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Supplementary Figure 2 - Patient characteristics in 6 cohorts analysed

Supplementary Figure 3 - Clinical characteristics of EphA2high and EphA2low patients in cohort 1 to 6

Purpose: EphA2 receptor 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. Colorectal cancer (CRC) EphA2 overexpression has also been correlated to stem-like properties of cells and tumor malignancy. We investigated the molecular cross-talk and miRNAs modulation of the EphA2 and EGFR pathways. We also explored the role of EphA2/EGFR pathway mediators as prognostic factors or predictors of cetuximab benefit in patients with CRC.Experimental Design: Gene expression analysis was performed in EphA2high cells isolated from CRC of the AOM/DSS murine model by FACS-assisted procedures. Six independent cohorts of patients were stratified by EphA2 expression to determine the potential prognostic role of a EphA2/EGFR signature and its effect on cetuximab treatment response.Results: We identified a gene expression pattern (EphA2, Efna1, Egfr, Ptpn12, and Atf2) reflecting the activation of EphA2 and EGFR pathways and a coherent dysregulation of mir-26b and mir-200a. Such a pattern showed prognostic significance in patients with stage I–III CRC, 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 treatment. Furthermore, EphA2 and EGFR overexpression showed a combined effect relative to cetuximab resistance, independently from KRAS mutation status.Conclusions: These results suggest that EphA2/Efna1/Egfr genes, linked to a possible control by miR-200a and miR-26b, could be proposed as novel CRC prognostic biomarkers. Moreover, EphA2 could be linked to a mechanism of resistance to cetuximab alternative to KRAS mutations. Clin Cancer Res; 23(1); 159–70. ©2016 AACR.

Supplementary Table 3 - Univariate and multivariate analyses of factors affecting PFS in patients who received Cetuximab monotherapy belonging to EphA2high group (cohort 5, N=40).

Kaplan-Meier survival curves of EGFRhigh (dashed line) versus EGFRlow (solid line) for cohort 1, 2 and 3. P-values were calculated using log-rank tests. Tick marks represent censored data. Expression value thresholds for determining high and low groups were found through maxstat R package. For the analysis conducted only for patients belonging to EphA2high group (below figures) Hazard Ratio and 95% Confidence Intervals are reported below each cohort analysed.

Supplementary Table 2A. KRAS mutational status vs. EphA2 expression (N = 70; P = 0.133{section sign}); 2B. Response rate vs. EphA2 expression (N = 68; P = 0.33{section sign}); 2C. Disease control rate vs. EphA2 expression (N = 68; P = 0.012{section sign}); 2D. Response rate vs. EphA2 expression (In KRAS WT patients; N = 39; P = 0.574{section sign}); 2E. Disease control rate vs. EphA2 expression (In KRAS WT patients; N = 39; P = 0.008{section sign})

Supplementary Table 1 - Univariate and multivariate analyses of factors affecting DFS of patients with EphA2high from pooled cohorts 1, 2, and 3 (N = 455).

Kaplan-Meier survival curves of (A) Ptpn12 (high versus low for EphA2high group) (B) Atf2 (high versus low for EphA2high group) and (C) Pik3CG (high versus low for EphA2high group) for cohorts in which results were significant. P-values were calculated using log-rank tests. Expression value thresholds for determining high and low groups were determined through maxstat R package. Tick marks represent censored data.

Supplementary Figure 1 - TaqMan assays ID (Applied Biosystems)

