Molecular-based targeted therapies in patients with hepatocellular carcinoma and hepato-cholangiocarcinoma refractory to atezolizumab/bevacizumab.

BACKGROUND & AIMS: The "French Medicine Genomic program 2025" has been designed to give patients with cancers that are refractory to systemic treatments access to off-label therapies adapted to their specific genomic profile. Herein, we reported the results of this program in patients with advanced hepatocellular carcinoma (HCC) and hepato-cholangiocarcinoma (H-CCK). METHODS: In one center, all patients with HCC or H-CCK who progressed under atezolizumab/bevacizumab with available tumor frozen samples benefited from whole-genome/-exome and RNA sequencing. Targeted therapies were matched to genomic alterations following the recommendations of a molecular tumor board and radiological response and overall survival were assessed. RESULTS: Among 135 patients with HCC and H-CCK treated by atezolizumab/bevacizumab, 20 patients benefited from genomic analysis after progression (16 HCC; 4 H-CCK). Nineteen patients had analyzable data, 70% were male, median age was 57 years, 65% had metastatic disease and 45% had vascular invasion. Among these 19 patients, 14 patients (76%) harbored at least one actionable genomic alteration and 9/14 received an adapted targeted therapy (45%). One patient with H-CCK showing CDK4 amplification was treated with palbociclib and achieved a partial radiological response for 16 months. Another patient with H-CCK, high HER2 overexpression and a high homologous recombination score was treated with trastuzumab/olaparib and had stable disease. One patient with an HCC and bi-allelic inactivation of TSC2 achieved a complete radiological response under everolimus. The remaining six treated patients (all HCC) had progressive disease, including three patients treated with trametinib, two with everolimus and one with olaparib. CONCLUSION: Molecular-based guided therapy is feasible in patients with HCC/H-CCK progressing under atezolizumab/bevacizumab and may be useful in a small subset of patients. IMPACT AND IMPLICATIONS: The use of whole-genome/-exome and RNA sequencing in clinical practice has not been reported in patients with hepatocellular carcinoma and hepato-cholangiocarcinoma. Herein, we performed a pilot study which suggested that whole-genome/-exome and RNA sequencing is feasible on tumor biopsies from patients refractory to atezolizumab/bevacizumab, with a small subset of patients exhibiting at least one actionable genomic alteration and receiving an adapted targeted therapy. This proof-of-concept study suggests that this clinical strategy could benefit a small subset of patients. Finally, validation of this approach will be required in a larger cohort of patients.

ROCK2 inhibition triggers the collective invasion of colorectal adenocarcinomas.

The metastatic progression of cancer is a multi-step process initiated by the local invasion of the peritumoral stroma. To identify the mechanisms underlying colorectal carcinoma (CRC) invasion, we collected live human primary cancer specimens at the time of surgery and monitored them ex vivo. This revealed that conventional adenocarcinomas undergo collective invasion while retaining their epithelial glandular architecture with an inward apical pole delineating a luminal cavity. To identify the underlying mechanisms, we used microscopy-based assays on 3D organotypic cultures of Caco-2 cysts as a model system. We performed two siRNA screens targeting Rho-GTPases effectors and guanine nucleotide exchange factors. These screens revealed that ROCK2 inhibition triggers the initial leader/follower polarization of the CRC cell cohorts and induces collective invasion. We further identified FARP2 as the Rac1 GEF necessary for CRC collective invasion. However, FARP2 activation is not sufficient to trigger leader cell formation and the concomitant inhibition of Myosin-II is required to induce invasion downstream of ROCK2 inhibition. Our results contrast with ROCK pro-invasive function in other cancers, stressing that the molecular mechanism of metastatic spread likely depends on tumour types and invasion mode.

A novel Chr1-miR-200 driven whole transcriptome signature shapes tumor immune microenvironment and predicts relapse in early-stage lung adenocarcinoma.

