• The Elucidation of Mutation-Specific Glycoprotein and Exosomal Non-small Cell Lung Cancer Biomarkers

      Clark, David Joseph; Mao, Li, M.D.; 0000-0003-0527-8469 (2015)
      Lung cancer is the leading cause of cancer-related death in the world. This high rate of mortality is often a result of late-stage diagnosis, which occurs in the majority of lung cancer cases. The high false positive rate associated with the current screening technique leads to unnecessary and invasive follow-up procedures, thus newer diagnostics must be developed. Here, we evaluated the potential role of exosomes as "biomarker vesicles" in non-small cell lung cancer (NSCLC). We employed quantitative proteomics to evaluate both N-linked glycoprotein expression patterns, and exosomal protein cargo abundance differences between an immortalized bronchial epithelial cell line and two NSCLC cell lines each harboring distinct, non-overlapping mutations in cell signaling molecules: Kirsten rat sarcoma viral oncogene homolog (KRAS) and epidermal growth factor receptor (EGFR). The inherent benefits of this strategy included: (i) the identification of several glycoproteins that were overexpressed in both NSCLC cells, as well as glycoproteins uniquely overexpressed in each profiled NSCLC cell line, and (ii) characterization of NSCLC exosomes to determine proteins enriched in NSCLC exosomes. These results allowed us to generate a preliminary panel of overexpressed glycoproteins that could be further validated as enriched NSCLC exosomal protein cargo, and identify several potential biomarkers of NSCLC to be examined in future clinical studies. We also developed a novel strategy to unbiasedly characterize the exosome proteome by pairing quantitative proteomics and multivariate cluster analysis. Using the breast cancer cell line SKBR3B as a proof-of-concept, we were able to calculate the probability of an identified protein being exosomal in origin. With this method, we were able to determine proteins enriched in the exosome fraction in an unbiased manner, and identify potential residual contaminant proteins that were identified in the final analyte. In summary, our study provides an in-depth characterization of NSCLC exosomes and the identification of several exosomal proteins that could serve as candidate markers of NSCLC In addition, we described a methodology to classify a protein as a "true" constituent of the exosome proteome. Taken together, these studies provide a foundation for the development of a platform that utilizes exosomes in a diagnostic setting.
    • 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.
    • Role of Cell Cycle Regulators Enhancer of Zeste Homolog 2 and Cell Division Cycle 25A in Tumorigenesis of the Aerodigestive Tract

      Younis, Rania Hassan M.; Mao, Li, M.D. (2011)
      Oral Squamous Cell Carcinoma (OSCC) and Non Small Cell Lung Cancer (NSCLC) represent the most common aerodigestive tract malignancies and are a major public health burden worldwide. The prognosis of NSCLC patients is dismal with a 5-year survival rate of 16%. Although the 5-year survival rate of patients with OSCC is more than 50%, many of the survivors suffer significant treatment side effects and disfigurement. Therefore, further understanding of the biologic basis of tumorigenic processes of these cancers is critical for development of novel diagnostic and therapeutic strategies. One of the common features of these cancers is the deregulation of cell cycle process during carcinogenesis. The transcriptional repressor Enhancer of Zeste Homolog 2 (EZH2) has been implicated in cell cycle regulation and tumorigenesis. While the cell cycle promoter Cell Division Cycle 25A (CDC25A) has been proposed as a critical cell cycle promoter linked to several malignancies. Here we investigated the role of the epigenetic EZH2 on cell cycle progression and malignant phenotypes in oral premalignancy and NSCLC. We also studied CDC25A expression and modulation of the cell cycle in NSCLC. Our results provide evidence that EZH2 promotes malignancy in Leuk-1 cell line and is a prognostic marker to OSCC onset in patients with oral premalignancy. EZH2 expression directly correlated with expression of cell cycle promoters in NSCLC NCI-H1299 and Leuk-1 cells. We also identified CDC25A-Q110del a stable isoform in NSCLC that confers CDC25A protein stability and promotes its activity. CDC25A-Q110del confers more cellular survival upon DNA damage and correlates with poor overall survival in NSCLC patients. These data unravel a novel role of EZH2 in cell cycle regulation of oral premalignancy and describe a new mode of regulation and expression of CDC25A in NSCLC. Given the potential role of the two molecules in the tumorigenic process, they may also serve as targets for blocking tumor development or progression. The link between EZH2 and CDC25A in cell cycle regulation may warrant additional investigation regarding the cross talk between the two molecules.
    • The Role of miRNAs in Early Detection of Oral Cancer

      Almubarak, Hussain Mohammed; Meiller, Timothy F.; Mao, Li, M.D. (2012)
      MicroRNAs, commonly noted as miRNAs (miRs), are evolutionary conserved small non-coding RNAs that negatively regulate gene expression post-transcriptionally. Recently, miRNAs have been identified as potentially important biomarkers in various cancers including oral squamous cell carcinoma. The hypothesis of this study was that the expression of miRNAs is different in squamous cell carcinoma in African Americans as compared to Caucasians; as well as when compared to Age, Sex, Location, Recurrence, Grade of cancer, Stage of cancer, Neural invasion, Muscular invasion, and HPV 16/18 or HPV 31/33/51 status. Fifty cases of oral squamous cell carcinoma were matched to the Maryland Cancer Registry (MCR) database and were selected for one Caucasian and one African American in matched sets for age, sex and location. To detect potentially significant miRNAs, two different epithelial cell lines were selected to represent the normal epithelium (HaCaT and NOK) and compared to the dysplastic epithelium (Leuk-1) and the oral cancer (SCC-9 and SCC-25). The miRNAs profiling was performed for the cell lines and several dysregulated miRNAs were identified. The microarray data were validated by the real-time PCR. The expression of miR-708, miR-193a-5p, miR-155, miR-584, miR-663, and miR-886-5p suggested their significant potential at the early stages of tumorigenesis. However, miR-30d, miR-34a, miR-34c-3p, miR-138, and miR-486-5p were dysregulated only in cancer cell lines reflecting their potential at the late stages. The prominently dysregulated miR-708 and miR-193a-5p were selected for further analysis. The lentiviral transduction targeting miR-708 and miR-193a-5p was used to reverse their originally detected expression levels. Immunoblotting was performed on Survivin, a miR-708's direct target in Renal Cell Carcinoma, and on previously studied biomarkers including Hexokinase II, Beclin 1, and LC3. The expression patterns of the detected miRNAs were matched with microarrays results of the 50 paraffin-embedded tissue samples. Significant difference was found based on race (miR-34c-3p), sex (miR-193a-5p), location (miR-193a-5p & miR-34a), grade (miR-486-5p), and HPV status (miR-584). The survival analysis showed a significant difference based on age, location, and grade. The bioinformatics analysis supported our findings and showed consistent results with other miRNAs studies in the literature. The high expressions of miR-584 and miR-486-5p deserve further investigation.