Ehf controls mammary alveolar lineage differentiation and is a putative suppressor of breast tumorigenesis.

The transcription factor EHF is highly expressed in the lactating mammary gland, but its role in mammary development and tumorigenesis is not fully understood. Utilizing a mouse model of Ehf deletion, herein, we demonstrate that loss of Ehf impairs mammary lobuloalveolar differentiation at late pregnancy, indicated by significantly reduced levels of milk genes and milk lipids, fewer differentiated alveolar cells, and an accumulation of alveolar progenitor cells. Further, deletion of Ehf increased proliferative capacity and attenuated prolactin-induced alveolar differentiation in mammary organoids. Ehf deletion also increased tumor incidence in the MMTV-PyMT mammary tumor model and increased the proliferative capacity of mammary tumor organoids, while low EHF expression was associated with higher tumor grade and poorer outcome in luminal A and basal human breast cancers. Collectively, these findings establish EHF as a non-redundant regulator of mammary alveolar differentiation and a putative suppressor of mammary tumorigenesis.

Loss of tumor-derived SMAD4 enhances primary tumor growth but not metastasis following BMP4 signalling.

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.

Inhibition of EphA3 Expression in Tumour Stromal Cells Suppresses Tumour Growth and Progression.

Tumour progression relies on interactions with untransformed cells in the tumour microenvironment (TME), including cancer-associated fibroblasts (CAFs), which promote blood supply, tumour progression, and immune evasion. Eph receptor tyrosine kinases are cell guidance receptors that are most active during development but re-emerge in cancer and are recognised drug targets. EphA3 is overexpressed in a wide range of tumour types, and we previously found expression particularly in stromal and vascular tissues of the TME. To investigate its role in the TME, we generated transgenic mice with inducible shRNA-mediated knockdown of EphA3 expression. EphA3 knockdown was confirmed in aortic mesenchymal stem cells (MSCs), which displayed reduced angiogenic capacity. In mice with syngeneic lung tumours, EphA3 knockdown reduced vasculature and CAF/MSC-like cells in tumours, and inhibited tumour growth, which was confirmed also in a melanoma model. Single cell RNA sequencing analysis of multiple human tumour types confirmed EphA3 expression in CAFs, including in breast cancer, where EphA3 was particularly prominent in perivascular- and myofibroblast-like CAFs. Our results thus indicate expression of the cell guidance receptor EphA3 in distinct CAF subpopulations is important in supporting tumour angiogenesis and tumour growth, highlighting its potential as a therapeutic target.

Single-cell RNA sequencing captures patient-level heterogeneity and associated molecular phenotypes in breast cancer pleural effusions.

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.

Estrogen receptor beta expression in triple negative breast cancers is not associated with recurrence or survival.

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 ERß1 having the most benefit. Recently, the antibodies commonly used to assess ERß1 in TNBC have been found to lack specificity, which calls into question available data regarding the proportion of TNBC that express ERß1 and any relationship to clinical outcome. METHODS: To confirm the true frequency of ERß1 in TNBC we performed robust ERß1 immunohistochemistry using the specific antibody CWK-F12 ERß1 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 ERß1 was not associated with increased recurrence or survival when assessed as percentage of ERß1 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 ERß1 expression in TNBC tumours does not associate with prognosis.

Innovative thiosemicarbazones that induce multi-modal mechanisms to down-regulate estrogen-, progesterone-, androgen- and prolactin-receptors in breast cancer.

The estrogen receptor-α (ER-α) is a key driver of breast cancer (BC) and the ER-antagonist, tamoxifen, is a central pillar of BC treatment. However, cross-talk between ER-α, other hormone and growth factor receptors enables development of de novo resistance to tamoxifen. Herein, we mechanistically dissect the activity of a new class of anti-cancer agents that inhibit multiple growth factor receptors and down-stream signaling for the treatment of ER-positive BC. Using RNA sequencing and comprehensive protein expression analysis, we examined the activity of di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), on the expression and activation of hormone and growth factor receptors, co-factors, and key resistance pathways in ER-α-positive BC. DpC differentially regulated 106 estrogen-response genes, and this was linked to decreased mRNA levels of 4 central hormone receptors involved in BC pathogenesis, namely ER, progesterone receptor (PR), androgen receptor (AR), and prolactin receptor (PRL-R). Mechanistic investigation demonstrated that due to DpC and Dp44mT binding metal ions, these agents caused a pronounced decrease in ER-α, AR, PR, and PRL-R protein expression. DpC and Dp44mT also inhibited activation and down-stream signaling of the epidermal growth factor (EGF) family receptors, and expression of co-factors that promote ER-α transcriptional activity, including SRC3, NF-κB p65, and SP1. In vivo, DpC was highly tolerable and effectively inhibited ER-α-positive BC growth. Through bespoke, non-hormonal, multi-modal mechanisms, Dp44mT and DpC decrease the expression of PR, AR, PRL-R, and tyrosine kinases that act with ER-α to promote BC, constituting an innovative therapeutic approach.

