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Abstract Background Bone morphogenetic protein 4 (BMP4) is a potent inhibitor of breast cancer metastasis. However, a tumor-promoting effect of BMP4 is reported in other tumor types, especially when SMAD4 is inactive. Methods To assess the requirement for SMAD4 in BMP4-mediated suppression of metastasis, we knocked down SMAD4 in two different breast tumors and enforced SMAD4 expression in a third line with endogenous SMAD4 deletion. In addition, we assessed the requirement for SMAD4 in tumor cell-specific BMP signalling by expression of a constitutively active BMP receptor. Delineation of genes regulated by BMP4 in the presence or absence of SMAD4 was assessed by RNA sequencing and a BMP4-induced gene, MYO1F was assessed for its role in metastasis. Genes regulated by BMP4 and/or SMAD4 were assessed in a publicly available database of gene expression profiles of breast cancer patients. Results In the absence of SMAD4, BMP4 promotes primary tumor growth that is accompanied by increased expression of genes associated with DNA replication, cell cycle, and MYC signalling pathways. Despite increased primary tumor growth, BMP4 suppresses metastasis in the absence of tumor cell expression of SMAD4. Consistent with the anti-metastatic activity of BMP4, enforced signalling through the constitutively active receptor in SMAD4 positive tumors that lacked BMP4 expression still suppressed metastasis, but in the absence of SMAD4, the suppression of metastasis was largely prevented. Thus BMP4 is required for suppression of metastasis regardless of tumor SMAD4 status. The BMP4 upregulated gene, MYO1F, was shown to be a potent suppressor of breast cancer metastasis. Gene signature upregulated by BMP4 in the absence of SMAD4 was associated with poor prognosis in breast cancer patients, whereas gene signature upregulated by BMP4 in the presence of SMAD4 was associated with improved prognosis. Conclusions BMP4 expression is required for suppression of metastasis regardless of the SMAD4 status of the tumor cells. Since BMP4 is a secreted protein, we conclude that it can act both in an autocrine manner in SMAD4-expressing tumor cells and in a paracrine manner on stromal cells to suppress metastasis. Deletion of SMAD4 from tumor cells does not prevent BMP4 from suppressing metastasis via a paracrine mechanism.

Purpose: Both local tumor control and distant metastasis are important indicators of the efficacy of radiation therapy treatment. Synchrotron microbeam radiation therapy (MRT), spatially fractionated radiation delivered at ultrahigh dose rates, shows remarkable normal tissue sparing with excellent local control in some models. Some MRT regimens trigger an antitumor immune response that contributes not only to the local but also to systemic treatment efficacy. Despite recent advances in the treatment of primary breast cancer, metastatic disease is still the major cause of treatment failure in the clinic. Here, in an aggressive preclinical triple-negative breast cancer model, we compared local tumor response and metastasis following different MRT treatment programs. Methods and Materials: 4T1.2 mouse mammary tumors were treated with 300 Gy peak/7 Gy valley dose MRT and/or 8 Gy broad beam (BB) radiation, all delivered as daily fractionated programs (3 consecutive daily sessions of either MRT or BB or 1 MRT combined with 2 BB sessions, the first or last of the 3 fractions). The mice were euthanized on day 9 post last irradiation, when unirradiated control animals reached an ethical endpoint. Primary tumors were collected to evaluate immune cell prevalence, while lungs, spinal cords, and locoregional lymph nodes were collected to measure metastatic burden. In parallel, local tumor growth and survival were monitored. Results: The combined MRT/BB treatment shifted the balance between pro- and antitumorigenic macrophages toward the accumulation of antitumorigenic macrophages in the tumor. Monitoring of the tumor volume and animal health indicated the benefit of the combined MRT/BB treatment for local control and treatment tolerance, while animal survival was only marginally longer for one combined schedule. The metastatic burden was similar for all 4 treatment schedules. Conclusions: The addition of a single MRT to BB treatment improved the primary tumor response. This provides a basis for future experiments incorporating adjuvant immunotherapy or chemotherapy to improve local and systemic treatment outcomes.

