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Supplementary Figure 1 from Matrix Metalloproteinase-8 Functions as a Metastasis Suppressor through Modulation of Tumor Cell Adhesion and Invasion

Collagenase-2 (matrix metalloproteinase-8, MMP-8) is an MMP mainly produced by neutrophils and associated with many inflammatory conditions. We have previously described that MMP-8 plays a protective role in cancer through its ability to regulate the inflammatory response induced by carcinogens. Moreover, it has been reported that experimental manipulation of the expression levels of this enzyme alters the metastatic behavior of human breast cancer cells. In this work, we have used mutant mice deficient in MMP-8 and syngenic melanoma and lung carcinoma tumor cells lines overexpressing this enzyme to further explore the putative antimetastatic potential of MMP-8. We report herein that MMP-8 prevents metastasis formation through the modulation of tumor cell adhesion and invasion. Thus, tumor cells overexpressing MMP-8 have an increased adhesion to extracellular matrix proteins, whereas their invasive ability through Matrigel is substantially reduced when compared with control cells. Analysis of MMP-8 in breast cancer patients revealed that the expression of this metalloproteinase by breast tumors correlates with a lower incidence of lymph node metastasis and confers good prognosis to these patients. On this basis, we propose that MMP-8 is a tumor protective factor, which also has the ability to reduce the metastatic potential of malignant cells in both mice and human. [Cancer Res 2008;68(8):2755–63]

The adoption of massively parallel short-read DNA sequencing methods has greatly expanded the scope and availability of genetic testing for inherited diseases. Indeed, the power of these methods has encouraged the integration of whole genome sequencing, the most comprehensive single approach to genomic analysis, into clinical practice. Despite these advances, diagnostic techniques that incompletely resolve the precise molecular boundaries of pathogenic sequence variants continue to be routinely deployed. This can present a barrier for certain prenatal diagnostic approaches. For example, the pre-referral workup for couples seeking preimplantation genetic diagnosis requires intragenic dosage variants to be characterised at nucleotide resolution.We sought to assess the use of long-read nanopore sequencing to rapidly characterise an apparent heterozygous RB1 exon 23 deletion that was initially identified by multiplex ligation-dependent probe amplification (MLPA), in a patient with bilateral retinoblastoma.Target enrichment was performed by long-range polymerase chain reaction (PCR) amplification prior to Flongle sequencing on a MinION long-read sequencer.Characterisation of the deletion breakpoint included an unexpected 85-bp insertion which duplicated RB1 exon 24 (and was undetected by MLPA). The long-read sequence permitted design of a multiplex PCR assay, which confirmed that the mutation arose de novo.Our experience demonstrates the diagnostic utility of long-read technology for the precise characterisation of structural variants, and highlights how this technology can be efficiently deployed to enable onward referral to reproductive medicine services.

Multicellular 3-dimensional (3D) in vitro models of normal human breast tissue to study cancer initiation are required. We present a model incorporating three of the major functional cell types of breast, detail the phenotype and document our breast cancer initiation studies. Myoepithelial cells and fibroblasts were isolated and immortalised from breast reduction mammoplasty samples. Tri-cultures containing non-tumorigenic luminal epithelial cells HB2, or HB2 overexpressing different HER proteins, together with myoepithelial cells and fibroblasts were established in collagen I. Phenotype was assessed morphologically and immunohistochemically and compared to normal breast tissue. When all three cell types were present, polarised epithelial structures with lumens and basement membrane production were observed, akin to normal human breast tissue. Overexpression of HER2 or HER2/3 caused a significant increase in size, while HER2 overexpression resulted in development of a DCIS-like phenotype. In summary, we have developed a 3D tri-cellular model of normal human breast, amenable to comparative analysis after genetic manipulation and with potential to dissect the mechanisms behind the early stages of breast cancer initiation.

The CUB and sushi multiple domains 1 (CSMD1) gene maps to chromosome 8p23, a region deleted in many cancers. Loss of CSMD1 expression is associated with poor prognosis in breast cancer suggesting that it acts as a tumour suppressor in this cancer. However, the function of CSMD1 is largely unknown. Herein, we investigated CSMD1 functions in cell line models. CSMD1 expression was suppressed in MCF10A and LNCaP cells using short hairpin RNA. Functional assays were performed focusing on the 'normal' MCF10A cell line. Suppression of CSMD1 significantly increased the proliferation, cell migration and invasiveness of MCF10A cells compared to shcontrols. shCSMD1 cells also showed significantly reduced adhesion to Matrigel and fibronectin. In a three-dimensional Matrigel model of MCF10A cells, reduced CSMD1 expression resulted in the development of larger and more poorly differentiated breast acini-like structures that displayed impaired lumen formation. Loss of CSMD1 expression disrupts a model of mammary duct formation while enhancing proliferation, migration and invasion. Our data suggest that CSMD1 is involved in the suppression of a transformed phenotype.

