An Early Neoplasia Index (ENI10), Based on Molecular Identity of CD10 Cells and Associated Stemness Biomarkers, is a Predictor of Patient Outcome in Many Cancers.

An accurate estimate of patient survival at diagnosis is critical to plan efficient therapeutic options. A simple and multiapplication tool is needed to move forward the precision medicine era. Taking advantage of the broad and high CD10 expression in stem and cancers cells, we evaluated the molecular identity of aggressive cancer cells. We used epithelial primary cells and developed a breast cancer stem cell­based progressive model. The superiority of the early-transformed isolated molecular index was evaluated by large-scale analysis in solid cancers. BMP2-driven cell transformation increases CD10 expression which preserves stemness properties. Our model identified a unique set of 159 genes enriched in G2­M cell-cycle phases and spindle assembly complex. Using samples predisposed to transformation, we confirmed the value of an early neoplasia index associated to CD10 (ENI10) to discriminate premalignant status of a human tissue. Using a stratified Cox model, a large-scale analysis (>10,000 samples, The Cancer Genome Atlas Pan-Cancer) validated a strong risk gradient (HRs reaching HR = 5.15; 95% confidence interval: 4.00­6.64) for high ENI10 levels. Through different databases, Cox regression model analyses highlighted an association between ENI10 and poor progression-free intervals for more than 50% of cancer subtypes tested, and the potential of ENI10 to predict drug efficacy. The ENI10 index constitutes a robust tool to detect pretransformed tissues and identify high-risk patients at diagnosis. Owing to its biological link with refractory cancer stem cells, the ENI10 index constitutes a unique way of identifying effective treatments to improve clinical care. SIGNIFICANCE: We identified a molecular signature called ENI10 which, owing to its biological link with stem cell properties, predicts patient outcome and drugs efficiency in breast and several other cancers. ENI10 should allow early and optimized clinical management of a broad number of cancers, regardless of the stage of tumor progression.

The differential role of the lipid raft-associated protein flotillin 2 for progression of myeloid leukemia.

Lipid raft-associated proteins play a vital role in membrane-mediated processes. The lipid microdomain-associated protein flotillin 2 (FLOT2), which has a scaffolding function, is involved in polarization, as well as in actin cytoskeletal organization of primitive and mature hematopoietic cells and has been associated with different malignancies. However, its involvement in myeloid leukemias is not well studied. Using murine transplantation models, we show here that the absence of FLOT2 from leukemia-initiating cells (LICs) altered the disease course of BCR-ABL1+ chronic myeloid leukemia (CML), but not of MLL-AF9-driven acute myeloid leukemia (AML). While FLOT2 was required for expression of the adhesion molecule CD44 on both CML- and AML-LIC, a defect in the cytoskeleton, cell polarity, and impaired homing ability of LIC was only observed in FLOT2-deficient BCR-ABL1+ compared with MLL-AF9+ cells. Downstream of CD44, BCR-ABL1 kinase-independent discrepancies were observed regarding expression, localization, and activity of cell division control protein 42 homolog (CDC42) between wild-type (WT) and FLOT2-deficient human CML and AML cells. Inhibition of CDC42 by ML141 impaired the homing of CML LIC and, thereby, CML progression. This suggested that alteration of both CD44 and CDC42 may be causative of impaired CML progression in the absence of FLOT2. In summary, our data suggest a FLOT2-CD44-CDC42 axis, which differentially regulates CML vs AML progression, with deficiency of FLOT2 impairing the development of CML.

A Human Bone Marrow 3D Model to Investigate the Dynamics and Interactions Between Resident Cells in Physiological or Tumoral Contexts.

The medullary niche is a complex ecosystem that is essential to maintain homeostasis for resident cells. Indeed, the bone marrow, which includes a complex extracellular matrix and various cell types, such as mesenchymal stem cells, osteoblasts, and endothelial cells, is deeply involved in hematopoietic stem cell regulation through direct cell-cell interactions, as well as cytokine production. To closely mimic this in vivo structure and conduct experiments reflecting the responses of the human bone marrow, several 3D models have been created based on biomaterials, relying primarily on primary stromal cells. Here, a protocol is described to obtain a minimal and standardized system that is easy to set up and provides features of bone marrow-like structure, which combines different cell populations including endothelial cells, and reflects the heterogeneity of in vivo bone marrow tissue. This 3D bone marrow-like structure-assembled using calcium phosphate-based particles and human cell lines, representative of the bone marrow microenvironment-allows the monitoring of a wide variety of biological processes by combining or replacing different primary cell populations within the system. The final 3D structures can then either be harvested for image analysis after fixation, paraffin-embedding, and histological/immunohistochemical staining for cell localization within the system, or dissociated to collect each cellular component for molecular or functional characterization.