Purpose: Although early detection and high-quality treatment options, metastatic colorectal cancer (mCRC) is the main cause of CRC-related mortality. Dissecting the mechanisms underlying CRC progression may identify new accurate markers for invasive tumors, which can also be used as druggable targets, providing further improvement in the patients clinical outcomes. YKL-40 is a glycosyl hydrolase functionally linked to increased proliferation, angiogenesis, migration, and invasion of tumor cells. Because in colorectal cancer (CRC), YKL-40 serological level may serve as a risk predictor and prognostic biomarker, we investigated the underlying mechanisms by which it may contribute to tumor progression and the clinical significance of its tissue expression in metastatic CRC. Experimental Design: HCT116 and Caco2 cells genetically engineered to achieve YKL-40 silencing/overexpression were used for cell motility, invasion and proliferation assays, and to measure EMT markers. The YKL-40 expression level was assessed in the early and late tumor phases obtained in the AOM/DSS mouse model, as well as in tumors and sera from CRC patients. Six independent cohorts of CRC patients were stratified by YKL-40 tissue expression to define its prognostic role and its effect on cetuximab and oxaliplatin treatment response. Results: YKL-40 high-expressing HCT116 and Caco2 cells showed increased motility, invasion and proliferation. YKL-40 up-regulation was associated with EMT signaling activation. In murine and human samples, elevated YKL-40 levels correlated with high-grade tumors. In retrospective analyses, YKL-40 elevated expression correlated with shorter survival in patients with advanced CRC. Strikingly, YKL-40 high tissue levels showed a predictive value for better response to cetuximab, even in patients with stage IV CRC and mutant KRAS, and worse sensitivity to oxaliplatin. Conclusions: Our study recognizes a novel role of YKL-40 tissue expression in promoting CRC metastatic potential, through EMT signaling activation, and provides significant clinical implications that may impact the risk prediction of patients with mCRC. YKL-40 high tissue levels also strengthen a predictive value for better cetuximab responsiveness, even in patients with KRAS mutations. Since resistance to cetuximab remains one of the greatest challenges in treating CRC, our findings may be crucial for developing novel YKL-40-targeted therapy approaches. Citation Format: Mariangela De Robertis, Maria Raffaella Greco, Rosa Angela Cardone, Tommaso Mazza, Flaviana Marzano, Nikolay Mehterov, Maria Kazakova, Nikolay Belev, Apollonia Tullo, Graziano Pesole, Victoria Sarafian, Emanuela Signori. YKL-40 overexpression enhances metastatic potential and is a novel prognostic candidate and predictive biomarker for patients with colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2465.

Carcinogenesis; Microbiome; Tumor evolution Carcinogènesi; Microbioma; Evolució del tumor Carcinogénesis; Microbioma; Evolución del tumor Preclinical forms of gastrointestinal stromal tumor (GIST), small asymptomatic lesions, called microGIST, are detected in approximately 30% of the general population. Gastrointestinal stromal tumor driver mutation can be already detected in microGISTs, even if they do not progress into malignant cancer; these mutations are necessary, but insufficient events to foster tumor progression. Here we profiled the tissue microbiota of 60 gastrointestinal specimens in three different patient cohorts—micro, low-risk, and high-risk or metastatic GIST—exploring the compositional structure, predicted function, and microbial networks, with the aim of providing a complete overview of microbial ecology in GIST and its preclinical form. Comparing microGISTs and GISTs, both weighted and unweighted UniFrac and Bray–Curtis dissimilarities showed significant community-level separation between them and a pronounced difference in Proteobacteria, Firmicutes, and Bacteroidota was observed. Through the LEfSe tool, potential microbial biomarkers associated with a specific type of lesion were identified. In particular, GIST samples were significantly enriched in the phylum Proteobacteria compared to microGISTs. Several pathways involved in sugar metabolism were also highlighted in GISTs; this was expected as cancer usually displays high aerobic glycolysis in place of oxidative phosphorylation and rise of glucose flux to promote anabolic request. Our results highlight that specific differences do exist in the tissue microbiome community between GIST and benign lesions and that microbiome restructuration can drive the carcinogenesis process. Francesca Goriini and Federica Zanotti have been supported by Fondazione Cassa di Risparmio di Bologna.

