Introduction

Hepatocellular carcinoma (HCC) is a well-known deadly malignancy, accounting for about 8.2% cancer-related death worldwide. Particularly, invasion and metastasis is still the main obstacle to improve the survival of advanced HCC patients. Although high-throughout screen, such as GWAS, whole exon-sequencing, RNA sequencing has uncovered some genetics changes, the driver genes are relatively low mutation ratio in HCC. On the other hand, accumulated evidence suggests that cancer cells can use the similar regulation mechanisms in mammalian development. Our previous work revealed that goosecoid, which plays critical roles in gastrulation and neural crest development could promote the metastasis of HCC by modulating the epithelial-mesenchymal transition. However, the underlying development-related mechanisms utilized by HCC cells remain largely unknown, particularly genes involving epigenetic changes.

Aims & Methods

Aims: Polycomb proteins, such as EZH2 and EED, have been widely revealed involving in development and cancer. However, the roles of another development-related polycomb protein suppressor of zeste 12 (SUZ12) in cancer remains largely unknown. Here, we investigated the roles of SUZ12 in hepatocellular carcinoma (HCC) and the underlyling mechanisms.
Methods: Tissue microarray based on 88 tumor tissues and 27 normal liver tissues was used to examine the expression level of SUZ12. The gain- and loss- of function analysis was conducted to evaluate the effects of SUZ12 on the proliferation, migration and invasion of HCC cells. Meanwhile, luciferase reporter assay and RT-PCR assay were conducted to examine the effect of SUZ12 on the transcriptional activity of chemokine receptors 7 (CXCR7).

Results

High frequency of 17q amplification including SUZ12 locus in HCC was revealed through genome sequencing. Further, SUZ12 expressed relatively higher in tumor tissues and HCC cell lines, particularly in HCC cells with highly metastatic potential. Clinically, high SUZ12 expression was an independent prognostic factor for poor overall survival (hazard ratio =2.540, p = 0.002) of HCC patients, which was further confirmed by analyses of TCGA based database. Functionally, in vitro assays revealed that up-regulation of SUZ12 promoted the migration and invasion of HCC cells, whereas down-regulation of SUZ12 inhibited the migration and invasion potentials. Furthermore, we found SUZ12 strongly upregulated the transcriptional level of CXCR7 screened from chemokine receptors family. Also, CXCR7 was revealed to contribute to the tumor-promoting roles of SUZ12. Of interest, luciferase reporter assay revealed SUZ12 positively controlled the CXCR7 via direct promoter combination but not epigenetic suppression. Moreover, high SUZ12 expression was positively correlated with CXCR7 in advanced patients with portal vein invasion.

Conclusion

These findings indicate that SUZ12 is associated with the poor survival of HCC, and promotes the invasion potential of HCC cells partially by activating CXCR7 transcription. This non-classical transcriptional control of SUZ12 in HCC warrants future exploration.

References

1. Yang, J. D.; Hainaut, P.; Gores, G. J.; Amadou, A.; Plymoth, A.; Roberts, L. R., A global view of hepatocellular carcinoma: trends, risk, prevention and management. Nat Rev Gastroenterol Hepatol 2019, 16, (10), 589-604.
2. Torimura, T.; Iwamoto, H., Treatment and the prognosis of hepatocellular carcinoma in Asia. Liver Int 2021.
3. Rebouissou, S.; Nault, J. C., Advances in molecular classification and precision oncology in hepatocellular carcinoma. J Hepatol 2020, 72, (2), 215-229.
4. Ding, X. X.; Zhu, Q. G.; Zhang, S. M.; Guan, L.; Li, T.; Zhang, L.; Wang, S. Y.; Ren, W. L.; Chen, X. M.; Zhao, J.; Lin, S.; Liu, Z. Z.; Bai, Y. X.; He, B.; Zhang, H. Q., Precision medicine for hepatocellular carcinoma: driver mutations and targeted therapy. Oncotarget 2017, 8, (33), 55715-55730.
5. Cancer Genome Atlas Research Network. Electronic address, w. b. e.; Cancer Genome Atlas Research, N., Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma. Cell 2017, 169, (7), 1327-1341 e23.

Disclosure

The authors have no conflict of interest.

