David Laperrière

Direct and Indirect Induction by 1,25-Dihydroxyvitamin D3 of the NOD2/CARD15-Defensin β2 Innate Immune Pathway Defective in Crohn Disease

Vitamin D signaling through its nuclear vitamin D receptor has emerged as a key regulator of innate immunity in humans. Here we show that hormonal vitamin D, 1,25-dihydroxyvitamin D3, robustly stimulates expression of pattern recognition receptor NOD2/CARD15/IBD1 gene and protein in primary human monocytic and epithelial cells. The vitamin D receptor signals through distal enhancers in the NOD2 gene, whose function was validated by chromatin immunoprecipitation and chromatin conformation capture assays. A key downstream signaling consequence of NOD2 activation by agonist muramyl dipeptide is stimulation of NF-κB transcription factor function, which induces expression of the gene encoding antimicrobial peptide defensin β2 (DEFB2/HBD2). Pretreatment with 1,25-dihydroxyvitamin D3 synergistically induced NF-κB function and expression of genes encoding DEFB2/HBD2 and antimicrobial peptide cathelicidin in the presence of muramyl dipeptide. Importantly, this synergistic response was also seen in macrophages from a donor wild type for NOD2 but was absent in macrophages from patients with Crohn disease homozygous for non-functional NOD2 variants. These studies provide strong molecular links between vitamin D deficiency and the genetics of Crohn disease, a chronic incurable inflammatory bowel condition, as Crohns pathogenesis is associated with attenuated NOD2 or DEFB2/HBD2 function.

Widespread Alu repeat-driven expansion of consensus DR2 retinoic acid response elements during primate evolution

BackgroundNuclear receptors are hormone-regulated transcription factors whose signaling controls numerous aspects of development and physiology. Many receptors recognize DNA hormone response elements formed by direct repeats of RGKTCA motifs separated by 1 to 5 bp (DR1-DR5). Although many known such response elements are conserved in the mouse and human genomes, it is unclear to which extent transcriptional regulation by nuclear receptors has evolved specifically in primates.ResultsWe have mapped the positions of all consensus DR-type hormone response elements in the human genome, and found that DR2 motifs, recognized by retinoic acid receptors (RARs), are heavily overrepresented (108,582 elements). 90% of these are present in Alu repeats, which also contain lesser numbers of other consensus DRs, including 50% of consensus DR4 motifs. Few DR2s are in potentially mobile AluY elements and the vast majority are also present in chimp and macaque. 95.5% of Alu-DR2s are distributed throughout subclasses of AluS repeats, and arose largely through deamination of a methylated CpG dinucleotide in a non-consensus motif present in AluS sequences. We find that Alu-DR2 motifs are located adjacent to numerous known retinoic acid target genes, and show by chromatin immunoprecipitation assays in squamous carcinoma cells that several of these elements recruit RARs in vivo. These findings are supported by ChIP-on-chip data from retinoic acid-treated HL60 cells revealing RAR binding to several Alu-DR2 motifs.ConclusionThese data provide strong support for the notion that Alu-mediated expansion of DR elements contributed to the evolution of gene regulation by RARs and other nuclear receptors in primates and humans.

A combined approach identifies a limited number of new thyroid hormone target genes in post-natal mouse cerebellum

Thyroid hormones act directly on gene transcription in the post-natal developing cerebellum, controlling neuronal, and glial cell differentiation. We have combined three experimental approaches to identify the target genes that are underlying this phenomenon: 1) a microarray analysis of gene expression to identify hormone responsive genes in the cerebellum of Pax8-/- mice, a transgenic mouse model of congenital hypothyroidism; 2) a similar microarray analysis on primary culture of cerebellum neurons; and 3) a bioinformatics screen of conserved putative-binding sites in the mouse genome. This identifies surprisingly a small set of target genes, which, for some of them, might be key regulators of cerebellum development and neuronal differentiation.

Evolution of the repertoire of nuclear receptor binding sites in genomes

Nuclear receptors (NRs) are ligand-dependent transcription factors with important roles in normal development and physiology and in a wide array of pathologies. While identification of natural or synthetic ligands for all human NRs has clarified their physiological roles and led to numerous therapeutic applications, much remains to be understood about the mechanisms by which NRs control transcription of specific networks of target genes. The DNA binding domain, composed of two C4 type zinc fingers, is the most conserved region in NRs. Binding motif selectivity stems from variations in dimerization interfaces in the DNA and ligand binding domains as well as from divergence in a few base-contacting residues in the first zinc finger. However, overlaps in DNA binding patterns suggest competition between different NRs at target sites in vivo, resulting in cross-regulation of subsets of target gene networks. As NRs can regulate transcription even when bound far from the transcription initiation sites of target genes, the considerable expansion in intergenic DNA in genomes throughout evolution likely contributes to the diversity and complexity of tissue-specific gene regulation by NRs. In particular, the presence of NR binding sites in the primate-specific Alu family of short-interspersed elements has been shown to confer transcriptional regulation by NRs to adjacent genes, although the impact on NR regulatory networks at the genome-wide scale remains to be more fully evaluated.

