Mono-ADP-ribosylation (MARylation) is emerging as an important regulator of anti-cancer immunity and immunosuppressive tumor microenvironment (TME). Our previous studies showed that PARP11, one of several enzymes that facilitate MARylation, regulates the activities of intratumoral cytotoxic T lymphocytes (CTLs) and regulatory T cells (Tregs). Here, we demonstrate that stimuli such as adenosine, epinephrine, or glucagon-like peptide-1 (GLP1) induced PARP11 in cancer cells. Upregulation of PARP11 in cancer cells led to PARP11-mediated MARylation, ubiquitination, and accelerated degradation of MHC-I through the autophagy-lysosomal pathway. Induction of PARP11 protected cancer cells from killing by specific CTLs and stimulated tumor growth and progression. Genetic ablation of PARP11 attenuated MHC-I MARylation, ubiquitination, and interaction with autophagy receptors. Pharmacologic inhibition of PARP11 in pancreatic ductal adenocarcinoma (PDAC) cells restored their MHC-I levels, sensitized them to killing by CTLs, inhibited tumor growth, and impeded their initial resistance to chemotherapy and their acquired resistance to targeted therapy with RAS inhibitors. Moreover, inhibition of PARP11 prevented hyperprogressive disease in a mouse melanoma model treated with immune checkpoint inhibitors (ICBs), suggesting that PARP11 is a major therapeutically actionable driver of immunosuppression in tumors. SYNOPSIS:Induction of PARP11 in the tumor microenvironment mediates immunosuppression. This study reports that PARP11-driven MARylation and ubiquitination of MHC-I in cancer cells drives immune evasion, tumor growth and resistance to therapies.

Immunosuppressive tumor microenvironment (TME) inactivates CD8+ cytotoxic lymphocytes (CTLs). Here, we identify SPTBN2 spectrin as a key immunosuppressive regulator induced in CTLs in response to nutritional deficit. In human pancreatic and colorectal cancers, SPTBN2 expression negatively correlated with CTL infiltration and patients' survival. In TME of mouse pancreatic and colorectal adenocarcinomas, SPTBN2 inactivated intratumoral CTLs, stimulated tumor growth and conferred cross-resistance to anti-cancer therapies. SPTBN2 knockout protected CAR T-cells from trogocytosis and increased their memory state. SPTBN2 maintained levels of cell surface proteins such as BTLA that undermine CAR T-cell cytotoxicity and promote exhaustion. Re-expression of BTLA largely reversed phenotypes in SPTBN2-deficient CAR T-cells. In manufactured CAR T cells, SPTBN2 was associated with their clinical failure in pediatric patients with leukemia. Accordingly, ablation of SPTBN2 in CAR T-cells increased their cytotoxicity, in vivo persistence and therapeutic effects indicating that SPTBN2 can be targeted to increase the efficacy of anti-cancer therapies.

Tumor-infiltrating regulatory T cells (TI-Tregs) elicit immunosuppressive effects in the tumor microenvironment (TME) leading to accelerated tumor growth and resistance to immunotherapies against solid tumors. Here, we demonstrate that poly-(ADP-ribose)-polymerase-11 (PARP11) is an essential regulator of immunosuppressive activities of TI-Tregs. Expression of PARP11 correlates with TI-Treg cell numbers and poor responses to immune checkpoint blockade (ICB) in human patients with cancer. Tumor-derived factors including adenosine and prostaglandin E2 induce PARP11 in TI-Tregs. Knockout of PARP11 in the cells of the TME or treatment of tumor-bearing mice with selective PARP11 inhibitor ITK7 inactivates TI-Tregs and reinvigorates anti-tumor immune responses. Accordingly, ITK7 decelerates tumor growth and significantly increases the efficacy of anti-tumor immunotherapies including ICB and adoptive transfer of chimeric antigen receptor (CAR) T cells. These results characterize PARP11 as a key driver of TI-Treg activities and a major regulator of immunosuppressive TME and argue for targeting PARP11 to augment anti-cancer immunotherapies.

