Nicotinamide N-methyltransferase sustains a core epigenetic program that promotes metastatic colonization in breast cancer.

Metastatic colonization of distant organs accounts for over 90% of deaths related to solid cancers, yet the molecular determinants of metastasis remain poorly understood. Here, we unveil a mechanism of colonization in the aggressive basal-like subtype of breast cancer that is driven by the NAD+ metabolic enzyme nicotinamide N-methyltransferase (NNMT). We demonstrate that NNMT imprints a basal genetic program into cancer cells, enhancing their plasticity. In line, NNMT expression is associated with poor clinical outcomes in patients with breast cancer. Accordingly, ablation of NNMT dramatically suppresses metastasis formation in pre-clinical mouse models. Mechanistically, NNMT depletion results in a methyl overflow that increases histone H3K9 trimethylation (H3K9me3) and DNA methylation at the promoters of PR/SET Domain-5 (PRDM5) and extracellular matrix-related genes. PRDM5 emerged in this study as a pro-metastatic gene acting via induction of cancer-cell intrinsic transcription of collagens. Depletion of PRDM5 in tumor cells decreases COL1A1 deposition and impairs metastatic colonization of the lungs. These findings reveal a critical activity of the NNMT-PRDM5-COL1A1 axis for cancer cell plasticity and metastasis in basal-like breast cancer.

Hepatic stellate cells suppress NK cell-sustained breast cancer dormancy.

The persistence of undetectable disseminated tumour cells (DTCs) after primary tumour resection poses a major challenge to effective cancer treatment1-3. These enduring dormant DTCs are seeds of future metastases, and the mechanisms that switch them from dormancy to outgrowth require definition. Because cancer dormancy provides a unique therapeutic window for preventing metastatic disease, a comprehensive understanding of the distribution, composition and dynamics of reservoirs of dormant DTCs is imperative. Here we show that different tissue-specific microenvironments restrain or allow the progression of breast cancer in the liver-a frequent site of metastasis4 that is often associated with a poor prognosis5. Using mouse models, we show that there is a selective increase in natural killer (NK) cells in the dormant milieu. Adjuvant interleukin-15-based immunotherapy ensures an abundant pool of NK cells that sustains dormancy through interferon-γ signalling, thereby preventing hepatic metastases and prolonging survival. Exit from dormancy follows a marked contraction of the NK cell compartment and the concurrent accumulation of activated hepatic stellate cells (aHSCs). Our proteomics studies on liver co-cultures implicate the aHSC-secreted chemokine CXCL12 in the induction of NK cell quiescence through its cognate receptor CXCR4. CXCL12 expression and aHSC abundance are closely correlated in patients with liver metastases. Our data identify the interplay between NK cells and aHSCs as a master switch of cancer dormancy, and suggest that therapies aimed at normalizing the NK cell pool might succeed in preventing metastatic outgrowth.

Activation of multiple proto-oncogenic tyrosine kinases in breast cancer via loss of the PTPN12 phosphatase.

Among breast cancers, triple-negative breast cancer (TNBC) is the most poorly understood and is refractory to current targeted therapies. Using a genetic screen, we identify the PTPN12 tyrosine phosphatase as a tumor suppressor in TNBC. PTPN12 potently suppresses mammary epithelial cell proliferation and transformation. PTPN12 is frequently compromised in human TNBCs, and we identify an upstream tumor-suppressor network that posttranscriptionally controls PTPN12. PTPN12 suppresses transformation by interacting with and inhibiting multiple oncogenic tyrosine kinases, including HER2 and EGFR. The tumorigenic and metastatic potential of PTPN12-deficient TNBC cells is severely impaired upon restoration of PTPN12 function or combined inhibition of PTPN12-regulated tyrosine kinases, suggesting that TNBCs are dependent on the proto-oncogenic tyrosine kinases constrained by PTPN12. Collectively, these data identify PTPN12 as a commonly inactivated tumor suppressor and provide a rationale for combinatorially targeting proto-oncogenic tyrosine kinases in TNBC and other cancers based on their profile of tyrosine-phosphatase activity.

Glucocorticoids promote breast cancer metastasis.

