Águeda Martínez-Barriocanal

Colorectal cancer causes >900,000 deaths every year and a deeper understanding of the molecular mechanisms underlying this disease will contribute to improve its clinical management and survival. Myosin Vb (MYO5B) regulates intracellular vesicle trafficking, and inactivation of this myosin disrupts the polarization and differentiation of intestinal epithelial cells causing microvillous inclusion disease (MVID), a rare congenital disorder characterized by intractable life-threatening diarrhea. Here, we show that the loss Myosin Vb interfered with the differentiation/polarization of colorectal cancer cells. Although modulation of Myosin Vb expression did not affect the proliferation of colon cancer cells, MYO5B inactivation increased their migration, invasion, and metastatic potential. Moreover, Myo5b inactivation in an intestine-specific knockout mouse model caused a >15-fold increase in the number of azoxymethane-initiated small intestinal tumors. Consistently, reduced expression of Myosin Vb in a cohort of 155 primary colorectal tumors was associated with shorter patient survival. In conclusion, we show here that loss of Myosin Vb reduces polarization/differentiation of colon cancer cells while enhancing their metastatic potential, demonstrating a tumor suppressor function for this myosin. Moreover, reduced expression of Myosin Vb in primary tumors identifies a subset of poor prognosis colorectal cancer patients that could benefit from more aggressive therapeutic regimens.

Gastric cancer constitutes the third leading cause of cancer mortality worldwide and tumors with diffuse histology have poor prognosis and low response rates to therapy. Missense mutations in the RHOA (Ras homolog family member A) GTPase have recently been identified in 24% of patients with diffuse gastric cancer. RHOA mutations are not randomly distributed along the coding sequence, but enriched in specific and recurrent hotspots. Substitution in codon 42 (Y42C) occurs in approximately 50% of the patients in which RHOA is mutated. RHOA orchestrates cellular processes such as proliferation, differentiation, migration and invasion, whose deregulation is essential for the onset, maintenance and progression of tumors. However, the functional role of wild type RHOA and RHOA mutants remains poorly characterized in gastric cancer. We used isogenic cell lines systems and animal models to study the impact of RHOA in the gastric tumorigenesis. RHOA loss strongly increased the proliferation, migration and invasion of diffuse gastric cancer cells in vitro, but also when growing as subcutaneous xenografts in immunodeficient mice, and in experimental mouse models of peritoneal and lung metastasis. Stomach tumor burden was also increased in transgenic mice conditionally expressing a dominant negative form of RHOA in the gastrointestinal tract (RHOA T19N). Moreover, cells overexpressing wild-type RHOA or G14V RHOA (a constitutive active form of the GTPase) exhibited reduced tumorigenic features both in vitro and in vivo. Conversely, diffuse gastric cancer cells engineered for overexpressing RHOA Y42C mutant, displayed enhanced growth and invasion in vitro. Importantly, stomach cancer tumors formed more efficiently in a transgenic mouse model in which we targeted the expression of Y42C RHOA mutant to the gastric epithelium. Collectively, these results demonstrate that RHOA has tumor-suppressive activity in diffuse gastric cancer, while the recurrent Y42C hotspot RHOA mutation found in these tumors is oncogenic. Citation Format: Agueda Martinez-Barriocanal, Higinio Dopeso, Lizbeth M. Jiménez-Flores, Juliana C. Santos, Estefania Anguita, Josipa Bilic, Rocio Nieto, Elia García-Vidal, Manuel Sánchez Martín, Stefania Landolfi, Kazuto Kobayashi, Javier Hernandez-Losa, Simo Schwartz, Santiago Ramón y Cajal, Diego Arango. Role of RHOA in diffuse gastric cancer tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 847.

Background: Cancer initiation and progression are driven by genetic and epigenetic changes. Although genome/exome sequencing has significantly contributed to the characterization of the genetic driver alterations, further investigation is required to systematically identify cancer driver genes regulated by promoter hypermethylation. Results: Using genome-wide analysis of promoter methylation in 45 colorectal cancer cell lines, we found that higher overall methylation levels were associated with microsatellite instability (MSI), faster proliferation and absence of APC mutations. Because epigenetically silenced genes could represent important oncogenic drivers, we used mRNA expression profiling of colorectal cancer cell lines and primary tumors to identify a subset of 382 (3.9%) genes for which promoter methylation was negatively associated with gene expression. Remarkably, a significant enrichment in zinc finger proteins was observed, including the transcriptional repressor ZBTB18. Re-introduction of ZBTB18 in colon cancer cells significantly reduced proliferation in vitro and in a subcutaneous xenograft mouse model. Moreover, immunohistochemical analysis revealed that ZBTB18 is frequently lost or reduced in colorectal tumors, and reduced ZBTB18 expression was found to be associated with lymph node metastasis and shorter survival of patients with locally advanced colorectal cancer. Conclusions: We identified a set of 382 genes putatively silenced by promoter methylation in colorectal cancer that could significantly contribute to the oncogenic process. Moreover, as a proof of concept, we demonstrate that the epigenetically silenced gene ZBTB18 has tumor suppressor activity and is a novel prognostic marker for patients with locally advanced colorectal cancer.