De novo and cell line models of human mammary cell transformation reveal an essential role for Yb-1 in multiple stages of human breast cancer.

Breast cancer heterogeneity has made it challenging to identify mechanisms critical to the initial stages of their genesis in vivo. Here, we sought to interrogate the role of YB-1 in newly arising human breast cancers as well as in established cell lines. In a first series of experiments, we found that short-hairpin RNA-mediated knockdown of YB-1 in MDA-MB-231 cells blocked both their local tumour-forming and lung-colonising activity in immunodeficient mice. Conversely, upregulated expression of YB-1 enhanced the poor in vivo tumorigenicity of T47D cells. We then found that YB-1 knockdown also inhibits the initial generation in mice of invasive ductal carcinomas and ductal carcinomas in situ from freshly isolated human mammary cells transduced, respectively, with KRASG12D or myristoylated-AKT1. Interestingly, increased expression of HIF1α and G3BP1, two YB-1 translational targets and elements of a stress-adaptive programme, mirrored the levels of YB-1 in both transformed primary and established MDA-MB-231 breast cancer cells.

A minimal standardized human bone marrow microphysiological system to assess resident cell behavior during normal and pathological processes.

Bone marrow is a complex and dynamic microenvironment that provides essential cues to resident cells. We developed a standardized three-dimensional (3D) model to decipher mechanisms that control human cells during hematological and non-hematological processes. Our simple 3D-model is constituted of a biphasic calcium phosphate-based scaffold and human cell lines to ensure a high reproducibility. We obtained a minimal well-organized bone marrow-like structure in which various cell types and secreted extracellular matrix can be observed and characterized by in situ imaging or following viable cell retrieval. The complexity of the system can be increased and customized, with each cellular component being independently modulated according to the issue investigated. Introduction of pathological elements in this 3D-system accurately reproduced changes observed in patient bone marrow. Hence, we have developed a handy and flexible standardized microphysiological system that mimics human bone marrow, allowing histological analysis and functional assays on collected cells.

Altered BMP2/4 Signaling in Stem Cells and Their Niche: Different Cancers but Similar Mechanisms, the Example of Myeloid Leukemia and Breast Cancer.

Understanding mechanisms of cancer development is mandatory for disease prevention and management. In healthy tissue, the microenvironment or niche governs stem cell fate by regulating the availability of soluble molecules, cell-cell contacts, cell-matrix interactions, and physical constraints. Gaining insight into the biology of the stem cell microenvironment is of utmost importance, since it plays a role at all stages of tumorigenesis, from (stem) cell transformation to tumor escape. In this context, BMPs (Bone Morphogenetic Proteins), are key mediators of stem cell regulation in both embryonic and adult organs such as hematopoietic, neural and epithelial tissues. BMPs directly regulate the niche and stem cells residing within. Among them, BMP2 and BMP4 emerged as master regulators of normal and tumorigenic processes. Recently, a number of studies unraveled important mechanisms that sustain cell transformation related to dysregulations of the BMP pathway in stem cells and their niche (including exposure to pollutants such as bisphenols). Furthermore, a direct link between BMP2/BMP4 binding to BMP type 1 receptors and the emergence and expansion of cancer stem cells was unveiled. In addition, a chronic exposure of normal stem cells to abnormal BMP signals contributes to the emergence of cancer stem cells, or to disease progression independently of the initial transforming event. In this review, we will illustrate how the regulation of stem cells and their microenvironment becomes dysfunctional in cancer via the hijacking of BMP signaling with main examples in myeloid leukemia and breast cancers.

The quiescent fraction of chronic myeloid leukemic stem cells depends on BMPR1B, Stat3 and BMP4-niche signals to persist in patients in remission.

