Macrophage depletion overcomes human hematopoietic cell engraftment failure in zebrafish embryo.

Zebrafish is widely adopted as a grafting model for studying human development and diseases. Current zebrafish xenotransplantations are performed using embryo recipients, as the adaptive immune system, responsible for host versus graft rejection, only reaches maturity at juvenile stage. However, transplanted primary human hematopoietic stem/progenitor cells (HSC) rapidly disappear even in zebrafish embryos, suggesting that another barrier to transplantation exists before the onset of adaptive immunity. Here, using a labelled macrophage zebrafish line, we demonstrated that engraftment of human HSC induces a massive recruitment of macrophages which rapidly phagocyte transplanted cells. Macrophages depletion, by chemical or pharmacological treatments, significantly improved the uptake and survival of transplanted cells, demonstrating the crucial implication of these innate immune cells for the successful engraftment of human cells in zebrafish. Beyond identifying the reasons for human hematopoietic cell engraftment failure, this work images the fate of human cells in real time over several days in macrophage-depleted zebrafish embryos.

MAGT1 Deficiency Dysregulates Platelet Cation Homeostasis and Accelerates Arterial Thrombosis and Ischemic Stroke in Mice.

BACKGROUND: MAGT1 (magnesium transporter 1) is a subunit of the oligosaccharide protein complex with thiol-disulfide oxidoreductase activity, supporting the process of N-glycosylation. MAGT1 deficiency was detected in human patients with X-linked immunodeficiency with magnesium defect syndrome and congenital disorders of glycosylation, resulting in decreased cation responses in lymphocytes, thereby inhibiting the immune response against viral infections. Curative hematopoietic stem cell transplantation of patients with X-linked immunodeficiency with magnesium defect causes fatal bleeding and thrombotic complications. METHODS: We studied the role of MAGT1 deficiency in platelet function in relation to arterial thrombosis and hemostasis using several in vitro experimental settings and in vivo models of arterial thrombosis and transient middle cerebral artery occlusion model of ischemic stroke. RESULTS: MAGT1-deficient mice (Magt1-/y) displayed accelerated occlusive arterial thrombus formation in vivo, a shortened bleeding time, and profound brain damage upon focal cerebral ischemia. These defects resulted in increased calcium influx and enhanced second wave mediator release, which further reinforced platelet reactivity and aggregation responses. Supplementation of MgCl2 or pharmacological blockade of TRPC6 (transient receptor potential cation channel, subfamily C, member 6) channel, but not inhibition of store-operated calcium entry, normalized the aggregation responses of Magt1-/y platelets to the control level. GP (glycoprotein) VI activation of Magt1-/y platelets resulted in hyperphosphorylation of Syk (spleen tyrosine kinase), LAT (linker for activation of T cells), and PLC (phospholipase C) γ2, whereas the inhibitory loop regulated by PKC (protein kinase C) was impaired. A hyperaggregation response to the GPVI agonist was confirmed in human platelets isolated from a MAGT1-deficient (X-linked immunodeficiency with magnesium defect) patient. Haploinsufficiency of TRPC6 in Magt1-/y mice could normalize GPVI signaling, platelet aggregation, and thrombus formation in vivo. CONCLUSIONS: These results suggest that MAGT1 and TRPC6 are functionally linked. Therefore, deficiency or impaired functionality of MAGT1 could be a potential risk factor for arterial thrombosis and stroke.

General transcription factor TAF4 antagonizes epigenetic silencing by Polycomb to maintain intestine stem cell functions.

