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Tumor heterogeneity and the presence of stem-like cells have been identified as key features for resistance to anticancer treatments including targeted therapy. The Eph receptors comprise a large family of receptor tyrosine kinases that marks stem-like cells in different tissues. In colorectal cancer (CRC), EphA2 receptor overexpression has been linked to stem-like properties of cells and malignancy. In particular, EphA2 is involved in multiple cross-talks with other cellular networks including EGFR, FAK and VEGF pathways, with which it collaborates to stimulate cell migration, invasion and metastasis. We investigated the molecular crosstalk and miRNAs modulation of the EphA2 and EGFR pathways in CRC. We also explored the role of EphA2/EGFR pathway mediators as prognostic factors or predictors of cetuximab benefit in CRC patients. We used a strategy to uncover in murine homogeneous tumor EphA2high cells a novel potential molecular signature involving EphA2 and EGFR pathways. Gene expression analysis was performed in EphA2high cells isolated from colorectal adenocarcinoma obtained from the AOM/DSS-induced CRC murine model. Six independent cohorts of patients were analyzed to determine the potential prognostic role of a EphA2/EGFR signature and its effect on cetuximab treatment response. We identified a gene expression pattern, including a coherent miRNAs dysregulation, reflecting the activation of EphA2 and EGFR pathways. Such pattern showed prognostic significance in stage I-III CRC patients, in both univariate and multivariate analysis. In patients with stage IV and WT KRAS, EphA2/Efna1/EGFR gene expression status was significantly associated with poor response to cetuximab. Furthermore, EphA2 and EGFR overexpression showed a combined effect relative to cetuximab resistance, independently from KRAS mutation status. Collectively, our data indicate that EphA2/Efna1/EGFR genes, linked to a possible control by miRNAs, could be proposed as novel prognostic biomarkers in CRC. Moreover, EphA2 could be linked to a mechanism of resistance to cetuximab alternative to KRAS mutations. Since cetuximab resistance, associated to an intrinsic genetic heterogeneity, remains the most critical issue in treating CRC, this study sheds light on new potential biomarkers and therapeutically actionable kinase targets in the EphA2/EGFR-linked pathway. Citation Format: Mariangela De Robertis, Luisa Loiacono, Caterina Fusilli, Maria Luana Poeta, Tommaso Mazza, Massimo Sanchez, Luigi Marchionni, Emanuela Signori, Giuseppe Lamorte, Angelo Luigi Vescovi, Jesus Garcia-Foncillas, Vito Michele Fazio. The EphA2/EGFR pathway dysregulation associates with poor prognosis and cetuximab treatment response in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4296.

Background: The definition of cancer stem cells (CSCs) as “tumor cells that contain self-renewal and multilineage differentiation capacity” still lacks conclusive experimental evidence. This is partly because the gold standard assay to identify CSCs, serial transplantation in animal models, is an operation that can undervalue total CSC numbers. Recently, the combination of mouse models that spontaneously develop tumors with genetic tracing provided exciting support for the CSC theory. Here, we took advantage of the use of cell-surface biomarkers, to purify, to some extent, murine cell populations with CSC phenotype by cell sorting on fresh spontaneous colorectal cancer (CRC). The aim of our study was to define a panel of miRNAs pathways with a potential function in the orchestration of selfrenewal and cell fate during cancer development using tissue of colorectal cancer from both mouse and human origin. Methods: Different populations of murine colon cells derived from AOM/DSS-induced carcinoma were isolated with cell sorting performed on a BD FACSAria system, by the expression of cell surface biomarkers (CD44, EphB2), then stem/progenitor cells-like property was analysed by the expression of stemness/differentiation genes (gene expression and IHC analysis). Specific regions of AOM/DSS treated colon mucosa containing dysplastic ACFs, microadenoma, adenoma, carcinoma compared to normal mucosa were isolated with Laser Capture Microdissection for RNA extraction. miRNAs expression profiling were performed using TaqMan Low Density Arrays (LifeTechnologies) both on cells and tissue samples. Microarray data were evaluated by state of the art statistics and bioinformatics tools. Single Real-Time PCR for selected miRNAs were performed on a training set of 60 FFPE human samples. Results: Global miRNAs expression profiles showed a total of ∼100 miRNAs with a significantly (p The analysis of miRNAs expression pattern in the sorted stem-like cell populations in comparison to miRNAs altered in the murine CRC initiation/progression steps revealed a significant cancer stemness signature, composed by miRNAs related to pluripotency and differentiation. Some of these changes, including miR-200b, mir-429, mir-365, mir-215, mir-26b, mir-135, were studied as well in the human samples, showing a similar pattern of that observed in the murine model. Conclusions: These data provide a comprehensive miRNAs signature implicated in the regulation of tumorigenesis, stemness, and cell fate determination that could be exploited for diagnosis and therapeutic design. Citation Format: Mariangela De Robertis, Eva Bandrés, Maria Luana Poeta, Luisa Loiacono, Giuseppe Lamorte, Massimo Sanchez, Luigi Marchionni, Maria Grazia Diodoro, Edoardo Pescarmona, Angelo Luigi Vescovi, Jesus Garcia-Foncillas, Vito Michele Fazio. Phase specific microRNA deregulation of oncogenesis and stemness in a mouse model of sporadic CRC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1465. doi:10.1158/1538-7445.AM2014-1465