Screening for Hepatitis Virus B Protein X (HBx) in Colitis-associated Cancer (CAC)

The human microbiota is a rich community of bacteria, viruses, fungi, protists, and archaea, and is an integral part of the body influencing its overall homeostasis. It colonizes all mucosal surfaces, with the gastrointestinal (GI) tract being the most densely populated. Although the most investigated gut colonizers are bacteria, emerging evidence highlights dysbiosis of the gut virome, made of both bacteriophages and eukaryotic targeting viruses, as responsible for influencing the overall intestinal microbiota composition and mucosal homeostasis by directly interacting with the other commensals, such as bacteria, or with the host, as for the eukaryotic-targeting viruses.

Eukaryotic viruses colonizing the mucosal surfaces may infect the host's cells without symptoms, and, even if asymptomatic, a virus-carrying host may hold a persistent immune response with a "continuum" of inflammatory mediators that might increase host susceptibility to disease. In parallel, eukaryotic viruses have been well-described as inducers of multiple mutational events, among which insertional mutagenesis, so viral infections are known to be responsible for about 20% of the global cancer burden. As an example, one of the most frequent infection-related cancers is hepatocarcinoma, mainly caused by the Hepatitis B virus (HBV).

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), the third most diagnosed malignancy and the fourth leading cause of cancer death in the world. UC and cancer share several factors influencing their etiogenesis, including intestinal dysbiosis. Of note, UC is a risk factor in developing colitis-associated colorectal cancer (CAC). Indeed, patients with UC have a risk of developing cancer approximately 2- to 3-fold more than the general population with poor prognosis. Dysplasia in CAC develops with different mechanisms in comparison to sporadic colorectal cancer. Patients affected by UC and CAC share a pro-inflammatory microenvironment within their mucosal tissues. Nevertheless, the mechanistic link between these two diseases is still missing.

It was recently reported that a factor derived from the Orthohepadnavirus genus, namely the Hepatitis B Virus (HBV) protein X (HBx), was the most enriched component of the virome in early-diagnosed UC patients, compared to both healthy subjects and Crohn's Disease patients. This factor was harbored by a specific cohort of UC patients (approximately 45% of the total analyzed), likely because of a zoonotic spillover event.

In vivo, HBx was shown as able to induce colitis per se independently of the other commensals, by interfering with the epithelial transcriptomic apparatus that causes the loss of appropriate barrier functions, in turn leading to the proinflammatory and immune sequelae of events within colonic mucosa, all these occurring independently of the microbiota. Indeed, HBx-treated mice displayed a reduction of neutrophils, dendritic and CD8+ T cells, indicating failure of the resolution phase (reduced neutrophils) and hampered defense against pathogens (dendritic and CD8+ T cells), all hallmarks consistent with the leading theory of UC pathogenesis. Moreover, it was shown that HBx shapes the transcriptomic state of the intestinal epithelium by binding regulatory DNA regions and activating the transcription of genes involved in the augmented epithelial cell proliferation and stemness. Notably, HBx overexpression in human colonic mucosa biopsies induced activation of biological processes related to the Wnt pathway, which is involved in colorectal carcinogenesis. This result is consistent with the HBx description in the context of HBV infection. Indeed, this viral protein is known to prevent the HBV-infected hepatocytes from efficiently repairing damaged DNA, thus leading to an accumulation of DNA mutations and, eventually, hepatocarcinogenesis.

Based on this evidence and preliminary results, it is believed that HBx-mediated epithelial alterations affect mucosal homeostasis, disrupting and driving intestinal chronic inflammation, which may progress to dysplastic lesions or adenocarcinoma.

For this reason, a multicenter retrospective study is being conducted to investigate the presence of HBx in patient-derived samples from individuals diagnosed with CAC. In detail, the study will employ reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing techniques to analyze Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples to detect HBx. A control group will be included, comprising CRC samples from patients without colitis and samples from patients with diverticulitis, indeterminate colitis (IC), and Crohn's disease (CD) without cancer.

The assessment of HBx in CAC samples could revolutionize clinical approaches by enabling earlier diagnosis and refining risk assessment for patients with UC. This finding may also lead to the development of personalized treatment strategies that specifically target HBx-related pathways, thereby advancing the understanding of the underlying disease mechanisms