Douglas Feldman

Reciprocal regulation by TLR4 and TGF-β in tumor-initiating stem-like cells

Tumor-initiating stem-like cells (TICs) are resistant to chemotherapy and associated with hepatocellular carcinoma (HCC) caused by HCV and/or alcohol-related chronic liver injury. Using HCV Tg mouse models and patients with HCC, we isolated CD133(+) TICs and identified the pluripotency marker NANOG as a direct target of TLR4, which drives the tumor-initiating activity of TICs. These TLR4/NANOG-dependent TICs were defective in the TGF-β tumor suppressor pathway. Functional oncogene screening of a TIC cDNA library identified Yap1 and Igf2bp3 as NANOG-dependent genes that inactivate TGF-β signaling. Mechanistically, we determined that YAP1 mediates cytoplasmic retention of phosphorylated SMAD3 and suppresses SMAD3 phosphorylation/activation by the IGF2BP3/AKT/mTOR pathway. Silencing of both YAP1 and IGF2BP3 restored TGF-β signaling, inhibited pluripotency genes and tumorigenesis, and abrogated chemoresistance of TICs. Mice with defective TGF-β signaling (Spnb2(+/-) mice) exhibited enhanced liver TLR4 expression and developed HCC in a TLR4-dependent manner. Taken together, these results suggest that the activated TLR4/NANOG oncogenic pathway is linked to suppression of cytostatic TGF-β signaling and could potentially serve as a therapeutic target for HCV-related HCC.

Pluripotency factor-mediated expression of the leptin receptor (OB-R) links obesity to oncogenesis through tumor-initiating stem cells

Misregulation of a pluripotency-associated transcription factor network in adult tissues is associated with the expansion of rare, highly malignant tumor-initiating stem cells (TISCs) through poorly understood mechanisms. We demonstrate that robust and selective expression of the receptor for the adipocyte-derived peptide hormone leptin (OB-R) is a characteristic feature of TISCs and of a broad array of embryonic and induced pluripotent stem cells and is mediated directly by the core pluripotency-associated transcription factors OCT4 and SOX2. TISCs exhibit sensitized responses to leptin, including the phosphorylation and activation of the pluripotency-associated oncogene STAT3 and induction of Oct4 and Sox2, thereby establishing a self-reinforcing signaling module. Exposure of cultured mouse embryonic stem cells to leptin sustains pluripotency in the absence of leukemia inhibitory factor. By implanting TISCs into leptin-deficient ob/ob mice or into comparably overweight Leprdb/db mice that produce leptin, we provide evidence of a central role for the leptin-TISC–signaling axis in promoting obesity-induced tumor growth. Differential responses to extrinsic, adipocyte-derived cues may promote the expansion of tumor cell subpopulations and contribute to oncogenesis.

Cancer stem cells generated by alcohol, diabetes, and hepatitis C virus

Cancer stem cells (tumor‐initiating stem‐like cells: TISCs) are resistant to chemotherapy and are associated with metastatic hepatocellular carcinoma (HCC), which is commonly observed in hepatitis C virus (HCV)‐infected patients with obesity or alcohol abuse. However, it is unknown whether the TLR4‐NANOG pathway serves as a universal oncogenic signaling in the genesis of TISCs and HCC. We aimed to determine whether Tlr4 is a putative proto‐oncogene for TISCs in liver oncogenesis due to different etiologies and how Tlr4 is regulated at the transcriptional and epigenetic levels. CD133+/CD49f+ TISCs were isolated using FACS from HCC developed in HCV Core Tg mice fed alcohol, diethylnitrosamine‐treated mice, and alcoholic patients with or without HCV infection. CD133+/CD49f+ cells isolated from the animal models and patients are tumorigenic both in vitro and in a xenograft model, and Tlr4 or Nanog silencing with shRNA attenuates their tumor initiating property. Functional oncogene screening of a cDNA library identified the organ size control pathway targets Yap1 and AKT activator Igf2bp3 as NANOG‐dependent genes that inhibit transforming growth factor‐β signaling in TISCs. Tlr4 expression is higher in TISCs compared with CD133‐/CD49f+ cells. Taken together, Tlr4 may be a universal proto‐oncogene responsible for the genesis of TLR4‐NANOG dependent TISCs, and this pathway serves as a novel therapeutic target for HCC.