Introduction

The complex signalling pathways of thyroid hormones (TH) has been recently involved in the liver diseases pathogenesis. Particularly, it has been proven that the expression of deiodinases type-1 and 3 (D1-3) changes during liver injury, but their role is still poorly understood in hepatocarcinogenesis process.

Aims & Methods

We aimed to evaluate the role of TH signalling pathway in liver carcinogenesis. In this prospective study, 49 patients underwent liver surgery for HCC in cirrhosis (31 cases) or for other non-neoplastic indication in non-cirrhotic context (18 controls). We evaluated genes and protein expression of the main TH metabolism factors (D1, Monocarboxylate transporter-MCT8, Thyroid receptors-TR alpha and beta, Kruppel-like factor-KLF-9 and TH-responsive SPOT-14 gene), with RT-PCR and Western blot analysis in HCC, cirrhotic tissue and healthy liver samples.

Results

RT-PCR analysis showed a progressive statistically significant decrease of D1 (p=0.003), MCT-8 (p=0.001), and TRβ (p=0.04) mRNA expression from healthy liver to HCC. The expression of KLF9, involved in cell differentiation and proliferation, decreased accordingly (p=0.01). Similarly, the expression of SPOT14 decreased in relation to reduced TH activation (p=0.009). Western Blot analysis showed a decreased expression of D1 protein in all cirrhotic samples (p=0.01), while D3 increased in 50% of HCC (p=0.02). Among HCC patients, D3 expression was associated with poor tumor differentiation compared to D3 negative HCC subjects (p=0.007). Furthermore, at Kaplan-Meyer analysis, overall survival of D3 positive HCC was significantly lower than D3 negative HCC (p=0.04).

Conclusion

These preliminary data showed that D3 expression could define a more severe phenotype of HCC with poor tumor differentiation and reduced survival. Moreover, the signalling pathway of TH is dysregulated in HCC, with intrahepatic hypothyroidism. However, these results need to be confirmed in large cohorts.

Introduction

Oncostatin M (OSM) is a pleiotropic cytokine of the IL‐6 family which can bind two different heterodimeric receptors: Glycoprotein 130 (GP130)/ Leukemia inhibitory factor receptor beta (LIFRβ) and GP130/ OSM receptor beta (OSMRβ) that is the receptor able to mediate the most relevant biological effects of OSM in mice. OSM was found to be involved in different aspects related to tumor development and progression in several cancers including hepatocellular carcinoma (HCC). High levels of OSM were found in cirrhotic patients with different etiology carrying HCC. In particular, patients with HCC developed on a metabolic dysfunction-associated steatotic liver disease (MASLD) background showed increased OSM serum levels that correlate with clinical parameters and disease outcome.

Aims & Methods

The aim of the study is to explore the role of OSM as a cytokine able to modulate the tumor microenvironment and affect crucial events of MASLD-related HCC development and progression taking advantage of: a) cohort of MASLD patients with HCC and related clinical data to correlate OSM levels with HCC incidence and severity; b) advanced molecular techniques to study gene expression changes on human THP1 macrophage cell lines and liver cancer cell lines (HepG2 cells); c) animal models to investigate the role of OSM/OSMRβ axis in the progression of MASLD-related HCC, in particular mice genetically manipulated to delete OSMRβ in hepatocytes (OSMRβ ^-/- mice) as well as control WT littermates, submitted to the experimental DEN/CDAA MASLD-related protocol of liver carcinogenesis.

Results

Data obtained indicate that liver tumor masses in OSMRβ ^-/- mice differ significantly when compared to those observed in WT mice, including: I) a significant reduction in the angiogenic switch; II) an impaired proliferation indexes; III) a marked decreases in the recruitment/function of TAMs and Tregs lymphocytes. A similar immunosuppressive trend was observed in a small cohort of human patients with MASLD exhibiting fibrosis or HCC. Multiplex immunoassay analyses revealed a significant serum increase of a subset of cytokines that may be related to the tumor immunosuppressive microenvironment (TIME) (like IL1β, CCL2, IL8, CXCL13), and transcript levels of these cytokines correlate with OSM expression in MASLD patients. Of interest, higher serum levels of CCL15, a cytokine involved in favoring a TIME, were found to be associated with a worse prognosis in the cohort of MASLD-related HCC patients examined. CCL15 expression was increased in MASLD-related HCC compared to F3/F4 MASLD patients, and correlated with OSM expression. Moreover, spatial transcriptomics analysis confirmed that CCL15 is mainly expressed by malignant hepatocytes in HCC patients of different etiologies. Furthermore, the transcript levels of CCL15 were increased in mice subjected to the DEN/CDAA carcinogenic protocol compared to mice fed a CSAA control diet or CDAA diet, and were drastic reduced in the DEN/CDAA OSMRβ^-/- mice.