To date there are several studies focusing on the importance of gut microbiome for human health, however the selection of a universal sampling matrix representative of the microbial biodiversity associated to the gastrointestinal (GI) tract, still represents a challenge. Here we present a study in which, through a deep metabarcoding analysis of the 16S rRNA gene, we compared two sampling matrices, feces (F) and colonic lavage liquid (LL), in order to evaluate their accuracy to represent the complexity of the human gut microbiome. A training set of 37 volunteers was attained and paired F and LL samples were collected from each subject. A preliminary absolute quantification of total 16S rDNA, performed by droplet digital PCR (ddPCR), confirmed that sequencing and taxonomic analysis were performed on same total bacterial abundance obtained from the two sampling methods. The taxonomic analysis of paired samples revealed that, although specific taxa were predominantly or exclusively observed in LL samples, as well as other taxa were detectable only or were predominant in stool, the microbiomes of the paired samples F and LL in the same subject hold overlapping taxonomic composition. Moreover, LL samples revealed a higher biodiversity than stool at all taxonomic ranks, as demonstrated by the Shannon Index and the Inverse Simpson’s Index. We also found greater inter-individual variability than intra-individual variability in both sample matrices. Finally, functional differences were unveiled in the gut microbiome detected in the F and LL samples. A significant overrepresentation of 22 and 13 metabolic pathways, mainly occurring in Firmicutes and Proteobacteria, was observed in gut microbiota detected in feces and LL samples, respectively. This suggests that LL samples may allow for the detection of microbes adhering to the intestinal mucosal surface as members of the resident flora that are not easily detectable in stool, most likely representative of a diet-influenced transient microbiota. This first comparative study on feces and LL samples for the study of the human gut microbiome demonstrates that the use of both types of sample matrices may represent a possible choice to obtain a more complete view of the human gut microbiota in response to different biological and clinical questions.

Simple Summary The mechanisms underlying the effects of exogenous factors on impaired intestinal stem niche homeostasis in colorectal cancer pathogenesis are an important and ongoing focus for stem cell research. The most recent findings indicate that dysbiosis (changes in the homeostatic gut microbiota composition) can induce an aberrant reprogramming of the intestinal stem cells (ISCs) through several mechanisms, such as impaired metabolism and abnormal activation of the immune system, as well as genetic and epigenetic instability. The review goes beyond the discussion of the involvement of gut dysbiosis in colorectal cancer development, mainly summarizing the most recent findings linking the gut microbiome to colorectal cancer pathogenesis through the ISC niche impairment. The most significant advances in this field are described, focusing on different “omics” strategies, with a particular interest for the multiomics approach which will be gradually included into the framework of precision medicine. Abstract Colorectal cancer (CRC) initiation is believed to result from the conversion of normal intestinal stem cells (ISCs) into cancer stem cells (CSCs), also known as tumor-initiating cells (TICs). Hence, CRC evolves through the multiple acquisition of well-established genetic and epigenetic alterations with an adenoma-carcinoma sequence progression. Unlike other stem cells elsewhere in the body, ISCs cohabit with the intestinal microbiota, which consists of a diverse community of microorganisms, including bacteria, fungi, and viruses. The gut microbiota communicates closely with ISCs and mounting evidence suggests that there is significant crosstalk between host and microbiota at the ISC niche level. Metagenomic analyses have demonstrated that the host-microbiota mutually beneficial symbiosis existing under physiologic conditions is lost during a state of pathological microbial imbalance due to the alteration of microbiota composition (dysbiosis) and/or the genetic susceptibility of the host. The complex interaction between CRC and microbiota is at the forefront of the current CRC research, and there is growing attention on a possible role of the gut microbiome in the pathogenesis of CRC through ISC niche impairment. Here we primarily review the most recent findings on the molecular mechanism underlying the complex interplay between gut microbiota and ISCs, revealing a possible key role of microbiota in the aberrant reprogramming of CSCs in the initiation of CRC. We also discuss recent advances in OMICS approaches and single-cell analyses to explore the relationship between gut microbiota and ISC/CSC niche biology leading to a desirable implementation of the current precision medicine approaches.

