Mechanisms of chronic alcohol exposure-induced aggressiveness in cellular model of HCC and recovery after alcohol withdrawal.

Alcohol-related liver disease is the most prevalent chronic liver disease worldwide, accounting for 30% of hepatocellular carcinoma (HCC) cases and HCC-specific deaths. However, the knowledge on mechanisms by which alcohol consumption leads to cancer progression and its aggressiveness is limited. Better understanding of the clinical features and the mechanisms of alcohol-induced HCC are of critical importance for prevention and the development of novel treatments. Early stage Huh-7 and advanced SNU449 liver cancer cell lines were subjected to chronic alcohol exposure (CAE), at different doses for 6 months followed by 1-month alcohol withdrawal period. ADH activity and ALDH expression were much lower in SNU449 compared with Huh-7 cells and at the 270 mM dose, CAE decreased cell viability by about 50% and 80%, respectively, in Huh-7 and SNU449 cells but induced mortality only in Huh-7 cells. Thus, Huh-7 may be more vulnerable to ethanol toxicity because of the higher levels of acetaldehyde. CAE induced a dose-dependent increase in cell migration and invasion and also in the expression of cancer stem cells markers (CD133, CD44, CD90). CAE in Huh-7 cells selectively activated ERK1/2 and inhibited GSK3ß signaling pathways. Most of the changes induced by CAE were reversed after alcohol withdrawal. Interestingly, we confirmed the increase in CD133 mRNA levels in the tumoral tissue of patients with ethanol-related HCC compared to other HCC etiologies. Our results may explain the benefits observed in epidemiological studies showing a significant increase of overall survival in abstinent compared with non-abstinent patients.

Rescuing SLAMF3 Expression Restores Sorafenib Response in Hepatocellular Carcinoma Cells through the Induction of Mesenchymal-to-Epithelial Transition.

BACKGROUND: Acquired resistance to sorafenib in hepatocellular carcinoma (HCC) patients results in poor prognosis. Epithelial-to-mesenchymal transition (EMT) is the major mechanism implicated in the resistance to sorafenib. We have reported the tumor suppressor role of SLAMF3 (signaling lymphocytic activation molecules family 3) in HCC progression and highlighted its implication in controlling the MRP-1 transporter activity. These data suggest the implication of SLAMF3 in sorafenib resistance mechanisms. METHODS: We evaluated the resistance to sorafenib in Huh-7 cells treated with progressive doses (Res cells). We investigated the link between acquired resistance to sorafenib and SLAMF3 expression by flow cytometry and Western blot methods. Furthermore, we analyzed the EMT and the stem cell potential of cells resistant to sorafenib. RESULTS: Sorafenib resistance was confirmed in Res cells by analyzing the cell viability in the presence of sorafenib. The mesenchymal transition, in Res cells, was confirmed by high migratory index and the expression of EMT antigens. Interestingly, we found that loss of SLAMF3 expression corresponded to sorafenib-resistant phenotypes. The overexpression of SLAMF3 reversed EMT, decreased metastatic potential and inhibited mTOR/ERK1/2 in Res cells. CONCLUSIONS: We propose that rescuing SLAMF3 expression in resistant cells could represent a potential therapeutic strategy to enhance sorafenib efficacy in HCC patients.

Signaling lymphocytic activation molecules Slam and cancers: friends or foes?

Signaling Lymphocytic Activation Molecules (SLAM) family receptors are initially described in immune cells. These receptors recruit both activating and inhibitory SH2 domain containing proteins through their Immunoreceptor Tyrosine based Switch Motifs (ITSMs). Accumulating evidence suggest that the members of this family are intimately involved in different physiological and pathophysiological events such as regulation of immune responses and entry pathways of certain viruses. Recently, other functions of SLAM, principally in the pathophysiology of neoplastic transformations have also been deciphered. These new findings may prompt SLAM to be considered as new tumor markers, diagnostic tools or potential therapeutic targets for controlling the tumor progression. In this review, we summarize the major observations describing the implications and features of SLAM in oncology and discuss the therapeutic potential attributed to these molecules.

Hepatocyte SLAMF3 reduced specifically the multidrugs resistance protein MRP-1 and increases HCC cells sensitization to anti-cancer drugs.

