Involvement of N-glycan in Multiple Receptor Tyrosine Kinases Targeted by Ling-Zhi-8 for Suppressing HCC413 Tumor Progression.

The poor prognosis of hepatocellular carcinoma (HCC) is resulted from tumor metastasis. Signaling pathways triggered by deregulated receptor tyrosine kinases (RTKs) were the promising therapeutic targets for prevention of HCC progression. However, RTK-based target therapy using conventional kinase-based inhibitors was often hampered by resistances due to compensatory RTKs signaling. Herein, we report that Ling-Zhi-8 (LZ-8), a medicinal peptide from Ganoderma lucidium, was effective in suppressing cell migration of HCC413, by decreasing the amount and activity of various RTKs. These led to the suppression of downstream signaling including phosphorylated JNK, ERK involved in HCC progression. The capability of LZ-8 in targeting multiple RTKs was ascribed to its simultaneous binding to these RTKs. LZ-8 may bind on the N-linked glycan motif of RTKs that is required for their maturation and function. Notably, pretreatment of the N-glycan trimming enzyme PNGase or inhibitors of the mannosidase (N-glycosylation processing enzyme), kifunensine (KIF) and swainsonine (SWN), prevented LZ-8 binding on the aforementioned RTKs and rescued the downstream signaling and cell migration suppressed by LZ-8. Moreover, pretreatment of KIF prevented LZ-8 triggered suppression of tumor growth of HCC413. Our study suggested that a specific type of N-glycan is the potential target for LZ-8 to bind on multiple RTKs for suppressing HCC progression.

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

Hepatocyte growth factor (HGF) and its receptor c-Met were frequently deregulated in hepatocellular carcinoma (HCC). Signaling pathways activated by HGF-c-Met are promising targets for preventing HCC progression. HGF can induce the reactive oxygen species (ROS) signaling for cell adhesion, migration and invasion of tumors including HCC. On the other hand, extracellular signal-regulated kinases (ERK), member of mitogen activated kinase, can be activated by ROS for a lot of cellular processes. As expected, HGF-induced phosphorylation of ERK and progression of HCC cell HepG2 were suppressed by ROS scavengers. By N-(biotinoyl)-N'-(iodoacetyl)-ethylenediamine (BIAM) labeling method, a lot of cysteine (-SH)-containing proteins with M.W. 50-75 kD were decreased in HepG2 treated with HGF or two other ROS generators, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and phenazine methosulfate. These redox sensitive proteins were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Among them, two chaperones, heat shock protein 60 (HSP60) and protein disulfide isomerase (PDI), were found to be the most common redox sensitive proteins in responding to all three agonists. Affinity blot of BIAM-labeled, immunoprecipitated HSP60 and PDI verified that HGF can decrease the cysteine (-SH) containing HSP60 and PDI. On the other hand, HGF and TPA increased cysteinyl glutathione-containing HSP60, consistent with the decrease of cysteine (-SH)-containing HSP60. Moreover, depletion of HSP60 and PDI or expression of dominant negative mutant of HSP60 with alteration of Cys, effectively prevented HGF-induced ERK phosphorylation and HepG2 migration.In conclusion, the redox sensitive HSP60 and PDI are required for HGF-induced ROS signaling and potential targets for preventing HCC progressions.

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

One of the signaling components involved in hepatocellular carcinoma (HCC) progression is the focal adhesion adaptor paxillin. Hydrogen peroxide inducible clone-5 (Hic-5), one of the paralogs of paxillin, exhibits many biological functions distinct from paxillin, but may cooperate with paxillin to trigger tumor progression. Screening of Hic-5 in 145 surgical HCCs demonstrated overexpression of Hic-5 correlated well with intra- and extra-hepatic metastasis. Hic-5 highly expressed in the patient derived HCCs with high motility such as HCC329 and HCC353 but not in the HCCs with low motility such as HCC340. Blockade of Hic-5 expression prevented constitutive migration of HCC329 and HCC353 and HGF-induced cell migration of HCC340. HCC329Hic-5(-), HCC353Hic-5(-), HCC372Hic-5(-), the HCCs stably depleted of Hic-5, exhibited reduced motility compared with each HCC expressing Scramble shRNA. Moreover, intra/extrahepatic metastasis of HCC329Hic-5(-) in SCID mice greatly decreased compared with HCC329Scramble. On the other hand, ectopic Hic-5 expression in HCC340 promoted its progression. Constitutive and HGF-induced Hic-5 expression in HCCs were suppressed by the reactive oxygen species (ROS) scavengers catalase and dithiotheritol and c-Jun N-terminal kinase (JNK) inhibitor SP600125. On the contrary, depletion of Hic-5 blocked constitutive and HGF-induced ROS generation and JNK phosphorylation in HCCs. Also, ectopic expression of Hic-5 enhanced ROS generation and JNK phosphorylation. These highlighted that Hic-5 plays a central role in the positive feedback ROS-JNK signal cascade. Finally, the Chinese herbal derived anti-HCC peptide LZ-8 suppressed constitutive Hic-5 expression and JNK phosphorylation. In conclusion, Hic-5 mediates ROS-JNK signaling and may serve as a therapeutic target for prevention of HCC progression.

PKCε-mediated c-Met endosomal processing directs fluctuant c-Met-JNK-paxillin signaling for tumor progression of HepG2.