COMMD3 loss drives invasive breast cancer growth by modulating copper homeostasis.

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 < 0.05). Consistent with this, depletion of COMMD3 induced invasive spheroid growth in ER + breast cancer cell lines in vitro, while Commd3 depletion in the relatively indolent 4T07 TNBC mouse cell line promoted tumour expansion in syngeneic Balb/c hosts. Notably, RNA sequencing revealed a role for COMMD3 in copper signalling, via regulation of the Na+/K+-ATPase subunit, ATP1B1. Treatment of COMMD3-depleted cells with the copper chelator, tetrathiomolybdate, significantly reduced invasive spheroid growth via induction of apoptosis. CONCLUSION: Overall, we found that COMMD3 loss promoted aggressive behaviour in breast cancer cells.

Functional and Phenotypic Characterisations of Common Syngeneic Tumour Cell Lines as Estrogen Receptor-Positive Breast Cancer Models.

Estrogen receptor-positive breast cancers (ER+ BCas) are the most common form of BCa and are increasing in incidence, largely due to changes in reproductive practices in recent decades. Tamoxifen is prescribed as a component of standard-of-care endocrine therapy for the treatment and prevention of ER+ BCa. However, it is poorly tolerated, leading to low uptake of the drug in the preventative setting. Alternative therapies and preventatives for ER+ BCa are needed but development is hampered due to a paucity of syngeneic ER+ preclinical mouse models that allow pre-clinical experimentation in immunocompetent mice. Two ER-positive models, J110 and SSM3, have been reported in addition to other tumour models occasionally shown to express ER (for example 4T1.2, 67NR, EO771, D2.0R and D2A1). Here, we have assessed ER expression and protein levels in seven mouse mammary tumour cell lines and their corresponding tumours, in addition to their cellular composition, tamoxifen sensitivity and molecular phenotype. By immunohistochemical assessment, SSM3 and, to a lesser extent, 67NR cells are ER+. Using flow cytometry and transcript expression we show that SSM3 cells are luminal in nature, whilst D2.0R and J110 cells are stromal/basal. The remainder are also stromal/basal in nature; displaying a stromal or basal Epcam/CD49f FACS phenotype and stromal and basal gene expression signatures are overrepresented in their transcript profile. Consistent with a luminal identity for SSM3 cells, they also show sensitivity to tamoxifen in vitro and in vivo. In conclusion, the data indicate that the SSM3 syngeneic cell line is the only definitively ER+ mouse mammary tumour cell line widely available for pre-clinical research.

Assessment of Potential Anti-Methanogenic and Antimicrobial Activity of Ethyl Nitroacetate, α-Lipoic Acid, Taurine and L-Cysteinesulfinic Acid In Vitro.

Livestock producers need new technologies to maintain the optimal health and well-being of their animals while minimizing the risks of propagating and disseminating pathogenic and antimicrobial-resistant bacteria to humans or other animals. Where possible, these interventions should contribute to the efficiency and profitability of animal production to avoid passing costs on to consumers. In this study, we examined the potential of nitroethane, 3-nitro-1-propionate, ethyl nitroacetate, taurine and L-cysteinesulfinic acid to modulate rumen methane production, a digestive inefficiency that results in the loss of up to 12% of the host's dietary energy intake and a major contributor of methane as a greenhouse gas to the atmosphere. The potential for these compounds to inhibit the foodborne pathogens, Escherichia coli O157:H7 and Salmonella Typhimurium DT104, was also tested. The results from the present study revealed that anaerobically grown O157:H7 and DT104 treated with the methanogenic inhibitor, ethyl nitroacetate, at concentrations of 3 and 9 mM had decreased (p < 0.05) mean specific growth rates of O157:H7 (by 22 to 36%) and of DT104 (by 16 to 26%) when compared to controls (0.823 and 0.886 h-1, respectively). The growth rates of O157:H7 and DT104 were decreased (p < 0.05) from controls by 31 to 73% and by 41 to 78% by α-lipoic acid, which we also found to inhibit in vitro rumen methanogenesis up to 66% (p < 0.05). Ethyl nitroacetate was mainly bacteriostatic, whereas 9 mM α-lipoic acid decreased (p < 0.05) maximal optical densities (measured at 600 nm) of O157:H7 and DT104 by 25 and 42% compared to controls (0.448 and 0.451, respectively). In the present study, the other oxidized nitro and organosulfur compounds were neither antimicrobial nor anti-methanogenic.