Conclusion

These results suggest a possible crucial role of OSM in the progression of MASLD, contributing to the formation of an immunosuppressive tumor microenvironment that would facilitate tumor evasion mechanisms by the immune system.

Introduction

Aims & Methods

Results

Conclusion

Introduction

Ileocolonoscopy and Rutgeerts endoscopic score remains the gold standard to evaluate post-operative recurrence (POR) in Crohn's disease (CD) patients (1).
Intestinal ultrasound (IUS) is proposed as a non-invasive alternative to ileocolonoscopy for the diagnosis of POR, with a sensitivity of 94% and specificity of 84% (2). There are several IUS scores that evaluate CD disease activity such as SUS-CD (validated), IBUS-SAS and Simple US Score, which have not yet been studied in the diagnosis of POR.

Aims & Methods

The aim of this study is to assess whether IUS parameters and ultrasound scores correlate with endoscopy in the diagnosis of POR. A unicentric retrospective study was performed. There were included patients with CD with both an ileocolonoscopy and intestinal ultrasound performed for the detection of POR, the time between tests was less than 6 months and there was no therapeutic change between tests.
Endoscopic POR was evaluated with Rutgeers score (RS), considering POR RS ≥i2b. In IUS, were used: Simple US Score, SUS-CD and IBUS-SAS.

Results

103 patients were included, the Montreal classification was: A1 n=6 (5.8%), A2 n=78 (75.7%), A3 n=18 (17.5%), L1 n=54 (52.4%), L2 n=2 (1.9%), L3 n=47 (50.6%), B1 n=10 (9.7%), B2 n=49 (47.6%) and B3 n=44 (42.7%).
30 patients (29.1%) had no endoscopic POR, 22 (21.4%) had RS i2a, 12 (11.6%) RS i2b and 39 (37.9%) had severe POR (RS i3-i4). The mean wall thickness measured by IUS in patients without endoscopic POR was 2.7 mm (SD +/- 1.3) versus 5.0 (SD +/- 1.7) in patients with RS ≥i2b (p 0.001). Hyperemia (Limberg >1) was present in 60 patients (82.2%) with RS ≥i2a vs in 4 patients (13.3%) without endoscopic POR (p 0.001). Both hyperemia and wall thickness had a sensitivity of 89.0% and specificity of 76.7% in the diagnosis of POR.
The mean values for IUS scores were: Simple US Score 5.5 (SD +/- 3.1), SUS-CD 2.1 (SD +/- 1.8) and IBUS-SAS 32.9 (SD +/- 26.3). There weren’t statistical differences between IUS scores in patients with RS <i2b and ≥i2b. Simple US Score had an area under the ROC curve (AUC) of 51%, IBUS-SAS presented an AUC of 48.4% and SUS-CD of 48.5%.

Conclusion

In our experience wall thickness and hyperemia are useful parameters to diagnosis POR, however IUS scores (IBUS-SAS, SUS-CD and Simple US Score) are less reliable. In the future new scores adapted to POR may be needed.

References

1. Domènech E, López-Sanromán A, Nos P, Vera M, Chaparro M, Esteve M, et al., Recommendations of the Spanish Working Group on Crohn’s Disease and Ulcerative Colitis (GETECCU) on the monitoring, prevention and treatment of post-operative recurrence in Crohn’s disease. Gastroen- terol Hepatol (English Ed [Internet]. 2017 Aug;40:472-83. Disponible en: https://linkinghub.elsevier.com/retrieve/pii/ S2444382417301190.
2. Rispo A, Imperatore N, Testa A, Nardone OM, Luglio G, Caporaso N, et al. Diagnostic accuracy of ultrasonography in the detec- tion of postsurgical recurrence in Crohn’s disease: A systematic review with meta-analysis. Inflamm Bowel Dis. 2018;24:977-88.