Multidrug resistance MDR proteins (MRPs) are members of the C family of a group of proteins named ATP binding cassette (ABC) transporters. MRPs can transport drugs including anticancer drugs, nucleoside analogs, antimetabolites and tyrosine kinase inhibitors. Drugs used in HCC therapy, such as tyrosine kinase inhibitor sorafenib, are substrates of uptake and/or efflux transporters. Variable expression of MRPs at the plasma membrane of tumor cells may contribute to drug resistance and subsequent clinical response. Recently, we reported that the hepatocyte SLAMF3 expression (Signaling Lymphocytic Activation Molecule Family member 3) was reduced in tumor cells from hepatocellular carcinoma (HCC) compared to its high expression in adjacent tissues. In the present study, we make a strong correlation between induced SLAMF3 overexpression and the specific loss of MRP-1 expression and its functionalities as a drugs resistance transporter. No changes were observed on expression of ABCG2 and MDR. More importantly, we highlight a strong inverse correlation between MRP-1 and SLAMF3 expression in patients with HCC. We propose that the SLAMF3 overexpression in cancerous cells could represent a potential therapeutic strategy to improve the drugs sensibility of resistant cells and thus control the therapeutic failure in HCC patients.

RB/PLK1-dependent induced pathway by SLAMF3 expression inhibits mitosis and control hepatocarcinoma cell proliferation.

Polo-like kinase PLK1 is a cell cycle protein that plays multiple roles in promoting cell cycle progression. Among the many roles, the most prominent role of PLK1 is to regulate the mitotic spindle formation checkpoint at the M-phase. Recently we reported the expression of SLAMF3 in Hepatocytes and show that it is down regulated in tumor cells of hepatocellular carcinoma (HCC). We also show that the forced high expression level of SLAMF3 in HCC cells controls proliferation by inhibiting the MAPK ERK/JNK and the mTOR pathways. In the present study, we provide evidence that the inhibitory effect of SLAMF3 on HCC proliferation occurs through Retinoblastoma (RB) factor and PLK1-dependent pathway. In addition to the inhibition of MAPK ERK/JNK and the mTOR pathways, expression of SLAMF3 in HCC retains RB factor in its hypophosphorylated active form, which in turn inactivates E2F transcription factor, thereby repressing the expression and activation of PLK1. A clear inverse correlation was also observed between SLAMF3 and PLK expression in patients with HCC. In conclusion, the results presented here suggest that the tumor suppressor potential of SLAMF3 occurs through activation of RB that represses PLK1. We propose that the induction of a high expression level of SLAMF3 in cancerous cells could control cellular mitosis and block tumor progression.

The retinoblastoma (Rb) protein regulates ferroptosis induced by sorafenib in human hepatocellular carcinoma cells.

Sorafenib is the treatment of reference for advanced hepatocellular carcinoma (HCC), the most frequent form of primary liver tumour. The loss of function of the retinoblastoma (Rb) protein is an important event during liver carcinogenesis, but it is unclear whether the Rb status modulates the response of HCC cells to sorafenib. Here, we examined this question in HCC cells with reduced levels of Rb achieved through stable RNA interference. We show that HCC cells with reduced levels of Rb exhibit a two- to threefold increase in cell death induction upon exposure to sorafenib compared with controls. Sorafenib treatment of Balb/c nude mice that received tumour xenografts derived from HCC cells with reduced Rb levels resulted in complete tumour regression in 50% of the animals treated, compared with tumour stabilization in mice that received control cells. We show that, upon exposure to sorafenib, the Rb-negative status of HCC cells promotes the occurrence of ferroptosis, a form of oxidative necrosis. The findings highlight the role of Rb in the response of HCC cells to sorafenib and the regulation of ferroptosis.

Maternally acquired genotoxic Escherichia coli alters offspring's intestinal homeostasis.

