Alteration of Developmental Genes in Breast Cancer

Institution: The Burnham Institute for Medical Research
Investigator(s): Fumiichiro Yamamoto, Ph.D. -
Award Cycle: 1998 (Cycle IV) Grant #: 4JB-0144 Award: $292,500
Award Type: IDEAS II
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (1998)
Cancer is the result of genetic accidents that disrupt the normal regulation of cell proliferation, differentiation and survival. Such a disruption may result from permanent changes (mutations) in genes, but there are other more transitory ways that the DNA can be altered, also resulting in a disruption of these functions. A process called DNA methylation is the best understood of these types of mechanisms. In this process, genes undergo a chemical modification where a methyl group is added to one of their nucleotides. Thus, some spots in genes may be "methylated" or "unmethylated". Usually, a gene that has many methyl groups added to it (hypermethylated) no longer makes its protein. Methylation can therefore be a method to alter the function of the genes involved in developing cancer.

There are few techniques that will efficiently scan and identify changes in gene methylation. We have developed a method called Methylation Sensitive-Amplified Fragment Length Polymorphism (MS-AFLP). This innovative technique allows the identification and isolation of DNA fragments showing methylation changes by looking for patterns called "DNA fingerprints". MS-AFLP pilot experiments using matched normal/tumor DNA of breast, prostate, and colon showed reproducible changes in gene methylation status, some of which were specifically associated with the tumors. In these pilot experiments we unexpectedly found that several of these fingerprint bands contained sequences derived from genes that are believed to be involved in developmental regulation called homeotic genes.

We believe that our observation of alterations in methylation in several homeotic genes by MS-AFLP fingerprinting may be the tip of the iceberg, and that DNA methylation changes in homeotic genes are very frequent in carcinogenesis in general and in breast cancer in particular. This hypothesis is consistent with the observation that cancer cells often possess characteristics specific to fetal cells. Because homeotic genes direct animal and cell maturation, inhibition of homeotic genes could result in a less mature cell. Thus, hypermethylation of homeotic genes may play a role in breast cancer by contributing to the escape of the cells from their normal constraints of cell maturation. The role of hypermethylation of some homeotic genes in the pathogenesis of breast cancer will be directly tested by introducing unmethylated forms of the homeotic genes into breast cancer cells in culture and monitoring their effects. This approach may open potential therapeutic avenues aiming at the reactivation of those genes.

Final Report (2000)
Cancer causes cells in the body to grow out of control to form a tumor. Cells of the tumor often break away and travel to other parts of the body and grow there to form additional tumors. Therefore, cancer is characterized by the defects in growth control and in cell positioning. During development, a group of transcription factor genes named homeotic genes switch on and off many genes and provide cells with information on their identity and positioning. We analyzed the genome wide changes in DNA methylation (a modification of DNA) in tumors and observed the alterations in several sequences that are related to homeotic genes. Since DNA methylation is an important mechanism to inactivate gene expression, this finding prompted us to believe that some of the homeotic genes may be involved in breast carcinogenesis.

In the first year of this project, we searched for DNA methylation alterations in homeotic genes in breast cancer. We identified the changes in two genes named Hox B13 and IPF I in a majority of breast cancer cases. We mapped these genes in the same chromosomal bands with already cloned genes involved in familial forms of breast cancers (Hox B13 and BRCA1 are on chromosome17q21, and IPF 1 and BRCA2 are on chromosome 13q12). We looked mutations in the coding sequence of the Hox B 13 gene in breast tumors, but no mutations were found. In the second year, we analyzed the gene expression of Hox B13. No changes in expression were observed. Neither normal nor tumor breast tissues expressed the Hox B13 gene. Since the purification of good quality RNA from clinical specimens turned out to be difficult, we shifted our research toward using breast cancer cell lines rather than clinical specimens in the later study. We analyzed the spectrum of the Hox gene expression in two breast carcinoma cell lines. We also examined the spectrum after treatment of those cells with estrogen or with a DNA methylation inhibitor. Although concomitant activation of gene expression was not observed with the Hox B 13 gene, treatment with the DNA methylation inhibitor was shown to change the patterns of Hox gene expression.

Further studies are needed to identify homeotic genes that may play an important role in breast carcinogenesis. DNA methylation inhibitors may not be good candidates for cancer therapy since the effects were so diverse.

Notl-Msell methylation-sensitive amplied fragment length polymorhism for DNA methylation analysis of human cancers
Index Medicus:
Authors: Yamamoto F, Yamamoto M, Soto JL, Kojima E, Wang EN, Perucho M, Sekiya T, Yamanaka H
Yr: 2001 Vol: 22 Nbr: 10 Abs: Pg:1946-56

A single targeted Ets allele restricts development of mammary tumors in transgenic mice.
Periodical:Cancer Research
Index Medicus: Cancer Res
Authors: Neznanov N, Man AK, Yamamoto H, Hauser CA, Cardiff RD, and oshima RG
Yr: 1999 Vol: 59 Nbr: 17 Abs: Pg:4242-4246