Vitamin C enhances co-localization of novel TET1 nuclear bodies with both Cajal and PML bodies in colorectal cancer cells.

Deregulation of ten-eleven Translocation protein 1 (TET1) is commonly reported to induce imbalances in gene expression and subsequently to colorectal cancer development (CRC). On the other hand, vitamin C (VitC) improves the prognosis of colorectal cancer by reprogramming the cancer epigenome and limiting chemotherapeutic drug resistance events. In this study, we aimed to characterize TET1-specific subcellular compartments and evaluate the effect of VitC on TET1 compartmentalization in colonic tumour cells. We demonstrated that TET1 is concentrated in coarse nuclear bodies (NB) and 5-hydroxymethylcytosine (5hmC) in foci in colorectal cancer cells (HCT116, Caco-2, and HT-29). To our knowledge, this is the first report of a novel intracellular localization profile of TET1 and its demethylation marker, 5hmC, in CRC cells. Interestingly, we found that TET1-NBs frequently interacted with Cajal bodies, but not with promyelocytic leukaemia (PML) bodies. In addition, we report that VitC treatment of HCT116 cells induces 5hmC foci biogenesis and triggers 5hmC marks to form active complexes with nuclear body components, including both Cajal and PML proteins. Our data highlight novel NB-concentrating TET1 in CRC cells and demonstrate that VitC modulates TET1-NBs' interactions with other nuclear structures. These findings reveal novel TET1-dependent cellular functions and potentially provide new insights for CRC management.

Comparison of the structures and topologies of plasma extracted circulating nuclear and mitochondrial cell-free DNA.

Introduction: The function, origin and structural features of circulating nuclear DNA (cir-nDNA) and mitochondrial DNA (cir-mtDNA) are poorly known, even though they have been investigated in numerous clinical studies, and are involved in a number of routine clinical applications. Based on our previous report disproving the conventional plasma isolation used for cirDNA analysis, this work enables a direct topological comparison of the circulating structures associated with nuclear DNA and mitochondrial cell-free DNA. Materials and methods: We used a Q-PCR and low-pass whole genome sequencing (LP-WGS) combination approach of cir-nDNA and cir-mtDNA, extracted using a procedure that eliminates platelet activation during the plasma isolation process to prevent mitochondria release in the extracellular milieu. Various physical procedures, such as filtration and differential centrifugation, were employed to infer their circulating structures. Results: DSP-S cir-mtDNA mean size profiles distributed on a slightly shorter range than SSP-S. SSP-S detected 40-fold more low-sized cir-mtDNA fragments (<90 bp/nt) and three-fold less long-sized fragments (>200 bp/nt) than DSP-S. The ratio of the fragment number below 90 bp over the fragment number above 200 bp was very homogenous among both DSP-S and SSP-S profiles, being 134-fold lower with DSP-S than with SSP-S. Cir-mtDNA and cir-nDNA DSP-S and SSP-S mean size profiles of healthy individuals ranged in different intervals with periodic sub-peaks only detectable with cir-nDNA. The very low amount of cir-mtDNA fragments of short size observed suggested that most of the cir-mtDNA is poorly fragmented and appearing longer than ∼1,000 bp, the readout limit of this LP-WGS method. Data suggested that cir-nDNA is, among DNA extracted in plasma, associated with ∼8.6% of large structures (apoptotic bodies, large extracellular vesicles (EVs), cell debris…), ∼27.7% in chromatin and small EVs and ∼63.7% mainly in oligo- and mono-nucleosomes. By contrast, cir-mtDNA appeared to be preponderantly (75.7%) associated with extracellular mitochondria, either in its free form or with large EVs; to a lesser extent, it was also associated with other structures: small EVs (∼18.4%), and exosomes or protein complexes (∼5.9%). Conclusion: This is the first study to directly compare the structural features of cir-nDNA and cir-mtDNA. The significant differences revealed between both are due to the DNA topological structure contained in the nucleus (chromatin) and in the mitochondria (plasmid) that determine their biological stability in blood. Although cir-nDNA and cir-mtDNA are principally associated with mono-nucleosomes and cell-free mitochondria, our study highlights the diversity of the circulating structures associated with cell-free DNA. They consequently have different pharmacokinetics as well as physiological functions. Thus, any accurate evaluation of their biological or diagnostic individual properties must relies on appropriate pre-analytics, and optimally on the isolation or enrichment of one category of their cirDNA associated structures.