Taf4 (TATA-box binding protein-associated factor 4) is a subunit of the general transcription factor TFIID, a component of the RNA polymerase II pre-initiation complex that interacts with tissue-specific transcription factors to regulate gene expression. Properly regulated gene expression is particularly important in the intestinal epithelium that is constantly renewed from stem cells. Tissue-specific inactivation of Taf4 in murine intestinal epithelium during embryogenesis compromised gut morphogenesis and the emergence of adult-type stem cells. In adults, Taf4 loss impacted the stem cell compartment and associated Paneth cells in the stem cell niche, epithelial turnover and differentiation of mature cells, thus exacerbating the response to inflammatory challenge. Taf4 inactivation ex vivo in enteroids prevented budding formation and maintenance and caused broad chromatin remodeling and a strong reduction in the numbers of stem and progenitor cells with a concomitant increase in an undifferentiated cell population that displayed high activity of the Ezh2 and Suz12 components of Polycomb Repressive Complex 2 (PRC2). Treatment of Taf4-mutant enteroids with a specific Ezh2 inhibitor restored buddings, cell proliferation and the stem/progenitor compartment. Taf4 loss also led to increased PRC2 activity in cells of adult crypts associated with modification of the immune/inflammatory microenvironment that potentiated Apc-driven tumorigenesis. Our results reveal a novel function of Taf4 in antagonizing PRC2-mediated repression of the stem cell gene expression program to assure normal development, homeostasis, and immune-microenvironment of the intestinal epithelium.

The Appendix Orchestrates T-Cell Mediated Immunosurveillance in Colitis-Associated Cancer.

BACKGROUND & AIMS: Although appendectomy may reduce colorectal inflammation in patients with ulcerative colitis (UC), this surgical procedure has been suggested to be associated with an increased risk of colitis-associated cancer (CAC). Our aim was to explore the mechanism underlying the appendectomy-associated increased risk of CAC. METHODS: Five-week-old male BALB/c mice underwent appendectomy, appendicitis induction, or sham laparotomy. They were then exposed to azoxymethane/dextran sodium sulfate (AOM/DSS) to induce CAC. Mice were killed 12 weeks later, and colons were taken for pathological analysis and immunohistochemistry (CD3 and CD8 staining). Human colonic tumors from 21 patients with UC who underwent surgical resection for CAC were immunophenotyped and stratified according to appendectomy status. RESULTS: Whereas appendectomy significantly reduced colitis severity and increased CAC number, appendicitis induction without appendectomy led to opposite results. Intratumor CD3+ and CD8+ T-cell densities were lower after appendectomy and higher after appendicitis induction compared with the sham laparotomy group. Blocking lymphocyte trafficking to the colon with the anti-α4ß7 integrin antibody or a sphingosine-1-phosphate receptor agonist suppressed the inducing effect of the appendectomy on tumors' number and on CD3+/CD8+ intratumoral density. CD8+ or CD3+ T cells isolated from inflammatory neo-appendix and intravenously injected into AOM/DSS-treated recipient mice increased CD3+/CD8+ T-cell tumor infiltration and decreased tumor number. In UC patients with a history of appendectomy, intratumor CD3+ and CD8+ T-cell densities were decreased compared with UC patients without history of appendectomy. CONCLUSIONS: In UC, appendectomy could suppress a major site of T-cell priming, resulting in a less efficient CAC immunosurveillance.

Regulation of the THRA gene, encoding the thyroid hormone nuclear receptor TRα1, in intestinal lesions.

The THRA gene, encoding the thyroid hormone nuclear receptor TRα1, is expressed in an increasing gradient at the bottom of intestinal crypts, overlapping with high Wnt and Notch activities. Importantly, THRA is upregulated in colorectal cancers, particularly in the high-Wnt molecular subtype. The basis of this specific and/or altered expression pattern has remained unknown. To define the mechanisms controlling THRA transcription and TRα1 expression, we used multiple in vitro and ex vivo approaches. Promoter analysis demonstrated that transcription factors important for crypt homeostasis and altered in colorectal cancers, such as transcription factor 7-like 2 (TCF7L2; Wnt pathway), recombining binding protein suppressor of hairless (RBPJ; Notch pathway), and homeobox protein CDX2 (epithelial cell identity), modulate THRA activity. Specifically, although TCF7L2 and CDX2 stimulated THRA, RBPJ induced its repression. In-depth analysis of the Wnt-dependent increase showed direct regulation of the THRA promoter in cells and of TRα1 expression in murine enteroids. Given our previous results on the control of the Wnt pathway by TRα1, our new results unveil a complex regulatory loop and synergy between these endocrine and epithelial-cell-intrinsic signals. Our work describes, for the first time, the regulation of the THRA gene in specific cell and tumor contexts.

