Tumour Microenvironment-Immune Cell Interactions Influencing Breast Cancer Heterogeneity and Disease Progression.

Breast cancer is a complex, dynamic disease that acquires heterogeneity through various mechanisms, allowing cancer cells to proliferate, survive and metastasise. Heterogeneity is introduced early, through the accumulation of germline and somatic mutations which initiate cancer formation. Following initiation, heterogeneity is driven by the complex interaction between intrinsic cellular factors and the extrinsic tumour microenvironment (TME). The TME consists of tumour cells and the subsequently recruited immune cells, endothelial cells, fibroblasts, adipocytes and non-cellular components of the extracellular matrix. Current research demonstrates that stromal-immune cell interactions mediated by various TME components release environmental cues, in mechanical and chemical forms, to communicate with surrounding and distant cells. These interactions are critical in facilitating the metastatic process at both the primary and secondary site, as well as introducing greater intratumoral heterogeneity and disease complexity by exerting selective pressures on cancer cells. This can result in the adaptation of cells and a feedback loop to the cancer genome, which can promote therapeutic resistance. Thus, targeting TME and immune-stromal cell interactions has been suggested as a potential therapeutic avenue given that aspects of this process are somewhat conserved between breast cancer subtypes. This mini review will discuss emerging ideas on how the interaction of various aspects of the TME contribute to increased heterogeneity and disease progression, and the therapeutic potential of targeting the TME.

Transcription Factors as Novel Therapeutic Targets and Drivers of Prostate Cancer Progression.

Prostate cancer is the second most diagnosed cancer among men worldwide. Androgen deprivation therapy, the most common targeted therapeutic option, is circumvented as prostate cancer progresses from androgen dependent to castrate-resistant disease. Whilst the nuclear receptor transcription factor, androgen receptor, drives the growth of prostate tumor during initial stage of the disease, androgen resistance is associated with poorly differentiated prostate cancer. In the recent years, increased research has highlighted the aberrant transcriptional activities of a small number of transcription factors. Along with androgen receptors, dysregulation of these transcription factors contributes to both the poorly differentiated phenotypes of prostate cancer cells and the initiation and progression of prostate carcinoma. As master regulators of cell fate decisions, these transcription factors may provide opportunity for the development of novel therapeutic targets for the management of prostate cancer. Whilst some transcriptional regulators have previously been notoriously difficult to directly target, technological advances offer potential for the indirect therapeutic targeting of these transcription factors and the capacity to reprogram cancer cell phenotype. This mini review will discuss how recent advances in our understanding of transcriptional regulators and material science pave the way to utilize these regulatory molecules as therapeutic targets in prostate cancer.

The Influence of Modifiable Factors on Breast and Prostate Cancer Risk and Disease Progression.

Breast and prostate cancers are among the most commonly diagnosed cancers worldwide, and together represented almost 20% of all new cancer diagnoses in 2020. For both cancers, the primary treatment options are surgical resection and sex hormone deprivation therapy, highlighting the initial dependence of these malignancies on the activity of both endogenous and exogenous hormones. Cancer cell phenotype and patient prognosis is not only determined by the collection of specific gene mutations, but through the interaction and influence of a wide range of different local and systemic components. While genetic risk factors that contribute to the development of these cancers are well understood, increasing epidemiological evidence link modifiable lifestyle factors such as physical exercise, diet and weight management, to drivers of disease progression such as inflammation, transcriptional activity, and altered biochemical signaling pathways. As a result of this significant impact, it is estimated that up to 50% of cancer cases in developed countries could be prevented with changes to lifestyle and environmental factors. While epidemiological studies of modifiable risk factors and research of the biological mechanisms exist mostly independently, this review will discuss how advances in our understanding of the metabolic, protein and transcriptional pathways altered by modifiable lifestyle factors impact cancer cell physiology to influence breast and prostate cancer risk and prognosis.