The neonatal gut is rapidly colonized by a newly dominant group of commensal Escherichia coli strains among which a large proportion produces a genotoxin called colibactin. In order to analyze the short- and long-term effects resulting from such evolution, we developed a rat model mimicking the natural transmission of E. coli from mothers to neonates. Genotoxic and non-genotoxic E. coli strains were equally transmitted to the offspring and stably colonized the gut across generations. DNA damage was only detected in neonates colonized with genotoxic E. coli strains. Signs of genotoxic stress such as anaphase bridges, higher occurrence of crypt fission and accelerated renewal of the mature epithelium were detected at adulthood. In addition, we observed alterations of secretory cell populations and gut epithelial barrier. Our findings illustrate how critical is the genotype of E. coli strains acquired at birth for gut homeostasis at adulthood.

The expression of the hepatocyte SLAMF3 (CD229) receptor enhances the hepatitis C virus infection.

Hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer worldwide. We recently characterized for the first time the expression of Signaling Lymphocyte Activating Molecule 3 (SLAMF3) in human hepatocytes and here, we report that SLAMF3 interacts with the HCV viral protein E2 and is implicated in HCV entry process. We found a strong correlation between SLAMF3 expression level and hepatocyte susceptibility to HCV infection. The use of specific siRNAs to down-modulate SLAMF3 expression and SLAMF3-blocking antibodies both decreased the hepatocytes susceptibility to HCV infection. Moreover, SLAMF3 over-expression significantly increased susceptibility to HCV infection. Interestingly, experiments with peptides derived from each SLAMF3 domain showed that the first N-terminal extracellular domain is essential for interaction with HCV particles. Finally, we showed that recombinant HCV envelop protein E2 can bind SLAMF3 and that anti-SLAMF3 antibodies inhibited specifically this interaction. Overall, our results revealed that SLAMF3 plays a role during HCV entry, likely by enhancing entry of viral particle within hepatocytes.

The genotoxin colibactin exacerbates lymphopenia and decreases survival rate in mice infected with septicemic Escherichia coli.

Sepsis is a life-threatening infection. Escherichia coli is the first known cause of bacteremia leading to sepsis. Lymphopenia was shown to predict bacteremia better than conventional markers of infection. The pks genomic island, which is harbored by extraintestinal pathogenic E. coli (ExPEC) and encodes the genotoxin colibactin, is epidemiologically associated with bacteremia. To investigate a possible relationship between colibactin and lymphopenia, we examined the effects of transient infection of lymphocytes with bacteria that were and those that were not producing the genotoxin. A mouse model of sepsis was used to compare the virulence of a clinical ExPEC isolate with its isogenic mutant impaired for the production of colibactin. We observed that colibactin induced double-strand breaks in the DNA of infected lymphocytes, leading to cell cycle arrest and to cell death by apoptosis. E. coli producing colibactin induced a more profound lymphopenia in septicemic mice, compared with the isogenic mutant unable to produce colibactin. In a sepsis model in which the mice were treated by rehydration and antibiotics, the production of colibactin by the bacteria was associated with a significantly lower survival rate. In conclusion, we demonstrate that production of colibactin by E. coli exacerbates lymphopenia associated with septicemia and could impair the chances to survive sepsis.

Identification of SLAMF3 (CD229) as an inhibitor of hepatocellular carcinoma cell proliferation and tumour progression.

Although hepatocellular carcinoma (HCC) is one of the most common malignancies and constitutes the third leading cause of cancer-related deaths, the underlying molecular mechanisms are not fully understood. In the present study, we demonstrate for the first time that hepatocytes express signalling lymphocytic activation molecule family member 3 (SLAMF3/CD229) but not other SLAMF members. We provide evidence to show that SLAMF3 is involved in the control of hepatocyte proliferation and in hepatocellular carcinogenesis. SLAMF3 expression is significantly lower in primary human HCC samples and HCC cell lines than in human healthy primary hepatocytes. In HCC cell lines, the restoration of high levels of SLAMF3 expression inhibited cell proliferation and migration and enhanced apoptosis. Furthermore, SLAMF3 expression was associated with inhibition of HCC xenograft progression in the nude mouse model. The restoration of SLAMF3 expression levels also decreased the phosphorylation of MAPK ERK1/2, JNK and mTOR. In samples from resected HCC patients, SLAMF3 expression levels were significantly lower in tumorous tissues than in peritumoral tissues. Our results identify SLAMF3 as a specific marker of normal hepatocytes and provide evidence for its potential role in the control of proliferation of HCC cells.

