Nathan Ungerleider

TGFβ signaling confers sorafenib resistance via induction of multiple RTKs in hepatocellular carcinoma cells

Transforming growth factor β (TGFβ) is a multifunctional cytokine which is importantly implicated in hepatocarcinogenesis. The current study provides novel evidence that TGFβ upregulates the expression of multiple receptor tyrosine kinases (RTKs), including IGF1R, EGFR, PDGFβR, and FGFR1 in human hepatocellular carcinoma (HCC) cells. This, in turn, sensitized HCC cells to individual cognate RTK ligands, leading to cell survival. Our data showed that the TGFβ‐mediated increase in growth factor sensitivity led to evasion of apoptosis induced by the mutikinase inhibitor, sorafenib. Conversely, we observed that inhibition of the TGFβ signaling pathway by LY2157299, a TGFβRI kinase inhibitor, enhanced sorafenib‐induced apoptosis, in vitro. Our findings disclose an important interplay between TGFβ and RTK signaling pathways, which is critical for hepatocellular cancer cell survival and resistance to therapy.

Explosive mutation accumulation triggered by heterozygous human Pol ε proofreading-deficiency is driven by suppression of mismatch repair

Tumors defective for DNA polymerase (Pol) ε proofreading have the highest tumor mutation burden identified. A major unanswered question is whether loss of Pol ε proofreading by itself is sufficient to drive this mutagenesis, or whether additional factors are necessary. To address this, we used a combination of next generation sequencing and in vitro biochemistry on human cell lines engineered to have defects in Pol ε proofreading and mismatch repair. Absent mismatch repair, monoallelic Pol ε proofreading deficiency caused a rapid increase in a unique mutation signature, similar to that observed in tumors from patients with biallelic mismatch repair deficiency and heterozygous Pol ε mutations. Restoring mismatch repair was sufficient to suppress the explosive mutation accumulation. These results strongly suggest that concomitant suppression of mismatch repair, a hallmark of colorectal and other aggressive cancers, is a critical force for driving the explosive mutagenesis seen in tumors expressing exonuclease-deficient Pol ε.

p53 Mediates Vast Gene Expression Changes That Contribute to Poor Chemotherapeutic Response in a Mouse Model of Breast Cancer

p53 is a transcription factor that regulates expression of genes involved in cell cycle arrest, senescence, and apoptosis. TP53 harbors mutations that inactivate its transcriptional activity in roughly 30% of breast cancers, and these tumors are much more likely to undergo a pathological complete response to chemotherapy. Thus, the gene expression program activated by wild-type p53 contributes to a poor response. We used an in vivo genetic model system to comprehensively define the p53- and p21-dependent genes and pathways modulated in tumors following doxorubicin treatment. We identified genes differentially expressed in spontaneous mammary tumors harvested from treated MMTV-Wnt1 mice that respond poorly (Trp53+/+) or favorably (Trp53-null) and those that lack the critical senescence/arrest p53 target gene Cdkn1a. Trp53 wild-type tumors differentially expressed nearly 10-fold more genes than Trp53-null tumors after treatment. Pathway analyses showed that genes involved in cell cycle, senescence, and inflammation were enriched in treated Trp53 wild-type tumors; however, no genes/pathways were identified that adequately explain the superior cell death/tumor regression observed in Trp53-null tumors. Cdkn1a-null tumors that retained arrest capacity (responded poorly) and those that proliferated (responded well) after treatment had remarkably different gene regulation. For instance, Cdkn1a-null tumors that arrested upregulated Cdkn2a (p16), suggesting an alternative, p21-independent route to arrest. Live animal imaging of longitudinal gene expression of a senescence/inflammation gene reporter in Trp53+/+ tumors showed induction during and after chemotherapy treatment, while tumors were arrested, but expression rapidly diminished immediately upon relapse.

The Epstein Barr virus circRNAome

Our appreciation for the extent of Epstein Barr virus (EBV) transcriptome complexity continues to grow through findings of EBV encoded microRNAs, new long non-coding RNAs as well as the more recent discovery of over a hundred new polyadenylated lytic transcripts. Here we report an additional layer to the EBV transcriptome through the identification of a repertoire of latent and lytic viral circular RNAs. Utilizing RNase R-sequencing with cell models representing latency types I, II, and III, we identified EBV encoded circular RNAs expressed from the latency Cp promoter involving backsplicing from the W1 and W2 exons to the C1 exon, from the EBNA BamHI U fragment exon, and from the latency long non-coding RPMS1 locus. In addition, we identified circular RNAs expressed during reactivation including backsplicing from exon 8 to exon 2 of the LMP2 gene and a highly expressed circular RNA derived from intra-exonic backsplicing within the BHLF1 gene. While expression of most of these circular RNAs was found to depend on the EBV transcriptional program utilized and the transcription levels of the associated loci, expression of LMP2 exon 8 to exon 2 circular RNA was found to be cell model specific. Altogether we identified over 30 unique EBV circRNAs candidates and we validated and determined the structural features, expression profiles and nuclear/cytoplasmic distributions of several predominant and notable viral circRNAs. Further, we show that two of the EBV circular RNAs derived from the RPMS1 locus are detected in EBV positive clinical stomach cancer specimens. This study increases the known EBV latency and lytic transcriptome repertoires to include viral circular RNAs and it provides an essential foundation and resource for investigations into the functions and roles of this new class of EBV transcripts in EBV biology and diseases.

