Abstract
Dickkopf-1 (DKK1), an inhibitor of the most frequently impaired signaling pathway in hepatocellular carcinoma (HCC), the Wnt/beta-catenin pathway, seems to fulfill contradictory functions in the process of tumorigenesis, acting either as an oncogenic promoter of metastasis or as a tumor suppressor. Elevated serum levels of DKK1 have been reported in HCC; however, little is known about its functional significance. In the current study, we treated HepG2/C3A and PLC/PRF/5 with the recombinant protein DKK1. Cytotoxicity was first determined by the WST-8 assay. AFP expression was measured at both the mRNA and protein levels. Expression of the oncogenes MYC, CCND1, hTERT, and MDM2 and the tumor suppressor genes TP53, P21 and RB was assessed. Western blot analysis of non-phosphorylated ẞ-catenin and Sanger sequencing were performed to explain the functional differences between the two cell lines. Subsequently, inflammation, migration and invasion were evaluated by qPCR, ELISA, the Boyden chamber assay, zymography, and MMP-2 and MMP-9 western blot analysis. Knockdown of DKK1 and TGF-β1 were also performed. Our results suggest that DKK1 exerts an oncogenic effect on HepG2/C3A cell line by upregulating the expression of oncogenes and downregulating that of tumor suppressor genes, whereas the opposite effect was demonstrated in PLC/PRF/5 cells. This differential impact of DKK1 can be explained by the mutations that affect the canonical Wnt pathway that were detected in exon 3 of the CTNNB1 gene in the HepG2 cell line. We further confirmed that DKK1 promotes inflammation, tumor invasion and migration in both cell types. The canonical pathway was not responsible for the DKK1 proinvasive effect, as indicated by the active ẞ-catenin levels in the two cell lines upon DKK1 treatment. Interestingly, knockdown of TGF-β1 negatively affected the DKK1 proinvasive effect. Taken together, DKK1 appears to facilitate tumor invasion and migration through TGF- β1 by remodeling the tumor microenvironment and inducing inflammation. This finding endorses the relevance of TGF-β1 as a therapeutic target.
Highlights
GLOBOCAN estimates that in 2018, approximately 841,000 new cases of liver cancer and 782,000 related deaths were reported, marking liver cancer as the sixth most commonly diagnosed cancer and the fourth leading cause of cancer-related death worldwide [1]
The cytotoxicity assay revealed that DKK1 was not cytotoxic towards hepatocellular carcinoma cell lines at any concentration tested
We evaluated the effect of DKK1 on AFP production in our cell lines
Summary
GLOBOCAN estimates that in 2018, approximately 841,000 new cases of liver cancer and 782,000 related deaths were reported, marking liver cancer as the sixth most commonly diagnosed cancer and the fourth leading cause of cancer-related death worldwide [1]. Fewer than 20% of patients are considered eligible for curative treatment modalities due to a late diagnosis that is established when the tumor has already invaded the liver parenchyma, spread through the portal venous system, or formed metastases [4]. In this context, elucidating the molecular basis governing the progression and metastasis of HCC is crucial for the development of new diagnostic and therapeutic tools to improve the prognosis. While most genetic alterations occur in genes with no known carcinogenic consequences, a small number of these alterations occur in pilot genes that are members of key signaling pathways involved in carcinogenesis [8]
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