Diversity within or between tumours and metastases (known as intra-patient tumour heterogeneity) that develops during disease progression is a serious hurdle for therapy1-3. Metastasis is the fatal hallmark of cancer and the mechanisms of colonization, the most complex step in the metastatic cascade4, remain poorly defined. A clearer understanding of the cellular and molecular processes that underlie both intra-patient tumour heterogeneity and metastasis is crucial for the success of personalized cancer therapy. Here, using transcriptional profiling of tumours and matched metastases in patient-derived xenograft models in mice, we show cancer-site-specific phenotypes and increased glucocorticoid receptor activity in distant metastases. The glucocorticoid receptor mediates the effects of stress hormones, and of synthetic derivatives of these hormones that are used widely in the clinic as anti-inflammatory and immunosuppressive agents. We show that the increase in stress hormones during breast cancer progression results in the activation of the glucocorticoid receptor at distant metastatic sites, increased colonization and reduced survival. Our transcriptomics, proteomics and phospho-proteomics studies implicate the glucocorticoid receptor in the activation of multiple processes in metastasis and in the increased expression of kinase ROR1, both of which correlate with reduced survival. The ablation of ROR1 reduced metastatic outgrowth and prolonged survival in preclinical models. Our results indicate that the activation of the glucocorticoid receptor increases heterogeneity and metastasis, which suggests that caution is needed when using glucocorticoids to treat patients with breast cancer who have developed cancer-related complications.

Imagine beyond: recent breakthroughs and next challenges in mammary gland biology and breast cancer research.

On 8 December 2022 the organizing committee of the European Network for Breast Development and Cancer labs (ENBDC) held its fifth annual Think Tank meeting in Amsterdam, the Netherlands. Here, we embraced the opportunity to look back to identify the most prominent breakthroughs of the past ten years and to reflect on the main challenges that lie ahead for our field in the years to come. The outcomes of these discussions are presented in this position paper, in the hope that it will serve as a summary of the current state of affairs in mammary gland biology and breast cancer research for early career researchers and other newcomers in the field, and as inspiration for scientists and clinicians to move the field forward.

Single-cell Analysis Reveals Inter- and Intratumour Heterogeneity in Metastatic Breast Cancer.

Metastasis is the leading cause of cancer-related deaths of breast cancer patients. Some cancer cells in a tumour go through successive steps, referred to as the metastatic cascade, and give rise to metastases at a distant site. We know that the plasticity and heterogeneity of cancer cells play critical roles in metastasis but the precise underlying molecular mechanisms remain elusive. Here we aimed to identify molecular mechanisms of metastasis during colonization, one of the most important yet poorly understood steps of the cascade. We performed single-cell RNA-Seq (scRNA-Seq) on tumours and matched lung macrometastases of patient-derived xenografts of breast cancer. After correcting for confounding factors such as the cell cycle and the percentage of detected genes (PDG), we identified cells in three states in both tumours and metastases. Gene-set enrichment analysis revealed biological processes specific to proliferation and invasion in two states. Our findings suggest that these states are a balance between epithelial-to-mesenchymal (EMT) and mesenchymal-to-epithelial transitions (MET) traits that results in so-called partial EMT phenotypes. Analysis of the top differentially expressed genes (DEGs) between these cell states revealed a common set of partial EMT transcription factors (TFs) controlling gene expression, including ZNF750, OVOL2, TP63, TFAP2C and HEY2. Our data suggest that the TFs related to EMT delineate different cell states in tumours and metastases. The results highlight the marked interpatient heterogeneity of breast cancer but identify common features of single cells from five models of metastatic breast cancer.

PI3K inhibition circumvents resistance to SHP2 blockade in metastatic triple-negative breast cancer.

The protein tyrosine phosphatase SHP2 activates oncogenic pathways downstream of most receptor tyrosine kinases (RTK) and has been implicated in various cancer types, including the highly aggressive subtype of triple-negative breast cancer (TNBC). Although allosteric inhibitors of SHP2 have been developed and are currently being evaluated in clinical trials, neither the mechanisms of the resistance to these agents, nor the means to circumvent such resistance have been clearly defined. The PI3K signaling pathway is also hyperactivated in breast cancer and contributes to resistance to anticancer therapies. When PI3K is inhibited, resistance also develops for example via activation of RTKs. We therefore assessed the effect of targeting PI3K and SHP2 alone or in combination in preclinical models of metastatic TNBC. In addition to the beneficial inhibitory effects of SHP2 alone, dual PI3K/SHP2 treatment decreased primary tumor growth synergistically, blocked the formation of lung metastases, and increased survival in preclinical models. Mechanistically, transcriptome and phospho-proteome analyses revealed that resistance to SHP2 inhibition is mediated by PDGFRß-evoked activation of PI3K signaling. Altogether, our data provide a rationale for co-targeting of SHP2 and PI3K in metastatic TNBC.

