UCLA

Tumor cells can adopt alternative metabolic pathways during oncogenesis. This is an event characterized by an enhanced utilization of glucose for rapid synthesis of macromolecules such as nucleotides, lipids and proteins. This phenomenon was also known as the `Warburg effect', distinguished by a shift from oxidative phosphorylation to increased aerobic glycolysis in many types of cancer cells. Increased aerobic glycolysis was also indicated with enhanced lactate production and glutamine consumption, and has been suggested to confer growth advantage for proliferating cells during oncogenic transformation. Development of a tracer-based methodology to determine de novo protein synthesis by tracing metabolic pathways from nutrient utilization may certainly enhance current understanding of nutrient gene interaction in cancer cells. We hypothesized that the metabolic phenotype of cancer cells as characterized by nutrient utilization for protein synthesis is significantly altered during oncogenesis, and 13C stable isotope tracers may incorporate 13C into non-essential amino acids of protein peptides during de novo protein synthesis to reflect the underlying mechanisms in cancer cell metabolism.

OBJECTIVE: The aims of this study are: 1) To examine the effects of using siRNA to knockdown metabolic enzymes, transketolase (TKT) and adenylate kinase 2 (AK2) of glycolysis and pentose phosphate pathway (PPP) in oral cancer cells. 2) To develop a 13C stable isotope tracer-based approach for studying de novo protein synthesis in oral squamous cell carcinoma. 3) To develop a 13C stable isotope tracer-based approach to investigate 13C labeled proteins in TKT siRNA and control scrambled siRNA transfected oral cancer cells. 4) To develop a 13C stable isotope tracer-based approach to determine differential 13C labeled proteins in normal human pancreatic duct epithelial (HPDE) cells and human pancreatic ductal adenocarcinoma (PDAC) cells via in-vitro and in-vivo.

METHODS: Knockdown of TKT or AK2 was achieved by transfecting oral cancer cells with small interfering RNA for TKT or AK2. 13C labeling of proteins was achieved by infusing cells in-vitro and in-vivo with [U -13C6]-glucose. Protein samples were analyzed using proteomic approach based on liquid chromatography mass spectrometry (LC-MS/MS). Protein identification was performed by MASCOT database search. Protein clustering and functional annotation was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) software.

RESULTS: Knockdown of TKT or AK2 in oral cancer cells inhibited cell proliferation and displayed enhanced glucose, glutamine consumption, and lactate production. Knockdown of TKT in oral cancer cells displayed a decrease in 13C labeled protein peptides for TKT compared to control. Overall, knockdown of TKT displayed lower frequency of 13C labeled protein peptides compared to control, but comparison of the same 13C labeled peptide displayed no significant difference in peptide mass isotopomer distribution. Enzymes involved in glycolysis including glyceraldehyde 3 phosphates, enolase, and phosphoglycerate mutase also displayed enhanced 13C labeling compared to control. 13C labeled proteins among HPDE cells, PDAC cells, and PDAC mouse tumor tissue displayed differential labeling frequency primarily in vimentin, β-tubulin and cytoskeletal keratin proteins. Peptide mass isotopomer distribution displayed mass shift in 13C labeled peptides compared to unlabeled (natural 12C) peptide mass isotopomer distribution. Overall, 13C enrichment of proteins achieved greater yield in cell lines compared to mouse tumor tissue. Structural proteins and metabolic enzymes involved in glycolysis, cell cycle, and apoptosis signaling pathways were predominately found labeled.

CONCLUSION: This study, we have developed a novel methodology based on 13C labeling of de novo synthesized proteins to study cancer cell metabolism. 13C labeling of cancer cells with truncated PPP due to knock down of TKT suggested robust metabolic adaptation for glucose utilization. Differential expression of 13C labeled proteins between HPDE cells and PDAC cells, suggested alteration in nutrient gene interaction and metabolic pathways for de novo synthesis during oncogenesis. Our findings demonstrated that 13C stable isotope may serve as a valuable tracer-based tool for studying metabolic regulation of de novo protein synthesis in cancer cells.