Epigenetic+Regulation+of+Tumor+Suppressor+Genes

By Sonya Chalaka and Lauren Ducote

@Glossary of Terms

=Purpose:= The purpose of our project is to examine the relationship between gene expression, exercise and cancer survival. toc =Introduction:= Our project explores the correlation between exercise and breast cancer survival by examining the results of a longitudinal study conducted (1998-2007) at the University of Turin in conjunction with Peking University, Yale and New York State University. The results suggest that physical activity significantly improves breast cancer survival by the activation of a tumor suppressor gene, L3MBTL1. The activation of L3MBTL1 is induced by epigenetic regulation. It is suggested that exercise promotes survival by preventing DNA methylation of L3MBTL1. This significant finding would suggest a correlation exists between exercise and gene regulation. Since gene regulation is central to the determination of cancer outcome, perhaps exercise is a determining factor. =Context:= LMBTL genes are a family of genes that regulate what genes are turned off. Specifically, LMBTL genes belong to a group of proteins called the polycomb group. Polycomb group proteins alter chromatin resulting in epigenetic silencing of genes and regulating transcription. Ultimately, transcription is what determines what genes are turned on and what genes are turned off. This is a means to modify gene expression without alteration to the genetic code. The alteration of chromatin is accomplished by methylation. Methylation is the process used for epigenetic gene regulation where a methyl group (–CH3) is usually added to a deoxycytidine 5´of a deoxyguanosine (5´–CpG–3´ or CpG). Proteins called the methylCpG binding domain proteins (MBD) bind to methylated deoxycytidine residues. These proteins enlist more chromatin remodeling proteins that may alter histones. This forms a compact chromatin that is inactive. Inactive chromatin is referred to as heterochromatin. Heterochromatin determines what genes will be expressed. Our project examined the L3MBTL1 gene's validity[?] as a tumor suppressor that[?] is activated by exercise. L3MBTL1 is located on the long arm of chromosome 20. Chromosome 20 is involved in cell cycle regulation. The disruption of normal cell growth and division is seen in cancer. The function of L3MBTL1 is to alter histone proteins. The alteration of histone proteins limits DNA's access to transcription factors and thereby preventing transcription. In order for L3MBTL1 to function, methylation levels must be minimal. L3MBTL1 target three histone proteins by binding to lysine residues on the surface of the three histones resulting in inactivation of chromatin. L3MBTL1 can inactivate chromatin in two ways. First, the direct binding of L3MBTL1 condenses chromatin and limits DNA mobility. Second, this binding prevents other proteins from binding. This greatly limits the transcription of DNA and chromatin expression. L3MBTL1 function is paramount to gene regulation. =Research:=

Methods:
The study was separated into two clinical trials. The first was a randomized clinical trial conducted at Yale University. The purpose of trial one was to examine the relationship between exercise, growth factors and sex hormones. The eligibility criteria for this first trial were physically inactive postmenopausal women diagnosed with stage 0-IIIA breast cancer. Additionally, these participants were required to have completed adjuvant treatment 6 months before enrollment into this study. The researchers randomly allocated the 75 participants into a control arm and an investigational arm. The investigational arm was composed of 37 women who were instructed to exercise by moderate-intensity brisk walking on a treadmill for 150 minutes every week for a 6-month duration. While, the control arm was instructed to maintain their standard routine. Upon enrollment into the study each woman provided a blood sample and at the end of 6 months another blood sample was collected. Moreover, a detailed account of physical activity was provided at both of these specific time points. The second random clinical trial was a follow up of the first study. It randomly selected six participants from both the control and investigational arms from study one to participate. This study was conducted at the Universtiy of Turin in Italy. The purpose of this study was to verify molecular linkage and the determinate causes of cancer. Additionally, 12 participants from study one were randomly selected from 348 patients. The 348 patients were required to have undergone surgery for breast cancer before enrollment. These new participants donated their surgically removed tumor samples. These tumor samples were tested to confirm tumor cell content and frozen in liquid nitrogen at -80 degree Celsius. The original study of 348 followed 302 participants for 10 years and randomly selected 204 frozen tumor samples for further analysis. Meanwhile, the blood samples from the first clinical trial were analyzed for methylation levels. First genomic DNA was extracted from each blood sample. Next, the [|Infinium Human Methylation 27 Bead Chip] analyzed Cytosine-Guanine regions to determine degree of methylation. The methylation levels at each CpG site were estimated by comparing the signal intensities between methylated and non-methylated regions. Then a beta value was calculated by taking the difference between the Beta value at 6 months and the Beta value at baseline to determine methylation changes before and after exercise treatment [what is beta value]. This process was repeated with the frozen tumor samples. The researchers used this data to determine the methylation changes before and after the exercise treatment.

