Introduction

The Wnt/β-catenin signalling is frequently deregulated in colorectal cancer (CRC); indeed, germ-line (as found in familial adenomatous polyposis – FAP) or somatic loss of function of the APC gene represent crucial events in CRC onset. Importantly the PI3K/mTOR pathway is frequently impaired in CRC development and closely involved together with Wnt/β-catenin signalling in self-renewal and homeostasis of intestinal stem cells (1,2). Numerous inhibitors of Wnt/β-catenin and PI3K/mTOR pathways have been proposed as potential anti-cancer treatments (3,4), but long-term exposure often fails due to toxicity or the acquisition of resistance, usually associated with the activation of the non-inhibited pathway due to their mutual regulation (5).

Aims & Methods

The aim of the study was to evaluate the effect of a combination (RDE) of Rapamycin, ω3-PUFA Docosahexaenoic Acid (DHA) and epigallocatechin-3-gallate (EGCG) on PI3K/mTOR and Wnt/β-catenin pathways, in order to define an effective combinatorial approach for CRC prevention through simultaneous suppression of both pathways.
For this purpose RDE was tested for 24 or 48 hours (in cell lines and intestinal organoids, respectively) in selected different genetic backgrounds: (i) APC-mutated settings consisting of cell lines SW480 and HT-29, and intestinal organoids from four FAP patients morphologically normal-appearing mucosa (NM); (ii) non-APC-mutated CRC settings comprising HCT116 (CTNNB1 mutated) cell line and sporadic CRC organoids (CRC1: TP53 mutated; CRC2: PI3KCA and TP53 mutated); HEK293STF cell line and three healthy patients NM-derived organoids as non-mutated settings (iii). We evaluated cells and organoids viability, markers of intestinal stemness (LGR5) and differentiation (KRT20), and we analyzed the expression of target genes and effector proteins of the Wnt/β-catenin and PI3K/mTOR pathways by RT-qPCR and Western-blot.

Results

We found that RDE strongly inhibited PI3K/mTOR by downregulating the protein expression of phospho-P70S6K and phospho-S6R in all tested genetic backgrounds. FAP NM organoids responded to RDE similarly to HEK293STF cell line and the healthy organoids, with increased translocation of β-catenin into the nucleus. In contrast, in the APC-mutated SW480 and HT-29 cell lines, RDE inactivated the Wnt/β-catenin pathway, inhibiting β-catenin nuclear translocation despite the downregulation of APC and AXIN1. These findings were replicated in HCT116 cell line, that also showed reduced cell viability and induction of apoptosis. On the other hand, in non-APC-mutated CRC organoids, RDE showed a mild downregulation of Wnt/β-catenin pathway. Importantly, RDE significantly suppressed LGR5 and induced KRT20 gene expression. Finally, independently from the genetic backgrounds, exposing organoids to RDE led to increased expression of c-MYC.

Conclusion

We found that the dual inhibition of PI3K/mTOR and Wnt/β-catenin pathways by RDE is influenced by genetic backgrounds. Our approach was effective in CRC cell lines and in organoids by affecting markers of intestinal stemness and tissue differentiation, suggesting a potential therapeutic approach for both preventive and therapeutic strategies against CRC.

References

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2. Mossmann D, Park S, Hall MN. mTOR signalling and cellular metabolism are mutual determinants in cancer. Nat Rev Cancer. 2018 Dec;18(12):744-757. doi: 10.1038/s41568-018-0074-8. PMID: 30425336.

3. Zhao H, Ming T, Tang S, Ren S, Yang H, Liu M, Tao Q, Xu H. Wnt signaling in colorectal cancer: pathogenic role and therapeutic target. Mol Cancer. 2022 Jul 14;21(1):144. doi: 10.1186/s12943-022-01616-7. PMID: 35836256.