Abstract Background Triple negative BCa (TNBC) is defined by a lack of expression of estrogen (ERα), progesterone (PgR) receptors and human epidermal growth factor receptor 2 (HER2) as assessed by protein expression and/or gene amplification. It makes up ~ 15% of all BCa and often has a poor prognosis. TNBC is not treated with endocrine therapies as ERα and PR negative tumors in general do not show benefit. However, a small fraction of the true TNBC tumors do show tamoxifen sensitivity, with those expressing the most common isoform of ERβ1 having the most benefit. Recently, the antibodies commonly used to assess ERβ1 in TNBC have been found to lack specificity, which calls into question available data regarding the proportion of TNBC that express ERβ1 and any relationship to clinical outcome. Methods To confirm the true frequency of ERβ1 in TNBC we performed robust ERβ1 immunohistochemistry using the specific antibody CWK-F12 ERβ1 on 156 primary TNBC cancers from patients with a median of 78 months (range 0.2–155 months) follow up. Results We found that high expression of ERβ1 was not associated with increased recurrence or survival when assessed as percentage of ERβ1 positive tumor cells or as Allred > 5. In contrast, the non-specific PPG5-10 antibody did show an association with recurrence and survival. Conclusions Our data indicate that ERβ1 expression in TNBC tumours does not associate with prognosis.

Abstract Background Despite overall improvement in breast cancer patient outcomes from earlier diagnosis and personalised treatment approaches, some patients continue to experience recurrence and incurable metastases. It is therefore imperative to understand the molecular changes that allow transition from a non-aggressive state to a more aggressive phenotype. This transition is governed by a number of factors. Methods As crosstalk with extracellular matrix (ECM) is critical for tumour cell growth and survival, we applied high throughput shRNA screening on a validated ‘3D on-top cellular assay’ to identify novel growth suppressive mechanisms. Results A number of novel candidate genes were identified. We focused on COMMD3, a previously poorly characterised gene that suppressed invasive growth of ER + breast cancer cells in the cellular assay. Analysis of published expression data suggested that COMMD3 is normally expressed in the mammary ducts and lobules, that expression is lost in some tumours and that loss is associated with lower survival probability. We performed immunohistochemical analysis of an independent tumour cohort to investigate relationships between COMMD3 protein expression, phenotypic markers and disease-specific survival. This revealed an association between COMMD3 loss and shorter survival in hormone-dependent breast cancers and in particularly luminal-A-like tumours (ER+/Ki67-low; 10-year survival probability 0.83 vs. 0.73 for COMMD3-positive and -negative cases, respectively). Expression of COMMD3 in luminal-A-like tumours was directly associated with markers of luminal differentiation: c-KIT, ELF5, androgen receptor and tubule formation (the extent of normal glandular architecture; p

Abstract Background Malignant pleural effusions (MPEs) are a common complication of advanced cancers, particularly those adjacent to the pleura, such as lung and breast cancer. The pathophysiology of MPE formation remains poorly understood, and although MPEs are routinely used for the diagnosis of breast cancer patients, their composition and biology are poorly understood. It is difficult to distinguish invading malignant cells from resident mesothelial cells and to identify the directionality of interactions between these populations in the pleura. There is a need to characterize the phenotypic diversity of breast cancer cell populations in the pleural microenvironment, and investigate how this varies across patients. Methods Here, we used single‐cell RNA‐sequencing to study the heterogeneity of 10 MPEs from seven metastatic breast cancer patients, including three Miltenyi‐enriched samples using a negative selection approach. This dataset of almost 65 000 cells was analysed using integrative approaches to compare heterogeneous cell populations and phenotypes. Results We identified substantial inter‐patient heterogeneity in the composition of cell types (including malignant, mesothelial and immune cell populations), in expression of subtype‐specific gene signatures and in copy number aberration patterns, that captured variability across breast cancer cell populations. Within individual MPEs, we distinguished mesothelial cell populations from malignant cells using key markers, the presence of breast cancer subtype expression patterns and copy number aberration patterns. We also identified pleural mesothelial cells expressing a cancer‐associated fibroblast‐like transcriptomic program that may support cancer growth. Conclusions Our dataset presents the first unbiased assessment of breast cancer‐associated MPEs at a single cell resolution, providing the community with a valuable resource for the study of MPEs. Our work highlights the molecular and cellular diversity captured in MPEs and motivates the potential use of these clinically relevant biopsies in the development of targeted therapeutics for patients with advanced breast cancer.

