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.

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.

Synergistic activation of genes promoting invasiveness by dual deprivation in oxygen and nutrients.

By depriving cancer cells of blood supplies of oxygen and nutrients, anti-angiogenic therapy is aimed at simultaneously asphyxiating and starving the cells. But in spite of its apparent logic, this strategy is generally counterproductive over the long term as the treatment seems to elicit malignancy. Since a defect of blood supply is expected to deprive tumours simultaneously of oxygen and nutrients naturally, we examine here these two deprivations, alone or in combination, on the phenotype and signalling pathways of moderately aggressive MCF7 cancer cells. Each deprivation induces some aspects of the aggressive and migratory phenotypes through activating several pathways, including HIF1-alpha as expected, but also SRF/MRTFA and TCF4/beta-catenin. Strikingly, the dual deprivation has strong cooperative effects on the upregulation of genes increasing the metastatic potential, such as four and a half LIM domains 2 (FHL2) and HIF1A-AS2 lncRNA, which have response elements for both pathways. Using anti-angiogenic agents as monotherapy is therefore questionable as it may give falsely promising short-term tumour regression, but could ultimately exacerbate aggressive phenotypes.

Hypoxia and ERα Transcriptional Crosstalk Is Associated with Endocrine Resistance in Breast Cancer.

Estrogen receptor-alpha (ERα) is the driving transcription factor in 70% of breast cancers and its activity is associated with hormone dependent tumor cell proliferation and survival. Given the recurrence of hormone resistant relapses, understanding the etiological factors fueling resistance is of major clinical interest. Hypoxia, a frequent feature of the solid tumor microenvironment, has been described to promote endocrine resistance by triggering ERα down-regulation in both in vitro and in vivo models. Yet, the consequences of hypoxia on ERα genomic activity remain largely elusive. In the present study, transcriptomic analysis shows that hypoxia regulates a fraction of ERα target genes, underlying an important regulatory overlap between hypoxic and estrogenic signaling. This gene expression reprogramming is associated with a massive reorganization of ERα cistrome, highlighted by a massive loss of ERα binding sites. Profiling of enhancer acetylation revealed a hormone independent enhancer activation at the vicinity of genes harboring hypoxia inducible factor (HIFα) binding sites, the major transcription factors governing hypoxic adaptation. This activation counterbalances the loss of ERα and sustains hormone-independent gene expression. We describe hypoxia in luminal ERα (+) breast cancer as a key factor interfering with endocrine therapies, associated with poor clinical prognosis in breast cancer patients.

A high-throughput drug screen reveals means to differentiate triple-negative breast cancer.

Plasticity delineates cancer subtypes with more or less favourable outcomes. In breast cancer, the subtype triple-negative lacks expression of major differentiation markers, e.g., estrogen receptor α (ERα), and its high cellular plasticity results in greater aggressiveness and poorer prognosis than other subtypes. Whether plasticity itself represents a potential vulnerability of cancer cells is not clear. However, we show here that cancer cell plasticity can be exploited to differentiate triple-negative breast cancer (TNBC). Using a high-throughput imaging-based reporter drug screen with 9 501 compounds, we have identified three polo-like kinase 1 (PLK1) inhibitors as major inducers of ERα protein expression and downstream activity in TNBC cells. PLK1 inhibition upregulates a cell differentiation program characterized by increased DNA damage, mitotic arrest, and ultimately cell death. Furthermore, cells surviving PLK1 inhibition have decreased tumorigenic potential, and targeting PLK1 in already established tumours reduces tumour growth both in cell line- and patient-derived xenograft models. In addition, the upregulation of genes upon PLK1 inhibition correlates with their expression in normal breast tissue and with better overall survival in breast cancer patients. Our results indicate that differentiation therapy based on PLK1 inhibition is a potential alternative strategy to treat TNBC.

Phenotypic plasticity during metastatic colonization.

Most solid cancer-related deaths result from metastasis, a multistep process in which cancer cells exit the primary site, intravasate into the bloodstream, extravasate, and colonize distant organs. Colonization is facilitated by clonal selection and the high phenotypic plasticity of cancer cells that creates reversible switching of cellular states. Cancer cell plasticity leads to intratumor heterogeneity and fitness, yielding cells with molecular and cellular programs that facilitate survival and colonization. While cancer cell plasticity is sometimes limited to the process of epithelial-to-mesenchymal transition (EMT), recent studies have broadened its definition. Plasticity arises from both cell-intrinsic and cell-extrinsic factors and is a major obstacle to efficacious anti-cancer therapies. Here, we discuss the multifaceted notion of cancer cell plasticity associated with metastatic colonization.

