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The complement cascade plays a “complementing” role in human immunity. However, the potential roles of complement system in impacting molecular and clinical features of hepatocellular carcinoma (HCC) remain unclear. In this study, eleven public datasets are analyzed to compare the complement status between normal and cancerous samples based on 18 classical complement-associated genes. The complement scores are constructed to quantify complement signatures of individual tumors. HCC patients in the The Cancer Genome Atlas (TCGA) cohort are focused to perform systematical analyses between complement status and immune infiltration, miRNA expression, DNA methylation, clinicopathological features, and drug response. The results show that the complement scores in normal tissues are dramatically higher than those of tumor tissues. Tumor samples in the TCGA cohort are classified into complement score-low and score-high groups. Pathway analysis reveals that tumor-promoting pathways are typically inhibited in complement score-high group. This study also shows that tumor-killing immune cells, such as CD8 + T cells and natural killer cells are abundant and tumor-suppressing miRNAs are upregulated in complement score-high samples. In addition, we identify that complement scores are negatively correlated with certain clinical features, including pathological grade, clinical-stage, and portal vein invasion. Moreover, various molecular features together with complement scores are found to be correlated with response to anti-cancer drugs. This study provides a comprehensive and multidimensional analysis conducive to understanding the role of complement in cancer.

Drug resistance has greatly limited the clinical efficacy of lenvatinib in hepatocellular carcinoma (HCC). However, the underlying molecular mechanisms of lenvatinib resistance remain largely undetermined. Further in-depth exploration of mechanisms underlying lenvatinib resistance is still required for the majority of HCC patients. In this study, an integrated unbiased whole-genome CRISPR-Cas9 screen with database analysis indicated LAPTM5 (lysosomal protein transmembrane 5) as the critical contributor to lenvatinib resistance in HCC. We revealed that LAPTM5 could promote intrinsic macroautophagic/autophagic flux by facilitating autolysosome formation to drive lenvatinib resistance. The upregulation of LAPTM5 in HCC was induced by both DNA hypomethylation and driver mutations like TP53. Inhibition of autolysosome formation by either hydroxychloroquine (HCQ) or LAPTM5 abrogation worked synergistically with lenvatinib to inhibit tumor growth. In HCC cell lines, patient-derived primary cell lines and organoids, as well as human HCC xenografts and immunocompetent mouse HCC model, the close association between LAPTM5 and sensitivity to lenvatinib was consistently verified. Importantly, in clinical HCC samples, where lenvatinib was used as the first line or adjuvant therapy, LAPTM5 expression negatively correlated with lenvatinib sensitivity, implying it as a biomarker to predict patient response to lenvatinib. In conclusion, the combinational therapy targeting autophagy represented a promising strategy to overcome lenvatinib resistance in HCC, and LAPTM5 expression could provide potential guidance for clinical interference. cld-CASP3: cleaved caspase 3; cld-PARP: cleaved PARP; DTP: drug tolerant persister; GO: Gene Ontology; GTEx: The Genotype-Tissue Expression; HCC: hepatocellular carcinoma; HCQ: hydroxychloroquine; IC50: half maximal inhibitory concentration value; KEGG: Kyoto Encyclopedia of Genes and Genomes; LAPTM5: lysosomal protein transmembrane 5; NT: non-targeting; PDC: patient-derived primary cell lines; PDO: patient-derived primary organoid; TCGA: The Cancer Genome Atlas. [ABSTRACT FROM AUTHOR]