Abstract MiR-21 was identified as a gene whose expression correlated with the extent of metastasis of murine mammary tumours. Since miR-21 is recognised as being associated with poor prognosis in cancer, we investigated its contribution to mammary tumour growth and metastasis in tumours with capacity for spontaneous metastasis. Unexpectedly, we found that suppression of miR-21 activity in highly metastatic tumours resulted in regression of primary tumour growth in immunocompetent mice but did not impede growth in immunocompromised mice. Analysis of the immune infiltrate of the primary tumours at the time when the tumours started to regress revealed an influx of both CD4+ and CD8+ activated T cells and a reduction in PD-L1+ infiltrating monocytes, providing an explanation for the observed tumour regression. Loss of anti-tumour immune suppression caused by decreased miR-21 activity was confirmed by transcriptomic analysis of primary tumours. This analysis also revealed reduced expression of genes associated with cell cycle progression upon loss of miR-21 activity. A second activity of miR-21 was the promotion of metastasis as shown by the loss of metastatic capacity of miR-21 knockdown tumours established in immunocompromised mice, despite no impact on primary tumour growth. A proteomic analysis of tumour cells with altered miR-21 activity revealed deregulation of proteins known to be associated with tumour progression. The development of therapies targeting miR-21, possibly via targeted delivery to tumour cells, could be an effective therapy to combat primary tumour growth and suppress the development of metastatic disease.

The visualisation of the bone metastasis process in a spatial temporal manner is lacking. Here, the authors use three-dimensional quantitative imaging and show that mouse mammary tumour cells preferentially home to endothelial subtype type H vessels within the bone marrow and remodel this vasculature by producing granulocyte-colony stimulating factor.

Abstract Background Human epidermal growth factor receptor-2 (HER2)-targeted therapies prolong survival in HER2-positive breast cancer patients. Benefit stems primarily from improved control of systemic disease, but up to 50% of patients progress to incurable brain metastases due to acquired resistance and/or limited permeability of inhibitors across the blood-brain barrier. Neratinib, a potent irreversible pan-tyrosine kinase inhibitor, prolongs disease-free survival in the extended adjuvant setting, and several trials evaluating its efficacy alone or combination with other inhibitors in early and advanced HER2-positive breast cancer patients are ongoing. However, its efficacy as a first-line therapy against HER2-positive breast cancer brain metastasis has not been fully explored, in part due to the lack of relevant pre-clinical models that faithfully recapitulate this disease. Here, we describe the development and characterisation of a novel syngeneic model of spontaneous HER2-positive breast cancer brain metastasis (TBCP-1) and its use to evaluate the efficacy and mechanism of action of neratinib. Methods TBCP-1 cells were derived from a spontaneous BALB/C mouse mammary tumour and characterised for hormone receptors and HER2 expression by flow cytometry, immunoblotting and immunohistochemistry. Neratinib was evaluated in vitro and in vivo in the metastatic and neoadjuvant setting. Its mechanism of action was examined by transcriptomic profiling, function inhibition assays and immunoblotting. Results TBCP-1 cells naturally express high levels of HER2 but lack expression of hormone receptors. TBCP-1 tumours maintain a HER2-positive phenotype in vivo and give rise to a high incidence of spontaneous and experimental metastases in the brain and other organs. Cell proliferation/viability in vitro is inhibited by neratinib and by other HER2 inhibitors, but not by anti-oestrogens, indicating phenotypic and functional similarities to human HER2-positive breast cancer. Mechanistically, neratinib promotes a non-apoptotic form of cell death termed ferroptosis. Importantly, metastasis assays demonstrate that neratinib potently inhibits tumour growth and metastasis, including to the brain, and prolongs survival, particularly when used as a neoadjuvant therapy. Conclusions The TBCP-1 model recapitulates the spontaneous spread of HER2-positive breast cancer to the brain seen in patients and provides a unique tool to identify novel therapeutics and biomarkers. Neratinib-induced ferroptosis provides new opportunities for therapeutic intervention. Further evaluation of neratinib neoadjuvant therapy is warranted.