Carcinoma-associated fibroblasts (CAFs) influence the behaviour of cancer cells but the roles of microRNAs in this interaction are unknown. We report microRNAs that are differentially expressed between breast normal fibroblasts and CAFs of oestrogen receptor-positive cancers, and explore the influences of one of these, miR-26b, on breast cancer biology. We identified differentially expressed microRNAs by expression profiling of clinical samples and a tissue culture model: miR-26b was the most highly deregulated microRNA. Using qPCR, miR-26b was confirmed as down-regulated in fibroblasts from 15 of 18 further breast cancers. Next, we examined whether manipulation of miR-26b expression changed breast fibroblast behaviour. Reduced miR-26b expression caused fibroblast migration and invasion to increase by up to three-fold in scratch-closure and trans-well assays. Furthermore, in co-culture with MCF7 breast cancer epithelial cells, fibroblasts with reduced miR-26b expression enhanced both MCF7 migration in trans-well assays and MCF7 invasion from three-dimensional spheroids by up to five-fold. Mass spectrometry was used to identify expression changes associated with the reduction of miR-26b expression in fibroblasts. Pathway analyses of differentially expressed proteins revealed that glycolysis/TCA cycle and cytoskeletal regulation by Rho GTPases are downstream of miR-26b. In addition, three novel miR-26b targets were identified (TNKS1BP1, CPSF7, COL12A1) and the expression of each in cancer stroma was shown to be significantly associated with breast cancer recurrence. MiR-26b in breast CAFs is a potent regulator of cancer behaviour in oestrogen receptor-positive cancers, and we have identified key genes and molecular pathways that act downstream of miR-26b in CAFs. © 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Integrin α9β1 is a receptor for ECM proteins, including Tenascin-C and the EDA domain of fibronectin, and has been shown to transduce TGFβ signalling. This study has examined the expression pattern of α9β1 in 141 frozen breast carcinoma samples and related expression to prognostic indices, molecular subtype and patient outcome. Effects of α9β1 on tumour cell migration and invasion were assessed using blocking antibody and gene transduction approaches. Integrin α9β1 localized to myoepithelial cells in normal ducts and acini, a pattern maintained in DCIS. A subset (17%) of invasive carcinomas exhibited tumour cell expression of α9β1, which related significantly to the basal-like phenotype, as defined by either CK5/6 or CK14 expression. Tumour expression of α9β1 showed a significant association with reduced overall patient survival (p < 0.0001; HR 5.94, 95%CI 3.26-10.82) and with reduced distant-metastasis-free survival (p < 0.0001; HR 6.37, CI 3.51-11.58). A series of breast cancer cell lines was screened for α9β1 with the highly invasive basal-like GI-101 cell line expressing significant levels. Both migration and invasion of this line were reduced significantly in the presence of α9-blocking antibody and following α9-knockdown with siRNA. Conversely, migratory and invasive behaviour of α9-negative MCF7 cells and α9-low MDA MB468 cells was enhanced significantly by over-expression of α9. Thus, α9β1 acts as a novel marker of the basal-like breast cancer subtype and expression is associated with reduced survival, while its ability to promote breast cancer cell migration and invasion suggests that it contributes to the aggressive clinical behaviour of this tumour subtype.

Contains fulltext : 69166.pdf (Publisher’s version ) (Closed access) Collagenase-2 (matrix metalloproteinase-8, MMP-8) is an MMP mainly produced by neutrophils and associated with many inflammatory conditions. We have previously described that MMP-8 plays a protective role in cancer through its ability to regulate the inflammatory response induced by carcinogens. Moreover, it has been reported that experimental manipulation of the expression levels of this enzyme alters the metastatic behavior of human breast cancer cells. In this work, we have used mutant mice deficient in MMP-8 and syngenic melanoma and lung carcinoma tumor cells lines overexpressing this enzyme to further explore the putative antimetastatic potential of MMP-8. We report herein that MMP-8 prevents metastasis formation through the modulation of tumor cell adhesion and invasion. Thus, tumor cells overexpressing MMP-8 have an increased adhesion to extracellular matrix proteins, whereas their invasive ability through Matrigel is substantially reduced when compared with control cells. Analysis of MMP-8 in breast cancer patients revealed that the expression of this metalloproteinase by breast tumors correlates with a lower incidence of lymph node metastasis and confers good prognosis to these patients. On this basis, we propose that MMP-8 is a tumor protective factor, which also has the ability to reduce the metastatic potential of malignant cells in both mice and human.