Background: SQSTM1/p62 is a selective autophagy receptor that regulates multiple signaling pathways participating in the initiation and progression of tumors. Metastasis is still the main cause for intrahepatic cholangiocarcinoma (ICC)‐associated mortality. Hence, this study aimed to explore the mechanism of p62 promoting the progression of ICC. Methods: Western blotting and immunohistochemical analyses were conducted to detect the expression level of protein p62 in ICC tissues and its correlation with prognosis. Subsequently, the loss‐of‐function experiments in vitro and in vivo were performed to define the role of p62 in ICC cell proliferation, invasion, and metastasis. Then, the effect of p62 knockdown on mitochondrial function and mitophagy was evaluated by measuring the oxygen consumption rate, and using immunofluorescence and western blotting analyses. Results: The expression of p62 was significantly upregulated in ICC specimens compared with normal tissues. We further illustrated that p62 expression positively correlated with lymph node metastasis and poor prognosis. The loss‐of‐function assays revealed that p62 not only promoted ICC cell proliferation, migration, and invasive capacities in vitro, but also induced lung metastasis in the xenograft mouse model. Mechanistically, high expression of p62‐induced epithelial–mesenchymal transition (EMT) with the upregulation of Snail, vimentin, N‐cadherin, and downregulation of E‐cadherin. Moreover, the autophagy‐dependent function of p62 might play a vital role in maintaining the mitochondrial function of ICC by mitophagy which might further promote EMT. Conclusion: These data provided new evidence for the mechanism by which abundant p62 expression promoted ICC progression, suggesting a promising therapeutic target for antimetastatic strategies in patients with ICC. [ABSTRACT FROM AUTHOR]

Intrahepatic cholangiocarcinoma (ICC) remains a highly heterogeneous disease with poor prognosis. Tumor‐infiltrating lymphocytes were predictive in various cancers, but their prognostic value in ICC is less clear. A total of 168 ICC patients who had received liver resection were enrolled and assigned to the derivation cohort. Sixteen immune markers in tumor and peritumor regions were examined by immunohistochemistry. A least absolute shrinkage and selection operator model was used to identify prognostic markers and to establish an immune signature for ICC (ISICC). An ISICC‐applied prediction model was built and validated in another independent dataset. Five immune features, including CD3peritumor (P), CD57P, CD45RAP, CD66bintratumoral (T) and PD‐L1P, were identified and integrated into an individualized ISICC for each patient. Seven prognostic predictors, including total bilirubin, tumor numbers, CEA, CA19‐9, GGT, HBsAg and ISICC, were integrated into the final model. The C‐index of the ISICC‐applied prediction model was 0.719 (95% CI, 0.660‐0.777) in the derivation cohort and 0.667 (95% CI, 0.581‐0.732) in the validation cohort. Compared with the conventional staging systems, the new model presented better homogeneity and a lower Akaike information criteria value in ICC. The ISICC‐applied prediction model may provide a better prediction performance for the overall survival of patients with resectable ICC in clinical practice. [ABSTRACT FROM AUTHOR]

Background: Various immune cell types in the tumor microenvironment (TME) of hepatocellular carcinoma (HCC) have been identified as important parameters associated with prognosis and responsiveness to immunotherapy. However, how various factors influence immune cell infiltration remains incompletely understood. Hence, we investigated the single cell multi-omics landscape of immune infiltration in HCC, particularly key gene and cell subsets that influence immune infiltration, thus potentially linking the immunotherapy response and immune cell infiltration., Methods: We grouped patients with HCC according to immune cell infiltration scores calculated by single sample gene set enrichment analysis (ssGSEA). Differential expression analysis, functional enrichment, clinical trait association, gene mutation analysis, tumor immune dysfunction and exclusion (TIDE) and prognostic model construction were used to investigate the immune infiltration landscape through multi-omics. Stepwise regression was further used to identify key genes regulating immune infiltration. Single cell analysis was performed to explore expression patterns of candidate genes and investigate associated cellular populations. Correlation analysis, ROC analysis, Immunotherapy cohorts were used to explore and confirm the role of key gene and cellular population in predicting immune infiltration state and immunotherapy response. Immunohistochemistry and multiplexed fluorescence staining were used to further validated our results., Results: Patients with HCC were clustered into high and low immune infiltration groups. Mutations of CTNNB1 and TTN were significantly associated with immune infiltration and altered enrichment of cell populations in the TME. TIDE analysis demonstrated that T cell dysfunction and the T cell exclusion score were elevated in the high and low infiltration groups, respectively. Six risk genes and five risk immune cell types were identified and used to construct risk scores and a nomogram model. CXCR6 and LTA, identified by stepwise regression, were highly associated with immune infiltration. Single cell analysis revealed that LTA was expressed primarily in tumor infiltrating T lymphocytes and partial B lymphocytes, whereas CXCR6 was enriched predominantly in T and NK cells. Notably, CXCR6 + CD8 T cells were characterized as tumor enriched cells that may be potential predictors of high immune infiltration and the immune-checkpoint blockade response, and may serve as therapeutic targets., Conclusion: We constructed a comprehensive single cell and multi-omics landscape of immune infiltration in HCC, and delineated key genes and cellular populations regulating immune infiltration and immunotherapy response, thus providing insights into the mechanisms of immune infiltration and future therapeutic control., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li, Gao, Chen, Feng, Luo, Shi, Tang, Liu, Zhang, Huang, Gao, Ke, Zhou, Fan, Fu and Ding.)