BACKGROUND: In Lung adenocarcinoma (LUAD), targeted therapies and immunotherapies have moved from metastatic to early stage and stratification of the relapse risk becomes mandatory. Here we identified a miR-200 based RNA signature that delineates Epithelial-to-mesenchymal transition (EMT) heterogeneity and predicts survival beyond current classification systems. METHODS: A miR-200 signature was identified using RNA sequencing. We scored the miR-200 signature by WISP (Weighted In Silico Pathology), used GSEA to identify pathway enrichments and MCP-counter to characterize immune cell infiltrates. We evaluate the clinical value of this signature in our series of LUAD and using TCGA and 7 published datasets. RESULTS: We identified 3 clusters based on supervised classification: I is miR-200-sign-down and enriched in TP53 mutations IIA and IIB are miR-200-sign-up: IIA is enriched in EGFR (p < 0.001), IIB is enriched in KRAS mutation (p < 0.001). WISP stratified patients into miR-200-sign-down (n = 65) and miR-200-sign-up (n = 42). Several biological processes were enriched in MiR-200-sign-down tumors, focal adhesion, actin cytoskeleton, cytokine/receptor interaction, TP53 signaling and cell cycle pathways. Fibroblast, immune cell infiltration and PDL1 expression were also significantly higher suggesting immune exhaustion. This signature stratified patients into high-vs low-risk groups, miR-200-sign-up had higher DFS, median not reached at 60 vs 41 months and within subpopulations with stage I, IA, IB, or II. Results were validated on TCGA data on 7 public datasets. CONCLUSION: This EMT and miR-200-related prognostic signature refines prognosis evaluation independently of tumor stage and paves the way towards assessing the predictive value of this LUAD clustering to optimize perioperative treatment.

Stratification of Pancreatic Ductal Adenocarcinomas Based on Tumor and Microenvironment Features.

BACKGROUND & AIMS: Genomic studies have revealed subtypes of pancreatic ductal adenocarcinoma (PDA) based on their molecular features, but different studies have reported different classification systems. It is a challenge to obtain high-quality, freshly frozen tissue for clinical analysis and determination of PDA subtypes. We aimed to redefine subtypes of PDA using a large number of formalin-fixed and paraffin-embedded PDA samples, which are more amenable to routine clinical evaluation. METHODS: We collected PDA samples from 309 consecutive patients who underwent surgery from September 1996 through December 2010 at 4 academic hospitals in Europe; nontumor tissue samples were not included. Samples were formalin fixed and paraffin embedded. DNA and RNA were isolated; gene expression, targeted DNA sequencing, and immunohistochemical analyses were performed. We used independent component analysis to deconvolute normal, tumor, and microenvironment transcriptome patterns in samples. We devised classification systems from an unsupervised analysis using a consensus clustering approach of our data set after removing normal contamination components. We associated subtypes with overall survival and disease-free survival of patients using Cox proportional hazards regression with estimation of hazard ratios and 95% confidence interval. We used The Cancer Genome Consortium and International Cancer Genome Consortium PDA data sets as validation cohorts. RESULTS: We validated the previously reported basal-like and classical tumor-specific subtypes of PDAs. We identified features of the PDA, including microenvironment gene expression patterns, that allowed tumors to be categorized into 5 subtypes, called pure basal like, stroma activated, desmoplastic, pure classical, and immune classical. These PDA subtypes have features of cancer cells and immune cells that could be targeted by pharmacologic agents. Tumor subtypes were associated with patient outcomes, based on analysis of our data set and the International Cancer Genome Consortium and The Cancer Genome Consortium PDA data sets. We also observed an exocrine signal associated with acinar cell contamination (from pancreatic tissue). CONCLUSIONS: We identified a classification system based on gene expression analysis of formalin-fixed PDA samples. We identified 5 PDA subtypes, based on features of cancer cells and the tumor microenvironment. This system might be used to select therapies and predict patient outcomes. We found evidence that the previously reported exocrine-like (called ADEX) tumor subtype resulted from contamination with pancreatic acinar cells. ArrayExpress accession number: E-MTAB-6134.

Association of IL-36γ with tertiary lymphoid structures and inflammatory immune infiltrates in human colorectal cancer.

IL-1 family cytokines play a dual role in the gut, with different family members contributing either protective or pathogenic effects. IL-36γ is an IL-1 family cytokine involved in polarizing type-1 immune responses. However, its function in the gut, including in colorectal cancer pathogenesis, is not well appreciated. In a murine model of colon carcinoma, IL-36γ controls tertiary lymphoid structure formation and promotes a type-1 immune response concurrently with a decrease in expression of immune checkpoint molecules in the tumor microenvironment. Here, we demonstrate that IL-36γ plays a similar role in driving a pro-inflammatory phenotype in human colorectal cancer. We analyzed a cohort of 33 primary colorectal carcinoma tumors using imaging, flow cytometry, and transcriptomics to determine the pattern and role of IL-36γ expression in this disease. In the colorectal tumor microenvironment, we observed IL-36γ to be predominantly expressed by M1 macrophages and cells of the vasculature, including smooth muscle cells and high endothelial venules. This pattern of IL-36γ expression is associated with a CD4+ central memory T cell infiltrate and an increased density of B cells in tertiary lymphoid structures, as well as with markers of fibrosis. Conversely, expression of the antagonist to IL-36 signaling, IL-1F5, was associated with intratumoral expression of checkpoint molecules, including PD-1, PD-L1, and CTLA4, which can suppress the immune response. These data support a role for IL-36γ in the physiologic immune response to colorectal cancer by sustaining inflammation within the tumor microenvironment.