Chronic myelogenous leukemia arises from the transformation of hematopoietic stem cells by the BCR-ABL oncogene. Though transformed cells are predominantly BCR-ABL-dependent and sensitive to tyrosine kinase inhibitor treatment, some BMPR1B+ leukemic stem cells are treatment-insensitive and rely, among others, on the bone morphogenetic protein (BMP) pathway for their survival via a BMP4 autocrine loop. Here, we further studied the involvement of BMP signaling in favoring residual leukemic stem cell persistence in the bone marrow of patients having achieved remission under treatment. We demonstrate by single-cell RNA-Seq analysis that a sub-fraction of surviving BMPR1B+ leukemic stem cells are co-enriched in BMP signaling, quiescence and stem cell signatures, without modulation of the canonical BMP target genes, but enrichment in actors of the Jak2/Stat3 signaling pathway. Indeed, based on a new model of persisting CD34+CD38- leukemic stem cells, we show that BMPR1B+ cells display co-activated Smad1/5/8 and Stat3 pathways. Interestingly, we reveal that only the BMPR1B+ cells adhering to stromal cells display a quiescent status. Surprisingly, this quiescence is induced by treatment, while non-adherent BMPR1B+ cells treated with tyrosine kinase inhibitors continued to proliferate. The subsequent targeting of BMPR1B and Jak2 pathways decreased quiescent leukemic stem cells by promoting their cell cycle re-entry and differentiation. Moreover, while Jak2-inhibitors alone increased BMP4 production by mesenchymal cells, the addition of the newly described BMPR1B inhibitor (E6201) impaired BMP4-mediated production by stromal cells. Altogether, our data demonstrate that targeting both BMPR1B and Jak2/Stat3 efficiently impacts persisting and dormant leukemic stem cells hidden in their bone marrow microenvironment.

MYC-induced human acute myeloid leukemia requires a continuing IL-3/GM-CSF costimulus.

Hematopoietic clones with leukemogenic mutations arise in healthy people as they age, but progression to acute myeloid leukemia (AML) is rare. Recent evidence suggests that the microenvironment may play an important role in modulating human AML population dynamics. To investigate this concept further, we examined the combined and separate effects of an oncogene (c-MYC) and exposure to interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) on the experimental genesis of a human AML in xenografted immunodeficient mice. Initial experiments showed that normal human CD34+ blood cells transduced with a lentiviral MYC vector and then transplanted into immunodeficient mice produced a hierarchically organized, rapidly fatal, and serially transplantable blast population, phenotypically and transcriptionally similar to human AML cells, but only in mice producing IL-3, GM-CSF, and SCF transgenically or in regular mice in which the cells were exposed to IL-3 or GM-CSF delivered using a cotransduction strategy. In their absence, the MYC+ human cells produced a normal repertoire of lymphoid and myeloid progeny in transplanted mice for many months, but, on transfer to secondary mice producing the human cytokines, the MYC+ cells rapidly generated AML. Indistinguishable diseases were also obtained efficiently from both primitive (CD34+CD38-) and late granulocyte-macrophage progenitor (GMP) cells. These findings underscore the critical role that these cytokines can play in activating a malignant state in normally differentiating human hematopoietic cells in which MYC expression has been deregulated. They also introduce a robust experimental model of human leukemogenesis to further elucidate key mechanisms involved and test strategies to suppress them.

Single-cell analysis of autophagy activity in normal and de novo transformed human mammary cells.

Assessment of autophagy activity has historically been limited to investigations of fixed tissue or bulk cell populations. To address questions of heterogeneity and relate measurements to functional properties of viable cells isolated from primary tissue, we created a lentiviral (RFP-GFP-MAP1LC3B) vector that allows the autophagosome and autolysosome content of transduced cells to be monitored at the single-cell level. Use of this strategy to analyze purified subsets of normal human mammary cells showed that both the luminal progenitor-containing (LP) subset and the basal cells (BCs) display highly variable but overall similar autophagic flux activity despite differences suggested by measurements of the proteins responsible (i.e., LC3B, ATG7 and BECLIN1) in bulk lysates. Autophagosome content was also highly variable in the clonogenic cells within both the LPs and BCs, but the proliferative response of the BCs was more sensitive to autophagy inhibition. In addition, use of this vector showed cells with the lowest autophagosome content elicited the fastest tumor growth in 2 different models of human mammary tumorigenesis. These results illustrate the utility of this vector to define differences in the autophagy properties of individual cells in primary tissue and couple these with their responses to proliferative and oncogenic stimuli.

Targeting BMP signaling in the bone marrow microenvironment of myeloid leukemia.