4. Magaway C, Kim E, Jacinto E. Targeting mTOR and Metabolism in Cancer: Lessons and Innovations. Cells. 2019 Dec 6;8(12):1584. doi: 10.3390/cells8121584. PMID: 31817676.

5. Prossomariti A, Piazzi G, Alquati C, Ricciardiello L. Are Wnt/β-Catenin and PI3K/AKT/mTORC1 Distinct Pathways in Colorectal Cancer? Cell Mol Gastroenterol Hepatol. 2020;10(3):491-506. doi: 10.1016/j.jcmgh.2020.04.007. Epub 2020 Apr 22. PMID: 32334125.

Introduction

Colorectal cancer (CRC) is the 3rd most common cancer worldwide. Approximately 85% of CRC develop on a background of Wnt/β-catenin signalling impairment. Familial adenomatous polyposis (FAP), caused by inherited APC gene mutations (part of the β-catenin destruction complex), is characterized by the development of hundreds to thousands adenomatous polyps. FAP patients have up to 100% risk of developing CRC during their lifetime. The Wnt/β-catenin together with the PI3K/mTOR signalling control the self-renewal and homeostasis of intestinal stem cells and are frequently deregulated in CRC therefore representing attractive targets for chemoprevention and treatment [1], [2]. It has been shown that each pathway may be deregulated by an altered composition of the gut microbiota [3], [4], which plays an important role in the onset, development and progression of CRC thus representing potential target for prevention and potentially biomarkers for the early detection of deranged Wnt/β-catenin-driven lesions [5].

Aims & Methods

The aim of the study was to identify potential targets for the prevention and early detection of CRC in the setting of Wnt/β-catenin disruption.
Transcriptional and translational levels of Wnt/β-catenin and PI3K/mTOR pathways were evaluated by RT-qPCR and immunohistochemical analysis, respectively, in FAP adenomas (n=17 patients) and in sporadic CRC tissues (tumor stages I-III; n=15 patients). In FAP adenomas germline and somatic APC mutations were confirmed by Sanger and Targeted Next Generation Sequencing. The composition of oral, fecal and mucosal-associated microbiota was sequenced by the bacterial 16S rRNA gene. Non-diseased Faecal Immunochemical Testing positive subjects (FIT+; n=17) were included as control group.

Results

We found increased expressions of AXIN2 and cMYC, downstream targets of Wnt/β-catenin, in FAP adenomas. The activation status of PI3K/mTOR was confirmed by an increase of RPS6 gene and pS6R protein expression, indicating that germline and somatic APC mutations of FAP adenomas induce an activation of both pathways. On the other hand, compared to FAP adenomas, a higher expression of AXIN2, cMYC and cytosolic and nuclear β-catenin was found in CRC, while no changes were detected in pS6R, suggesting that the hyperactivation of Wnt/β-catenin in sporadic CRC contributes to the impairment of mTOR signalling. Importantly, we analyzed the microbiome composition in FAP and CRC settings. The oral microbiota of FAP was significantly enriched of Proteobacteria (p<0.01), which decreased in FAP adenomatous mucosal samples compared to FIT+ and CRC. In contrast, Firmicutes showed an opposite trend in FAP patients, with a decrease in the oral composition (p<0.01) and an increase in the adenomatous mucosa (p<0.05). Interestingly, a significant decrease of the CRC-protective phylum Actinobacteria was found in oral and adenomas microbiota of FAP patients compared to FIT+. In contrast, CRC fecal microbiota was enriched of Proteobacteria and a higher abundance of the well-known cancer associated families Fusobacteriaceae, Clostridiaceae and Gemellaceae was found in CRC mucosal samples compared to FIT+.

Conclusion

These findings reveal new insights into the role of Wnt/β-catenin and PI3K/mTOR pathways in influencing the homeostasis of precancerous CRC lesions. Moreover, by defining different microbial signatures in FAP patients, these results will contribute to the enhancement of non-invasive screening tools for the early detection of WNT signalling-driven colorectal cancers.