HSP110 T17 simplifies and improves the microsatellite instability testing in patients with colorectal cancer.

BACKGROUND: Every colorectal cancer (CRC) patient should be tested for microsatellite instability (MSI, a marker for defective DNA mismatch repair) as a first screen for Lynch syndrome (LS). In this study, we investigated whether it may be possible to improve the detection of MSI in CRC. We examined whether the HT17 DNA repeat (critical for correct splicing of the chaperone HSP110) might constitute a superior marker for diagnosis of the MSI phenotype in patients with CRC compared with the standard panel of markers (pentaplex). METHODS: The HT17 polymorphism was analysed in germline DNA from 1037 multi-ethnic individuals. We assessed its sensitivity and specificity for detecting MSI in a multicentre, population-based cohort of 685 patients with CRC and an additional series of 70 patients with CRC considered to be at-risk of LS. All cases were screened earlier for MSI using pentaplex markers. Cases showing discordant HT17/pentaplex results were further examined for the expression of mismatch repair proteins. RESULTS: HT17 status was analysed independently and blinded to previous results from pentaplex genotyping. HT17 showed no germline allelic variation outside a very narrow range. Compared with the pentaplex panel, HT17 showed better sensitivity (0.984 (95% CI 0.968 to 0.995) vs 0.951 (95% CI 0.925 to 0.972)) and similar specificity (0.997 (95% CI 0.989 to 1.000) for both) for the detection of MSI. Furthermore, HT17 alone correctly classified samples judged to be uncertain with the pentaplex panel and showed excellent ability to detect MSI in patients with LS. CONCLUSIONS: HT17 simplifies and improves the current standard molecular methods for detecting MSI in CRC.

Patients with colorectal tumors with microsatellite instability and large deletions in HSP110 T17 have improved response to 5-fluorouracil­based chemotherapy.

BACKGROUND & AIMS: Patients with colorectal tumors with microsatellite instability (MSI) have better prognoses than patients with tumors without MSI, but have a poor response to 5-fluorouracil­based chemotherapy. A dominant-negative form of heat shock protein (HSP)110 (HSP110DE9) expressed by cancer cells with MSI, via exon skipping caused by somatic deletions in the T(17) intron repeat, sensitizes the cells to 5-fluorouracil and oxaliplatin.We investigated whether HSP110 T(17) could be used to identify patients with colorectal cancer who would benefit from adjuvant chemotherapy with 5-fluorouracil and oxaliplatin. METHODS: We characterized the interaction between HSP110 and HSP110DE9 using surface plasmon resonance. By using polymerase chain reaction and fragment analysis, we examined how the size of somatic allelic deletions in HSP110 T(17) affected the HSP110 protein expressed by tumor cells. We screened 329 consecutive patients with stage II­III colorectal tumors with MSI who underwent surgical resection at tertiary medical centers for HSP110 T(17). RESULTS: HSP110 and HSP110DE9 interacted in a1:1 ratio. Tumor cells with large deletions in T(17) had increased ratios of HSP110DE9:HSP110, owing to the loss of expression of full-length HSP110. Deletions in HSP110 T(17) were mostly biallelic in primary tumor samples with MSI. Patients with stage II­III cancer who received chemotherapy and had large HSP110 T(17) deletions (≥5 bp; 18 of 77 patients, 23.4%) had longer times of relapse-free survival than patients with small or no deletions (≤4 bp; 59 of 77 patients, 76.6%) in multivariate analysis (hazard ratio, 0.16; 95% confidence interval, 0.012­0.8; P = .03). We found a significant interaction between chemotherapy and T17 deletion (P =.009). CONCLUSIONS: About 25% of patients with stages II­III colorectal tumors with MSI have an excellent response to chemotherapy, due to large, biallelic deletions in the T(17) intron repeat of HSP110 in tumor DNA.

