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Barriers to Cancer Development
by Yoshiaki Ito

ancer is a disease of the old. Before cancer becomes symptomatic, it is believed that there usually exist a long latency period, often about 20-30 years. In other words, the initial trigger that induces cancer development occurs many years before the actual development of cancer. It is well known that the treatment of cancer is an uphill task. Many major pharmaceutical companies are spending huge amounts of money, yet the development of a successful anti-cancer drug is very rare. So there is something that we must think about. If cancer develops years after the initial trigger, then we would in theory have a long period within which we can try to catch the signs of future cancer development while the patients are still healthy. Developing methods to detect early stages of cancer and to be able to halt further progression of the cancer before overt cancer develops would be perhaps ideal ways to control cancer. Therefore, it is important to study early stage of cancer development.

We have been studying the gene called RUNX3 and its involvement in human cancer for some time. As will be discussed below, RUNX3 might be one of the genes that may contribute to early diagnosis.

RUNX gene is a mammalian homolog of Drosophila segmentation gene, Runt. The cDNA sequence of Runt was described first by Peter Gergen. Then Misao Ohki identified AML1 gene on chromosome 21 that is involved in chromosomal translocation, t(8;21), which is the most frequently observed chromosome translocation in acute myeloid leukemia. We were studying transcription factors that regulate gene expression of polyomavirus in developmentally regulated fashion. One of them was polyomavirus enhancer binding protein 2 (PEBP2). Nancy Speck identified a transcription factor called Core Binding Factor (CBF) that regulates retrovirus gene expression. PEBP2 and CBF turned out to be identical and they are heterodimeric transcription factors. The cDNA sequences of Runt, AML1 and DNA binding subunit of PEBP2 and CBF are highly related and we now call them as the RUNX family. From the sequence comparison, it was clear that the gene family is a developmental regulator, involved in human cancer and functions as a transcription factor (Figure1). There are three mammalian RUNX genes, called RUNX1, RUNX2 and RUNX3. These genes are evolutionarily very old and regulate cell specification in development. Developmental regulation and cancer are two different sides of the same coin. We are interested in studying these genes, since our primary interest is to uncover the mechanism by which cancer develops.

We began working on RUNX3 because we found its expression in stomach and intestine and there was a hint that this gene may be involved in stomach and colorectal cancer. To study the gene function, we disrupted Runx3 gene in mouse and examined the changes in intestine and stomach. To analyze intestinal phenotype, we study our Runx3+/- mouse side by side with Min mouse. Min mouse is a mouse line in which one of the allele of APC (adenomatous polyposis coli) gene is inactivated. APC gene is a well-known colon cancer gene and most of the colon cancer patients usually have an impaired APC gene. Min mouse is a standard mouse model to study the mechanism by which APC gene induces colon cancer. In human, colon cancer is common but cancer in small intestine is rare, whereas mouse develops intestinal tumors but only rarely in colon. Tumor formation in intestine in mouse is considered to be a model of human colon cancer. Usually, the function of APC gene is studied in one of the inbred mouse strains, C57BL/6. In this strain, intestinal tumors develop in Min mouse in several months. However, in different mouse strains, the same genetic change in APC gene causes tumors to develop very slowly. The reason is that there is a gene that inhibits the tumor growth when APC gene is inactivated. C57BL/6 strain does not have the function of this gene. We studied Min mutation and Runx3 heterozygous mutation in parallel in BALB/c strain. The reason why we used BALB/c strain was because Runx3-/- mice in C57BL/6 strain die soon after birth, whereas significant percentage of BALB/c strain with Runx3-/- genotype can survive to adulthood. Ironically, Runx3-/- mice did not spontaneously develop tumors. Other investigators also observed that biallelic inactivation of well-known tumor suppressor genes some times do not induce tumors. However, we found that both Min mice and Runx3+/- mice developed several small adenomas, premalignant tumors, at around 15 months after birth: tumor morphology and incidence are remarkably similar. Genomic studies showed that adenomas that developed in Min mice showed homozygous mutation in APC gene, while Runx3 locus did not show any change. On the other hand, in adenomas developed in Runx3+/- mice, there was no sign of genetic alteration in APC gene. Instead, both alleles of Runx3 were inactivated by promoter hypermethylation. This is reminiscent of the frequent observations of RUNX3 hypermethylation in human colorectal cancer. Interestingly, analysis of very small adenomas formed in the compound mice showed either biallelic mutation of APC gene without affecting Runx3 gene or biallelic silencing of Runx3 gene without affecting APC gene. These results strongly suggest that heterozygous mutation of both APC and Runx3 genes are nearly equivalent in inducing adenomas in mouse intestine. Importantly, analysis of human sporadic adenomas revealed similar results. We found that Runx3 attenuates the oncogenic Wnt signaling pathway and that the human gastric cancer-derived mutation R122C is deficient in attenuation of Wnt signaling. These are strong evidence of tumor suppressor activity in Runx3. The results clearly indicate that APC gene and Runx3 gene are similar in inducing early stage of cancer development. APC is characterized as a “gatekeeper” of colon carcinogenesis. Runx3 also functions in the early stage of colon carcinogenesis.

