Unique molecular characteristics of NAFLD-associated liver cancer accentuate ß-catenin/TNFRSF19-mediated immune evasion.

BACKGROUND & AIMS: The hepatic manifestation of the metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), can lead to the development of hepatocellular carcinoma (HCC). Despite a strong causative link, NAFLD-HCC is often underrepresented in systematic genome explorations. METHODS: Herein, tumor-normal pairs from 100 patients diagnosed with NAFLD-HCC were subject to next-generation sequencing. Bioinformatic analyses were performed to identify key genomic, epigenomic and transcriptomic events associated with the pathogenesis of NAFLD-HCC. Establishment of primary patient-derived NAFLD-HCC culture was used as a representative human model for downstream in vitro investigations of the underlying CTNNB1 S45P driver mutation. A syngeneic immunocompetent mouse model was used to further test the involvement of CTNNB1mutand TNFRSF19 in reshaping the tumor microenvironment. RESULTS: Mutational processes operative in the livers of patients with NAFLD inferred susceptibility to tumor formation through defective DNA repair pathways. Dense promoter mutations and dysregulated transcription factors accentuated activated transcriptional regulation in NAFLD-HCC, in particular the enrichment of MAZ-MYC activities. Somatic events common in HCCs arising from NAFLD and viral hepatitis B infection underscore similar driver pathways, although an incidence shift highlights CTNNB1mut dominance in NAFLD-HCC (33%). Immune exclusion correlated evidently with CTNNB1mut. Chromatin immunoprecipitation-sequencing integrated with transcriptome and immune profiling revealed a unique transcriptional axis, wherein CTNNB1mut leads to an upregulation of TNFRSF19 which subsequently represses senescence-associated secretory phenotype-like cytokines (including IL6 and CXCL8). This phenomenon could be reverted by the Wnt-modulator ICG001. CONCLUSIONS: The unique mutational processes in the livers of patients with NAFLD and NAFLD-HCC allude to a "field effect" involving a gain-of-function role of CTNNB1 mutations in immune exclusion. LAY SUMMARY: The increasing prevalence of metabolic syndrome in adult populations means that NAFLD is poised to be the major cause of liver cancer in the 21st century. We showed a strong "field effect" in the livers of patients with NAFLD, wherein activated ß-catenin was involved in reshaping the tumor-immune microenvironment.

Manipulation of the Nanoscale Presentation of Integrin Ligand Produces Cancer Cells with Enhanced Stemness and Robust Tumorigenicity.

Developing strategies for efficient expansion of cancer stem-like cells (CSCs) in vitro will help investigate the mechanism underlying tumorigenesis and cancer recurrence. Herein, we report a dynamic culture substrate tethered with integrin ligand-bearing magnetic nanoparticles via a flexible polymeric linker to enable magnetic manipulation of the nanoscale ligand tether mobility. The cancer cells cultured on the substrate with high ligand tether mobility develop into large semispherical colonies with CSCs features, which can be abrogated by magnetically restricting the ligand tether mobility. Mechanistically, the substrate with high ligand tether mobility suppresses integrin-mediated mechanotransduction and histone-related methylation, thereby enhancing cancer cell stemness. The culture-derived high-stemness cells can generate tumors both locally and at the distant lung and uterus much more efficiently than the low-stemness cells. We believe that this magnetic nanoplatform provides a promising strategy for investigating the dynamic interaction between CSCs and the microenvironment and establishing a cost-effective tumor spheroid model.

Lethal (3) malignant brain tumor-like 2 (L3MBTL2) protein protects against kidney injury by inhibiting the DNA damage-p53-apoptosis pathway in renal tubular cells.

DNA damage contributes to renal tubular cell death during kidney injury, but how DNA damage in tubular cells is regulated is not fully understood. Lethal (3) malignant brain tumor-like 2 (L3MBTL2), a novel polycomb group protein, has been implicated in regulating chromatin architecture. However, the biological functions of L3MBTL2 are largely undefined. Here we found that L3MBTL2 was expressed in the nuclei of renal tubular epithelial cells in mice. Ablation of L3mbtl2 in renal tubular cells resulted in increases in nuclear DNA damage, p53 activation, apoptosis, tubular injury and kidney dysfunction after cisplatin treatment or unilateral ureteral obstruction. In vitro, inhibition of L3MBTL2 sequentially promoted histone γH2AX expression, p53 activation and apoptosis in cisplatin-treated mouse proximal tubular TKPTS cells. Inhibition of p53 activity attenuated the apoptosis induced by L3mbtl2 deficiency after cisplatin treatment both in vivo and in vitro. Intriguingly, unlike other polycomb proteins, L3MBTL2 was not recruited to DNA damage sites, but instead increased nuclear chromatin density and reduced initial DNA damage load. Thus, L3MBTL2 plays a protective role in kidney injury, in part by inhibiting the DNA damage-p53-apoptosis pathway.

Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.

CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which, however, has focused on HDR-based strategies and was proven inefficient. Here, we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs, and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy, integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells, and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells.

Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors.

The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around -120 to -80 bp, while highly effective sgRNAs targeted from -147 to -89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells.

Overexpression of nucleotide metabolic enzyme DUT in hepatocellular carcinoma potentiates a therapeutic opportunity through targeting its dUTPase activity.

Uracil misincorporation during DNA replication is a major cell toxic event, of which cancer cells overcome by activating the dUTPase enzyme. The DUT gene is the only known dUTPase in human. Despite reports on common upregulations in cancers, the role of DUT in human hepatocellular carcinoma (HCC) remains largely undetermined. In this study, we investigated the mechanism underlying DUT biology in HCC and tumor susceptibility to drug targeting dUTPase. Overexpression of DUT was found in 42% of HCC tumors and correlated with advanced stage HCC. Knockout of DUT in HCC cell lines showed suppressed proliferation through cell cycle arrest and a spontaneous induction of DNA damage. A protective effect from oxidative stress was also demonstrated in both knockout and overexpression DUT assays. Transcriptome analysis highlighted the NF-κB survival signaling as the downstream effector pathway of DUT in overriding oxidative stress-induced cell death. Interestingly, stably expressed DUT in liver progenitor organoids conferred drug resistance to TKI Sorafenib. Targeting dUTPase activity by TAS-114, could potentiate suppression of HCC growth that synergized with Sorafenib for better treatment sensitivity. In conclusion, upregulated DUT represents a nucleotide metabolic weakness and therapeutic opportunity in HCC.

Nuclear Receptor LRH-1 Functions to Promote Castration-Resistant Growth of Prostate Cancer via Its Promotion of Intratumoral Androgen Biosynthesis.

Targeting of steroidogenic enzymes (e.g., abiraterone acetate targeting CYP17A1) has been developed as a novel therapeutic strategy against metastatic castration-resistant prostate cancer (CRPC). However, resistance to steroidal inhibitors inevitably develops in patients, the mechanisms of which remain largely unknown. Liver receptor homolog-1 (LRH-1, NR5A2) is a nuclear receptor, originally characterized as an important regulator of some liver-specific metabolic genes. Here, we report that LRH-1, which exhibited an increased expression pattern in high-grade prostate cancer and CRPC xenograft models, functions to promote de novo androgen biosynthesis via its direct transactivation of several key steroidogenic enzyme genes, elevating intratumoral androgen levels and reactivating AR signaling in CRPC xenografts as well as abiraterone-treated CRPC tumors. Pharmacologic inhibition of LRH-1 activity attenuated LRH-1-mediated androgen deprivation and anti-androgen resistance of prostate cancer cells. Our findings not only demonstrate the significant role of LRH-1 in the promotion of intratumoral androgen biosynthesis in CRPC via its direct transcriptional control of steroidogenesis, but also suggest targeting LRH-1 could be a potential therapeutic strategy for CRPC management.Significance: These findings not only demonstrate the significant role of the nuclear receptor LRH-1 in the promotion of intratumoral androgen biosynthesis in CRPC via its direct transcriptional control of steroidogenesis, but also suggest targeting LRH-1 could be a potential therapeutic strategy for CRPC management. Cancer Res; 78(9); 2205-18. ©2018 AACR.

Orphan nuclear receptor TLX contributes to androgen insensitivity in castration-resistant prostate cancer via its repression of androgen receptor transcription.

The metastatic castration-resistant prostate cancer (CRPC) is a lethal form of prostate cancer, in which the expression of androgen receptor (AR) is highly heterogeneous. Indeed, lower AR expression and attenuated AR signature activity is shown in CRPC tissues, especially in the subset of neuroendocrine prostate cancer (NEPC) and prostate cancer stem-like cells (PCSCs). However, the significance of AR downregulation in androgen insensitivity and de-differentiation of tumor cells in CRPC is poorly understood and much neglected. Our previous study shows that the orphan nuclear receptor TLX (NR2E1), which is upregulated in prostate cancer, plays an oncogenic role in prostate carcinogenesis by suppressing oncogene-induced senescence. In the present study, we further established that TLX exhibited an increased expression in metastatic CRPC. Further analyses showed that overexpression of TLX could confer resistance to androgen deprivation and anti-androgen in androgen-dependent prostate cancer cells in vitro and in vivo, whereas knockdown of endogenous TLX could potentiate the sensitivity to androgen deprivation and anti-androgen in prostate cancer cells. Our study revealed that the TLX-induced resistance to androgen deprivation and anti-androgen was mediated through its direct suppression of AR gene transcription and signaling in both androgen-stimulated and -unstimulated prostate cancer cells. We also characterized that TLX could bind directly to AR promoter and repress AR transcription by recruitment of histone modifiers, including HDAC1, HDAC3, and LSD1. Together, our present study shows, for the first time, that TLX can contribute to androgen insensitivity in CRPC via repression of AR gene transcription and signaling, and also implicates that targeting the druggable TLX may have a potential therapeutic significance in CRPC management, particularly in NEPC and PCSCs.