TLR4 Signaling via NANOG Cooperates With STAT3 to Activate Twist1 and Promote Formation of Tumor-initiating Stem-like Cells in Livers of Mice.

BACKGROUND & AIMS Obesity and alcohol consumption contribute to steatohepatitis, which increases the risk for hepatitis C virus (HCV)-associated hepatocellular carcinomas (HCCs). Mouse hepatocytes that express HCV-NS5A in liver up-regulate the expression of Toll-like receptor 4 (TLR4), and develop liver tumors containing tumor-initiating stem-like cells (TICs) that express NANOG. We investigated whether the TLR4 signals to NANOG to promote the development of TICs and tumorigenesis in mice placed on a Western diet high in cholesterol and saturated fat (HCFD). METHODS We expressed HCV-NS5A from a transgene (NS5A Tg) in Tlr4-/- (C57Bl6/10ScN), and wild-type control mice. Mice were fed a HCFD for 12 months. TICs were identified and isolated based on being CD133+, CD49f+, and CD45-. We obtained 142 paraffin-embedded sections of different stage HCCs and adjacent nontumor areas from the same patients, and performed gene expression, immunofluorescence, and immunohistochemical analyses. RESULTS A higher proportion of NS5A Tg mice developed liver tumors (39%) than mice that did not express HCV NS5A after the HCFD (6%); only 9% of Tlr4-/- NS5A Tg mice fed HCFD developed liver tumors. Livers from NS5A Tg mice fed the HCFD had increased levels of TLR4, NANOG, phosphorylated signal transducer and activator of transcription (pSTAT3), and TWIST1 proteins, and increases in Tlr4, Nanog, Stat3, and Twist1 messenger RNAs. In TICs from NS5A Tg mice, NANOG and pSTAT3 directly interact to activate expression of Twist1. Levels of TLR4, NANOG, pSTAT3, and TWIST were increased in HCC compared with nontumor tissues from patients. CONCLUSIONS HCFD and HCV-NS5A together stimulated TLR4-NANOG and the leptin receptor (OB-R)-pSTAT3 signaling pathways, resulting in liver tumorigenesis through an exaggerated mesenchymal phenotype with prominent Twist1-expressing TICs.

NUMB phosphorylation destabilizes p53 and promotes self-renewal of tumor-initiating cells by a NANOG-dependent mechanism in liver cancer

Stem cell populations are maintained through self‐renewing divisions in which one daughter cell commits to a particular fate whereas the other retains the multipotent characteristics of its parent. The NUMB, a tumor suppressor, in conjunction with another tumor‐suppressor protein, p53, preserves this property and acts as a barrier against deregulated expansion of tumor‐associated stem cells. In this context, NUMB‐p53 interaction plays a crucial role to maintain the proper homeostasis of both stem cells, as well as differentiated cells. Because the molecular mechanism governing the assembly and stability of the NUMB‐p53 interaction/complex are poorly understood, we tried to identify the molecule(s) that govern this process. Using cancer cell lines, tumor‐initiating cells (TICs) of liver, the mouse model, and clinical samples, we identified that phosphorylations of NUMB destabilize p53 and promote self‐renewal of TICs in a pluripotency‐associated transcription factor NANOG‐dependent manner. NANOG phosphorylates NUMB by atypical protein kinase C zeta (aPKCζ), through the direct induction of Aurora A kinase (AURKA) and the repression of an aPKCζ inhibitor, lethal (2) giant larvae. By radioactivity‐based kinase activity assays, we showed that NANOG enhances kinase activities of both AURKA and aPKCζ, an important upstream process for NUMB phosphorylation. Phosphorylation of NUMB by aPKCζ destabilizes the NUMB‐p53 interaction and p53 proteolysis and deregulates self‐renewal in TICs. Conclusion: Post‐translational modification of NUMB by the NANOG‐AURKA‐aPKCζ pathway is an important event in TIC self‐renewal and tumorigenesis. Hence, the NANOG‐NUMB‐p53 signaling axis is an important regulatory pathway for TIC events in TIC self‐renewal and liver tumorigenesis, suggesting a therapeutic strategy by targeting NUMB phosphorylation. Further in‐depth in vivo and clinical studies are warranted to verify this suggestion. (Hepatology 2015;62:1466‐1479)