References

[1] X. Cheng, X. Xu, D. Chen, F. Zhao, and W. Wang, “Therapeutic potential of targeting the Wnt/β-catenin signaling pathway in colorectal cancer,” Biomed. Pharmacother., vol. 110, pp. 473–481, 2019, doi: https://doi.org/10.1016/j.biopha.2018.11.082.
[2] J. Zhang, T. M. Roberts, and R. A. Shivdasani, “Targeting PI3K Signaling as a Therapeutic Approach for Colorectal Cancer,” Gastroenterology, vol. 141, no. 1, pp. 50–61, 2011, doi: https://doi.org/10.1053/j.gastro.2011.05.010.
[3] M. H. Noureldein and A. A. Eid, “Gut microbiota and mTOR signaling: Insight on a new pathophysiological interaction,” Microb. Pathog., vol. 118, no. March 2018, pp. 98–104, 2018, doi: 10.1016/j.micpath.2018.03.021.
[4] O. Silva-García, J. J. Valdez-Alarcón, and V. M. Baizabal-Aguirre, “Wnt/β-catenin signaling as a molecular target by pathogenic bacteria,” Front. Immunol., vol. 10, no. SEP, pp. 1–14, 2019, doi: 10.3389/fimmu.2019.02135.
[5] J. H. Kim, Y. J. Kim, G. M. Oh, W. Jung, and S. J. Park, “How is gut microbiome of patients with familial adenomatous polyposis different from healthy people?,” Medicine (Baltimore)., vol. 101, no. 49, p. e32194, Dec. 2022, doi: 10.1097/MD.0000000000032194.

Introduction

In recent years, emphasis has been shifted on to understanding the role of N1-methyladenosine (m1A) in tumor progression. While little is known about its regulatory effect on mRNA and its role in metastatic colorectal cancer (CRC).

Aims & Methods

We performed methylated RNA immunoprecipitation sequencing with tumor tissues and tumor-adjacent normal tissues from three CRC patients to determine the profile of m1A methylation in CRC. Expression of Diaphanous-related formin 3 (DIAPH3) and its correlation with clinicopathological characteristics of CRC were evaluated using immunohistochemistry and online datasets. The role of DIAPH3 in the migration and invasion of CRC cells was evaluated by wound healing and transwell assays. Besides, the downstream targets of DIAPH3 were screened by mass spectrometry analysis. By co-transfecting DIAPH3 siRNA and Keratin 19 (KRT19) ectopic plasmid in CRC cells, the role of DIAPH3-KRT19 signaling axis was confirmed.

Results

The mRNA level of DIAHP3 and its m1A modification were increased simultaneously in CRC. Besides, high expression of DIAPH3 in CRC tissues was significantly associated with metastasis, and tended to progress to an advanced stage. After knocking down the expression of DIAPH3, the migration and invasion capabilities of CRC cells suffered notable decline, which can be rescued by overexpressing KRT19. Besides, the degradation of KRT19 might be regulated by DIAPH3 in a proteasome-dependent pathway.

Conclusion

DIAPH3 mRNA was upregulated in CRC cells through m1A methylation. Targeting DIAPH3 results in suppressed migration and invasion of CRC cells, potentially through proteasome-dependent degradation of downstream KRT19.

Introduction

Canopy FGF signaling regulator 3 (CNPY3) is a protein predominantly localized in the endoplasmic reticulum. Its expression is increased in several cancers, including colorectal cancer (CRC). However, how CNPY3 affects CRC progression and the function of CNPY3 translocation to the nucleus in CRC is not clear.

Aims & Methods

This study aimed to investigate the role of CNPY3 in the development of CRC. In vitro, the impacts of CNPY3 on CRC growth were evaluated by 5-Ethynyl-2′-deoxyuridine staining, colony formation, wound healing, transwell migration, and apoptosis assay. Tumor xenograft mouse models and orthotopic mouse models of colorectal cancer metastasis were used to assess in vivo. Furthermore, the underlying mechanism of CNPY3 in CRC was revealed by RT-qPCR, western blot, LC-MS/MS analysis, immunofluorescence staining, coimmunoprecipitation, GST-pulldown, confocal microscopy, ubiquitination assays, and rescue experiments.

