• Elucidating the Role of Fatty Acid Synthase in Oral Carcinogenesis and Potential Therapeutics

      Wisniewski, David John; Schneider, Abraham; 0000-0002-7514-7918 (2018)
      The 5-year overall survival rate in oral squamous cell carcinoma (OSCC) has remained relatively unchanged over decades, due to late stage diagnosis and high recurrence rates. This work investigates two potential contributing risk factors associated with OSCC development: nicotine, present in traditional combustible tobacco cigarettes and electronic nicotine delivery systems, and high glucose as associated with Type II diabetes and hyperglycemia. A novel therapeutic, TVB-3166, for OSCC treatment was also studied through in vitro experiments, which may help improve clinical treatments for fully developed, often late-stage OSCC. Through cell viability and growth assays, scratch assays to mimic in vitro migration, and western blotting, we determined that both nicotine and high glucose caused oral dysplastic keratinocytes to exhibit an increase in malignant-like behavior. High expression levels of fatty acid synthase (FASN), a key de novo lipogenic enzyme, have been implicated in OSCC, and this work presents the first evidence that both nicotine and high glucose markedly increase oral dysplastic keratinocyte FASN expression, which drives epidermal growth factor receptor (EGFR) signaling, a key pro-oncogenic signaling pathway commonly associated with oral carcinogenesis. We also demonstrate that TVB-3166, a novel selective FASN inhibitor, induces apoptosis and reduces in vitro OSCC cell migration. Moreover, TVB-3166 inhibits basal EGFR activity and several other oncogenic signaling proteins. This further establishes a potential role for FASN and EGFR not only in the progression of oral epithelial dysplastic pre-malignant lesions, but in fully-developed OSCC tumors. Overall, this work suggests that both nicotine and high glucose play a role in OSCC progression, specifically as it relates to FASN-dependent EGFR activation. Further, the novel drug TVB-3166 should be investigated in future pre-clinical animal models as a potential adjunct to OSCC therapeutics. Through an improved understanding of risk factors for OSCC development, as well as determination of novel therapeutic strategies, this work aims to improve overall patient survival through prevention of OSCC development, as well as discovery of new adjunctive treatments for fully established tumors.
    • Metformin and Mesenchymal Stem Cell Osteogenic Differentiation: Role of Organic Cation Transporters

      Aljofi, Faisal Egal; Schneider, Abraham; 000-0002-1652-5325 (2017)
      Objective: The long-term goal of these studies is to develop novel tissue engineering strategies to enhance craniofacial bone regeneration by combining human umbilical cord derived mesenchymal stem cells (UC-MSCs) and other potential MSC with either systemically or locally delivered metformin. Metformin is a first line, well-tolerated antidiabetic drug with potential osteogenic actions most likely mediated by the activation of the AMP-activated protein kinase (AMPK) signaling pathway. As a highly hydrophilic cationic drug, metformin requires active intracellular uptake via polyspecific cell membrane organic cation transporters (OCTs) encoded by the SLC22A gene family. Despite their critical involvement in hepatic and renal cellular transport, the role played by OCTs in metformin-induced AMPK pathway activation and osteogenic differentiation in UC-MSCs, remains largely unexplored. Here, we hypothesize that to effectively induce AMPK activation and osteogenic differentiation, metformin must gain intracellular access into functional OCT-expressing UC-MSCs. Methods: Immunoblotting was used to assess OCT expression in human-derived UCMSCs. UC-MSCs were treated in vitro with metformin to determine its intracellular uptake, AMPK pathway activation, mineralized nodule formation, and induction of osteogenic markers. Results: Immunoblotting and cellular uptake assays demonstrate that one or more of the OCT isoforms are highly expressed in UC-MSCs and mediate responses to metformin. Treatment of UC-MSCs with clinically relevant doses of metformin (10 μM) resulted in activation of the AMPK signaling pathway. Use of chemical inhibitors targeting OCT function (10 μM quinidine) or AMPK activation (10 μM compound C) markedly inhibited these responses. Metformin significantly enhanced UC-MSC mineralized nodule formation and increased expression and nuclear localization of the osteogenic transcription factor RUNX2. Collectively, these findings indicate that both OCTs and the AMPK signaling pathway play an important role in mediating metformin-induced UCMSC osteogenic differentiation. Conclusions: By gaining a mechanistic insight into the role played by OCTs on metformin-induced MSC osteogenic differentiation mediated by AMPK/RUNX2 signaling, our work may lead to future tissue engineering platforms where metformin together with functional, OCT expressing UC-MSCs may be used as a novel autogenous therapeutic option to enhance bone regeneration. In particular, these treatment strategies might benefit pediatric patients affected with congenital malformations that compromise orofacial skeletal tissues.
    • Metformin Induces Pro-angiogenic Responses in Dental Pulp Stem Cells: Potential Applications in Craniofacial Bone Regeneration

      Ge, Sheng; Schneider, Abraham (2020)
      The present study was conducted to determine whether metformin, a low-cost drug widely prescribed to control type 2 diabetes mellitus, stimulates production of angiogenic factors to potentially enhance vascularization of dental pulp stem cell (DPSC)-based craniofacial tissue engineered bone. Bone tissue engineering utilizing stem cells, growth factors and scaffolds offer an attractive alternative for regenerating large craniofacial osseous defects versus autologous bone grafts. Yet, successful stem cell-based bone regeneration highly depends on proper adaptation of cells to hypoxia and reestablishment of a functional microvasculature. Recent reports show that metformin induces DPSC’s osteogenic differentiation; however, it remains unknown whether metformin stimulates DPSC-derived, pro-angiogenic responses to support bone regeneration. We found that metformin induced a marked but variable increase in DPSC-derived angiogenic factors, including VEGF and angiogenin, which were further amplified by hypoxia. These results point to a novel, pro-angiogenic action of metformin to potentially enhance DPSC-based vascularized craniofacial skeletal regeneration.
    • Novel effects of piRNAs and pfeRNAs in lung somatic cells

