Sorted-Cell Sequencing on HCC Specimens Reveals EPS8L3 as a Key Player in CD24/CD13/EpCAM-Triple Positive, Stemness-Related HCC Cells.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a heterogeneous cancer with varying levels of liver tumor initiating or cancer stem cells in the tumors. We aimed to investigate the expression of different liver cancer stem cell (LCSC) markers in human HCCs and identify their regulatory mechanisms in stemness-related cells. METHODS: We used an unbiased, single-marker sorting approach by flow cytometry, fluorescence-activated cell sorting, and transcriptomic analyses on HCC patients' resected specimens. Knockdown approach was used, and relevant functional assays were conducted on the identified targets of interest. RESULTS: Flow cytometry on a total of 60 HCC resected specimens showed significant heterogeneity in the expression of LCSC markers, with CD24, CD13, and EpCAM mainly contributing to this heterogeneity. Concomitant expression of CD24, CD13, and EpCAM was detected in 32 HCC samples, and this was associated with advanced tumor stages. Transcriptomic sequencing on the HCC cells sorted for these individual markers identified epidermal growth factor receptor kinase substrate 8-like protein 3 (EPS8L3) as a common gene associated with the 3 markers and was functionally validated in HCC cells. Knocking down EPS8L3 suppressed the expression of all 3 markers. To search for the upstream regulation of EPS8L3, we found SP1 bound to EPS8L3 promoter to drive EPS8L3 expression. Furthermore, using Akt inhibitor MK2206, we showed that Akt signaling-driven SP1 drove the expression of the 3 LCSC markers. CONCLUSIONS: Our findings suggest that Akt signaling-driven SP1 promotes EPS8L3 expression, which is critical in maintaining the downstream expression of CD24, CD13, and EpCAM. The findings provide insight into potential LCSC-targeting therapeutic strategies.

Multimodal Integrative Genomics and Pathology Analyses in Neoadjuvant Nivolumab Treatment for Intermediate and Locally Advanced Hepatocellular Carcinoma.

Introduction: Immunotherapy has resulted in pathologic responses in hepatocellular carcinoma (HCC), but the benefits and molecular mechanisms of neoadjuvant immune checkpoint blockade are largely unknown. Methods: In this study, we evaluated the efficacy and safety of preoperative nivolumab (anti-PD-1) in patients with intermediate and locally advanced HCC and determined the molecular markers for predicting treatment response. Results: Between July 2020 and November 2021, 20 treatment-naive HCC patients with intermediate and locally advanced tumors received preoperative nivolumab at 3 mg/kg for 3 cycles prior to surgical resection. Nineteen patients underwent surgical resection on trial. Seven (36.8%) of the 19 patients had major pathologic tumor necrosis (≥60%) in the post-nivolumab resection specimens, with 3 having almost complete (>90%) tumor necrosis. The tumor necrosis was hemorrhagic and often accompanied by increased or dense immune cell infiltrate at the border of the tumors. None of the patients developed major adverse reactions contradicting hepatectomy. RNA-sequencing analysis on both pre-nivolumab tumor biopsies and post-nivolumab resected specimens showed that, in cases with major pathologic necrosis, the proportion of CD8 T cells in the HCC tissues predominantly increased after treatment. Moreover, to investigate noninvasive biomarker for nivolumab response, we evaluated the copy number variation (CNV) using target-panel sequencing on plasma cell-free DNA of the patients and derived a CNV-based anti-PD-1 score. The score correlated with the extent of tumor necrosis and was validated in a Korean patient cohort with anti-PD-1 treatment. Conclusion: Neoadjuvant nivolumab demonstrated promising clinical activity in intermediate and locally advanced HCC patients. We also identified useful noninvasive biomarker predicting responsiveness.

Lymphoepithelioma-like neoplasm of the biliary tract with 'probable low malignant potential'.

AIMS: Lymphoepithelioma-like carcinomas (LELCs) are uncommon epithelial cancers characteristically showing two distinct components consisting of malignant epithelial cells and prominent dense lymphoid infiltrate. Hepatic LELCs consist of two types, the lymphoepithelioma-like hepatocellular carcinoma and lymphoepithelioma-like cholangiocarcinoma (LEL-CCA), with the latter being strongly associated with Epstein-Barr virus (EBV). METHODS AND RESULTS: We present a series of three cases of intrahepatic biliary EBV-associated LEL tumours in which the biliary epithelial component showed a distinctly benign appearance, instead of the usual malignant epithelial features of a typical CCA or EBV-associated LEL-CCA. In the lesions, the biliary epithelium showed interconnecting glands or cords of cells. All had a very low proliferation (Ki-67) index. Immunohistochemistry for IDH1 and TP53 performed on two cases was negative and molecular tests for EGFR and KRAS gene mutations performed on one were negative. Prognosis was very good in all three cases, with patients alive with no evidence of disease 24-62 months after surgery. Intriguingly, all three cases had co-infection of HBV and EBV. These cases are also discussed in the context of the 63 cases of LEL-CCA available in the literature, with a focus on epidemiology, clinicopathological features and potential research interests. CONCLUSIONS: Based on the distinct clinicopathological features and unique survival benefits, we believe these tumours represent the benign end of the spectrum of EBV-associated lymphoepithelial biliary carcinomas. Whether these tumours require a revision of the current nomenclature to 'lymphoepithelioma-like neoplasm of the biliary tract with probable low malignant potential' will require more detailed analysis with larger case-series.