Background 3D cell cultures are emerging as more physiologically meaningful alternatives to monolayer cultures for many biological applications. They are attractive because they more closely mimic in vivo morphology, especially when co-cultured with stromal fibroblasts. Methodology/Principal Findings We compared the efficacy of 3 different 3D cell culture systems; collagen I, low attachment culture vessels and a modification of Fibrolife®, a specialised humanised cell culture medium devoid of animal-derived components, using breast cancer cell lines representative of the different molecular subtypes of breast cancer, cultured alone or with human mammary fibroblasts with a view to developing matrix-free humanised systems. 3D collagen I culture supported the growth of a range of breast cancer cell lines. By modifying the composition of Fibrolife® to epiFL, matrix-free cell culture was possible. During sequential transfer to epiFL breast cancer cells gradually detached from the flask, growing progressively as spheroids. Phenotype was stable and reversible with cells remaining actively proliferating and easily accessible throughout culture. They could also be revived from frozen stocks. To achieve co-culture with fibroblasts in epiFL required use of low attachment culture vessels instead of standard plastic as fibroblasts remained adherent in epiFL. Here, cancer cell spheroids were allowed to form before adding fibroblasts. Immunohistochemical examination showed fibroblasts scattered throughout the epithelial spheroid, not dissimilar to the relationship of tumour stroma in human breast cancer. Conclusions Because of its ease of handling, matrix-free 3D cell culture may be a useful model to study the influence of fibroblasts on breast cancer epithelial cells with use of epiFL culture medium taking this a step further towards a fully humanised 3D model. This methodology could be applied to other types of cancer cell lines, making this a versatile technique for cancer researchers wishing to use in vitro systems that better reflect cancer in vivo.

Models considering breast cancer complexity cannot be easily or accurately replicated in routine cell line or animal models. We aimed to evaluate the practicality of organotypic tissue slice culture in breast cancer. Following ethical approval, 250 µm thick sections from surplus breast tumours (n=10) were prepared using a vibrating blade microtome. Triplicate tissue slices were placed in 6-well plates and cultured for up to 7 days ± tamoxifen (1 nM) or doxorubicin (1 µM). Tissue slices were fixed and embedded before sectioning for morphological evaluation and immunohistochemistry. H&E showed good preservation of tissue morphology. Collagen production was evident. Biomarkers of proliferation and apoptosis could be evaluated using immunohistochemistry and used as surrogates to quantify drug effects. In summary, breast cancer tissue slices can be cultured in vitro as organotypic models. Nevertheless, although simple in concept, the delicacy of the model with regard to handling makes subsequent analytical processes challenging.

Loss of caveolin-1 (Cav-1) expression in breast cancer-associated fibroblasts (CAFs) is predictive of poor prognosis in breast cancer, but its function has not been established. Our study tested the hypotheses that loss of Cav-1 expression in breast fibroblasts was associated with poor prognosis in breast cancer, through promotion of breast cancer cell invasion. Cav-1 stromal expression was immunohistochemically assessed in 358 breast cancers. Cav-1 expression in primary breast fibroblasts was analysed by western blot. Modified Boyden chamber assays determined fibroblast ability to promote invasion of breast cancer cells. The impact of siRNA silencing of Cav-1 in fibroblasts was evaluated using invasion assays and 3D co-culture assays. Loss of Cav-1 expression in breast stroma was significantly associated with decreased breast cancer-specific and disease-free survival (p = 0.01). Mean survival was 72 months (Cav-1(+) group) versus 29.5 months (Cav-1(-) group). This was confirmed in multivariate analysis. Cav-1 expression was significantly decreased in CAFs compared to normal fibroblasts (p = 0.01) and was associated with increased invasion-promoting capacity. Cav-1 siRNA-treated fibroblasts promoted significantly increased invasion of MDA-MB-468 and T47D breast cancer cells from 27% (control) to 67% (p = 0.006) and from 37% to 56%, respectively (p = 0.01). 3D co-cultures of MDA-MB-468 cells with myoepithelial cells led to the formation of organized cohesive structures when cultured with conditioned media from fibroblasts but resulted in a disorganized appearance in the presence of conditioned media from Cav-1 siRNA-treated fibroblasts, accompanied by loss of E-cadherin expression in tumour cells. Our data confirm that loss of stromal Cav-1 in breast cancer predicts poor outcome. At a functional level, Cav-1-deficient CAFs are capable of significantly increasing the invasive capacity of breast cancer cells.