We also studied the knockout phenotype in stomach. In this case, we treated mice with chemical carcinogen which is known to induce gastric cancer. When we give mice N-methyl-N- nitrosourea (MNU) for 10 weeks from 8 weeks of age in drinking water, wild type mice will develop cancer in 1 year of age. We used a smaller dose which will not cause tumor development in wild type mice. When Runx3-/- mice were treated similarly, 100% of the mice developed adenocarcinoma. Runx3+/- mice showed intermediate value, namely showing adenoma and dysplasia. These results show that absence of Runx3 makes mice prone to induce gastric cancer.

From the study of intestine and stomach, we found that Runx3 functions at the beginning of cancer development. This notion is consistent with the results obtained from different studies by others. It has been shown that all Runx1, Runx2 and Runx3 genes induce cellular senescence, that is, permanent cessation of cell division. When activation of oncogene, such as Ras oncogene occurs in normal cells, the cell growth would be strongly stimulated. When this happens in normal cells, however, cellular fail-safe mechanism is activated to stop abnormal cell growth, since such aberrant growth of cells would be harmful to a healthy body. Therefore, Runx genes are at least a part of the fail-safe machinery in our body. If Runx genes are inactivated, the cell would lose a fail-safe mechanism and cells in which oncogenic Ras is activated would keep growing. This is the basis of the first step of carcinogenesis. When these cells acquire additional genetic changes, the cells would gradually become cancer.

It has been known that DNA damage response is a barrier for cancer development. That is to say that when DNA, which stores hereditary information and gets transmitted from generation to generation, is damaged by radiation or toxic substance, our body is equipped with multiple levels of repair mechanisms to rectify such damages. It is now known that the DNA damage response is also activated as an anti-cancer barrier in response to oncogenes such as Ras. This is because oncogenes can also induce DNA damage by increasing the rate of DNA replication. Thus, if this system becomes defective, then damaged DNA would remain in the body which would gradually cause the genomic instability. When genomic instability persists, activation of oncogene or inactivation of tumor suppressor genes could be generated to eventually induce cancer. Therefore, DNA damage response is considered to be an essential barrier to prevent carcinogenesis from being initiated. We found that Runx3 is also involved in DNA damage response.

All these evidence support the notion that Runx3 functions at the initial stage of cancer development. Obviously, cells in our body are protected by multi-levels of protective mechanisms. RUNX family genes seem to be a part of this protective machinery to play a role as a barrier to protect normal cells from becoming malignant. We believe this is the basis of the tumor suppressor activity of RUNX3.

Our studies revealed that RUNX3 is one of the genes that will be worthwhile to study for the purpose of identifying clues of very early stage of cancer. One of the points to note is that mutations in RUNX3 gene in various cancers are rare. RUNX3 is frequently inactivated by methylation of the promoter region. Promoter methylation is the structural modification of the DNA in the regulatory region of the gene to control the expression of the gene. Many tumor suppressor genes are known to be inactivated by promoter methylation. When this structural modification occurs, gene expression is silenced. This is functionally equivalent to mutation of the gene to inactivate it. Interestingly, there have been reports that RUNX3 methylation is a good diagnostic tool for a certain type of colon cancer. Technology to detect methylation of genes is improving. Since RUNX3 is a transcription factor which activates or represses target genes, there is a possibility to detect one of the target genes as a surrogate marker for inactivation of RUNX3. Some of them could be useful tool to detect RUNX3 inactivation.

RUNX genes are multi-functional. We must still continue to study the roles of the genes in the various biological systems. At the same time, we are studying RUNX3 to find out how we can contribute to improve the diagnosis of the precancerous state of the patients.

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