High-resolution analysis of DNA copy number alterations in rectal cancer: correlation with metastasis, survival, and mRNA expression.

BACKGROUND AND PURPOSE: This study aimed to determine the candidate genes and chromosomal imbalances capable of predicting occurrences of metastasis in patients with rectal cancer. PATIENTS AND METHODS: Fresh frozen tumor tissues from 80 patients with rectal cancer were prospectively collected and analyzed using Affymetrix HG-U133 Plus 2.0 gene expression arrays and high-resolution Illumina single-nucleotide polymorphism (SNP) arrays. Endpoints of the study were metastasis-free survival (MFS) and cancer-specific survival (CSS). RESULTS: The median follow-up was 102 months (1-146). Deletions of 8p and 1p36-35 correlated with worse MFS (p = 0.005 and p = 0.01, respectively) and CSS (p = 0.001 and p = 0.01, respectively). Multivariate analysis identified 8p deletion as an independent prognostic factor for MFS (p = 0.04) and CSS (p = 0.003); 97 genes located on the 8p chromosome were significantly underexpressed in tumors with 8p deletion. CONCLUSION: This study shows for the first time in rectal cancer an independent correlation of 8p deletion with MFS and CSS and highlights potential new tumor suppressor genes.

Extensive characterization of sphere models established from colorectal cancer cell lines.

Links between cancer and stem cells have been proposed for many years. As the cancer stem cell (CSC) theory became widely studied, new methods were developed to culture and expand cancer cells with conserved determinants of "stemness". These cells show increased ability to grow in suspension as spheres in serum-free medium supplemented with growth factors and chemicals. The physiological relevance of this phenomenon in established cancer cell lines remains unclear. Cell lines have traditionally been used to explore tumor biology and serve as preclinical models for the screening of potential therapeutic agents. Here, we grew cell-forming spheres (CFS) from 25 established colorectal cancer cell lines. The molecular and cellular characteristics of CFS were compared to the bulk of tumor cells. CFS could be isolated from 72 % of the cell lines. Both CFS and their parental CRC cell lines were highly tumorigenic. Compared to their parental cells, they showed similar expression of putative CSC markers. The ability of CRC cells to grow as CFS was greatly enhanced by prior treatment with 5-fluorouracil. At the molecular level, CFS and parental CRC cells showed identical gene mutations and very similar genomic profiles, although microarray analysis revealed changes in CFS gene expression that were independent of DNA copy-number. We identified a CFS gene expression signature common to CFS from all CRC cell lines, which was predictive of disease relapse in CRC patients. In conclusion, CFS models derived from CRC cell lines possess interesting phenotypic features that may have clinical relevance for drug resistance and disease relapse.

A de novo germline pathogenic BRCA1 variant identified following an osteosarcoma pangenomic molecular analysis.

De novo germline pathogenic variants (gPV) of the BReast CAncer 1 (BRCA1) gene are very rare. Only a few have been described up to date, usually in patients with a history of ovarian or breast cancer. Here, we report the first case of an incidental de novo BRCA1 germline pathogenic variant which was identified within the framework of the Plan France Médecine Génomique (PFMG) 2025 French national tumor sequencing program. The proband was a 29-year-old man diagnosed with metastatic osteosarcoma. Tumor whole exome sequencing identified a BRCA1 c.3756_3759del p.(Ser1253Argfs*10) pathogenic variant without loss-of-heterozygosity. A low genomic instability score and the absence of single base substitution signatures of homologous recombination deficiency suggested that the BRCA1 variant was not driver in the osteosarcoma tumorigenesis. Germline whole genome sequencing asserted the germline nature of this variant, with a 36% allele frequency, suggesting a mosaicism caused by a post-zygotic mutational event. The proband's family (parents and siblings) were not carriers of this variant confirming the de novo occurrence. Tumor sequencing programs like the French PFMG 2025 have been implemented worldwide and may help identify new gPV, including de novo variants.

Gene expression classification of colon cancer into molecular subtypes: characterization, validation, and prognostic value.