A straightforward method to quantify circulating mRNAs as biomarkers of colorectal cancer.

Optimizing the biomarker combination to be analyzed in liquid biopsies should improve personalized medicine. We developed a method to purify circulating cell-free mRNAs from plasma samples and to quantify them by RT-qPCR. We selected three candidate colorectal cancer biomarkers (B2M, TIMP-1, and CLU). Their mRNA levels were significantly higher in plasma of patients with metastatic colorectal cancer patients (mCRC) (n = 107) than in healthy individuals (HI) (n = 53). To increase the discriminating performance of our method, we analyzed the sum of the three mRNA levels (BTC index). The area under the ROC curve (AUC) to estimate the BTC index capacity to discriminate between mCRC and HI plasma was 0.903. We also determined the optimal BTC index cut-off to distinguish between plasma samples, with 82% of sensitivity and 93% of specificity. By using mRNA as a novel liquid biopsy analytical parameter, our method has the potential to facilitate rapid screening of CRCm.

Prenatal exposure to low doses of fungicides corrupts neurogenesis in neonates.

Neurogenesis plays a crucial role during neurodevelopment and its dysfunction can lead to neurodevelopmental disorders. A recent hypothesis stipulates that exogenous factors could corrupt this process and predispose to neurodegenerative disorders later in life. The presence of pesticide residues in the diet represents a threat of which we have recently become aware of. Indeed, they could corrupt neurogenesis, especially during gestation, potentially leading to impaired neuronal and synaptic functions. Since the effects of this low-noise contamination have not yet been evaluated on the neurodevelopment, we investigated the impact of fungicide residues on WT mice exposed throughout gestation. Thus, mice were exposed to fungicides, cyprodinil, mepanipyrim and pyrimethanil, alone at 0.1 µg/L during gestation until P3. Besides, another group was exposed to a cocktail of these three fungicides (0.1 µg/L each) for the same time. Exposure was performed through drinking water at the regulatory limit dose of the European countries (0.1 µg/L). No general toxicity was observed in neonates on body and brain weight upon fungicide exposure. However, results showed that gestational exposure to fungicide residues substantially promoted an increase of neural precursor cells at P3. This corrupted neurogenesis was linked to increased levels of ß-catenin, likely through the crosstalk of the PI3K/Akt and Wnt/ß-catenin pathways, both involved in cell proliferation. Fungicide exposure also altered protein expression of PSD95 and NMDA receptors in P3 neonates, two targets of the ß-catenin signaling pathway. Adult neural stem cell extractions from mice treated with the fungicide cocktail, showed an increase proliferation and differentiation combined with a reduction of their migration properties. In addition, in vitro studies on hippocampal primary cell cultures treated with various concentrations of fungicides showed neurotoxic effects. To conclude, corruption of neurogenesis by this chemical assault could be a fertile ground for the development of neurological diseases later in life.

Blood contains circulating cell-free respiratory competent mitochondria.

Mitochondria are considered as the power-generating units of the cell due to their key role in energy metabolism and cell signaling. However, mitochondrial components could be found in the extracellular space, as fragments or encapsulated in vesicles. In addition, this intact organelle has been recently reported to be released by platelets exclusively in specific conditions. Here, we demonstrate for the first time, that blood preparation with resting platelets, contains whole functional mitochondria in normal physiological state. Likewise, we show, that normal and tumor cultured cells are able to secrete their mitochondria. Using serial centrifugation or filtration followed by polymerase chain reaction-based methods, and Whole Genome Sequencing, we detect extracellular full-length mitochondrial DNA in particles over 0.22 µm holding specific mitochondrial membrane proteins. We identify these particles as intact cell-free mitochondria using fluorescence-activated cell sorting analysis, fluorescence microscopy, and transmission electron microscopy. Oxygen consumption analysis revealed that these mitochondria are respiratory competent. In view of previously described mitochondrial potential in intercellular transfer, this discovery could greatly widen the scope of cell-cell communication biology. Further steps should be developed to investigate the potential role of mitochondria as a signaling organelle outside the cell and to determine whether these circulating units could be relevant for early detection and prognosis of various diseases.

Hypoxia differently modulates the release of mitochondrial and nuclear DNA.

