Time-resolved single-cell analysis of Brca1 associated mammary tumourigenesis reveals aberrant differentiation of luminal progenitors

Abstract: It is unclear how genetic aberrations impact the state of nascent tumour cells and their microenvironment. BRCA1 driven triple negative breast cancer (TNBC) has been shown to arise from luminal progenitors yet little is known about how BRCA1 loss-of-function (LOF) and concomitant mutations affect the luminal progenitor cell state. Here we demonstrate how time-resolved single-cell profiling of genetically engineered mouse models before tumour formation can address this challenge. We found that perturbing Brca1/p53 in luminal progenitors induces aberrant alveolar differentiation pre-malignancy accompanied by pro-tumourigenic changes in the immune compartment. Unlike alveolar differentiation during gestation, this process is cell autonomous and characterised by the dysregulation of transcription factors driving alveologenesis. Based on our data we propose a model where Brca1/p53 LOF inadvertently promotes a differentiation program hardwired in luminal progenitors, highlighting the deterministic role of the cell-of-origin and offering a potential explanation for the tissue specificity of BRCA1 tumours

Time-resolved single-cell analysis of Brca1 associated mammary tumourigenesis reveals aberrant differentiation of luminal progenitors

Abstract: It is unclear how genetic aberrations impact the state of nascent tumour cells and their microenvironment. BRCA1 driven triple negative breast cancer (TNBC) has been shown to arise from luminal progenitors yet little is known about how BRCA1 loss-of-function (LOF) and concomitant mutations affect the luminal progenitor cell state. Here we demonstrate how time-resolved single-cell profiling of genetically engineered mouse models before tumour formation can address this challenge. We found that perturbing Brca1/p53 in luminal progenitors induces aberrant alveolar differentiation pre-malignancy accompanied by pro-tumourigenic changes in the immune compartment. Unlike alveolar differentiation during gestation, this process is cell autonomous and characterised by the dysregulation of transcription factors driving alveologenesis. Based on our data we propose a model where Brca1/p53 LOF inadvertently promotes a differentiation program hardwired in luminal progenitors, highlighting the deterministic role of the cell-of-origin and offering a potential explanation for the tissue specificity of BRCA1 tumours

Activation of the latent form of Transforming Growth Factor-beta occurs at the surface of human regulatory T cells

Regulatory T cells (Tregs) are a subset of CD4+ T cells specialized in the inhibition of immune responses. Tregs are required for the maintenance of self-tolerance, but an excess of Treg function may inhibit anti-tumor immunity. Human Tregs are difficult to study due to the lack of a specific marker. To circumvent that issue, we derived stable human Treg clones from cancerous and non-cancerous patients. Treg clones were defined on the basis of in vitro suppressive function and were characterized by a stable epigenetic mark not found in other types of T cells. To gain insight into the suppression mechanisms by which Tregs inhibit immune responses, we performed gene expression profiling. We compared transcriptomes of Treg and T helper (Th) clones after stimulation through the T cell receptor (TCR). The only feature we found to distinguish stimulated Treg clones from Th clones was a signature suggestive of autocrine Transforming Growth Factor-beta (TGF-b) signaling. We showed that although both Treg and Th clones produce the latent, inactive form of TGF-b, only Treg clones produce active TGF-b after TCR stimulation. Furthermore, we provided evidence that Treg-derived TGF-b has paracrine actions on neighboring T cells, and that this effect requires cell-cell contact. Finally, we demonstrated that the in vitro suppressive function of Tregs relies at least in part on the production of active TGF-b. TGF-b is initially produced as a latent, inactive form, in which the mature cytokine is associated to the so-called Latency-Associated Peptide (LAP), preventing binding of the cytokine to its receptor. A subsequent step of activation is required to release active TGF-b from the LAP. How Tregs activate latent TGF-b is presently unknown. We showed that stimulated Treg clones, but not Th clones, bear LAP at their surface. Searching for a LAP receptor on Tregs, we observed that transmembrane protein GARP is expressed in stimulated Treg clones, but not in Th clones. We also found that GARP binds LAP and mature TGF-b, i.e. latent TGF-b. Forced GARP expression induced by lentiviral transduction in Th cells is sufficient to induce the binding of latent TGF-b at the cell surface, confirming that GARP is a receptor for latent TGF-b. However, GARP-transduced Th cells do not produce active TGFb, indicating that GARP expression is not sufficient to induce the transformation of latent TGF-b into the active cytokine. In summary, our results show that human Tregs, like other types of T cells, produce latent TGF-b. Latent TGF-b localizes at the Treg surface through binding to GARP, a receptor that is expressed upon TCR stimulation in Tregs, but not in other T cells. Stimulated Tregs transform latent TGF-b into the active cytokine, which exerts paracrine actions that require cell-cell contact. We propose therefore that a unique functional feature of human Tregs, by comparison to other T cells, consists in the ability to activate latent TGF-b, and this occurs close to the cell surface. GARP may be implicated in the activation of latent TGF-b by Tregs, but it is not sufficient. Altogether, our findings open the way for the discovery of the mechanism by which Tregs activate latent TGF-b.(SBIM 3) -- UCL, 201

Characterization of stable human CD4+ T cell clones that constitutively express high levels of CD25 and FOXP3.