Id Proteins Promote a Cancer Stem Cell Phenotype in Mouse Models of Triple Negative Breast Cancer via Negative Regulation of Robo1.

Breast cancers display phenotypic and functional heterogeneity and several lines of evidence support the existence of cancer stem cells (CSCs) in certain breast cancers, a minor population of cells capable of tumor initiation and metastatic dissemination. Identifying factors that regulate the CSC phenotype is therefore important for developing strategies to treat metastatic disease. The Inhibitor of Differentiation Protein 1 (Id1) and its closely related family member Inhibitor of Differentiation 3 (Id3) (collectively termed Id) are expressed by a diversity of stem cells and are required for metastatic dissemination in experimental models of breast cancer. In this study, we show that ID1 is expressed in rare neoplastic cells within ER-negative breast cancers. To address the function of Id1 expressing cells within tumors, we developed independent murine models of Triple Negative Breast Cancer (TNBC) in which a genetic reporter permitted the prospective isolation of Id1+ cells. Id1+ cells are enriched for self-renewal in tumorsphere assays in vitro and for tumor initiation in vivo. Conversely, depletion of Id1 and Id3 in the 4T1 murine model of TNBC demonstrates that Id1/3 are required for cell proliferation and self-renewal in vitro, as well as primary tumor growth and metastatic colonization of the lung in vivo. Using combined bioinformatic analysis, we have defined a novel mechanism of Id protein function via negative regulation of the Roundabout Axon Guidance Receptor Homolog 1 (Robo1) leading to activation of a Myc transcriptional programme.

Proteogenomic analysis of Inhibitor of Differentiation 4 (ID4) in basal-like breast cancer.

BACKGROUND: Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies. Inhibitor of differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator required for mammary gland development. ID4 is overexpressed in a subset of BLBC patients, associating with a stem-like poor prognosis phenotype, and is necessary for the growth of cell line models of BLBC through unknown mechanisms. METHODS: Here, we have defined unique molecular insights into the function of ID4 in BLBC and the related disease high-grade serous ovarian cancer (HGSOC), by combining RIME proteomic analysis, ChIP-seq mapping of genomic binding sites and RNA-seq. RESULTS: These studies reveal novel interactions with DNA damage response proteins, in particular, mediator of DNA damage checkpoint protein 1 (MDC1). Through MDC1, ID4 interacts with other DNA repair proteins (γH2AX and BRCA1) at fragile chromatin sites. ID4 does not affect transcription at these sites, instead binding to chromatin following DNA damage. Analysis of clinical samples demonstrates that ID4 is amplified and overexpressed at a higher frequency in BRCA1-mutant BLBC compared with sporadic BLBC, providing genetic evidence for an interaction between ID4 and DNA damage repair deficiency. CONCLUSIONS: These data link the interactions of ID4 with MDC1 to DNA damage repair in the aetiology of BLBC and HGSOC.

ELF5 modulates the estrogen receptor cistrome in breast cancer.

Acquired resistance to endocrine therapy is responsible for half of the therapeutic failures in the treatment of breast cancer. Recent findings have implicated increased expression of the ETS transcription factor ELF5 as a potential modulator of estrogen action and driver of endocrine resistance, and here we provide the first insight into the mechanisms by which ELF5 modulates estrogen sensitivity. Using chromatin immunoprecipitation sequencing we found that ELF5 binding overlapped with FOXA1 and ER at super enhancers, enhancers and promoters, and when elevated, caused FOXA1 and ER to bind to new regions of the genome, in a pattern that replicated the alterations to the ER/FOXA1 cistrome caused by the acquisition of resistance to endocrine therapy. RNA sequencing demonstrated that these changes altered estrogen-driven patterns of gene expression, the expression of ER transcription-complex members, and 6 genes known to be involved in driving the acquisition of endocrine resistance. Using rapid immunoprecipitation mass spectrometry of endogenous proteins, and proximity ligation assays, we found that ELF5 interacted physically with members of the ER transcription complex, such as DNA-PKcs. We found 2 cases of endocrine-resistant brain metastases where ELF5 levels were greatly increased and ELF5 patterns of gene expression were enriched, compared to the matched primary tumour. Thus ELF5 alters ER-driven gene expression by modulating the ER/FOXA1 cistrome, by interacting with it, and by modulating the expression of members of the ER transcriptional complex, providing multiple mechanisms by which ELF5 can drive endocrine resistance.