Most cancer patients with solid tumors who succumb to their illness die of metastatic disease. While early detection and improved treatment have led to reduced mortality, even for those with metastatic cancer, some patients still respond poorly to treatment. Understanding the mechanisms of metastasis is important to improve prognostication, to stratify patients for treatment, and to identify new targets for therapy. We have shown previously that expression of nephronectin (NPNT) is correlated with metastatic propensity in breast cancer cell lines. In the present study, we provide a comprehensive analysis of the expression pattern and distribution of NPNT in breast cancer tissue from 842 patients by immunohistochemical staining of tissue microarrays from a historic cohort. Several patterns of NPNT staining were observed. An association between granular cytoplasmic staining (in

Estrogen stimulates breast development during puberty and mammary tumors in adulthood through estrogen receptor-α (ERα). These effects are proposed to occur via ERα+ luminal cells and not the mammary stem cells (MaSCs) that are ERαneg. Since ERα+ luminal cells express stem cell antigen-1 (SCA-1), we sought to determine if SCA-1 could define an ERα+ subset of EpCAM+/CD24+/CD49fhi MaSCs. We show that the MaSC population has a distinct SCA-1+ population that is abundant in pre-pubertal mammary glands. The SCA-1+ MaSCs have less stem cell markers and less in vivo repopulating activity than their SCA-1neg counterparts. However, they express ERα and specifically enter the cell cycle at puberty. Using estrogen-deficient aromatase knockouts (ArKO), we showed that the SCA-1+ MaSC could be directly modulated by estrogen supplementation. Thus, SCA-1 enriches for an ERα+, estrogen-sensitive subpopulation within the CD24+/CD49fhi MaSC population that may be responsible for the hormonal sensitivity of the developing mammary gland.

A reactive tumour stroma is associated with poor prognosis. Here, the authors show that in patients with triple negative breast cancer resident monocytes activate cancer-associated fibroblasts and induce production of CXCL16, which acts as a monocyte chemoattractant, resulting in an amplificatory feedback loop.

Breast cancer brain metastases remain largely incurable. Although several mouse models have been developed to investigate the genes and mechanisms regulating breast cancer brain metastasis, these models often lack clinical relevance since they require the use of immunocompromised mice and/or are poorly metastatic to brain from the mammary gland. We describe the development and characterisation of an aggressive brain metastatic variant of the 4T1 syngeneic model (4T1Br4) that spontaneously metastasises to multiple organs, but is selectively more metastatic to the brain from the mammary gland than parental 4T1 tumours. As seen by immunohistochemistry, 4T1Br4 tumours and brain metastases display a triple-negative phenotype, consistent with the high propensity of this breast cancer subtype to spread to brain. In vitro assays indicate that 4T1Br4 cells have an enhanced ability to adhere to or migrate across a brain-derived endothelial monolayer and greater invasive response to brain-derived soluble factors compared to 4T1 cells. These properties are likely to contribute to the brain selectivity of 4T1Br4 tumours. Expression profiling and gene set enrichment analyses demonstrate the clinical relevance of the 4T1Br4 model at the transcriptomic level. Pathway analyses implicate tumour-intrinsic immune regulation and vascular interactions in successful brain colonisation, revealing potential therapeutic targets. Evaluation of two histone deacetylase inhibitors, SB939 and 1179.4b, shows partial efficacy against 4T1Br4 metastasis to brain and other sites in vivo, and potent radio-sensitising properties in vitro. The 4T1Br4 model provides a clinically relevant tool for mechanistic studies and to evaluate novel therapies against brain metastasis. This article has an associated First Person interview with Soo-Hyun Kim, joint first author of the paper.