Exosome-like nanoparticles (ELNs) are attracting interest as important vehicles of intercellular communication, both in prokaryotes and eukaryotes. Recently, dietary nanoparticles similar to mammalian exosomes have attracted attention for these features. In particular they appear to be relevant in the modulation of several cellular processes as well as candidate carriers of bioactive molecules (proteins, lipids, and nucleic acids, including miRNAs) with therapeutic value. Herein, we investigated the cellular uptake of blueberry-derived ELNs (B-ELNs) by a human stabilized endothelial cell line (EA.hy926) and the ability of B-ELNs to modulate the expression of inflammatory genes as the response of tumor necrosis factor-&alpha, (TNF-&alpha, ). Our results indicate that 1) EA.hy926 cells internalize B-ELNs in a dose-dependent manner, 2) pretreatment with B-ELNs counters TNF-&alpha, induced reactive oxygen species (ROS) generation and loss of cell viability and modulates the differential expression of 29 genes (fold change &gt, 1.5) induced by TNF-&alpha, compared to control, 3) pathway analysis reveals their involvement in a total of 340 canonical pathways, 121 KEGG pathways, and 121 GO Biological processes, and 4) the intersection between differentially expressed (DE) genes and miRNAs contained in B-ELNs unveils a set of candidate target genes, such as prostaglandin I2 synthase (PTGIS), mitogen-activated protein kinase 14 (MAPK14), and phosphodiesterase 7A (PDE7A), for ELNs-contained cargo. In conclusion, our study indicates that B-ELNs can be considered candidate therapeutic carriers of bioactive compounds potentially able to protect vascular system against various stressors.

Gene Electro-Transfer (GET) is a powerful method of DNA delivery with great potential for medical applications. Although GET has been extensively studied in vitro and in vivo, the optimal parameters remain controversial. 2D cell cultures have been widely used to investigate GET protocols, but have intrinsic limitations, whereas 3D cultures may represent a more reliable model thanks to the capacity of reproducing the tumor architecture. Here we applied two GET protocols, using a plate or linear electrode, on 3D-cultured HCC1954 and MDA-MB231 breast cancer cell lines grown on a novel collagen-free 3D scaffold and compared results with conventional 2D cultures. To evaluate the electrotransfer efficiency, we used the plasmid pEGFP-C3 encoding the enhanced green fluorescent protein (EGFP) reporter gene. The novel 3D scaffold promoted extracellular matrix deposition, which particularly influences cell behavior in both in vitro cell cultures and in vivo tumor tissue. While the transfection efficiency was similar in the 2D-cultures, we observed significant differences in the 3D-model. The transfection efficiency in the 3D vs 2D model was 44% versus 15% (p &lt, 0.01) and 24% versus 17% (p &lt, 0.01) in HCC1954 and MDA-MB231 cell cultures, respectively. These findings suggest that the novel 3D scaffold allows reproducing, at least partially, the peculiar morphology of the original tumor tissues, thus allowing us to detect meaningful differences between the two cell lines. Following GET with plate electrodes, cell viability was higher in 3D-cultured HCC1954 (66%) and MDA-MB231 (96%) cell lines compared to their 2D counterpart (53% and 63%, respectively, p &lt, 0.001). Based on these results, we propose the novel 3D scaffold as a reliable support for the preparation of cell cultures in GET studies. It may increase the reliability of in vitro assays and allow the optimization of GET parameters of in vivo protocols.

Epigenetic modifications of glyco-genes have been documented in different types of cancer and are tightly linked to proliferation, invasiveness, metastasis, and drug resistance. This study aims to investigate the diagnostic, prognostic, and therapy-response predictive value of the glyco-gene B4GALT1 in colorectal cancer (CRC) patients. A Kaplan&ndash, Meier analysis was conducted in 1418 CRC patients (GEO and TCGA datasets) to assess the prognostic and therapy-response predictive values of the aberrant expression and methylation status of B4GALT1. Quantitative methylation-specific PCR (QMSP) and droplet digital quantitative methylation-specific PCR (dd-QMSP) were respectively used to detect hypermethylated B4GALT1 in metastasis and plasma in four cohorts of metastatic CRC cases (mCRC). Both the downregulated expression and promoter hypermethylation of B4GALT1 have a negative prognostic impact on CRC. Interestingly a low expression level of B4GALT1 was significantly associated with poor cetuximab response (progression-free survival (PFS) p = 0.01) particularly in wild-type (WT)-KRAS patients (p = 0.03). B4GALT1 promoter was aberrantly methylated in liver and lung metastases. The detection of hypermethylated B4GALT1 in plasma of mCRC patients showed a highly discriminative receiver operating characteristic (ROC) curve profile (area under curve (AUC) value 0.750, 95% CI: 0.592&ndash, 0.908, p = 0.008), clearly distinguishing mCRC patients from healthy controls. Based on an optimal cut-off value defined by the ROC analysis, B4GALT1 yield a 100% specificity and a 50% sensitivity. These data support the potential value of B4GALT1 as an additional novel biomarker for the prediction of cetuximab response, and as a specific and sensitive diagnostic circulating biomarker that can be detected in CRC.