BACKGROUND: We investigated the influence of hypoxia on the concentration of mitochondrial and nuclear cell-free DNA (McfDNA and NcfDNA, respectively). METHOD: By an ultra-sensitive quantitative PCR-based assay, McfDNA and NcfDNA were measured in the supernatants of different colorectal cell lines, and in the plasma of C57/Bl6 mice engrafted with TC1 tumour cells, in normoxic or hypoxic conditions. RESULTS: Our data when setting cell culture conditions highlighted the higher stability of McfDNA as compared to NcfDNA and revealed that cancer cells released amounts of nuclear DNA equivalent to the mass of a chromosome over a 6-h duration of incubation. In cell model, hypoxia induced a great increase in NcfDNA and McfDNA concentrations within the first 24 h. After this period, cfDNA total concentrations remained stable in hypoxia consecutive to a decrease of nuclear DNA release, and noteworthy, to a complete inhibition of daily mitochondrial DNA release. In TC1-engrafted mice submitted to intermittent hypoxia, plasma NcfDNA levels are much higher than in mice bred in normoxia, unlike plasma McfDNA concentration that is not impacted by hypoxia. CONCLUSION: This study suggests that hypoxia negatively modulates nuclear and, particularly, mitochondrial DNA releases in long-term hypoxia, and revealed that the underlying mechanisms are differently regulated.

A Short SOX9 Peptide Mimics SOX9 Tumor Suppressor Activity and Is Sufficient to Inhibit Colon Cancer Cell Growth.

Differently from cytotoxic chemotherapies, targeted therapies do not necessarily drive cancer cells toward death, but reduce cell proliferation, angiogenesis, and/or prevent metastasis without affecting healthy cells. Oncogenic proteins that are hyperactivated and/or overexpressed in cancer cells are prime targets for such therapies. On the other hand, the activity of tumor suppressor proteins is more difficult to harness. Here, we identified a short SOX9 sequence (S9pep) located at the hinge between the HMG DNA-binding domain and the SOX-E central conserved domain that mimics SOX9 tumor-suppressive properties. Doxycycline-induced S9pep expression in DLD-1 colorectal cancer cells inhibited the growth potential of these cells, including colorectal cancer stem cells, restored cell-cell contact inhibition, and inhibited the activity of the oncogenic Wnt/ß-catenin signaling pathway. It also significantly decreased tumor growth in BALB/cAnNCrl mice grafted with mouse doxycycline-inducible CT26 colorectal cancer cells in which S9pep was induced by treating them with doxycycline. As the Wnt/ß-catenin signaling pathway is constitutively activated in 80% of colorectal cancer and SOX9-inactivating mutations are present in up to 11% of colorectal cancer, S9pep could be a promising starting point for the development of a peptide-based therapeutic approach to restore a SOX9-like tumor suppressor function in colorectal cancer.

Modulating PKCα Activity to Target Wnt/ß-Catenin Signaling in Colon Cancer.

Inactivating mutations of the tumor suppressor Adenomatosis Polyposis Coli (APC), which are found in familial adenomatosis polyposis and in 80% of sporadic colorectal cancers (CRC), result in constitutive activation of the Wnt/ß-catenin pathway and tumor development in the intestine. These mutations disconnect the Wnt/ß-catenin pathway from its Wnt extracellular signal by inactivating the APC/GSK3-ß/axin destruction complex of ß-catenin. This results in sustained nuclear accumulation of ß-catenin, followed by ß-catenin-dependent co-transcriptional activation of Wnt/ß-catenin target genes. Thus, mechanisms acting downstream of APC, such as those controlling ß-catenin stability and/or co-transcriptional activity, are attractive targets for CRC treatment. Protein Kinase C-α (PKCα) phosphorylates the orphan receptor RORα that then inhibits ß-catenin co-transcriptional activity. PKCα also phosphorylates ß-catenin, leading to its degradation by the proteasome. Here, using both in vitro (DLD-1 cells) and in vivo (C57BL/6J mice) PKCα knock-in models, we investigated whether enhancing PKCα function could be beneficial in CRC treatment. We found that PKCα is infrequently mutated in CRC samples, and that inducing PKCα function is not deleterious for the normal intestinal epithelium. Conversely, di-terpene ester-induced PKCα activity triggers CRC cell death. Together, these data indicate that PKCα is a relevant drug target for CRC treatment.

A Cell Model Suitable for a High-Throughput Screening of Inhibitors of the Wnt/ß-Catenin Pathway.

A constitutive activation of the Wnt/ß-catenin pathway is an initiating event in colon carcinogenesis. We developed colon cancer cells models that highlight the non-selectivity of previously described inhibitors of the Wnt pathway and we propose our model as a suitable screening system for inhibitors of the pathway.

