Heterogeneity, inherent and acquired drug resistance in patient-derived organoid models of primary liver cancer.

PURPOSE: We aimed to elucidate the applicability of tumor organoids for inherent drug resistance of primary liver cancer (PLC) and mechanisms of acquired drug resistance. METHODS: PLC tissues were used to establish organoids, organoid-derived xenograft (ODX) and patient-derived xenograft (PDX) models. Acquired drug resistance was induced in hepatocellular carcinoma (HCC) organoids. Gene expression profiling was performed by RNA-sequencing. RESULTS: Fifty-two organoids were established from 153 PLC patients. Compared with establishing PDX models, establishing organoids of HCC showed a trend toward a higher success rate (29.0% vs. 23.7%) and took less time (13.0 ± 4.7 vs. 25.1 ± 5.4 days, p = 2.28 × 10-13). Larger tumors, vascular invasion, higher serum AFP levels, advanced stages and upregulation of stemness- and proliferation-related genes were significantly associated with the successful establishment of HCC organoids and PDX. Organoids and ODX recapitulated PLC histopathological features, but were enriched in more aggressive cell types. PLC organoids were mostly resistant to lenvatinib in vitro but sensitive to lenvatinib in ODX models. Stemness- and epithelial-mesenchymal transition (EMT)-related gene sets were found to be upregulated, whereas liver development- and liver specific molecule-related gene sets were downregulated in acquired sorafenib-resistant organoids. Targeting the mTOR signaling pathway was effective in treating acquired sorafenib-resistant HCC organoids, possibly via inducing phosphorylated S6 kinase. Genes upregulated in acquired sorafenib-resistant HCC organoids were associated with an unfavorable prognosis. CONCLUSIONS: HCC organoids perform better than PDX for drug screening. Acquired sorafenib resistance in organoids promotes HCC aggressiveness via facilitating stemness, retro-differentiation and EMT. Phosphorylated S6 kinase may be predictive for drug resistance in HCC.

circKCNN2 suppresses the recurrence of hepatocellular carcinoma at least partially via regulating miR-520c-3p/methyl-DNA-binding domain protein 2 axis.

BACKGROUND: Recurrence is the major cause of hepatocellular carcinoma (HCC) death. We aimed to identify circular RNA (circRNA) with predictive and therapeutic value for recurrent HCC. METHODS: Tissue samples from recurrent and non-recurrent HCC patients were subjected to circRNA sequencing and transcriptome sequencing. circKCNN2 was identified through multi-omics analyses. The effects of circKCNN2 on HCC were evaluated in cells, animals, database of The Cancer Genome Atlas, and a cohort with 130 HCC patients. circRNA precipitation, chromatin immunoprecipitation assay, RNA pull-down, luciferase assay, and cell experiments were applied to evaluate the interaction of circKCNN2 with miRNAs and proteins. The association between circKCNN2 and the therapeutic effect of lenvatinib was investigated in HCC cell lines and HCC tissue-derived organoids. RESULTS: The expression of circKCNN2 was downregulated in HCC tissues and predicted a favorable overall survival and recurrence-free survival. The expression of circKCNN2 was positively correlated with the parental gene, potassium calcium-activated channel subfamily N member (KCNN2). Nuclear transcription factor Y subunit alpha (NFYA) was proven to inhibit the promoter activity of KCNN2, downregulate the expression of KCNN2 and circKCNN2, and predict an unfavorable recurrence-free survival. Ectopic expression of circKCNN2 inhibited HCC cell proliferation, colony formation, migration, and tumor formation in a mouse model. miR-520c-3p sponged by circKCNN2 could reverse the inhibitory effect of circKCNN2 on HCC cells and down-regulate the expression of methyl-DNA-binding domain protein 2 (MBD2). The intratumoral expression of MBD2 predicted a favorable recurrence-free survival. circKCNN2 down-regulated the expression of fibroblast growth factor receptor 4 (FGFR4), which can be reversed by miR-520c-3p and knockdown of MBD2. Lenvatinib inhibited the expression of FGFR4 and upregulated the expression of circKCNN2 and MBD2. Ectopic expression of circKCNN2 in HCC cells enhanced the therapeutic effect of lenvatinib. However, the high inherent level of circKCNN2 in HCC cells was associated with lenvatinib resistance. CONCLUSIONS: circKCNN2, transcriptionally repressed by NFYA, suppresses HCC recurrence via the miR-520c-3p/MBD2 axis. Inherent level of circKCNN2 in HCC cells predisposes anti-tumor effect of lenvatinib possibly because both circKCNN2 and lenvatinib repress the expression of FGFR4. circKCNN2 may be a promising predictive biomarker and therapeutic agent for HCC recurrence.

Polyamines Involved in Regulating Self-Incompatibility in Apple.

