REGγ-proteasome system represents an emerging pathway recently recognized to be involved in cancer development. This study provides further links between REGγ and multiple cancer related pathways by a combination of bioinfomatic analysis and molecular biological approach.
To our knowledge, this is the first computational study so far in REGγ association with multiple cancers. We are also the first to demonstrate high expression of REGγ in lung and liver cancers despite that overexpression of REGγ in thyroid and colon cancer were reported [6
]. Tissue array analyses of four different human cancers, including lung, colon, thyroid, and liver cancers, revealed significant increase of REGγ protein in over 50% of these cancer samples. Bioinformatic analysis of human microarray gene expression profiles indicates that REGγ gene expression is also increased in most of these human cancers, providing new evidence that REGγ-proteasome pathway may be involved in the development of multiple cancers.
Computational analysis of datasets from thyroid cancer with thyroid non-cancer disease and liver cancer with HCC clinical stage information indicated a potential correlation of gradual increase of REGγ level with cancer stages (Figure ). Although the sample size and numbers are relatively small, the results suggest a potential of REGγ as a prognostic cancer marker and hinted some molecular mechanisms linking REGγ to development of cancers toward later stages or malignancy.
Our meta-analysis disclosed significant correlation between REGγ and many genes in cancer and cancer related pathways from ingenuity analysis, including colorectal cancer, lung carcinoma, sarcoma, lymphoma, tumorigenesis, cell division and apoptosis related pathways etc. Importantly, genes downstream of the previously identified REGγ regulated proteins [24
], p53, was found highly correlated with REGγ expression. Despite that p53 mutation in different cancer may complicate the correlation status of its downstream target genes with REGγ, the overall high correlation values strongly support the previous finding that REGγ-mediated regulation of p53 may play an important role in cancer development. Annotation analysis indicated significant correlation of REGγ with many different proteasome components, suggesting that REGγ may be elevated and function together with other proteasome complexes. Recent studies have indicated important roles of the ubiquitin-mediated protein degradation pathway in cancer and application of proteasome inhibitors as a promising anti-cancer therapeutic approach [28
]. An additional interesting finding is that numerous REGγ-correlated genes are involved in metabolism, particularly in energy metabolism (Additional file 8
: Table S7). The link between cell metabolism and cancers has been well documented [29
]. The result concurs a recent notion that cancer cell metabolisms are controlled by oncogenes and tumor suppressor genes [30
The mathematical approaches of bioinformatics used in this study are quite standard [18
]. To minimize the chance of acquiring false-positive results and ensure that most of the strong candidate genes are selected, we set rigorous criteria for all studies performed. The significance of our computational analysis has been underlined by laboratory validation experiments in 4 pairs of cancer cell lines in which differential expression of REGγ was created in the same background to facilitate correlation studies. Results from quantitative analysis of selected genes were largely consistent with predicted correlations, suggesting powerful combination of bioinformatics and molecular biological studies in disclosing potentially novel functions of REGγ-proteasome in cancer progression. It is not unlikely that REGγ could serve as a cancer marker, particularly for cancers with aggressive behavior.
Given that REGγ mainly functions as a proteasome activator to induce protein degradation, the biological links between REGγ and its correlated genes may reflect a result of direct or indirect regulation on transcription. Ingenuity analysis of validated gene network led our attention to the correlation between REGγ and Myc gene. Previous research documented overexpression of both genes in colorectal cancers [31
]. Coincident with the largest amount of datasets and highest REGγ differential expression from colon cancers, the positive correlation between REGγ and Myc was validated specifically in HTC116-shN and -shR cells. Since Myc functions as a transcription factor, we searched REGγ promoter and found numerous Myc binding sites within 1.5 KB upstream REGγ transcriptional initiation site (data not shown). Yet we could not exclude the possibility that REGγ may target a negative regulator of Myc for degradation. Further experiments will be carried out to understand the molecular detail of these hypotheses. It is likely that elevated expression of Myc in certain cancer cells is one of the potential mechanisms contributing to higher expression of REGγ.