Breast cancer is a complex and heterogeneous disease. Gene expression profiling has contributed significantly to our understanding of this heterogeneity at a molecular level, refining taxonomy based on simple measures such as histological type, tumour grade, lymph node status and the presence of predictive markers like oestrogen receptor and human epidermal growth factor receptor 2 (HER2) to a more sophisticated classification comprising luminal A, luminal B, basal-like, HER2-positive and normal subgroups. In the laboratory, breast cancer is often modelled using established cell lines. In the present review we discuss some of the issues surrounding the use of breast cancer cell lines as experimental models, in light of these revised clinical classifications, and put forward suggestions for improving their use in translational breast cancer research.

While the events leading to breast cancer development are not fully understood, a pre-invasive lesion, ductal carcinoma in situ (DCIS), is recognised as the main precursor of invasive disease. Understanding how pre-invasive lesions develop into invasive breast cancer is critical, since currently there is no way of predicting which tumours are likely to progress, leading to unnecessary surgical intervention or chemotherapy. With a lack of good animal models able to mimic DCIS progression in a laboratory setting, there has been a shift toward developing in vitro human models which more accurately represent human disease. By manipulating individual cell populations in these models, we can recapitulate the complex cellular interactions involved in disease progression, an essential step in understanding breast cancer behaviour.

A high percentage of stroma predicts poor survival in triple-negative breast cancers but is diminished in studies of unselected cases. We determined the prognostic significance of tumour-stroma ratio (TSR) in oestrogen receptor (ER)-positive male and female breast carcinomas.TSR was measured in haematoxylin and eosin-stained tissue sections (118 female and 62 male). Relationship of TSR (cutoff 49%) to overall survival (OS) and relapse-free survival (RFS) was analysed.Tumours with ≥49% stroma were associated with better survival in female (OS P=0.008, HR=0.2-0.7; RFS P=0.006, HR=0.1-0.6) and male breast cancer (OS P=0.005, HR=0.05-0.6; RFS P=0.01, HR=0.87-5.6), confirmed in multivariate analysis.High stromal content was related to better survival in ER-positive breast cancers across both genders, contrasting data in triple-negative breast cancer and highlighting the importance of considering ER status when interpreting the prognostic value of TSR.

Integrin α9β1 is a receptor for ECM proteins, including Tenascin-C and the EDA domain of fibronectin, and has been shown to transduce TGFβ signalling. This study has examined the expression pattern of α9β1 in 141 frozen breast carcinoma samples and related expression to prognostic indices, molecular subtype and patient outcome. Effects of α9β1 on tumour cell migration and invasion were assessed using blocking antibody and gene transduction approaches. Integrin α9β1 localized to myoepithelial cells in normal ducts and acini, a pattern maintained in DCIS. A subset (17%) of invasive carcinomas exhibited tumour cell expression of α9β1, which related significantly to the basal-like phenotype, as defined by either CK5/6 or CK14 expression. Tumour expression of α9β1 showed a significant association with reduced overall patient survival (p0.0001; HR 5.94, 95%CI 3.26-10.82) and with reduced distant-metastasis-free survival (p0.0001; HR 6.37, CI 3.51-11.58). A series of breast cancer cell lines was screened for α9β1 with the highly invasive basal-like GI-101 cell line expressing significant levels. Both migration and invasion of this line were reduced significantly in the presence of α9-blocking antibody and following α9-knockdown with siRNA. Conversely, migratory and invasive behaviour of α9-negative MCF7 cells and α9-low MDA MB468 cells was enhanced significantly by over-expression of α9. Thus, α9β1 acts as a novel marker of the basal-like breast cancer subtype and expression is associated with reduced survival, while its ability to promote breast cancer cell migration and invasion suggests that it contributes to the aggressive clinical behaviour of this tumour subtype.

Estrogen receptor (ER) action is modulated by posttranslational modifications. Although ERalpha phosphorylation correlates with patient outcome, ERbeta is similarly phosphorylated but its significance in breast cancer has not been addressed. We investigated whether ERbeta that is phosphorylated at serine 105 (S105-ERbeta) is expressed in breast cancer and assessed potential clinical implications of this phosphorylation. Following antibody validation, S105-ERbeta expression was studied in tissue microarrays comprising 108 tamoxifen-resistant and 351 tamoxifen-sensitive cases and analyzed against clinical data. S105-ERbeta regulation in vitro was assessed by Western blot, flow cytometry, and immunofluorescence. Nuclear S105-ERbeta was observed in breast carcinoma and was associated with better survival (Allred scoreor =3), even in tamoxifen-resistant cases, and additionally correlated with ERbeta1 and ERbeta2 expression. Distinct S105-ERbeta nuclear speckles were seen in some higher grade tumors. S105-ERbeta levels increased in MCF-7 cells in response to 17beta-estradiol, the ERbeta-specific agonist diarylpropionitrile, and the partial ERbeta-agonist genistein. S105-ERbeta nuclear speckles were also seen in MCF-7 cells and markedly increased in size and number at 24 hours following 17beta-estradiol and, in particular diarylpropionitrile, treatment. These speckles were coexpressed with ERbeta1 and ERbeta2. Presence of S105-ERbeta in breast cancer and association with improved survival, even in endocrine resistant breast tumors suggest S105-ERbeta might be a useful additional prognostic marker in this disease.