The TBC1D15 oncoprotein controls stem cell self-renewal through destabilization of the Numb-p53 complex.

Stem cell populations are maintained through self-renewing divisions in which one daughter cell commits to a specific fate while the other retains the multipotent characteristics of its parent. The p53 tumor suppressor, in conjunction with its interacting partner protein Numb, preserves this asymmetry and functions as a vital barrier against the unchecked expansion of tumor stem cell pools; however, little is known about the biological control of the Numb-p53 interaction. We show here that Numb and p53 are the constituents of a high molecular mass complex, which is disintegrated upon activation of aPKCζ, a Numb kinase. Using large-scale affinity purification and tandem mass spectrometry, we identify TBC1D15 as a Numb-associated protein and demonstrate that its amino-terminal domain disengages p53 from Numb, triggering p53 proteolysis and promoting self-renewal and pluripotency. Cellular levels of TBC1D15 are diminished upon acute nutrient deprivation through autophagy-mediated degradation, indicating that TBC1D15 serves as a conduit through which cellular metabolic status is linked to self-renewal. The profound deregulation of TBC1D15 expression exhibited in a diverse array of patient tumors underscores its proposed function as an oncoprotein.

Cancer stem cells generated by alcohol, diabetes, and HCV

Cancer stem cells (tumor‐initiating stem‐like cells: TISCs) are resistant to chemotherapy and are associated with metastatic hepatocellular carcinoma (HCC), which is commonly observed in hepatitis C virus (HCV)‐infected patients with obesity or alcohol abuse. However, it is unknown whether the TLR4‐NANOG pathway serves as a universal oncogenic signaling in the genesis of TISCs and HCC. We aimed to determine whether Tlr4 is a putative proto‐oncogene for TISCs in liver oncogenesis due to different etiologies and how Tlr4 is regulated at the transcriptional and epigenetic levels. CD133+/CD49f+ TISCs were isolated using FACS from HCC developed in HCV Core Tg mice fed alcohol, diethylnitrosamine‐treated mice, and alcoholic patients with or without HCV infection. CD133+/CD49f+ cells isolated from the animal models and patients are tumorigenic both in vitro and in a xenograft model, and Tlr4 or Nanog silencing with shRNA attenuates their tumor initiating property. Functional oncogene screening of a cDNA library identified the organ size control pathway targets Yap1 and AKT activator Igf2bp3 as NANOG‐dependent genes that inhibit transforming growth factor‐β signaling in TISCs. Tlr4 expression is higher in TISCs compared with CD133‐/CD49f+ cells. Taken together, Tlr4 may be a universal proto‐oncogene responsible for the genesis of TLR4‐NANOG dependent TISCs, and this pathway serves as a novel therapeutic target for HCC.

Erasure of Tet-Oxidized 5-Methylcytosine by a SRAP Nuclease

Enzymatic oxidation of 5-methylcytosine (5mC) in DNA by the Tet dioxygenases reprograms genome function in embryogenesis and postnatal development. Tet-oxidized derivatives of 5mC such as 5-hydroxymethylcytosine (5hmC) act as transient intermediates in DNA demethylation or persist as durable marks, yet how these alternative fates are specified at individual CpGs is not understood. Here, we report that the SOS response-associated peptidase (SRAP) domain protein Srap1, the mammalian ortholog of an ancient protein superfamily associated with DNA damage response operons in bacteria, binds to Tet-oxidized forms of 5mC in DNA and catalyzes turnover of these bases to unmodified cytosine by an autopeptidase-coupled nuclease. Biallelic inactivation of murine Srap1 causes embryonic sublethality associated with widespread accumulation of ectopic 5hmC. These findings establish a function for a class of DNA base modification-selective nucleases and position Srap1 as a determinant of 5mC demethylation trajectories during mammalian embryonic development.

