HCC is a very aggressive tumor with frequent intrahepatic and distant metastasis, which is also the main reason for the high recurrence and low survival rate after surgical resection of HCC (Yang et al., 2019). However, the exact mechanism of HCC progression is not yet completely clear, and an increasing number of studies have attempted to clarify the detailed mechanism of HCC progression. In this study, we found that the expression levels of Egr1 and PAR1 were significantly increased in HCC clinical samples, and their mRNA levels were positively correlated. Using PLC/PRF/5 cells and HCCLM3 cells, we further found that Egr1 regulates the expression of PAR1 and cell proliferation, invasion and migration. Furthermore, we elucidated that Egr1 can transcriptionally regulate the expression of PAR1 and activate the MAPK/ERK signaling pathway through PAR1 to promote the proliferation, invasion and migration of HCC cells. Finally, we demonstrated that the regulation of PAR1 by Egr1 was not affected by the PAR1 inhibitor thrombin, and clarified the important role of the Egr1/PAR1/MAPK pathway in the progression of HCC.
The transcription factor Egr1 plays an important regulatory role in the occurrence, metastasis and angiogenesis of various malignant tumors (De Mestre et al., 2005). However, its role in HCC remains controversial. Some studies have reported that Egr1 can inhibit the progression of HCC (Wang et al., 2016; Hao et al., 2002), while more studies believe that Egr1 promotes HCC (Lee et al., 2009; Archer et al., 2009; Archer et al. 2016; Bi et al., 2019). We found that Egr1 expression was significantly increased in clinical HCC specimens and could stimulate the proliferation, invasion and migration of cancer cells. The results of this study showed that Egr1 expression was upregulated in HCC and promoted tumor progression, which is consistent with the findings that Egr1 is an oncogene in HCC.
PAR1 is a GPCR with 7 transmembrane units. Abnormal expression of PAR1 can promote the growth and metastasis of various types of tumors (Kaufmann et al., 2007; Ramachandran et al., 2012). Abnormally high expression of PAR1 in HCC promotes the proliferation and invasion of cancer cells (Kaufmann et al., 2007; Xiao et al., 2018; Mußbach et al., 2015). However, the upstream regulatory mechanism of PAR1 in HCC is unclear. Previous studies have shown that in prostate cancer, Egr1 can directly bind to the PAR1 promoter region and regulate the transcription of PAR1 (Salah et al., 2007). In this study, the mRNA expression levels of the Egr1 and PAR1 genes in 18 HCC clinical specimens were positively correlated. Therefore, does Egr1 promote the progression of HCC by regulating the expression of PAR1? In HCC, is Egr1 an upstream transcriptional regulator of PAR1? To answer these questions, we performed dual luciferase reporter assays and in vitro gain-of-function and loss-of-function experiments. The results showed that Egr1 regulates the transcription of PAR1 by directly binding to the promoter region of PAR1, thereby promoting the proliferation, invasion and migration of cancer cells.
The MAPK/ERK signaling pathway is activated by signal transduction from cell surface receptors such as receptor tyrosine kinases (RTKs) or GPCRs (Delire et al., 2015). The MAPK/ERK pathway plays a key role in regulating cell survival and proliferation, and its abnormal activation is closely related to cell transformation and carcinogenesis (Guo et al., 2020). MAPK/ERK signaling is considered to be activated in approximately 50% of early HCC patients and almost all advanced patients (Neuzillet et al., 2014). Previous studies have shown that PAR1, a GPCR, can activate the MAPK/ERK pathway to promote tumor progression (Rabinovitch et al. 2021; Darmoul et al., 2003). Therefore, does Egr1 promote HCC progression by activating the MAPK/ERK pathway through PAR1? We found that in PLC/PRF/5 cells, silencing PAR1 restored the upregulation of ERK1/2 phosphorylation caused by Egr1 overexpression; in HCCLM3 cells, overexpression of PAR1 restored the downregulation of ERK1/2 phosphorylation caused by Egr1 downregulation. This finding suggested that Egr1 promotes HCC progression by regulating PAR1 to activate the MAPK/ERK pathway.
Thrombin is a clear regulator of PAR1 that promotes the expression of PAR1 at the posttranscriptional level by recognizing a specific site in the extracellular N-terminus of PAR1 (Ramachandran et al., 2012). Therefore, in HCC, is thrombin involved in the regulation of PAR1 by Egr1? We used the thrombin inhibitor R-hirudin (Wakui et al., 2019) for in vitro experiments and found that R-hirudin had no significant effect on the activation of the MAPK/ERK pathway caused by Egr1 overexpression through the upregulation of PAR1 transcription.