Epigenetic Regulation of Breast Cancer Stem Cells

Breast cancer arises as a result of abnormal breast cells forming at an uncontrolled rate. Death in this case of breast cancer is due to the ability of cancer cells to adapt so that it can have an effect on metastasis and recurrence of cancer that was previously thought to have been resolved. The results showed, there is a stem cell population in breast cancer cases which will cause breast cancer to become increasingly difficult to treat. Such cells are known as breast cancer stem cells. Breast cancer cells have the ability to differentiate and contribute greatly to the breast cancer program, as well as to resistance to therapy. Therefore, epigenetic regulation of breast cancer cells is important to study in order to overcome cancer so that it can overcome progression and resistance to cancer therapy being carried out. Epigenetic regulation that has been known in cancer cases includes DNA methylation, histone acetylation, histone methylation and epigenetic regulation by miRNA. DNA methylation is the addition of a methyl group to the nitrogen base of DNA cytosine which will force the DNA transcription process. Acetylation of the addition of an acetyl group at the end of the histone causes reduced chromatin condensation so that it will activate the transcription process. Methylation histones will also suppress transcription so that genes cannot be expressed. In addition, there is also a small RNA molecule known as miRNA which can bind to the transcribed mRNA. This binding will cause the mRNA to degrade or inhibit its translation.


Introduction
Cancer is a disease that has become a common concern for people in Indonesia and in the world. Every year, 12 million people worldwide suffer from cancer and 7.6 million of them die from cancer 1

. The World
Helath Organization (WHO) states that if adequate action is not taken, by 2030 it is estimated that 26 million people will suffer from cancer and 17 million of them will die from cancer 2 . This incident will happen more rapidly in poor and developing countries. Indonesia. 3 Breast cancer is a neoplasm that originates from the parenchyma. Breast cancer arises as a result of abnormal breast cells forming at an uncontrolled rate. This is due to mutations in the genes contained in the breast so that uncontrolled cell proliferation can occur.
Breast cancer can spread to other organs such as the lungs, liver and brain through blood vessels. Many deaths in breast cancer cases are experienced because of the ability of cancer cells to adapt so that they can have an effect on metastasis.
The study of epigenetic mechanisms in cancer such as DNA methylation and histone modification revealed a number of events that contribute to cancer, especially those related to the stabilization of certain gene expression so that it will be closely related in the

Biomedical Journal of Indonesia
Journal Homepage: https://www.jurnalkedokteranunsri.id/index.php/BJI/index pathway of transforming normal cells into cancer cells.
Therefore, it is important to know the epigenetic regulation of cancer cells in order to find better cancer management techniques.
In addition, several studies have shown that there are stem cell populations in breast cancer cases that will make breast cancer more difficult to treat4. Such cells are known as breast cancer stem cells. Breast cancer stem cells have the ability to differentiate and contribute substantially to breast cancer progression, as well as to therapeutic resistance5. This paper will also briefly discuss the hypothesized epigenetic regulation that may occur in breast cancer stem cells.

Epigenetic Regulation
Genetic expression is a complex set of processes involving many factors. One of the important features of the living body system is the regularity of the system. Therefore, in genetic expression the control process becomes an important fundamental part. Control of the regulation of genetic expression in eukaryotes is carried out at many control points and can generally be divided into genetic regulation and epigenetic regulation 6 .
Epigenetic changes are part of changes in genetic expression without changing the structure in DNA. The prefix epi in epigenetic is taken from Ancient Greek which means to cover. So, literally epigenetic means covering or disguising the genetic process. An example of the presence of epigenetic regulation is the process of cell differentiation. The process of cell differentiation goes hand in hand with the process of individual growth and development. Along with the mitosis process, when forming new cells, the process of specialization of the daughter cells occurs by differentiating the genes that will be expressed in the two daughter cells. Thus, there is a selection of genes that have permanent expression and genes that are permanently unexpressed. The differentiated cell nucleus is difficult to return to the initial cell condition.
However, the cell nucleus does not lose the total potential of its genes.
Epigenetic regulation is possible because the DNA in each cell is wrapped in a specific dynamic structure called chromatin. Chromatin consists of DNA wrapped in histone proteins. When the chromatin structure around the genome region is tightly wrapped, regardless of the DNA sequence, gene expression is suppressed. In contrast, exposed chromatin, so DNA and histones interact more loosely, causes access to transcription factors and transcription engines in gene regulators initiating gene expression 6 .
The history of epigenetics is related to the study of evolution and development, but the term epigenetics has changed as understanding of the molecular mechanisms underlying the regulation of gene expression in eukaryotes has increased. Until the 1950s, the term epigenetics was used differently, namely to classify all developmental events starting from the zygote to the adult organism, in this case all regulatory processes, starting with the genetic material which then formed the final product.
Conrad Waddington expressed the discovery of the term epigenetic in 1942 which means the branch of Biology that studies the possible interactions of genes to become their products. The epigenetic field has become a bridge between genetics and the environment. In the 21st century, epigenetics is defined as the study of inherited changes in genome function, which occur without changes in the arrangement of DNA sequences.

