Alterations in redox and energy metabolism in Ras-transformed cells: Mechanisms and therapeutic implications

Increasing attention has been given to the connection between metabolism and cancer. Under aerobic conditions, normal cells predominantly use oxidative phosphorylation for ATP generation. In contrast, increase of glycolytic activity has been observed in various tumor cells, which is known as Warburg effect. Cancer cells, compared to normal cells, produce high levels of Reactive Oxygen Species (ROS) and hence are constantly under oxidative stress. Increase of oxidative stress and glycolytic activity in cancer cells represent major biochemical alterations associated with malignant transformation. Despite prevalent upregulation of ROS production and glycolytic activity observed in various cancer cells, underlying mechanisms still remain to be defined. Oncogenic signals including Ras has been linked to regulation of energy metabolism and ROS production. Current study was initiated to investigate the mechanism by which Ras oncogenic signal regulates cellular metabolism and redox status. A doxycycline inducible gene expression system with oncogenic K-ras transfection was constructed to assess the role played by Ras activation in any given studied parameters. Data obtained here reveals that K-ras activation directly caused mitochondrial dysfunction and ROS generation, which appeared to be mechanistically associated with translocation of K-ras to mitochondria and the opening of the mitochondrial permeability transition pore. K-ras induced mitochondrial dysfunction led to upregulation of glycolysis and constitutive activation of ROS-generating NAD(P)H Oxidase (NOX). Increased oxidative stress, upregulation of glycolytic activity, and constitutive activated NOX were also observed in the pancreatic K-ras transformed cancer cells compared to their normal counterparts. Compared to non-transformed cells, the pancreatic K-ras transformed cancer cells with activated NOX exhibited higher sensitivity to capsaicin, a natural compound that appeared to target NOX and cause preferential accumulation of oxidative stress in K-ras transformed cells. Taken together, these findings shed new light on the role played by Ras in the road to cancer in the context of oxidative stress and metabolic alteration. The mechanistic relationship between K-ras oncogenic signals and metabolic alteration in cancer will help to identify potential molecular targets such as NAD(P)H Oxidase and glycolytic pathway for therapeutic intervention of cancer development. ^

Alterations in cell adhesion and migration are transcriptionally regulated by the tumor suppressor, TP53

The p53 transcription factor is a tumor suppressor and a master regulator of apoptosis and the cell cycle in response to cell stress. In some advanced tumors, such as prostate cancers, the loss of p53 correlates with an increase in the occurrence of metastases. In addition, several groups have suggested that p53 status correlates with changes in cell migration and cell morphology associated with a migratory phenotype. Others have identified several genes with roles in cell migration that are directly transcriptionally regulated by p53. Even so, modulation of cell migration is not widely recognized as a p53 stress response. ^ In an effort to identify novel p53 target genes and expand our knowledge of the p53 transcriptional response, we performed Affymetrix gene expression analysis in p53-null PC3 prostate cancer cells following infection with a control virus or adenoviral construct expressing wild-type p53. Over 300 genes that had not been previously recognized as p53 target genes were identified. Of these genes, 224 were upregulated and 111 were downregulated (p<0.05). Functional over-representation analysis identified cell migration as a significantly over-represented biological function of p53. Further analysis identified two genes that are critical for the control of cell migration as potential p53 targets. One, hyaluronan mediated motility receptor (HMMR), has recently been shown to be a p53 target important for regulation of the cell cycle. Here, we show that HMMR is downregulated by p53 in several cell lines, and HMMR's regulation is dependent on the presence of the cdk inhibitor, p21, and histone deactelyase activity. The other gene, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), itself a tumor suppressor, is shown here, for the first time, as a p53 direct target by ChIP analysis. We next determined the effect of p53 activation on cell migration and found that p53 significantly slows the rate of cell migration in Boyden chamber migration assays and digital videomicroscopy wound healing studies. Further, our studies established the specific roles of CEACAM1 and HMMR in cell migration and determine that loss of CEACAM1 and overexpression of HMMR independently contribute to increased cell migration. Taken together, these studies provide a direct mechanistic link between p53 to the regulatory control of specific target genes that mediate cell adhesion and migration. ^

Alterations in the ras oncogene and the p53 tumor suppressor gene in ultraviolet radiation-induced skin carcinogenesis

A rapid increase of the ultraviolet radiation (UVR)-related skin cancer incidence has attracted more and more public attention during the last few decades. Prevention and treatment of UVR-related skin cancer has become an important public health issue in the United States. Recent studies indicate that mutations in ras and/or p53 genes may be involved in UVR-induced skin tumor development but the precise molecular mechanism remains unclear. In this study, alterations of H-ras and p53 genes were investigated in different stages of carcinogenesis in a chronic UVR (solar simulator) exposure-induced Sencar mouse skin carcinogenesis model in order to clarify the role of the alterations of these genes during the skin carcinogenesis process and to further understand the mechanisms by which UVR causes skin cancer.^ Positive ras-p21 staining in cell membranes and cytosol were detected in 18/33 (55%) of squamous cell carcinomas (SCCs), but were not detected in UV-exposed skin, papillomas, or spindle cell tumors (SCTs). Positive staining of the malignant progression marker K13 was found in 17/33 (52%) of SCCs only. A significant positive correlation was observed between the K13 and the ras-p21 expression. Polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) analysis and gene sequencing analysis revealed three point mutations, one (codon 56) in UV-exposed non-tumor bearing skin and the other two (codons 21 and 13) in SCCs. No UV-specific mutation patterns were found.^ Positive p53 nuclear staining was found in 10/37 (27%) of SCCs and 12/24 (50%) of SCTs, but was not detected in normal skin or papillomas. PCR-based SSCP and sequencing analysis revealed eight point mutations in exons 5 and 6 (four in SCTs, two in SCCs, and two in UV-exposed skin) including six C-T or C-A transitions. Four of the mutations occurred at a dipyrimidine (CC) sequence. The pattern of the mutations indicated that the mutagenic lesions were induced by UVR.^ These results indicate that overexpression of ras-p21 in conjunction with aberrant expression of K13 occurred frequently in UVR-induced SCCs in Sencar mouse skin. The point mutation in the H-ras gene appeared to be a rare event in UVR skin carcinogenesis and may not be responsible for overexpression of ras-p21. UVR-induced P53 gene alteration is a frequent event in UVR-induced SCCs and later stage SCT tumors in Sencar mice skin, suggesting the p53 gene mutation plays an important role in skin tumor malignant progression. ^