A novel interaction of nucleophosmin with BCL2-associated X protein regulating death evasion and drug sensitivity in human hepatoma cells.

UNLABELLED: Death evasion is crucial for both carcinogenesis and resistance to anticancer therapies. Recently, we identified nucleophosmin (NPM) as a key factor counteracting death stimuli in human hepatocellular carcinoma (HCC) cells. Here we report the identification of a novel NPM-BCL2-associated X protein (BAX) pathway orchestrating death evasion in human HCC cells. Silencing of NPM expression significantly sensitized HCC cells-particularly those bearing inactivated p53 gene (Huh7, Hep3B, and Mahlavu)-to ultraviolet irradiation, mitomycin C, doxorubicin, cisplatin, sorafenib, and lapatinib. This sensitizing effect was not changed further, as p53 expression had been simultaneously silenced. Following cell stress, NPM and BAX were induced and exported out of the nucleoli and nucleus, respectively. BAX was translocated to cytoplasm in cells with relatively high NPM level, or accumulated in the mitochondria in cells with relatively low NPM level and undergoing apoptosis. Subcellular fractionation revealed that silencing of NPM expression greatly enhanced mitochondrial translocation and oligomerization of BAX in Huh7 and Mahlavu cells. In situ proximity ligation assays and reciprocal co-immunoprecipitation revealed a direct interaction between NPM and BAX in the cytoplasm. Silencing of BAX expression abolished the sensitization effect exerted by silencing of NPM in HCC cells. Clinically, up-regulation of NPM was significantly associated with advanced tumor stage and poor prognosis. CONCLUSION: By directly blockading BAX mitochondrial translocation and activation, NPM helps human HCC cells evade death induction independently of p53-mediated cell death. Silencing of NPM significantly sensitized HCC cells to anticancer therapies. NPM is a potential cotarget in combination with other therapies for HCC, particularly those that harbor inactivated p53 gene. Our findings are of clinical significance because NPM up-regulation and p53 mutations are usually found in advanced human cancers, including HCC.

Secreted ERBB3 isoforms are serum markers for early hepatoma in patients with chronic hepatitis and cirrhosis.

Most hepatocellular carcinoma (HCC) is generated from chronic hepatitis and cirrhosis. To discover new markers for early HCC in patients with chronic hepatitis and cirrhosis, we initiated our search in the interstitial fluid of tumor (TIF) via differential gel electrophoresis and antibody arrays and identified secreted ERBB3 isoforms (sERBB3). The performance of serum sERBB3 in diagnosis of HCC was analyzed using receiver operating characteristic curves (ROC). The serum sERBB3 level was significantly higher in HCC than in cirrhosis (p < 0.001) and chronic hepatitis (p < 0.001). The accuracy of serum sERBB3 in detection of HCC was further validated in two independent sets of patients. In discrimination of early HCC from chronic hepatitis or cirrhosis, serum sERBB3 had a better performance than alpha-fetoprotein (AFP) (areas under ROC [AUC]: sERBB3 vs AFP = 93.1 vs 81.0% from chronic hepatitis and 70.9 vs 62.7% from cirrhosis). Combination of sERBB3 and AFP further improved the accuracy in detection of early HCC from chronic hepatitis (AUC = 97.1%) or cirrhosis (AUC = 77.5%). Higher serum sERBB3 levels were associated with portal-vein invasion and extrahepatic metastasis of HCC (p = 0.017). Therefore, sERBB3 are serum markers for early HCC in patients with chronic hepatitis and cirrhosis.

Aberrant DNA methylation profile and frequent methylation of KLK10 and OXGR1 genes in hepatocellular carcinoma.

Investigating aberrant DNA methylation in the cancer genome may identify genes that play an important role in tumor progression. In this study, we combined differential methylation hybridization and a CpG microarray platform to characterize methylation profiles and identify novel candidate genes associated with hepatocellular carcinoma (HCC). The genomic DNA of 21 paired adjacent normal and HCC samples was used, and results were analyzed by hierarchical clustering. Twenty-seven hypermethylated candidates and 38 hypomethylated candidates were obtained. Six candidate genes from the hypermethylated group were validated by combined bisulfite restriction analysis; two genes, human kallikrein 10 gene (KLK10) and oxoglutarate (alpha-ketoglutarate) receptor 1 gene (OXGR1), were further analyzed by bisulfite sequencing. The DNA hypermethylation status of KLK10 and OXGR1 were subsequently examined in HCC cell lines and clinical samples using methylation-specific PCR. In 49 HCC samples, 46 (94%) showed that at least one of these two genes was highly methylated. Moreover, KLK10 and OXGR1 mRNA levels were inversely correlated (r = -0.435 and -0.497, P < 0.05) with DNA methylation as examined in paired adjacent normal and tumor samples. Statistical analyses further indicated that KLK10 hypermethylation was significantly associated with cirrhosis (P = 0.042) and HCV infection (P = 0.017) as well as inversely associated with HBV infection (P = 0.023). Furthermore, restoration of KLK10 and OXGR1 expression reduced the ability of anchorage-independent growth, and sensitized HCC cells to doxorubicin- or 5-fluorouracil-induced cytotoxicity. Our results suggest that the hypermethylated KLK10 and OXGR1 are frequent in HCC and may be useful as markers for clinical application.

Identifying apoptosis-evasion proteins/pathways in human hepatoma cells via induction of cellular hormesis by UV irradiation.

Evading apoptosis is pivotal in both of carcinogenesis and resistance to anticancer therapy. We investigated the molecules and pathways of apoptosis evasion in human hepatoma cells by irradiating hepatoma cells with optimized UV (so-called "hormetic responses"). Proteins and pathways related to hormetic responses were identified via proteomic approaches followed by reconstruction of function-networks. Of the 2326 defined protein spots, 42 distinct proteins significantly changed their expression. Eleven hormetic response proteins (HINT1, PHB, CTSD, ANXA1, LGASL1, TPT1, NPM, PRDX2, UCHL1, CERK, and C1QBP) were involved in 5 death-regulatory pathways, including the p53-dependent apoptotic pathway, protein ubiquinization, cellular redox, calcium-mediated signaling pathway, and sphingomyelin-metabolism pathway. Knockdown of HINT1 expression via RNA interference increased tumor cell resistance to apoptosis induction, while silencing NPM, UCHL1, or CERK greatly sensitized tumor cells to apoptosis induction. In conclusion, NPM, UCHL1, and CERK act as apoptosis-evasion proteins that may serve as therapeutic targets for hepatoma. Silencing their expression would increase therapeutic efficacy, thereby reducing the corresponding doses and side-effects of anticancer therapy. This model of induction of cellular hormetic responses to identify apoptosis-evasion molecules/pathways via proteomic approaches can be applied to other modalities of anticancer therapy.