Concurrent CDX2 cis-deregulation and UBTF::ATXN7L3 fusion define a novel high-risk subtype of B-cell ALL.

Oncogenic alterations underlying B-cell acute lymphoblastic leukemia (B-ALL) in adults remain incompletely elucidated. To uncover novel oncogenic drivers, we performed RNA sequencing and whole-genome analyses in a large cohort of unresolved B-ALL. We identified a novel subtype characterized by a distinct gene expression signature and the unique association of 2 genomic microdeletions. The 17q21.31 microdeletion resulted in a UBTF::ATXN7L3 fusion transcript encoding a chimeric protein. The 13q12.2 deletion resulted in monoallelic ectopic expression of the homeobox transcription factor CDX2, located 138 kb in cis from the deletion. Using 4C-sequencing and CRISPR interference experiments, we elucidated the mechanism of CDX2 cis-deregulation, involving PAN3 enhancer hijacking. CDX2/UBTF ALL (n = 26) harbored a distinct pattern of additional alterations including 1q gain and CXCR4 activating mutations. Within adult patients with Ph- B-ALL enrolled in GRAALL trials, patients with CDX2/UBTF ALL (n = 17/723, 2.4%) were young (median age, 31 years) and dramatically enriched in females (male/female ratio, 0.2, P = .002). They commonly presented with a pro-B phenotype ALL and moderate blast cell infiltration. They had poor response to treatment including a higher risk of failure to first induction course (19% vs 3%, P = .017) and higher post-induction minimal residual disease (MRD) levels (MRD ≥ 10-4, 93% vs 46%, P < .001). This early resistance to treatment translated into a significantly higher cumulative incidence of relapse (75.0% vs 32.4%, P = .004) in univariate and multivariate analyses. In conclusion, we discovered a novel B-ALL entity defined by the unique combination of CDX2 cis-deregulation and UBTF::ATXN7L3 fusion, representing a high-risk disease in young adults.

CDX2 controls genes involved in the metabolism of 5-fluorouracil and is associated with reduced efficacy of chemotherapy in colorectal cancer.

Most patients affected with colorectal cancers (CRC) are treated with 5-fluorouracil (5-FU)-based chemotherapy but its efficacy is often hampered by resistance mechanisms linked to tumor heterogeneity. A better understanding of the molecular determinants involved in chemoresistance is critical for precision medicine and therapeutic progress. Caudal type homeobox 2 (CDX2) is a master regulator of intestinal identity and acts as tumor suppressor in the colon. Here, using a translational approach, we examined the role of CDX2 in CRC chemoresistance. Unexpectedly, we discovered that the prognosis value of CDX2 for disease-free survival of patients affected with CRC is lost upon chemotherapy and that CDX2 expression enhances resistance of colon cancer cells towards 5-FU. At the molecular level, we found that CDX2 expression correlates with higher levels of genes regulating the bioavailability of 5-FU through efflux (ABCC11) and catabolism (DPYD) in patients affected with CRC and CRC cell lines. We further showed that CDX2 directly regulates the expression of ABCC11 and that the inhibition of ABCC11 improves 5-FU-sensitivity of CDX2-expressing colon cancer cells. Thus, this study illustrates how biological functions are hijacked in CRC cells and reveals the therapeutic interest of CDX2/ABCC11/DPYD to improve systemic chemotherapy in CRC.

Mesalazine initiates an anti-oncogenic ß-catenin / MUCDHL negative feed-back loop in colon cancer cells by cell-specific mechanisms.