A mutation in the viral sensor 2'-5'-oligoadenylate synthetase 2 causes failure of lactation.

We identified a non-synonymous mutation in Oas2 (I405N), a sensor of viral double-stranded RNA, from an ENU-mutagenesis screen designed to discover new genes involved in mammary development. The mutation caused post-partum failure of lactation in healthy mice with otherwise normally developed mammary glands, characterized by greatly reduced milk protein synthesis coupled with epithelial cell death, inhibition of proliferation and a robust interferon response. Expression of mutant but not wild type Oas2 in cultured HC-11 or T47D mammary cells recapitulated the phenotypic and transcriptional effects observed in the mouse. The mutation activates the OAS2 pathway, demonstrated by a 34-fold increase in RNase L activity, and its effects were dependent on expression of RNase L and IRF7, proximal and distal pathway members. This is the first report of a viral recognition pathway regulating lactation.

Rac-ing from Epithelial Secretor to Eater.

Mammary epithelial phagocytosis is critical for removal of apoptotic cells during involution, but the mechanisms governing this process are largely unknown. In this issue of Developmental Cell, Akhtar et al. (2016) provide insight into mechanisms regulating involution, demonstrating that Rac1 drives the switch from differentiation to phagocytosis in mammary epithelium.

ELF5 Drives Lung Metastasis in Luminal Breast Cancer through Recruitment of Gr1+ CD11b+ Myeloid-Derived Suppressor Cells.

During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mammary luminal progenitor cell. In breast cancer, ELF5 is a key transcriptional determinant of tumor subtype and has been implicated in the development of insensitivity to anti-estrogen therapy. In the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT) model of luminal breast cancer, induction of ELF5 levels increased leukocyte infiltration, angiogenesis, and blood vessel permeability in primary tumors and greatly increased the size and number of lung metastasis. Myeloid-derived suppressor cells, a group of immature neutrophils recently identified as mediators of vasculogenesis and metastasis, were recruited to the tumor in response to ELF5. Depletion of these cells using specific Ly6G antibodies prevented ELF5 from driving vasculogenesis and metastasis. Expression signatures in luminal A breast cancers indicated that increased myeloid cell invasion and inflammation were correlated with ELF5 expression, and increased ELF5 immunohistochemical staining predicted much shorter metastasis-free and overall survival of luminal A patients, defining a group who experienced unexpectedly early disease progression. Thus, in the MMTV-PyMT mouse mammary model, increased ELF5 levels drive metastasis by co-opting the innate immune system. As ELF5 has been previously implicated in the development of antiestrogen resistance, this finding implicates ELF5 as a defining factor in the acquisition of the key aspects of the lethal phenotype in luminal A breast cancer.

ID4 controls mammary stem cells and marks breast cancers with a stem cell-like phenotype.

Basal-like breast cancer (BLBC) is a heterogeneous disease with poor prognosis; however, its cellular origins and aetiology are poorly understood. In this study, we show that inhibitor of differentiation 4 (ID4) is a key regulator of mammary stem cell self-renewal and marks a subset of BLBC with a putative mammary basal cell of origin. Using an ID4GFP knock-in reporter mouse and single-cell transcriptomics, we show that ID4 marks a stem cell-enriched subset of the mammary basal cell population. ID4 maintains the mammary stem cell pool by suppressing key factors required for luminal differentiation. Furthermore, ID4 is specifically expressed by a subset of human BLBC that possess a very poor prognosis and a transcriptional signature similar to a mammary stem cell. These studies identify ID4 as a mammary stem cell regulator, deconvolute the heterogeneity of BLBC and link a subset of mammary stem cells to the aetiology of BLBC.