Microenvironmental factors are fundamental in the regulation of normal and tumour breast tissue. Two cell types have been implicated in having opposing effects on breast tumour cell behaviour: myoepithelial cells exhibit broad tumour-suppressor activity, whilst fibroblasts frequently promote tumour growth and invasion. Previous work has described the development of a physiologically relevant three-dimensional heterotypic culture system containing tumour, myoepithelial and fibroblast cells. The data showed organisation of the cells into co-unit structures recapitulating ductal carcinoma in situ breast, with homing of myoepithelial cells around luminal cells, and highlighted a central role for tumour-associated fibroblasts in disrupting ductal carcinoma in situ structures. This study describes further manipulation of the model to include tumour cells that represent the heterogeneity of breast cancer. MCF-7 (ER+), MDA-MB-468, MDA-MB-231 (basal) and MDA-MB-453 (Her2+) were cultured in collagen for 7 days in the presence or absence of normal myoepithelial cells. Gels were fixed in formalin, paraffin embedded and immunohistochemistry was performed for a series of markers recognising the cell types along with basal polarity and basement membrane proteins. Initial morphological analysis of the cultures has been performed to assess the degree of co-unit formation, based on a visual description of the size and shape of the co-units. Co-unit formation has been employed as a representative measure of tumour progression as it is known to be a key feature in early breast cancer invasion. When cultured alone, MCF-7 and MDA-MB-468 cells formed spherical co-unit structures and this was maintained in the presence of myoepithelial cells. In contrast, MDA-MB-231 and MDA-MB-453 cells show a more scattered appearance. The presence of myoepithelial cells induced polarity in the MDA-MB-231 cells and a more ordered appearance. This study is the first time that the co-culture of tumour cell populations with myoepithelial cells has been investigated in three-dimensional collagen gels showing differences in morphology that may relate to tumour progression.

CUB and Sushi multiple domain protein 1 (CSMD1) is a candidate tumour suppressor gene of unknown function. CSMD1 maps to chromosome 8p23, a region deleted in 50% of breast cancers (BC). We have examined the contribution of CSMD1 to the tumorigenic phenotype of mammary acini and evaluated its prognostic value in BC patients.

Yes-associated protein (YAP) has been shown to positively regulate p53 family members and to be negatively regulated by the AKT proto-oncogene product in promoting apoptosis. On the basis of this function and its location at 11q22.2, a site of frequent loss of heterozygosity (LOH) in breast cancer, we investigated whether YAP is a tumor suppressor in breast. Examination of tumors by immunohistochemistry demonstrated significant loss of YAP protein. LOH analysis revealed that protein loss correlates with specific deletion of the YAP gene locus. Functionally, short hairpin RNA knockdown of YAP in breast cell lines suppressed anoikis, increased migration and invasiveness, inhibited the response to taxol and enhanced tumor growth in nude mice. This is the first report indicating YAP as a tumor suppressor, revealing its decreased expression in breast cancer as well as demonstrating the functional implications of YAP loss in several aspects of cancer signaling.

Purpose: Polymorphisms within the promoter region of several matrix metalloproteinase (MMP) genes have been linked to alterations in the level of transcription. We hypothesized that an individual's MMP genotype and haplotype will influence breast tumor progression and help predict prognosis.Experimental Design: This study has evaluated the association between single-nucleotide polymorphisms (SNP) in the promoter regions of MMP-1, MMP-3, MMP-7, MMP-9, MMP-12, and MMP-13 and metastatic spread of breast cancer in 128 lymph node–negative and 93 lymph node–positive patients. The study cohort was of mixed ethnicity, with Caucasian patients comprising 65%. Associations between genotype and lymph node status were estimated by logistic regression and with overall survival using the method of Kaplan-Meier and log-rank test. Associations between haplotype and lymph node status were also investigated.Results: The data show a significant and independent association of the C/T genotype for MMP-9 [mixed ethnicities odds ratio 3.6, 95% confidence interval (95% CI) 1.2-11.1; Caucasian odds ratio 9.1, 95% CI 1.7-48.4] and the 2G/2G genotype for MMP-1 (mixed ethnicities odds ratio 3.9, 95% CI 1.7-9.4; Caucasian odds ratio 2.6, 95% CI 1.0-6.9) with lymph node–positive disease. MMP-1 2G/2G was associated with reduced survival (hazard ratio 3.1, 95% CI 1.1-8.7), although this is dependent on lymph node status. Two haplotypes, driven by the MMP-1 2G allele, were significantly associated with lymph node–positive disease and survival.Conclusions: These results suggest that MMP single-nucleotide polymorphisms influence breast cancer behavior and that the MMP-1 2G/2G genotype increases the risk of lymph node metastasis and predicts poor prognosis.