Microbeam Radiation Therapy Controls Local Growth of Radioresistant Melanoma and Treats Out-of-Field Locoregional Metastasis.

PURPOSE: Synchrotron-generated microbeam radiation therapy (MRT) represents an innovative preclinical type of cancer radiation therapy with an excellent therapeutic ratio. Beyond local control, metastatic spread is another important endpoint to assess the effectiveness of radiation therapy treatment. Currently, no data exist on an association between MRT and metastasis. Here, we evaluated the ability of MRT to delay B16F10 murine melanoma progression and locoregional metastatic spread. METHODS AND MATERIALS: We assessed the primary tumor response and the extent of metastasis in sentinel lymph nodes in 2 cohorts of C57BL/6J mice, one receiving a single MRT and another receiving 2 MRT treatments delivered with a 10-day interval. We compared these 2 cohorts with synchrotron broad beam-irradiated and nonirradiated mice. In addition, using multiplex quantitative platforms, we measured plasma concentrations of 34 pro- and anti-inflammatory cytokines and frequencies of immune cell subsets infiltrating primary tumors that received either 1 or 2 MRT treatments. RESULTS: Two MRT treatments were significantly more effective for local control than a single MRT. Remarkably, the second MRT also triggered a pronounced regression of out-of-radiation field locoregional metastasis. Augmentation of CXCL5, CXCL12, and CCL22 levels after the second MRT indicated that inhibition of melanoma progression could be associated with increased activity of antitumor neutrophils and T-cells. Indeed, we demonstrated elevated infiltration of neutrophils and activated T-cells in the tumors after the second MRT. CONCLUSIONS: Our study highlights the importance of monitoring metastasis after MRT and provides the first MRT fractionation schedule that promotes local and locoregional control with the potential to manage distant metastasis.

MicroRNA-21 is immunosuppressive and pro-metastatic via separate mechanisms.

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.

Computational Screening of Anti-Cancer Drugs Identifies a New BRCA Independent Gene Expression Signature to Predict Breast Cancer Sensitivity to Cisplatin.

The development of therapies that target specific disease subtypes has dramatically improved outcomes for patients with breast cancer. However, survival gains have not been uniform across patients, even within a given molecular subtype. Large collections of publicly available drug screening data matched with transcriptomic measurements have facilitated the development of computational models that predict response to therapy. Here, we generated a series of predictive gene signatures to estimate the sensitivity of breast cancer samples to 90 drugs, comprising FDA-approved drugs or compounds in early development. To achieve this, we used a cell line-based drug screen with matched transcriptomic data to derive in silico models that we validated in large independent datasets obtained from cell lines and patient-derived xenograft (PDX) models. Robust computational signatures were obtained for 28 drugs and used to predict drug efficacy in a set of PDX models. We found that our signature for cisplatin can be used to identify tumors that are likely to respond to this drug, even in absence of the BRCA-1 mutation routinely used to select patients for platinum-based therapies. This clinically relevant observation was confirmed in multiple PDXs. Our study foreshadows an effective delivery approach for precision medicine.

Epithelial de-differentiation triggered by co-ordinate epigenetic inactivation of the EHF and CDX1 transcription factors drives colorectal cancer progression.

