Research Grants Awarded
Critical Roles Of Dmp1 In The Prevention Of Cyclin D1-Induced Breast Cancer
(1) The scientific rationale supporting the proposed research.
Dmp1 (cyclin D binding myb-like protein 1) is a novel transcription factor that causes p19Arf-, p53-dependent cell cycle arrest in primary cells. Both Dmp1-/- and Dmp1+/- mice are prone to tumor development, especially adenocarcinomas including breast cancer when left untreated or when they are neonatally treated with carcinogen. Also our preliminary findings have indicated critical roles of Dmp1 in normal mammary gland development. Mice lacking Dmp1 have abnormal breast phenotype with little lobuloalveolar development with very less milk in their ducts. This is supported by our evidence of GeneChip Microarray and immunohistochemistry data for the EGFR ligands, amphiregulin and EGF, were significantly downregulated in the mammary glands of Dmp1-/- mice as compared to Dmp1+/+ mice. HER2 is a trans-membrane glycoprotein that belongs to the EGF receptor protein tyrosine kinases. HER2 is overexpressed in ~30% of breast tumors, primarily due to gene amplification, and is associated with very poor prognosis of patients. We recently found that HER2 overexpression selectively activates both the Dmp1 and Arf promoters, suggesting the specific roles of the Dmp1-Arf pathway in breast cancer prevention. It has been reported that cyclin D1 is a critical target in MMTV-cyclin D1- and MMTV-ras-induced breast carcinogenesis. The human Cyclin D1 gene is amplified in ~15% of breast cancers and is associated with shorter survival of patients. On the other hand, the Cyclin D1 protein is overexpressed in >50% of human breast cancers and Cyclin D1 overexpression without gene amplification has been reported to be a favorable prognostic factor of breast cancer. We recently reported that Dmp1 and cyclin D1 collaborates to activate the Arf promoter to neutralize the oncogenic effects of cyclin D1. Our recent data show that both Dmp1-/- and Dmp1+/- mouse mammary epithelial cells are resistant to cyclin D1-induced cell cycle arrest in culture.
(2) The specific hypotheses to be tested.
We hypothesize that Dmp1 will play essential roles in preventing breast tumorigenesis caused by overexpression of cyclin D1. We also hypothesize that Dmp1 will be haplo-insufficient for breast tumor suppression. We hypothesize that loss of heterozygosity (LOH) of the hDMP1 gene will occur simultaneously with overexpression of Cyclin D1 without gene amplification in human breast cancer. Thus, hDMP1 LOH(+) breast cancer patients might have better prognosis than hDMP1 LOH(-) cases.
(3) The research aims, design, and the expected results.
In order to demonstrate the activity of Dmp1 in preventing cyclin D1-driven breast carcinogenesis in vivo, we will cross mice that overexpress cyclin D1 (MMTV-cyclin D1 mice) with Dmp1-knockout mice to determine the time length until the mice get breast cancer (Aim 1). We expect that one allelic or biallelic deletion of Dmp1 will lead to accelerated cyclin D1-induced breast carcinogenesis. When mice get breast tumors, DNA, RNA, and proteins will be extracted from tumors to investigate the integrity of major tumor suppressor pathways, i.e. the Rb and the p53 pathways, and the Ink4a/Arf modulators (Tbx2/3, Bmi1, Twist, and Pokemon), all of which are frequently disrupted in human cancers.
The second Aim we have proposed is to cross the MMTV-Dmp1 mice with MMTV-cyclin D1 mice. We expect to show that the breast tumorigenesis will be significantly inhibited or completely prevented in Dmp1, cyclin D1-double transgenic mice, dependent on the levels of expression of transgenic Dmp1. This will directly prove the potential of Dmp1 in the prevention of breast cancer.
The third Aim is to study the correlation of inactivation of the human DMP1 (hDMP1) gene and overexpression of Cyclin D1 in human breast cancer samples. We expect to show that the hDMP1 gene is hemizygously deleted (LOH) or mutated in more than 50% of human breast cancers, especially those retain wild-type INK4a/ARF and/or P53 locus. We expect to show that deletion of hDMP1 is frequently associated with Cyclin D1 overexpression without gene amplification, thus with favorable prognosis of breast cancer patients. Therefore, decreased levels of hDMP1 protein expression might be a novel prognostic factor of human breast cancer.
(4) How the project uniquely advances our understanding of breast cancer and leads to reductions in incidence and/or mortality.
