A complex of cadherin 17 with desmocollin 1 and p120-catenin regulates colorectal cancer migration and invasion according to the cell phenotype.

BACKGROUND: Cadherin-17 (CDH17), a marker of differentiation in intestinal cells, binds and activates α2ß1 integrin to promote cell adhesion and proliferation in colorectal cancer (CRC) metastasis. Furthermore, CDH17 associates with p120- and ß-catenin in a manner yet to be fully elucidated. In this report, we explored the molecular mediators involved in this association, their contribution to CRC dissemination and potential therapeutic implications. METHODS: Proteomic and confocal analyses were employed to identify and validate CDH17 interactors. Functional characterization involved the study of proliferation, migration, and invasion in cell lines representative of various phenotypes. Immunohistochemistry was conducted on CRC tissue microarrays (TMA). In vivo animal experiments were carried out for metastatic studies. RESULTS: We found that desmocollin-1 (DSC1), a desmosomal cadherin, interacts with CDH17 via its extracellular domain. DSC1 depletion led to increased or decreased invasion in CRC cells displaying epithelial or mesenchymal phenotype, respectively, in a process mediated by the association with p120-catenin. Down-regulation of DSC1 resulted in an increased expression of p120-catenin isoform 1 in epithelial cells or a shift in cellular location in mesenchymal cells. Opposite results were observed after forced expression of CDH17. DSC1 is highly expressed in budding cells at the leading edge of the tumor and associates with poor prognosis in the stem-like, mesenchymal CRC subtypes, while correlates with a more favorable prognosis in the less-aggressive subtypes. In vivo experiments demonstrated that DSC1 silencing reduced tumor growth, liver homing, and metastasis in CRC mesenchymal cells. Furthermore, a synthetic peptide derived from CDH17, containing the NLV motif, effectively inhibited invasion and liver homing in vivo, opening up new possibilities for the development of novel therapies focused on desmosomal cadherins. CONCLUSIONS: These findings shed light on the multifaceted roles of CDH17, DSC1, and p120-catenin in CRC metastasis, offering insights into potential therapeutic interventions for targeting desmosomal cadherins in poorly-differentiated carcinomas.

Molecular Classification of Colorectal Cancer by microRNA Profiling: Correlation with the Consensus Molecular Subtypes (CMS) and Validation of miR-30b Targets.

Colorectal cancer consensus molecular subtypes (CMSs) are widely accepted and constitutes the basis for patient stratification to improve clinical practice. We aimed to find whether miRNAs could reproduce molecular subtypes, and to identify miRNA targets associated to the High-stroma/CMS4 subtype. The expression of 939 miRNAs was analyzed in tumors classified in CMS. TALASSO was used to find gene-miRNA interactions. A miR-mRNA regulatory network was constructed using Cytoscape. Candidate gene-miR interactions were validated in 293T cells. Hierarchical-Clustering identified three miRNA tumor subtypes (miR-LS; miR-MI; and miR-HS) which were significantly associated (p < 0.001) to the reported mRNA subtypes. miR-LS correlated with the low-stroma/CMS2; miR-MI with the mucinous-MSI/CMS1 and miR-HS with high-stroma/CMS4. MicroRNA tumor subtypes and association to CMSs were validated with TCGA datasets. TALASSO identified 1462 interactions (p < 0.05) out of 21,615 found between 176 miRs and 788 genes. Based on the regulatory network, 88 miR-mRNA interactions were selected as candidates. This network was functionally validated for the pair miR-30b/SLC6A6. We found that miR-30b overexpression silenced 3'-UTR-SLC6A6-driven luciferase expression in 293T-cells; mutation of the target sequence in the 3'-UTR-SLC6A6 prevented the miR-30b inhibitory effect. In conclusion CRC subtype classification using a miR-signature might facilitate a real-time analysis of the disease course and treatment response.

Association of miR-21 and miR-335 to microsatellite instability and prognosis in stage III colorectal cancer.

BACKGROUND: MicroRNAs (miRs) are frequently altered in colorectal cancer (CRC) and can be used as prognostic factors. OBJECTIVE: To confirm in stage III CRC patients a reported miR signature that was associated to the presence of metastatic disease. To correlate miR expression with microsatellite instability (MSI) and mutations in RAS and BRAF. METHODS: miR-21, miR-135a, miR-206, miR-335 and miR-Let-7a expression was analyzed by RT-qPCR in 150 patients out of the 329 patients used to analyze MSI and RAS and BRAF mutations. Association with disease free survival (DFS) and overall survival (OS) was analyzed. Data was confirmed by a multivariate analysis. RESULTS: MiR-21 high expression (p= 0.034) and miR-335 low expression (p= 0.0061) were significantly associated with MSI-H. A positive trend (p= 0.0624) between miR-135a high expression and RAS mutations was found. Lower miR-21 expression levels are associated with DFS (HR = 2.654, 95% CI: 1.066-6.605, p= 0.036) and a trend with OS (HR = 2.419, 95% CI: 0.749-7.815, p= 0.140). MiR-21 high expression significantly improves DFS of the poor prognosis group (T4 or N2) (p= 0.03). CONCLUSIONS: Association of increased expression of miR-21 and better prognosis in the poor prognostic group may be of interest and could be explored in future prospective clinical trials.