The Hippo kinases LATS1 and 2 control human breast cell fate via crosstalk with ERα.

Cell fate perturbations underlie many human diseases, including breast cancer. Unfortunately, the mechanisms by which breast cell fate are regulated are largely unknown. The mammary gland epithelium consists of differentiated luminal epithelial and basal myoepithelial cells, as well as undifferentiated stem cells and more restricted progenitors. Breast cancer originates from this epithelium, but the molecular mechanisms that underlie breast epithelial hierarchy remain ill-defined. Here, we use a high-content confocal image-based short hairpin RNA screen to identify tumour suppressors that regulate breast cell fate in primary human breast epithelial cells. We show that ablation of the large tumour suppressor kinases (LATS) 1 and 2 (refs 5, 6), which are part of the Hippo pathway, promotes the luminal phenotype and increases the number of bipotent and luminal progenitors, the proposed cells-of-origin of most human breast cancers. Mechanistically, we have identified a direct interaction between Hippo and oestrogen receptor-α (ERα) signalling. In the presence of LATS, ERα was targeted for ubiquitination and Ddb1-cullin4-associated-factor 1 (DCAF1)-dependent proteasomal degradation. Absence of LATS stabilized ERα and the Hippo effectors YAP and TAZ (hereafter YAP/TAZ), which together control breast cell fate through intrinsic and paracrine mechanisms. Our findings reveal a non-canonical (that is, YAP/TAZ-independent) effect of LATS in the regulation of human breast cell fate.

Breast Tumor Heterogeneity: Source of Fitness, Hurdle for Therapy.

Tumor heterogeneity impinges on prognosis, response to therapy, and metastasis. As such, heterogeneity is one of the most important and clinically relevant areas of cancer research. Breast cancer displays frequent intra- and inter-tumor heterogeneity as the result of genetic and non-genetic alterations that often enhance the vigor of cancer cells. In-depth characterization and understanding of the origin of this phenotypic and molecular diversity is paramount to improving diagnosis, the definition of prognostic and predictive biomarkers, and the design of therapeutic strategies. Here, we summarize current knowledge about sources of breast cancer heterogeneity, its consequences, and possible counter-measures. We discuss especially the impact on tumor heterogeneity of the differentiation state of the cell-of-origin, cancer cell plasticity, the microenvironment, and genetic evolution. Factors that enhance cancer cell vigor are clearly detrimental for patients.

Thirteenth Annual ENBDC Workshop: Methods in Mammary Gland Biology and Breast Cancer.

The thirteenth annual workshop of the European Network for Breast Development and Cancer (ENBDC) Laboratories Annual Workshop took place on the 28-30 April 2022 in Weggis, Switzerland and focused on methods in mammary gland biology and breast cancer. Sixty scientists participated in the ENBDC annual workshop which had not been held in person since 2019 due to the global COVID-19 pandemic. Topics spanned the mammary gland biology field, ranging from lactation biology and embryonic development, single cell sequencing of the human breast, and stunning cutting-edge imaging of the mouse mammary gland and human breast as well as breast cancer research topics including invasive progression of the pre-invasive DCIS stage, metabolic determinants of endocrine therapy resistance, models for lobular breast cancer, and how mutational landscapes of normal breast during age and pregnancy determine cancer risk. The latest findings from participating researchers were presented through oral presentations and poster sessions and included plenty of unpublished work.

Protein Tyrosine Phosphatase SHP2 Controls Interleukin-8 Expression in Breast Cancer Cells.