A novel TGF-β and H19 signaling axis in tumor-initiating hepatocytes that regulates hepatic carcinogenesis.

Functions of transforming growth factor‐β (TGF‐β) in the liver vary depending on specific cell types and their temporal response to TGF‐β during different stages of hepatocarcinogenesis (HCG). Through analysis of tumor tissues from hepatocellular carcinoma (HCC) patients, we were able to cluster hepatic epithelial cell‐derived TGF‐β gene signatures in association with distinct clinical prognoses. To delineate the role of hepatic epithelial TGF‐β signaling in HCC development, we used an experimental system in which tumor‐initiating hepatocytes (TICs) were isolated from TGF‐β receptor II floxed mice (Tgfbr2fl/fl) and transplanted into syngeneic C57BL/6J mice by splenic injection. Recipient mice were then administered Cre‐expressing adenovirus (Ad‐Cre) to inactivate Tgfbr2 in transplanted TICs. After latency, Tgfbr2‐inactivated TICs formed larger and more tumor nodules in recipient livers compared to TICs without Tgfbr2 inactivation. In vitro analyses revealed that treatment of cultured TICs with TGF‐β inhibited expression of progenitor cell factors (including SRY (sex determining region Y)‐box 2 [Sox2]). RNA sequencing (RNA‐seq) analysis identified H19 as one of the most up‐regulated long noncoding RNA (lncRNA) in association with Tgfbr2 inactivation in TICs. Tgfbr2 inactivation by Ad‐Cre led to a 5‐fold increase of H19 expression in TICs. Accordingly, TGF‐β treatment reduced H19 expression. We observed that forced overexpression of Sox2 in TICs increased transcription of H19, whereas knockdown of Sox2 decreased it. Furthermore, depletion of H19 reduced the progenitor property of TICs in vitro and decreased their tumorigenic potential in vivo. Finally, we observed a low level of H19 mRNA expression in human HCC tissues from patients with the epithelial TGF‐β gene signature in association with favorable prognosis. Conclusion: Our findings describe a TGF‐β and H19 signaling axis by Sox2 in TICs that importantly regulates HCG.

Loss of FOXO3 facilitates inflammatory colon cancer: transcriptome profiling of the immune landscape and novel targets

Background & Aims Diminished forkhead box O3 (FOXO3) function drives inflammation and cancer growth; however, mechanisms fostering these pathobiologies are unclear. Here, we aimed to identify in colon loss of FOXO3-dependent cellular and molecular changes that facilitate inflammation-mediated tumor growth. Methods FOXO3 knockout (KO) and wild-type (WT) mice were used in the AOM/DSS model of inflammation-mediated colon cancer. Bioinformatics were used for profiling of mRNA sequencing data from human and mouse colon and tumors; specific targets were validated in human colon cancer cells (shFOXO3). Results In mice, FOXO3 deficiency led to significantly elevated colonic tumor burden (incidence and size) compared with WT (P < .05). In FOXO3 KO colon, activated molecular pathways overlapped with those associated with mouse and human colonic inflammation and cancer, especially human colonic tumors with inflammatory microsatellite instability (false discovery rate < 0.05). FOXO3 KO colon, similar to tumors, had increased neutrophils, macrophages, B cells, T cells, and decreased natural killer cells (false discovery rate < 0.05). Moreover, in KO colon differentially expressed transcripts were linked to activation of inflammatory nuclear factor kappa B, tumorigenic cMyc, and bacterial Toll-like receptor signaling. Among differentially expressed transcripts, we validated altered expression of integrin subunit alpha 2 (ITGA2), ADAM metallopeptidase with thrombospondin type 1 motif 12, and ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 5 in mouse WT and FOXO3 KO colon and tumors (P < .05). Similarly, their altered expression was found in human inflammatory bowel disease and colon cancer tissues and linked to poor patient survival. Ultimately, in human colon cancer cells, FOXO3 knockdown (shFOXO3) led to significantly increased ITGA2, and silencing ITGA2 (siRNA) alone diminished cell growth. Conclusions We identified the loss of FOXO3-mediated immune landscape, pathways, and transcripts that could serve as biomarkers and new targets for inflammatory colon cancer treatment.

