Chi Han Li

A small-molecule modulator of the tumor-suppressor miR34a inhibits the growth of hepatocellular carcinoma.

Small molecules that restore the expression of growth-inhibitory microRNAs (miRNA) downregulated in tumors may have potential as anticancer agents. miR34a functions as a tumor suppressor and is downregulated or silenced commonly in a variety of human cancers, including hepatocellular carcinoma (HCC). In this study, we used an HCC cell-based miR34a luciferase reporter system to screen for miR34a modulators that could exert anticancer activity. One compound identified as a lead candidate, termed Rubone, was identified through its ability to specifically upregulate miR34a in HCC cells. Rubone activated miR34a expression in HCC cells with wild-type or mutated p53 but not in cells with p53 deletions. Notably, Rubone lacked growth-inhibitory effects on nontumorigenic human hepatocytes. In a mouse xenograft model of HCC, Rubone dramatically inhibited tumor growth, exhibiting stronger anti-HCC activity than sorafenib both in vitro and in vivo. Mechanistic investigations showed that Rubone decreased expression of cyclin D1, Bcl-2, and other miR34a target genes and that it enhanced the occupancy of p53 on the miR34a promoter. Taken together, our results offer a preclinical proof of concept for Rubone as a lead candidate for further investigation as a new class of HCC therapeutic based on restoration of miR34a tumor-suppressor function.

Hepatitis B virus X protein promotes hepatocellular carcinoma transformation through interleukin-6 activation of microRNA-21 expression

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and chronic hepatitis B virus (HBV) infection is the major risk factor of HCC. The virus encodes HBV X (HBx) protein that plays a critical role in the development of HCC. Studies have revealed numerous HBx-altered genes and signalling pathways that heavily contribute to tumourigenesis of non-tumour hepatocytes. However, the role of HBx in regulating other critical gene regulators such as microRNAs is poorly understood, which impedes the exploration of a complete HBx-associated carcinogenic network. Besides, critical microRNAs that drive the transformation of non-tumour hepatocytes are yet to be identified. Here, we overexpressed C-terminal truncated HBx protein in a non-tumour hepatocyte cell line MIHA, and measured a panel of cancer-associated miRNAs. We observed that oncogenic miR-21 was upregulated upon ectopic expression of this viral protein variant. HBx-miR-21 pathway was prevalent in HCC cells as inhibition of HBx in Hep3B and PLC/PRF/5 cells significantly suppressed miR-21 expression. Subsequently, we showed that the upregulation of miR-21 was mediated by HBx-induced interleukin-6 pathway followed by activation of STAT3 transcriptional factor. The high dependency of miR-21 expression to HBx protein suggested a unique viral oncogenic pathway that could aberrantly affect a network of gene expression. Importantly, miR-21 was essential in the HBx-induced transformation of non-tumour hepatocytes. Inhibition of miR-21 effectively attenuated anchorage-independent colony formation and subcutaneous tumour growth of MIHA cells. Our study suggested that overexpression of miR-21 was critical to promote early carcinogenesis of hepatocytes upon HBV infection.

A potential antitumor ellagitannin, davidiin, inhibited hepatocellular tumor growth by targeting EZH2

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is the third most common cause of cancer-related deaths. Currently available treatment options for HCC patients are scarce resulting in an urgent need to develop a novel effective cure. Polygonum capitatum is a medicinal herb which has been used to treat inflammatory diseases in Miao nationality of China. We recently isolated a pure compound davidiin from P. capitatum extract. Four HCC cell lines were treated with davidiin. Cell viability was recorded by MTT assay. siRNAs targeting enhancer of zeste homolog 2 (EZH2) were applied to modulate the expression of EZH2. Established xenograft mice models of HCC were applied to evaluate the in vivo anticancer activity of davidiin. We investigated the anticancer activity and the underlying mechanism of davidiin. The compound inhibited HCC cell growth and also suppressed tumor growth in xenografted HCC mouse. Such inhibition was facilitated by specifically downregulation on EZH2. The compound possesses anticancer activity both in vitro and in vivo which warrants further clinical investigation as a potential anti-HCC agent.

Role of microRNA-95 in the anticancer activity of Brucein D in hepatocellular carcinoma

Brucea javanica fruit has been used to treat amebic dysentery, malaria and various parasites and it has been applied as an anti-cancer agent in Traditional Chinese Medicine. Brucein D (BD) is a naturally occurring compound extracted from Brucea javanica fruit which shows anti-cancer activity against pancreatic cancer. Here, we further demonstrated that BD inhibited hepatocellular carcinoma (HCC) cell growth in vitro and tumor growth in vivo that were attributed to the induction of cell apoptosis. BD did not exert growth inhibition on non-tumorigenic human hepatocytes. MTT assay was used to measure cell viability. Annexin V and TUNEL assay were applied to identify apoptotic cells in cell suspension and in tissue section respectively. Downstream micro-RNA (miRNA) targets of BD were screened out by miRNA array. miRNAs and their target proteins were identified by bioinformatics analysis and luciferase reporter assay. 39 miRNAs regulated by BD in HCC were identified. miR-95 was found to be a potential drug target of BD. We further identified CUG triplet repeat RNA-binding protein 2 (CUGBP2) as the downstream target of miR-95. Our data suggested that BD exerted its anti-cancer activity against HCC through modulation of miR-95 expression.

