Distinct tumor protein p53 mutants in breast cancer subgroups.

Tumor protein p53 (TP53) is mutated in approximately 30% of breast cancers, but this frequency fluctuates widely between subclasses. We investigated the p53 mutation status in 572 breast tumors, classified into luminal, basal and molecular apocrine subgroups. As expected, the lowest mutation frequency was observed in luminal (26%), and the highest in basal (88%) tumors. Luminal tumors showed significantly higher frequency of substitutions (82 vs. 65%), notably A/T to G/C transitions (31 vs. 15%), whereas molecular apocrine and basal tumors presented much higher frequencies of complex mutations (deletions/insertions) (36 and 33%, respectively, vs. 18%). Accordingly, missense mutations were significantly more frequent in luminal tumors (75 vs. 54%), whereas basal tumors displayed significantly increased rates of TP53 truncations (43 vs. 25%), resulting in loss of function and/or expression. Interestingly, as basal tumors, molecular apocrine tumors presented with a high rate of complex mutations, but paradoxically, these were not associated with increased frequency of p53 truncation. As in luminal tumors, this could reflect a selective pressure for p53 gain of function, possibly through P63/P73 inactivation. Collectively, these observations point not only to different mechanisms of TP53 alterations, but also to different functional consequences in the different breast cancer subtypes.

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.

Fecal microbiota transplantation in a rodent model of short bowel syndrome: A therapeutic approach?

Extensive intestinal resection leads to Short Bowel Syndrome (SBS), the main cause of chronic intestinal failure. Colon preservation is crucial for spontaneous adaptation, to improve absorption and reduce parenteral nutrition dependence. Fecal microbiota transplantation (FMT), a promising approach in pathologies with dysbiosis as the one observed in SBS patients, was assessed in SBS rats with jejuno-colonic anastomosis. The evolution of weight and food intake, the lenght of intestinal villi and crypts and the composition of fecal microbiota of Sham and SBS rats, transplanted or not with high fat diet rat microbiota, were analyzed. All SBS rats lost weight, increased their food intake and exhibited jejunal and colonic hyperplasia. Microbiota composition of SBS rats, transplanted or not, was largely enriched with Lactobacillaceae, and α- and ß-diversity were significantly different from Sham. The FMT altered microbiota composition and α- and ß-diversity in Sham but not SBS rats. FMT from high fat diet rats was successfully engrafted in Sham, but failed to take hold in SBS rats, probably because of the specific luminal environment in colon of SBS subjects favoring aero-tolerant over anaerobic bacteria. Finally, the level of food intake in SBS rats was positively correlated with their Lactobacillaceae abundance. Microbiota transfer must be optimized and adapted to this specific SBS environment.

Human BAHD1 promotes heterochromatic gene silencing.

Gene silencing via heterochromatin formation plays a major role in cell differentiation and maintenance of homeostasis. Here we report the identification and characterization of a novel heterochromatinization factor in vertebrates, bromo adjacent homology domain-containing protein 1 (BAHD1). This nuclear protein interacts with HP1, MBD1, HDAC5, and several transcription factors. Through electron and immunofluorescence microscopy studies, we show that BAHD1 overexpression directs HP1 to specific nuclear sites and promotes the formation of large heterochromatic domains, which lack acetyl histone H4 and are enriched in H3 trimethylated at lysine 27 (H3K27me3). Furthermore, ectopically expressed BAHD1 colocalizes with the heterochromatic inactive X chromosome (Xi). The BAH domain is required for BAHD1 colocalization with H3K27me3, but not with the Xi chromosome. As highlighted by whole genome microarray analysis of BAHD1 knockdown cells, BAHD1 represses several proliferation and survival genes, in particular the insulin-like growth factor II gene (IGF2). When overexpressed, BAHD1 specifically binds the CpG-rich P3 promoter of IGF2, which increases MBD1 and HDAC5 targeting at this locus. This region contains DNA-binding sequences for the transcription factor SP1, with which BAHD1 coimmunoprecipitates. Collectively, these findings provide evidence that BAHD1 acts as a silencer by recruiting at specific promoters a set of proteins that coordinate heterochromatin assembly.

The Heterochromatin protein 1 is a regulator in RNA splicing precision deficient in ulcerative colitis.

Defects in RNA splicing have been linked to human disorders, but remain poorly explored in inflammatory bowel disease (IBD). Here, we report that expression of the chromatin and alternative splicing regulator HP1γ is reduced in ulcerative colitis (UC). Accordingly, HP1γ gene inactivation in the mouse gut epithelium triggers IBD-like traits, including inflammation and dysbiosis. In parallel, we find that its loss of function broadly increases splicing noise, favoring the usage of cryptic splice sites at numerous genes with functions in gut biology. This results in the production of progerin, a toxic splice variant of prelamin A mRNA, responsible for the Hutchinson-Gilford Progeria Syndrome of premature aging. Splicing noise is also extensively detected in UC patients in association with inflammation, with progerin transcripts accumulating in the colon mucosa. We propose that monitoring HP1γ activity and RNA splicing precision can help in the management of IBD and, more generally, of accelerated aging.

