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

1. T. Zuo et al., Gut. 68, 1169–1179 (2019).
2. L. Massimino, S. Lovisa, L. Antonio Lamparelli, S. Danese, F. Ungaro, Comput. Struct. Biotechnol. J. 19, 16–28 (2021).
3. L. Massimino et al., Gut (2023), doi:10.1136/gutjnl-2022-328375.
4. E. Morgan et al., Gut. 72, 338–344 (2023).
5. F. Ungaro, S. D’Alessio, S. Danese, Cancers (Basel). 12 (2020), doi:10.3390/cancers12082060.
6. C. Fiorentini et al., Int. J. Mol. Sci. 21 (2020), doi:10.3390/ijms21176201.
7. F. Ungaro, L. Massimino, S. D’Alessio, S. Danese, United European Gastroenterol. J. 7, 999–1007 (2019).
8. E. M. Schatoff, B. I. Leach, L. E. Dow, Curr. Colorectal Cancer Rep. 13, 101–110 (2017).
9. J. Gantchev et al., J. Cell Commun. Signal. 16, 159–177 (2022).
10. S. Aristin Revilla, O. Kranenburg, P. J. Coffer, Front. Immunol. 13, 903564 (2022).
11. N. Mukaida, Y.-Y. Wang, Y.-Y. Li, Cancer Sci. 102, 1437–1442 (2011).