Whole genome and transcriptome sequencing technologies have led to the identification of many long non-coding RNAs (lncRNAs) and stimulated the research of their role in health and disease. LncRNAs participate in the regulation of critical signaling pathways including cell growth, motility, apoptosis and differentiation; and their expression has been found dysregulated in human tumors. Thus, lncRNAs have emerged as new players in the initiation, maintenance and progression of tumorigenesis. PVT1 (plasmacytoma variant translocation 1) lncRNA is located on chromosomal 8q24.21, a large locus frequently amplified in human cancers and predictive of increased cancer risk in genome-wide association studies (GWAS). Combined, colorectal and gastric adenocarcinomas are the most frequent tumor malignancies and also the leading cause of cancer-related deaths worldwide. PVT1 expression is elevated in gastrointestinal tumors and correlates with poor patient prognosis. In this review, we discuss the mechanisms of action underlying PVT1 oncogenic role in colorectal and gastric cancer such as MYC upregulation, miRNA production, competitive endogenous RNA (ceRNA) function, protein stabilization and epigenetic regulation. We also illustrate the potential role of PVT1 as prognostic biomarker and its relationship with resistance to current chemotherapeutic treatments.

T cell bispecific antibodies against an isoform of HER2 effectively target many HER2-expressing tumors but not normal tissues.

Reduced RHOA signalling has been shown to increase the growth/metastatic potential of colorectal tumours. However, the mechanisms of inactivation of RHOA signalling in colon cancer have not been characterised. A panel of colorectal cancer cell lines and large cohorts of primary tumours were used to investigate the expression and activity of RHOA, as well as the presence of RHOA mutations/deletions and promoter methylation affecting RHOA. Changes in RHOA expression were assessed by western blotting and qPCR after modulation of microRNAs, SMAD4 and c-MYC. We show here that RHOA point mutations and promoter hypermethylation do not significantly contribute to the large variability of RHOA expression observed among colorectal tumours. However, RHOA copy number loss was observed in 16% of colorectal tumours and this was associated with reduced RHOA expression. Moreover, we show that miR-200a/b/429 downregulates RHOA in colorectal cancer cells. In addition, we found that TGF-β/SMAD4 upregulates the RHOA promoter. Conversely, RHOA expression is transcriptionally downregulated by canonical Wnt signalling through the Wnt target gene c-MYC that interferes with the binding of SP1 to the RHOA promoter in colon cancer cells. We demonstrate a complex pattern of inactivation of the tumour suppressor gene RHOA in colon cancer cells through genetic, transcriptional and post-transcriptional mechanisms.

Herein, we have used bioinformatics tools to predict five clusters defining ligand-binding sites on the extracellular domain of human CD300b receptor, presumably involved in the formation of both homodimers and heterodimers with other CD300 family members. Site-directed mutagenesis revealed residues glutamic acid 28 and glutamine 29 in cluster 5 to be necessary for the formation of CD300b complexes. Surprisingly, the disruption of cluster 2 and 4 reconstituted the binding capability lost by the mutation of residues glutamic acid 28 to alanine, glutamine 29 to alanine (E28A-Q29G). We identified a missense mutation arginine 33 to glutamine (R33Q) in CD300f by direct sequencing of exon 2 in peripheral blood samples from 50 patients with multiple sclerosis (MS). Levels of expression of CD300f were almost undetectable on monocytes from the patient bearing the R33Q mutation compared with healthy individuals. Whereas R33Q mutation had no effect in the formation of CD300f complexes, the inhibition of protein synthesis with cycloheximide indicated that CD300f R33Q is less stable than native CD300f. Finally, we report that the levels of expression of CD300f on the surface of classical and intermediate monocytes from MS patients are significantly lower when compared to the same cell populations in healthy individuals.

EPH signaling deregulation has been shown to be important for colorectal carcinogenesis and genome-wide sequencing efforts have identified EPHA3 as one of the most frequently mutated genes in these tumors. However, the role of EPHA3 in colorectal cancer has not been thoroughly investigated. We show here that ectopic expression of wild type EPHA3 in colon cancer cells did not affect their growth, motility/invasion or metastatic potential in vivo. Moreover, overexpression of mutant EPHA3 or deletion of the endogenous mutant EPHA3 in colon cancer cells did not affect their growth or motility. EPHA3 inactivation in mice did not initiate the tumorigenic process in their intestine, and had no effects on tumor size/multiplicity after tumor initiation either genetically or pharmacologically. In addition, immunohistochemical analysis of EPHA3 tumor levels did not reveal associations with survival or clinicopathological features of colorectal cancer patients. In conclusion, we show that EPHA3 does not play a major role in colorectal tumorigenesis. These results significantly contribute to our understanding of the role of EPH signaling during colorectal carcinogenesis, and highlighting the need for detailed functional studies to confirm the relevance of putative cancer driver genes identified in sequencing efforts of the cancer genome.