Purpose: Targeting the protein neddylation pathway has become an attractive anticancer strategy; however, the role of death receptor-mediated extrinsic apoptosis during treatment remained to be determined.Experimental Design: The activation of extrinsic apoptosis and its role in MLN4924 treatment of human esophageal squamous cell carcinoma (ESCC) were evaluated both in vitro and in vivo. The expression of the components of extrinsic apoptotic pathway was determined by immunoblotting analysis and downregulated by siRNA silencing for mechanistic studies.Results: Pharmaceutical or genetic inactivation of neddylation pathway induced death receptor 5 (DR5)-mediated apoptosis and led to the suppression of ESCC in murine models. Mechanistically, neddylation inhibition stabilized activating transcription factor 4 (ATF4), a Cullin-Ring E3 ubiquitin ligases (CRL) substrate. Transcription factor CHOP was subsequently transactivated by ATF4 and further induced the expression of DR5 to activate caspase-8 and induce extrinsic apoptosis. Moreover, the entire neddylation pathway was hyperactivated in ESCC and was negatively associated with patient overall survival.Conclusions: Our findings highlight a critical role of ATF4-CHOP-DR5 axis-mediated extrinsic apoptosis in neddylationtargeted cancer therapy and support the clinical investigation of neddylation inhibitors (e.g., MLN4924) for the treatment of ESCC, a currently treatment-resistant disease with neddylation hyperactivation.

In current study, we investigated the anti-tumor effect of luteolin in human ESCC cell lines in vitro and in vivo and tried to explore the potential mechanisms. Results from flow cytometry showed that luteolin could induce apoptosis and caspase-3 activation and induce cell cycle arrest at G2/M phase in a dose- and time-dependent manner in EC1 and KYSE450 cells. JC-1 test results showed that membrane potential of mitochondria after luteolin treatment was down-regulated and this was an indicator for intrinsic apoptosis. Western Blot results showed the expression of cell cycle regulatory protein p21 and p53 increased and three apoptosis related proteins that participate in mitochondrial apoptotic pathway, namely, Bim, CYT-c and cPARP, also increased in luteolin treated cells compared with control groups. We further confirmed that luteolin could significantly inhibit the growth of ESCC tumors in xenograft mouse models and no evidence of systemic toxicity was observed. Our results suggest that luteolin can induce cell apoptosis and cell cycle arrest in G2/M phase through mitochondrial pathway in EC1 and KYSE450 cell lines and proper utilization of luteolin might be a practical approach in ESCC chemotherapy.

p62/IMP2 is an oncofetal protein that was first reported as a tumor-associated antigen in hepatocellular carcinoma (HCC). In our previous studies, we demonstrated a high frequency of p62/IMP2 autoantibodies appearing in various types of cancer. Therefore, we hypothesize that p62/IMP2 plays an important role in the progression of HCC, although the mechanism remains to be explored. In this study, we evaluated the expression of p62/IMP2 protein both in human tissues and liver cancer cell lines by immunohistochemistry and western blotting analysis and found that p62/IMP2 protein is overexpressed in human HCC tissue in comparison to normal human liver tissue. To explore the role that p62/IMP2 plays in HCC, p62/IMP2 was knocked out in two p62/IMP2-positive liver cancer cell lines (SNU449 and HepG2). Due to the low expression level of p62/IMP2 in SNU449, we overexpressed p62/IMP2 in this cell line. We subsequently demonstrated that high expression of p62/IMP2 in both cell lines can promote cell migration and invasion abilities in vitro by activating the Wnt/β-catenin pathway. We also used the Wnt/β-catenin pathway inhibitor, XAV 939, and a phosphoproteome assay to confirm our findings. Conclusion: Our results suggest that p62/IMP2 is an essential regulator of Wnt signaling pathways and plays an important role in HCC progression and metastasis.Cancers 2020, 12, 7 2 of 16 with various mRNA to regulate their stability, translatability, and localization [9]. IMPs are considered oncofetal proteins, and their overexpression in various types of cancers has been reported [8,9]. p62/IMP2 was originally reported as a tumor-associated antigen (TAA) in HCC [10]. The high frequency of anti-p62/IMP2 autoantibody occurrence in the serum samples from various types of cancer patients implies that p62/IMP2 may play a role in cancer progression [11][12][13][14]. Several cancer-associated p62/IMP2 target mRNAs have been identified. They include CCN2 mRNA [15], IGF2 mRNA [7], and c-Myc mRNA [15]. Some studies have reported that p62/IMP2 is involved in carcinogenesis. When phosphorylated by mTOR, p62/IMP2 stabilizes HMGA1 and stimulates the expression of IGF2, potentially leading to increased cancer cell proliferation [16]. By activating the IGF2/PI3K/Akt pathway, p62/IMP2 can conceivably promote proliferation, migration, invasion, and epithelial to mesenchymal transition (EMT) of glioblastoma cells [17]. Recently, p62/IMP2 has been shown to enhance the migration ability of cancer cells and promote cancer metastasis. A cluster of p62/IMP2 targets (LIMS2, TRIM54, and LAMB2) are considered to be involved in the downstream effects of p62/IMP2 expression that indeed increased cell migration, cell adhesion, and cytoskeleton remodeling, which can promote the metastatic behavior of cancer cells [18]. A recent study from our group demonstrated that the overexpressed p62/IMP2 enhances the expression of β-catenin and CTGF, which therefore heightens cell migration and reduces cell adhesion in breast cancer cells [15]. Another group also reported that p...

