Inhibition of glypican 3 facilitates hepatocellular carcinoma cells to acquire cancer stem cell phenotype by activating epithelial mesenchymal transition pathway

Abstract

Hepatocellular carcinoma (HCC) is the most common primary liver cancer. In 2012, 782,000 HCC patients were diagnosed worldwide, and among them 746,000 died. Currently, HCC is the 6th most common and the 3rd most fatal cancer globally. Patients with the advanced HCC have few treatment options. Sorafenib is the first line systemic therapy but it only prolongs patients’ survival time by two months. As a second line therapy, regorafenib could be used to treat the patients with sorafenib resistance, however, this agent also has limited efficacy as it only extends patients survival by <3 months. Despite of advances in the early detection and therapeutic strategies over the recent years, the overall prognosis of HCC patients remains extremely unsatisfactory.

Glypican 3 (GPC3) has been thought to be a specific biomarker for HCC because it is almost exclusively and highly expressed by HCC tissues. As such, many therapeutic strategies targeting GPC3 including vaccines, antibodies and chimeric antigen receptor T cells have been developed. However, these treatment options have not been shown to significantly improve the outcomes of HCC patients. Over the past few years, published studies including our own data have indicated that cancer stem cells (CSCs) are the main culprit for cancer initiation, progression, metastasis and relapse. CSCs are also closely linked to drug resistance. Whether the unsatisfactory results of GPC3 targeting therapies are related to their inability to remove CSCs is a practical question remaining to be answered.

In this project, we aimed to unveil the possible mechanisms related to the poor efficacy of GPC3 targeted therapies. We started by performing bioinformatic pathway analysis for GPC3 using two large scale public databases including Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA). We found that GPC3 was positively correlated with cell cycle related pathways but was negatively correlated with epithelial mesenchymal transition (EMT) pathway, a key mechanism for the generation and maintenance of CSCs. We then performed protein binding predictions and found that GPC3 could bind to SHH, a ligand for sonic hedgehog pathway. As previously published, GPC3 could inhibit the activation of sonic hedgehog pathway by competing with PTCH1, a natural receptor for SHH binding during development. We hypothesized that GPC3 may inhibit the generation of CSCs in HCC by the inhibiting sonic hedgehog pathway and subsequent EMT pathway. Hence, inhibition of GPC3 expression in HCC tumour bulk may facilitate the generation of CSCs. To validate our hypothesis, we demonstrated that GPC3 and CD133 are expressed by different population of cells in the HCC tumour bulk. Based on our studies, we propose that the GPC3 targeted therapies may be able to kill the majority of HCC cells, these therapies are unable to eliminate liver cancer stem cells (LCSCs) such as CD133+ cells. Our further analysis by IHC revealed that GPC3/PTCH1/SHH axis follows a paracrine pattern. We then performed in vitro studies using recombinant human SHH protein to treat HCC cells to mimic the HCC tumour biology in microenvironment. We generated stable GPC3 knockdown cells (Huh7 and Hep3B) using specific short hairpin RNA (shRNA) against GPC3 and found that activation of sonic hedgehog pathway and EMT pathway occurred only when the cells were treated with recombinant human SHH. By using the co-immunoprecipitation (co-IP) experiments in human HEK293T cells, we confirmed that GPC3 could compete with PTCH1 for SHH binding in a dose dependent manner. Functional studies have revealed that the stable GPC3 knockdown cells treated with SHH exhibited stronger metastatic and stemness phenotypes.

In order to further demonstrate the possible interaction between GPC3 and stem cell features, we cultured the isolated LCSCs from Huh7 and Hep3B cells and tested the expression level of GPC3 by using Western blotting, qPCR and immunofluorescence. We also extracted the data from two public datasets using other stem cell models and analysed the expression pattern of GPC3 in these models. All the data showed that the expression level of GPC3 in CSCs is significantly lower than it is in the differentiated cells.

Based on the published data and our own studies, we proposed that within HCC tumour bulk, high expression level of GPC3 promotes the proliferation of cancer cells but inhibition of GPC3 endows cancer cells with a CSC phenotype via EMT pathway. Our results demonstrate that CSCs targeting strategies should be an integral part of GPC3 targeted therapies in HCC.