The Estrogen Receptor Cofactor SPEN Functions as a Tumor Suppressor and Candidate Biomarker of Drug Responsiveness in Hormone-Dependent Breast Cancers

The treatment of breast cancer has benefitted tremendously from the generation of estrogen receptor-α (ERα)-targeted therapies, but disease relapse continues to pose a challenge due to intrinsic or acquired drug resistance. In an effort to delineate potential predictive biomarkers of therapy responsiveness, multiple groups have identified several uncharacterized cofactors and interacting partners of ERα, including Split Ends (SPEN), a transcriptional corepressor. Here, we demonstrate a role for SPEN in ERα-expressing breast cancers. SPEN nonsense mutations were detectable in the ERα-expressing breast cancer cell line T47D and corresponded to undetectable protein levels. Further analysis of 101 primary breast tumors revealed that 23% displayed loss of heterozygosity at the SPEN locus and that 3% to 4% harbored somatically acquired mutations. A combination of in vitro and in vivo functional assays with microarray-based pathway analyses showed that SPEN functions as a tumor suppressor to regulate cell proliferation, tumor growth, and survival. We also found that SPEN binds ERα in a ligand-independent manner and negatively regulates the transcription of ERα targets. Moreover, we demonstrate that SPEN overexpression sensitizes hormone receptor-positive breast cancer cells to the apoptotic effects of tamoxifen, but has no effect on responsiveness to fulvestrant. Consistent with these findings, two independent datasets revealed that high SPEN protein and RNA expression in ERα-positive breast tumors predicted favorable outcome in patients treated with tamoxifen alone. Together, our data suggest that SPEN is a novel tumor-suppressor gene that may be clinically useful as a predictive biomarker of tamoxifen response in ERα-positive breast cancers.

Alu repeats as transcriptional regulatory platforms in macrophage responses to M. tuberculosis infection

To understand the epigenetic regulation of transcriptional response of macrophages during early-stage M. tuberculosis (Mtb) infection, we performed ChIPseq analysis of H3K4 monomethylation (H3K4me1), a marker of poised or active enhancers. De novo H3K4me1 peaks in infected cells were associated with genes implicated in host defenses and apoptosis. Our analysis revealed that 40% of de novo regions contained human/primate-specific Alu transposable elements, enriched in the AluJ and S subtypes. These contained several transcription factor binding sites, including those for members of the MEF2 and ATF families, and LXR and RAR nuclear receptors, all of which have been implicated in macrophage differentiation, survival, and responses to stress and infection. Combining bioinformatics, molecular genetics, and biochemical approaches, we linked genes adjacent to H3K4me1-associated Alu repeats to macrophage metabolic responses against Mtb infection. In particular, we show that LXRα signaling, which reduced Mtb viability 18-fold by altering cholesterol metabolism and enhancing macrophage apoptosis, can be initiated at response elements present in Alu repeats. These studies decipher the mechanism of early macrophage transcriptional responses to Mtb, highlighting the role of Alu element transposition in shaping human transcription programs during innate immunity.

MiSTIC, an integrated platform for the analysis of heterogeneity in large tumour transcriptome datasets.

Abstract Genome-wide transcriptome profiling has enabled non-supervised classification of tumours, revealing different sub-groups characterized by specific gene expression features. However, the biological significance of these subtypes remains for the most part unclear. We describe herein an interactive platform, Minimum Spanning Trees Inferred Clustering (MiSTIC), that integrates the direct visualization and comparison of the gene correlation structure between datasets, the analysis of the molecular causes underlying co-variations in gene expression in cancer samples, and the clinical annotation of tumour sets defined by the combined expression of selected biomarkers. We have used MiSTIC to highlight the roles of specific transcription factors in breast cancer subtype specification, to compare the aspects of tumour heterogeneity targeted by different prognostic signatures, and to highlight biomarker interactions in AML. A version of MiSTIC preloaded with datasets described herein can be accessed through a public web server (http://mistic.iric.ca); in addition, the MiSTIC software package can be obtained (github.com/iric-soft/MiSTIC) for local use with personalized datasets.