The bone morphogenetic protein (BMP) pathway regulates the fate and proliferation of normal hematopoietic stem cells (HSC) as well as interactions with their niche. While BMP2 and BMP4 promote HSC differentiation, only BMP4 maintains HSC pool and favors interactions with their niche. In myeloid leukemia, we have identified intrinsic and extrinsic dysregulations of the BMP pathway in Chronic Myeloid Leukemia (CML) and Acute Myeloid leukemia (AML) responsible for leukemic stem cells (LSC) survival. In AML, BMP pathway alterations sustain and promote resistant immature-like leukemic cells by activating a new signaling cascade. Binding of BMP4 to BMPR1A leads to ΔNp73 expression, which in turn induces NANOG, altogether associated with a poor patient's prognosis. Despite efficient targeted therapies, like Tyrosine Kinase Inhibitors (TKI) in CML, many patients retain LSCs. Our laboratory demonstrated that the BMP pathway sustains a permanent pool of LSCs expressing high levels of BMPR1B receptor, that evolve upon treatment to progressively implement a BMP4 autocrine loop, leading to TKI-resistant cells. Single cell RNA-Seq analysis of TKI-persisting LSCs showed a co-enrichment of BMP with Jak2-signaling, quiescence and stem cell (SC) signatures. Using a new model of persisting LSCs, we recently demonstrated that BMPR1B+ cells display co-activated Smad1/5/8 and Stat3 pathways and could be targeted by blocking BMPR1B/Jak2 signal. Lastly, a specific BMPR1B inhibitor impaired BMP4-mediated LSC protection against TKIs. Altogether, data based on various studies including ours, indicate that BMP targeting could eliminate leukemic cells within a protective bone marrow microenvironment to efficiently impact residual resistance or persistence of LSCs in myeloid leukemia.

Bone marrow niche-derived extracellular matrix-degrading enzymes influence the progression of B-cell acute lymphoblastic leukemia.

Specific and reciprocal interactions with the bone marrow microenvironment (BMM) govern the course of hematological malignancies. Matrix metalloproteinase-9 (MMP-9), secreted by leukemia cells, facilitates tumor progression via remodeling of the extracellular matrix (ECM) of the BMM. Hypothesizing that leukemias may instruct the BMM to degrade the ECM, we show, that MMP-9-deficiency in the BMM prolongs survival of mice with BCR-ABL1-induced B-cell acute lymphoblastic leukemia (B-ALL) compared with controls and reduces leukemia-initiating cells. MMP-9-deficiency in the BMM leads to reduced degradation of proteins of the ECM and reduced invasion of B-ALL. Using various in vivo and in vitro assays, as well as recipient mice deficient for the receptor for tumor necrosis factor (TNF) α (TNFR1) we demonstrate that B-ALL cells induce MMP-9-expression in mesenchymal stem cells (MSC) and possibly other cells of the BMM via a release of TNFα. MMP-9-expression in MSC is mediated by activation of nuclear factor kappa B (NF-κB) downstream of TNFR1. Consistently, knockdown of TNF-α in B-ALL-initiating cells or pharmacological inhibition of MMP-9 led to significant prolongation of survival in mice with B-ALL. In summary, leukemia cell-derived Tnfα induced MMP-9-expression by the BMM promoting B-ALL progression. Inhibition of MMP-9 may act as an adjunct to existing therapies.

Initiation of human mammary cell tumorigenesis by mutant KRAS requires YAP inactivation.

High YAP activity is associated with poor prognosis human breast cancers, but its role during the initial stage of mammary cell transformation is unknown. To address this question, we designed experiments that exploit the ability of KRASG12D-transduced subsets of freshly isolated normal human mammary cells to form invasive tumors rapidly and efficiently when transplanted into immunodeficient mice. Initial examination of the newly developing tumors thus generated revealed a consistent marked loss of nuclear YAP, independent of the initial primary human mammary cell type transduced. Conversely, co-transduction of the same subsets of primary human mammary cells with KRASG12D plus the constitutively active YAPS127A prevented tumor formation. These findings contrast with the enhanced display of transformed properties obtained when the immortalized, but non-tumorigenic MCF10A cells are transduced just with YAPS127A. In addition, we show that YAPS127A-transduction of the human MDA-MB-231 breast cancer cell line (that carry a similar KRAS mutation) enhances their metastatic activity in vivo. We also discover that the KRASG12D-induced early loss of YAP in primary human mammary cells is associated with their induced secretion of amphiregulin. Collectively, these findings suggest that YAP can differentially affect the acquisition of malignant properties by human mammary cells at different stages of their transformation.

Transcriptional regulation of normal human mammary cell heterogeneity and its perturbation in breast cancer.

