• 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.