Accurate transcription is required for the faithful expression of genetic information. However, relatively little is known about the molecular mechanisms that control the fidelity of transcription, or the conservation of these mechanisms across the tree of life. To address these issues, we measured the error rate of transcription in five organisms of increasing complexity and found that the error rate of RNA polymerase II ranges from 2.9 × 10 −6 ± 1.9 × 10 −7 /bp in yeast to 4.0 × 10 −6 ± 5.2 × 10 −7 /bp in worms, 5.69 × 10 −6 ± 8.2 × 10 −7 /bp in flies, 4.9 × 10 −6 ± 3.6 × 10 −7 /bp in mouse cells and 4.7 × 10 −6 ± 9.9 × 10 −8 /bp in human cells. These error rates were modified by various factors including aging, mutagen treatment and gene modifications. For example, the deletion or modification of several related genes increased the error rate substantially in both yeast and human cells. This research highlights the evolutionary conservation of factors that control the fidelity of transcription. Additionally, these experiments provide a reasonable estimate of the error rate of transcription in human cells and identify disease alleles in a subunit of RNA polymerase II that display error-prone transcription. Finally, we provide evidence suggesting that the error rate and spectrum of transcription co-evolved with our genetic code.

Enhanced activation of the transcription factor MYC and of the receptor tyrosine kinase MET are among the events frequently occurring in hepatocellular carcinoma (HCC). Both genes individually act as drivers of liver cancer initiation and progression. However, their concomitant alteration in HCC has not been explored, nor functionally documented. Here, we analysed databases of five independent human HCC cohorts and found a subset of patients with high levels of MYC and MET (MYChigh/METhigh) characterised by poor prognosis. This clinical observation drove us to explore the functionality of MYC and MET co-occurrence in vivo, combining hydrodynamic tail vein injection for MYC expression in the R26stopMet genetic setting, in which wild-type MET levels are enhanced following the genetic deletion of a stop cassette. Results showed that increased MYC and MET expression in hepatocytes is sufficient to induce liver tumorigenesis even in the absence of pre-existing injuries associated with a chronic disease state. Intriguingly, ectopic MYC in MET tumours increases expression of the Mki67 proliferation marker, and switches them into loss of Afp, Spp1, Gpc3, Epcam accompanied by an increase in Hgma1, Vim, and Hep-Par1 levels. We additionally found a switch in the expression of specific immune checkpoints, with an increase in the Ctla-4 and Lag3 lymphocyte co-inhibitory responses, and in the Icosl co-stimulatory responses of tumour cells. We provide in vitro evidence on the vulnerability of some human HCC cell lines to combined MYC and MET targeting, which are otherwise resistant to single inhibition. Mechanistically, combined blockage of MYC and MET converts a partial cytostatic effect, triggered by individual blockage of MYC or MET, into a cytotoxic effect. Together, these findings highlight a subgroup of HCC characterised by MYChigh/METhigh, and document functional cooperativity between MYC and MET in liver tumorigenesis. Thus, the MYC-R26Met model is a relevant setting for HCC biology, patient classification and treatment.

Hepatocellular carcinoma (HCC) is the predominant primary cancer arising from the liver and is one of the major causes of cancer-related mortality worldwide. The cellular origin of HCC has been a topic of great interest due to conflicting findings regarding whether it originates in hepatocytes, biliary cells, or facultative stem cells. These cell types all undergo changes during liver injury, and there is controversy about their contribution to regenerative responses in the liver. Most HCCs emerge in the setting of chronic liver injury from viral hepatitis, fatty liver disease, alcohol, and environmental exposures. The injuries are marked by liver parenchymal changes such as hepatocyte regenerative nodules, biliary duct cellular changes, expansion of myofibroblasts that cause fibrosis and cirrhosis, and inflammatory cell infiltration, all of which may contribute to carcinogenesis. Addressing the cellular origin of HCC is the key to identifying the earliest events that trigger it. Herein, we review data on the cells of origin in regenerating liver and HCC and the implications of these findings for prevention and treatment. We also review the origins of childhood liver cancer and other rare cancers of the liver.

