Clinical relevance and functional role of galectin-1 in hepatocellular carcinoma

Abstract

Abstract of thesis entitled “Clinical Relevance and Functional Role of Galectin-1 in Hepatocellular Carcinoma “ Submitted by Zoe LEUNG for the Degree of Doctor of Philosophy At the University of Hong Kong June 2018

Hepatocellular carcinoma (HCC) is one the most prevalent human cancers in the world and is responsible for the third leading cause of cancer deaths. This reinforces the need for improved understanding in its development and advanced therapeutic options. Galectin-1 (Gal-1) is a widely expressed carbohydrate-binding protein and influences a plethora of cellular processes. Gal-1 overexpression has therefore been related to enhanced oncogenesis in many cancers. However, its regulation and mechanistic role in HCC development is yet to be fully determined. In this study, aberrantly elevated Gal-1 expression was first observed in human HCC clinical samples and in patient blood sera, compared to their control counterparts. Clinical correlation analysis revealed the significance of Gal-1 overexpression to be associated with the absence of tumour encapsulation and the presence of microsatellites. Moreover, patients with Gal-1 overexpression tend to have a poorer prognosis. Secondly, the role of Gal-1 was determined in Gal-1 knockdown experiments in MHCC97L and BEL7402 cells. Knocking down Gal-1 markedly reduced cell migration, invasion and anchorage independent growth in in vitro experiments, as well as, abolished tumour growth and metastasis in in vivo mouse experiments. Given the notoriety of hypoxia in exacerbating tumour growth, the impact of hypoxia on Gal-1 was explored. MIHA, a normal liver cell line, along with HCC cell lines, Hep3B and MHCC97L revealed an evident increase in Gal-1 expression under hypoxia. Functionally, Gal-1 knockdown in MHCC97L cells demonstrated the increased invasive potential in shCtl cells under hypoxia. However, such enhancement was diminished in shGal-1 cells. This implies that Gal-1 exerts an enhanced oncogenic ability under hypoxia. MicroRNAs (miRNA) are small non-coding RNA that regulate gene expression at the post transcriptional level by binding to target mRNA 3’UTR. Lastly, to elucidate the regulatory mechanism on Gal-1, miR-22 was identified to target Gal-1 3’UTR, with this direct regulation confirmed by a dual luciferase reporter assay. Furthermore, this negative relationship was examined by stable miR-22 mimic and inhibitor transfected HCC cells to reduce or decrease Gal-1 expression, respectively, and its subsequent impact on HCC cell activity. Since miR-22 was found to be underexpressed in HCC, this could explain one of the reasons for Gal-1 overexpression in HCC. In a comparable experimental condition established in MHCC97L cells, inhibition of miR-22 resulted in increased Gal-1-induced in vitro cell activities. This led to the investigation of the efficacy of a Gal-1 specific inhibitor, a calixarene derivative, OTX008, to reduce Gal-1 levels in these cells. It was found that OTX008 significantly diminished Gal-1 functional activity in miR-22 inhibitor stable cells with elevated Gal-1 expression. Overall, the major findings of this study have enriched our understanding in the consequences of Gal-1 overexpression in the clinical and functional aspects of HCC. We highlighted the regulatory role of miR-22 on Gal-1 function and also the enhanced oncogenic activities of Gal-1 in response to hypoxia. The efficacy of targeted therapeutic inhibition of Gal-1 was demonstrated through Gal-1 specific inhibitor, which could prove to be a potential therapeutic option for cancer overexpressing Gal-1.