Introduction

Inflammation is a recognized hallmark of colorectal cancer (CRC), contributing to its development and progression. Therefore, targeting cancer-associated inflammation may offer new avenues for cancer treatment. Anti-inflammatory drugs currently used for the treatment of patients with CRC show many adverse side effects that prompted researchers to propose the specialized pro-resolving mediators (SPMs), derived from omega-3 polyunsaturated fatty acids, as promoters of resolution of cancer-associated inflammation.
We previously demonstrated that the endothelial Major Facilitator Superfamily Domain-containing 2A (MFSD2A) promotes the resolution of chronic intestinal inflammation by overseeing the release of SPMs.

Aims & Methods

Considering the direct connection between inflammation and cancer development that mutually impact on each other, we hypothesize that defective MFSD2A-dependent pathways in endothelial cells may fail to resolve tumor-associated intestinal inflammation, thus promoting CRC development, growth and metastasization. Therefore, enhancing the expression of MFSD2A in the vascular compartment may counteract CRC-associated inflammation, ultimately resulting in anti-cancer effects.
Based on these premises, our project aims to identify the MFSD2A-dependent pathways responsible for the resolution of tumor-associated intestinal inflammation, paving the way for a new avenue for the treatment of CRC.
To test our hypothesis, we derived Human Intestinal Microvascular endothelial cells (HIMEC) from surgical specimens of CRC patients and healthy subjects. We evaluated the biological effects of MFSD2A overexpression in CRC and healthy HIMEC in terms of transcriptomic and lipidomic profiles. To better elucidate how the enhancement of MFSD2A expression in vitro shapes cancer cell growth, we performed co-colture experiments with HIMEC and human epithelial adenocarcinoma cell line (Caco2).

Results

To test our hypothesis, we performed a transcriptomic analysis on HIMEC. Besides the increased levels of MFSD2A transcript in CRC by comparison with the healthy, this analysis pointed out the activation of the resolution phase-related biological processes in CRC as compared to the healthy, proposing the endothelium as a player also in CRC pathogenesis. Furthermore, by lipidomics, CRC HIMEC displayed increased release of SPMs and reduced levels of the pro-inflammatory lipids by comparison with the healthy control cells. Interestingly, upon MFSD2A silencing, CRC HIMEC switched to a pro-inflammatory phenotype, characterized by the activation of pro-inflammatory biological processes and the increased release of pro-inflammatory lipids. Notably, the enhancement of MFSD2A expression in vitro reduces cancer cell growth.

Conclusion

These data suggested that MFSD2A might contrast the tumor-associated inflammation and ensure the correct balance between pro-inflammatory and pro-resolving milieu, offering novel insights into the development of innovative therapies suitable for CRC patient management.

References

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Introduction

Colorectal cancer, the third most prevalent malignancy globally[1], exhibits strong associations with aberrant DNA methylation patterning. While DNA methyltransferase inhibitors (DNMTis) demonstrate therapeutic efficacy in hematologic malignancies, their clinical application in solid tumors remains constrained by dose-limiting genomic instability secondary to genome-wide hypomethylation[2]. Recent mechanistic studies have established methyl-CpG-binding domain protein 2 (MBD2) as a pivotal mediator of methylation-dependent transcriptional repression; however, its potential involvement in context-dependent oncogene activation remains unresolved. This study elucidates that pharmacological MBD2 inhibition achieves dual epigenetic rectification – reactivating tumor suppressor genes through promoter demethylation while selectively attenuating WNT16 overexpression via methylation-sensitive suppression, thereby establishing a targeted therapeutic paradigm that circumvents the pleiotropic toxicity associated with conventional DNMTis.

Aims & Methods

A multi-dimensional platform encompassing clinical specimens, cell lines, patient-derived organoids, and PDX models was employed. Integrative whole-genome bisulfite sequencing and RNA-seq analyses delineated methylation-driven transcriptional networks. Chromatin immunoprecipitation sequencing characterized MBD2 binding at differentially methylated regions, with CRISPR interference validating functional regulatory elements.

Results

CRC cells exhibited pronounced hypomethylation at the WNT16 promoter accompanied by diminished MBD2 occupancy. MBD2 inhibitor treatment downregulated WNT16 expression at both RNA and protein levels, concomitant with suppression of Wnt/β-catenin signaling. In PDX models, MBD2 inhibition reduced tumor burden without inducing genomic instability—a hallmark toxicity of conventional DNMTis.

Conclusion

This study unveils a novel epigenetic mechanism wherein MBD2 facilitates oncogenic WNT16 activation via its "methylation reader" function. Compared to broad-spectrum DNMTis, MBD2-targeted therapy achieves precise correction of carcinogenic epigenetic lesions while avoiding genome-wide hypomethylation risks, advancing a paradigm for targeted CRC treatment.

