RNA N⁶-METHYLADENOSINE READER YTHDF1 PROMOTES STEMNESS AND DRUG RESISTANCE OF COLORECTAL CANCER THROUGH INDUCING NOTCH1 AND IS A THERAPEUTIC TARGET

Background: Immune checkpoint blockade has revolutionized cancer treatment by harnessing the patient’s native anti-tumor T cells, but most colorectal cancers do not respond, for unclear reasons. A major challenge in the field is understanding how patient charactistics, such as age, modify cancer cell-mediated antigen presentation and T cell activation. Patient age across cancer types is associated with increased tumor mutational burden, increased expression of immune checkpoint genes, and increased pro-inflammatory signaling. Here, we investigated the effects of aging on immune pathways in an inducible, genetically engineered mouse model of colorectal cancer.

Materials and Methods: We induced Apc-null tumors in the distal colons of young (age 2-3 months) and old (age 18-22 months) Apc^fl/fl;Villin^CreERT2 mice (C57BL/6J background) via colonoscopy-guided injection of 4-hydroxytamoxifen. Tumor size was measured by ex vivo weight. We used flow cytometry to measure the proportions of T cell subtypes, monocytes, macrophages and dendritic cells. Epithelial (Epcam+) cells were isolated from young and old tumors with flow cytometry, and then analyzed with RNA-Seq and ATAC-Seq.
Results: Tumor weight was significantly lower in old mice compared to tumors from young mice (N=6 in each group) (0.36 gr vs 0.46 grams, P=0.04). Compared to tumors from young mice (N=3), tumors from old mice (N=3) demonstrated a significantly higher percentage of dendritic cells ( 36.5 vs. 23.5, P=0.03), CD3+ T cells ( 27.6 vs. 15.4, P=0.005) (Figure 1), and IFN-gamma producing Th17 cells (22.1 vs. 14.9, P=0.02), and lower proportion of Tregs ( 25.4 vs. 45.6, P=0.02). CD4+, CD8+, and exhausted T cell population frequencies were not significantly different between old and young cohorts. Gene ontology (GO) analysis from RNA-Seq data (Figure 2) showed that differentially expressed genes in old vs. young colon tumor epithelial cells were significantly enriched in multiple immune functions, including inflammatory pathways, T-cell function, and antigen presentation (MHC class II). We found increased expression and chromatin accessibility of Ciita (a master regulator of MHC class II) , and increased expression of H2-Ab1 (which encodes the MHC class II protein complex), in cancer epithelial cells from old vs. young mice. Higher expression of MHC-II-related genes in tumor epithelial cells from old vs. young mice was confirmed by flow cytometry and in situ hybridization.

Conclusion: Results from this study demonstrate that aging promotes an anti-tumor immune microenvironment in an inducible, genetically engineered mouse model of colorectal cancer, possibly by enhancing cancer cell-mediated antigen presentation by MHC class II machinery.

Gastric cancer (GC) is one of the most common cancers in humans. Despite advances in GC therapy, it is still among the world's highest-mortality tumours due to metastases and recurrences, which usually occur within 2 years. Recently, a high ratio of GC, metastasis and cancer recurrence has been associated with asymptomatic Helicobacter pylori (Hp) infections. Hp penetrates into the deeper layers of the mucosa, including the gastric glands, where it interacts with mucous, parietal or progenitor cells and with non-glandular stroma cells, including fibroblasts. Gaseous mediator hydrogen sulfide (H₂S) has been shown to have anti-inflammatory and antioxidant properties and to inhibit NFκB signaling in conjunction with the reduction of pro-inflammatory cytokines such as IL-6, IL-1β and TNFα. We have undertaken study to determine whether the H₂S-released from its fast donor, NaHS, is able to inhibit the activity of Hp-infected cancer-associated fibroblasts (CAF). Such activated fibroblasts (Hp-AGF) have recently been shown to induce phenotypic reprogramming of normal rat gastric epithelial cells towards a propluripotent and highly invasive phenotype. Herein, we attempted to assess the effect of Hp infection on activation of human gastric fibroblasts and to define the possible mechanisms of this activation. Human fibroblasts isolated from biopsies of patients without systemic inflammatory, autoimmune and Hp infection, who underwent the laparoscopic sleeve gastrectomy, were infected with 1x10⁹ live Hp (cagA+;vacA+) per plate and incubated for 96 hrs. To determine inhibition of TGFβ signaling, a non-toxic dose of SB-431542 (10µM, ALK5/TGF-β type I receptor inhibitor) alone or in combination with NaHS (non-toxic dose 50µM) was used. Fibroblast activation markers and their corresponding signaling pathways were determined by RT-PCR, Western Blot and immunofluorescence. Hp-infection upregulated a subset of genes characterizing phenotype of CAFs. The α-SMA was upregulated and incorporated into stress fibres, which was mimicked by co-incubation with TGFβ1 (p<0.05). Hp-infection triggered inflammatory pathways upregulating TLR2, TLR4, STAT3 and NFκB (relA) signaling (p<0.05), resulting in a Snail⁺Twist⁺ phenotype with subsequent incorporation of Twist and Snail into the nucleus. Expression of TLR2, relA and STAT3 genes was dependent on autocrine TGFβ1 signaling. Co-incubation with NaHS evoked a transcriptomic downregulation of components of proinflammatory pathway including TLR2 and TLR4, STAT3 and NFκB (p65) with subsequent downregulation of Twist (p<0.05). We conclude that H₂S donors deserve a great attention as promising anti-inflammatory drugs capable of reducing fibroblasts activation during Hp-infection, thus limiting the risk of Hp-induced GC development and possibly constituting novel candidates acting as adjuvants in future eradication pharmacotherapy.