Results

CNPY3 was significantly upregulated in CRC tissue and cell lines, correlating with their poor prognosis. Functional experiments demonstrated that CNPY3 enhances CRC proliferation, migration, invasion, and tumor growth. Notably, our investigation reveals a nucleus translocation of cnpy3 by activation of nuclear localization sequences through protein conformational changes. Meanwhile, nuclear localization of CNPY3 promotes the binding of TRIM21 and p53 and forms a CNPY3-TRIM21-p53 complex, thereby promoting the ubiquitination-proteasomal degradation of wild-type p53 protein, inhibiting the related genes of the TP53 signaling pathway, and promoting apoptosis of tumor cells. In vivo and in vitro, inhibiting the degradation of p53 was able to suppress CNPY3 in cancer-promoting tumors, whereas the knockdown of CNPY3 in p53^-/- HCT116 cells did not significantly inhibit CRC.

Conclusion

CNPY3, after nuclear translocated, binds to TRIM21 and p53 and stabilizes the TRIM21–p53 interaction. This CNPY3–TRIM21–p53 complex facilitates TRIM21‐mediated p53 ubiquitinoylation and causes p53 proteasome degradation. Collectively, these findings support that CNPY3, an oncogene with CRC progression and poor prognosis, serves as a biomarker and therapeutic target correlated to suppress p53 function.

References

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3. Xiao L, Li XX, Chung HK, Kalakonda S, et al. RNA-Binding Protein HuR Regulates Paneth Cell Function by Altering Membrane Localization of TLR2 via Post-transcriptional Control of CNPY3. Gastroenterology. 2019;157(3):731-743.
4. Ghait M, Husain RA, Duduskar SN, et al. The TLR-chaperone CNPY3 is a critical regulator of NLRP3-inflammasome activation. Eur J Immunol. 2022;52(6):907-923.
5. Zhou YQ, Bao TS, Xie JX, et al. The SLITRK4-CNPY3 axis promotes liver metastasis of gastric cancer by enhancing the endocytosis and recycling of TrkB in tumour cells. Cell Oncol (Dordr). 2023;46(4):1049-1067.
6. Reddy BA, van der Knaap JA, Bot AG, et al. Nucleotide biosynthetic enzyme GMP synthase is a TRIM21-controlled relay of p53 stabilization. Mol Cell. 2014;53(3):458-70.
7. Liu J, Zhang C, Xu D, et al. The ubiquitin ligase TRIM21 regulates mutant p53 accumulation and gain of function in cancer. J Clin Invest. 2023;133(6):e164354.

Introduction

Chemoresistance is common in colorectal cancer (CRC) and it contributes to treatment failure and relapse. Cancer stem cells (CSCs) is a sub-population of tumor-initiating cells that drive chemoresistance in CRC. However, the interaction between gut microbiome and CSCs remains unclear.

Aims & Methods

In this study, we investigated the effect of a CRC pathogen, Peptostreptococcus anaerobius, in promoting colorectal CSC stemness and chemoresistance, and elucidated its molecular mechanism of action. The effect of P. anaerobius on CRC stemness and chemoresistance were investigated in human CSCs, and validated in Apc^Min/+ and AOM/DSS induced CRC tumorigenesis mouse models. Attachment of P. anaerobius on human CSCs was evaluated by transmission electron microscopy and scanning electron microscopy. P. anaerobius proteins interacting with CSC receptors were identified by GST pulldown and surface plasmon resonance (SPR) assays. The protein binding sites were determined by crosslinking mass spectrometry (MS) and docking.

