Multiple-level copy number variations in cell-free DNA for prognostic prediction of HCC with radical treatments.

Copy number variations (CNVs) in cell-free DNA (cfDNA) are emerging as noninvasive biomarkers for various cancers. However, multiple-level analysis of cfDNA CNVs for hepatocellular carcinoma (HCC) patients with radical treatments remains uninvestigated. Here, CNVs at genome-wide, chromosomal-arm, and bin levels were analyzed in cfDNA from 117 HCC patients receiving radical treatments. Then, the relationship between cfDNA CNVs and clinical outcomes was explored. Our results showed that a concordant profile of CNVs was observed between cfDNA and tumor tissue DNA. Three genome-wide CNV indicators including tumor fraction (TFx), prediction score (P-score), and stability score (S-score) were calculated and demonstrated to exhibit significant correlation with poorer overall survival (OS) and recurrence-free survival (RFS). Furthermore, the high-frequency cfDNA CNVs at chromosomal-arm level including the loss of 4q, 17p, and 19p and the gain of 8q and 1q clearly predicted HCC prognosis. Finally, a bin-level risk score was constructed to improve the ability of CNVs in predicting prognosis. Altogether, our study indicates that the multiple-level cfDNA CNVs are significantly associated with OS and RFS in HCC patients with radical treatments, suggesting that cfDNA CNVs detected by low-coverage whole-genome sequencing (WGS) may be used as potential prognostic biomarkers of HCC patients.

Machine learning-based genome-wide interrogation of somatic copy number aberrations in circulating tumor DNA for early detection of hepatocellular carcinoma.

BACKGROUND: DNAs released from tumor cells into blood (circulating tumor DNAs, ctDNAs) carry tumor-specific genomic aberrations, providing a non-invasive means for cancer detection. In this study, we aimed to leverage somatic copy number aberration (SCNA) in ctDNA to develop assays to detect early-stage HCCs. METHODS: We conducted low-depth whole-genome sequencing (WGS) to profile SCNAs in 384 plasma samples of hepatitis B virus (HBV)-related HCC and cancer-free HBV patients, using one discovery and two validation cohorts. To fully capture the robust signals of WGS data from the complete genome, we developed a machine learning-based statistical model that is focused on detection accuracy in early-stage HCC. FINDINGS: We built the model using a discovery cohort of 209 patients, achieving an overall area under curve (AUC) of 0.893, with 0.874 for early-stage (Barcelona clinical liver cancer [BCLC] stage 0-A) and 0.933 for advanced-stage (BCLC stage B-D). The performance of the model was then assessed in two validation cohorts (76 and 99 patients) that only consisted of patients with stage 0-A HCC. Our model exhibited a robust predictive performance, with an AUC of 0.920 and 0.812 for the two validation cohorts. Further analyses showed the impact of tumor sample heterogeneity in model training on detecting early-stage tumors, and a refined model addressing the heterogeneity in the discovery cohort significantly increased model performance in validation. INTERPRETATION: We developed an SCNA-based, machine learning-driven model in the non-invasive detection of early-stage HCC in HBV patients and demonstrated its performance through strict independent validations.

Awakening p53 in vivo by D-peptides-functionalized ultra-small nanoparticles: Overcoming biological barriers to D-peptide drug delivery.

Peptides are a rapidly growing class of therapeutics with many advantages over conventional small molecule drugs. Dextrorotary (D)-peptides, with increased enzymatic stability and prolonged plasma half-life in comparison with natural L-peptides, are considered to have great potential as recognition molecules and therapeutic agents. However, the in vivo efficacy of current therapeutic D-peptides is hindered by their inefficient cellular uptake in diseased tissues. Methods: To overcome physiological and cellular barriers to D-peptides, we designed a gold-based ultra-small nanocarrier coupled with polylysine (PLL) and a receptor-targeted peptide to deliver therapeutic D-peptides. Using a D-peptide p53 activator (DPA) as a proof of concept, we synthesized, functionalized and characterized gold- and DPA-based nanoparticles termed AuNP-DPA. Results: AuNP-DPA were effectively enriched in tumor sites and subsequently internalized by cancer cells, thereby suppressing tumor growth via reactivating p53 signaling. More importantly, through a series of in vivo experiments, AuNP-DPA showed excellent biosafety without the common side effects that hinder p53 therapies in clinic trials. Conclusion: The present study not only sheds light on the development of AuNP-DPA as a novel class of antitumor agents for drugging the p53 pathway in vivo, but also supplies a new strategy to use D-peptides as intracellular PPI inhibitors for cancer-targeted therapy.

Ferritin level prospectively predicts hepatocarcinogenesis in patients with chronic hepatitis B virus infection.