Chronic inflammation associated with intestinal architecture and barrier disruption puts patients with inflammatory bowel disease (IBD) at increased risk of developing colorectal cancer (CRC). Widely used to reduce flares of intestinal inflammation, 5-aminosalicylic acid derivatives (5-ASAs) such as mesalazine appear to also exert more direct mucosal healing and chemopreventive activities against CRC. The mechanisms underlying these activities are poorly understood and may involve the up-regulation of the cadherin-related gene MUCDHL (CDHR5). This atypical cadherin is emerging as a new actor of intestinal homeostasis and opposes colon tumorigenesis. Here, we showed that mesalazine increase mRNA levels of MUCDHL and of other genes involved in the intestinal barrier function in most intestinal cell lines. In addition, using gain / loss of function experiments (agonists, plasmid or siRNAs transfections), luciferase reporter genes and chromatin immunoprecipitation, we thoroughly investigated the molecular mechanisms triggered by mesalazine that lead to the up-regulation of MUCDHL expression. We found that basal transcription of MUCDHL in different CRC cell lines is regulated positively by CDX2 and negatively by ß-catenin through a negative feed-back loop. However, mesalazine-stimulation of MUCDHL transcription is controlled by cell-specific mechanisms, involving either enhanced activation of CDX2 and PPAR-γ or repression of the ß-catenin inhibitory effect. This work highlights the importance of the cellular and molecular context in the activity of mesalazine and suggests that its efficacy against CRC depends on the genetic alterations of transformed cells.

A Core Response to the CDX2 Homeoprotein During Development and in Pathologies.

Whether a gene involved in distinct tissue or cell functions exerts a core of common molecular activities is a relevant topic in evolutionary, developmental, and pathological perspectives. Here, we addressed this question by focusing on the transcription factor and regulator of chromatin accessibility encoded by the Cdx2 homeobox gene that plays important functions during embryonic development and in adult diseases. By integrating RNAseq data in mouse embryogenesis, we unveiled a core set of common genes whose expression is responsive to the CDX2 homeoprotein during trophectoderm formation, posterior body elongation and intestinal specification. ChIPseq data analysis also identified a set of common chromosomal regions targeted by CDX2 at these three developmental steps. The transcriptional core set of genes was then validated with transgenic mouse models of loss or gain of function of Cdx2. Finally, based on human cancer data, we highlight the relevance of these results by displaying a significant number of human orthologous genes to the core set of mouse CDX2-responsive genes exhibiting an altered expression along with CDX2 in human malignancies.

CDX2 inducible microRNAs sustain colon cancer by targeting multiple DNA damage response pathway factors.

Meta-analysis of transcripts in colon adenocarcinoma patient tissues led to the identification of a DNA damage responsive miR signature called DNA damage sensitive miRs (DDSMs). DDSMs were experimentally validated in the cancerous colon tissues obtained from an independent cohort of colon cancer patients and in multiple cellular systems with high levels of endogenous DNA damage. All the tested DDSMs were transcriptionally upregulated by a common intestine-specific transcription factor, CDX2. Reciprocally, DDSMs were repressed via the recruitment of HDAC1/2-containing complexes onto the CDX2 promoter. These miRs downregulated multiple key targets in the DNA damage response (DDR) pathway, namely BRCA1, ATM, Chk1 (also known as CHEK1) and RNF8. CDX2 directly regulated the DDSMs, which led to increased tumor volume and metastasis in multiple preclinical models. In colon cancer patient tissues, the DDSMs negatively correlated with BRCA1 levels, were associated with decreased probability of survival and thereby could be used as a prognostic biomarker. This article has an associated First Person interview with the first author of the paper.

CDX2 expression in the hematopoietic lineage promotes leukemogenesis via TGFß inhibition.

