in vivo extravasation only in 21NT cells; whereas overexpression increased migration of both 21PT and 21NT cells. WNT5A overexpression also increased RHOA expression of both cell lines and subsequent RHOA knockdown blocked WNT5A-induced migration, but only partially blocked WNT5A-induced invasion of 21NT cells. PCP can signal through VANGL1 to modulate AP-1 target genes (e.g. MMP3) and induce invasion. VANGL1 knockdown inhibited WNT5A-induced invasion of 21NT cells, but had no effect on WNT5A-induced migration of either 21PT or 21NT cells. WNT5A-induced MMP3 expression was seen only in 21NT cells, an effect that was VANGL1 dependent, but independent of AP-1. We thus provide evidence that PCP signaling can act in a context dependent manner to promote breast cancer progression.

Tumor heterogeneity confounds cancer diagnosis and the outcome of therapy, necessitating analysis of tumor cell subsets within the tumor mass. Elevated expression of hyaluronan (HA) and HA receptors, receptor for HA-mediated motility (RHAMM)/HA-mediated motility receptor and cluster designation 44 (CD44), in breast tumors correlates with poor outcome. We hypothesized that a probe for detecting HA-HA receptor interactions may reveal breast cancer (BCa) cell heterogeneity relevant to tumor progression. A fluorescent HA (F-HA) probe containing a mixture of polymer sizes typical of tumor microenvironments (10-480 kDa), multiplexed profiling, and flow cytometry were used to monitor HA binding to BCa cell lines of different molecular subtypes. Formulae were developed to quantify binding heterogeneity and to measure invasion in vivo. Two subsets exhibiting differential binding (HA(-/low) vs. HA(high)) were isolated and characterized for morphology, growth, and invasion in culture and as xenografts in vivo. F-HA-binding amounts and degree of heterogeneity varied with BCa subtype, were highest in the malignant basal-like cell lines, and decreased upon reversion to a nonmalignant phenotype. Binding amounts correlated with CD44 and RHAMM displayed but binding heterogeneity appeared to arise from a differential ability of HA receptor-positive subpopulations to interact with F-HA. HA(high) subpopulations exhibited significantly higher local invasion and lung micrometastases but, unexpectedly, lower proliferation than either unsorted parental cells or the HA(-/low) subpopulation. Querying F-HA binding to aggressive tumor cells reveals a previously undetected form of heterogeneity that predicts invasive/metastatic behavior and that may aid both early identification of cancer patients susceptible to metastasis, and detection/therapy of invasive BCa subpopulations.

The analysis of dynamic events in the tumor microenvironment during cancer progression is limited by the complexity of current in vivo imaging models. This is coupled with an inability to rapidly modulate and visualize protein activity in real time and to understand the consequence of these perturbations in vivo. We developed an intravital imaging approach that allows the rapid induction and subsequent depletion of target protein levels within human cancer xenografts while assessing the impact on cell behavior and morphology in real time. A conditionally stabilized fluorescent E-cadherin chimera was expressed in metastatic breast cancer cells, and the impact of E-cadherin induction and depletion was visualized using real-time confocal microscopy in a xenograft avian embryo model. We demonstrate the assessment of protein localization, cell morphology and migration in cells undergoing epithelial-mesenchymal and mesenchymal-epithelial transitions in breast tumors. This technique allows for precise control over protein activity in vivo while permitting the temporal analysis of dynamic biophysical parameters.

Early breast cancer progression involves advancement through specific morphological stages including atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive mammary carcinoma (IMC), although not necessarily always in a linear fashion. Observational studies have examined genetic, epigenetic and gene expression differences in breast tissues representing these stages of progression, but model systems which would allow for experimental testing of specific factors influencing transition through these stages are scarce. The 21T series cell lines, all originally derived from the same patient with metastatic breast cancer, have been proposed to represent a mammary tumor progression series. We report here that three of the 21T cell lines indeed mimic specific stages of human breast cancer progression (21PT-derived cells, ADH; 21NT-derived cells, DCIS; 21MT-1 cells, IMC) when grown in the mammary fat pad of nude mice, albeit after a year. To develop a more rapid, readily manipulatable in vitro assay for examining the biological differences between these cell lines, we have used a 3D Matrigel system. When the three cell lines were grown in 3D Matrigel, they showed characteristic morphologies, in which quantifiable aspects of stage-specific in vivo behaviors (ie, differences in acinar structure formation, cell polarization, colony morphology, cell proliferation, cell invasion) were recapitulated in a reproducible fashion. Gene expression profiling revealed a characteristic pattern for each of the three cell lines. Interestingly, Wnt pathway alterations are particularly predominant in the early transition from 21PTci (ADH) to 21NTci (DCIS), whereas alterations in expression of genes associated with control of cell motility and invasion phenomena are more prominent in the later transition of 21NTci (DCIS) to 21MT-1 (IMC). This system thus reveals potential therapeutic targets and will provide a means of testing the influences of identified genes on transitions between these stages of pre-malignant to malignant growth.

Introduction: Breast cancer is the commonest female cancer in Singapore. It is steadily rising with an incidence of 53.1 cases per 100,000 persons per year among women. Screening for detection of early lesions which are highly curable helps to reduce mortality., Methods: Over three afternoon sessions in December 2003, 224 female patients aged 40-65 years, participated in interviews conducted by the National Healthcare Group Polyclinics, Singapore. The survey sought information on mammographic screening history, the time interval since the previous mammographic screening, and the reasons for not going for the screening., Results: The survey found that only 26.4 percent (28 out of 106) among those aged 40 to 49 years had mammographic screening done within the past one year, and 43.2 percent (51 out of 118) among those aged 50 to 65 years had screening done within the last two years. Chinese women were twice more likely than Malay women to have a mammogram done. The commonest reasons for not wanting to have mammographic screening among women who did not have a mammogram done or had mammogram done more than two years ago, were lack of time (42.5 percent), fear of pain during the procedure (26.9 percent), and the belief that cancer would not happen to them (24.6 percent)., Conclusion: Despite publicity on breast cancer being the commonest cancer among women in Singapore and cure being possible if the malignancy was detected early, close to half of the women aged 40-65 years old who attended the National Healthcare Group Polyclinics did not have mammographic screening done. One-quarter of the women who did not have mammogram screening did not do so as they did not think cancer would happen to them.