Results:
The results provide evidence that exercise after the diagnosis of breast cancer may increase survival outcome. After comparison of the methylation profiles before and after exercise 43 genes illustrated significant differences in methylation levels. The researchers proceeded to compare these results to the genes expressed in the 204 frozen tumor samples. This new data was used to select 6 genes from 43 genes[what happened to the other 37 genes?]. These 6 genes displayed a significant correlation with survival. Next, the researchers proceeded to select L3MBTL1 because it illustrates the pattern of decreased methylation after exercise. Moreover, L3MBTL1 is being investigated as a possible tumor suppressor gene. The other five genes did not have the characteristics the researchers believed would demonstrate a relationship between exercise and cancer survival outcome. The P values are strong for all 6 genes suggesting the data is statistically signficant. Finally, an examination of the relationship between methylation and expression of L3MBTL1 and breast cancer survival revealed that higher expression of L3MBTL1 is correlated with better survival outcome and improved disease free survival (Fig 1,2).

Discussion:
According to our study a decrease in methylation results in an increase in gene expression. However, this paradigm is not always accurate. While, a strong correlation between a decrease in methylation and gene silencing exist because 97% of the time this occurs, still instances where this is not the case occur. This discrepancy is a matter of what region of the gene is methylated. Our study attempted to circumvent this issue by examining the promotor region of the 6 genes of interest. However, one wonders what the researchers would have discovered in the 43 genes. Moreover, questions arise as to the the methodology of the elimination criteria and L3MBTL1 being a significant tumor suppressor because this is the first study to suggest this finding.

  The paradigm [?] hypothesis that exercise decreases methylation is used to narrow down the 6 genes being examined to three possibilities. Arguably, this is an over simplification.[| "Epigenetic regulation on gene expression induced by physical exercise"] suggest that exercise may result in hypermethylation of some genes and hypomethylation of other genes. Hypermethylation induced by exercise would be evident in oncogenes.The result of hypermethylation would be a decreased expression of the oncogene which would improve survival outcome. While, hypomethylation would lead to an increase in expression and be evident in a tumor suppressor gene. This over simplification dismisses the idea that exercise may result in silencing of oncogenes. In reviewing the results of the study, it becomes apparent why the researchers dismissed the other genes and examined L3MBTL1 exclusively. The other five genes have no substantial correlation to anti-cancer activity. Notably, of the 43 genes anaylzed CXCL10 illustrates a correlation with anti-tumor activity when hypermethylated. This gene illustrated the greatest increase in methylation by 5%. The elimination of this gene may be motivated by the failure of the gene to reappear significantly in the 204 frozen tissue samples.

 A more valid reason for the elimination of genes that illustrated hypermethylation are the significant studies showing an association between hypomethylation and exercise. A recent preliminary study entitled,[| "Physical Activity and Differenital Methylation of Breast Cancer Genes Assayed from Saliva: A Preliminary Investigation"] ,suggested that physical fitness level is associated with lower levels of DNA methylation. This study indicates that physical exercise may have a greater affect on lowering methylation than increasing methylation. Therefore, the results of hypermethylation are not as strongly associated with exercise. Moreover, a previous study entitled, [|"Physical Exercise as an Epigenetic Modulator: Eustress, The Positive Stress as an Effector of Gene expression"], discovered a relationship between TP53 and exercise[vague]. TP53 is a well documented tumor suppressor gene and exercise appears to increase the expression of TP53. This pattern is consistent with the results of our study.

 The second assertion is L3MBTL1 is a tumor suppressor. This concept is critical to the validity of the study [why?]. A robust argument may be formulated by the comparison of a paralogue genes and patterns of spectrum mutations. L3MBTL1 may be compared to its paralogue L3MBTL4 to determine the validity of L3MBTL1 as a tumor suppressor gene. A paralogue is a duplicate gene that has evolved a new function.L3MBTL1 and L3MBTL4 both belong to the L3MBTL family and are polycomb group proteins which can alter chromatin and epigenetically silence genes. L3MBTL1 has evolved the ability to repress p53 and Rb. While, L3MBTL4 represses transcription.