Background In recent years there has been an increasing awareness of the role of the microenvironment surrounding breast cancer epithelium, particularly the carcinoma-associated fibroblast (CAF), in modulating the behaviour of breast tumours. MicroRNAs, a family of small non-coding RNAs that are key in the post-transcriptional regulation of mRNAs, have a role in controlling the behaviour of cancers and have been studied extensively in breast cancer epithelial cells. We hypothesised that miRNAs have important roles in the behaviour of breast CAFs and, in turn, affect the behaviour of malignant breast epithelial cells. Methods We used laser capture microdissected normal fibroblast and CAFs from clinical samples, and a tissue co-culture model, to examine expression of miRNAs in breast CAFs. Deregulated miRNAs that were identified with this screening strategy were further assessed in a larger set of paired laser capture microdissected normal fibroblasts and CAFs. We assessed functional effects on fibroblasts by overexpressing, or knocking down miRNAs (or both) in immortalised fibroblast cell lines and using various growth, migration, and invasion assays. Functional effects of fibroblasts with reduced miRNA on breast cancer epithelial cells were examined in co-cultures. To identify potential miRNA targets and pathways downstream, we used mass spectrometry and in-silico analysis. The clinical relevance of these targets was examined by interrogating publicly available datasets. Findings Six miRNAs were consistently downregulated in CAFs compared with normal fibroblasts with a fold change of more that ten in both the tissue co-culture model and in patient samples. Of these miRNAs, miR-26b was downregulated in 12 of 14 cases of microdissected matched normal fibroblasts and CAFs from clinical formalin fixed paraffin embedded samples (Wilcoxon signed rank test, p=0·04), and consistently in a further four of four cases of matched primary cultures of normal fibroblasts and CAFs. Reduction of the level of miR-26b in immortalised breast fibroblasts showed a small decrease in proliferation but a very notable increase in migration (p COL12A1 was identified as a target of miR-26b. Increased stromal mRNA expression of COL12A1 was significantly associated with increased recurrence of breast cancer on multivariate analysis in publically available datasets (hazard ratio 17, 95% CI 1·9–159; p=0·01). Interpretation We have shown that downregulation of miR-26b in breast cancer fibroblasts increases the migration and invasion of breast cancer epithelial cells, and we have identified molecular changes that are downstream of miR-26b. These findings add to the growing body of evidence that epithelial-stromal interactions are important in modulating the behaviour of breast cancer. Funding UK Medical Research Council, Pathological Society of Great Britain and Ireland, Breast Cancer Campaign, National Institute of Health Research.

The mechanisms involved in breast cancer initiation are not well understood. This may in part be due to a lack of an in vitro model that faithfully recapitulates the morphology, phenotype and in vivo architecture of the human mammary gland. Most in vitro models of normal breast have relied on the use of reconstituted basement membrane gels to induce luminal epithelial cell polarity and have neglected the role of myoepithelial cells and fibroblasts in this process. We aimed to develop a 3D in vitro culture system of normal breast which includes three of the major functional cell types embedded in a more physiologically relevant collagen 1 matrix. We used this system to investigate the mechanisms behind breast cancer initiation via genetic manipulation of some well known oncogenes and tumour suppressors involved in breast cancer progression. Myoepithelial cells (Myo1089) and fibroblasts (generated in house) were isolated and immortalised from breast reduction mammoplasty samples collected with ethical approval. Following characterisation, these were virally transfected with Enhanced Green Fluorescent Protein (EGFP) and dsRed protein respectively to enable tracking. 3D cultures were established in collagen 1 and included the non-tumorigenic luminal epithelial cell line HB2, EGFP Myo1089 cells and dsRed fibroblasts. Cells were cultured for 3 weeks in Transwell™ cell culture inserts. Following fixation these were analysed by H&E, confocal microscopy and immunohistochemistry. Morphology and immunostaining profiles were compared to sections of normal breast tissue. Immunohistochemical characterisation using the following antibodies: E-cadherin, epithelial membrane antigen, vimentin, laminin 5, collagen 4 plus luminal and basal cytokeratins, demonstrated polarised epithelial structures with lumen formation and basement membrane production with a similar immunostaining profile to normal breast tissue. The importance of including myoepithelial cells and fibroblasts in maintaining these structures was demonstrated. We established this model is amenable to genetic manipulation by overexpressing Her2 in HB2 cells, and knocking out ERβ1 in EGFP Myo1089 cells and Rac1 and Dock4 in dsRed fibroblast cell lines using shRNA techniques. These were included in separate models with morphological and phenotypic effects determined by confocal microscopy. In summary we have developed an in vitro model of normal breast tissue that includes three of the major functional breast cell types cultured in a physiologically relevant 3D matrix. We have validated the morphology and protein expression profile against human breast tissue specimens and confirm that this is a suitable model of normal breast. The model is suitable for experimental manipulation and cell behaviour can be easily visualised using standard laboratory techniques. We conclude this is a robust in vitro model of normal breast tissue offering an alternative cost-effective method of studying genes involved in breast cancer initiation. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-02-06.