BACKGROUND: Colon cancer (CC) pathological staging fails to accurately predict recurrence, and to date, no gene expression signature has proven reliable for prognosis stratification in clinical practice, perhaps because CC is a heterogeneous disease. The aim of this study was to establish a comprehensive molecular classification of CC based on mRNA expression profile analyses. METHODS AND FINDINGS: Fresh-frozen primary tumor samples from a large multicenter cohort of 750 patients with stage I to IV CC who underwent surgery between 1987 and 2007 in seven centers were characterized for common DNA alterations, including BRAF, KRAS, and TP53 mutations, CpG island methylator phenotype, mismatch repair status, and chromosomal instability status, and were screened with whole genome and transcriptome arrays. 566 samples fulfilled RNA quality requirements. Unsupervised consensus hierarchical clustering applied to gene expression data from a discovery subset of 443 CC samples identified six molecular subtypes. These subtypes were associated with distinct clinicopathological characteristics, molecular alterations, specific enrichments of supervised gene expression signatures (stem cell phenotype-like, normal-like, serrated CC phenotype-like), and deregulated signaling pathways. Based on their main biological characteristics, we distinguished a deficient mismatch repair subtype, a KRAS mutant subtype, a cancer stem cell subtype, and three chromosomal instability subtypes, including one associated with down-regulated immune pathways, one with up-regulation of the Wnt pathway, and one displaying a normal-like gene expression profile. The classification was validated in the remaining 123 samples plus an independent set of 1,058 CC samples, including eight public datasets. Furthermore, prognosis was analyzed in the subset of stage II-III CC samples. The subtypes C4 and C6, but not the subtypes C1, C2, C3, and C5, were independently associated with shorter relapse-free survival, even after adjusting for age, sex, stage, and the emerging prognostic classifier Oncotype DX Colon Cancer Assay recurrence score (hazard ratio 1.5, 95% CI 1.1-2.1, p = 0.0097). However, a limitation of this study is that information on tumor grade and number of nodes examined was not available. CONCLUSIONS: We describe the first, to our knowledge, robust transcriptome-based classification of CC that improves the current disease stratification based on clinicopathological variables and common DNA markers. The biological relevance of these subtypes is illustrated by significant differences in prognosis. This analysis provides possibilities for improving prognostic models and therapeutic strategies. In conclusion, we report a new classification of CC into six molecular subtypes that arise through distinct biological pathways.

The atypical cadherin MUCDHL antagonizes colon cancer formation and inhibits oncogenic signaling through multiple mechanisms.

Cadherins form a large and pleiotropic superfamily of membranous proteins sharing Ca2+-binding repeats. While the importance of classic cadherins such as E- or N-cadherin for tumorigenesis is acknowledged, there is much less information about other cadherins that are merely considered as tissue-specific adhesion molecules. Here, we focused on the atypical cadherin MUCDHL that stood out for its unusual features and unique function in the gut. Analyses of transcriptomic data sets (n > 250) established that MUCDHL mRNA levels are down-regulated in colorectal tumors. Importantly, the decrease of MUCDHL expression is more pronounced in the worst-prognosis subset of tumors and is associated with decreased survival. Molecular characterization of the tumors indicated a negative correlation with proliferation-related processes (e.g., nucleic acid metabolism, DNA replication). Functional genomic studies showed that the loss of MUCDHL enhanced tumor incidence and burden in intestinal tumor-prone mice. Extensive structure/function analyses revealed that the mode of action of MUCDHL goes beyond membrane sequestration of ß-catenin and targets through its extracellular domain key oncogenic signaling pathways (e.g., EGFR, AKT). Beyond MUCDHL, this study illustrates how the loss of a gene critical for the morphological and functional features of mature cells contributes to tumorigenesis by dysregulating oncogenic pathways.

Intratumor CMS Heterogeneity Impacts Patient Prognosis in Localized Colon Cancer.