Breast cancer survival predicted by TP53 mutation status differs markedly depending on treatment

BackgroundPrevious studies on the role of TP53 mutation in breast cancer treatment response and survival are contradictory and inconclusive, limited by the use of different endpoints to determine clinical significance and by small sample sizes that prohibit stratification by treatment.MethodsWe utilized large datasets to examine overall survival according to TP53 mutation status in patients across multiple clinical features and treatments.ResultsConfirming other studies, we found that in all patients and in hormone therapy-treated patients, TP53 wild-type status conferred superior 5-year overall survival, but survival curves crossed at 10 or more years. In contrast, further stratification within the large dataset revealed that in patients receiving chemotherapy and no hormone therapy, wild-type TP53 status conferred remarkably poor overall survival. This previously unrecognized inferior survival is consistent with p53 inducing arrest/senescence instead of apoptosis. Addition of hormone therapy to chemotherapy improved survival notably in patients with TP53 wild-type tumors, but not mutant, suggesting hormone therapy could eradicate arrested/senescent cells. Testing this, we found that estrogen receptor-positive, TP53 wild-type breast cancer cells that were made senescent by doxorubicin treatment were sensitive to tamoxifen.ConclusionsThe poor survival of chemotherapy-treated patients with TP53 wild-type tumors may be improved by strategies to eliminate senescent cells, including the addition of hormone therapy when appropriate.

Abstract 981: Loss of TGFβ signaling leads to increased susceptibility to HCC in mice: Evidences of lncRNA H19's effects in HCC promotion

The output of a TGF-β response is highly contextual depending on specific tissue types and diseases. This is especially true in hepatocellular carcinoma (HCC) initiation and progression, where the tumor-initialing and malignant cells are highly influenced by the background liver environments. To delineate the role of TGF-β in the process of hepatocarcinogenesis, we employed a novel tumor-initiating cell transplantation system that avoids the possible effects incurred from genetic manipulation of the background liver. Specifically, TGF-β receptor II (Tgfbr2) flox/flox mice at 14 days of age were given one dose (25μg/g bodyweight) of the hepatic carcinogen diethylnitrosamine (DEN) via intraperitoneal injection. Three months later, the initiated hepatocytes from Tgfbr2 flox/flox mice were isolated and transplanted to the same strain (C57Bl/6) wild type recipient mice. Four weeks after transplantation, the recipient mice received one does of Cre recombinase adenovirus (Ad-Cre) through tail vein injection to delete Tgfbr2 in the transplanted tumor-initiating hepatocytes; separate group of the recipient mice were injected with the control adenovirus (Ad-GFP). The recipient mice were closely monitored for liver tumor burden. We observed that deletion of Tgfbr2 in tumor-initiating hepatocytes by tail vein injection of Ad-Cre led to formation of more and bigger liver tumors (compared to Ad-GFP group). This finding suggests that TGF-β inhibits the malignant potential of tumor-initiating hepatocytes. In parallel, we further analyzed the effect of TGF-β in tumor initiating hepatocytes in vitro. Specifically, tumor-initiating hepatocytes isolated from DEN-treated Tgfbr2 flox/flox mice were infected with Ad-Cre or Ad-GFP prior to TGF-β1 treatment in vitro; RNAs were then isolated for transcriptome sequencing analysis. We identified a group of lncRNAs that were noticeably regulated by TGF-β, including the lncRNA H19. We found that deletion of Tgfbr2 by Ad-Cre in tumor initiating hepatocytes led to a 5-fold increase of H19 expression. We observed that deletion of H19 by siRNA decreased the tumor-initiating cell property, whereas forced overexpression of H19 enhanced it. Chromatin immunoprecipitation assay showed that SOX2 bound to the promoter region of H19 gene. While SOX2 overexpression in tumor-initiating hepatocytes enhanced H19 transcription, SOX2 knockdown reduced it. Furthermore, TGF-β treatment reduced the protein level of SOX2 in tumor-initiating hepatocytes. Taken together, our findings disclose a novel mechanism that importantly regulates hepatocarcinogenesis — TGF-β inhibits H19 expression via SOX2 in tumor-initiating hepatocytes and this effect leads to inhibition of HCC development. Citation Format: jinqiang Zhang, Chang Han, Weina Chen, Kyoungsub Song, Ying Wang, Lu Yao, Nathan Ungerleider, Hyunjoo Kwon, Tong Wu. Loss of TGFβ signaling leads to increased susceptibility to HCC in mice: Evidences of lncRNA H199s effects in HCC promotion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 981.

