Nanog is a transcription factor that is well-established as a key regulator of embryonic stem cell (ESC) maintenance. Recent evidence demonstrates that Nanog is dysregulated and intimately involved in promoting tumorigenesis in part through regulation of the cancer stem cell (CSC) population. Elevated Nanog is associated with poorer outcome in numerous epithelial malignancies. Nanog is enriched in CSCs and ablation of Nanog is sufficient to reduce the CSC pool. Nanog has also been implicated to promote chemoresistance and epithelial-mesenchymal transition (EMT). Insight into the Nanog signaling cascade, upstream regulators and downstream effectors, is beginning to emerge but remains to be fully elucidated. This review highlights the current literature on the emerging role of Nanog in tumorigenesis and CSCs.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide with about 600,000 new cases diagnosed in the last year. Our laboratory showed that miR-107 expression is reduced and functions as a tumor suppressor gene in HNSCC suggesting the potential application of miR-107 as a novel anticancer therapeutic. In this study, we determined the efficiency and efficacy of cationic lipid nanoparticles to deliver pre-miR-107 (NP/pre-miR-107) in HNSCC cells in vitro and in vivo. NP/pre-miR-107 increased delivery of miR-107 into HNSCC cells by greater than 80,000-fold compared to free pre-miR-107. Levels of known miR-107 targets, protein kinase Cε (PKCε), cyclin-dependent kinase 6 (CDK6), and hypoxia-inducible factor 1-β (HIF1-β), decreased following NP/pre-miR-107 treatment. Clonogenic survival, cell invasion, and cell migration of HNSCC cells was inhibited with NP/pre-miR-107. Moreover, NP/pre-miR-107 reduced the cancer-initiating cell (CIC) population and dampened the expression of the core embryonic stem cell transcription factors, Nanog, Oct3/4, and Sox2. In a preclinical mouse model of HNSCC, systemic administration of NP/pre-miR-107 significantly retarded tumor growth by 45.2% compared to NP/pre-miR-control (P < 0.005, n = 7). Kaplan-Meier analysis showed a survival advantage for the NP/pre-miR-107 treatment group (P = 0.017). Our results demonstrate that cationic lipid nanoparticles are an effective carrier approach to deliver therapeutic miRs to HNSCC.

The incidence of human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) has rapidly increased over the past 30 years prompting the suggestion that an epidemic may be on the horizon. Therefore, there is a clinical need to develop alternate therapeutic strategies to manage the growing number of HPV-positive HNSCC patients. High-risk HPV E6 inactivates p53 through two distinct mechanisms; association with E6AP to degrade p53 and association with p300 to block p300-mediated p53 acetylation and activation. In this study, we determined if targeting the E6-p300 interaction is an effective approach to reactivate p53 in HPV-positive HNSCC. Ectopic expression of the CH1 domain of p300 in HPV-positive HNSCC blocks the association between E6 and p300, increases total and acetylated p53 levels, and enhances p53 transcriptional activity. Moreover, expression of p21, miR-34a, and miR-200c are increased demonstrating functional p53 reactivation. CH1 overexpression in HPV-positive HNSCC has a global anti-cancer effect resulting in a decrease in cell proliferation and clonogenic survival and an increase in apoptosis. The in vivo tumor initiating ability of HPV-positive HNSCC is severely compromised with CH1 overexpression, in part through a reduction in the cancer initiating cell population. A novel small molecule CH1 inhibitor, CH1iB, reactivates p53 and potentiates the anti-cancer activity of cis-platinum in HPV-positive HNSCC cells. Our work shows that CH1 domain inhibitors represent a novel class of p53 reactivation therapeutics for managing HPV-positive HNSCC patients.

Background Human papillomavirus 16 (HPV16) is a major risk factor for the development of head and neck squamous cell carcinoma (HNSCC), in particular, oropharyngeal SCC (OPSCC). Cancer stem cells (CSCs) are resistant to conventional therapies and postulated to be responsible for disease recurrence and/or progression. Since the prognosis of HPV16-positive and HPV-negative HNSCC are distinct, we determine if differences in the CSC number may account for this clinical observation. Methods CSC population in HPV16-positive and HPV-negative HNSCC was assessed using the ALDEFLUOR assay, in vitro tumorsphere formation assay, and in vivo limiting cell dilution in NOD/SCID mice. A high-density tissue microarray was stained with ALDH1, a CSC marker, to determine the association between CSCs and HPV16-positive/HPV-negative OPSCC. Results HPV16-positive HNSCC had a higher intrinsic CSC pool than HPV-negative HNSCC. Inactivation of p53 is a major mechanism for elevated CSC population in HPV16-positive HNSCC. In vivo limiting cell dilution experiments using HPV16-positive and HPV-negative OPSCC patient tumors indicated that the CSC frequency is 62.5-fold higher in the HPV16-positive OPSCC tumor than in the HPV-negative OPSCC tumor. Primary tumors from HPV16-positive OPSCC patients were associated with elevated tumor ALDH1 staining further extending the association between HPV16 and CSC. Conclusions Our data and the clinical observation that HPV16-positive HNSCC patients respond more favorably than HPV-negative HNSCC patients to current treatment paradigms support the suggestion that CSC phenotype is not homogeneous. Therefore, the reliance on CSC number may be insufficient to accurately assess the potential of a particular tumor for disease recurrence and/or progression.