Epigenetic modifications of glyco-genes have been documented in different types of cancer and are tightly linked to proliferation, invasiveness, metastasis, and drug resistance. This study aims to investigate the diagnostic, prognostic, and therapy-response predictive value of the glyco-gene B4GALT1 in colorectal cancer (CRC) patients. A Kaplan–Meier analysis was conducted in 1418 CRC patients (GEO and TCGA datasets) to assess the prognostic and therapy-response predictive values of the aberrant expression and methylation status of B4GALT1. Quantitative methylation-specific PCR (QMSP) and droplet digital quantitative methylation-specific PCR (dd-QMSP) were respectively used to detect hypermethylated B4GALT1 in metastasis and plasma in four cohorts of metastatic CRC cases (mCRC). Both the downregulated expression and promoter hypermethylation of B4GALT1 have a negative prognostic impact on CRC. Interestingly a low expression level of B4GALT1 was significantly associated with poor cetuximab response (progression-free survival (PFS) p = 0.01) particularly in wild-type (WT)-KRAS patients (p = 0.03). B4GALT1 promoter was aberrantly methylated in liver and lung metastases. The detection of hypermethylated B4GALT1 in plasma of mCRC patients showed a highly discriminative receiver operating characteristic (ROC) curve profile (area under curve (AUC) value 0.750; 95% CI: 0.592–0.908, p = 0.008), clearly distinguishing mCRC patients from healthy controls. Based on an optimal cut-off value defined by the ROC analysis, B4GALT1 yield a 100% specificity and a 50% sensitivity. These data support the potential value of B4GALT1 as an additional novel biomarker for the prediction of cetuximab response, and as a specific and sensitive diagnostic circulating biomarker that can be detected in CRC.

Background: Degenerative forms of mitral valve diseases (MVDs) are complex pathologies. Thus, it is difficult to make generalizations about MVD pathways or genetic risk factors. However, a key role of metalloproteinases (MMPs) in their pathophysiology is emerging. Thus, we performed a study to assess eventual associations of some functional SNPs in MMP-2 and MMP-9 genes with MVD risk, symptom severity and short- and long-term (4.8 years) complications. Methods: For this purpose, 90 patients and two control groups were genotyped for MMP-2 and MMP-9 gene SNPs, and systemic levels of proatrial natriuretic peptide (ANP), and two enzymes were quantified and correlated to the MMP-2 and MMP-9 SNPs. In addition, associations between these SNPs and symptom severity and short- and long-term complications were evaluated. Results: Interestingly, rs3918242 MMP-9 and rs2285053 MMP-2 SNPs were significantly represented in cases compared to two control groups, and were associated with a higher MVD risk. Cases stratified for New York Heart Association (NYHA) symptoms, and particularly NYHA III+IV, with rs3918242 CT+TT MMP-9 and rs2285053CT+TT genotypes also showed higher severity related to significant higher systemic levels of MMP enzymes and pro-ANP at enrollment and 4.8-year follow-up times. In addition, cases with these genotypes, and particularly NYHAIII+IV, had a very significant percentage of complications, particularly at the 4.8-year follow-up. Surprisingly, 20% of patient controls developed MVD at 4.8- year follow-up and were carriers of these genotypes. Conclusions: The associations observed seem to suggest that the two SNPs might represent useful biomarkers and targets for preventing and monitoring MVDs, leading to a more appropriate management and outcome

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.