Hepatocellular carcinoma (HCC) is the most prevalent cancer of the liver and is a leading cause of cancer deaths worldwide. The multikinase inhibitor sorafenib has long been used to treat HCC because it was shown to extend survival by 3 months.1 Although other drugs have recently been approved for therapy of HCC based on slightly improved outcomes,2 there is still a pressing need for new and effective treatments. Sequencing technologies have identified hundreds of prognostic genes in HCC.3 Nevertheless, correlation does not equate to functionality.

The idea that it may be possible to “cure” AIDS is beginning to be accepted. Whether that is realistic or a pipe dream remains to be seen. The impediment is HIV latency. Thinking is that (1) if we could wake up the virus and (2) kill it without allowing it to infect naive cells, the battle would be won. To our knowledge no sure-fire way of doing either currently exists. Not surprising, different drugs activate silent virus differentially in cell cultures or in patients. HIV latency essentially is transcriptional block—resulting from block of transcriptional initiation due to limiting transcriptional factors (eg, NFkB); suppression of transcriptional elongation due insufficiency of Tat; epigenetic modification of the viral genome by promoter methylation; or chromatin remodeling by histone acetylation. It makes sense then to target multiple blocks by a combinatorial approach. We have tested this idea in a cell culture model of latency where provirus carries reporter GFP gene and its activation is scored by GFP flow cytometry. We have used (1) prostratin to activate NFkB, (2) Tat to promote transcript elongation, (3) Aza CdR to demethylate the viral promoter, and (4) HDAC inhibitor SAHA to uncondense the chromatin. The results confirm that combination of agents is more effective than single agents. For example, prostratin alone and prostratin plus Tat increased GFP expression by 20 and 100 folds, respectively. Tat alone only minimally activated. Tat being an elongation factor, unless transcription has been initiated, there is nothing to elongate. This work was supported by the intramural program of NCI (2011–2012). The opinions expressed here are those of the authors and not of the National Cancer Institute.

Transcription errors occur in all living cells; however, it is unknown how these errors affect cellular health. To answer this question, we monitor yeast cells that are genetically engineered to display error-prone transcription. We discover that these cells suffer from a profound loss in proteostasis, which sensitizes them to the expression of genes that are associated with protein-folding diseases in humans; thus, transcription errors represent a new molecular mechanism by which cells can acquire disease phenotypes. We further find that the error rate of transcription increases as cells age, suggesting that transcription errors affect proteostasis particularly in aging cells. Accordingly, transcription errors accelerate the aggregation of a peptide that is implicated in Alzheimer's disease, and shorten the lifespan of cells. These experiments reveal a previously unappreciated role for transcriptional fidelity in cellular health and aging.

Claudins have been reported to be differentially regulated in malignancies and implicated in the process of carcinogenesis and tumor progression. Claudin-1 has been described as key factor in the entry of hepatitis C virus (HCV) into hepatocytes and as promoter of epithelial-mesenchymal transition in liver cells. The objective of the current study was to characterize claudin expression in hepatocellular carcinoma (HCC) as well as HCC-surrounding and normal liver samples with respect to cirrhosis and HCV infection. Expression of claudin-1, -2, -3, -4, and -7 was measured by morphometric analysis of immunohistochemistry, and Western blotting in 30 HCCs with 30 corresponding non-tumorous tissues and 6 normal livers. Claudin-1 and -7 protein expression was found significantly elevated in cirrhosis when compared with non-cirrhotic liver. HCCs developed in cirrhotic livers showed even higher expression of claudin-1 contrary to decreased claudin-7 expression when compared with cirrhosis. With reference to HCV status, HCCs or surrounding livers of HCV-infected samples did not show significant alterations in claudin expression when compared with HCV-negative specimens. Cirrhotic transformation associates with elevated claudin-1 and -7 expressions in both non-tumorous liver and HCC. The fact that no significant differences in claudin expression were found regarding HCV-positivity in our sample set suggests that HCV infection alone does not induce a major increase in the total amount of its entry co-factor claudin-1. Increased expression of claudin-1 seems to be a consequence of cirrhotic transformation and might contribute to a more effective HCV entry and malignant transformation.