CUB and Sushi multiple domains protein 1 (CSMD1) maps to 8p23, a region deleted in many cancers, and thought to be a tumour suppressor gene. Loss of CSMD1 expression is associated with reduced survival in breast cancer patients. CSMD1’s function is unknown; however, CSMD1‘s structure suggests it is involved in signal transduction. Here, we have investigated the function of CSMD1. CSMD1 expression was silenced in MCF10A, MDA-MB435 and LNCaP cell lines by shRNA and functional assays were performed. Loss of CSMD1 expression disrupted cell morphology and caused 30% (p < 0.001), 32% (p = 0.03) and 56% (p < 0.001) increase in cell proliferation of MDA-MB-435, LNCaP and MCF10A, respectively, compared to controls. Also MDA-MB-435 and MCF10A shCSMD1 cells showed reduced adhesion to matrigel (32%, p = 0.0005 and 44%, p = 0.0006, respectively), and to fibronectin (39%, p = 0.004 and 32%, p < 0.001, respectively). Moreover, loss of CSMD1 expression enhanced cell migration of MDA-MB-435 and MCF10A and caused 33% (p < 0.001) increase in cell invasion of MCF10A, compared to control. The MCF10A 3D model revealed that loss of CSMD1 expression resulted in the development of larger poorly differentiated breast acini and impaired lumen formation. Loss of CSMD1 expression induced behaviour consistent with cellular transformation. Our data supports the concept that CSMD1 participates in signaling pathways that regulate a range of key cellular processes involved in the suppression of a transformed phenotype.

Background: Estrogen receptor (ER) α predicts response to hormonal therapy in breast carcinoma. The role of ERβ is less well understood. ER activities can be modulated by post-translational modifications including phosphorylation and phosphorylated ERα has been correlated with patient outcome. We investigated whether ERβ phosphorylated at Serine 105 (P-S105-ERβ) is expressed in breast carcinoma and assessed its potential clinical implications.Material and Methods: Tissue microarrays comprising 427 breast tumours (23% grade 1, 43% grade 2, 34% grade 3) with median follow up 118 months and 106 endocrine resistant breast tumours (15% grade 1, 41% grade 2, 44% grade 3) with median follow up 71 months were used in this study. Expression of P-S105-ERβ was studied by immunohistochemistry and analysed against clinical data. Regulation of P-S105-ERβ in vitro was assessed by immunofluorescence and Western blotting.Results: Expression of P-S105-ERβ was mainly nuclear and could be abolished by phosphatase pre-incubation, indicating specificity. In the first cohort Allred scoring ranged from 0 to 8 (median 6). Nuclear speckling was observed in 45% of cases. P-S105-ERβ correlated with ERβ1 (Allred score ≥ 3) and was associated with better overall survival (OS) and disease free survival (DFS) (p=0.028 and p=0.027, respectively). In a multivariate Cox hazard analysis, P-S105-ERβ overexpression was a significant predictor of better survival independent of tumor grade, lymph node status, size or ERα. In the endocrine resistant cohort Allred scoring ranged from 0 to 8 (median of 4) and the overexpression of P-S105-ERβ was also associated with improved OS and DFS (p=0.044 and p=0.033, respectively). Nuclear speckling was present in only 11% of cases. The difference in expression of P-S105-ERβ and association with survival outcome between the two cohorts was statistically significant. P-S105-ERβ was expressed in MCF-7 cells and in response to 17β-estradiol (E2); levels were raised within 30 min, and sustained for 24 hours. ERβ1 expression was unaffected by this treatment. Nuclear speckling was also observed and was markedly increased at 24 hours following E2 but not Tamoxifen.Conclusion: This is the first time to our knowledge that the expression of P-S105-ERβ in breast carcinoma has been investigated. Its presence in breast carcinoma and association with improved survival suggest P-S105-ERβ might be a useful additional prognostic marker in breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4141.