Purpose: To uncover the biological role of LINC00355 in regulating the proliferative and apoptotic potentials in hepatocellular carcinoma (HCC), and the underlying mechanism., Methods: LINC00355 levels in HCC tissues and cell lines were detected by quantitative real-time polymerase chain reaction (qRT-PCR). After knockdown of LINC00355 or miR-217-5p in Hub7 and Hep3B cells, proliferative and apoptotic potentials were assessed by cell counting kit-8 (CCK-8), colony formation assay and flow cytometry. The interaction between LINC00355 and miR-217-5p was determined by dual-luciferase reporter assay and Pearson correlation test. Western blot analysis was conducted to illustrate the regulatory effects of LINC00355 and miR-217-5p on the Wnt/β-catenin signaling., Results: LINC00355 was upregulated in HCC tissues and cell lines. Knockdown of LINC00355 reduced viability in Hub7 and Hep3B cells, which was much pronounced on days 3 and 4. Clonality was attenuated by transfection of shLINC00355 as well. In addition, apoptosis rate increased by knockdown of LINC00355 in HCC cells. Protein levels of β-catenin, GSK3β, c-myc and cyclin D1 were downregulated in Hub7 and Hep3B cells transfected with shLINC00355. MiR-217-5p was the target gene binding LINC00355. It displayed exactly opposite regulations on HCC cell phenotypes and protein levels of vital genes in the Wnt/β-catenin signaling to those of LINC00355., Conclusions: LINC00355 is upregulated in HCC specimens, LINC00355 triggers proliferative rate and inhibits apoptosis in HCC cells by negatively regulating miR-217-5p and activating the Wnt/β-catenin signaling.

Lysyl oxidase like 4 (LOXL4), a member of the secreted copper-dependent amine oxidases that contribute to the assemble and maintenance of the extracellular matrix (ECM), was found to be up-regulated or down-regulated in different cancer types, suggesting its paradoxical roles in cancer. The specific role of LOXL4 in hepatocellular carcinoma (HCC), however, is still yet to be defined. Twenty-eight pairs of HCC specimens were used for LOXL4 mRNA expression analysis. The mRNA expression in HCC cell lines was examined, and HepG2 was selected for LOXL4 small interfering RNA (siRNA) interference to investigate the biological function of LOXL4, LOXL4 immunohistochemical staining was performed using a tissue microarray containing 298 HCC patients. The prognostic and diagnostic value of LOXL4 was evaluated using Cox regression and Kaplan-Meier analysis. LOXL4 mRNA or protein expression was significantly lower in HCC tissues than peritumoral tissues (LOXL4 mRNA expression, P = 0.018; LOXL4 protein expression, P < 0.001). Low LOXL4 expression was associated with lower overall survival (OS) rates and higher cumulative recurrence rates. Multivariate analysis indicated that LOXL4 was an independent prognostic indicator for OS and time to recurrence (TTR). Our results revealed that LOXL4 was down-regulated in HCC and correlated with aggressive tumors and a worse clinical outcome. LOXL4 may be a potential biomarker to identify the HCC patients with a higher risk of recurrence.