The intestine-specific caudal-related homeobox gene-2 (CDX2) homeobox gene, while being a tumor suppressor in the gut, is ectopically expressed in a large proportion of acute leukemia and is associated with poor prognosis. Here, we report that turning on human CDX2 expression in the hematopoietic lineage of mice induces acute monoblastic leukemia, characterized by the decrease in erythroid and lymphoid cells at the benefit of immature monocytic and granulocytic cells. One of the highly stimulated genes in leukemic bone marrow cells was BMP and activin membrane-bound inhibitor (Bambi), an inhibitor of transforming growth factor-ß (TGF-ß) signaling. The CDX2 protein was shown to bind to and activate the transcription of the human BAMBI promoter. Moreover, in a leukemic cell line established from CDX2-expressing mice, reducing the levels of CDX2 or Bambi stimulated the TGF-ß-dependent expression of Cd11b, a marker of monocyte maturation. Taken together, this work demonstrates the strong oncogenic potential of the homeobox gene CDX2 in the hematopoietic lineage, in contrast with its physiological tumor suppressor activity exerted in the gut. It also reveals, through BAMBI and TGF-ß signaling, the involvement of CDX2 in the perturbation of the interactions between leukemia cells and their microenvironment.

Temporal multiomic modeling reveals a B-cell receptor proliferative program in chronic lymphocytic leukemia.

B-cell receptor (BCR) signaling is crucial for the pathophysiology of most mature B-cell lymphomas/leukemias and has emerged as a therapeutic target whose effectiveness remains limited by the occurrence of mutations. Therefore, deciphering the cellular program activated downstream this pathway has become of paramount importance for the development of innovative therapies. Using an original ex vivo model of BCR-induced proliferation of chronic lymphocytic leukemia cells, we generated 108 temporal transcriptional and proteomic profiles from 1 h up to 4 days after BCR activation. This dataset revealed a structured temporal response composed of 13,065 transcripts and 4027 proteins, comprising a leukemic proliferative signature consisting of 430 genes and 374 proteins. Mathematical modeling of this complex cellular response further highlighted a transcriptional network driven by 14 early genes linked to proteins involved in cell proliferation. This group includes expected genes (EGR1/2, NF-kB) and genes involved in NF-kB signaling modulation (TANK, ROHF) and immune evasion (KMO, IL4I1) that have not yet been associated with leukemic cells proliferation. Our study unveils the BCR-activated proliferative genetic program in primary leukemic cells. This approach combining temporal measurements with modeling allows identifying new putative targets for innovative therapy of lymphoid malignancies and also cancers dependent on ligand-receptor interactions.

Murine intestinal stem cells are highly sensitive to modulation of the T3/TRα1-dependent pathway.

The thyroid hormone T3 and its nuclear receptor TRα1 control gut development and homeostasis through the modulation of intestinal crypt cell proliferation. Despite increasing data, in-depth analysis on their specific action on intestinal stem cells is lacking. By using ex vivo 3D organoid cultures and molecular approaches, we observed early responses to T3 involving the T3-metabolizing enzyme Dio1 and the transporter Mct10, accompanied by a complex response of stem cell- and progenitor-enriched genes. Interestingly, specific TRα1 loss-of-function (inducible or constitutive) was responsible for low ex vivo organoid development and impaired stem cell activity. T3 treatment of animals in vivo not only confirmed the positive action of this hormone on crypt cell proliferation but also demonstrated its key action in modulating the number of stem cells, the expression of their specific markers and the commitment of progenitors into lineage-specific differentiation. In conclusion, T3 treatment or TRα1 modulation has a rapid and strong effect on intestinal stem cells, broadening our perspectives in the study of T3/TRα1-dependent signaling in these cells.

Deciphering the Role of Intestinal Crypt Cell Populations in Resistance to Chemotherapy.