Background Stromal fibroblasts can contribute to tumor invasion through the release of matrix metalloproteinases (MMPs). Population studies have suggested that single nucleotide polymorphisms (SNPs) in MMP genes influence levels of expression and may be associated with breast cancer risk and with disease progression. This study directly examined the impact of MMP SNP genotype on the ability of host fibroblasts to promote tumor cell invasion. Methods Primary breast fibroblasts were isolated from patients with (n = 13) or without (n = 19) breast cancer, and their ability to promote breast cancer cell invasion was measured in in vitro invasion assays. Fibroblast invasion-promoting capacity (IPC) was analyzed in relation to donor type (tumor or non-tumor patient), MMP-1, MMP-3, and MMP-9 SNP genotype and MMP activity using independent samples t test and analysis of variance. All statistical tests were two-sided. Results Tumor-derived fibroblasts promoted higher levels of invasion than normal fibroblasts (p = 0.041). When IPC was related to genotype, higher levels of IPC were generated by tumor fibroblasts with the high-expressing MMP-3 5A/5A genotype compared with the 5A/6A and 6A/6A genotypes (p = 0.05 and 0.07, respectively), and this was associated with enhanced MMP-3 release. The functional importance of MMP-3 was demonstrated by enhanced invasion in the presence of recombinant MMP-3, whereas reduction occurred in the presence of a specific MMP-3 inhibitor. An inverse relationship was demonstrated between fibroblast IPC and the high-expressing MMP-1 genotype (p = 0.031), but no relationship was seen with MMP-9 SNP status. In contrast, normal fibroblasts showed no variation in IPC in relation to MMP genotype, with MMP-3 5A/5A fibroblasts exhibiting significantly lower levels of IPC than their tumor-derived counterparts (p = 0.04). Conclusion This study has shown that tumor-derived fibroblasts exhibit higher levels of IPC than normal fibroblasts and that the MMP-3 5A/5A genotype contributes to this through enhanced MMP-3 release. Despite a high-expressing genotype, normal fibroblasts do not exhibit higher IPC or enhanced MMP release. This suggests that more complex changes occur in tumor-derived fibroblasts, enabling full expression of the MMP SNP genotype and these possibly are epigenetic in nature. The results do suggest that, in women with breast cancer, a high-expressing MMP-3 genotype may promote tumor progression more effectively.

Introduction There is increasing recognition of the role of the microenvironment in the control of both normal and tumour cell behaviour. In the breast, myoepithelial cells and fibroblasts can influence tumour cell behaviour, with myoepithelial cells exhibiting a broad tumour-suppressor activity while fibroblasts frequently promote tumour growth and invasion. This study describes the development of physiologically relevant three-dimensional heterotypic culture systems containing mixed normal or tumour-derived breast populations and shows how such models can be used to dissect the interactions that influence cell behaviour. Methods Populations of luminal cells, myoepithelial cells and fibroblasts were isolated from normal and malignant breast tissue, characterised and compared with immortalised cell lines. Co-localisation of normal and malignant luminal cells with myoepithelial cells alone or with either normal or tumour-derived fibroblasts was studied. Cultures were grown for seven days, and then gels were fixed and whole gel immunofluorescence carried out to assess co-localisation and polarisation. The potential role of matrix metalloproteinases (MMP) or hepatocyte growth factor(HGF)-c-met signalling in disrupting cellular organisation was investigated by incorporating inhibitors into cultures either alone or in combination. Results Over a culture period of seven days, myoepithelial cells organised themselves around luminal cell populations forming dual-cell co-units. Characterisation of co-units showed established basal polarity and differentiation analogous to their in vivo counterparts. Tumour cell co-units revealed subtle differences to normal co-units including disruption of basement membrane and loss of β4-integrin, as described in ductal carcinoma in situ (DCIS) in vivo. Inclusion of normal fibroblasts had no influence on co-unit formation; however, inclusion of tumour-associated fibroblasts lead to disruption of co-unit organisation, and this was significantly inhibited in the presence of MMP and/or c-met inhibitors. Conclusions To the best of the authors' knowledge, this study describes for the first time a co-culture model comprising three major components of normal and malignant breast: luminal cells, myoepithelial cells and stromal fibroblasts. These cells organise into structures recapitulating normal and DCIS breast, with homing of myoepithelial cells around the luminal population. Importantly, differences are exhibited between these systems reflecting those described in tissues, including a central role for tumour-associated fibroblasts and MMPs in mediating disruption of normal structures. These findings support the value of these models in dissecting normal and tumour cell behaviour in an appropriate microenvironment.