Previous studies have detected a higher level of ferritin in patients with hepatocellular carcinoma (HCC), but a potential causal association between serum ferritin level and hepatocarcinogenesis remains to be clarified. Using a well-established prospective cohort and longitudinally collected serial blood samples, the association between baseline ferritin levels and HCC risk were evaluated in 1,152 patients infected with hepatitis B virus (HBV), a major risk factor for HCC. The association was assessed by Cox proportional hazards regression model using univariate and multivariate analyses and longitudinal analysis. It was demonstrated that HBV patients who developed HCC had a significantly higher baseline ferritin level than those who remained cancer-free (188.00 vs. 108.00 ng/ml, P<0.0001). The patients with a high ferritin level (≥200 ng/ml) had 2.43-fold increased risk of HCC compared to those with lower ferritin levels [hazard ratio (HR), 2.43; 95% confidence interval, 1.63-3.63]. A significant trend of increasing HRs along with elevated ferritin levels was observed (P for trend <0.0001). The association was still significant after multivariate adjustment. Incorporating ferritin into the α-fetoprotein (AFP) model significantly improved the performance of HCC prediction (the area under the curve from 0.74 to 0.77, P=0.003). Longitudinal analysis showed that the average ferritin level in HBV patients who developed HCC was persistently higher than in those who were cancer-free during follow-up. HCC risk reached a peak at approximately the fifth year after baseline ferritin detection. Moreover, stratified analyses showed that the association was noted in both males and females, and was prominent in patients with a low AFP value. In short, serum ferritin level could independently predict the risk of HBV-related HCC and may have a complementary role in AFP-based HCC diagnosis. Future studies are warranted to validate these findings and test its clinical applicability in HCC prevention and management.

Novel peptide GX1 inhibits angiogenesis by specifically binding to transglutaminase-2 in the tumorous endothelial cells of gastric cancer.

The clinical application of GX1, an optimal gastric cancer (GC) targeting peptide, is greatly limited because its receptor in the GC vasculature is unknown. In this study, we screened the candidate receptor of GX1, transglutaminase-2(TGM2), by co-immunoprecipitation (co-IP) combined with mass spectrometry. We found that TGM2 was up-regulated in GC vascular endothelial cells and that GX1 receptor expression was suppressed correspondingly after TGM2 downregulation. A highly consistent co-localization of GX1 receptor and TGM2 was detected at both the cellular and tissue levels. High TGM2 expression was evident in GC tissues from patients with poor prognosis. After TGM2 downregulation, the GX1-mediated inhibition of proliferation and migration and the induction of the apoptosis of GC vascular endothelial cells were weakened or even reversed. Finally, we observed that GX1 could inhibit the GTP-binding activity of TGM2 by reducing its intracellular distribution and downregulating its downstream molecular targets (nuclear factor-kappa B, NF-κB; hypoxia-inducible factor 1-α, HIF1α) in GC vascular endothelial cells. Our study confirms that peptide GX1 can inhibit angiogenesis by directly binding to TGM2, subsequently reducing the GTP-binding activity of TGM2 and thereby suppressing its downstream pathway(NF-κB/HIF1α). Our conclusions suggest that GX1/TGM2 may provide a new target for the diagnosis and treatment of GC.

Multi-regional sequencing reveals intratumor heterogeneity and positive selection of somatic mtDNA mutations in hepatocellular carcinoma and colorectal cancer.

Recent studies have revealed significant intratumor heterogeneity (ITH) of nuclear genome mutations and highlighted its function in tumor progression and treatment resistance. However, the ITH of somatic mitochondrial DNA (mtDNA) mutations detected in cancers remains unknown. In this study, we performed multiregional mtDNA sequencing of tumor and paratumor tissue samples from 12 hepatocellular carcinoma (HCC) and 13 colorectal cancer (CRC) patients. A substantial level of mtDNA mutations was found in paired non-HCC inflammatory tissues, suggesting that these tissues might not be mtDNA-genetically "normal." Moreover, our data indicated that the ITH of somatic mtDNA mutations was a common feature in HCC and CRC patients. In addition, we found that shared mutations which were observed in at least 2 samples in each patient exhibited a significantly higher heteroplasmic level than mutations that were private to a specific tumor region from both HCC (p = 0.039) and CRC patients (p = 0.001). The heteroplasmic level of shared mutations was positively correlated with intratumoral recurrence of mtDNA mutations. We also found that shared mutations in tumor tissues with a higher degree of pathogenicity risk exhibited a higher heteroplasmic level and intratumoral recurrence in both HCC and CRC patients. These findings suggest that some mtDNA mutations may undergo positive selection during the clonal expansion. Taken together, our analyses identified various levels of ITH of somatic mtDNA mutations in HCC and CRC patients and provided evidence supporting the positive selection working on some somatic mtDNA mutations in tumor tissues.