Colorectal cancers (CRCs) often display histological features indicative of aberrant differentiation but the molecular underpinnings of this trait and whether it directly drives disease progression is unclear. Here, we identify co-ordinate epigenetic inactivation of two epithelial-specific transcription factors, EHF and CDX1, as a mechanism driving differentiation loss in CRCs. Re-expression of EHF and CDX1 in poorly-differentiated CRC cells induced extensive chromatin remodelling, transcriptional re-programming, and differentiation along the enterocytic lineage, leading to reduced growth and metastasis. Strikingly, EHF and CDX1 were also able to reprogramme non-colonic epithelial cells to express colonic differentiation markers. By contrast, inactivation of EHF and CDX1 in well-differentiated CRC cells triggered tumour de-differentiation. Mechanistically, we demonstrate that EHF physically interacts with CDX1 via its PNT domain, and that these transcription factors co-operatively drive transcription of the colonic differentiation marker, VIL1. Compound genetic deletion of Ehf and Cdx1 in the mouse colon disrupted normal colonic differentiation and significantly enhanced colorectal tumour progression. These findings thus reveal a novel mechanism driving epithelial de-differentiation and tumour progression in CRC.

Can preclinical drug development help to predict adverse events in clinical trials?

The development of novel therapeutics is associated with high rates of attrition, with unexpected adverse events being a major cause of failure. Serious adverse events have led to organ failure, cancer development and deaths that were not expected outcomes in clinical trials. These life-threatening events were not identified during therapeutic development due to the lack of preclinical safety tests that faithfully represented human physiology. We highlight the successful application of several novel technologies, including high-throughput screening, organs-on-chips, microbiome-containing drug-testing platforms and humanised mouse models, for mechanistic studies and prediction of toxicity. We propose the incorporation of similar preclinical tests into future drug development to reduce the likelihood of hazardous therapeutics entering later-stage clinical trials.

Immunomodulatory effects of G-CSF in cancer: Therapeutic implications.

Numerous preclinical studies have reported a pro-tumour role for granulocyte colony-stimulating factor (G-CSF) that is predominantly mediated by neutrophils and MDSCs, the major G-CSF receptor expressing populations. In the presence of G-CSF (either tumour-derived or exogenous) these myeloid populations commonly exhibit a T cell suppressive phenotype. However, the direct effects of this cytokine on other immune lineages, such as T and NK cells, are not as well established. Herein we discuss the most recent data relating to the effect of G-CSF on the major immune populations, exclusively in the context of cancer. Recent publications have drawn attention to the other tumour-promoting effects of G-CSF on myeloid cells, including NETosis, promotion of cancer stemness and skewed differentiation of bone marrow progenitors towards myelopoiesis. Although G-CSF is safely and commonly used as a supportive therapy to prevent or treat chemotherapy-associated neutropenia in cancer patients, we also discuss the potential impacts of G-CSF on other anti-cancer treatments. Importantly, considerations for immune checkpoint blockade are highlighted, as many publications report a T cell suppressive effect of G-CSF that may diminish the effectiveness of this immunotherapy.

Mouse Breast Carcinoma Monocytic/Macrophagic Myeloid-Derived Suppressor Cell Infiltration as a Consequence of Endothelial Dysfunction in Shb-Deficient Endothelial Cells Increases Tumor Lung Metastasis.

Metastasis reflects both the inherent properties of tumor cells and the response of the stroma to the presence of the tumor. Vascular barrier properties, either due to endothelial cell (EC) or pericyte function, play an important role in metastasis in addition to the contribution of the immune system. The Shb gene encodes the Src homology-2 domain protein B that operates downstream of tyrosine kinases in both vascular and immune cells. We have investigated E0771.lmb breast carcinoma metastasis in mice with conditional deletion of the Shb gene using the Cdh5-CreERt2 transgene, resulting in inactivation of the Shb-gene in EC and some hematopoietic cell populations. Lung metastasis from orthotopic tumors, tumor vascular and immune cell characteristics, and immune cell gene expression profiles were determined. We found no increase in vascular leakage that could explain the observed increase in metastasis upon the loss of Shb expression. Instead, Shb deficiency in EC promoted the recruitment of monocytic/macrophagic myeloid-derived suppressor cells (mMDSC), an immune cell type that confers a suppressive immune response, thus enhancing lung metastasis. An MDSC-promoting cytokine/chemokine profile was simultaneously observed in tumors grown in mice with EC-specific Shb deficiency, providing an explanation for the expanded mMDSC population. The results demonstrate an intricate interplay between tumor EC and immune cells that pivots between pro-tumoral and anti-tumoral properties, depending on relevant genetic and/or environmental factors operating in the microenvironment.

Mammary tumour cells remodel the bone marrow vascular microenvironment to support metastasis.