In humans, FOXP3 expression is stable and constitutively high in the CD4+CD25+ T cells that display suppressive function in vitro, but it is also transiently upregulated in activated CD4+CD25- T cells. It was recently shown that CpGs located in FOXP3 intron 1 are unmethylated in both murine and human CD4+CD25+ regulatory T cells, in contrast to what is observed in activated conventional T cells. We selected FOXP3hi clones derived from cancerous or non-cancerous patients on the basis of the unmethylated status of the intronic region of gene FOXP3. These human FOXP3hi clones produce no IL-2, IFNg, TNFa, IL-4, IL-5 or IL-10. They suppress the proliferation of activated T cells in vitro, and at least part of this suppressive function can be attributed to the reduction of growth factor availability in the co-culture. We analyzed the gene expression profiles of activated FOXP3hi or conventional CD4+ clones by Affymetrix microarray. Approximately half of the genes specifically up-regulated in activated FOXP3hi T clones are also upregulated by TGFbeta in conventional CD4+ T cell clones. Moreover, half of the genes upregulated as a result of activation in conventional CD4+ but not in FOXP3hi clones, can be downregulated by TGFbeta. It appears therefore that a significant proportion of the gene expression profile of activated human FOXP3hi clones corresponds to a TGFbeta signature. In addition, a “suppressed” T clone co-cultured with a FOXP3hi clone also shows a TGFbeta signature. We propose that activated human FOXP3hi T cells suppress the proliferation of neighbouring T cells via a combination of growth factor consumption and bioactive TGFbeta production. Interestingly, TGFbeta also acts in an autocrine manner on FOXP3hi cells, potentially participating to their inability to produce growth factors

Role of GARP in the activation of latent TGF-β1.

TGF-β1, 2 and 3 cytokines are involved in many cellular processes including cell proliferation, differentiation, migration and survival. Whereas TGF-β2 and 3 play important roles in embryonic development, TGF-β1 is mostly implicated in controlling immune responses after birth. The production of TGF-β1 is a tightly regulated process, occurring mostly at a post-translational level. Virtually all cells produce the latent, inactive form of TGF-β1. In latent TGF-β1, the mature TGF-β1 dimer is non-covalently associated to the Latency Associated Peptide, or LAP, which prevents binding to the TGF-β1 receptor. Activation of the cytokine implies release of mature TGF-β1 from LAP. Only a few cell types activate latent TGF-β1, via mechanisms that are cell type specific. Proteins such as integrins, proteases and thrombospondin-1 activate TGF-β1 in epithelial cells, fibroblasts and dendritic cells. More recently, the protein GARP was shown to be involved in TGF-β1 activation by regulatory T cells (Treg), a subset of CD4+ T lymphocytes specialized in suppression of immune responses. GARP is a transmembrane protein that binds latent-TGF-β1 and tethers it on the Treg surface. The role of GARP was studied mostly in Tregs, and this was recently reviewed in L. Sun, H. Jin and H. Li, Oncotarget, 2016, 7, 42826-42836. However, GARP is also expressed in non-immune cells. This review focuses on the roles of GARP in latent TGF-β1 activation by immune and non-immune cells

Comparison of stable human Treg and Th clones by transcriptional profiling

Objectives: Our long-term objective is to assess the contribution of regulatory T cells (Treg) in the immune suppressive environment that seems to prevail in human tumors, and notably in melanoma metastases. Because FOXP3 mRNA and protein are not specific markers of human Tregs, this question has remained difficult to address rigorously. We attempted to derive stable human Treg clones, and used them as a model to gain insight into human Treg biology. Methods : From cancerous and non-cancerous patients, we derived stable clones of CD4+ Treg, defined as clones that expressed high CD25 at rest, were anergic in vitro, and suppressed the proliferation of co-cultured CD4+ cells. We analyzed the methylation status of a conserved regulatory region in FOXP3 intron 1 by methyl specific qPCR on bisulfite-treated gDNA in a panel of Treg and T helper (Th) clones. We also compared Treg and Th clones by expression microarrays. Results : A conserved region of FOXP3 intron 1 was demethylated in all Treg clones, whereas it was methylated in non-regulatory Th and cytotoxic T cell (CTL) clones. In our panel of human clones, this stable epigenetic mark correlated better with suppressive activity than did FOXP3 mRNA or protein expression. We used expression microarrays to compare Treg and Th clones after activation, which is required for suppressive function. The transcriptional profile that is specific of activated Treg clones includes a TGF-b signature. Both activated Treg and Th clones produced the latent form of TGF-b. However, SMAD2 phosphorylation was observed after activation in the Treg but not in the Th clones, indicating that only activated Treg clones produced the bioactive form of TGF-b. A TGF-b signature was also displayed by a Th clone ''suppressed'' by a Treg clone. Conclusion : The hallmark of our panel of activated human Treg clones is to produce bioactive TGF-b which has autocrine actions on Tregs and can have paracrine actions on other T cells

Membrane protein GARP is a receptor for latent TGF-beta on the surface of activated human Treg.