Abstract Objective: Primary human liver cancer (PLC), the third most lethal cancer worldwide, is classified into biologically distinct subgroups, which suggests origin from different hepatic lineage cells. The existence of cancer stem cells (CSCs) was reported in PLC, but the cellular origin of liver CSCs has not been elucidated. Our aim was to investigate the contribution of different hepatic lineage cells to the evolution of CSCs and the phenotypic and genetic heterogeneity of PLC. Methods: Three cell types at different levels of differentiation, including primary mouse hepatic progenitor cells (HPCs), lineage committed hepatoblasts (HBs) and terminally differentiated hepatocytes (AHs) were co-transduced with lentiviral vectors carrying oncogenic H-Ras-Luciferase/EGFP and SV40 large T (LT)-mCherry. CSC properties of FACS sorted H-Ras-EGFP+/SV40LT-mCherry+ cells were tested by standard in vitro and in vivo assays. Individual liver tumors derived from intrasplenic injection of transduced HPCs, HBs and AHs were subjected to immunohistochemistry and whole transcriptome profiling. Results: HPCs, HBs and AHs were susceptible to transformation albeit with a different efficiency as shown by the frequency of tumor initiating cells (1/7, 1/26 and 1/42, respectively). All transduced cells acquired similar attributes of liver CSCs in vitro as judged by self-renewal ability, expression of CSC marker CD133, CD24, CD44 and CD90 and high percentage of side population cells. HPC-, HB- and AH-initiated liver tumors commonly showed a multi-lineage differentiation expressing hepatocyte (HNFα), hepatic progenitor cell (EpCAM, cytokeratin 19, A6) and mesenchymal (vimentin) markers and resembled human PLC. Nevertheless, tumors displayed distinct morphophenotypes according to their cell-of origin: AH tumors showed predominantly hepatocellular carcinoma, HB tumors cholangicarcinoma and HPC tumors epithelial-mesenchymal transition (EMT)-like features. Gene expression analyses revealed the activation of EMT- and embryonic cell-related transcriptional programs in all tumors with the highest number of significant genetic changes in AH (2826) versus HB (574) and HPC tumors (906). Hierarchical clustering distinguished tumors of different cellular origin underscoring the contribution of lineage-stage-dependent genetic changes in malignant transformation. Notably, AH-derived tumors showed specific enrichment of c-Myc target genes. Stable knockdown of c-Myc in transformed AHs reduced their CSC properties and delayed tumor growth. Conclusions: Our results indicate that liver tumors with dominant CSC features can originate from any cell in the hepatocytic lineage. Identification of common and cell-of-origin specific phenotypic and genetic changes may provide novel therapeutic targets for treatment of PLC. Citation Format: Agnes Holczbauer, Valentina M. Factor, Jesper B. Andersen, David E. Kleiner, Jens U. Marquardt, Chiara Raggi, Mitsuteru Kitade, Daekwan Seo, Akita Hirofumi, Marian E. Durkin, Snorri S. Thorgeirsson. Direct oncogenic reprogramming of adult mouse hepatocytes into cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2653. doi:10.1158/1538-7445.AM2013-2653

Reversal of DNA hypermethylation and associated gene silencing is an emerging cancer therapy approach. Here we addressed the impact of epigenetic alterations and cellular context on functional and transcriptional reprogramming of hepatocellular carcinoma (HCC) cells. Our strategy employed a 3-day treatment of established and primary human HCC-derived cell lines grown as a monolayer at various cell densities with the DNMT1 inhibitor zebularine (ZEB) followed by a 3D culture to identify cells endowed with self-renewal potential. Differences in self-renewal, gene expression, tumorigenicity, and metastatic potential of spheres at generations G1-G5 were examined. Transient ZEB exposure produced differential cell density-dependent responses. In cells grown at low density, ZEB caused a remarkable increase in self-renewal and tumorigenicity associated with long-lasting gene expression changes characterized by a stable overexpression of cancer stem cell-related and key epithelial-mesenchymal transition genes. These effects persisted after restoration of DNMT1 expression. In contrast, at high cell density, ZEB caused a gradual decrease in self-renewal and tumorigenicty, and up-regulation of apoptosis- and differentiation-related genes. A permanent reduction of DNMT1 protein using short hairpin RNA (shRNA)-mediated DNMT1 silencing rendered HCC cells insensitive both to cell density and ZEB effects. Similarly, WRL68 and HepG2 hepatoblastoma cells expressing low DNMT1 basal levels also possessed a high self-renewal, irrespective of cell density or ZEB exposure. Spheres formed by low-density cells treated with ZEB or shDNMT1 displayed a high molecular similarity which was sustained through consecutive generations, confirming the essential role of DNMT1 depletion in the enhancement of cancer stem cell properties. Conclusion: These results identify DNA methylation as a key epigenetic regulatory mechanism determining the pool of cancer stem cells in liver cancer and possibly other solid tumors. (Hepatology 2014;59:2251–2262)