Intracellular virion traffic to the endosome driven by cell type specific sialic acid receptors determines parvovirus tropism.

Parvoviruses are promising anticancer and gene therapy agents, but a deep knowledge of the entry process is crucial to exploit their therapeutic potential. We addressed this issue while attempting to retarget the oncolytic parvovirus minute virus of mice (MVMp) to the tumor vasculature. Residues at three functional domains of the icosahedral capsid were substituted by rational design with peptides competing with the vascular endothelial growth factor. Most substitutions impaired virus maturation, though some yielded infectious chimeric virions, and substitutions in a dimple at the twofold axis that allocates sialic acid (SIA) receptors altered viral tropism. One dimple-modified chimeric virion was efficiently attached as MVMp to α2-linked SIA moieties, but the infection was impaired by the binding to some inhibitory α2-3,-6,-8 SIA pseudoreceptors, which hampers intracellular virus traffic to the endosome in a cell type-dependent manner. Infectious from nonproductive traffic could be mechanistically discriminated by an endosomal drastic capsid structural transition comprising the cleavage of some VP2-Nt sequences and its associated VP1-Nt exposure. Correspondingly, neuraminidase removal of inhibitory SIA moieties enhanced the infection quantitatively, correlating to the restored virus traffic to the endosome and the extent of VP2-Nt cleavage/VP1-Nt exposure. This study illustrates (i) structural constraints to retarget parvoviruses with evolutionary adopted narrow grooves allocating small SIA receptors, (ii) the possibility to enhance parvovirus oncolysis by relaxing the glycan network on the cancer cell surface, and (iii) the major role played by the attachment to cell type-specific SIAs in the intracellular virus traffic to the endosome, which may determine parvovirus tropism and host range.

Antiangiogenic Vascular Endothelial Growth Factor-Blocking Peptides Displayed on the Capsid of an Infectious Oncolytic Parvovirus: Assembly and Immune Interactions.

As many tumor cells synthetize vascular endothelial growth factors (VEGF) that promote neo-vascularization and metastasis, frontline cancer therapies often administer anti-VEGF (α-VEGF) antibodies. To target the oncolytic parvovirus minute virus of mice (MVM) to the tumor vasculature, we studied the functional tolerance, evasion of neutralization, and induction of α-VEGF antibodies of chimeric viruses in which the footprint of a neutralizing monoclonal antibody within the 3-fold capsid spike was replaced by VEGF-blocking peptides: P6L (PQPRPL) and A7R (ATWLPPR). Both peptides allowed viral genome replication and nuclear translocation of chimeric capsid subunits. MVM-P6L efficiently propagated in culture, exposing the heterologous peptide on the capsid surface, and evaded neutralization by the anti-spike monoclonal antibody. In contrast, MVM-A7R yielded low infectious titers and was poorly recognized by an α-A7R monoclonal antibody. MVM-A7R showed a deficient assembly pattern, suggesting that A7R impaired a transitional configuration that the subunits must undergo in the 3-fold axis to close up the capsid shell. The MVM-A7R chimeric virus consistently evolved in culture into a mutant carrying the P6Q amino acid substitution within the A7R sequence, which restored normal capsid assembly and infectivity. Consistent with this finding, anti-native VEGF antibodies were induced in mice by a single injection of MVM-A7R empty capsids, but not by MVM-A7R virions. This fundamental study provides insights to endow an infectious parvovirus with immune antineovascularization and evasion capacities by replacing an antibody footprint in the capsid 3-fold axis with VEGF-blocking peptides, and it also illustrates the evolutionary capacity of single-stranded DNA (ssDNA) viruses to overcome engineered capsid structural restrictions.IMPORTANCE Targeting the VEGF signaling required for neovascularization by vaccination with chimeric capsids of oncolytic viruses may boost therapy for solid tumors. VEGF-blocking peptides (VEbp) engineered in the capsid 3-fold axis endowed the infectious parvovirus MVM with the ability to induce α-VEGF antibodies without adjuvant and to evade neutralization by MVM-specific antibodies. However, these properties may be compromised by structural restraints that the capsid imposes on the peptide configuration and by misassembly caused by the heterologous peptides. Significantly, chimeric MVM-VEbp resolved the structural restrictions by selecting mutations within the engineered peptides that restored efficient capsid assembly. These data show the promise of antineovascularization vaccines using chimeric VEbp-icosahedral capsids of oncolytic viruses but also raise safety concerns regarding the genetic stability of manipulated infectious parvoviruses in cancer and gene therapies.