PURPOSE: The consensus molecular subtypes (CMS) represent a significant advance in the understanding of intertumor heterogeneity in colon cancer. Intratumor heterogeneity (ITH) is the new frontier for refining prognostication and understanding treatment resistance. This study aims at deciphering the transcriptomic ITH of colon cancer and understanding its potential prognostic implications. EXPERIMENTAL DESIGN: We deconvoluted the transcriptomic profiles of 1,779 tumors from the PETACC8 trial and 155 colon cancer cell lines as weighted sums of the four CMSs, using the Weighted In Silico Pathology (WISP) algorithm. We assigned to each tumor and cell line a combination of up to three CMS subtypes with a threshold above 20%. RESULTS: Over 55% of tumors corresponded to mixtures of at least two CMSs, demonstrating pervasive ITH in colon cancer. Of note, ITH was associated with shorter disease-free survival (DFS) and overall survival, [HR, 1.34; 95% confidence interval (CI; 1.12-1.59), 1.40, 95% CI (1.14-1.71), respectively]. Moreover, we uncovered specific combinations of CMS associated with dismal prognosis. In multivariate analysis, ITH represents the third parameter explaining DFS variance, after T and N stages. At a cellular level, combined WISP and single-cell transcriptomic analysis revealed that most colon cancer cell lines are a mixture of cells falling into different CMSs, indicating that ITH may correspond to distinct functional statuses of colon cancer cells. CONCLUSIONS: This study shows that CMS-based transcriptomic ITH is frequent in colon cancer and impacts its prognosis. CMS-based transcriptomic ITH may correspond to distinct functional statuses of colon cancer cells, suggesting plasticity between CMS-related cell populations. Transcriptomic ITH deserves further assessment in the context of personalized medicine.

Genomic consequences of cytochrome P450 2C9 overexpression in human hepatoma cells.

Cytochrome P450 2C9 (P450 2C9) is one of the most important P450 isoforms in the human liver, as it metabolizes numerous exogenous and endogenous substrates. Moreover, it is inducible by several compounds, such as rifampicin, phenobarbital, and NSAIDs (nonsteroidal anti-inflammatories). The aim of this study was to investigate the global cellular consequences of P450 2C9 overexpression at the transcriptional level using an untargeted approach: pangenomic microarrays. Recombinant adenovirus was used to express P450 2C9 instead of an inducer to prevent a per se effect of inducer or its metabolites. P450 2C9 overexpression induced endoplasmic reticulum (ER) stress and regulated genes implicated in the unfolded protein response (UPR) as heat shock protein (HSP) (we studied particurlarly HSPA5 and HSPB1) and in the endoplasmic reticulum associated degradation (ERAD) system as Sec61 and ubiquitin and proteasome pathways. UPR and ERAD are two mechanisms of adaptative response to ER stress. Moreover, activation of Akt was observed in HepG2 cells that overexpress P450 2C9 and might participate in the cellular adaptive response to stress, thus leading to the activation of cell survival pathways. UPR and ERAD should be caused by accumulation of native and misfolded P450 2C9 protein. Our results indicated that P450 2C9 overexpression did not lead to toxicity but induced an ER stress due to protein overexpression rather than mono-oxygenase activity. The ER stress triggered activation of the adaptative response and of pathways leading to cell survival.

Genomewide and biochemical analyses of DNA-binding activity of Cdc6/Orc1 and Mcm proteins in Pyrococcus sp.

The origin of DNA replication (oriC) of the hyperthermophilic archaeon Pyrococcus abyssi contains multiple ORB and mini-ORB repeats that show sequence similarities to other archaeal ORB (origin recognition box). We report here that the binding of Cdc6/Orc1 to a 5 kb region containing oriC in vivo was highly specific both in exponential and stationary phases, by means of chromatin immunoprecipitation coupled with hybridization on a whole genome microarray (ChIP-chip). The oriC region is practically the sole binding site for the Cdc6/Orc1, thereby distinguishing oriC in the 1.8 M bp genome. We found that the 5 kb region contains a previously unnoticed cluster of ORB and mini-ORB repeats in the gene encoding the small subunit (dp1) for DNA polymerase II (PolD). ChIP and the gel retardation analyses further revealed that Cdc6/Orc1 specifically binds both of the ORB clusters in oriC and dp1. The organization of the ORB clusters in the dp1 and oriC is conserved during evolution in the order Thermococcales, suggesting a role in the initiation of DNA replication. Our ChIP-chip analysis also revealed that Mcm alters the binding specificity to the oriC region according to the growth phase, consistent with its role as a licensing factor.

The cellular prion protein controls the mesenchymal-like molecular subtype and predicts disease outcome in colorectal cancer.