References

[1] Jiexi Li, Xingdi Ma, Deepavali Chakravarti, Shabnam Shalapour, Ronald A DePinho. Genetic and biological hallmarks of colorectal cancer. Genes Dev.
2021;35(11-12):787-820
[2] Marie Lopez, Julie Gilbert, Jean Contreras, Ludovic Halby, Paola B Arimondo. Inhibitors of DNA Methylation. Adv Exp Med Biol. 2022;1389:471-513
[3] Yang Xie, Feng Wang, Jun Yu, Jing Zhang, Yuting Liu, Mengying Li, Jian Qi. Silencing of MBD2 and EZH2 inhibits the proliferation of colorectal carcinoma cells by rescuing the expression of SFRP. Oncol Rep. 2021;46(6):250.

Introduction

Colorectal cancer (CRC) is the third-most common malignancy worldwide. Lysyl oxidase-like protein 2 (LOXL2), which promotes crosslinking of collagen and elastin, contributes to tumor progression in different tumour entities, but its role in CRC has not been evaluated. Here, we unraveled the role of LOXL2 in regulating tumour immune microenvironment of CRC and identified LOXL2 as a potential target for immunotherapy.

Aims & Methods

We analyzed the expression of LOXL2 in the Cancer Genome Atlas (TCGA) data. The effect of LOXL2 on CRC cell growth was determined by cell viability and colony formation assays. In vivo models were established by subcutaneous injection of colorectal cancer cell lines into immunodeficient NOD-SCID-γ (NSG) mice and immunocompetent wild-type BALB/c mice. Flow cytometry and immunohistochemistry were used to analyze immune cell composition. The collagen fibers in the extracellular matrix (ECM) were observed by masson staining, second harmonic generation imaging (SHG), and two-photon fluorescence imaging.

Results

Survival prognosis analysis revealed that high LOXL2 expression was correlated with poor survival of patients with CRC in TCGA cohort. However, we found that LOXL2 overexpression in human (HCT116 and DLD1) and mouse (CT26 and MC38) CRC cell lines had little effect on tumor growth in vitro and in immunodeficient mice. But its overexpression in mouse CRC cell lines could significantly promote tumor growth in immunocompetent mice (p<0.05). Overexpression of LOXL2 led to CXCL1 upregulation in the supernatant of CRC cells and CRC tumor tissues; subsequently, upregulation of CXCL1 induced more migration of myeloid-derived suppressor cells (MDSCs) in vitro and recruited more MDSCs in the tumor microenvironment. On the other hand, LOXL2 increased the crosslinking of collagen fibers in vitro and in the tumor microenvironment, which subsequently activated the function of MDSCs (up-expression of Arg1, IDO1, and INOS) and increased the oncostatin M (OSM) secreted by MDSCs. In turn, OSM secreted by MDSCs induced the expression of LOXL2 in CRC cells. The accumulation and activation of MDSCs in the tumor microenvironment inhibited the infiltration and function of CD8+ T cells, which suppressed the anti-tumour immunity of the host. Targeting of LOXL2 with a neutralizing antibody or inhibitor (PAT-1251) significantly impaired tumor growth (p<0.05) and MDSC infiltration and activation (p<0.05) in immunodeficient mice. Finally, PAT-1251 boosted anti-PD1 efficacy in colorectal cancer, suggesting that it could be repurposed to improve immunotherapy efficacy.

Conclusion

Our findings revealed that LOXL2 exerted a pro-tumorigenic role in CRC. LOXL2 recruited and activated MDSCs through CXCL1 and crosslinking of collagen fibers, respectively, subsequently reducing infiltration and activation of anti-tumor CD8⁺ T cells in the tumor microenvironment. Targeting this axis with PAT-1251 improved the efficacy of immunotherapy in colorectal cancer.

Introduction

Endoscopic surveillance after surgery for colorectal cancer (CRC) has the main objectives of early identification of metachronous lesions and neoplastic recurrence. This study aimed to evaluate the possible benefit of intensified endoscopic surveillance compared to standard recommendations.

Aims & Methods

In this retrospective cohort study, all patients who underwent follow-up colonoscopy after CRC surgery over a period of 6 months were included. Patients with endoscopically treated CRC and patients with postoperative complications were excluded. The recommended follow-up period was considered the 1st year, the 4th year, and the 9th year after surgery and a time period of +/- 6 months to the recommended date was considered. Statistical analysis: Chi-square test; t-student test; Mann-Whitney test.

Results

102 patients (38 women) were included, aged 72±11 years. Patients were divided into two groups, those who followed the recommended protocol (55.9%; n=57) and those who underwent intensified surveillance (44.1%; n=45). No statistically significant differences were identified between the two groups regarding demographics, location of the neoplasm or type of surgery performed. There was only a higher adenoma detection in the intensive surveillance subgroup in the 1st year after surgery (p=0.042), with no differences in the remaining endoscopic controls. None of the advanced adenomas identified in this group required earlier endoscopic surveillance. No advanced adenomas were identified in additional colonoscopies carried out outside the recommended period. The adenoma detection rate didn’t show statistically significant differences between the two groups. An excess of 156 colonoscopies were performed over a total of 361 years of follow-up.