Results

Intratumoral P. anaerobius abundance was positively correlated with stemness-related genes, including CD133 and LGR5 in CRC patients (n=73). P. anaerobius gavage promoted CRC formation and elevated Cd133⁺ and Lgr5⁺ cell populations in CRC tissues from Apc^Min/+ mice and AOM/DSS-induced CRC models. Consistently, P. anaerobius co-culture increased the self-renewal capacity and sphere formation in human colorectal CSCs, along with increased expression of CD133 and LGR5. In line with its stemness boosting effect, P. anaerobius promoted resistance to oxaliplatin and 5-fluorouracil treatment in human CSCs in vitro, and in AOM/DSS-induced CRC tumorigenesis model. Mechanistically, we revealed that the outer membrane protein NlpC/P60 from P. anaerobius interacts with the Annexin A2 receptor on colorectal CSCs. Co-immunoprecipitation, SPR assays, and crosslinking MS revealed that NlpC/P60 competed with Yes-associated protein 1 (YAP) for binding to Annexin A2 (ANXA2). This interaction disrupted the intracellular binding between ANXA2 and YAP, leading to the release of YAP from the cell membrane, promoting YAP translocation into nucleus to activate Hippo signaling pathway. Anti-ANXA2 neutralizing antibody blocks the binding between P. anaerobius and colorectal CSCs and abrogated the effect of P. anaerobius on promoting stemness and chemoresistance. Corrobrating these pre-clinical observations, Levels of P. anaerobius were found to be increased in chemotherapy non-responders as compared to responders in CRC patients.

Conclusion

P. anaerobius drives CRC stemness and chemoresistance via NlpC/P60- Annexin A2 interaction, leading to YAP1-driven activation of Hippo signaling pathway. Blocking NlpC/P60-Annexin interaction using Anti-ANXA2 antibody could overcome P. anaerobius-mediated chemoresistance in CRC.

Introduction

One of major obstacles of treatment in colorectal cancer (CRC) is drug resistance, specially 5-Fu chemoresistance which is a fundamental drug in CRC chemotherapy. Also, there is no reliable biomarkers in order to identify chemoresistance patients and improve their treatment. There are several factors that cause 5-FU chemoresistance include autophagy. This cell death phenomenon is played key role in 5-FU chemoresistance in CRC patients by providing nutritarians for CRC cells in hard circumstances; also, autophagy has regulated by different factors like genes and non-coding RNAs(ncRNA).

Aims & Methods

Verifying key gene in autophagy pathway at KEGG database. Moreover, evaluation the expression of key gene in CRC patients at TCGA database. Investigation on the key gene related LncRNAs and miRNAs in CRC at LNCpedia and mirTARBase database respectively, in order to find most relevant ncRNAs of this gene and obtain proper biomarkers. Developing 5-FU chemoresistance Caco2 and SW480 cell lines by increasing dosage of drug and MTT assay was performed to confirm the chemoresistance cell lines and western blot was performed to confirm autophagy in cell lines. Finally, evaluating the expression of MAP1LC3A and its related ncRNAs in 5-FU chemoresistant CRC cell lines by qRT-PCR.

Results

MAP1LC3A was confirmed as key gene based on KEGG analysis; also, FAM225A, NCK1-DT, LINC01825, LINC02188 and miR-335-5p have nominated as most relevant ncRNAs based on bioinformatic analysis. Furthermore, qRT-PCR analysis of MAP1LC3A and its related ncRNAs have shown differential expression between chemoresistant and chemoresistant CRC cell lines.

Conclusion

These findings indicate that FAM225A, NCK1-DT, LINC01825, LINC02188 and miR-335-5p participate in autophagy by effecting the expression of MAP1LC3A gene and they can function as proper biomarkers for 5-FU chemoresistance CRC patients. Not only they can function as biomarkers but also, they can attend in target therapy by designing siRNAs to knockdown this ncRNAs and enhance chemotherapy in CRC patients.