      Gable, Tyler; Mao, Li, M.D.; Schneider, Abraham (2017)
      Aberrant expression and function of PIWI-interacting RNAs (piRNAs) and piRNA-Likes (pilRNAs or piR-Ls) have been reported in various cancers. The majority of current reports have identified or assumed roles of these pilRNA that require association with PIWI family proteins to affect either transposable element silencing or mRNA transcript silencing through base pair matching. However, new reports are describing pilRNA which are capable of regulating physiological and pathological conditions through interactions with non-PIWI and non-PIWI-related proteins and whose interaction is necessary for the function of the binding partner protein. Specific cases examined in lung tissue have also found evidence that such interactions may be occurring at critical phosphorylated residues on the target proteins and suggests an early mechanism through which such interactions may occur. Therefore, we hypothesized that somatic expression of piRNA and piRNA-Like non-coding RNAs play active and dynamic roles in the progression of lung squamous cell and adenocarcinoma via phosphorylation-site interactions of PIWI-independent mechanisms. We began our investigations with RNA sequencing profiles of differentially expressed piRNA and pilRNA in lung somatic cells. Guided by expression data, we explored the phenotype and mechanism of action of potentially significant pilRNA species. We began with our study investigating a piR-L species which induces chemoresistance to cisplatin-based therapy by inhibiting apoptosis in lung squamous cell carcinoma. Next, we examined mitochondrial piRNA57125 which associates with Far Upstream Element Binding Protein 1 (FUBP1) to promote lung adenocarcinoma tumorigenesis. Finally, we performed a phosphorylation-wide sncRNA screen which reveals Protein Functional Effector sncRNAs (pfeRNAs) in human lung somatic cells. Collectively, these studies have supported the hypothesis that pilRNA and pfeRNA do play critical roles in the progression of lung squamous cell and adenocarcinomas and may, in fact, do so through phosphorylation-site interactions. As a result of these studies, we have re-named these pilRNA as pfeRNA, a more encompassing and descriptive terminology which is described below.
    • Phenformin and Oral Cancer: The Role of Organic Cation Transporters

      Irhama, Hussein; Schneider, Abraham (2015)
      Abstract: Background: Compelling preclinical evidence indicates that the biguanide metformin, the most widely prescribed oral antidiabetic drug in the United States, prevents the progression of oral premalignant lesions into oral squamous cell carcinoma (OSCC). Metformin triggers antitumoral responses in part by inhibiting the oncogenic mammalian target of rapamycin (mTOR) pathway through activation of one of its key negative regulators and critical sensor of cellular bioenergetics, the AMP-activated protein kinase (AMPK). As a small hydrophilic cationic compound, metformin enters cells through cell membrane organic cation transporters (OCTs) belonging to the SLC22A gene family. Emerging evidence also shows that OCT expression is significantly reduced or absent as OSCC tumors become more aggressive and less differentiated. Therefore, we hypothesized that phenformin, another biguanide compound with a more hydrophobic structure and possibly more potent antineoplastic activity, might exert its antitumoral effects through an OCT-independent manner. This hypothesis was tested through the following specific aims: (1) To determine whether phenformin induces OSCC growth inhibitory actions; (2) To determine whether OCT function is necessary for phenformin-induced activation of the AMPK signaling pathway. Methods and Materials: Cell viability assays and immunoblotting techniques were conducted by using human-derived OSCC cell lines HN6 and HN13. Results: Our results demonstrated that: (1) phenformin significantly reduced OSCC cell viability, and (2) phenformin appeared to activate AMPK signaling through an OCT-independent manner. Conclusion: This in vitro study shows that phenformin, in contrast to metformin, may exert a more potent antineoplastic effect since it decreased OSCC cell viability with much lower doses. In addition, phenformin appears not to be dependent on OCT expression and activity to activate AMPK signaling. Collectively, these studies suggest that phenformin might become an alternative biguanide, alone or in combination with conventional chemotherapy agents, when treating OCT-deficient OSCC tumors.
    • Potential Role of Metformin in Mesenchymal Stem Cell-based Osteogenic and Angiogenic Coupling

      Alshawaf, Nour; Schneider, Abraham (2017)
      Objective: Metformin is a first-line medication used to treat type 2 diabetes. Non-antidiabetic effects of metformin have recently included its osteogenic effect on bone marrow mesenchymal stem cells (BMSCs). The purpose of the present work was to study the potential effects of metformin in the osteogenic/angiogenic coupling. Results: Clinically relevant doses of metformin exerted both direct and indirect effects on endothelial cells capillary tube formation, a well-known assay used to analyze angiogenic effects in vitro. Also, metformin directly induced BMSCs to express and secrete vascular endothelial growth factor (VEGF). Metformin induced these responses in both BMSCs and endothelial cells due to the fact that they express functional organic cation transporters (OCTs), critical cellular determinants of metformin intracellular uptake and activity. This study provides preliminary data and further research is required to gain a better understanding on the role of metformin as a potential osteogenic/angiogenic factor in skeletal regeneration.