Apple exhibits typical gametophytic self-incompatibility, in which self-S-RNase can arrest pollen tube growth, leading to failure of fertilization. To date, there have been few studies on how to resist the toxicity of self-S-RNase. In this study, pollen tube polyamines were found to respond to self-S-RNase and help pollen tubes defend against self-S-RNase. In particular, the contents of putrescine, spermidine, and spermine in the pollen tube treated with self-S-RNase were substantially lower than those treated with non-self-S-RNase. Further analysis of gene expression of key enzymes in the synthesis and degradation pathways of polyamines found that the expression of DIAMINE OXIDASE 4 (MdDAO4) as well as several polyamine oxidases such as POLYAMINE OXIDASES 3 (MdPAO3), POLYAMINE OXIDASES 4 (MdPAO4), and POLYAMINE OXIDASES 6 (MdPAO6) were significantly up-regulated under self-S-RNase treatment, resulting in the reduction of polyamines. Silencing MdPAO6 in pollen tubes alleviates the inhibitory effect of self-S-RNase on pollen tube growth. In addition, exogenous polyamines also enhance pollen tube resistance to self-S-RNase. Transcriptome sequencing data found that polyamines may communicate with S-RNase through the calcium signal pathway, thereby regulating the growth of the pollen tubes. To summarize, our results suggested that polyamines responded to the self-incompatibility reaction and could enhance pollen tube tolerance to S-RNase, thus providing a potential way to break self-incompatibility in apple.

Genetic Polymorphisms Predisposing the Interleukin 6-Induced APOBEC3B-UNG Imbalance Increase HCC Risk via Promoting the Generation of APOBEC-Signature HBV Mutations.

PURPOSE: APOBEC3-UNG imbalance contributes to hepatitis B virus (HBV) inhibition and somatic mutations. We aimed to explore the associations between hepatocellular carcinoma (HCC) risk and genetic polymorphisms predisposing the imbalance.Experimental Design: Genetic polymorphisms at APOBEC3 promoter and UNG enhancer regions were genotyped in 5,621 participants using quantitative PCR. HBV mutations (nt.1600-nt.1945, nt.2848-nt.155) were determined by Sanger sequencing. Dual-luciferase reporter assay was applied to detect the transcriptional activity. Effects of APOBEC3B/UNG SNPs and expression levels on HCC prognosis were evaluated with a cohort of 400 patients with HCC and public databases, respectively. RESULTS: APOBEC3B rs2267401-G allele and UNG rs3890995-C allele significantly increased HCC risk. rs2267401-G allele was significantly associated with the generation of APOBEC-signature HBV mutation whose frequency consecutively increased from asymptomatic HBV carriers to patients with HCC. Multiplicative interaction of rs2267401-G allele with rs3890995-C allele increased HCC risk, with an adjusted OR (95% confidence interval) of 1.90 (1.34-2.81). rs2267401 T-to-G and rs3890995 T-to-C conferred increased activities of APOBEC3B promoter and UNG enhancer, respectively. IL6 significantly increased APOBEC3B promoter activity and inhibited UNG enhancer activity, and these effects were more evident in those carrying rs2267401-G and rs3890995-C, respectively. APOBEC3B rs2267401-GG genotype, higher APOBEC3B expression, and higher APOBEC3B/UNG expression ratio in HCCs indicated poor prognosis. APOBEC-signature somatic mutation predicts poor prognosis in HBV-free HCCs rather than in HBV-positive ones. CONCLUSIONS: Polymorphic genotypes predisposing the APOBEC3B-UNG imbalance in IL6-presenting microenvironment promote HCC development, possibly via promoting the generation of high-risk HBV mutations. This can be transformed into specific prophylaxis of HBV-caused HCC.

A critical role of toll-like receptor 2 (TLR2) and its' in vivo ligands in radio-resistance.

The role of Toll-like receptor-2 (TLR2) in radio-resistance remained largely unknown. TLR2 knockout (TLR2(-/-)) mice received radiation of 6.5 Gy, and then were studied. We found that radiation resulted in more severe mortality and morbidity rates in TLR2(-/-) mice. The cause of death in TLR2(-/-) mice may be severe and persistent bone marrow cell loss. Injection of the TLR2 agonist Pam3CSK4 into wild type (WT) mice induced radio-resistance. Myd88(-/-) mice were more susceptible to radiation. In conclusion, our data indicate that, similar to TLR4, TLR2 plays a critical role in radio-resistance.

Radiation-driven lipid accumulation and dendritic cell dysfunction in cancer.

Dendritic cells (DCs) play important roles in the initiation and maintenance of the immune response. The dysfunction of DCs contributes to tumor evasion and growth. Here we report our findings on the dysfunction of DCs in radiation-induced thymic lymphomas, and the up-regulation of the expression of the lipoprotein lipase (LPL) and the fatty acid binding protein (FABP4), and the level of triacylglycerol (TAG) in serum after total body irradiation, which contribute to DCs lipid accumulation. DCs with high lipid content showed low expression of co-stimulatory molecules and DCs-related cytokines, and were not able to effectively stimulate allogeneic T cells. Normalization of lipid abundance in DCs with an inhibitor of acetyl-CoA carboxylase restored the function of DCs. A high-fat diet promoted radiation-induced thymic lymphoma growth. In all, our study shows that dysfunction of DCs in radiation-induced thymic lymphomas was due to lipid accumulation and may represent a new mechanism in radiation-induced carcinogenesis.