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Single-cell analysis reveals the landscape of tumor heterogeneity and identifies MLXIPL as a potential biomarker in tumor evolution trajectory of hepatocellular carcinoma

ABSTRACT: Hepatocellular carcinoma (HCC) is a globally prevailing cancer with a low five-year survival rate. Little is known about its intricate gene expression profile and tumor evolutionary traits. Single-cell RNA sequencing (scRNA-seq) is an indispensable tool to explore the genetic characteristics at the single-cell level. In this study, we profiled gene expression of single cells from human HCC tumor and para-tumor tissues using Smart-seq 2 sequencing method. Based on DEGs, we identified heterogeneous subclones in HCC tissues, including 4 HCC and 2 hepatocyte subclones. Then, we carried out hub-gene co-network and functional annotations analysis to further explain tumor heterogeneity. Next, we conducted the evolution trajectory by pseudo-time analysis and its regulated transcriptional factors (TF) gene network. In the following research, we found that MLXIPL, as a TF, was commonly upregulated in HCC tissues and cells at both the mRNA and protein levels, promoting cell proliferation and inhibiting its apoptosis. Mechanistically, MLXIPL activation is crucial for accelerating HCC cell glycolysis. Taken together, our work identified the heterogeneity of HCC cell subclones, and MLXIPL might be a promising therapeutic target of HCC.

ORGANISM(S): Homo sapiens

PROVIDER: GSE154906 | GEO | 2021/01/27

REPOSITORIES: GEO

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Project description:Increasing evidences have been recently reported on tumor cells heterogeneity within several malignancies, but data on liver tumours are still lacking. The aim of this study was to investigate and characterize tumor initiating cell (TIC) compartments within human hepatocellular carcinoma (HCC). After long term culture, we were able to identify 3 morphologically different tumor cell populations from an individual HCC specimen. These cell populations were extensively characterized by flow cytometry, fluorescence microscopy, single cell cloning, xenotransplantation in NOD/SCID IL2Rgamma -/- mice, karyotyping and microarray analyses. The 3 primary cell populations, hcc-1, -2 and -3, and the 2 clones (clone-1/7 and -1/8) generated by limiting dilution from hcc-1 showed different expression profiles for several tumor associated stem cell markers, including EpCAM, CD49f, CD44, CD133, CD56, Thy-1, ALDH and CK19. Moreover, they showed different doubling time and drug resistance. The tumorigenic potential was higher for hcc-1 and clone-1/7. Karyotyping analyses revealed a clonal evolution of cell populations and clones within the primary tumor. Importantly, we noticed that the primary tumor cell population with a higher tumorigenic potential and drug resistance was that showing more chromosomal alterations and containing different clones with both epithelial and mesenchymal features. Individual HCC can harbor different self-renewing tumorigenic cell types expressing a variety of morphology and phenotypic markers, karyotypic evolution and different gene expression profiles. This finding suggests that the therapeutic approach for HCC eradication should take into account the possible TIC heterogeneity due to intratumor clonal evolution. The aim was to analyse the whole genome expression profile of 3 HCC cell lines (Hcc-1, Hcc-2 and Hcc-3) and two clones (Clone-1/7 and Clone-1/8) cells obtained from one patient. Tumor sample was dissociated and cells cultured on collagen coated Petri dishes. When cell colonies appeared in culture, they were detached and cultured separately; clones were obtained fron HCC-1 cell lines by means of limiting dilution. Total Five independent total RNA extractions were performed for each cell line and clone and after checked the quality, concentration and integrity of the purified RNA, only samples showing the better results were chosen to be further processed. After the acquisition of the raw intensity data matrix with the corresponding annotations, probes corresponding to "predicted" genes or not associated to a Entrez ID were discarded from the analysis; moreover, different probe signals corresponding to the same Entrez ID were converted into unique intensity signal, corresponding to the median of the values for each array, in order to obtain a single signal for each Entrez ID. After this process, we generated a boxplot of 16964 intensity signals whose values were log2-transformed and quantile normalized using the aroma.light package for Bioconductor. Genes with a differential expression value of at least 2 fold (on log2 transformed scale) were considered differentially expressed.

Project description:Background: Tumor necrosis factor α-induced protein 1 (TNFAIP1) is frequently downregulated in cancer cell lines and promotes cancer cell apoptosis. However, its role, clinical significance and molecular mechanisms in hepatocellular carcinoma (HCC) are unknown. Methods: The expression of TNFAIP1 in HCC tumor tissues and cell lines was measured by Western blot and immunohistochemistry. The effects of TNFAIP1 on HCC proliferation, apoptosis, metastasis, angiogenesis and tumor formation were evaluated by Cell Counting Kit-8 (CCK8), Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL), transwell, tube formation assay in vitro and nude mice experiments in vivo. The interaction between TNFAIP1 and CSNK2B was validated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), Co-immunoprecipitation and Western blot. The mechanism of how TNFAIP1 regulated nuclear factor-kappaB (NF-κB) pathway was analyzed by dual-luciferase reporter, immunofluorescence, quantitative Real-time polymerase chain reaction (RT-qPCR) and Western blot. Findings: The TNFAIP1 expression is significantly decreased in HCC tissues and cell lines, and negatively correlated with the increased HCC histological grade. Overexpression of TNFAIP1 inhibits HCC cell proliferation, metastasis, angiogenesis and promotes cancer cell apoptosis both in vitro and in vivo, whereas the knockdown of TNFAIP1 in HCC cell displays opposite effects. Mechanistically, TNFAIP1 interacts with CSNK2B and promotes its ubiquitin-mediated degradation with Cul3, causing attenuation of CSNK2B-dependent NF-κB trans-activation in HCC cell. Moreover, the enforced expression of CSNK2B counteracts the inhibitory effects of TNFAIP1 on HCC cell proliferation, migration, and angiogenesis in vitro and in vivo. Interpretation: Our results support that TNFAIP1 can act as a tumor suppressor of HCC by modulating TNFAIP1/CSNK2B/NF-κB pathway, implying that TNFAIP1 may represent a potential marker and a promising therapeutic target for HCC. Fund: This work was supported in part by the financial support from the China Natural Science Foundation (No. 81972642, No. 81601122 and No. 81770389), Hunan Natural Science Foundation (No. 2017JJ3205), Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (No. 20134486), and the Scientific Research Fund of Hunan Provincial Education Department (17B162).

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Single-cell analysis reveals the landscape of tumor heterogeneity and identifies MLXIPL as a potential biomarker in tumor evolution trajectory of hepatocellular carcinoma

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