The mammary gland in adult women consists of biologically distinct cell types that differ in their surface phenotypes. Isolation and molecular characterization of these subpopulations of mammary cells have provided extensive insights into their different transcriptional programs and regulation. This information is now serving as a baseline for interpreting the heterogeneous features of human breast cancers. Examination of breast cancer mutational profiles further indicates that most have undergone a complex evolutionary process even before being detected. The consequent intra-tumoral as well as inter-tumoral heterogeneity of these cancers thus poses major challenges to deriving information from early and hence likely pervasive changes in potential therapeutic interest. Recently described reproducible and efficient methods for generating human breast cancers de novo in immunodeficient mice transplanted with genetically altered primary cells now offer a promising alternative to investigate initial stages of human breast cancer development. In this review, we summarize current knowledge about key transcriptional regulatory processes operative in these partially characterized subpopulations of normal human mammary cells and effects of disrupting these processes in experimentally produced human breast cancers.

A new signaling cascade linking BMP4, BMPR1A, ΔNp73 and NANOG impacts on stem-like human cell properties and patient outcome.

In a significant number of cases cancer therapy is followed by a resurgence of more aggressive tumors derived from immature cells. One example is acute myeloid leukemia (AML), where an accumulation of immature cells is responsible for relapse following treatment. We previously demonstrated in chronic myeloid leukemia that the bone morphogenetic proteins (BMP) pathway is involved in stem cell fate and contributes to transformation, expansion, and persistence of leukemic stem cells. Here, we have identified intrinsic and extrinsic dysregulations of the BMP pathway in AML patients at diagnosis. BMP2 and BMP4 protein concentrations are elevated within patients' bone marrow with a BMP4-dominant availability. This overproduction likely depends on the bone marrow microenvironment, since MNCs do not overexpress BMP4 transcripts. Intrinsically, the receptor BMPR1A transcript is increased in leukemic samples with more cells presenting this receptor at the membrane. This high expression of BMPR1A is further increased upon BMP4 exposure, specifically in AML cells. Downstream analysis demonstrated that BMP4 controls the expression of the survival factor ΔNp73 through its binding to BMPR1A. At the functional level, this results in the direct induction of NANOG expression and an increase of stem-like features in leukemic cells, as shown by ALDH and functional assays. In addition, we identified for the first time a strong correlation between ΔNp73, BMPR1A and NANOG expression with patient outcome. These results highlight a new signaling cascade initiated by tumor environment alterations leading to stem-cell features and poor patients' outcome.

The BMP pathway: A unique tool to decode the origin and progression of leukemia.

The microenvironment (niche) governs the fate of stem cells (SCs) by balancing self-renewal and differentiation. Increasing evidence indicates that the tumor niche plays an active role in cancer, but its important properties for tumor initiation progression and resistance remain to be identified. Clinical data show that leukemic stem cell (LSC) survival is responsible for disease persistence and drug resistance, probably due to their sustained interactions with the tumor niche. Bone morphogenetic protein (BMP) signaling is a key pathway controlling stem cells and their niche. BMP2 and BMP4 are important in both the normal and the cancer context. Several studies have revealed profound alterations of the BMP signaling in cancer SCs, with major deregulations of the BMP receptors and their downstream signaling elements. This was illustrated in the hematopoietic system by pioneer studies in chronic myelogenous leukemia that may now be expanded to acute myeloid leukemia and lymphoid leukemia, as reviewed here. At diagnosis, cells from the leukemic microenvironment are the major providers of soluble BMPs. Conversely, LSCs display altered receptors and downstream BMP signaling elements accompanied by altered functional responses to BMPs. These studies reveal the role of BMPs in tumor initiation, in addition to their known effects in later stages of transformation and progression. They also reveal the importance of BMPs in fueling cell transformation and expansion by overamplifying a natural SC response. This mechanism may explain the survival of LSCs independently of the initial oncogenic event and therefore may be involved in resistance processes.

Barcoding reveals complex clonal dynamics of de novo transformed human mammary cells.

Most human breast cancers have diversified genomically and biologically by the time they become clinically evident. Early events involved in their genesis and the cellular context in which these events occur have thus been difficult to characterize. Here we present the first formal evidence of the shared and independent ability of basal cells and luminal progenitors, isolated from normal human mammary tissue and transduced with a single oncogene (KRAS(G12D)), to produce serially transplantable, polyclonal, invasive ductal carcinomas within 8 weeks of being introduced either subrenally or subcutaneously into immunodeficient mice. DNA barcoding of the initial cells revealed a dramatic change in the numbers and sizes of clones generated from them within 2 weeks, and the first appearance of many 'new' clones in tumours passaged into secondary recipients. Both primary and secondary tumours were phenotypically heterogeneous and primary tumours were categorized transcriptionally as 'normal-like'. This system challenges previous concepts that carcinogenesis in normal human epithelia is necessarily a slow process requiring the acquisition of multiple driver mutations. It also presents the first description of initial events that accompany the genesis and evolution of malignant human mammary cell populations, thereby contributing new understanding of the rapidity with which heterogeneity in their properties can develop.

