Mass spectrometry-based cancer biomarker discovery

The aim of the projects in this thesis was to identify biomarkers for clear cell renal cell carcinomas (ccRCC) and head and neck/oral squamous cell carcinoma (HNOSCC), using quantitative or qualitative proteomics. Comparative analysis of cancerous and normal tissue homogenates, or secretome analysis of cancer cell cultures using liquid chromatography - mass spectrometry (LC-MS) and immunoassay techniques, allowed the identification of different types of biomarkers: diagnostic or prognostic, biofluid- or tissue-based. Chapter 1 of this thesis provides general information on cancer and cancer biomarker discovery. Chapter 2 gives a brief introduction to the techniques used in this work and theories behind them. Chapters 3 - 5 are papers that resulted from the cancer biomarker discovery research performed here, and Chapter 6 contains the conclusions, the author's comments and the final remarks. The papers on the identification of biomarkers for different diseases, to which the author of this thesis contributed, are listed in the Appendix

Nuclear heterogeneous nuclear ribonucleoprotein D is associated with poor prognosis and interactome analysis reveals its novel binding partners in oral cancer

Abstract Background Post-transcriptional regulation by heterogeneous ribonucleoproteins (hnRNPs) is an important regulatory paradigm in cancer development. Our proteomic analysis revealed hnRNPD overexpression in oral dysplasia as compared with normal mucosa; its role in oral carcinogenesis remains unknown. Here in we determined the hnRNPD associated protein networks and its clinical significance in oral squamous cell carcinoma (OSCC). Methods Immunoprecipitation (IP) followed by tandem mass spectrometry was used to identify the binding partners of hnRNPD in oral cancer cell lines. Ingenuity pathway analysis (IPA) was carried out to unravel the protein interaction networks associated with hnRNPD and key interactions were confirmed by co-IP-western blotting. hnRNPD expression was analyzed in 183 OSCCs, 44 oral dysplasia and 106 normal tissues using immunohistochemistry (IHC) and correlated with clinico-pathological parameters and follow up data over a period of 91 months. Kaplan–Meier survival and Cox-multivariate-regression analyses were used to evaluate the prognostic significance of hnRNPD in OSCC. Results We identified 345 binding partners of hnRNPD in oral cancer cells. IPA unraveled novel protein–protein interaction networks associated with hnRNPD and suggested its involvement in multiple cellular processes: DNA repair, replication, chromatin remodeling, cellular proliferation, RNA splicing and stability, thereby directing the fate of oral cancer cells. Protein–protein interactions of hnRNPD with 14-3-3ζ, hnRNPK and S100A9 were confirmed using co-IP-western blotting. IHC analysis showed significant overexpression of nuclear hnRNPD in oral dysplasia [p = 0.001, Odds ratio (OR) = 5.1, 95 % CI = 2.1–11.1) and OSCCs (p = 0.001, OR = 8.1, 95 % CI = 4.5–14.4) in comparison with normal mucosa. OSCC patients showing nuclear hnRNPD overexpression had significantly reduced recurrence free survival [p = 0.026, Hazard ratio = 1.95, 95 % CI = 1.0–3.5] by Kaplan–Meier survival and Cox-multivariate-regression analyses and has potential to define a high-risk subgroup among OSCC patients with nodal negative disease. Conclusions Our findings suggest novel functions of hnRNPD in cellular proliferation and survival, besides RNA splicing and stability in oral cancer. Association of nuclear hnRNPD with poor prognosis in OSCC patients taken together with its associated protein networks in oral cancer warrant future studies designed to explore its potential as a plausible novel target for molecular therapeutics

Lactate Dehydrogenase A is a potential prognostic marker in clear cell renal cell carcinoma

Abstract Background Over 90% of cancer-related deaths in clear cell renal cell carcinoma (RCC) are caused by tumor relapse and metastasis. Thus, there is an urgent need for new molecular markers that can potentiate the efficacy of the current clinical-based models of prognosis assessment. The objective of this study is to evaluate the potential significance of lactate dehydrogenase A (LDHA), assessed by immunohistochemical staining, as a prognostic marker in clear cell renal cell carcinoma in relation to clinicopathological features and clinical outcome. Methods We assessed the expression of LDHA at the protein level, by immunohistochemistry, and correlated its expression with multiple clinicopathological features including tumor size, clinical stage, histological grade, disease-free and overall survival in 385 patients with primary clear cell renal cell carcinoma. We also correlated the LDHA expression with overall survival, at mRNA level, in an independent data set of 170 clear cell renal cell carcinoma cases from The Cancer Genome Atlas databases. Cox proportional hazards models adjusted for the potential clinicopathological factors were used to test for associations between the LDHA expression and both disease-free survival and overall survival. Results There is statistically significant positive correlation between LDHA level of expression and tumor size, clinical stage and histological grade. Moreover, LDHA expression shows significantly inverse correlation with both disease-free survival and overall survival in patients with clear cell renal cell carcinoma. Our results are validated by examining LDHA expression, at the mRNA level, in the independent data set of clear cell renal cell carcinoma cases from The Cancer Genome Atlas databases which also shows that higher lactate dehydrogenase A expression is associated with significantly shorter overall survival. Conclusion Our results indicate that LDHA up-regulation can be a predictor of poor prognosis in clear cell renal cell carcinoma. Thus, it represents a potential prognostic biomarker that can boost the accuracy of other prognostic models in patients with clear cell renal cell carcinoma

Additional file 2: of Nuclear heterogeneous nuclear ribonucleoprotein D is associated with poor prognosis and interactome analysis reveals its novel binding partners in oral cancer

Figure S2. Spearman rank correlation co-efficient test:Graphs represent a positive correlation between (A) hnRNPD expression level and OSCC tumor size, (B) OSCC tumor stage

Additional file 1: of Nuclear heterogeneous nuclear ribonucleoprotein D is associated with poor prognosis and interactome analysis reveals its novel binding partners in oral cancer

Figure S1. (A) SDS-PAGE analysis of hnRNPD immunoprecipitates from OSCC samples. Lanes shows separation of IP-hnRNPD from oral cancer cells SCC4 and MDA1986, and beads only; PMW is protein molecular weight marker. (B) Binding partners of hnRNPD classified on basis of cellular function. Pie-chart demonstrating the distribution of binding partners of hnRNPD identified using IP-LC–MS/MS on the basis of their cellular functions

Identification of Differentially Regulated Secretome Components During Skeletal Myogenesis*

Myogenesis is a well-characterized program of cellular differentiation that is exquisitely sensitive to the extracellular milieu. Systematic characterization of the myogenic secretome (i.e. the ensemble of secreted proteins) is, therefore, warranted for the identification of novel secretome components that regulate both the pluripotency of these progenitor mesenchymal cells, and also their commitment and passage through the differentiation program. Previously, we have successfully identified 26 secreted proteins in the mouse skeletal muscle cell line C2C12 (1). In an effort to attain a more comprehensive picture of the regulation of myogenesis by its extracellular milieu, quantitative profiling employing stable isotope labeling by amino acids in cell culture was implemented in conjunction with two parallel high throughput online reverse phase liquid chromatography-tandem mass spectrometry systems