Conclusion

Intensified endoscopic surveillance after CRC surgery does not appear to confer benefit and is a significant burden to endoscopy units, in addition to unnecessary risks and discomfort for patients. Adherence to international recommendations should be promoted among referring physicians and a strict colonoscopy vetting process should be enforced.

Introduction

Colorectal cancer, with an estimated two million new cases each year worldwide, poses a significant socioeconomic burden on modern society. It is the second leading cause of cancer-related deaths, accounting for nearly one million premature deaths annually.

Aims & Methods

The role of Signal Transducer and Activator of Transcription 2 (STAT2) in this context is not yet understood, and no therapies targeting this transcription factor are currently being pursued. We explored the function of STAT2 in colorectal cancer by employing experimental mouse models, as well as conducting functional assays with three-dimensional tumoroids in the context of anticancer therapy.

Results

In vivo studies showed that Stat2KO mice were more resistant to developing tumors in both inflammation-driven and inflammation-independent models of experimental colorectal cancer. This unexpected outcome could not be reconciled with the known role of STAT2 in inducing cell death downstream of type I interferon signaling. We consequently performed ex vivo experiments with tumoroids derived from Stat2KO mice carrying the multiple intestinal neoplasia (Min) mutant allele of the adenomatous polyposis coli (Apc) gene. These tumoroids exhibited slower growth, were generally smaller in size, and showed a higher degree of differentiation compared to those derived from ApcMin/+ wildtype mice. Remarkably, when treated with anti-tumor agents that induce cell death through various mechanisms, tumoroids derived from ApcMin/+ Stat2KO mice were found to be highly sensitive. Additional functional studies indicated that the underlying mechanism might involve a STAT2-dependent disruption of innate immune DNA sensing.

Conclusion

Our results establish STAT2 as a promoter of colorectal cancer and a contributor to resistance against anti-tumor therapy. These findings suggest that targeting STAT2-dependent signaling could be a promising therapeutic strategy for patients with colorectal cancer.

Introduction

Eukaryotic-targeting viruses have recently attracted considerable interest in gastrointestinal diseases such as ulcerative colitis (UC), a chronic inflammatory condition of the colonic tract, and colorectal cancer (CRC)^1–3, the third most commonly diagnosed malignancy and the fourth leading cause of cancer death in the world⁴. Both UC and CRC share several factors influencing their etiogenesis, including intestinal dysbiosis^{2, 5–7}. Our group has recently published a work pinpointing a gut virome-associated Orthohepadnaviridae protein, namely Hepatitis B protein X (HBx) to correlate with UC pathogenesis and promote intestinal inflammation in mice by disrupting the epithelial barrier and shaping the gut mucosal immune environment, independently of the microbiota³. Moreover, we showed that HBx is able to induce DNA damage-related biological processes and active biological processes related to the Wnt pathway³ known to be involved in colorectal carcinogenesis⁸.

Aims & Methods

Based on this evidence we hypothesize that HBx may fill the gap existing between intestinal inflammation and CRC onset by stimulating pro-inflammatory and/or pro-carcinogenic effects.
To verify this hypothesis, we address the following Aims:
1. To evaluate the presence of HBx-transcript in CRC-derived mucosal samples
2. To evaluate the pro-carcinogenic properties of HBx in vitro
3. To investigate the role of HBx in CRC onset and development in vivo
HBx positivity of CRC patient-derived gut mucosa was assessed by RT-PCR and Sanger.
Multidimensional flow cytometry analysis was performed on HBx-overexpressing Caco-2 cells, an in vitro model of the gut barrier.
C57BL/6 mice underwent intramucosal injections of liposome-conjugated HBx-encoding plasmids or the control. Multidimensional flow cytometry analysis and transcriptomic analysis were performed on colonic samples from HBx-treated and control animals.

Results

Metatranscriptomic analysis revealed that HBx is significantly abundantly present in CRC-derived mucosal samples compared with healthy individuals, strengthening the link between a virome protein-induced inflammation and tumor development in patients.
Moreover, HBx overexpression in Caco-2 cells directly provokes DNA damage, as assessed by the significant increase in the number of γH2AX+ cells, a marker of double-strand DNA breaks which identifies genomic instability that can potentially contribute to cancer initiation and progression⁹.
Furthermore, we already showed that HBx was able to induce colitis-like symptoms in mice after 15-20 days-long administration³. Interestingly, experiments conducted in C57BL/6 mice at longer time points (from 40 to 60 days-long observation) revealed that animals developed polypoid structures in the colonic mucosa, characterized by decreased abundance of T cells, including effector T cells and NK T cells¹⁰. Of note, in vivo HBx modulated the expression of genes involved in the regulation of transcription and activation of the proto-oncogene PIM3¹¹, further supporting the role of a viral protein on CRC onset.

Conclusion

This study introduces unconventional and innovative insights for giving CRC studies a new direction for the scientific and clinical investigations, looking at the gut virome as an active component and not as a mere bystander entity of the microbiota, ultimately promoting a new standpoint in the CRC studies and management in the short-term future.

References

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8. E. M. Schatoff, B. I. Leach, L. E. Dow, Curr. Colorectal Cancer Rep. 13, 101–110 (2017).
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