Human regulatory T cell (Treg) and T helper (Th) clones secrete the latent form of transforming growth factor beta (TGF-beta), in which the mature TGF-beta protein is bound to the latency associated peptide (LAP), and is thereby prevented from binding to the TGF-beta receptor. We previously showed that upon T cell receptor (TCR) stimulation, human Treg clones but not Th clones produce active TGF-beta and bear LAP on their surface. Here, we show that latent TGF-beta, i.e. both LAP and mature TGF-beta, binds to GARP, a transmembrane protein containing leucine rich repeats which is present on the surface of stimulated Treg clones but not on Th clones. Membrane localization of latent TGF-beta mediated by binding to GARP may be necessary for the ability of Treg to activate TGF-beta upon TCR stimulation. However, it is not sufficient as lentiviral mediated expression of GARP in human Th cells induces binding of latent TGF-beta to the cell surface, but does not result in the production of active TGF-beta upon stimulation of these Th cells

Targeting immunosuppression by Tregs with monoclonal antibodies against GARP.

Reducing Treg function in cancer patients should augment antitumor immune responses. We recently uncovered a mechanism of immunosuppression by human Tregs that implies transmembrane protein GARP and production of active TGF-ß1. We obtained monoclonal antibodies that block this process and could thus serve as a novel approach for cancer immunotherapy

Role of glycolysis inhibition and poly(ADP-ribose) polymerase activation in necrotic-like cell death caused by ascorbate/menadione-induced oxidative stress in K562 human chronic myelogenous leukemic cells.

Among different features of cancer cells, two of them have retained our interest: their nearly universal glycolytic phenotype and their sensitivity towards an oxidative stress. Therefore, we took advantage of these features to develop an experimental approach by selectively exposing cancer cells to an oxidant insult induced by the combination of menadione (vitamin K(3)) and ascorbate (vitamin C). Ascorbate enhances the menadione redox cycling, increases the formation of reactive oxygen species and kills K562 cells as shown by more than 65% of LDH leakage after 24 hr of incubation. Since both lactate formation and ATP content are depressed by about 80% following ascorbate/menadione exposure, we suggest that the major intracellular event involved in such a cytotoxicity is related to the impairment of glycolysis. Indeed, NAD(+) is rapidly and severely depleted, a fact most probably related to a strong Poly(ADP-ribose) polymerase (PARP) activation, as shown by the high amount of poly-ADP-ribosylated proteins. The addition of N-acetylcysteine (NAC) restores most of the ATP content and the production of lactate as well. The PARP inhibitor dihydroxyisoquinoline (DiQ) was able to partially restore both parameters as well as cell death induced by ascorbate/menadione. These results suggest that the PARP activation induced by the oxidative stress is a major but not the only intracellular event involved in cell death by ascorbate/menadione. Due to the high energetic dependence of cancer cells on glycolysis, the impairment of such an essential pathway may explain the effectiveness of this combination to kill cancer cells

Oxidative stress by ascorbate/menadione association kills K562 human chronic myelogenous leukaemia cells and inhibits its tumour growth in nude mice.

The effect of oxidative stress induced by the ascorbate/menadione-redox association was examined in K562 cells, a human erythromyeloid leukaemia cell line. Our results show that ascorbate enhances menadione redox cycling, leading to the formation of intracellular reactive oxygen species (as shown by dihydrorhodamine 123 oxidation). The incubation of cells in the presence of both ascorbate/menadione and aminotriazole, a catalase inhibitor, resulted in a strong decrease of cell survival, reinforcing the role of H(2)O(2) as the main oxidizing agent killing K562 cells. This cell death was not caspase-3-dependent. Indeed, neither procaspase-3 and PARP were processed and only a weak cytochrome c release was observed. Moreover, we observed only 23% of cells with depolarized mitochondria. In ascorbate/menadione-treated cells, DNA fragmentation was observed without any sign of chromatin condensation (DAPI and TUNEL tests). The cell demise by ascorbate/menadione is consistent with a necrosis-like cell death confirmed by both cytometric profile of annexin-V/propidium iodide labeled cells and by light microscopy examination. Finally, we showed that a single i.p. administration of the association of ascorbate and menadione is able to inhibit the growth of K562 cells by about 60% (in both tumour size and volume) in an immune-deficient mice model. Taken together, these results reinforced our previous claims about a potential application of the ascorbate/menadione association in cancer therapy