Treatment of metastasis remains a clinical challenge and the majority of breast cancer-related deaths are the result of drug-resistant metastases. The protein tyrosine phosphatase SHP2 encoded by the proto-oncogene PTPN11 promotes breast cancer progression. Inhibition of SHP2 has been shown to decrease metastases formation in various breast cancer models, but specific downstream effectors of SHP2 remain poorly characterized. Certain cytokines in the metastatic cascade facilitate local invasion and promote metastatic colonization. In this study, we investigated cytokines affected by SHP2 that could be relevant for its pro-tumorigenic properties. We used a cytokine array to investigate differentially released cytokines in the supernatant of SHP2 inhibitor-treated breast cancer cells. Expression of CXCL8 transcripts and protein abundance were assessed in human breast cancer cell lines in which we blocked SHP2 using shRNA constructs or an allosteric inhibitor. The impact of SHP2 inhibition on the phospho-tyrosine-proteome and signaling was determined using mass spectrometry. From previously published RNAseq data (Aceto et al. in Nat. Med. 18:529-37, 2012), we computed transcription factor activities using an integrated system for motif activity response analysis (ISMARA) (Balwierz et al. in Genome Res. 24:869-84, 2014). Finally, using siRNA against ETS1, we investigated whether ETS1 directly influences CXCL8 expression levels. We found that IL-8 is one of the most downregulated cytokines in cell supernatants upon SHP2 blockade, with a twofold decrease in CXCL8 transcripts and a fourfold decrease in IL-8 protein. These effects were also observed in preclinical tumor models. Analysis of the phospho-tyrosine-proteome revealed that several effectors of the mitogen-activated protein kinase (MAPK) pathway are downregulated upon SHP2 inhibition in vitro. MEK1/2 inhibition consistently reduced IL-8 levels in breast cancer cell supernatants. Computational analysis of RNAseq data from SHP2-depleted tumors revealed reduced activity of the transcription factor ETS1, a direct target of ERK and a transcription factor reported to regulate IL-8 expression. Our work reveals that SHP2 mediates breast cancer progression by enhancing the production and secretion of the pro-metastatic cytokine IL-8. We also provide mechanistic insights into the effects of SHP2 inhibition and its downstream repercussions. Overall, these results support a rationale for targeting SHP2 in breast cancer.

Twelfth Annual ENBDC Workshop: Methods in Mammary Gland Biology and Breast Cancer.

The twelfth annual workshop of the European Network for Breast Development and Cancer focused on methods in mammary gland biology and breast cancer, was scheduled to take place on March 26-28, 2020, in Weggis, Switzerland. Due to the COVID-19 pandemic, the meeting was rescheduled twice and eventually happened as a virtual meeting on April 22 and 23, 2021. The main topics of the meeting were branching and development of the mammary gland, tumor microenvironment, circulating tumor cells, tumor dormancy and breast cancer metastasis. Novel and unpublished findings related to these topics were presented, with a particular focus on the methods used to obtain them. Virtual poster sessions were a success, with many constructive and fruitful interactions between researchers and covered many areas of mammary gland biology and breast cancer.

PIK3CA(H1047R) induces multipotency and multi-lineage mammary tumours.

The adult mouse mammary epithelium contains self-sustained cell lineages that form the inner luminal and outer basal cell layers, with stem and progenitor cells contributing to its proliferative and regenerative potential. A key issue in breast cancer biology is the effect of genomic lesions in specific mammary cell lineages on tumour heterogeneity and progression. The impact of transforming events on fate conversion in cancer cells of origin and thus their contribution to tumour heterogeneity remains largely elusive. Using in situ genetic lineage tracing and limiting dilution transplantation, we have unravelled the potential of PIK3CA(H1047R), one of the most frequent mutations occurring in human breast cancer, to induce multipotency during tumorigenesis in the mammary gland. Here we show that expression of PIK3CA(H1047R) in lineage-committed basal Lgr5-positive and luminal keratin-8-positive cells of the adult mouse mammary gland evokes cell dedifferentiation into a multipotent stem-like state, suggesting this to be a mechanism involved in the formation of heterogeneous, multi-lineage mammary tumours. Moreover, we show that the tumour cell of origin influences the frequency of malignant mammary tumours. Our results define a key effect of PIK3CA(H1047R) on mammary cell fate in the pre-neoplastic mammary gland and show that the cell of origin of PIK3CA(H1047R) tumours dictates their malignancy, thus revealing a mechanism underlying tumour heterogeneity and aggressiveness.

Cessation of CCL2 inhibition accelerates breast cancer metastasis by promoting angiogenesis.

Secretion of C-C chemokine ligand 2 (CCL2) by mammary tumours recruits CCR2-expressing inflammatory monocytes to primary tumours and metastatic sites, and CCL2 neutralization in mice inhibits metastasis by retaining monocytes in the bone marrow. Here we report a paradoxical effect of CCL2 in four syngeneic mouse models of metastatic breast cancer. Surprisingly, interruption of CCL2 inhibition leads to an overshoot of metastases and accelerates death. This is the result of monocyte release from the bone marrow and enhancement of cancer cell mobilization from the primary tumour, as well as blood vessel formation and increased proliferation of metastatic cells in the lungs in an interleukin (IL)-6- and vascular endothelial growth factor (VEGF)-A-dependent manner. Notably, inhibition of CCL2 and IL-6 markedly reduced metastases and increased survival of the animals. CCL2 has been implicated in various neoplasias and adopted as a therapeutic target. However, our results call for caution when considering anti-CCL2 agents as monotherapy in metastatic disease and highlight the tumour microenvironment as a critical determinant of successful anti-metastatic therapy.