The dose-dependent effect of SOX9 and its incidence in colorectal cancer.

A member of the Sry-related HMG-box family of transcription factors (SOX9) is a transcription factor that belongs to the superfamily of High Mobility Group (HMG) domain transcription factors. SOX9 is expressed in a variety of tissues, including as the intestinal epithelium, where it is now recognised as an important actor for homeostasis. Beside, a high level of SOX9 has recently been correlated with a good prognosis for stage II colorectal cancers. However, growing evidence indicates that deciphering the function of SOX9 in the intestine has to take into account a dose-dependent effect of SOX9. Given the recurrent controversies and the lack of a state of the art as to whether SOX9 behaves like a tumour suppressor or an oncogen in the intestine epithelium, it is time to provide an update of the accumulated knowledge about the biological function of SOX9 in the intestine and about the role of SOX9 in colorectal cancers.

SOX9 is an atypical intestinal tumor suppressor controlling the oncogenic Wnt/ß-catenin signaling.

SOX9 inactivation is frequent in colorectal cancer (CRC) due to SOX9 gene mutations and/or to ectopic expression of MiniSOX9, a dominant negative inhibitor of SOX9. In the present study, we report a heterozygous L142P inactivating mutation of SOX9 in the DLD-1 CRC cell line and we demonstrate that the conditional expression of a wild type SOX9 in this cell line inhibits cell growth, clonal capacity and colonosphere formation while decreasing both the activity of the oncogenic Wnt/ß-catenin signaling pathway and the expression of the c-myc oncogene. This activity does not require SOX9 transcriptional function but, rather, involves an interaction of SOX9 with nuclear ß-catenin. Furthermore, we report that SOX9 inhibits tumor development when conditionally expressed in CRC cells injected either subcutaneous or intraperitoneous in BALB/c mice as an abdominal metastasis model. These observations argue in favor of a tumor suppressor activity for SOX9. As an siRNA targeting SOX9 paradoxically also inhibits DLD-1 and HCT116 CRC cell growth, we conclude that there is a critical level of endogenous active SOX9 needed to maintain CRC cell growth.

Applying ecological and evolutionary theory to cancer: a long and winding road.

Since the mid 1970s, cancer has been described as a process of Darwinian evolution, with somatic cellular selection and evolution being the fundamental processes leading to malignancy and its many manifestations (neoangiogenesis, evasion of the immune system, metastasis, and resistance to therapies). Historically, little attention has been placed on applications of evolutionary biology to understanding and controlling neoplastic progression and to prevent therapeutic failures. This is now beginning to change, and there is a growing international interest in the interface between cancer and evolutionary biology. The objective of this introduction is first to describe the basic ideas and concepts linking evolutionary biology to cancer. We then present four major fronts where the evolutionary perspective is most developed, namely laboratory and clinical models, mathematical models, databases, and techniques and assays. Finally, we discuss several of the most promising challenges and future prospects in this interdisciplinary research direction in the war against cancer.

A new mechanism of SOX9 action to regulate PKCalpha expression in the intestine epithelium.

Variations of protein kinase C (PKC) expression greatly influence the proliferation-to-differentiation transition (PDT) of intestinal epithelial cells and might have an important impact on intestinal tumorigenesis. We demonstrate here that the expression of PKCalpha in proliferating intestinal epithelial cells is repressed both in vitro and in vivo by the SOX9 transcription factor. This repression does not require DNA binding of the SOX9 high-mobility group (HMG) domain but is mediated through a new mechanism of SOX9 action requiring the central and highly conserved region of SOXE members. Because SOX9 expression is itself upregulated by Wnt-APC signaling in intestinal epithelial cells, the present study points out this transcription factor as a molecular link between the Wnt-APC pathway and PKCalpha. These results provide a potential explanation for the decrease of PKCalpha expression in colorectal cancers with constitutive activation of the Wnt-APC pathway.

A disrupted expression in cancers: multiple potential causes.

Tumor cells exhibit significant variations in the rate of pro- or anti-tumoral proteins that provide them a selective advantage of growth over normal cells. The control of these rates occurs at the three DNA, RNA and protein levels, and is determined by the structure of each of these three actors for the implementation of the molecular mechanisms involved in the control of the synthesis, maturation and stability of the mRNA and the protein itself. We give here an overview of the main events that can lead to a disruption of these mechanisms.