Runx2 is a novel regulator of mammary epithelial cell fate in development and breast cancer.

Regulators of differentiated cell fate can offer targets for managing cancer development and progression. Here, we identify Runx2 as a new regulator of epithelial cell fate in mammary gland development and breast cancer. Runx2 is expressed in the epithelium of pregnant mice in a strict temporally and hormonally regulated manner. During pregnancy, Runx2 genetic deletion impaired alveolar differentiation in a manner that disrupted alveolar progenitor cell populations. Conversely, exogenous transgenic expression of Runx2 in mammary epithelial cells blocked milk production, suggesting that the decrease in endogenous Runx2 observed late in pregnancy is necessary for full differentiation. In addition, overexpression of Runx2 drove epithelial-to-mesenchymal transition-like changes in normal mammary epithelial cells, whereas Runx2 deletion in basal breast cancer cells inhibited cellular phenotypes associated with tumorigenesis. Notably, loss of Runx2 expression increased tumor latency and enhanced overall survival in a mouse model of breast cancer, with Runx2-deficient tumors exhibiting reduced cell proliferation. Together, our results establish a previously unreported function for Runx2 in breast cancer that may offer a novel generalized route for therapeutic interventions. Cancer Res; 74(18); 5277-86. ©2014 AACR.

Deletion of Cd151 reduces mammary tumorigenesis in the MMTV/PyMT mouse model.

BACKGROUND: Tetraspanins are transmembrane proteins that serve as scaffolds for multiprotein complexes containing, for example, integrins, growth factor receptors and matrix metalloproteases, and modify their functions in cell adhesion, migration and transmembrane signaling. CD151 is part of the tetraspanin family and it forms tight complexes with ß1 and ß4 integrins, both of which have been shown to be required for tumorigenesis and/or metastasis in transgenic mouse models of breast cancer. High levels of the tetraspanin CD151 have been linked to poor patient outcome in several human cancers including breast cancer. In addition, CD151 has been implicated as a promoter of tumor angiogenesis and metastasis in various model systems. METHODS: Here we investigated the effect of Cd151 deletion on mammary tumorigenesis by crossing Cd151-deficient mice with a spontaneously metastasising transgenic model of breast cancer induced by the polyoma middle T antigen (PyMT) driven by the murine mammary tumor virus promoter (MMTV). RESULTS: Cd151 deletion did not affect the normal development and differentiation of the mammary gland. While there was a trend towards delayed tumor onset in Cd151-/- PyMT mice compared to Cd151+/+ PyMT littermate controls, this result was only approaching significance (Log-rank test P-value =0.0536). Interestingly, Cd151 deletion resulted in significantly reduced numbers and size of primary tumors but did not appear to affect the number or size of metastases in the MMTV/PyMT mice. Intriguingly, no differences in the expression of markers of cell proliferation, apoptosis and blood vessel density was observed in the primary tumors. CONCLUSION: The findings from this study provide additional evidence that CD151 acts to enhance tumor formation initiated by a range of oncogenes and strongly support its relevance as a potential therapeutic target to delay breast cancer progression.

Structural and mechanical functions of integrins.

Integrins are ubiquitously expressed cell surface receptors that play a critical role in regulating the interaction between a cell and its microenvironment to control cell fate. These molecules are regulated either via their expression on the cell surface or through a unique bidirectional signalling mechanism. However, integrins are just the tip of the adhesome iceberg, initiating the assembly of a large range of adaptor and signalling proteins that mediate the structural and signalling functions of integrin. In this review, we summarise the structure of integrins and mechanisms by which integrin activation is controlled. The different adhesion structures formed by integrins are discussed, as well as the mechanical and structural roles integrins play during cell migration. As the function of integrin signalling can be quite varied based on cell type and context, an in depth understanding of these processes will aid our understanding of aberrant adhesion and migration, which is often associated with human pathologies such as cancer.