Nuclear translocation of MRTFA in MCF7 breast cancer cells shifts ERα nuclear/genomic to extra-nuclear/non genomic actions.

The Myocardin-related transcription factor A [MRTFA, also known as Megakaryoblastic Leukemia 1 (MKL1))] is a major actor in the epithelial to mesenchymal transition (EMT). We have previously shown that activation and nuclear accumulation of MRTFA mediate endocrine resistance of estrogen receptor alpha (ERα) positive breast cancers by initiating a partial transition from luminal to basal-like phenotype and impairing ERα cistrome and transcriptome. In the present study, we deepen our understanding of the mechanism by monitoring functional changes in the receptor's activity. We demonstrate that MRTFA nuclear accumulation down-regulates the expression of the unliganded (Apo-)ERα and causes a redistribution of the protein localization from its normal nuclear place to the entire cell volume. This phenomenon is accompanied by a shift in Apo-ERα monomer/dimer ratio towards the monomeric state, leading to significant functional consequences on ERα activities. In particular, the association of Apo-ERα with chromatin is drastically decreased, and the remaining ERα binding sites are substantially less enriched in ERE motifs than in control conditions. Monitored by proximity Ligation Assay, ERα interactions with P160 family coactivators are partly impacted when MRTFA accumulates in the nucleus, and those with SMRT and NCOR1 corepressors are abolished. Finally, ERα interactions with kinases such as c-src and PI3K are increased, thereby enhancing MAP Kinase and AKT activities. In conclusion, the activation and nuclear accumulation of MRTFA in ERα positive breast cancer cells remodels both ERα location and functions by shifting its activity from nuclear genome regulation to extra-nuclear non-genomic signaling.

The NFIB-ERO1A axis promotes breast cancer metastatic colonization of disseminated tumour cells.

Metastasis is the main cause of deaths related to solid cancers. Active transcriptional programmes are known to regulate the metastatic cascade but the molecular determinants of metastatic colonization remain elusive. Using an inducible piggyBac (PB) transposon mutagenesis screen, we have shown that overexpression of the transcription factor nuclear factor IB (NFIB) alone is sufficient to enhance primary mammary tumour growth and lung metastatic colonization. Mechanistically and functionally, NFIB directly increases expression of the oxidoreductase ERO1A, which enhances HIF1α-VEGFA-mediated angiogenesis and colonization, the last and fatal step of the metastatic cascade. NFIB is thus clinically relevant: it is preferentially expressed in the poor-prognostic group of basal-like breast cancers, and high expression of the NFIB/ERO1A/VEGFA pathway correlates with reduced breast cancer patient survival.

Nuclear accumulation of MKL1 in luminal breast cancer cells impairs genomic activity of ERα and is associated with endocrine resistance.

Estrogen receptor (ERα) is central in driving the development of hormone-dependent breast cancers. A major challenge in treating these cancers is to understand and overcome endocrine resistance. The Megakaryoblastic Leukemia 1 (MKL1, MRTFA) protein is a master regulator of actin dynamic and cellular motile functions, whose nuclear translocation favors epithelial-mesenchymal transition. We previously demonstrated that nuclear accumulation of MKL1 in estrogen-responsive breast cancer cell lines promotes hormonal escape. In the present study, we confirm through tissue microarray analysis that nuclear immunostaining of MKL1 is associated with endocrine resistance in a cohort of breast cancers and we decipher the underlining mechanisms using cell line models. We show through gene expression microarray analysis that the nuclear accumulation of MKL1 induces dedifferentiation leading to a mixed luminal/basal phenotype and suppresses estrogen-mediated control of gene expression. Chromatin immunoprecipitation of DNA coupled to high-throughput sequencing (ChIP-Seq) shows a profound reprogramming in ERα cistrome associated with a massive loss of ERα binding sites (ERBSs) generally associated with lower ERα-binding levels. Novel ERBSs appear to be associated with EGF and RAS signaling pathways. Collectively, these results highlight a major role of MKL1 in the loss of ERα transcriptional activity observed in certain cases of endocrine resistances, thereby contributing to breast tumor cells malignancy.

