Tumor-initiating stem-like cells (TICs) are defective in maintaining asymmetric cell division and responsible for tumor recurrence. Cell-fate-determinant molecule NUMB-interacting protein (TBC1D15) is overexpressed and contributes to p53 degradation in TICs. Here we identify TBC1D15-mediated oncogenic mechanisms and tested the tumorigenic roles of TBC1D15 in vivo. We examined hepatocellular carcinoma (HCC) development in alcohol Western diet-fed hepatitis C virus NS5A Tg mice with hepatocyte-specific TBC1D15 deficiency or expression of non-phosphorylatable NUMB mutations. Liver-specific TBC1D15 deficiency or non-p-NUMB expression reduced TIC numbers and HCC development. TBC1D15-NuMA1 association impaired asymmetric division machinery by hijacking NuMA from LGN binding, thereby favoring TIC self-renewal. TBC1D15-NOTCH1 interaction activated and stabilized NOTCH1 which upregulated transcription of NANOG essential for TIC expansion. TBC1D15 activated three novel oncogenic pathways to promote self-renewal, p53 loss, and Nanog transcription in TICs. Thus, this central regulator could serve as a potential therapeutic target for treatment of HCC.
Key Points• Rare and pathogenic germline variants, including in IKZF1, contribute to acute lymphoblastic leukemia in children with Down syndrome.
Stem cell markers such as Nanog have been implicated in various cancer, but whether they are functionally contributing to cancer pathogenesis has remained unclear. Tumor-initiating stem-like cells (TICs) were isolated from mouse hepatocellular carcinoma (HCC) and human HCC patients. Using knowledge gained from the oncogenic signaling of TICs, we addressed new mechanistic hypotheses based on the novel discovery that the pluripotent transcription factor, NANOG, can reprogram the metabolism of HCC-derived TICs. These studies will motivate novel strategies for targeting and removing TICs and suggest new avenues to treat malignancies associated with human HCV and alcoholism and/or obesity. We determined novel targets of NANOG in TICs from patient and mouse models of HCC using genome-wide NANOG-binding site analysis (ChIP-seq) and how Nanog is regulated at the transcriptional to promote oncogenesis and self-renewal in TICs. NANOG-mediated metabolic reprogramming through suppression of mitochondria function in both experimental and clinical HCC downstream of TLR4/NANOG generates TICs and drives liver tumorigenesis. The p53 tumor suppressor acts as a barrier against stem cell proliferation, and inactivation of p53, or its stabilizing partner NUMB (cell fate decision molecule), leads to expansion of TICs. Although treatment efficacy of HCV has been dramatically improved in recent years, the incidence of HCV-associated HCC keeps rising due to the prevalence of obesity, alcoholism and illicit drug usage in HCV patients. RNA-binding protein MSI2 is elevated in several cancers and is linked to poor prognosis. We also performed RIP-seq using anti-MSI2 antibody in TICs and subsequent validation by qPCR analysis. Among the MSI2-bound RNAs, MYC mRNA and long noncoding RNA (LncRNA) were identified by RIP-qPCR analysis. MSI2 promotes liver tumorigenesis by maintenance of MYC expression by inhibition of LncRNA processing. This newly discovered mechanistic framework for TIC proliferation represents a key innovation and holds significant potential as a therapeutic target.
Tumor-initiating stem-like cells (TICs) are defective in their control of asymmetric division. Stem cells are maintained through asymmetric 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 interacting protein NUMB preserves this intrinsic cellular asymmetry and functions as a vital barrier against unchecked expansion as seen in TICs. How TICs overcome this control of asymmetric division to cause cancer is unknown. The hypothesis is that accentuated TLR4-NANOG-mediated NUMB phosphorylation and TBC1D15 upregulation promote self-renewal of TICs in liver oncogenesis caused by HCV and alcohol. Here, we determined the causal roles of NUMB phosphorylation and TBC1D15 oncoprotein in alcohol-induced liver tumorigenesis in HCV-Tg mice. Liver-specific TBC1D15 deficiency or non-phosphorylatable mutations of NUMB, reduce liver tumor incidence and tumor-associated NANOG+ TICs in HCV NS5A Tg mice fed alcohol Western diet. TICs lost asymmetric division capacity and overexpressed TBC1D15, which associated with NUMB to promote aPKCζ activity and NUMB phosphorylation. Thus, TIC self-renewal is dependent on TBC1D15 and NUMB phosphorylation. Similarly, aPKCζ-mediated NUMB phosphorylation and induced TBC1D15 are mutually required for self-renewal. Furthermore, TBC1D15 activated NOTCH via binding to NOTCH intracellular domain to trigger its proteolytic. TBC1D15 interacts with all NOTCH isoforms, activates NOTCH pathway, and induces Nanog and TIC self-renewal in a NOTCH-dependent manner. TBC1D15 cooperates with the NOTCH pathway to support TIC tumorigenic activity and an inhibitor of this interaction is potentially therapeutic. Candidate small molecule antagonists have been tested for their ability to block the interaction of TBC1D15-NOTCH/NICD. The originality of this findings lies within the discovery of the novel oncoprotein TBC1D15 and its unique oncogenic activities involving p53 degradation and cooperation with the NOTCH pathway in TICs and CD133+ Huh7 cells. These mechanistic findings have laid down the foundation for a new translational path for discovery of new therapeutic targets. Citation Format: Keigo Machida, Hifzur R. Siddique, Mengmei Zheng, Peleg Winer, Dinesh Babu Uthaya Kumar, Ahmed Rokan, Linda Sher, Stanley M. Tahara, Michael Elowitz, Chengyu Liang, Hidekazu Tsukamoto. Cell fate reprogramming of liver tumor-initiating stem-like cells via phosphorylated NUMB and TBC1D15 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1984.
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