MiRNA Genes Constitute New Targets for Microsatellite Instability in Colorectal Cancer

Mismatch repair-deficient colorectal cancers (CRC) display widespread instability at DNA microsatellite sequences (MSI). Although MSI has been reported to commonly occur at coding repeats, leading to alterations in the function of a number of genes encoding cancer-related proteins, nothing is known about the putative impact of this process on non-coding microRNAs. In miRbase V15, we identified very few human microRNA genes with mono- or di-nucleotide repeats (n = 27). A mutational analysis of these sequences in a large series of MSI CRC cell lines and primary tumors underscored instability in 15 of the 24 microRNA genes successfully studied at variable frequencies ranging from 2.5% to 100%. Following a maximum likelihood statistical method, microRNA genes were separated into two groups that differed significantly in their mutation frequencies and in their tendency to represent mutations that may or may not be under selective pressures during MSI tumoral progression. The first group included 21 genes that displayed no or few mutations in CRC. The second group contained three genes, i.e., hsa-mir-1273c, hsa-mir-1303 and hsa-mir-567, with frequent (≥80%) and sometimes bi-allelic mutations in MSI tumors. For the only one expressed in colonic tissues, hsa-mir-1303, no direct link was found between the presence or not of mono- or bi-allelic alterations and the levels of mature miR expression in MSI cell lines, as determined by sequencing and quantitative PCR respectively. Overall, our results provide evidence that DNA repeats contained in human miRNA genes are relatively rare and preserved from mutations due to MSI in MMR-deficient cancer cells. Functional studies are now required to conclude whether mutated miRNAs, and especially the miR-1303, might have a role in MSI tumorigenesis

Extracellular HSP110 skews macrophage polarization in colorectal cancer

IF 7.644International audienceHSP110 is induced by different stresses and, through its anti-apoptotic and chaperoning properties, helps the cells to survive these adverse situations. In colon cancers, HSP110 is abnormally abundant. We have recently showed that colorectal cancer (CRC) patients with microsatellite instability (MSI) had an improved response to chemotherapy because they harbor an HSP110 inactivating mutation (HSP110DE9). In this work, we have used patients' biopsies and human CRC cells grown in vitro and in vivo (xenografts) to demonstrate that (1) HSP110 is secreted by CRC cells and that the amount of this extracellular HSP110 is strongly decreased by the expression of the mutant HSP110DE9, (2) Supernatants from CRC cells overexpressing HSP110 or purified recombinant human HSP110 (LPS-free) affect macrophage differentiation/polarization by favoring a pro-tumor, anti-inflammatory profile, (3) Conversely, inhibition of HSP110 (expression of siRNA, HSP110DE9 or immunodepletion) induced the formation of macrophages with a cytotoxic, pro-inflammatory profile. (4) Finally, this effect of extracellular HSP110 on macrophages seems to implicate TLR4. These results together with the fact that colorectal tumor biopsies with HSP110 high were infiltrated with macrophages with a pro-tumoral profile while those with HSP110 low were infiltrated with macrophages with a cytotoxic profile, suggest that the effect of extracellular HSP110 function on macrophages may also contribute to the poor outcomes associated with HSP110 expression

Classification of miRNAs with MNR according to their mutation frequencies in MSI CRCs.

<p>Two distinct groups of miRNAs with MNR are established based on their mutation frequencies in MSI primary tumors. The cut-off value is calculated by the ratio of likelihood statistical method and is marked by a dashed vertical line. Note that <i>hsa-mir-644</i> is included in the group of miRNAs rarely or not mutated in MSI CRCs (<i>n</i> = 18, frequency of mutation <25%) whereas <i>hsa-mir-1273c</i>, <i>hsa-mir-567</i> and <i>hsa-mir-1303</i> constitute the group of miRNAs frequently altered (<i>n</i> = 3, frequency of mutation >75%).</p

Secondary structures of WT and mutated <i>hsa-mir-1303</i> and expression levels of miR-1303 in CRC cell lines.