The Basal Level of Gene Expression Associated with Chromatin Loosening Shapes Waddington Landscapes and Controls Cell Differentiation.

The baseline level of transcription, which is variable and difficult to quantify, seriously complicates the normalization of comparative transcriptomic data, but its biological importance remains unappreciated. We show that this currently neglected ingredient is essential for controlling gene network multistability and therefore cellular differentiation. Basal expression is correlated to the degree of chromatin loosening measured by DNA accessibility and systematically leads to cellular dedifferentiation as assessed by transcriptomic signatures, irrespective of the molecular and cellular tools used. Modeling gene network motifs formally involved in developmental bifurcations reveals that the epigenetic landscapes of Waddington are restructured by the level of nonspecific expression, such that the attractors of progenitor and differentiated cells can be mutually exclusive. This mechanism is universal and holds beyond the particular nature of the genes involved, provided the multistable circuits are correctly described with autonomous basal expression. These results explain the relationships long established between gene expression noise, chromatin decondensation and cellular dedifferentiation, and highlight how heterochromatin maintenance is essential for preventing pathological cellular reprogramming, age-related diseases, and cancer.

A model of dynamic stability of H3K9me3 heterochromatin to explain the resistance to reprogramming of differentiated cells.

Despite their dynamic nature, certain chromatin marks must be maintained over the long term. This is particulary true for histone 3 lysine 9 (H3K9) trimethylation, that is involved in the maintenance of healthy differentiated cellular states by preventing inappropriate gene expression, and has been recently identified as the most efficient barrier to cellular reprogramming in nuclear transfer experiments. We propose that the capacity of the enzymes SUV39H1/2 to rebind to a minor fraction of their products, either directly or via HP1α/ß, contributes to the solidity of this mark through (i) a positive feedback involved in its establishment by the mutual enforcement of H3K9me3 and SUV39H1/2 and then (ii) a negative feedback sufficient to strongly stabilize H3K9me3 heterochromatin in post-mitotic cells by generating local enzyme concentrations capable of counteracting transient bursts of demethylation. This model does not require direct molecular interactions with adjacent nucleosomes and is favoured by a series of additional mechanisms including (i) the protection of chromatin-bound SUV39H1/2 from the turnovers of soluble proteins, which can explain the uncoupling between the cellular contents in SUV39H1 mRNA and protein; (ii) the cooperative dependence on the local density of the H3K9me3 of HP1α/ß-dependent heterochomatin condensation and, dispensably (iii) restricted enzyme exchanges with chromocenters confining the reactive bursts of SUV39H1/2 in heterochromatin. This mechanism illustrates how seemingly static epigenetic states can be firmly maintained by dynamic and reversible modifications.

Envisioning metastasis as a transdifferentiation phenomenon clarifies discordant results on cancer.

Cancer is generally conceived as a dedifferentiation process in which quiescent post-mitotic differentiated cells acquire stem-like properties and the capacity to proliferate. This view holds for the initial stages of carcinogenesis but is more questionable for advanced stages when the cells can transdifferentiate into the contractile phenotype associated to migration and metastasis. Singularly from this perspective, the hallmark of the most aggressive cancers would correspond to a genuine differentiation status, even if it is different from the original one. This seeming paradox could help reconciling discrepancies in the literature about the pro- or anti-tumoral functions of candidate molecules involved in cancer and whose actual effects depend on the tumoral grade. These ambiguities which are likely to concern a myriad of molecules and pathways, are illustrated here with the selected examples of chromatin epigenetics and myocardin-related transcription factors, using the human MCF10A and MCF7 breast cancer cells. Self-renewing stem like cells are characterized by a loose chromatin with low levels of the H3K9 trimetylation, but high levels of this mark can also appear in cancer cells acquiring a contractile-type differentiation state associated to metastasis. Similarly, the myocardin-related transcription factor MRTF-A is involved in metastasis and epithelial-mesenchymal transition, whereas this factor is naturally enriched in the quiescent cells which are precisely the most resistant to cancer: cardiomyocytes. These seeming paradoxes reflect the bistable epigenetic landscape of cancer in which dedifferentiated self-renewing and differentiated migrating states are incompatible at the single cell level, though coexisting at the population level.