Crohn's Disease: Is the Cold Chain Hypothesis Still Hot?

Crohn's disease [CD] is an inflammatory bowel disease of unknown aetiology. During recent decades, significant technological advances led to development of -omic datasets allowing a detailed description of the disease. Unfortunately these have not, to date, resolved the question of the aetiology of CD. Thus, it may be necessary to [re]consider hypothesis-driven approaches to resolve the aetiology of CD. According to the cold chain hypothesis, the development of industrial and domestic refrigeration has led to frequent exposure of human populations to bacteria capable of growing in the cold. These bacteria, at low levels of exposure, particularly those of the genus Yersinia, are believed to be capable of inducing exacerbated inflammation of the intestine in genetically predisposed subjects. We discuss the consistency of this working hypothesis in light of recent data from epidemiological, clinical, pathological, microbiological, and molecular studies.

Is Crohn's Disease the Price to Pay Today for Having Survived the Black Death?

BACKGROUND AND AIMS: Nucleotide Oligomerisation Domain 2 [NOD2] is a key gene of innate immunity which participates in the host defence against pathogens. Several loss-of-function NOD2 mutations are associated with Crohn's disease [CD]. Their high frequencies in populations of European ancestry suggest a model of balancing selection. Because NOD2 deficiency has been associated with a resistance to Yersinia pseudotuberculosis in mice, we hypothesised that NOD2 mutations have been selected during past plague outbreaks due to the closely related bacterium Yersinia pestis. METHODS: Contemporary frequencies of the main CD-associated NOD2 mutations [R702W, G908R, and 1007fs], measured in healthy people from European and Mediterranean countries, were collected from 60 studies via a PubMed search. Plague exposure was calculated from a dataset providing outbreaks from 1346 to 1860 in Europe and the Mediterranean Bassin. A plague index was built to capture the intensity of plague exposure in the studied geographical areas. RESULTS: NOD2 mutation frequencies were associated with the past exposure to plague. Statistical significance was obtained for the most frequent mutation [R702W, p = 0.03] and for the pooled three mutations [p = 0.023]. The association remained significant when putative demographic biases were considered. CONCLUSIONS: This result argues for a selection of CD-associated NOD2 mutations by plague outbreaks and further questioned the role of exposure to enteropathogenic Yersinia species in CD.

Prevalence of Yersinia Species in the Ileum of Crohn's Disease Patients and Controls.

Yersinia are common contaminants of food products, but their prevalence in the human gut is poorly documented. Yersinia have been implicated in Crohn's Disease (CD, an inflammatory bowel disease) however their role in CD is controversial. We performed highly sensitive PCR assays of specific sequences for the gyrB gene of Y. aldovae, Y. bercovieri, Y. enterocolitica, Y. intermedia, Y. mollaretii and the inv gene of Y. pseudotuberculosis. We analyzed a total of 470 ileal samples taken from 338 participants (262 CD patients and 76 controls) belonging to three independent cohorts. All patients and controls were phenotyped and genotyped for the main CD susceptibility variants: NOD2, ATG16L1, and IRGM. Yersinia were found in 7.7% of ileal samples (respectively 7.9 and 7.6% in controls and CD patients) corresponding to 10% of participants (respectively 11.8 and 9.5% in controls and CD patients). Y. enterocolitica, Y. pseudotuberculosis and Y. intermedia were the most frequently identified species. The bacteria were more frequent in resected specimens, lymph nodes and Peyer's patches. Yersinia were no more likely to be detected in CD tissues than tissues from inflammatory and non-inflammatory controls. CD patients treated with immunosuppressants were less likely to be Yersinia carriers. In conclusion, this work shows that Yersinia species are frequently found at low levels in the human ileum in health and disease. The role of Yersinia species in this ecosystem should now be explored.

The superoxide dismutase inhibitor diethyldithiocarbamate has antagonistic effects on apoptosis by triggering both cytochrome c release and caspase inhibition.

Tumor necrosis factor-alpha (TNF-alpha) and etoposide both trigger a large and rapid production of reactive oxygen species (ROS) in HeLa cells. This occurs before translocations of the proapoptotic Bax and cytochrome c proteins, the loss of mitochondrial membrane potential (DeltaPsim), and apoptosis. We have used diethyldithiocarbamate (DDC), a well-known inhibitor of Cu, Zn superoxide dismutase to study the role of ROS in this system. We report that DDC strongly inhibits caspase activation, loss of DeltaPsim, and cell death induced by TNF-alpha or etoposide. Surprisingly, DDC does not inhibit Bax and cytochrome c translocations. On the contrary, we have observed that DDC can trigger the translocations of these proteins by itself, without altering DeltaPsim. Here, we report that DDC has at least two antagonistic apoptosis regulation functions. First, DDC triggers ROS-dependent Bax and cytochrome c translocations, which are potentially proapoptotic, and second, DDC inhibits caspase activation and activity, loss of DeltaPsim, and cell death, in a ROS-independent manner. Our results suggest an interesting model in which ROS-dependent Bax and cytochrome c translocations can be studied without interference from later apoptotic events.