 A relationship between breast cancer survival and the role of L3MBTL1 and L3MBTL4 expression may be inferred. In [|"Loss, mutation and deregulation of L3MBTL4 in breast cancers"], researchers determined that L3MBTL4 loss occurs in the more aggressive breast cancer phenotypes. There is a 9% decrease in 5 year metastasis free survival and a 5% decrease in overall specific survival. According to our study the higher expression of L3MBTL1 is associated with a less aggressive cancer phenotypes and better survival outcome.These studies convey the presence and absence of L3MBTL family is associated with breast cancer survival outcomes. The absence of L3MBTL4 appears to negatively affect survival outcomes and the presence of L3MBTL1 appears to positively affect survival outcomes. It may be inferred that L3MBTL family is a determinate factor in breast cancer survival outcome. L3MBTL1 expression is critical to breast cancer survival outcome suggesting it is a tumor suppressor.

 Further analysis by histograms from [|Cosmic Database] supports this conclusion because the pattern of the spectrum mutations resembles a tumor suppressor. Tumor suppressors and oncogenes display different patterns of mutations. Therefore, a comparison of L3MBTL1 to accepted models will allow for an inference on its status. Consequently, a comparison between [|KRAS], [|TP53] and [|L3MBTL1] reveals that L3MBTL1 resembles TP53 pattern of mutations. L3MBTL1 fits the accepted paradigm of a tumor suppressor gene with sporadic mutations. Sporadic mutations are necessary to promote a loss of function. Moreover, the function is suggestive of a tumor suppressor known as L3MBTL1 function which is used to regulate chromatin. Chromatin is essential for genetic regulation, DNA replication, prevention of DNA damage and is related to mitosis. The loss of this function would result in uncontrollable cell growth and cell proliferation.

 For a more elaborate comparison between L3MBTL1 and L3MBTL4 click [|here].  For a more elaborate comparison between mutation spectrum click [|here]. =Ramifications:= In considering the value of our study it is imperative to consider application. One valuable application of exercise for breast cancer patients could be as a complementary therapy. The incidence of breast cancer has only been increasing and many regions of the developing world are unable to meet the needs of patients because of the cost of care. An estimated 1 million women were diagnosed in 2008 alone. This map illustrates the global incidence of breast cancer.

  Exercise may be a cost effective means to address this global concern. An examination of the differences in the cost of standard care are imperative to the issue of pragmatic interventions. In the article, [|"Costs and Health Effects of Breast Cancer Interventions in Epidemiological Different Regions of Africa, North America, and Asia"] it discusses the disparities in terms of cost of treatment and examines the [|DALYs]. The table below examines the disparities and allows for several key conclusions to be formulated.  <span style="font-family: Arial,Helvetica,sans-serif; line-height: 1.5;"> First, the patients in North America received treatment at earlier stages than patients in Africa. An examination of the amount spent on screening explains this trend because North America spends significantly more than Africa. Second, the disparity in treatment and economic resources is evident in the cost spent per patient for treatment of stage I breast cancer. North America spends approximately 20 times more than Asia and 13 times more than Africa. Third, the CER (cost effectiveness ratio) implies that the most cost effective treatment is during the earliest stages of the disease. <span style="font-family: Arial,Helvetica,sans-serif; line-height: 1.5;"> It was noted in the study that to adequately care for a nation the size of the "Netheralnds" $ 50 million is required for 95% of geographic coverage. In addressing the needs of developing countries their resources are best directed for treatment at the early stage disease and therefore generating less expensive means to treat. This implies that any intervention with the capability to prevent the progression of the disease in a cost effective manner will alleviate the treatment disparities and increase survival outcome is optimal for addressing a global concern. Consequently, exercise would be an effective complementary therapy because it may be implemented with minimal cost. This would be ideal and may mitigate some of the disparity in treatment between the developing world and the developed world. <span style="font-family: Arial,Helvetica,sans-serif; line-height: 1.5;"> For more information please click [|here]. =Conclusion:= <span style="font-family: Arial,Helvetica,sans-serif; line-height: 1.5;">The evidence would suggest exercise is capable of increasing survival outcome by inducing a tumor suppressor gene. The study uses a valid means to eliminate genes in order to examine a single gene in greater detail. This gene appears to fit the paradigm of a tumor suppressor gene. However, this evidence is highly suggestive still, it is not definitive. An alternative explanation is individuals with increased expression of L3MBTL1 may have had less advanced breast cancer meaning better prognosis. Moreover, these individuals may have been recovering by the end of the 6 months resulting in less silencing of tumor suppressor genes. The reduction in hypermethylation would mean a greater expression of all tumor suppressor genes and not just L3MBTL1. Despite these reservations, the mounting evidence that exercise is capable of increasing survival outcome by epigenetic regulation is considerable.Further analysis of L3MBTL1 relationship with exercise and breast cancer is required before major conclusions are formulated. Perhaps, future research may include the cost effectiveness of exercise as a complementary therapy.