The Eleventh ENBDC Workshop: Advances in Technology Help to Unveil Mechanisms of Mammary Gland Development and Cancerogenesis.

The eleventh annual workshop of the European Network for Breast Development and Cancer, Methods in mammary gland biology and breast cancer, took place on the 16th to 18th of May 2019 in Weggis, Switzerland. The main topics of the meeting were high resolution genomics and proteomics for the study of mammary gland development and cancer, breast cancer signaling, tumor microenvironment, preclinical models of breast cancer, and tissue morphogenesis. Exciting novel findings in, or highly relevant to, mammary gland biology and breast cancer field were presented, with insights into the methods used to obtain them. Among others, the discussed methods included single-cell RNA sequencing, genetic barcoding, lineage tracing, spatial transcriptomics, optogenetics, genetic mouse models and organoids.

The ninth ENBDC Weggis meeting: growth and in-depth characterisation of normal and neoplastic breast cells.

Mammary gland biologists gathered for the ninth annual workshop of the European Network for Breast Development and Cancer (ENBDC) at Weggis on the shores of Lake Lucerne in March 2017. The main themes were oestrogen receptor alpha signalling, new techniques for mammary cell culture, CRISPR screening and proteogenomics.

Patient-derived xenograft (PDX) models in basic and translational breast cancer research.

Patient-derived xenograft (PDX) models of a growing spectrum of cancers are rapidly supplanting long-established traditional cell lines as preferred models for conducting basic and translational preclinical research. In breast cancer, to complement the now curated collection of approximately 45 long-established human breast cancer cell lines, a newly formed consortium of academic laboratories, currently from Europe, Australia, and North America, herein summarizes data on over 500 stably transplantable PDX models representing all three clinical subtypes of breast cancer (ER+, HER2+, and "Triple-negative" (TNBC)). Many of these models are well-characterized with respect to genomic, transcriptomic, and proteomic features, metastatic behavior, and treatment response to a variety of standard-of-care and experimental therapeutics. These stably transplantable PDX lines are generally available for dissemination to laboratories conducting translational research, and contact information for each collection is provided. This review summarizes current experiences related to PDX generation across participating groups, efforts to develop data standards for annotation and dissemination of patient clinical information that does not compromise patient privacy, efforts to develop complementary data standards for annotation of PDX characteristics and biology, and progress toward "credentialing" of PDX models as surrogates to represent individual patients for use in preclinical and co-clinical translational research. In addition, this review highlights important unresolved questions, as well as current limitations, that have hampered more efficient generation of PDX lines and more rapid adoption of PDX use in translational breast cancer research.

SHP2 regulates proliferation and tumorigenicity of glioma stem cells.

SHP2 is a cytoplasmic protein tyrosine phosphatase (PTPase) involved in multiple signaling pathways and was the first identified proto-oncogene PTPase. Previous work in glioblastoma (GBM) has demonstrated the role of SHP2 PTPase activity in modulating the oncogenic phenotype of adherent GBM cell lines. Mutations in PTPN11, the gene encoding SHP2, have been identified with increasing frequency in GBM. Given the importance of SHP2 in developing neural stem cells, and the importance of glioma stem cells (GSCs) in GBM oncogenesis, we explored the functional role of SHP2 in GSCs. Using paired differentiated and stem cell primary cultures, we investigated the association of SHP2 expression with the tumor stem cell compartment. Proliferation and soft agar assays were used to demonstrate the functional contribution of SHP2 to cell growth and transformation. SHP2 expression correlated with SOX2 expression in GSC lines and was decreased in differentiated cells. Forced differentiation of GSCs by removal of growth factors, as confirmed by loss of SOX2 expression, also resulted in decreased SHP2 expression. Lentiviral-mediated knockdown of SHP2 inhibited proliferation. Finally, growth in soft-agar was similarly inhibited by loss of SHP2 expression. Our results show that SHP2 function is required for cell growth and transformation of the GSC compartment in GBM.