Inhibition of autophagy as a new means of improving chemotherapy efficiency in high-LC3B triple-negative breast cancers.

The triple-negative breast cancer (TN BC) subtype is the most aggressive form of invasive BC. Despite intensive efforts to improve BC treatments, patients with TN BC continue to exhibit poor survival, with half developing resistance to chemotherapy. Here we identify autophagy as a key mechanism in the progression and chemoresistance of a subset of TN tumors. We demonstrate that LC3B, a protein involved in autophagosome formation, is a reliable marker of poor prognosis in TN BC, validating this prognostic value at both the mRNA and protein levels in several independent cohorts. We also show that LC3B has no prognostic value for other BC subtypes (Luminal or HER2 BC), thus revealing a specific impact of autophagy on TN tumors. Autophagy is essential for the proliferative and invasive properties in 3D of TN BC cells characterized by high LC3B levels. Interestingly, the activity of the transcriptional co-activator YAP1 (Yes-associated protein 1) is regulated by the autophagy process and we identify YAP1 as a new actor in the autophagy-dependent proliferative and invasive properties of high-LC3B TN BC. Finally, inhibiting autophagy by silencing ATG5 or ATG7 significantly impaired high-LC3B TN tumor growth in vivo. Moreover, using a patient-derived TN tumor transplanted into mice, we show that an autophagy inhibitor, chloroquine, potentiates the effects of chemotherapeutic agents. Overall, our data identify LC3B as a new prognostic marker for TN BC and the inhibition of autophagy as a promising therapeutic strategy for TN BC patients.

Repression of interferon-α stimulated genes expression by Kaposi's sarcoma-associated herpesvirus K-bZIP protein.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a transcriptional repressor, K-bZIP. We previously demonstrated that K-bZIP suppresses interferon (IFN)-ß expression. Here, we provide evidence that K-bZIP affects IFN signaling, resulting in impaired IFN-stimulated genes expression. Inhibition by K-bZIP is independent of the phosphorylation of signal transducers and activators of transcription proteins or the binding of the IFN-stimulated gene factor 3 to the IFN-stimulated responsive element. Relative to the wild-type protein, K-bZIPK158R mutant exhibited reduced inhibitory activity, indicating that sumoylation on K158 is likely important for this effect. A new sumoylation site present within a newly identified K-bZIP variant and absent from the fully spliced K-bZIP was also identified. Furthermore, K-bZIP is acetylated on lysine residues and CBP enhances K-bZIP acetylation. Combined with its ability to negatively modulate IFN-ß production and inhibit type I IFN signaling, K-bZIP represents a viral immunomodulator that contributes to curtail immune defense mechanisms and favor KSHV's persistence.

Oxidative stress promotes myofibroblast differentiation and tumour spreading.

JunD regulates genes involved in antioxidant defence. We took advantage of the chronic oxidative stress resulting from junD deletion to examine the role of reactive oxygen species (ROS) in tumour development. In a model of mammary carcinogenesis, junD inactivation increased tumour incidence and revealed an associated reactive stroma. junD-inactivation in the stroma was sufficient to shorten tumour-free survival rate and enhance metastatic spread. ROS promoted conversion of fibroblasts into highly migrating myofibroblasts through accumulation of the hypoxia-inducible factor (HIF)-1alpha transcription factor and the CXCL12 chemokine. Accordingly, treatment with an antioxidant reduced the levels of HIF and CXCL12 and numerous myofibroblast features. CXCL12 accumulated in the stroma of HER2-human breast adenocarcinomas. Moreover, HER2 tumours exhibited a high proportion of myofibroblasts, which was significantly correlated to nodal metastases. Interestingly, this subset of tumours exhibited a significant nuclear exclusion of JunD and revealed an associated oxido-reduction signature, further demonstrating the relevance of our findings in human cancers. Collectively, our data uncover a new mechanism by which oxidative stress increases the migratory properties of stromal fibroblasts, which in turn potentiate tumour dissemination.