Abstract 949: Epigenetic silencing of microRNA-34a promotes cholangiocarcinoma growth by regulating Notch pathway

Aberrant expression and regulation of microRNAs (miRNAs) have been found in multiple cancers and their dysregulation importantly contribute to the tumorigenic processes. Among these miRNAs, miRNA-34a (miR-34a) functions as a tumor suppressor and its dysregulation has been reported in several cancers. In this study, we investigated epigenetic regulation and biological function of miR-34a in human cholangiocarcinoma cells. Our data indicate that miR-34a expression is epigenetically silenced in human cholangiocarcinoma cells (CCLP1, SG231, Hucct1 and TFK1) compared to the nonmalignant biliary epithelial cells (H69). Treatment of human cholangiocarcinoma cells with the DNA methylation inhibitor, 5’aza-2’deoxycytidine (5-Aza-CdR), or the EZH2 (enhancer of zeste 2 polycomb repressive complex 2) inhibitor, GSK126, enhanced the expression of miR-34a. In parallel, knockdown of EZH2 by shRNA also significantly decreased miR-34a expression in human cholangiocarcinoma cells. We further showed that DNA methylation and EZH2-mediated histone H3 lysine 27 trimethylation (H3K27Me3) repressed miR-34a gene expression, as determined by methylation-specific PCR and chromatin immunoprecipitation. The DNA methylation and EZH2-mediated H3K27Me3 independently repress miR-34a expression in cholangiocarcinoma cells since treatment of 5-Aza-CdR and GSK126 had no effect on H3K27me3 and DNMT1 levels, respectively. Functional analyses revealed that miR-34a mimic decreased cholangiocarcinoma cell proliferation, colony formation and migration, in vitro. Moreover, we identified Notch1, Notch2 and Jagged 1, which are the major receptors and ligands of the Notch pathway, as miR-34a target genes in cholangiocarcinoma cells. Accordingly, forced overexpression of miR-34a significantly decreased the expression of Notch1, Notch2 and Jagged1, as determined by Western blotting and qRT-PCR analyses. Our results provide novel evidence that miR-34a expression is epigenetically silenced by DNA methylation and EZH2 in human cholangiocarcinoma cells. Thus, epigenetic induction of miR-34a or miR-34a replacement therapy may represent a novel therapeutic approach for the treatment of cholangiocarcinoma. Citation Format: Hyunjoo Kwon, Kyoungsub Song, Chang Han, Jinqing Zhang, Nathan Ungerleider, Lu Yao, Tong Wu. Epigenetic silencing of microRNA-34a promotes cholangiocarcinoma growth by regulating Notch pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 949.

Abstract 3921: TGFβ sensitizes hepatocellular carcinoma cells to growth factors, preventing sorafenib-induced apoptosis

Many hepatocellular carcinoma (HCC) patients have a poor prognosis due to an acquired resistance of tumors to chemotherapeutic treatment. Transforming growth factor β (TGFβ) has important roles in the control of cell differentiation, cell cycle progression, and apoptosis. An upregulation in the TGFβ signalling pathway is associated with poor prognosis in HCC patients. In this study, we aim to investigate the mechanisms by which TGFβ contributes to HCC tumor progression. We found that TGFβ significantly increases mRNA and protein levels of several mitogenic receptor tyrosine kinases, including insulin-like growth factor 1 receptor (IGF1R) and epidermal growth factor receptor (EGFR) in both time-dependent and dose-dependent manners in PLC/PRF/5 cells. Furthermore, TGFβ pretreatment significantly increases the acute cellular response to IGF1, IGF2, and EGF treatment, as measured by levels of pAKT and pERK also in both time-dependent and dose-dependent manners. We hypothesized that this increase in growth factor sensitivity would render cells resistant to sorafenib, the only approved targeted HCC chemotherapeutic agent. To test this, we pretreated PLC/PRF/5 cells with TGFβ for 48 hours then incubated the cells with sorafenib for up to 24 hours. We found that TGFβ pretreatment led to a reduction of cleaved caspases 3, 7, 9, and PARP, which are cleaved as cells undergo apoptosis. We confirmed an increase in cell viability via WST-1 assays and by counting viable cells. Lastly, we found that the TGFβRI kinase inhibitor, LY2157299, synergized with sorafenib to promote apoptosis. Taken together, our data suggests that TGFβ can enhance the survival of HCC cells by upregulating cell sensitivity to pro-survival growth factors. Furthermore, the use of both sorafenib and an inhibitor of the TGFβ signalling pathway may one day prove to be a more effective therapy for patients with advanced HCC. Citation Format: Nathan Ungerleider, Chang Han, Tong Wu. TGFβ sensitizes hepatocellular carcinoma cells to growth factors, preventing sorafenib-induced apoptosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3921. doi:10.1158/1538-7445.AM2015-3921