Epigenetic Regulation in Breast Cancer
Haris et al 7   The presence of a special enzyme that plays a role in DNA methylation raises the suspicion that an enzyme is also involved in the demethylation process.
In addition, there are other enzymes that also play a role in changes due to methylation, namely enzymes that play a role in the demethylation process. In 2009, the ten eleven translocation (TET) gene that plays a role in the conversion of 5mC to 5hydroxymethylcytosine (5hmC) was identified.
Overexpression on TET1 resulted in a decrease in the 5mC level, while knock-down could reduce 5hmC by up to 40%. In bacte ria, TET also plays a role in the demethylation process of DNA, 5mC will be converted to 5 hmC 12 . Demethylation that occurs in mammals, its activity will be induced by cytidine deaminase, an

Figure 2. Methylation in Normal Cells and Cancer Cells 10
In normal cells, there is relative hypermetylation of CpG islands, whereas in cancer cells there is an increase in the methylation process to produce hypermetylated CpG islands. The hypermetylation that occurs silences various groups of genes that play a role in tumor suppression as well as genes involved in DNA repair, cell cycle control, apoptosis, adhesion, and metastasis13. Table  1 shows some hypermetylated and hypomethylated genes in breast cancer. Folate and methionine cannot be synthesized in the body, thus a diet low in these components will be able to minimize the possibility of methylation in DNA 15 .
In addition, agents present in the environment such as arsenic, nickel, chromium, and cadmium can have a profound effect on the epigenetics of DNA.
Arsenic causes hypomethylation of the RAS gene whereas cadmium induces hypomethylation in the genome by deactivating the DNMT1 enzyme. In addition, arsenic exposure has been shown to influence methylation rates that occur in DNA in general and in certain gene promoters.

b. Histone Modification
The Thus, modifications to the N-terminal histone will affect the density and pattern of chromatin formed. Histone Acetylation

Histone Acetylation in Normal Cells
Each histone modification that occurs is a unique

Figure 3. Histone Acetylation and Deacetylation
In the histone acetylation process, the acetyl groups incorporate to the lysine in the histone tail.
When lysine is acetylated, the positive charge is neutralized and the histone tail no longer binds to the adjacent nucleosome. In fact, in the non-acetylated condition, this bond will encourage the curling of the chromatin into a denser form. When this binding cannot occur, the chromatin structure loosens. As a result, transcription proteins have an easier time accessing genes in the acetylated region. A number of enzymes that acetylate or deacetylate histones are associated with transcription factors that bind to promoters, or are even components of these factors.
Thus, the histone acetylation enzyme will promote the initiation of transcription not only through remodeling the chromatin structure but also the binding of components to the transcription mechanism. Figure 4 shows the effect of histone acetylation on the chromatin structure.

Histone Acetylation in Breast Cancer Cells
Deregulation of the acetylation process occurs in breast cancer cells. Proteins that play a role include the H4K16, H4K20, and H3K56 13 proteins. Under normal conditions, these proteins will undergo acetylation. The acetylation of these proteins is related to the apoptosis process and DNA repair mechanisms.
In breast cancer, it was found that these three proteins were actually deacetylated. As a result, the process of apoptosis and DNA repair will be disrupted.
The interesting thing about the histone acetylation process is that there is a connectivity between the acetylation and methylation processes of the amino acid lysine which is part of the histone protein. The process that occurs is a substitution that interferes with one another. Histone proteins that are known to undergo this type of regulation are H3K9 and H3K27.
It is known that acetylation and methylation are opposite processes related to the epigenetic regulation of gene expression. The acetylation that occurs will hinder the methylation process and vice versa. Thus, genes that are not expressed will undergo a lot of methylation while those that are not expressed will undergo a lot of acetylation.
In addition, it has also been identified that the deacetylation process is an oncogenic mediator that greatly influences the incidence of breast cancer.
Deacetylation will be catalyzed by the enzyme histone deacetylase. It was recently discovered that the substrate for this enzyme was not only histone proteins, but also non-histone proteins. Non-histone proteins that can be substrate for deacetylase enzymes include proteins that play a role in the transcription process (p53, p73, E2F1, STAT1, STAT3, and GATA1) and in the DNA repair process (Ku70) 16 .
Some time ago, the processes of DNA methylation and histone deacetylation were studied as separate mechanisms both of which were able to independently modulate chromatin structure and gene expression. It is now recognized that the two are interrelated mechanisms. DNA methylation will be complemented by deacetylation of histone proteins mediated by methyl group binding proteins such as MeCP2 which are able to recognize methylated DNA sites and activate histone deacetylase to act on these sites 10 . Figure 5 shows the interaction of these two processes in normal cells and in cancer cells.
Apart from its role in DNA replication and chromatin assembly, histone acetylation also plays a role in the timing of DNA replication and the activities that initiate the replication process. In general, the increase in histone acetylation of chromatin at the Ori site will encourage Ori to initiate replication when compared to Ori which is in a hypoacetylation state.
Thus, maintaining a balance between acetylation and deacetylation is essential for proper DNA repair and cell survival.