Bone marrow is a preferred metastatic site for multiple solid tumours and is associated with poor prognosis and significant morbidity. Accumulating evidence indicates that cancer cells colonise specialised niches within the bone marrow to support their long-term propagation, but the precise location and mechanisms that mediate niche interactions are unknown. Using breast cancer as a model of solid tumour metastasis to the bone marrow, we applied large-scale quantitative three-dimensional imaging to characterise temporal changes in the bone marrow microenvironment during disease progression. We show that mouse mammary tumour cells preferentially home to a pre-existing metaphyseal domain enriched for type H vessels. Metastatic lesion outgrowth rapidly remodelled the local vasculature through extensive sprouting to establish a tumour-supportive microenvironment. The evolution of this tumour microenvironment reflects direct remodelling of the vascular endothelium through tumour-derived granulocyte-colony stimulating factor (G-CSF) in a hematopoietic cell-independent manner. Therapeutic targeting of the metastatic niche by blocking G-CSF receptor inhibited pathological blood vessel remodelling and reduced bone metastasis burden. These findings elucidate a mechanism of 'host' microenvironment hijacking by mammary tumour cells to subvert the local microvasculature to form a specialised, pro-tumorigenic niche.

PTU, a novel ureido-fatty acid, inhibits MDA-MB-231 cell invasion and dissemination by modulating Wnt5a secretion and cytoskeletal signaling.

Migration and invasion promote tumor cell metastasis, which is the leading cause of cancer death. At present there are no effective treatments. Epidemiological studies have suggested that ω-3 polyunsaturated fatty acids (PUFA) may decrease cancer aggressiveness. In recent studies epoxide metabolites of ω-3 PUFA exhibited anti-cancer activity, although increased in vivo stability is required to develop useful drugs. Here we synthesized novel stabilized ureido-fatty acid ω-3 epoxide isosteres and found that one analogue - p-tolyl-ureidopalmitic acid (PTU) - inhibited migration and invasion by MDA-MB-231 breast cancer cells in vitro and in vivo in xenografted nu/nu mice. From proteomics analysis of PTU-treated cells major regulated pathways were linked to the actin cytoskeleton and actin-based motility. The principal finding was that PTU impaired the formation of actin protrusions by decreasing the secretion of Wnt5a, which dysregulated the Wnt/planar cell polarity (PCP) pathway and actin cytoskeletal dynamics. Exogenous Wnt5a restored invasion and Wnt/PCP signalling in PTU-treated cells. PTU is the prototype of a novel class of agents that selectively dysregulate the Wnt/PCP pathway by inhibiting Wnt5a secretion and actin dynamics to impair MDA-MB-231 cell migration and invasion.

The site of breast cancer metastases dictates their clonal composition and reversible transcriptomic profile.

Intratumoral heterogeneity is a driver of breast cancer progression, but the nature of the clonal interactive network involved in this process remains unclear. Here, we optimized the use of optical barcoding to visualize and characterize 31 cancer subclones in vivo. By mapping the clonal composition of thousands of metastases in two clinically relevant sites, the lungs and liver, we found that metastases were highly polyclonal in lungs but not in the liver. Furthermore, the transcriptome of the subclones varied according to their metastatic niche. We also identified a reversible niche-driven signature that was conserved in lung and liver metastases collected during patient autopsies. Among this signature, we found that the tumor necrosis factor-α pathway was up-regulated in lung compared to liver metastases, and inhibition of this pathway affected metastasis diversity. These results highlight that the cellular and molecular heterogeneity observed in metastases is largely dictated by the tumor microenvironment.

Targeting a cell surface vitamin D receptor on tumor-associated macrophages in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive tumor with limited treatment options and poor prognosis. We applied the in vivo phage display technology to isolate peptides homing to the immunosuppressive cellular microenvironment of TNBC as a strategy for non-malignant target discovery. We identified a cyclic peptide (CSSTRESAC) that specifically binds to a vitamin D receptor, protein disulfide-isomerase A3 (PDIA3) expressed on the cell surface of tumor-associated macrophages (TAM), and targets breast cancer in syngeneic TNBC, non-TNBC xenograft, and transgenic mouse models. Systemic administration of CSSTRESAC to TNBC-bearing mice shifted the cytokine profile toward an antitumor immune response and delayed tumor growth. Moreover, CSSTRESAC enabled ligand-directed theranostic delivery to tumors and a mathematical model confirmed our experimental findings. Finally, in silico analysis showed PDIA3-expressing TAM in TNBC patients. This work uncovers a functional interplay between a cell surface vitamin D receptor in TAM and antitumor immune response that could be therapeutically exploited.