Intestinal crypts are composed of heterogeneous and highly plastic cell populations. Lgr5high-stem cells (SC) are responsible for homeostatic renewal, but other cells can revert to an SC-like phenotype to maintain epithelial integrity. Despite their distinct roles in orchestrating homeostasis, both populations have been designated as the putative "cell-of-origin" of colorectal cancer. However, their respective involvement in the emergence of drug-resistant cancer SCs (CSC), responsible for tumor relapse and associated with poor outcome of colorectal cancer, remains elusive. In this context, the intestinal SC/progenitor-marker Musashi1 (MSI1) is interesting as it plays important functions in intestinal homeostasis and is frequently overexpressed in human colorectal cancer. Therefore, our aims were: (i) to study the impact of chemotherapy on Lgr5-expressing and MSI1-expressing cell populations, (ii) to explore the effect of increased MSI1 levels in response to treatment, and (iii) to evaluate the relevance in human colorectal cancer. Engineered mouse models treated with the therapeutic agent 5-fluorouracil showed that upon increased MSI1 levels, Lgr5high SCs remain sensitive while Lgr5low progenitors reprogram to a drug-resistant phenotype. This resulted in the expansion of an MSI1-expressing cell subpopulation with improved resistance to DNA damage and increased detoxification, typical properties of dormant-CSCs that can reactivate after chemotherapy. Analysis in patients with colorectal cancer revealed a correlation between MSI1 levels and tumor grading, CSC phenotype, and chemoresistance. Altogether, these results shed new light on the biology and plasticity of normal crypt and cancer cell populations and also open new perspectives to target MSI1 to improve chemotherapy outcome. SIGNIFICANCE: This study unveils paradoxical roles for MSI1, underlining its importance in facilitating intestinal regeneration upon injury but also unraveling its new function in drug-resistant colorectal cancer stem cells.

Renin-angiotensin system is involved in embryonic emergence of hematopoietic stem/progenitor cells.

Angiotensin-converting enzyme (ACE), a key element of the renin-angiotensin system (RAS), has recently been identified as a new marker of both adult and embryonic human hematopoietic stem/progenitor cells (HSPCs). However, whether a full renin-angiotensin pathway is locally present during the hematopoietic emergence is still an open question. In the present study, we show that this enzyme is expressed by hematopoietic progenitors in the developing mouse embryo. Furthermore, ACE and the other elements of RAS-namely angiotensinogen, renin, and angiotensin II type 1 (AT1) and type 2 (AT2) receptors-are expressed in the paraaortic splanchnopleura (P-Sp) and in its derivative, the aorta-gonad-mesonephros region, both in human and mouse embryos. Their localization is compatible with the existence of a local autocrine and/or paracrine RAS in these hemogenic sites. in vitro perturbation of the RAS by administration of a specific AT1 receptor antagonist inhibits almost totally the generation of blood CD45-positive cells from dissected P-Sp, implying that angiotensin II signaling is necessary for the emergence of hematopoietic cells. Conversely, addition of exogenous angiotensin II peptide stimulates hematopoiesis in culture, with an increase in the number of immature c-Kit+ CD41+ CD31+ CD45+ hematopoietic progenitors, compared to the control. These results highlight a novel role of local-RAS during embryogenesis, suggesting that angiotensin II, via activation of AT1 receptor, promotes the emergence of undifferentiated hematopoietic progenitors.

The atypical cadherin MUCDHL antagonizes colon cancer formation and inhibits oncogenic signaling through multiple mechanisms.

Cadherins form a large and pleiotropic superfamily of membranous proteins sharing Ca2+-binding repeats. While the importance of classic cadherins such as E- or N-cadherin for tumorigenesis is acknowledged, there is much less information about other cadherins that are merely considered as tissue-specific adhesion molecules. Here, we focused on the atypical cadherin MUCDHL that stood out for its unusual features and unique function in the gut. Analyses of transcriptomic data sets (n > 250) established that MUCDHL mRNA levels are down-regulated in colorectal tumors. Importantly, the decrease of MUCDHL expression is more pronounced in the worst-prognosis subset of tumors and is associated with decreased survival. Molecular characterization of the tumors indicated a negative correlation with proliferation-related processes (e.g., nucleic acid metabolism, DNA replication). Functional genomic studies showed that the loss of MUCDHL enhanced tumor incidence and burden in intestinal tumor-prone mice. Extensive structure/function analyses revealed that the mode of action of MUCDHL goes beyond membrane sequestration of ß-catenin and targets through its extracellular domain key oncogenic signaling pathways (e.g., EGFR, AKT). Beyond MUCDHL, this study illustrates how the loss of a gene critical for the morphological and functional features of mature cells contributes to tumorigenesis by dysregulating oncogenic pathways.