Hepatocyte growth factor (HGF) induced c-Met signaling play critical roles in the progression of hepatocellular carcinoma (HCC). However, c-Met targeting approaches suffered resistance and side effect, thus identification of more suitable downstream targets is needed. Recently, we demonstrated HGF-induced fluctuant ERK/paxillin signaling within 24h. We further examined the underlying mechanisms for fluctuant c-Met/JNK/paxillin signal cascade within 12h. HGF-induced phosphorylation of c-Met, JNK, and paxillin (Ser178) shared a common fluctuation pattern characterized by an initial peak at 0.5h, a middle drop at 4h, and a later peak at 10h. Dynasore, the inhibitor of dynamin, suppressed HGF-induced c-Met internalization and phosphorylation of JNK and paxillin (Ser178) at 0.5h, indicating that endosome formation is required for initial signal enhancement. Further, depletion of PKCε not only enhanced HGF-induced phosphorylation of JNK and paxillin (Ser178) but also prevented c-Met degradation at 0.5h, suggesting that PKCε mediated c-Met degradation for signal declination. On the other hand, HGF induced colocalizations of both phosphorylated JNK and paxillin with the endosomal recycling protein GGA3 at 10h and depletion of GGA3 abolished membrane recycling of c-Met and phosphorylation of JNK/paxillin at the same time point. Interestingly, HGF induced GGA3 phosphorylation in a PKCε-dependent manner during 0.5-4h, which is associated with c-Met degradation in the same period. Finally, HGF-induced cell migration, invasion and intrahepatic metastasis of HepG2 were prevented by the inhibitors of endocytosis. Our results suggest that critical endosomal components are promising therapeutic targets for preventing HGF-induced progression of HCC.

Preclinical trials for prevention of tumor progression of hepatocellular carcinoma by LZ-8 targeting c-Met dependent and independent pathways.

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.

PKC mediates fluctuant ERK-paxillin signaling for hepatocyte growth factor-induced migration of hepatoma cell HepG2.

Hepatocyte growth factor (HGF) is critical for triggering metastasis of hepatocellular carcinoma cell (HCC). Extracellular signal-regulated kinase (ERK) mediates HGF-induced cell migration via focal adhesion signaling. Protein kinase C (PKC) is a negative regulator of ERK activation, however, both PKC and ERK were required for HGF-induced cell migration. To address this intriguing issue, the signal mechanisms for HGF-induced HepG2 cell migration were investigated in a long-term fashion. HGF-induced phosphorylations of ERK, Src (at Tyr 416) and paxillin (at Ser178 and Tyr31) were up and down for 3 times within 24h. HGF also induced fluctuant PKC activation and Rac degradation. Consistently, HGF induced intermittent actin polarization within 24h, which can be blocked by the inhibitors of PKC (Bisindolymaleimide) and ERK. Inhibitor studies revealed that ERK was required for HGF-induced paxillin phosphorylation at Ser178, whereas PKC and Rac-1 may suppress HGF-induced phosphorylation of ERK and paxillin (at Ser178) and upregulate phosphorylation of paxillin at Tyr31. Based on shRNA technique, PKCα and δ were responsible for suppressing HGF-induced phosphorylation of ERK and paxillin (at Ser178), whereas PKC ε and ζ were required for phosphorylation of paxillin at Tyr31. The HGF-induced fluctuant signaling is reminiscent of c-Met endocytosis. Using Concanavalin A, an inhibitor of endocytosis, we found that c-Met endocytosis was required for PKC to suppress ERK phosphorylation. Moreover, HGF-induced c-Met degradation was also fluctuant, which can be prevented by Bisindolymaleimide. In conclusion, PKC is critical for mediating HGF-induced fluctuant ERK-paxillin signaling during cell migration, probably via triggering endosomal degradation of c-Met.

Reactive oxygen species-mediated PKC and integrin signaling promotes tumor progression of human hepatoma HepG2.

The poor prognosis and recurrence of HCC are majorly caused by intrahepatic metastasis. Delineating the molecular pathways mediating these processes may benefit developing effective targeting therapies. Using human hepatoma HepG2 as a model, we have found reactive oxygen species (ROS) may cooperate with protein kinase C (PKC) for sustained ERK phosphorylation and migration of HepG2 induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We further investigated whether integrin signaling is involved. Various antagonists of integrin signaling prevented TPA-induced activation of ERK and PKC, ROS generation and migration of HepG2. On the other hand, TPA-induced phosphorylation of integrin signaling components including focal adhesion kinase (FAK), Src (Tyr416) and paxillin (Tyr31 and Ser178) can be prevented by PKC inhibitor Bisindolylmaleimides (BIS) and antioxidant dithiotheritol (DTT). HepG2 overexpressing PKCα contained elevated phosphorylated paxillin. Also, ROS generator phenazine methosulfate and tert-Butyl hydroperoxide may induce phosphorylation of paxillin and activation of PKC. Taken together, ROS mediated cross talk of PKC and integrin for migration of HepG2 induced by TPA. Furthermore, TPA induced intrahepatic metastasis of HepG2 in SCID mice, which was prevented by BIS or (BIS plus DTT). Elevated phosphorylation of paxillin was observed in tumor of mice treated with TPA as compared with those co-treated with TPA/BIS. In summary, the signal pathways for tumor progression of hepatoma induced by TPA can be established both in vitro and in vivo.