<p>A: Alterations in repeat sequences of <i>hsa-mir-1303</i> (A) and its variant (delA) did not seem to affect overall the secondary structure of the hairpin but the dimension of the loop (annoted inside) is slightly reduced as determined by mfold software (<a href="http://mfold.rna.albany.edu/" target="_blank">http://mfold.rna.albany.edu/</a>). Mature miR (bold letters) and MNR (underlined letters) are shown in both hairpin sequences. The arrows indicate the potential positions of an Adenine deletion that leads to an enlargement of the loop. B: Comparison of the relative expressions of mature miR-1303 in MSS (unaltered MNR) and MSI CRC cell lines with none, mono- or bi-allelic mutations of <i>hsa-mir-1303</i>. MiR expression was normalized to the expression of RNU48. Means are shown for each group (black horizontal line). A significant increase in the expression of miR-1303 was observed between MSS cell lines and normal colonic mucosae (<i>p</i> = 0.012). C: Absence of correlation between the size of mir-1303 loop and the levels of mature miR-1303 expression in MSI cell lines with no (HCT-8, TC7) or bi-allelic mutations (LS411, RKO, LIM2405, KM12, LoVo, HCT116) in MNR of <i>hsa-mir-1303</i>. Note cell lines that produce hairpin precursors with the same size of the loop do express mature miR-1303 at various levels.</p

MNR instabilities in <i>hsa-mir-1273c</i> (T11), <i>hsa-mir-567</i> (A13) and <i>hsa-mir-1303</i> (T13).

<p>Allelic profiles for several MSI CRC cell lines and primary tumors are shown. Normal profiles are defined in LBL and MSS cell lines and primary tumors. For monomorphic genes, a dashed vertical line indicates the unique allele. The polymorphic zone for <i>hsa-mir-1303</i> is defined between two dashed vertical lines going along the 2 alleles (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031862#pone.0031862.s001" target="_blank">Figure S1</a>). Sizes (bp) are indicated in a box below each profile. Various allelic deletions ranging from 1 to 4 bp were observed in MSI CRC cell lines and primary tumors and are indicated in bold. The observed deletions were sometimes bi-allelic in MSI CRC cell lines. In MSI primary tumors, the allelic profiles were also highly suggestive of bi-allelic mutations. Due to the inherent polymorphism that can modify the length of the sequence, the hairpin sequence of <i>hsa-mir-1303</i> was determined for a correct and reliable evaluation of the alterations in MSI CRC cell lines (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031862#pone.0031862.s005" target="_blank">Table S2</a>).</p

Polymorphism and somatic mutation frequency of microsatellite repeats in miRNA genes.

<p>NS, not significant;</p>a<p>the polymorphism rate is the percentage of normal samples showing length variations when compared to the major peak (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031862#pone.0031862.s004" target="_blank">Table S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031862#pone.0031862.s001" target="_blank">Figure S1</a>);</p>b<p>mutation rates were estimated by taking into account sizes that diverge from the normal polymorphism (refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031862#pone.0031862.s001" target="_blank">Figure S1</a>).</p

Correlation between the lengths of mononucleotide repeats in miRNAs and their mutation rates in MSI CRCs.

<p>Note the highly significant correlation observed.</p

Representative scheme of miRNA hairpins with repeats spaning different locations.

