Background and Aims: Ambiguous understanding of tumors and the tumor microenvironments (TMEs) hinders accurate diagnosis and available treatment for multifocal hepatocellular carcinoma (HCC) covering intrahepatic metastasis (IM) and multicentric occurrence (MO). Here, we characterized the diverse TMEs of IM and MO identified by whole-exome sequencing (WES) at single-cell resolution. Approach and Results: We performed parallel WES and single-cell RNA sequencing (scRNA-seq) on twenty-three samples from seven patients to profile their TMEs when major results were validated by immunohistochemistry in the additional cohort. Integrative analysis of WES and scRNA-seq found that malignant cells in IM showed higher intra-tumor heterogeneity, stemness and more activated metabolism than those in MO. Tumors from IM shared similar TMEs while distinct TMEs were noticed in those from MO. Furthermore, CD20+ B cells, plasma cells and conventional type II dendritic cells (cDC2s) were decreased in IM relative to MO while T cells in IM exhibited a more terminally exhausted capacity with a higher proportion of proliferative/exhausted T cells than that in MO. Both CD20 and CD1C correlated with better prognosis in multifocal HCC. Additionally, MMP9+ tumor-associated macrophages were enriched across IM and MO which formed cellular niches with regulatory T cells (Tregs) and proliferative/exhausted T cells. Conclusions: Our findings deeply decipher the heterogeneous TMEs between IM and MO, which provide a comprehensive landscape of multifocal HCC. Export

Hepatocellular carcinoma (HCC) is the most prevalent type of malignant liver tumor with high morbidity and mortality and severely threatens human health and life quality. Thus, it is of great significance to investigate the molecular mechanism underlying the pathogenesis of HCC and seek biomarkers for early diagnosis. Neddylation, one of the most conserved post-translational modification types in eukaryotes, plays vital roles in the progression of HCC. During the process of neddylation, NEDD8 is covalently conjugated to its substrate proteins, thereby modulating multiple necessary biological processes. Currently, increasing evidence shows that the aberrant activation of neddylation is positively correlated with the occurrence and development of tumors and the poor clinical prognosis of HCC patients. Based on the current investigations, neddylation modification has been reported to target both the cullins and non-cullin substrates and subsequently affect HCC progression, including the virus infection, malignant transformation, tumor cell proliferation, migration and invasion ability, and tumor microenvironment. Therefore, inhibitors targeting the neddylation cascade have been developed and entered clinical trials, indicating satisfactory anti-HCC treatment effects. This review aims to summarize the latest progress in the molecular mechanism of pathologically aberrant neddylation in HCC, as well as the advances of neddylation-targeted inhibitors as potential drugs for HCC treatment.

Biliary tract cancer (BTC) is an aggressive tumor with poor prognosis and limited treatment options. Epithelial-mesenchymal transition (EMT) significantly contributes to BTC metastasis, yet the critical regulators and underlying molecular mechanisms of EMT remain poorly unclear. In our study, we conducted an integrative analysis of single-cell RNA sequencing (scRNA-seq) data from 47 BTC samples, identifying two EMT-enriched epithelial subpopulations and a BTC specific EMT gene set. Using the EMT gene set, we classified BTC patients and noticed that those with elevated EMT scores had worse prognoses. Additionally, multi-omics analyses identified PMEPA1 as a pivotal EMT regulator, with its high expression levels correlating with adverse prognosis and distant metastasis. Functional assays revealed that PMEPA1 knockdown inhibits, while its overexpression promotes EMT progression by modulating Hippo-YAP signaling, specifically YAP1 nuclear localization. Moreover, we forecast and validated topoisomerase inhibitor SN-38 as a potential EMT-targeting agent. SN-38 effectively inhibited BTC cell migration and metastasis in vivo, likely by attenuating the transcriptional activity of PMEPA1 and its upstream regulator FOS. Overall, this research elucidates EMT-related molecular features in BTC, reveals PMEPA1 as a critical EMT driver via the Hippo-YAP1 pathway, and proposes SN-38 as a promising therapeutic candidate, offering new insights into BTC metastasis and precision therapy.