Remote limb ischemic postconditioning protects against cerebral ischemia-reperfusion injury by activating AMPK-dependent autophagy.

Remote limb ischemic postconditioning (RIPoC) is a promising adjunct treatment for cerebral ischemia-reperfusion (IR) injury. However, the underlying mechanisms have not been fully elucidated yet. The present study aims to investigate potential involvement and regulatory mechanisms of autophagy in RIPoC treatment against cerebral IR injury in mice. Mice were subjected to 2 h middle cerebral artery occlusion (MCAO) then treated with vehicle, 3-methyladenine (3-MA, an autophagy inhibitor), or compound C (an AMPK inhibitor) at the onset of reperfusion. RIPoC was carried out by 3 cycles of 10-min occlusion-reperfusion of bilateral femoral artery at the beginning of the reperfusion. Infarct volume, neurological score, and brain water content of the mice were assessed after 12 h reperfusion. Autophagy markers, cell apoptosis markers, and AMPK pathway activity were also evaluated. Our results indicated that RIPoC treatment reduced neurological deficits, brain water content, and infarct volume after IR. Meanwhile, RIPoC was proved to induce autophagy and activate AMPK pathway. Furthermore, the RIPoC-induced autophagy and neuroprotection were abolished by 3-MA and partially blocked by compound C. In conclusion, the present study suggests that RIPoC attenuates cerebral IR injury by activating AMPK-dependent autophagy.

High MRPS23 expression contributes to hepatocellular carcinoma proliferation and indicates poor survival outcomes.

Hepatocellular carcinoma is one of the most prevalent neoplasms and the leading cause of cancer-related mortality worldwide. Mitochondrial ribosomal protein S23 is encoded by a nuclear gene and participates in mitochondrial protein translation. Mitochondrial ribosomal protein S23 overexpression has been found in many types of cancer. In this study, we explored mitochondrial ribosomal protein S23 expression in primary hepatocellular carcinoma tissues compared with matched adjacent non-tumoral liver tissues using mitochondrial ribosomal protein S23 messenger RNA and protein levels collected from public databases and clinical samples. Immunohistochemistry was performed to analyze the relationship between mitochondrial ribosomal protein S23 and various clinicopathological features. The results indicated that mitochondrial ribosomal protein S23 was significantly overexpressed in hepatocellular carcinoma. High mitochondrial ribosomal protein S23 expression was correlated with the tumor size and tumor-metastasis-node stage. Moreover, patients with high mitochondrial ribosomal protein S23 expression levels presented poorer survival rates. Mitochondrial ribosomal protein S23 was an independent prognostic factor for survival, especially at the early stage of hepatocellular carcinoma. In addition, the downregulation of mitochondrial ribosomal protein S23 decreased the proliferation of hepatocellular carcinoma in vitro and in vivo. In conclusion, we verified for the first time that mitochondrial ribosomal protein S23 expression was upregulated in hepatocellular carcinoma. High mitochondrial ribosomal protein S23 levels can predict poor clinical outcomes in hepatocellular carcinoma, and this protein plays a key role in tumor proliferation. Therefore, mitochondrial ribosomal protein S23 may be a potential therapeutic target for hepatocellular carcinoma.

Tg737 regulates epithelial-mesenchymal transition and cancer stem cell properties via a negative feedback circuit between Snail and HNF4α during liver stem cell malignant transformation.

Determining the origin of liver cancer stem cells is important for treating hepatocellular carcinoma. Tg737 deficiency plays an important role in the malignant transformation of liver stem cells, but the underlying mechanism remains unclear. Here we established a chemical-induced mouse hepatoma model and found that Tg737 and hepatocyte nuclear factor 4-alpha (HNF4α) expression decreased and epithelial-mesenchymal transition (EMT)-related marker expression increased during liver cancer development. To investigate the underlying mechanism, we knocked down Tg737 in WB-F344 (WB) rat hepatic oval cells. Loss of Tg737 resulted in nuclear ß-catenin accumulation and activation of the Wnt/ß-catenin pathway, which further promoted EMT and the malignant phenotype. XAV939, a ß-catenin inhibitor, attenuated WB cell malignant transformation due to Tg737 knockdown. To clarify the relationships of Tg737, the ß-catenin pathway, and HNF4α, we inhibited Snail and overexpressed HNF4α after Tg737 knockdown in WB cells and found that Snail and HNF4α comprise a negative feedback circuit. Taken together, the results showed that Tg737 regulates a Wnt/ß-catenin/Snail-HNF4α negative feedback circuit, thereby blocking EMT and the malignant transformation of liver stem cells to liver cancer stem cells.