Interplay between siderophores and colibactin genotoxin biosynthetic pathways in Escherichia coli.

In Escherichia coli, the biosynthetic pathways of several small iron-scavenging molecules known as siderophores (enterobactin, salmochelins and yersiniabactin) and of a genotoxin (colibactin) are known to require a 4'-phosphopantetheinyl transferase (PPTase). Only two PPTases have been clearly identified: EntD and ClbA. The gene coding for EntD is part of the core genome of E. coli, whereas ClbA is encoded on the pks pathogenicity island which codes for colibactin. Interestingly, the pks island is physically associated with the high pathogenicity island (HPI) in a subset of highly virulent E. coli strains. The HPI carries the gene cluster required for yersiniabactin synthesis except for a gene coding its cognate PPTase. Here we investigated a potential interplay between the synthesis pathways leading to the production of siderophores and colibactin, through a functional interchangeability between EntD and ClbA. We demonstrated that ClbA could contribute to siderophores synthesis. Inactivation of both entD and clbA abolished the virulence of extra-intestinal pathogenic E. coli (ExPEC) in a mouse sepsis model, and the presence of either functional EntD or ClbA was required for the survival of ExPEC in vivo. This is the first report demonstrating a connection between multiple phosphopantetheinyl-requiring pathways leading to the biosynthesis of functionally distinct secondary metabolites in a given microorganism. Therefore, we hypothesize that the strict association of the pks island with HPI has been selected in highly virulent E. coli because ClbA is a promiscuous PPTase that can contribute to the synthesis of both the genotoxin and siderophores. The data highlight the complex regulatory interaction of various virulence features with different functions. The identification of key points of these networks is not only essential to the understanding of ExPEC virulence but also an attractive and promising target for the development of anti-virulence therapy strategies.

Genotoxicity of Escherichia coli Nissle 1917 strain cannot be dissociated from its probiotic activity.

Oral administration of the probiotic bacterium Escherichia coli Nissle 1917 improves chronic inflammatory bowel diseases, but the molecular basis for this therapeutic efficacy is unknown. E. coli Nissle 1917 harbors a cluster of genes coding for the biosynthesis of hybrid nonribosomal peptide-polyketide(s). This biosynthetic pathway confers the ability for bacteria to induce DNA double strand breaks in eukaryotic cells. Here we reveal that inactivation of the clbA gene within this genomic island abrogated the ability for the strain to induce DNA damage and chromosomal abnormalities in non-transformed cultured rat intestinal epithelial cells but is required for the probiotic activity of E. coli Nissle 1917. Thus, evaluation of colitis severity induced in rodent fed with E. coli Nissle 1917 or an isogenic non-genotoxic mutant demonstrated the need for a functional biosynthetic pathway both in the amelioration of the disease and in the modulation of cytokine expression. Feeding rodents with a complemented strain for which genotoxicity was restored confirmed that this biosynthetic pathway contributes to the health benefits of the probiotic by modulating its immunomodulatory properties. Our data provide additional evidence for the benefit of this currently used probiotic in colitis but remind us that an efficient probiotic may also have side effects as any other medication.

Escherichia coli induces DNA damage in vivo and triggers genomic instability in mammalian cells.

Escherichia coli is a normal inhabitant of the human gut. However, E. coli strains of phylogenetic group B2 harbor a genomic island called "pks" that codes for the production of a polyketide-peptide genotoxin, Colibactin. Here we report that in vivo infection with E. coli harboring the pks island, but not with a pks isogenic mutant, induced the formation of phosphorylated H2AX foci in mouse enterocytes. We show that a single, short exposure of cultured mammalian epithelial cells to live pks(+) E. coli at low infectious doses induced a transient DNA damage response followed by cell division with signs of incomplete DNA repair, leading to anaphase bridges and chromosome aberrations. Micronuclei, aneuploidy, ring chromosomes, and anaphase bridges persisted in dividing cells up to 21 d after infection, indicating occurrence of breakage-fusion-bridge cycles and chromosomal instability. Exposed cells exhibited a significant increase in gene mutation frequency and anchorage-independent colony formation, demonstrating the infection mutagenic and transforming potential. Therefore, colon colonization with these E. coli strains harboring the pks island could contribute to the development of sporadic colorectal cancer.