Disclosure

No conflict of interest

Introduction

The disease course of ileal and ileocolonic Crohn’s disease (CD) is highly heterogeneous, and continuous objective measures of disease activity are needed. Intestinal Ultrasound (IUS) is a non-invasive modality assessing disease activity in IBD. The International Bowel Ultrasound Segmental Activity Score (IBUS-SAS) is an index for grading CD activity. However, importance of the ultrasonic findings in the terminal ileum (TI) at diagnosis and the responsiveness of ultrasonic findings to different treatments is still limited. We thus aimed to investigate the IUS characteristics of the TI at diagnosis and changes in IUS parameters in response to treatment 3 months after inclusion.

Aims & Methods

Patients with new-onset IBD are recruited in the ongoing multicentre prospective inception cohort study, the IBD Prognosis Study. IUS is performed at diagnosis and after three months (3M). IBUS-SAS (0-100) is scored for the TI and incorporates the bowel wall thickness (BWT), bowel wall stratification (BWS), colour Doppler signal (CDS), and inflammatory fat (I-FAT). Only patients with ileal and ileocolonic CD who had an available IBUS-SAS of the TI at diagnosis were included. Patient without subsequent assessment at 3M were excluded (except resected patients). Transmural remission was defined as IBUS-SAS < 12 (corresponding to BWT < 3 mm and no signs of inflammation). If the operator assessed the TI as complete transmural remission at 3M without providing numeric BWT values, the IBUS-SAS was set to 8. Patients treated with corticosteroids for > 72 hours or with any IBD treatment > two weeks prior to baseline IUS were excluded from analysis.

Results

Baseline IBUS-SAS at TI was available in 46 CD patients (ileal: 21, ileocolonic: 25). According to the Montreal classification, 31 patients had B1 (non-stricturing, non-penetrating disease), 9 patients had B2 (stricturing disease), and 6 patients had B3 (penetrating disease).
Among all patients, the mean IBUS-SAS was 60.7 at diagnosis vs. 33.7 at 3M (p<0.001). Mean baseline BWT decreased from 5.8 to 4.6 (p=0.011) at 3M. The other IBUS-SAS parameters also had significant reductions (p=0.021, p=0.001, and p<0.001 for BWS, CDS, and I-FAT, respectively). After three months, 12/39 (30.8%) of non-resected patients were in transmural remission.
Changes in IBUS-SAS between diagnosis and 3M stratified by initiated treatment are presented in Figure 1 (​https://ibb.co/mBcVF9b​​​). In total, 6 (13.0%) patients started only systemic steroids with a mean IBUS-SAS reduction of 20.6 (p=0.025), 5 (10.9%) patients started systemic steroids + biological therapy with a mean reduction in IBUS-SAS of 37.6 (p=0.022), 11 (23.9%) patients started on systemic steroids + immunomodulator with a mean reduction in IBUS-SAS of 35.6 (p<0.001), 4 (8.7%) patients started on immunomodulator + biological therapy with a mean reduction in IBUS-SAS of 24.8 (p=0.271), and 9 (19.6%) patients were started on budesonide with a mean reduction in IBUS-SAS of 9.4 (p=0.287). Finally, 4 (8.7%) patients did not commence on any treatment and these patients increased their IBUS-SAS by 6.75 (p=0.536), and 7 (15.2%) patients were resected as primary treatment with 1, 2, and 4 having the B1, B2, and B3 Montreal classification, respectively. Baseline IBUS-SAS was significantly higher for patients undergoing resection than patients without resection, (87.9 vs. 55.8, P<0.001).

Conclusion

IUS performed at diagnosis and after three months can effectively identify patients achieving ultrasonic response and / or transmural healing to multiple drug classes. All four IBUS-SAS activity parameters demonstrated responsiveness to treatment and patients without treatment increased their IBUS-SAS non-significantly. Transmural remission was achieved in up to 30 % of non-resected patients within 3M.

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.