Cancer stem cells generated by alcohol, diabetes, and hepatitis C virus: Toll-like receptor signaling in liver diseases

Cancer stem cells (tumor‐initiating stem‐like cells: TISCs) are resistant to chemotherapy and are associated with metastatic hepatocellular carcinoma (HCC), which is commonly observed in hepatitis C virus (HCV)‐infected patients with obesity or alcohol abuse. However, it is unknown whether the TLR4‐NANOG pathway serves as a universal oncogenic signaling in the genesis of TISCs and HCC. We aimed to determine whether Tlr4 is a putative proto‐oncogene for TISCs in liver oncogenesis due to different etiologies and how Tlr4 is regulated at the transcriptional and epigenetic levels. CD133+/CD49f+ TISCs were isolated using FACS from HCC developed in HCV Core Tg mice fed alcohol, diethylnitrosamine‐treated mice, and alcoholic patients with or without HCV infection. CD133+/CD49f+ cells isolated from the animal models and patients are tumorigenic both in vitro and in a xenograft model, and Tlr4 or Nanog silencing with shRNA attenuates their tumor initiating property. Functional oncogene screening of a cDNA library identified the organ size control pathway targets Yap1 and AKT activator Igf2bp3 as NANOG‐dependent genes that inhibit transforming growth factor‐β signaling in TISCs. Tlr4 expression is higher in TISCs compared with CD133‐/CD49f+ cells. Taken together, Tlr4 may be a universal proto‐oncogene responsible for the genesis of TLR4‐NANOG dependent TISCs, and this pathway serves as a novel therapeutic target for HCC.

Abstract 2447: Novel TLR4-Nanog stemness pathway in liver cancer stem cells confers resistance to TGF-β-mediated tumor suppression through YAP1 and IGF2BP3-AKT-mTOR

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Cancer stem cells (CSCs) confer resistance to chemotherapy and are associated with metastatic hepatocellular carcinoma (HCC) commonly observed in hepatitis C virus (HCV)-infected patients with alcohol abuse. We recently identified pluripotency marker Nanog is induced by Toll-like receptor 4 (TLR4) signal activated by alcohol-associated endotoxemia in CD133+/CD49f+ CSCs from liver cancers in HCV Ns5a transgenic mice fed alcohol. We propose the novel oncogenic TLR4 signaling reciprocally regulates tumor suppressive TGF-β pathway. Molecular mechanisms for the genesis of CSCs through TLR4 signaling and defective TGF-β pathway, however, are unknown. CD133+/CD49f+ CSCs were isolated from liver tumors of alcohol-fed HCV Ns5a Tg mice. Lentiviral cDNA library were established from the CSCs for screening genes responsible for defective TGF-β signaling. GFP-labeled CSCs were injected in nude or NOG mice with or without genetic manipulation of TGF-β signaling components or newly identified genes to monitor tumor growth. Interactions of proteins encoded by identified genes with TGF-β pathway components were examined. Here we demonstrate TLR4/Nanog-dependent CSCs are indeed defective in TGF-β signaling. Functional oncogene screening of a cDNA library identified the organ size control pathway target Yap1 and AKT activator Igf2bp3 as Nanog-dependent genes that inhibit TGF-β signaling in CSCs through Yap1/Smad7/Smad3 association and suppressed Smad3 phospho-activation due to AKT-mTOR pathway, respectively. Silencing of both Yap1 and Igf2bp3 restores TGF-β signaling, inhibits pluripotency gene expression, and sensitizes CSCs to autophagy and cell death by the conventional chemotherapy (rapamycin and sorafenib). Therefore, a combined therapy targeting both YAP1 and AKT-mTOR pathways to restore TGF-β pathway should improve therapeutic efficacy for HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2447. doi:10.1158/1538-7445.AM2011-2447