Cancer vaccines based on plasmid DNA represent a good therapeutic perspective, despite their low potency. Animal-derived hyaluronidases (Hyals) are employed in oncological clinical practice. Hyal has been also demonstrated to be a good enhancer of intramuscular Gene Electro-Transfer (GET) efficiency in anti-cancer preclinical protocols, with increased transfected cells and higher expression of the encoded genes. Nevertheless, the use of animal-derived Hyals results limited respect to their potentialities, since such preparations could be affected by low purity, variable potency and uncertain safety. To improve the delivery of intramuscular GET-based protocols in mouse, we investigated a new recombinant Hyal, the rHyal-sk, to assess in vivo safety and activity of this treatment at cellular and biochemical levels. We evaluated the cellular events and the inflammation chemical mediators involved at different time points after rHyal-sk administration plus GET. Our results demonstrated the in vivo safety and efficacy of rHyal-sk when injected once intramuscularly in association with GET, with no toxicity, good plasmid in-take ability, useful inflammatory response activation, and low immunogenicity. Following these findings, we would recommend the use of the new rHyal-sk for the delivery of DNA-based vaccines and immunotherapy, as well as into clinical practice, for tumor disease treatments.

EphB2 and EphA2 control stemness and differentiation in the intestinal mucosa, but the way they cooperate with the complex mechanisms underlying tumor heterogeneity and how they affect the therapeutic outcome in colorectal cancer (CRC) patients, remain unclear. MicroRNA (miRNA) expression profiling along with pathway analysis provide comprehensive information on the dysregulation of multiple crucial pathways in CRC. Through a network-based approach founded on the characterization of progressive miRNAomes centered on EphA2/EphB2 signaling during tumor development in the AOM/DSS murine model, we found a miRNA-dependent orchestration of EphB2-specific stem-like properties in earlier phases of colorectal tumorigenesis and the EphA2-specific control of tumor progression in the latest CRC phases. Furthermore, two transcriptional signatures that are specifically dependent on the EphA2/EphB2 signaling pathways were identified, namely EphA2, miR-423-5p, CREB1, ADAMTS14, and EphB2, miR-31-5p, mir-31-3p, CRK, CXCL12, ARPC5, SRC. EphA2- and EphB2-related signatures were validated for their expression and clinical value in 1663 CRC patients. In multivariate analysis, both signatures were predictive of survival and tumor progression. The early dysregulation of miRs-31, as observed in the murine samples, was also confirmed on 49 human tissue samples including preneoplastic lesions and tumors. In light of these findings, miRs-31 emerged as novel potential drivers of CRC initiation. Our study evidenced a miRNA-dependent orchestration of EphB2 stem-related networks at the onset and EphA2-related cancer-progression networks in advanced stages of CRC evolution, suggesting new predictive biomarkers and potential therapeutic targets. Electronic supplementary material The online version of this article (10.1186/s12943-018-0912-z) contains supplementary material, which is available to authorized users.

Cancer stem cells (CSCs) in colorectal tumorigenesis are suggested to be responsible for initiation, development and propagation of colorectal cancer (CRC) and have been extensively characterized by the expression of phenotypic determinants, such as surface or intracellular proteins. The generation of CSCs is likely due to a dysregulation of the signaling pathways that principally control self-renewal and pluripotency in normal intestinal stem cells (ISCs) through different (epi)genetic changes that define cell fate, identity, and phenotype of CSCs. These aspects are currently under intense investigation. In the framework of the oncogenic signaling pathways controlled by microRNAs (miRNAs) during CRC development, a plethora of data suggests that miRNAs can play a key role in several regulatory pathways involving CSCs biology, epithelial-mesenchymal transition (EMT), angiogenesis, metastatization, and pharmacoresistance. This review examines the most relevant evidences about the role of miRNAs in the etiology of CRC, through the regulation of colon CSCs and the principal differences between colorectal CSCs and benign stem cells. In this perspective, the utility of the principal CSCs-related miRNAs changes is explored, emphasizing their use as potential biomarkers to aid in diagnosis, prognosis and predicting response to therapy in CRC patients, but also as promising targets for more effective and personalized anti-CRC treatments.