Background: Tumor-associated antigens (TAAs) have been investigated for many years as potential early diagnosis tools, especially for hepatocellular carcinoma (HCC). Nonetheless, very few studies have focused on the Hispanic HCC group that may be associated with distinct etiological risk factors. In the present study, we investigated novel anti-TAA autoantibodies as diagnostic biomarkers for Hispanic HCC patients. Methods: Novel TAA targets were identified by the serological proteome analysis (SERPA) and from differentially expressed HCC driver genes via bioinformatics. The autoantibody levels were validated by enzyme-linked immunosorbent assay (ELISA). Results: Among 19 potential TAA targets, 4 anti-TAA autoantibodies were investigated as potential diagnostic biomarkers with significantly high levels in Hispanic HCC sera, including DNA methyltransferase 3A (DNMT3A), p16, Hear shock protein 60 (Hsp60), and Heat shock protein A5 (HSPA5). The area under the ROC curve (AUC) value of the single autoantibodies varies from 0.7505 to 0.8885. After combining all 4 autoantibodies, the sensitivity of the autoantibody panel increased to 75% compared to the single one with the highest value of 45.8%. In a separate analysis of the Asian cohort, autoantibodies against HSPA5 and p16 showed significantly elevated levels in HCC compared to normal healthy controls, but not for DNMT3A or HSP60. Conclusion: Anti-DNMT3A, p16, HSPA5, and HSP60 autoantibodies have the potential to be diagnostic biomarkers for Hispanic HCC patients, of which DNMT3A and HSP60 might be exclusive for Hispanic HCC diagnosis.

The aim of this study was to explore the value of autoantibody to GNAS in the early detection of hepatocellular carcinoma (HCC). In a large-scale sample set of 912 participants (228 cases in each of HCC, liver cirrhosis (LC), chronic hepatitis B (CHB), and normal controls (NCs) groups), autoantibody to GNAS was detected with a positive result in 47.8% of HCC patients, which was significantly higher than that in patients with LC (35.1%), CHB (19.7%), and NCs (19.7%). Further analysis showed that the frequency of autoantibody to GNAS started increasing in compensated cirrhosis patients (37.0%) with a jump in decompensated cirrhosis patients (53.2%) and reached a peak in early HCC patients (62.4%). The increasing autoantibody response to GNAS in patients at different stages was closely associated with the progression of chronic liver lesions. The result from 44 human serial sera demonstrated that 5 of 11 (45.5%) HCC patients had elevated autoantibody to GNAS before and/or at diagnosis of HCC. Moreover, 46.1% and 62.4% of high positive rates in alpha-fetoprotein (AFP) negative and early-stage HCC patients can supplement AFP in early detection of HCC. These findings suggest that autoantibody to GNAS could be used as a potential biomarker for the early detection of HCC.