Triple-negative breast cancer (TNBC) represents 10-20% of all human ductal adenocarcinomas and has a poor prognosis relative to other subtypes. Hence, new molecular targets for therapeutic intervention are necessary. Analyses of panels of human or mouse cancer lines derived from the same individual that differ in their cellular phenotypes but not in genetic background have been instrumental in defining the molecular players that drive the various hallmarks of cancer. To determine the molecular regulators of metastasis in TNBC, we completed a rigorous in vitro and in vivo characterisation of four populations of the MDA-MB-231 human breast cancer line ranging in aggressiveness from non-metastatic to spontaneously metastatic to lung, liver, spleen and lymph node. Single nucleotide polymorphism (SNP) array analyses and genome-wide mRNA expression profiles of tumour cells isolated from orthotopic mammary xenografts were compared between the four lines to define both cell autonomous pathways and genes associated with metastatic proclivity. Gene set enrichment analysis (GSEA) demonstrated an unexpected association between both ribosome biogenesis and mRNA metabolism and metastatic capacity. Differentially expressed genes or families of related genes were allocated to one of four categories, associated with either metastatic initiation (e.g. CTSC, ENG, BMP2), metastatic virulence (e.g. ADAMTS1, TIE1), metastatic suppression (e.g. CST1, CST2, CST4, CST6, SCNNA1, BMP4) or metastatic avirulence (e.g. CD74). Collectively, this model system based on MDA-MB-231 cells should be useful for the assessment of gene function in the metastatic cascade and also for the testing of novel experimental therapeutics for the treatment of TNBC. This article has an associated First Person interview with the first author of the paper.

The translation of basic research into improved therapies for breast cancer patients requires relevant preclinical models that incorporate spontaneous metastasis. We have completed a functional and molecular characterisation of a new isogenic C57BL/6 mouse model of breast cancer metastasis, comparing and contrasting it with the established BALB/c 4T1 model. Metastatic EO771.LMB tumours were derived from poorly metastatic parental EO771 mammary tumours. Functional differences were evaluated using both in vitro assays and spontaneous metastasis assays in mice. Results were compared to non-metastatic 67NR and metastatic 4T1.2 tumours of the 4T1 model. Protein and transcript levels of markers of human breast cancer molecular subtypes were measured in the four tumour lines, as well as p53 (Tp53) tumour-suppressor gene status and responses to tamoxifen in vivo and in vitro. Array-based expression profiling of whole tumours identified genes and pathways that were deregulated in metastatic tumours. EO771.LMB cells metastasised spontaneously to lung in C57BL/6 mice and displayed increased invasive capacity compared with parental EO771. By immunohistochemical assessment, EO771 and EO771.LMB were basal-like, as was the 4T1.2 tumour, whereas 67NR had a luminal phenotype. Primary tumours from all lines were negative for progesterone receptor, Erb-b2/Neu and cytokeratin 5/6, but positive for epidermal growth factor receptor (EGFR). Only 67NR displayed nuclear estrogen receptor alpha (ERα) positivity. EO771 and EO771.LMB expressed mutant p53, whereas 67NR and 4T1.2 were p53-null. Integrated molecular analysis of both the EO771/EO771.LMB and 67NR/4T1.2 pairs indicated that upregulation of matrix metalloproteinase-3 (MMP-3), parathyroid hormone-like hormone (Pthlh) and S100 calcium binding protein A8 (S100a8) and downregulation of the thrombospondin receptor (Cd36) might be causally involved in metastatic dissemination of breast cancer.

Caveolae and caveolin-1 (CAV1) have been linked to several cellular functions. However, a model explaining their roles in mammalian tissues in vivo is lacking. Unbiased expression profiling in several tissues and cell types identified lipid metabolism as the main target affected by CAV1 deficiency. CAV1−/− mice exhibited impaired hepatic peroxisome proliferator-activated receptor α (PPARα)-dependent oxidative fatty acid metabolism and ketogenesis. Similar results were recapitulated in CAV1-deficient AML12 hepatocytes, suggesting at least a partial cell-autonomous role of hepatocyte CAV1 in metabolic adaptation to fasting. Finally, our experiments suggest that the hepatic phenotypes observed in CAV1−/− mice involve impaired PPARα ligand signaling and attenuated bile acid and FXRα signaling. These results demonstrate the significance of CAV1 in (1) hepatic lipid homeostasis and (2) nuclear hormone receptor (PPARα, FXRα, and SHP) and bile acid signaling.