Background The definition of cancer stem cells (CSC) still lacks conclusive experimental evidence. Colorectal cancer (CRC) EphB2 and EphA2 cells have been correlated to stem-like properties and tumor malignancy. Here, we investigated a panel of miRNA involved in the self-renewal and cell fate during cancer development using murine CRC EphA2 and EphB2 sorted cells and tumor tissues as well as public datasets of hCRC data. Methods FACS-isolated murine EphA2high and EphB2high cells were analyzed by gene expression and IHC analyses in order to characterize the stem-like/differentiation phenotype. miRNAs expression profiling was performed using TaqMan LDA both on murine sorted cells and tissue and validated on public hCRC datasets (TCGA and GEO). Results miRNAs differentially expressed in murine tumor EphA2/EphB2 cells and tissues were found to orchestrate functions related to stem-like properties or to proliferation, metastasis and drug-resistance. Particularly, the EphA2 cells showed a molecular pattern reflecting the activation of EphA2 and EGFR pathways and a coherent dysregulation of mir26b and mir-200a. This pattern displayed prognostic significance for stage I–III CRC. In patients with stage IV and WT KRAS, EphA2/Efna1/Egfr gene expression status was significantly associated with poor response to cetuximab treatment. EphA2 and EGFR overexpression showed a combined effect relative to cetuximab resistance, independently from KRAS mutations. The EphA2/EphB2-specific signatures resulted to be clinically relevant. Conclusions These data provide a comprehensive miRNAs signature implicated in the regulation of tumorigenesis, stemness and drug resistance that could be exploited for diagnosis, therapeutic design and could be proposed as novel CRC prognostic biomarkers.

Eradication of cancer cells is imperative for a successful treatment of tumours. In addition to the existent chemotherapy or radiation therapy, other novel immunotherapeutic strategies to fight tumours are currently under investigation. One of these is cancer vaccination, an approach aimed at inducing effective immune responses in the host against defined tumour antigens. Among several classes of cancer vaccines, the subunit vaccines based on the single and multi epitope-approach are worthy of note as they offer an exquisite specificity in targeting only tumour cells. In this review we will focus on the significant advances made in the development and use of epitope-based cancer vaccines, reporting a selection of important and recent patents on tumour antigen discovery and epitope design for immunotherapy of cancer.

Vaccines represent one of the most successful strategies in medical science. From the mechanistic perspective, vaccination works by manipulating the immune response through selecting, activating and expanding the memory of B and T cells. To determine the magnitude and quality of immune response, suitable vaccine adjuvants are required; therefore, much effort is going into finding new, effective and non-toxic adjuvant formulations focussed on the activation of key immune targets for inducing a long-term, potent and safe immune response. Significant research is being done in this area, to develop new classes of vaccines for use not only against infectious diseases, but also in the treatment of autoimmune disorders, allergies, chronic inflammatory diseases and cancer. Here the authors review and classify some of the main vaccine adjuvants on the basis of their immunomodulating properties on the immune system.

Epigenetic alterations, such as CpG islands methylation and histone modifications, are recognized key characteristics of cancer. Glycogenes are a group of genes which epigenetic status was found to be changed in several tumors. In this study, we determined promoter methylation status of the glycogene beta-1,4-galactosyltransferase 1 (B4GALT1) in colorectal cancer patients. Methylation status of B4GALT1 was assessed in 130 colorectal adenocarcinomas, 13 adenomas, and in paired normal tissue using quantitative methylation specific PCR (QMSP). B4GALT1 mRNA expression was evaluated in methylated/unmethylated tumor and normal specimens. We also investigated microsatellite stability and microsatellite instability status and KRAS/BRAF mutations. Discriminatory power of QMSP was assessed by receiving operating curve (ROC) analysis on a training set of 24 colorectal cancers and paired mucosa. The area under the ROC curve (AUC) was 0.737 (95% confidence interval [CI]:0.591-0.881, P = 0.005) with an optimal cutoff value of 2.07 yielding a 54% sensitivity (95% CI: 35.1%-72.1%) and a specificity of 91.7% (95% CI: 74.1%-97.7%). These results were confirmed in an independent validation set where B4GALT1 methylation was detected in 52/106 patients. An inverse correlation was observed between methylation and B4GALT1 mRNA expression levels (r = -0.482, P = 0.037). Significant differences in methylation levels and frequencies was demonstrated in invasive lesions as compared with normal mucosa (P = 0.0001) and in carcinoma samples as compared with adenoma (P = 0.009). B4GALT1 methylation is a frequent and specific event in colorectal cancer and correlates with downregulation of mRNA expression. These results suggest that the glycogene B4GALT1 represent a valuable candidate biomarker of invasive phenotype of colorectal cancer.