Centrosomal protein TAX1BP2 inhibits centrosome-microtubules aberrations induced by hepatitis B virus X oncoprotein.

Liver cancer (hepatocellular carcinoma, HCC) is one of the most prevalent cancers worldwide. Several etiological factors of HCC, including hepatitis B or hepatitis C virus infection, liver cirrhosis and aflatoxin B1 intake has been identified. HBx, which is an oncogenic protein encoded by the hepatitis B virus, is strongly associated with hepatocarcinogenesis. Using stable HBx-expressing cell, we showed that HBx induced chromosome gain, with amplification of centrosomes numbers and deregulation of centrosome ultrastructure. To dissect the mechanism for chromosome instability, our result revealed that HBx contributed to a hyperactive centrosome-microtubule dynamics by accelerating microtubule nucleation and polymerization. Further investigations suggested that HBx interacted with a centrosome linker protein TAX1BP2, which has previously been shown to function as an intrinsic block of centrosome amplification and a tumour suppressor in HCC. Restoring TAX1BP2 was able to block HBx-mediated centrosome amplification and abolish the HBx-mediated centrosome aberration, thereby suppressing chromosome instability. Thus, we demonstrate here a mechanism by which HBx deregulates centrosome-microtubule dynamics through interacting with TAX1BP2, which underlines the possibility of restoration of TAX1BP2 to rescue cells from chromosome instability.

Hypoxia regulates the mitochondrial activity of hepatocellular carcinoma cells through HIF/HEY1/PINK1 pathway.

Hypoxia is commonly found in cancers. Hypoxia, due to the lack of oxygen (O2) as the electron recipient, causes inefficient electron transfer through the electron transport chain at the mitochondria leading to accumulation of reactive oxygen species (ROS) which could create irreversible cellular damages. Through hypoxia-inducible factor 1 (HIF-1) which elicits various molecular events, cells are able to overcome low O2. Knowledge about the new molecular mechanisms governed by HIF-1 is important for new therapeutic interventions targeting hypoxic tumors. Using hepatocellular carcinoma (HCC) as a model, we revealed that the HIF-1 and the Notch signaling pathways cross-talk to control mitochondrial biogenesis of cancer cells to maintain REDOX balance. From transcriptome sequencing, we found that HEY1, a transcriptional repressor, in the NOTCH pathway was consistently induced by hypoxia in HCC cell lines. We identified a strong hypoxia response element (HRE) in HEY1 by chromatin immunoprecipitation (ChIP) and luciferase reporter assays. Transcriptome and ChIP sequencing further identified PINK1, a gene essential for mitochondrial biogenesis, as a novel transcriptional target of HEY1. HCC cells with HEY1 knockdown re-expressed PINK1. HEY1 and PINK1 expressions inversely correlated in human HCC samples. Overexpression of HEY1 and under-expression of PINK1 were detected in human HCC and associated with poor clinical outcomes. Functionally, we found that overexpression of HEY1 or knockdown of PINK1 consistently reduced mitochondrial cristae, mitochondrial mass, oxidative stress level, and increased HCC growth.

SERPINA5 inhibits tumor cell migration by modulating the fibronectin-integrin ß1 signaling pathway in hepatocellular carcinoma.

In our previous study, we identified 1241 loci with somatic copy number alterations in human hepatocellular carcinoma (HCC) using Affymetrix SNP 6.0 arrays, and a putative cancer gene SERPINA5 was uncovered in a novel chromosomal region with recurrent copy number loss at 14q31.1-32.13. The SERPINA5 was reported to be deregulated in renal, breast, prostate and ovarian cancers. However, the roles of SERPINA5 in cancer remain greatly elusive. In this study, we found that the DNA dosage and expression level of the SERPINA5 gene were significantly decreased in HCC by quantitative real-time PCR. Notably, the expression levels of SERPINA5 negatively correlated with malignant progression of HCC. The SERPINA5 gene was further observed to reduce in vitro and in vivo metastatic potential of HCC cells. Moreover, secreted SERPINA5 protein also could inhibit the metastatic ability of HCC cells. Finally, we discovered that one of the mechanisms explaining SERPINA5 inhibition of HCC metastasis is through direct interaction with fibronectin and disruption of the fibronectin-integrin signaling pathway. These findings highlight an important role of SERPINA5 in the regulation of migratory and metastatic potentials of HCC and suggest a potential application of SERPINA5 in cancer treatment.