MCM2: An alternative to Ki-67 for measuring breast cancer cell proliferation

Breast cancer is a heterogeneous disease comprising a diversity of tumor subtypes that manifest themselves in a wide variety of clinical, pathological, and molecular features. One important subset, luminal breast cancers, comprises two clinically distinct subtypes luminal A and B each of them endowed with its own genetic program of differentiation and proliferation. Luminal breast cancers were operationally defined as follows: Luminal A: ER+, PR+, HER2-, Ki-67<14% and Luminal B: ER+ and/or PR+, HER2-,Ki-67≥14% or, alternatively ER+ and/or PR+, HER2+, any Ki-67. There is currently a need for a clinically robust and validated immunohistochemical assay that can help distinguish between luminal A and B breast cancer. MCM2 is a family member of the minichromosome maintenance protein complex whose role in DNA replication and cell proliferation is firmly established. As MCM2 appears to be an attractive alternative to Ki-67, we sought to study the expression of MCM2 and Ki-67 in different histological grades and molecular subtypes of breast cancer focusing primarily on ER-positive tumors. MCM2 and Ki-67 mRNA expression were studied using in silico analysis of available DNA microarray and RNA-sequencing data of human breast cancer. We next used immunohistochemistry to evaluate protein expression of MCM2 and Ki-67 on tissue microarrays of invasive breast carcinoma. We found that MCM2 and Ki-67 are highly expressed in breast tumors of high histological grades, comprising clinically aggressive tumors such as triple-negative, HER2-positive and luminal B subtypes. MCM2 expression was detected at higher levels than that of Ki-67 in normal breast tissues and in breast cancers. The bimodal distribution of MCM2 scores in ER+/HER2- breast tumors led to the identification of two distinct subgroups with different relapse-free survival rates. In conclusion, MCM2 expression can help sorting out two clinically important subsets of luminal breast cancer whose treatment and clinical outcomes are likely to diverge.

Deregulated Expression of ANXA1 in Human High-Grade Breast Cancers

Background: Breast cancer is a heterogeneous disease comprising a diversity of tumor subtypes. All of them differ from each other resulting in a wide array of clinical pictures, risks of recurrence and response to treatment. In recent years, tumor biomarkers have changed the way breast cancers are diagnosed and treated. In this paper we have sought to investigate the differential expression of ANXA1, a multifunctional calcium binding protein, among various molecular subtypes of breast cancers and in particular triple negative tumors. Methods: ANXA1 was first studied using in-silico analysis on available DNA microarray and RNA sequencing data of human breast tissues. Next we ascertained ANXA1 expression on cell lines and breast carcinoma tissue microarrays along with cognate normal breast tissue. Results: Whereas ANXA1 expression is normally restricted to the normal myoepithelial cell layer it becomes ectopically and aberrantly expressed in tumor cells of a significant minority of aggressive breast cancers. Specifically, we found that ANXA1 expression is severely deregulated in high-grade breast cancers that comprise clinically aggressive tumors such as triple-negative and, to some extent, HER2-positive breast cancers. Conclusion: Our results indicate that ANXA1 is a valuable breast cancer biomarker that can help to segregate and dissect out subsets of high histological grade breast cancers paving the way to a better understanding of breast cancer progression and metastasis.

Abstract A084: Identification of mutation-reactive T cells in patients with gastrointestinal cancers

New immunotherapeutic advances for common epithelial cancers relies on our ability to stimulate T lymphocytes against specific tumour antigens (Ag). Next-generation sequencing now allows rapid identification of somatic cancer mutations that can lead to the expression of mutated Ag. We hypothesize that gastrointestinal cancer metastases are infiltrated by T cells recognizing tumour mutated Ag. Our aim is to set up an experimental platform to screen for and study the frequency and function of mutation-reactive T cells, while characterizing novel tumour mutated Ag. To do this, we used two cancer cell lines generated from a liver metastasis of a gastric cancer patient. The cell line A was recognized by an autologous CD8+ T cell clone infiltrating the patient9s metastasis, restricted by HLA C*0701, while cell line B was not recognized by the same clone despite its expression of the HLA C*0701. The CD8+ T cell clone was not reactive to a large panel of HLA-C*0701 expressing gastrointestinal cancer cell lines, reinforcing the hypothesis that the Ag recognized was unique to the autologous gastric cancer cell line. Exome and transcriptome sequencing was performed to compare the mutated genes differentially expressed by cancer cell line A and B. A total of 27 mutated Ag were selected as candidates: 26 Ag only expressed by cell line A, and one Ag overexpressed by cell line A. To screen for reactivity of the CD8+ T cell clone to mutated Ag, 25 amino acid (aa) mini-genes containing the mutation flanked upstream and downstream by normal aa were synthesised and cloned in tandem into 3 expression plasmids. A control sequence from the MAGE-A12 gene containing an epitope restricted by HLA-C*0701was included in each tandem minigene (TMG) construct. The mRNA from these 3 TMG constructs were in vitro transcribed and electroporated into CD40-activated B cells expressing HLA C*0701, used as antigen presenting cells. Co-culture assays are ongoing, using the CD8+ T cell clones as effectors and T cells transduced with a MAGE-A12-specific TCR as control. For the detection of mutation-reactive T cells, the sensitivity of TMG expression is compared to pulsing mutated peptides on B cells. Taking advantage of the differential recognition of two cancer cell lines by an autologous CD8+ T cell clone, we have established a bioinformatics approach based on next-generation sequencing to obtain a list of candidate mutated Ag and we have designed an experimental system to assess their recognition by T cells. This platform will allow us to study the function of T cells reactive against metastatic gastrointestinal cancers and should lead to the discovery of new tumour Ag. Gaining a better understanding of T cells reactive to gastrointestinal cancers should ultimately contribute to the development of immunotherapies for these common malignancies. Citation Format: Melissa Mathieu, Sandy Pelletier, David Laperriere, Sylvie Mader, Simon Turcotte. Identification of mutation-reactive T cells in patients with gastrointestinal cancers. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A084.