After more than 15 years of experimentation, DNA vaccines have become a promising perspective for tumour diseases, and animal models are widely used to study the biological features of human cancer progression and to test the efficacy of vaccination protocols. In recent years, immunisation with naked plasmid DNA encoding tumour-associated antigens or tumour-specific antigens has revealed a number of advantages: antigen-specific DNA vaccination stimulates both cellular and humoral immune responses; multiple or multi-gene vectors encoding several antigens/determinants and immune-modulatory molecules can be delivered as single administration; DNA vaccination does not induce autoimmune disease in normal animals; DNA vaccines based on plasmid vectors can be produced and tested rapidly and economically. However, DNA vaccines have shown low immunogenicity when tested in human clinical trials, and compared with traditional vaccines, they induce weak immune responses. Therefore, the improvement of vaccine efficacy has become a critical goal in the development of effective DNA vaccination protocols for anti-tumour therapy. Several strategies are taken into account for improving the DNA vaccination efficacy, such as antigen optimisation, use of adjuvants and delivery systems like electroporation, co-expression of cytokines and co-stimulatory molecules in the same vector, different vaccination protocols. In this review we discuss how the combination of these approaches may contribute to the development of more effective DNA vaccination protocols for the therapy of lymphoma in a mouse model.

To date, vaccination is an active area of investigation for its application to a great variety of human diseases including infections and cancer. In particular, naked-DNA vaccination has arisen as effective strategy in the preventive medicine field with promising future prospects. The ability of plasmid DNA to activate the humoural and the cellular arms of the immune system against the encoded antigen have resulted in intensive study of new strategies aimed at increasing the DNA vaccine immunogenicity. Nevertheless, plasmid-based vaccines emerged as a safer and advantageous alternative with respect to viral vector vaccines. Recent advances in both the immunological and biotechnological research field made it possible to enhance significantly the DNA vaccine potency. Most of these approaches are based on both the discovery of novel delivery systems and the implementation of plasmid constructs, achieved through genetic engineering. In this review, we will describe some of the most relevant patents issued in the last ten years, supporting the progress made in naked-DNA vaccination against infectious diseases.

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

Purpose: EphA2 receptor 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. Colorectal cancer (CRC) EphA2 overexpression has also been correlated to stem-like properties of cells and tumor malignancy. We investigated the molecular cross-talk and miRNAs modulation of the EphA2 and EGFR pathways. We also explored the role of EphA2/EGFR pathway mediators as prognostic factors or predictors of cetuximab benefit in patients with CRC. Experimental Design: Gene expression analysis was performed in EphA2high cells isolated from CRC of the AOM/DSS murine model by FACS-assisted procedures. Six independent cohorts of patients were stratified by EphA2 expression to determine the potential prognostic role of a EphA2/EGFR signature and its effect on cetuximab treatment response. Results: We identified a gene expression pattern (EphA2, Efna1, Egfr, Ptpn12, and Atf2) reflecting the activation of EphA2 and EGFR pathways and a coherent dysregulation of mir-26b and mir-200a. Such a pattern showed prognostic significance in patients with stage I–III CRC, 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 treatment. Furthermore, EphA2 and EGFR overexpression showed a combined effect relative to cetuximab resistance, independently from KRAS mutation status. Conclusions: These results suggest that EphA2/Efna1/Egfr genes, linked to a possible control by miR-200a and miR-26b, could be proposed as novel CRC prognostic biomarkers. Moreover, EphA2 could be linked to a mechanism of resistance to cetuximab alternative to KRAS mutations. Clin Cancer Res; 23(1); 159–70. ©2016 AACR.