A single-cell atlas enables mapping of homeostatic cellular shifts in the adult human breast.

Here we use single-cell RNA sequencing to compile a human breast cell atlas assembled from 55 donors that had undergone reduction mammoplasties or risk reduction mastectomies. From more than 800,000 cells we identified 41 cell subclusters across the epithelial, immune and stromal compartments. The contribution of these different clusters varied according to the natural history of the tissue. Age, parity and germline mutations, known to modulate the risk of developing breast cancer, affected the homeostatic cellular state of the breast in different ways. We found that immune cells from BRCA1 or BRCA2 carriers had a distinct gene expression signature indicative of potential immune exhaustion, which was validated by immunohistochemistry. This suggests that immune-escape mechanisms could manifest in non-cancerous tissues very early during tumor initiation. This atlas is a rich resource that can be used to inform novel approaches for early detection and prevention of breast cancer.

TGFß-mediated MMP13 secretion drives myoepithelial cell dependent breast cancer progression.

Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer. Virtually all women with DCIS are treated, despite evidence suggesting up to half would remain with stable, non-threatening, disease. Overtreatment thus presents a pressing issue in DCIS management. To understand the role of the normally tumour suppressive myoepithelial cell in disease progression we present a 3D in vitro model incorporating both luminal and myoepithelial cells in physiomimetic conditions. We demonstrate that DCIS-associated myoepithelial cells promote striking myoepithelial-led invasion of luminal cells, mediated by the collagenase MMP13 through a non-canonical TGFß - EP300 pathway. In vivo, MMP13 expression is associated with stromal invasion in a murine model of DCIS progression and is elevated in myoepithelial cells of clinical high-grade DCIS cases. Our data identify a key role for myoepithelial-derived MMP13 in facilitating DCIS progression and point the way towards a robust marker for risk stratification in DCIS patients.

Mechanostimulation of breast myoepithelial cells induces functional changes associated with DCIS progression to invasion.

Women with ductal carcinoma in situ (DCIS) have an increased risk of progression to invasive breast cancer. Although not all women with DCIS will progress to invasion, all are treated as such, emphasising the need to identify prognostic biomarkers. We have previously shown that altered myoepithelial cells in DCIS predict disease progression and recurrence. By analysing DCIS duct size in sections of human breast tumour samples, we identified an associated upregulation of integrin ß6 and an increase in periductal fibronectin deposition with increased DCIS duct size that associated with the progression of DCIS to invasion. Our modelling of the mechanical stretching myoepithelial cells undergo during DCIS progression confirmed the upregulation of integrin ß6 and fibronectin expression in isolated primary and cell line models of normal myoepithelial cells. Our studies reveal that this mechanostimulated DCIS myoepithelial cell phenotype enhances invasion in a TGFß-mediated upregulation of MMP13. Immunohistochemical analysis identified that MMP13 was specifically upregulated in DCIS, and it was associated with progression to invasion. These findings implicate tissue mechanics in altering the myoepithelial cell phenotype in DCIS, and that these alterations may be used to stratify DCIS patients into low and high risk for invasive progression.

The curcumin analog HO-3867 selectively kills cancer cells by converting mutant p53 protein to transcriptionally active wildtype p53.

p53 is an important tumor-suppressor protein that is mutated in more than 50% of cancers. Strategies for restoring normal p53 function are complicated by the oncogenic properties of mutant p53 and have not met with clinical success. To counteract mutant p53 activity, a variety of drugs with the potential to reconvert mutant p53 to an active wildtype form have been developed. However, these drugs are associated with various negative effects such as cellular toxicity, nonspecific binding to other proteins, and inability to induce a wildtype p53 response in cancer tissue. Here, we report on the effects of a curcumin analog, HO-3867, on p53 activity in cancer cells from different origins. We found that HO-3867 covalently binds to mutant p53, initiates a wildtype p53-like anticancer genetic response, is exclusively cytotoxic toward cancer cells, and exhibits high anticancer efficacy in tumor models. In conclusion, HO-3867 is a p53 mutant-reactivating drug with high clinical anticancer potential.

Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors.

Aberrant promoter DNA hypermethylation is a hallmark of cancer; however, whether this is sufficient to drive cellular transformation is not clear. To investigate this question, we use a CRISPR-dCas9 epigenetic editing tool, where an inactive form of Cas9 is fused to DNA methyltransferase effectors. Using this system, here we show simultaneous de novo DNA methylation of genes commonly methylated in cancer, CDKN2A, RASSF1, HIC1 and PTEN in primary breast cells isolated from healthy human breast tissue. We find that promoter methylation is maintained in this system, even in the absence of the fusion construct, and this prevents cells from engaging senescence arrest. Our data show that the key driver of this phenotype is repression of CDKN2A transcript p16 where myoepithelial cells harbour cancer-like gene expression but do not exhibit anchorage-independent growth. This work demonstrates that hit-and-run epigenetic events can prevent senescence entry, which may facilitate tumour initiation.

A 3D in vitro model of the human breast duct: a method to unravel myoepithelial-luminal interactions in the progression of breast cancer.

