Introduction

The purpose of this study is to identify target proteins that may play important functional roles in oral cancer stem-like cells (CSCs) using mass spectrometry-based quantitative proteomics.

Methods

Sphere-formation assays were performed on highly invasive UM1 and lowly invasive UM2 oral cancer cell lines, which were derived from the same tongue squamous cell carcinoma, to enrich CSCs. Quantitative proteomic analysis of CSC-like and non-CSC UM1 cells was carried out using tandem mass tagging and two-dimensional liquid chromatography with Orbitrap mass spectrometry.

Results

CSC-like cancer cells were found to be present in the highly invasive UM1 cell line but absent in the lowly invasive UM2 cell line. Stem cell markers SOX2, OCT4, SOX9 and CD44 were up-regulated, whereas HIF-1 alpha and PGK-1 were down-regulated in CSC-like UM1 cells versus non-CSC UM1 cells. Quantitative proteomic analysis indicated that many proteins in cell cycle, metabolism, G protein signal transduction, translational elongation, development, and RNA splicing pathways were differentially expressed between the two cell phenotypes. Both CREB-1-binding protein (CBP) and phosphorylated CREB-1 were found to be significantly over-expressed in CSC-like UM1 cells.

Conclusions

CSC-like cells can be enriched from the highly invasive UM1 oral cancer cell line but not from the lowly invasive UM2 oral cancer cell line. There are significant proteomic alterations between CSC-like and non-CSC UM1 cells. In particular, CBP and phosphorylated CREB-1 were significantly up-regulated in CSC-like UM1 cells versus non-CSC UM1 cells, suggesting that the CREB pathway is activated in the CSC-like cells.

Background

Cancer cells may undergo metabolic adaptations that support their growth as well as drug resistance properties. The purpose of this study is to test if oral cancer cells can overcome the metabolic defects introduced by using small interfering RNA (siRNA) to knock down their expression of important metabolic enzymes.

Methods

UM1 and UM2 oral cancer cells were transfected with siRNA to transketolase (TKT) or siRNA to adenylate kinase (AK2), and Western blotting was used to confirm the knockdown. Cellular uptake of glucose and glutamine and production of lactate were compared between the cancer cells with either TKT or AK2 knockdown and those transfected with control siRNA. Statistical analysis was performed with student T-test.

Results

Despite the defect in the pentose phosphate pathway caused by siRNA knockdown of TKT, the survived UM1 or UM2 cells utilized more glucose and glutamine and secreted a significantly higher amount of lactate than the cells transferred with control siRNA. We also demonstrated that siRNA knockdown of AK2 constrained the proliferation of UM1 and UM2 cells but similarly led to an increased uptake of glucose/glutamine and production of lactate by the UM1 or UM2 cells survived from siRNA silencing of AK2.

Conclusions

Our results indicate that the metabolic defects introduced by siRNA silencing of metabolic enzymes TKT or AK2 may be compensated by alternative feedback metabolic mechanisms, suggesting that cancer cells may overcome single defective pathways through secondary metabolic network adaptations. The highly robust nature of oral cancer cell metabolism implies that a systematic medical approach targeting multiple metabolic pathways may be needed to accomplish the continued improvement of cancer treatment.

XRCC4-like factor (XLF), also known as Cernunnos, is a protein encoded by the human NHEJ1 gene and an important repair factor for DNA double-strand breaks. In this study, we have found that XLF is over-expressed in HPV(+) versus HPV(-) head and neck squamous cell carcinoma (HNSCC) and significantly down-regulated in the HNSCC cell lines expressing high level of mutant p53 protein versus those cell lines harboring wild-type TP53 gene with low p53 protein expression. We have also demonstrated that Werner syndrome protein (WRN), a member of the NHEJ repair pathway, binds to both mutant p53 protein and NHEJ1 gene promoter, and siRNA knockdown of WRN leads to the inhibition of XLF expression in the HNSCC cells. Collectively, these findings suggest that WRN and p53 are involved in the regulation of XLF expression and the activity of WRN might be affected by mutant p53 protein in the HNSCC cells with aberrant TP53 gene mutations, due to the interaction of mutant p53 with WRN. As a result, the expression of XLF in these cancer cells is significantly suppressed. Our study also suggests that XLF is over-expressed in HPV(+) HNSCC with low expression of wild type p53, and might serve as a potential biomarker for HPV(+) HNSCC. Further studies are warranted to investigate the mechanisms underlying the interactive role of WRN and XLF in NHEJ repair pathway.