Actomyosin, vimentin and LINC complex pull on osteosarcoma nuclei to deform on micropillar topography.

Cell deformation occurs in many critical biological processes, including cell extravasation during immune response and cancer metastasis. These cells deform the nucleus, their largest and stiffest organelle, while passing through narrow constrictions in vivo and the underlying mechanisms still remain elusive. It is unclear which biochemical actors are responsible and whether the nucleus is pushed or pulled (or both) during deformation. Herein we use an easily-tunable poly-L-lactic acid micropillar topography, mimicking in vivo constrictions to determine the mechanisms responsible for nucleus deformation. Using biochemical tools, we determine that actomyosin contractility, vimentin and nucleo-cytoskeletal connections play essential roles in nuclear deformation, but not A-type lamins. We chemically tune the adhesiveness of the micropillars to show that pulling forces are predominantly responsible for the deformation of the nucleus. We confirm these results using an in silico cell model and propose a comprehensive mechanism for cellular and nuclear deformation during confinement. These results indicate that microstructured biomaterials are extremely versatile tools to understand how forces are exerted in biological systems and can be useful to dissect and mimic complex in vivo behaviour.

A redox ruthenium compound directly targets PHD2 and inhibits the HIF1 pathway to reduce tumor angiogenesis independently of p53.

Targeting specific tumor metabolic needs represents an actively investigated therapeutic strategy to bypass tumor resistance mechanisms. In this study, we describe an original approach to impact the cancer metabolism by exploiting the redox properties of a ruthenium organometallic compound. This organometallic complex induced p53-independent cytotoxicity and reduced size and vascularization of patients-derived tumor explants that are resistant to platinum drugs. At the molecular level, the ruthenium complex altered redox enzyme activities and the intracellular redox state by increasing the NAD+/NADH ratio and ROS levels. Pathway analysis pointed to HIF-1 as a top deregulated metabolite pathway. Unlike cisplatin, treatment with the ruthenium complex decreased HIF1A protein levels and expression of HIF1A target genes. The rapid downregulation of HIF1A protein levels involved a direct interaction of the ruthenium compound with the redox enzyme PHD2, a HIF1A master regulator. HIF1A inhibition led to decreased angiogenesis in patient-derived xenografted using fragments of primary human colon tumors. Altogether, our results show that a ruthenium compound impacts metabolic pathways acting as anticancer agents in colon cancer via an original mechanism of action that affects redox enzymes differently than platinum-based drugs.

Chromatin de-condensation by switching substrate elasticity.

Mechanical properties of the cellular environment are known to influence cell fate. Chromatin de-condensation appears as an early event in cell reprogramming. Whereas the ratio of euchromatin versus heterochromatin can be increased chemically, we report herein for the first time that the ratio can also be increased by purely changing the mechanical properties of the microenvironment by successive 24 h-contact of the cells on a soft substrate alternated with relocation and growth for 7 days on a hard substrate. An initial contact with soft substrate caused massive SW480 cancer cell death by necrosis, whereas approximately 7% of the cells did survived exhibiting a high level of condensed chromatin (21% heterochromatin). However, four consecutive hard/soft cycles elicited a strong chromatin de-condensation (6% heterochromatin) correlating with an increase of cellular survival (approximately 90%). Furthermore, cell survival appeared to be reversible, indicative of an adaptive process rather than an irreversible gene mutation(s). This adaptation process is associated with modifications in gene expression patterns. A completely new approach for chromatin de-condensation, based only on mechanical properties of the microenvironment, without any drug mediation is presented.