A spatiotemporally coordinated cascade of protein kinase C activation controls isoform-selective translocation.

In pituitary GH3B6 cells, signaling involving the protein kinase C (PKC) multigene family can self-organize into a spatiotemporally coordinated cascade of isoform activation. Indeed, thyrotropin-releasing hormone (TRH) receptor activation sequentially activated green fluorescent protein (GFP)-tagged or endogenous PKCbeta1, PKCalpha, PKCepsilon, and PKCdelta, resulting in their accumulation at the entire plasma membrane (PKCbeta and -delta) or selectively at the cell-cell contacts (PKCalpha and -epsilon). The duration of activation ranged from 20 s for PKCalpha to 20 min for PKCepsilon. PKCalpha and -epsilon selective localization was lost in the presence of Gö6976, suggesting that accumulation at cell-cell contacts is dependent on the activity of a conventional PKC. Constitutively active, dominant-negative PKCs and small interfering RNAs showed that PKCalpha localization is controlled by PKCbeta1 activity and is calcium independent, while PKCepsilon localization is dependent on PKCalpha activity. PKCdelta was independent of the cascade linking PKCbeta1, -alpha, and -epsilon. Furthermore, PKCalpha, but not PKCepsilon, is involved in the TRH-induced beta-catenin relocation at cell-cell contacts, suggesting that PKCepsilon is not the unique functional effector of the cascade. Thus, TRH receptor activation results in PKCbeta1 activation, which in turn initiates a calcium-independent but PKCbeta1 activity-dependent sequential translocation of PKCalpha and -epsilon. These results challenge the current understanding of PKC signaling and raise the question of a functional dependence between isoforms.

Targeting of PKCalpha and epsilon in the pituitary: a highly regulated mechanism involving a GD(E)E motif of the V3 region.

Protein kinase C (PKC) has been implicated in the control of intercellular adhesion. Our previous observation demonstrating that activated PKC alpha (PKCalpha is selectively targeted to cell-cell contacts of pituitary GH3B6 cells supports these findings. The relevance of this observation is further strengthened by the present data establishing that this targeting selectivity also occurs in the pituitary gland. Moreover, a new mechanism involved in the control of PKC targeting is unravelled. We demonstrate that a three amino acid motif located in the V3 region of alpha and epsilon (epsilon (GDE/GEE respectively) is essential for the targeting selectivity of these isoforms because: (1) this motif is absent in delta (delta) and mutated in the natural D294GPKCalpha mutant, which do not exhibit such selectivity, and (2) a GEE to GGE mutation abolishes the selectivity of targeting to cell-cell contacts for epsilon, as it does for the D294G PKCalpha mutant. Thus the GD(E)E motif may be part of a consensus sequence able to interact with shuttle and/or anchoring proteins. GFP-tagged deletion mutants also reveal a new function for the pseudosubstrate in the cytoplasmic sequestration. Together, these data underline the complexity of PKC subcellular targeting in the pituitary, determined by the cell-cell contact, at least for alpha and epsilon

Site-directed perturbation of protein kinase C- integrin interaction blocks carcinoma cell chemotaxis.

Polarized cell movement is an essential requisite for cancer metastasis; thus, interference with the tumor cell motility machinery would significantly modify its metastatic behavior. Protein kinase C alpha (PKC alpha) has been implicated in the promotion of a migratory cell phenotype. We report that the phorbol ester-induced cell polarization and directional motility in breast carcinoma cells is determined by a 12-amino-acid motif (amino acids 313 to 325) within the PKC alpha V3 hinge domain. This motif is also required for a direct association between PKC alpha and beta 1 integrin. Efficient binding of beta 1 integrin to PKC alpha requires the presence of both NPXY motifs (Cyto-2 and Cyto-3) in the integrin distal cytoplasmic domains. A cell-permeant inhibitor based on the PKC-binding sequence of beta 1 integrin was shown to block both PKC alpha-driven and epidermal growth factor (EGF)-induced chemotaxis. When introduced as a minigene by retroviral transduction into human breast carcinoma cells, this inhibitor caused a striking reduction in chemotaxis towards an EGF gradient. Taken together, these findings identify a direct link between PKC alpha and beta 1 integrin that is critical for directed tumor cell migration. Importantly, our findings outline a new concept as to how carcinoma cell chemotaxis is enhanced and provide a conceptual basis for interfering with tumor cell dissemination.