Integrins in development and cancer.

The correct control of cell fate decisions is critical for metazoan development and tissue homeostasis. It is established that the integrin family of cell surface receptors regulate cell fate by mediating cell-cell and cell-extracellular matrix (ECM) interactions. However, our understanding of how the different family members control discrete aspects of cell biology, and how this varies between tissues and is temporally regulated, is still in its infancy. An emerging area of investigation aims to understand how integrins translate changes in tension in the surrounding microenvironment into biological responses. This is particularly pertinent due to changes in the mechanical properties of the ECM having been linked to diseases, such as cancer. In this review, we provide an overview of the roles integrins play in important developmental processes, such as proliferation, polarity, apoptosis, differentiation and maintenance of "stemness". We also discuss recent advances in integrin mechanobiology and highlight the involvement of integrins and aberrant ECM in cancer.

Breast cancer stem cells.

Cancer metastasis, resistance to therapies and disease recurrence are significant hurdles to successful treatment of breast cancer. Identifying mechanisms by which cancer spreads, survives treatment regimes and regenerates more aggressive tumors are critical to improving patient survival. Substantial evidence gathered over the last 10 years suggests that breast cancer progression and recurrence is supported by cancer stem cells (CSCs). Understanding how CSCs form and how they contribute to the pathology of breast cancer will greatly aid the pursuit of novel therapies targeted at eliminating these cells. This review will summarize what is currently known about the origins of breast CSCs, their role in disease progression and ways in which they may be targeted therapeutically.

ß1 integrin deletion enhances progression of prostate cancer in the TRAMP mouse model.

ß1 integrin regulates the response of both normal and cancer cells to their local environment. Although mis-localised in prostate cancer, the role ß1 integrin plays in prostate development and carcinogenesis remains unknown. To assess the role of ß1 integrin in vivo, we conditionally deleted ß1 integrin from prostate epithelium and subsequently crossed these mice to the TRAMP prostate carcinogenesis model. Deletion of ß1 integrin following castration and subsequent androgen supplementation resulted in an expansion of the p63-positive basal cell population and decreased differentiation. Consistent with these findings, deletion of ß1 integrin in TRAMP mice decreased animal survival, decreased retention of normal prostate morphology, increased the percentage of tissue with poorly differentiated carcinoma, and increased cell proliferation. This study demonstrates that ß1 integrin regulates several aspects of normal prostate development and in contrast to its role in several other tissues, its loss is associated with increased rates of prostate tumour progression.

ELF5 suppresses estrogen sensitivity and underpins the acquisition of antiestrogen resistance in luminal breast cancer.

We have previously shown that during pregnancy the E-twenty-six (ETS) transcription factor ELF5 directs the differentiation of mammary progenitor cells toward the estrogen receptor (ER)-negative and milk producing cell lineage, raising the possibility that ELF5 may suppress the estrogen sensitivity of breast cancers. To test this we constructed inducible models of ELF5 expression in ER positive luminal breast cancer cells and interrogated them using transcript profiling and chromatin immunoprecipitation of DNA followed by DNA sequencing (ChIP-Seq). ELF5 suppressed ER and FOXA1 expression and broadly suppressed ER-driven patterns of gene expression including sets of genes distinguishing the luminal molecular subtype. Direct transcriptional targets of ELF5, which included FOXA1, EGFR, and MYC, accurately classified a large cohort of breast cancers into their intrinsic molecular subtypes, predicted ER status with high precision, and defined groups with differential prognosis. Knockdown of ELF5 in basal breast cancer cell lines suppressed basal patterns of gene expression and produced a shift in molecular subtype toward the claudin-low and normal-like groups. Luminal breast cancer cells that acquired resistance to the antiestrogen Tamoxifen showed greatly elevated levels of ELF5 and its transcriptional signature, and became dependent on ELF5 for proliferation, compared to the parental cells. Thus ELF5 provides a key transcriptional determinant of breast cancer molecular subtype by suppression of estrogen sensitivity in luminal breast cancer cells and promotion of basal characteristics in basal breast cancer cells, an action that may be utilised to acquire antiestrogen resistance.