Figure 5. DNA Methylation and Histone Deacetylation in Normal Cells and Tumor Cells 10
Histone Methylation

Histone Methylation in Normal Cells
Apart from acetyl, several other chemical groups can also be reversibly attached to the amino acids present in the histone tail. For example, the addition of metal groups to histone proteins is known as histone methylation. Methylation is the addition of a methyl group at a specific site on the histone protein. Figure 6 shows the methylation that occurs in the histones.

Figure 6. Histone Protein Methylation
Methylation of histones occurs mostly in lysine and arginine residues which are catalyzed by the enzyme histone methyltransferase. Arginine can accept one or two methyl groups while lysine can accept up to three methyl groups. Methylation on histones will encourage chromatin condensation or in other words will inhibit the transcription process.

Histone Methylation in Breast Cancer Cells
In breast cancer cells, part of the histone protein that is not normally methylated under normal conditions becomes methylated, and on the other hand, the histone that is normally methylated becomes unmethylated. Just like in DNA, methylated histones will make genes inaccessible to transcription factors which will hinder the process of genetic expression. In breast cancer, there are many demethylations that occur in H3K4 12 , H3K9 and H3K27.
The three most prominent methylation events were in H3K4, H3K27, and H3K9 12 . H3K4me3 and H3K27me3 and H3K9me3 will suppress transcription.
Methylation of histone arginine residues has not known the mechanism directly to activate or suppress gene expression. Methylated arginine will affect the binding of effector molecules in the transcription process. Interestingly, many of the arginine methylation sites are close to the methylation sites of lysine, such as the proximity of H3R2 to H3K4, H3R8 to H3K9, and H3R26 to H3K27. Cross talk occurs in these adjacent methylated residues. This suggests that arginine methylation can act as a switch, regulating lysine methylation events which are important in epigenetic regulation of gene expression 12 .
In addition, the methylation process that occurs in H3-K9 from histone proteins will induce DNA methylation to regulate gene expression. Figure 7 shows the occurrence of these positively correlated epigenetic regulatory processes. Methylation of the lysine residue will stimulate the binding of the HP1 protein which will also bind the DNA metal transferase enzyme so that DNA methylation can occur.

Figure 7. Histone Methylation will Activate Methylation in DNA
This demethylation is a process catalyzed by specific enzymes. Excessive presence of these demethylating enzymes indicates an overexpression of the coding genes. This can also be used as a marker for carcinogenesis. Thus, this histone methylation mechanism will support the process that causes genes that are normally suppressed to become activated.

Micro RNA (miRNA) as a molecule that plays a role in epigenetic regulation
MiRNA molecules were first discovered in 1993.
These molecules consist of small, single-stranded RNA molecules that are able to bind to complementary sequences in the mRNA molecule. miRNA is formed from RNA precursors that are longer and bound to each other to form a short hairpin structure that is double-stranded and each linked by a hydrogen bond.
After that, the formation of miRNA will be catalyzed by  The formation of cancer cell heterogeneity is shown in Figure 9. Figure 9a shows that the diversity of cancer cells can be formed from various phenotypes of cells that have the ability to proliferate. This approach is the initial approach used in explaining the heterogeneity of cancer cells. Figure 9b shows  Cancer stem cells are resistant to chemotherapy and radiotherapy. Therefore, the treatment with chemotherapy or radiotherapy will fail if the cancer stem cells have not been eliminated ( Figure 10).
Cancer stem cells also have higher transport membrane activity which is related to the ability to migrate and metastasize.  Figure 11 shows that epigenetic modification can regulate both embryonic stem cells and adult stem cells. methylation is the addition of a methyl group to the nitrogen base of DNA cytosine which will suppress the DNA transcription process. Acetylation is the addition of an acetyl group at the end of the histone which causes a reduction in the chromatin condensation so that it will activate the transcription process. Histone methylation will also suppress transcription so that genes cannot be expressed. In addition, there is also a small RNA molecule known as miRNA which can bind to the transcribed mRNA. This binding will cause the mRNA to degrade or inhibit its translation.
In cancer cells, there is deregulation of these processes, causing many conditions that lead to cancer cell resistance. The process occurs in the same mechanism both physiologically and pathologically.
However, the aberration that occurs in cancer cells is the expression of genes that should be silenced under normal conditions. Conversely, cancer cells also express certain genes even though under normal conditions these genes will not be expressed. Epigenetic deregulation is also very possible to occur in breast cancer stem cells although not many research results have reported on epigenetic regulation of breast cancer stem cells.