Hepatitis B virus (HBV) infection could cause hepatitis, liver cirrhosis, and hepatocellular carcinoma. HBV-mediated pathogenesis is only partially understood, but X protein (HBx) reportedly possesses oncogenic potential. Exosomes are small membrane vesicles with diverse functions released by various cells including hepatocytes, and HBV harnesses cellular exosome biogenesis and export machineries for virion morphogenesis and secretion. Therefore, HBV infection might cause changes in exosome contents with functional implications for both virus and host. In this work, exosome protein content changes induced by HBV and HBx were quantitatively analyzed by SILAC/LC-MS/MS. Exosomes prepared from SILAC-labeled hepatoma cell line Huh-7 transfected with HBx, wildtype, or HBx-null HBV replicon plasmids were analyzed by LC-MS/MS. Systematic analyses of MS data and confirmatory immunoblotting showed that HBx overexpression and HBV, with or without HBx, replication in Huh-7 cells indeed caused marked and specific changes in exosome protein contents. Furthermore, specific changes in protein contents were also detected in exosomes purified from HBV-infected patients' sera compared with control sera negative for HBV markers. These results illustrate a new aspect of interactions between HBV and the host and provide the foundation for future research into roles played by exosomes in HBV infection and pathogenesis.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death. However, the underlying mechanism during hepatocarcinogenesis remains unclarified. Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative strategy for proteome-wide discovery of novel biomarkers in cancers. Hippocalcin-like 1 (HPCAL1) is a calcium sensor protein. However, the biological function of HPCAL1 is poorly understood in cancers, including HCC. Herein, HPCAL1 was identified by SILAC as a novel hepatocarcinogenesis suppressor down-regulated in HCC cell lines and tissues. Importantly, lost expression of HPCAL1 was associated with worse prognosis of HCC patients. Interestingly, secreted HPCAL1 protein in the plasma dropped dramatically in HCC patients compared with healthy donors. Receiver operating characteristic curve analysis showed that serum HPCAL1 at a concentration of 8.654 ng/mL could better predict HCC. Furthermore, ectopic expression of HPCAL1 suppresses cell proliferation, while depletion of HPCAL1 led to increased cell growth both in vitro and in vivo. Mechanistically, HPCAL1 directly interacted with p21 Waf/Cip1 in the nucleus, which requires the EF-hand 4 motif of HPCAL1 and the Cy1 domain of p21. This interaction stabilized p21 Waf/Cip1 in an extracellular signal-regulated kinase 1/2-mitogen-activated protein kinase-dependent manner, which subsequently prevented p21 Waf/Cip1 proteasomal degradation by disrupting SCF Skp2 and CRL4 Cdt2 E3 ligase complexes, resulting in increased protein stability and inhibitory effect of p21 Waf/Cip1 . Notably, the tumor suppressive function of HPCAL1 was dependent on p21 in vitro and in vivo. Consistent with this observation, expression of HPCAL1 and p21 Waf/Cip1 was positively correlated in HCC tissues. Conclusion: These findings highlight a novel tumor suppressor upstream of p21 Waf/Cip1 in attenuating cell cycle progression and provide a promising diagnostic and prognostic factor, as well as a potential therapeutic target for HCC. (HEPATOLOGY 2016;63:880-897) H epatocellular carcinoma (HCC) is one of the most common malignancies worldwide and the third leading cause of cancer-related death. (1) In the past decades, despite the availability of improved diagnostic techniques and progress in surgical and nonsurgical therapies, the prognosis of HCC has remained poor due to easy metastasis and high recurrence rates after surgical resection. (2) Hepatocarcinogenesis is characterized in part by deregulated cell cycle progression, which contributes to excessive Abbreviations: CDK, cyclin-dependent kinase; CHX, cycloheximide; co-IP, coimmunoprecipitation; ERK, extracellular signal-regulated kinase; GSK, glycogen synthase kinase; GST, glutathione S-transferase; HCC, hepatocellular carcinoma; HPCAL1, hippocalcin-like 1; IHC, immunohistochemistry; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MAPK, mitogen-activated protein kinase; mRNA, messenger RNA; MS, mass spectrometry; qRT-PCR, quantitative reverse-transcription polymerase chain re...

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Background and Aims The wide prevalence of chemoresistance and compromised early diagnosis of gallbladder cancer (GBC) has led to poor patient prognosis, requiring sustained efforts for the identification of effective biomarkers and therapeutic intervention. Ceramides have emerged as intracellular signaling molecules linked to tumorigenesis and therapeutic response in cancers. However, the clinical relevance of ceramides with GBC has not been investigated. Approach and Results In the present study, we revealed aberrant gene expressions (e.g., serine palmitoyltransferase 1 [SPTLC1] and ceramide synthase 2 [CERS2]) of de novo ceramide biosynthesis and length‐specific ceramide production in GBC tissues. Analyses of serum ceramide pattern in healthy controls, gallbladder stone, and GBC patients identified C24‐Ceramide as a potential diagnostic biomarker for patients with GBC. Importantly, elevation of SPTLC1, CERS2, and its product, C24‐Ceramide, was associated with tumor staging, distal metastasis, and worse prognosis. In line with this, C24‐Ceramide promoted GBC cell proliferation and migration in vitro and in vivo. Mechanistically, C24‐Ceramide directly bound to phosphatidylinositol 5‐phosphate 4‐kinase type‐2 gamma (PIP4K2C), a regulator of mammalian target of rapamycin (mTOR), to facilitate mTOR complex formation and activation. C6‐Ceramide, an analogue of natural ceramide, competed with C24‐Ceramide for PIP4K2C binding, thereby abrogating C24‐Ceramide–mediated mTOR signaling activation and oncogenic activity. Furthermore, stimulation with C6‐Ceramide significantly suppressed the proliferative and metastatic capacity of GBC cells in vitro and in vivo, which was dependent on PIP4K2C. Conclusions Our findings highlight the clinical relevance of ceramide metabolism with GBC progression and identify C24‐Ceramide as a diagnostic biomarker for GBC. We propose that PIP4K2C is indispensable for C6‐Ceramide as a potential therapeutic intervention for GBC through a direct competition with C24‐Ceramide.