BACKGROUND: Comprehensive transcriptomic analyses have shown that colorectal cancer (CRC) is heterogeneous and have led to the definition of molecular subtypes among which the stem-cell, mesenchymal-like group is associated with poor prognosis. The molecular pathways orchestrating the emergence of this subtype are incompletely understood. In line with the contribution of the cellular prion protein PrPC to stemness, we hypothesize that deregulation of this protein could lead to a stem-cell, mesenchymal-like phenotype in CRC. METHODS: We assessed the distribution of the PrPC-encoding PRNP mRNA in two large CRC cohorts according to molecular classification and its association with patient survival. We developed cell-based assays to explore the impact of gain and loss of PrPC function on markers of the mesenchymal subtype and to delineate the signalling pathways recruited by PrPC. We measured soluble PrPC in the plasmas of 325 patients with metastatic CRC and probed associations with disease outcome. FINDINGS: We found that PRNP gene expression is enriched in tumours of the mesenchymal subtype and is associated with poor survival. Our in vitro analyses revealed that PrPC controls the expression of genes that specify the mesenchymal subtype through the recruitment of the Hippo pathway effectors YAP and TAZ and the TGFß pathway. We showed that plasma levels of PrPC are elevated in metastatic CRC and are associated with poor disease control. INTERPRETATION: Our findings define PrPC as a candidate driver of the poor-prognosis mesenchymal subtype of CRC. They suggest that PrPC may serve as a potential biomarker for patient stratification in CRC. FUNDING: Grant support was provided by the following: Cancéropôle Ile de France (grant number 2016-1-EMERG-36-UP 5-1), Association pour la Recherche sur le Cancer (grant number PJA 20171206220), SATT Ile de France Innov (grant number 415) as well as INSERM.

MAnGO: an interactive R-based tool for two-colour microarray analysis.

UNLABELLED: MAnGO (Microarray Analysis at the Gif/Orsay platform) is an interactive R-based tool for the analysis of two-colour microarray experiments. It is a compilation of various methods, which allows the user (1) to control data quality by detecting biases with a large number of visual representations, (2) to pre-process data (filtering and normalization) and (3) to carry out differential analyses. MAnGO is not only a 'turn-key' tool, oriented towards biologists but also a flexible and adaptable R script oriented towards bioinformaticians. AVAILABILITY: http://bioinfome.cgm.cnrs-gif.fr/.

The Cdx2 homeobox gene suppresses intestinal tumorigenesis through non-cell-autonomous mechanisms.

Developmental genes contribute to cancer, as reported for the homeobox gene Cdx2 playing a tumor suppressor role in the gut. In this study, we show that human colon cancers exhibiting the highest reduction in CDX2 expression belong to the serrated subtype with the worst evolution. In mice, mosaic knockout of Cdx2 in the adult intestinal epithelium induces the formation of imperfect gastric-type metaplastic lesions. The metaplastic knockout cells do not spontaneously become tumorigenic. However, they induce profound modifications of the microenvironment that facilitate the tumorigenic evolution of adjacent Cdx2-intact tumor-prone cells at the surface of the lesions through NF-κB activation, induction of inducible nitric oxide synthase, and stochastic loss of function of Apc This study presents a novel paradigm in that metaplastic cells, generally considered as precancerous, can induce tumorigenesis from neighboring nonmetaplastic cells without themselves becoming cancerous. It unveils the novel property of non-cell-autonomous tumor suppressor gene for the Cdx2 gene in the gut.

Increased expression of the thyroid hormone nuclear receptor TRα1 characterizes intestinal tumors with high Wnt activity.

Our previous work demonstrated a key function of the thyroid hormone nuclear receptor TRα1, a T3-modulated transcription factor, in controlling intestinal development and homeostasis via the Wnt and Notch pathways. Importantly, increased expression of TRα1 in the intestinal epithelium in a mutated Apc genetic background (vil-TRα1/Apc+/1638N mice) accelerated tumorigenesis and contributed to a more aggressive tumor phenotype compared to that of the Apc mutants alone. Therefore, the aim of this study was to determine the relevance of this synergistic effect in human colorectal cancers and to gain insights into the mechanisms involved. We analyzed cohorts of patients by in silico and experimental approaches and observed increased TRα1 expression and a significant correlation between TRα1 levels and Wnt activity. TRα1 loss-of-function and gain-of-function in Caco2 cell lines not only confirmed that TRα1 levels control Wnt activity but also demonstrated the role of TRα1 in regulating cell proliferation and migration. Finally, upon investigation of the molecular mechanisms responsible for the Wnt-TRα1 association, we described the repression by TRα1 of several Wnt inhibitors, including Frzb, Sox17 and Wif1. In conclusion, our results underline an important functional interplay between the thyroid hormone nuclear receptor TRα1 and the canonical Wnt pathway in intestinal cancer initiation and progression. More importantly, we show for the first time that the expression of TRα1 is induced in human colorectal cancers.