Emerging evidence indicates that Nanog is intimately involved in tumorigenesis in part through regulation of the cancer initiating cell population. However, the regulation and role of Nanog in tumorigenesis are still poorly understood. In this study, human Nanog was identified to be phosphorylated by human PKCε at multiple residues including T200 and T280. Our work indicated that phosphorylation at T200 and T280 modulates Nanog function through several regulatory mechanisms. Results with phosphorylation-insensitive and phosphorylation-mimetic mutant Nanog revealed that phosphorylation at T200 and T280 enhance Nanog protein stability. Moreover, phosphorylation-insensitive T200A and T280A mutant Nanog had a dominant-negative function to inhibit endogenous Nanog transcriptional activity. Inactivation of Nanog was due to impaired homodimerization, DNA binding, promoter occupancy, and p300, a transcriptional co-activator, recruitment resulting in a defect in target gene promoter activation. Ectopic expression of phosphorylation-insensitive T200A or T280A mutant Nanog reduced cell proliferation, colony formation, invasion, migration, and the cancer initiating cell population in head and neck squamous cell carcinoma (HNSCC) cells. The in vivo cancer initiating ability was severely compromised in HNSCC cells expressing phosphorylation-insensitive T200A or T280A mutant Nanog; 87.5% (14/16), 12.5% (1/8), and 0% (0/8) for control, T200A, and T280A, respectively. Nanog occupied the Bmi1 promoter to directly transactivate and regulate Bmi1. Genetic ablation and rescue experiments demonstrated that Bmi1 is a critical downstream signaling node for the pleiotropic, pro-oncogenic effects of Nanog. Taken together, our study revealed, for the first time, that post-translational phosphorylation of Nanog is essential to regulate Bmi1 and promote tumorigenesis.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide with about 600,000 new cases diagnosed in the last year. The incidence of human papillomavirus-positive head and neck squamous cell carcinoma (HPV-positive HNSCC) has rapidly increased over the past 30 years prompting the suggestion that an epidemic may be on the horizon. Therefore, there is a clinical need to develop alternate therapeutic strategies to manage the growing number of HPV-positive HNSCC patients. TriCurin is a composition of three food-derived polyphenols in unique stoichiometric proportions consisting of curcumin from the spice turmeric, resveratrol from red grapes, and epicatechin gallate from green tea. Cell viability, clonogenic survival, and tumorsphere formation were inhibited and significant apoptosis was induced by TriCurin in UMSCC47 and UPCI:SCC090 HPV-positive HNSCC cells. Moreover, TriCurin decreased HPV16E6 and HPV16E7 and increased p53 levels. In a pre-clinical animal model of HPV-positive HNSCC, intra-tumoral injection of TriCurin significantly inhibited tumor growth by 85.5% compared to vehicle group (P < 0.05, n = 7). Our results demonstrate that TriCurin is a potent anti-tumor agent for HPV-positive HNSCC. Further development of TriCurin as a novel anti-cancer therapeutic to manage the HPV-positive HNSCC population is warranted.

SummaryVascular smooth muscle cell (VSMC) proliferation promotes intimal hyperplasia (IH) in occluding vascular diseases. Here we identified a positive role of ALDH1A3 (an aldehyde dehydrogenase) in this pro-IH process. The expression of ALDH1A3, but not that of 18 other isoforms of the ALDH family, was substantially increased in cytokine-stimulated VSMCs. PDGF(BB) stimulated VSMC total ALDH activity and proliferation, whereas ALDH1A3 silencing abolished this effect. ALDH1A3 silencing also diminished the expression of two matricellular proteins (TNC1 and ESM1), revealing a previously unrecognized ALDH1A3 function. Loss-of-function experiments demonstrated that TNC1 and ESM1 mediated ALDH1A3's pro-proliferative function via activation of AKT/mTOR and/or MEK/ERK pathways. Furthermore, ALDH inhibition with disulfiram blocked VSMC proliferation/migration in vitro and decreased TNC1 and ESM1 and IH in angioplasty-injured rat carotid arteries. Thus, ALDH1A3 promotes VSMC proliferation at least partially through TNC1/ESM1 upregulation; dampening excessive ALDH1A3 activity represents a potential approach to IH mitigation.

The sigma-2 receptor (S2R) has long been pharmacologically targeted for antipsychotic treatment and tumor imaging. Only recently was it known for its coding gene and for its role implicated in cholesterol homeostasis. Here, we have investigated the transcriptional control of S2R by the Bromo/ExtraTerminal epigenetic reader family (BETs, including BRD2, 3, and 4) upon cholesterol perturbation. Cholesterol deprivation was induced in ARPE19 cells using a blocker of lysosomal cholesterol export. This condition up-regulated S2R mRNA and protein, and also SREBP2 but not SREBP1, both transcription factors key to cholesterol/fatty acid metabolism. Silencing BRD2 but not BRD3 or BRD4 (though widely deemed a master regulator) averted S2R up-regulation that was induced by cholesterol deprivation. Silencing SREBP2 but not SREBP1 diminished S2R expression. Furthermore, endogenous BRD2 co-immunoprecipitated with the transcription-active N-terminal half of SREBP2, and chromatin immunoprecipitation-qPCR signified co-occupancy of BRD2, H3K27ac (histone acetylation), and SREBP2Nterm at the S2R gene promoter. In summary, this study reveals a previously unrecognized BRD2/SREBP2 cooperative regulation of S2R transcription, thus shedding new light on signaling in response to cholesterol deprivation.