Transcript profiling of Elf5+/- mammary glands during pregnancy identifies novel targets of Elf5.

BACKGROUND: Elf5, an epithelial specific Ets transcription factor, plays a crucial role in the pregnancy-associated development of the mouse mammary gland. Elf5(-/-) embryos do not survive, however the Elf5(+/-) mammary gland displays a severe pregnancy-associated developmental defect. While it is known that Elf5 is crucial for correct mammary development and lactation, the molecular mechanisms employed by Elf5 to exert its effects on the mammary gland are largely unknown. PRINCIPAL FINDINGS: Transcript profiling was used to investigate the transcriptional changes that occur as a result of Elf5 haploinsufficiency in the Elf5(+/-) mouse model. We show that the development of the mouse Elf5(+/-) mammary gland is delayed at a transcriptional and morphological level, due to the delayed increase in Elf5 protein in these glands. We also identify a number of potential Elf5 target genes, including Mucin 4, whose expression, is directly regulated by the binding of Elf5 to an Ets binding site within its promoter. CONCLUSION: We identify novel transcriptional targets of Elf5 and show that Muc4 is a direct target of Elf5, further elucidating the mechanisms through which Elf5 regulates proliferation and differentiation in the mammary gland.

Molecular dissection of integrin signalling proteins in the control of mammary epithelial development and differentiation.

Cell-matrix adhesion is essential for the development and tissue-specific functions of epithelia. For example, in the mammary gland, beta1-integrin is necessary for the normal development of alveoli and for the activation of endocrine signalling pathways that determine cellular differentiation. However, the adhesion complex proteins linking integrins with downstream effectors of hormonal signalling pathways are not known. To understand the mechanisms involved in connecting adhesion with this aspect of cell phenotype, we examined the involvement of two proximal beta1-integrin signalling intermediates, integrin-linked kinase (ILK) and focal adhesion kinase (FAK). By employing genetic analysis using the Cre-LoxP system, we provide evidence that ILK, but not FAK, has a key role in lactogenesis in vivo and in the differentiation of cultured luminal epithelial cells. Conditional deletion of ILK both in vivo and in primary cell cultures resulted in defective differentiation, by preventing phosphorylation and nuclear translocation of STAT5, a transcription factor required for lactation. Expression of an activated RAC (RAS-related C3 botulinum substrate) in ILK-null acini restored the lactation defect, indicating that RAC1 provides a mechanistic link between the integrin/ILK adhesion complex and the differentiation pathway. Thus, we have determined that ILK is an essential downstream component of integrin signalling involved in differentiation, and have identified a high degree of specificity within the integrin-based adhesome that links cell-matrix interactions with the tissue-specific function of epithelia.

The Ets transcription factor Elf5 specifies mammary alveolar cell fate.

Hormonal cues regulate mammary development, but the consequent transcriptional changes and cell fate decisions are largely undefined. We show that knockout of the prolactin-regulated Ets transcription factor Elf5 prevented formation of the secretory epithelium during pregnancy. Conversely, overexpression of Elf5 in an inducible transgenic model caused alveolar differentiation and milk secretion in virgin mice, disrupting ductal morphogenesis. CD61+ luminal progenitor cells accumulated in Elf5-deficient mammary glands and were diminished in glands with Elf5 overexpression. Thus Elf5 specifies the differentiation of CD61+ progenitors to establish the secretory alveolar lineage during pregnancy, providing a link between prolactin, transcriptional events, and alveolar development.