The poor prognosis of hepatocellular carcinoma (HCC), one of the most devastating cancers worldwide, is due to frequent recurrence and metastasis. Among the metastatic factors in the tumor microenvironment, hepatocyte growth factor (HGF) has been well known to play critical roles in tumor progression, including HCC. Therefore, c-Met is now regarded as the most promising therapeutic target for the treatment of HCC. However, there are still concerns about resistance and the side effects of using conventional inhibitors of c-Met, such as tyrosine kinase inhibitors. Recently, many alternative strategies of c-Met targeting have been emerging. These include targeting the downstream effectors of c-Met, such as hydrogen peroxide-inducible clone 5 (Hic-5), to block the reactive oxygen species (ROS)-mediated signaling for HCC progression. Also, inhibition of endosomal regulators, such as PKCε and GGA3, may perturb the c-Met endosomal signaling for HCC cell migration. On the other hand, many herbal antagonists of c-Met-dependent signaling, such as saponin, resveratrol, and LZ-8, were identified. Taken together, it can be anticipated that more effective and safer c-Met targeting strategies for preventing HCC progression can be established in the future.

Snail is a multifunctional transcriptional factor that has been described as a repressor in many different contexts. It is also proposed as an activator in a few cases relevant to tumor progression and cell‐cycle arrest. This study investigated the detailed mechanisms by which Snail upregulates gene expression of the CDK inhibitor p15^INK4b in HepG2 induced by the tumor promoter tetradecanoyl phorbol acetate (TPA). Using deletion mapping, the TPA‐responsive element on the p15^INK4b promoter was located between 77 and 228 bp upstream of the transcriptional initiation site, within which the putative binding regions of early growth response gene 1 (EGR‐1) and stimulatory protein 1 (SP‐1) were found. Gene expression of EGR‐1, Snail and SP‐1 can be induced by TPA within 0.5–6 h. In addition, basal levels of SP‐1, but not of the other two transcriptional factors, were observed. Blockade of TPA‐induced gene expression of Snail, EGR‐1 or SP‐1 suppressed activation of the p15–pro228 reporter plasmid harboring the TPA‐responsive element. More detailed deletion mapping and site‐directed mutagenesis further concluded that the overlapping EGR‐1/SP‐1‐binding site was required for TPA‐induced p15–pro228 activation. In an EMSA, a DNA–protein complex was elevated by TPA, which can be blocked by antibodies against EGR‐1, SP‐1 or Snail at 6 h. Immunoprecipitation/western blotting demonstrated that TPA could trigger the association of EGR‐1 with Snail or SP‐1. Furthermore, a double chromatin immunoprecipitation assay verified that EGR‐1 could form a complex with Snail or SP‐1 on the TPA‐responsive element after treatment with TPA for 2–6 h. Finally, we demonstrated a novel Snail‐target region which could be bound by Snail and was also required for TPA‐induced p15–pro228 activation. In conclusion, Snail associates with EGR‐1 and SP‐1 to mediate TPA‐induced transcriptional upregulation of p15^INK4b in HepG2.Structured digital abstract MINT‐7384899: Snail (uniprotkb:O95863) physically interacts (MI:0915) with EGR‐1 (uniprotkb:P18146) by anti bait coimmunoprecipitation (MI:0006) MINT‐7384908: SP‐1 (uniprotkb:P08047) physically interacts (MI:0915) with EGR‐1 (uniprotkb:P18146) by anti bait coimmunoprecipitation (MI:0006)

Oxidation of heat shock protein 60 and protein disulfide isomerase activates ERK and migration of human hepatocellular carcinoma HepG2

Hepatocellular carcinoma (HCC) is among the most lethal cancers. Mounting studies highlighted the essential role of the HGF/c-MET axis in driving HCC tumor progression. Therefore, c-Met is a potential therapeutic target for HCC. However, several concerns remain unresolved in c-Met targeting. First, the status of active c-Met in HCC must be screened to determine patients suitable for therapy. Second, resistance and side effects have been observed frequently when using conventional c-Met inhibitors. Thus, a preclinical system for screening the status of c-Met signaling and identifying efficient and safe anti-HCC agents is urgently required. In this study, immunohistochemical staining of phosphorylated c-Met (Tyr1234) on tissue sections indicated that HCCs with positive c-Met signaling accounted for approximately 46% in 26 cases. Second, many patient-derived HCC cell lines were established and characterized according to motility and c-Met signaling status. Moreover, LZ8, a medicinal peptide purified from the herb Lingzhi, featuring immunomodulatory and anticancer properties, was capable of suppressing cell migration and slightly reducing the survival rate of both c-Met positive and negative HCCs, HCC372, and HCC329, respectively. LZ8 also suppressed the intrahepatic metastasis of HCC329 in SCID mice. On the molecular level, LZ8 suppressed the expression of c-Met and phosphorylation of c-Met, ERK and AKT in HCC372, and suppressed the phosphorylation of JNK, ERK, and AKT in HCC329. According to receptor array screening, the major receptor tyrosine kinase activated in HCC329 was found to be the epidermal growth factor receptor (EGFR). Moreover, tyrosine-phosphorylated EGFR (the active EGFR) was greatly suppressed in HCC329 by LZ8 treatment. In addition, LZ8 blocked HGF-induced cell migration and c-Met-dependent signaling in HepG2. In summary, we designed a preclinical trial using LZ8 to prevent the tumor progression of patient-derived HCCs with c-Met-positive or -negative signaling.

Hydrogen peroxide inducible clone-5 mediates reactive oxygen species signaling for hepatocellular carcinoma progression