The protein phosphatase 2A regulatory subunit PR70 is a gonosomal melanoma tumor suppressor gene.

Male gender is independently and significantly associated with poor prognosis in melanoma of all clinical stages. The biological underpinnings of this sex difference remain largely unknown, but we hypothesized that gene expression from gonosomes (sex chromosomes) might play an important role. We demonstrate that loss of the inactivated X chromosome in melanomas arising in females is strongly associated with poor distant metastasis-free survival, suggesting a dosage benefit from two X chromosomes. The gonosomal protein phosphatase 2 regulatory subunit B, beta (PPP2R3B) gene is located on the pseudoautosomal region (PAR) of the X chromosome in females and the Y chromosome in males. We observed that, despite its location on the PAR that predicts equal dosage across genders, PPP2R3B expression was lower in males than in females and was independently correlated with poor clinical outcome. PPP2R3B codes for the PR70 protein, a regulatory substrate-recognizing subunit of protein phosphatase 2A. PR70 decreased melanoma growth by negatively interfering with DNA replication and cell cycle progression through its role in stabilizing the cell division cycle 6 (CDC6)-chromatin licensing and DNA replication factor 1 (CDT1) interaction, which delays the firing of origins of DNA replication. Hence, PR70 functionally behaves as an X-linked tumor suppressor gene.

DNA double-strand break repair signalling: the case of RAD51 post-translational regulation.

DNA double-strand breaks (DSBs) are the major lethal lesion induced by ionizing radiation or by replication block. However, cells can take advantage of DSB-induced recombination in order to generate genetic diversity in physiological processes such as meiosis and V(D)J recombination. Two main alternative pathways compete for DSB repair: homologous recombination (HR) and non-homologous end-joining (NHEJ). This review will briefly present the mechanisms and the enzymatic complex for HR and NHEJ. The signalling of the DSB through the ATM pathway will be presented. Then, we will focus on the case of the RAD51 protein, which plays a pivotal role in HR and is conserved from bacteria to humans. Post-translational regulation of RAD51 is presented. Two contrasting situations are discussed: one with up-regulation (expression of the oncogene BCR/ABL) and one with a down-regulation (expression of the oncogene BCL-2) of RAD51, associated with apoptosis inhibition and tumour predisposition.

p53 in breast cancer subtypes and new insights into response to chemotherapy.

Despite an obvious central role of p53 in the hallmarks of cancer, TP53 status is not yet used for the management of breast cancer. Recent findings may lead to reconsider the role of p53 in breast cancer. TP53 mutations are the most frequent genetic alterations in breast cancer, observed in 30% of breast carcinomas. Their distribution is highly linked to molecular tumor subtypes found in 26% of luminal tumors (17% of luminal A, 41% of luminal B), in 50% of HER2 amplified tumors, in 69% of molecular apocrine breast carcinomas and in 88% of basal-like carcinomas. The type of mutation is linked to the tumor subtype with higher frequency of base-pair substitutions in luminal tumors, whereas molecular apocrine and basal-like tumors present much higher frequency of complex mutations (deletions/insertions). The timing of TP53 mutation also depends on the tumor subtype, being the first important event in luminal tumors but occurring after PTEN loss in basal-like tumors. Regarding response to cytotoxic chemotherapy, the situation is far from the p53-dependent apoptosis paradigm with subsequent clinical response. We reported that TP53 mutated non inflammatory locally advanced breast carcinomas had a high rate of complete pathological response to dose-dense doxorubicin-cyclophosphamide chemotherapy, while TP53 wild-type (WT) tumors never achieved complete response. Using human breast cancer xenograft models, we suggested that this could be due to the induction of senescence in TP53 WT tumor cells. A recent work confirmed these findings in MMTV-Wnt1 mammary tumors, showing that growth arrest and senescent phenotype, not apoptosis, were induced in TP53 WT tumors following doxorubicin treatment, while lack of arrest in mutant tumors resulted in aberrant mitoses, cell death and a superior clinical response. Furthermore, in ER positive (ER(+)) breast tumors, it has been recently reported that ER represses the p53-mediated apoptotic response induced by DNA damage. Taken together, these data can help to better understand p53-mediated response to doxorubicin-based chemotherapy in breast cancer: in ER(+) TP53 WT breast cancers, ER-induced inhibition of p53 apoptotic response would lead preferentially to tumor cell senescence and subsequent resistance to treatment. Conversely, in ER negative (ER(-)) TP53 mutated breast cancers, accumulation of genetic abnormalities would lead to mitotic catastrophe and subsequent better response. In view of these recent results, p53 impact in breast cancer should be reconsidered.