Background: RNA N⁶-Methyladenosine (m⁶A) modification is closely associated with tumor progression and therapy resistance. In this study, we aim to investigate the function and mechanism of m⁶A reader YTHDF1 in CRC stemness and chemoresistance.
Methods: The protein expression of YTHDF1 and cancer stem cells (CSCs) markers (CD133 and LGR5) was evaluated in human CRC tissue microarray (n=174) by immunohistochemistry staining. The effects of YTHDF1 on CRC stemness and chemoresistance were investigated in colon-specific YTHDF1 knockin mice (Ythdf1^KILgr5-Cre^ER) and in human colorectal CSC spheroids. Molecular targets and pathways targeted by YTHDF1 were identified by integrated m⁶A-seq, Ribo-seq, and RNA-seq, and further verified by RNA immunoprecipitation, qPCR and Western blot.
Results: YTHDF1 protein expression was positively correlated with colorectal CSCs markers (CD133 and LGR5) in human CRC tissues (P<0.001 for both markers). Functionally, YTHDF1 overexpression increased self-renewal capacity and sphere formation of human colorectal CSC spheroids in vitro, whereas YTHDF1 knockdown exerted opposite effects. In line with in vitro findings, YTHDF1-overexpression increased the tumor-initiating ability of CRC, while YTHDF1-knockdown suppressed tumor initiation ability, as evidenced by limiting dilution assay in mice. Moreover, colon-stem cell specific conditional Ythdf1 knockin mice accelerated azoxymethane/dextran sodium sulfate (AOM/DSS)-induced colorectal tumorigenesis with increased tumor multiplicity (P<0.05), implying Ythdf1 initiated and promoted CRC pathogenesis in a stem cell-specific manner. Mechanistically, integrated analysis of RNA-seq, m⁶A-seq, and Ribo-seq on YTHDF1-overexpressing colorectal CSCs revealed that YTHDF1 upregulated NOTCH signaling pathway. YTHDF1 could recognize and directly bind to m⁶A-modified NOTCH1 mRNA, leading to the up-regulation of NOTCH1 in CSCs. Furthermore, knockdown of NOTCH1 or NOTCH1 inhibitor DAPT abolished promoting effect of YTHDF1 on the self-renewal capacity of colorectal CSC spheroids, indicating a m⁶A-YTHDF1-NOTCH1 axis underlies CRC stemness. Consistently, YTHDF1 protein expression was positively correlated with NOTCH1 protein expression in human CRC tissues (n=174, P<0.01). In keeping with the increased stemness, YTHDF1 overexpressing colorectal CSCs showed significant resistance to the chemotherapies of oxaliplatin and fluorouracil. This was further confirmed in colon-stem cell specific Ythdf1 knockin mice, that showed resistance to oxaliplatin treatment as compared to their wildtype counterparts.
Conclusion: YTHDF1 promotes CRC stemness and chemoresistance via binding to m⁶A-modified NOTCH1 mRNA and inducing m6A-YTHDF1-NOTCH1 axis. YTHDF1 is a potential novel therapeutic target for CRC patients with chemoresistance.