BACKGROUND: 3D modelling fulfils a critical role in research, allowing for complex cell behaviour and interactions to be studied in physiomimetic conditions. With tissue banks becoming established for a number of cancers, researchers now have access to primary patient cells, providing the perfect building blocks to recreate and interrogate intricate cellular systems in the laboratory. The ducts of the human breast are composed of an inner layer of luminal cells supported by an outer layer of myoepithelial cells. In early-stage ductal carcinoma in situ, cancerous luminal cells are confined to the ductal space by an intact myoepithelial layer. Understanding the relationship between myoepithelial and luminal cells in the development of cancer is critical for the development of new therapies and prognostic markers. This requires the generation of new models that allows for the manipulation of these two cell types in a physiological setting. METHODS: Using access to the Breast Cancer Now Tissue Bank, we isolated pure populations of myoepithelial and luminal cells from human reduction mammoplasty specimens and placed them into 2D culture. These cells were infected with lentiviral particles encoding either fluorescent proteins, to facilitate cell tracking, or an inducible human epidermal growth factor receptor 2 (HER2) expression construct. Myoepithelial and luminal cells were then recombined in collagen gels, and the resulting cellular structures were analysed by confocal microscopy. RESULTS: Myoepithelial and luminal cells isolated from reduction mammoplasty specimens can be grown separately in 2D culture and retain their differentiated state. When recombined in collagen gels, these cells reform into physiologically reflective bilayer structures. Inducible expression of HER2 in the luminal compartment, once the bilayer has formed, leads to robust luminal filling, recapitulating ductal carcinoma in situ, and can be blocked with anti-HER2 therapies. CONCLUSIONS: This model allows for the interaction between myoepithelial and luminal cells to be investigated in an in-vitro environment and paves the way to study early events in breast cancer development with the potential to act as a powerful drug discovery platform.

Expression of CDK7, Cyclin H, and MAT1 Is Elevated in Breast Cancer and Is Prognostic in Estrogen Receptor-Positive Breast Cancer.

PURPOSE: CDK-activating kinase (CAK) is required for the regulation of the cell cycle and is a trimeric complex consisting of cyclin-dependent kinase 7 (CDK7), Cyclin H, and the accessory protein, MAT1. CDK7 also plays a critical role in regulating transcription, primarily by phosphorylating RNA polymerase II, as well as transcription factors such as estrogen receptor-α (ER). Deregulation of cell cycle and transcriptional control are general features of tumor cells, highlighting the potential for the use of CDK7 inhibitors as novel cancer therapeutics. EXPERIMENTAL DESIGN: mRNA and protein expression of CDK7 and its essential cofactors cyclin H and MAT1 were evaluated in breast cancer samples to determine if their levels are altered in cancer. Immunohistochemical staining of >900 breast cancers was used to determine the association with clinicopathologic features and patient outcome. RESULTS: We show that expressions of CDK7, cyclin H, and MAT1 are all closely linked at the mRNA and protein level, and their expression is elevated in breast cancer compared with the normal breast tissue. Intriguingly, CDK7 expression was inversely proportional to tumor grade and size, and outcome analysis showed an association between CAK levels and better outcome. Moreover, CDK7 expression was positively associated with ER expression and in particular with phosphorylation of ER at serine 118, a site important for ER transcriptional activity. CONCLUSIONS: Expressions of components of the CAK complex, CDK7, MAT1, and Cyclin H are elevated in breast cancer and correlate with ER. Like ER, CDK7 expression is inversely proportional to poor prognostic factors and survival. Clin Cancer Res; 22(23); 5929-38. ©2016 AACR.

Rigidity sensing and adaptation through regulation of integrin types.

Tissue rigidity regulates processes in development, cancer and wound healing. However, how cells detect rigidity, and thereby modulate their behaviour, remains unknown. Here, we show that sensing and adaptation to matrix rigidity in breast myoepithelial cells is determined by the bond dynamics of different integrin types. Cell binding to fibronectin through either α5ß1 integrins (constitutively expressed) or αvß6 integrins (selectively expressed in cancer and development) adapts force generation, actin flow and integrin recruitment to rigidities associated with healthy or malignant tissue, respectively. In vitro experiments and theoretical modelling further demonstrate that this behaviour is explained by the different binding and unbinding rates of both integrin types to fibronectin. Moreover, rigidity sensing through differences in integrin bond dynamics applies both when integrins bind separately and when they compete for binding to fibronectin.

Altered microenvironment promotes progression of preinvasive breast cancer: myoepithelial expression of αvß6 integrin in DCIS identifies high-risk patients and predicts recurrence.

PURPOSE: This study investigated the functional and clinical significance of integrin αvß6 upregulation in myoepithelial cells of ductal carcinoma in situ (DCIS). EXPERIMENTAL DESIGN: Archival samples of DCIS and DCIS with associated invasion (n = 532) were analyzed for expression of αvß6 by immunohistochemistry and ability to predict recurrence and progression assessed in an independent, unique cohort of DCIS cases with long-term follow-up. Primary myoepithelial cells and myoepithelial cell lines, with and without αvß6 expression, were used to measure the effect of αvß6 on growth and invasion of tumor cell lines in vitro and in a xenograft mouse model. Involvement of TGFß signaling was established using mink lung epithelial cell (MLEC) assay and antibody inhibition, and expression and activation of matrix metalloproteinase (MMP)-9 established by Real Time-PCR and zymography. RESULTS: Expression of αvß6 is significantly associated with progression to invasive cancer (P < 0.006) and with recurrence over a median follow-up of 114 months in a series of matched DCIS cases treated with local excision. We show that expression of αvß6 drives myoepithelial cells to promote tumor cell invasion in vitro and enhances mammary tumor growth in vivo. The tumor-promoting effect of αvß6-positive myoepithelial cells is dependent on TGFß-driven upregulation of MMP9 and can be abrogated by inhibiting this pathway. CONCLUSION: These findings indicate that altered myoepithelial cells in DCIS predict disease progression and recurrence and show that upregulation of αvß6 on myoepithelial cells generates a tumor promoter function through TGFß upregulation of MMP-9. These data suggest that expression of αvß6 may be used to stratify patients with DCIS.