<p>The basal segment (BS, single-stranded RNA), stem (S, double-stranded RNA) and terminal loop (L) are designated. The duplex (D, containing one or two potential miRs) is considered as a different entity and therefore distinguished from the stem region. Regions of the hairpin covered by MNRs or DNRs are noted for each miRNAs. To the left of the scheme are miRNA genes whose sequence repeats overlap two regions.</p

Counteracting tryptophan metabolism alterations as a new therapeutic strategy for rheumatoid arthritis

International audienceObjectives Alterations in tryptophan (Trp) metabolism have been reported in inflammatory diseases, including rheumatoid arthritis (RA). However, understanding whether these alterations participate in RA development and can be considered putative therapeutic targets remains undetermined. In this study, we combined quantitative Trp metabolomics in the serum from patients with RA and corrective administration of a recombinant enzyme in experimental arthritis to address this question. Methods Targeted quantitative Trp metabolomics was performed on the serum from 574 previously untreated patients with RA from the ESPOIR (Etude et Suivi des POlyarthrites Indifférenciées Récentes) cohort and 98 healthy subjects. A validation cohort involved 69 established patients with RA. Dosages were also done on the serum of collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA) mice and controls. A proof-of-concept study evaluating the therapeutic potency of targeting the kynurenine pathway was performed in the CAIA model. Results Differential analysis revealed dramatic changes in Trp metabolite levels in patients with RA compared with healthy controls. Decreased levels of kynurenic (KYNA) and xanthurenic (XANA) acids and indole derivatives, as well as an increased level of quinolinic acid (QUIN), were found in the serum of patients with RA. They correlated positively with disease severity (assessed by both circulating biomarkers and disease activity scores) and negatively with quality-of-life scores. Similar profiles of kynurenine pathway metabolites were observed in the CAIA and CIA models. From a mechanistic perspective, we demonstrated that QUIN favours human fibroblast-like synoviocyte proliferation and affected their cellular metabolism, through inducing both mitochondrial respiration and glycolysis. Finally, systemic administration of the recombinant enzyme aminoadipate aminotransferase, responsible for the generation of XANA and KYNA, was protective in the CAIA model. Conclusions Altogether, our preclinical and clinical data indicate that alterations in the Trp metabolism play an active role in the pathogenesis of RA and could be considered as a new therapeutic avenue

The Balance Between Cytotoxic T-cell Lymphocytes and Immune Checkpoint Expression in the Prognosis of Colon Tumors

IF 12.589International audienceBackgroundImmune checkpoint (ICK) expression might represent a surrogate measure of tumor-infiltrating T cell (CTL) exhaustion and therefore be a more accurate prognostic biomarker for colorectal cancer (CRC) patients than CTL enumeration as measured by the Immunoscore.MethodsThe expression of ICKs, Th1, CTLs, cytotoxicity-related genes, and metagenes, including Immunoscore-like metagenes, were evaluated in three independent cohorts of CRC samples (260 microsatellite instable [MSI], 971 non-MSI). Their associations with patient survival were analyzed by Cox models, taking into account the microsatellite instability (MSI) status and affiliation with various Consensus Molecular Subgroups (CMS). PD-L1 and CD8 expression were examined on a subset of tumors with immunohistochemistry. All statistical tests were two-sided.ResultsThe expression of Immunoscore-like metagenes was statistically significantly associated with improved outcome in non-MSI tumors displaying low levels of both CTLs and immune checkpoints (ICKs; CMS2 and CMS3; hazard ratio [HR] = 0.63, 95% confidence interval [CI] = 0.43 to 0.92, P = .02; and HR = 0.55, 95% CI = 0.34 to 0.90, P = .02, respectively), but clearly had no prognostic relevance in CRCs displaying higher levels of CTLs and ICKs (CMS1 and CMS4; HR = 0.46, 95% CI = 0.10 to 2.10, P = .32; and HR = 1.13, 95% CI = 0.79 to 1.63, P = .50, respectively), including MSI tumors. ICK metagene expression was statistically significantly associated with worse prognosis independent of tumor staging in MSI tumors (HR = 3.46, 95% CI = 1.41 to 8.49, P = .007). ICK expression had a negative impact on the proliferation of infiltrating CD8 T cells in MSI neoplasms (median = 0.56 in ICK low vs median = 0.34 in ICK high, P = .004).ConclusionsICK expression cancels the prognostic relevance of CTLs in highly immunogenic colon tumors and predicts a poor outcome in MSI CRC patients