The relative contribution of hepatocyte growth factor (HGF)/MET and epidermal growth factor (EGF)/EGF receptor (EGFR), two key signal transduction systems in the normal and diseased liver, to fate decisions of adult hepatic progenitor cells (HPCs) has not been resolved. Here, we developed a robust culture system that permitted expansion and genetic manipulation of cells capable of multilineage differentiation in vitro and in vivo to examine the individual roles of HGF/MET and EGF/EGFR in HPC self-renewal and binary cell fate decision. By employing loss-of-function and rescue experiments in vitro, we showed that both receptors collaborate to increase the self-renewal of HPCs through activation of the extracellular signal-regulated kinase (ERK) pathway. MET was a strong inducer of hepatocyte differentiation by activating AKT and signal transducer and activator of transcription (STAT3). Conversely, EGFR selectively induced NOTCH1 to promote cholangiocyte specification and branching morphogenesis while concomitantly suppressing hepatocyte commitment. Furthermore, unlike the deleterious effects of MET deletion, the liver-specific conditional loss of Egfr facilitated rather than suppressed progenitor-mediated liver regeneration by switching progenitor cell differentiation toward hepatocyte lineage. These data provide new insight into the mechanisms regulating the stemness properties of adult HPCs and reveal a previously unrecognized link between EGFR and NOTCH1 in directing cholangiocyte differentiation.

BACKGROUND & AIMS:Human primary liver cancer is classified into biologically distinct subgroups based on cellular origin. Liver cancer stem cells (CSCs) have been recently described. We investigated the ability of distinct lineages of hepatic cells to become liver CSCs and the phenotypic and genetic heterogeneity of primary liver cancer.METHODS:We transduced mouse primary hepatic progenitor cells, lineage-committed hepatoblasts, and differentiated adult hepatocytes with transgenes encoding oncogenic H-Ras and SV40LT. The CSC properties of transduced cells and their ability to form tumors were tested by standard in vitro and in vivo assays and transcriptome profiling.RESULTS:Irrespective of origin, all transduced cells acquired markers of CSC/progenitor cells, side populations, and self-renewal capacity in vitro. They also formed a broad spectrum of liver tumors, ranging from cholangiocarcinoma to hepatocellular carcinoma, which resembled human liver tumors, based on genomic and histologic analyses. The tumor cells coexpressed hepatocyte (hepatocyte nuclear factor 4α), progenitor/biliary (keratin 19, epithelial cell adhesion molecule, A6), and mesenchymal (vimentin) markers and showed dysregulation of genes that control the epithelial-mesenchymal transition. Gene expression analyses could distinguish tumors of different cellular origin, indicating the contribution of lineage stage-dependent genetic changes to malignant transformation. Activation of c-Myc and its target genes was required to reprogram adult hepatocytes into CSCs and for tumors to develop. Stable knockdown of c-Myc in transformed adult hepatocytes reduced their CSC properties in vitro and suppressed growth of tumors in immunodeficient mice.CONCLUSIONS:Any cell type in the mouse hepatic lineage can undergo oncogenic reprogramming into a CSC by activating different cell type-specific pathways. Identification of common and cell of origin-specific phenotypic and genetic changes could provide new therapeutic targets for liver cancer.