G protein-coupled estrogen receptor inhibits the P2Y receptor-mediated Ca2+ signaling pathway in human airway epithelia

P2Y receptor activation causes the release of inflammatory cytokines in the bronchial epithelium, whereas G protein-coupled estrogen receptor (GPER), a novel estrogen (E2) receptor, may play an anti-inflammatory role in this process. We investigated the cellular mechanisms underlying the inhibitory effect of GPER activation on the P2Y receptor-mediated Ca2+ signaling pathway and cytokine production in airway epithelia. Expression of GPER in primary human bronchial epithelial (HBE) or 16HBE14o- cells was confirmed on both the mRNA and protein levels. Stimulation of HBE or 16HBE14o- cells with E2 or G1, a specific agonist of GPER, attenuated the nucleotide-evoked increases in [Ca2+]i, whereas this effect was reversed by G15, a GPER-specific antagonist. G1 inhibited the secretion of two proinflammatory cytokines, interleukin (IL)-6 and IL-8, in cells stimulated by adenosine 5′-(γ-thio)triphosphate (ATPγS). G1 stimulated a real-time increase in cAMP levels in 16HBE14o- cells, which could be inhibited by adenylyl cyclase inhibitors. The inhibitory effects of E2 or G1 on P2Y receptor-induced increases in Ca2+ were reversed by treating the cells with a protein kinase A (PKA) inhibitor. These results demonstrated that the inhibitory effects of G1 or E2 on P2Y receptor-mediated Ca2+ mobilization and cytokine secretion were due to GPER-mediated activation of a cAMP-dependent PKA pathway. This study has reported, for the first time, the expression and function of GPER as an anti-inflammatory component in human bronchial epithelia, which may mediate through its opposing effects on the pro‐inflammatory pathway activated by the P2Y receptors in inflamed airway epithelia.

Berberine induces miR-373 expression in hepatocytes to inactivate hepatic steatosis associated AKT-S6 kinase pathway

Abstract Berberine is a Chinese herbal medicine extracted from rhizoma coptidis that functions to improve insulin resistance, hyperlipidemia, hepatosteatosis and inflammation. Berberine can modify the activity of cell metabolism and signaling pathways by regulating expression of genes. However, the roles and effects of differential microRNA (miRNA) expression induced by berberine treatment are largely unexplored. It is believed that miRNAs expression modified by berberine contributes to its therapeutic effects to diseases such as hepatosteatosis and non‐alcoholic fatty liver disease. By identifying novel miRNAs and their putative gene targets associated with abnormal hepatic lipid deposition, the underlying mechanism of these diseases could be established and effective therapies against the diseases could be developed. Here, we used the immortalized hepatocyte cell line MIHA as a model to study the effect of berberine on global miRNA expression profile of hepatocytes. Global miRNA expression levels were measured in berberine‐treated MIHA cells by quantitative reverse transcription PCR miRNA panel, and the potential berberine regulated miRNAs were then validated in MIHA and HepG2 cells. MicroRNA‐373 (MiR‐373) was consistently upregulated in both cell lines upon berberine treatments. Gene expression microarray showed that berberine upregulated Early Growth Response 1 (EGR1) level which functioned to transactivate miR‐373 expression. Subsequently, we showed that upregulation of miR‐373 depleted its target gene AKT serine/threonine kinase 1 (AKT1) mRNA level, which led to the inhibition of AKT‐mTOR‐S6K signaling pathway in hepatocytes that was critical in the development of hepatosteatosis. Study of the therapeutic effect of manipulating miR‐373 against abnormal lipid deposition in liver is warranted.

Abstract 2844: Identification of genes associated with pancreatic cancer metastasis by genome-wide CRISPR Cas9 screening

Pancreatic cancer is an extremely lethal cancer with a 5-year survival rate of 7%. One major reason why pancreatic cancer is hard to treat is its metastasis nature. Genes that related to the tumorigenesis and progression of pancreatic cancer were identified after decades of efforts. Several researches had revealed that pancreatic cancer originated from the successive accumulation of gene mutations, especially KRAS2, CNKD2A, TP53 and SMAD4. However, little is known about genes that regulate pancreatic cancer metastasis. Here we used a genome wide CRISPR Cas9 screening to identify the gene(s) associated with the metastasis of pancreatic cancer. We transduced pancreatic cancer cell line with lentivirus-based genome wide CRISPR sgRNA library to establish mutated cell library, and orthotopically injected the cell library into nude mice and waited for primary tumor growth and metastasis, then collected primary and metastatic tumors after several months. By next generation sequencing and analysis, we identified some gene candidates which may play important roles on pancreatic cancer metastasis. Note: This abstract was not presented at the meeting. Citation Format: Youjia Li, Huiyi Feng, Guangjie Liu, Chi Hin Wong, Chi Han Li, Yangchao Chen. Identification of genes associated with pancreatic cancer metastasis by genome-wide CRISPR Cas9 screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2844. doi:10.1158/1538-7445.AM2017-2844