Since both hDMP1 deletion (40-50%) and Cyclin D1 overexpression (>50%) are very commonly found in human breast cancers, crossing Dmp1-deficient mice with MMTV-cyclin D1 mice will generate valuable data to understand the pathogenesis of human breast carcinoma. We may be able to predict the treatment outcomes of breast cancer patients by studying LOH of hDMP1 and/or immunohistochemical staining of breast cancer tissue with hDMP1 and Cyclin D1-specific antibodies. The results obtained with Dmp1, cyclin D1 bi-transgenic mice will directly prove the therapeutic potential of Dmp1 in breast cancer. One possible strategy for cancer therapeutics is to restore the function of inactivated tumor-suppressor proteins in cancer cells. Tumor suppressors, such as DMP1, may be particularly useful in achieving this goal, because even cancer cells retain one allele of the tumor suppressor gene. Thus, if we invent drugs that reactivate hDMP1 in tumor cells, they might be very effective for the treatment and/or prevention of human breast cancer.
Breast cancer is the most common cancer among women. It is the second leading cause of cancer death in women, only after lung cancer. Every year around 200,000 new cases are diagnosed, and more than 40,000 deaths are caused by metastatic breast cancer. Breast cancer involves several complex signal transduction pathways of oncogenes (genes that cause cancer), growth factors, steroid hormones, and inactivation of tumor suppressor genes (genes that suppress cancer). In this proposal, we will investigate the roles of the novel tumor suppressor gene named Dmp1 in the progression and prevention of breast cancer by using mouse models that lack (Dmp1-knockout mice) or overexpress Dmp1 (Dmp1-transgenic mice). Dmp1 is a novel DNA-binding protein that stops cell growth by activating the Arf-p53 tumor suppressor pathway. Arf and p53 are the most frequently inactivated tumor suppressor genes in human cancer. Mice that lack Dmp1 often develop breast cancer, and thus, Dmp1 has been considered to be a tumor suppressor for breast cancer. Importantly, our preliminary data show that the human Dmp1 gene is inactivated in nearly 50% of human breast cancers by gene loss, the frequency of which is even higher than those of Arf or p53 in the same samples. Thus, it is highly possible that Dmp1 plays critical roles in human breast cancer prevention.
The cyclin D1 protein is overexpressed in approximately 50% of human breast cancers and is associated with shorter survival of patients when the gene copy number is abnormally increased. Importantly, our preliminary data indicate that the Arf tumor suppressor gene expression is significantly activated by collaboration of the Dmp1 tumor suppressor and the cyclin D1 oncogene expression, suggesting that Dmp1 plays critical roles to neutralize the breast cancer-causing activity of cyclin D1.
In order to prove this point in living animals, we will cross mice that overexpress cyclin D1 (cyclin D1-transgenic mice) with Dmp1-deficient mice to determine the time length until the mice get breast cancer. We expect that deficiency of Dmp1 will dramatically accelerate breast cancer formation in mice. When mice get breast cancer, the tumor tissues will be removed from mice to study how the tumor cells look like and what kind of genetic alterations have caused the breast cancer. This will be an essential step to understand the cause of breast cancer.
The second experiment we have proposed is to create mice that overexpress Dmp1 tumor suppressor at high levels in the breast (Dmp1-transgenic mice) and to cross the Dmp1-transgenic mice with breast cancer prone cyclin D1-transgenic mice. We expect that the breast cancer formation will be significantly inhibited or completely prevented in transgenic mice that express both Dmp1 and cyclin D1. This experiment will directly prove the potential of the Dmp1 tumor suppressor protein to prevent and/or treat breast cancer.
The third experiment we have proposed is to study the frequency and the mechanism of inactivation of the Dmp1 tumor suppressor gene in human breast cancer samples. We expect to show that the Dmp1 gene is inactivated by DNA loss in more than 50% of human breast cancers. We also expect to show that this happens simultaneously with high expression of the cyclin D1 oncogene. We will study the therapeutic response and survival of breast cancer patients whose tumor cells have lost the Dmp1 gene and/or express high levels of cyclin D1 in comparison to those people without alterations of Dmp1 or cyclin D1. We will also compare the prognostic values of Dmp1 deletion with other markers of human breast cancer. The data analysis will be conducted with a professor in the Cancer Center. The analyses of human breast cancer samples will be very important to translate the mouse studies to clinical medicine.
Every cell has two copies of each gene. In order to inactivate most tumor suppressor genes, both copies have to be inactivated. The Dmp1 tumor suppressor gene is very unique in that it is inactivated just by losing one copy of the gene. This means that human breast cancer cells still have one copy of Dmp1. A potentially important strategy for breast cancer therapeutics is to restore the function of inactivated tumor-suppressor proteins in cancer cells. Tumor suppressors, such as Dmp1, will be particularly useful in achieving this goal, because even breast cancer cells have one copy of the tumor suppressor gene. Thus, we will be able to start screening of novel drugs by choosing Dmp1 as a therapeutic target. Since both Dmp1 loss and high expression of cyclin D1 occurs very frequently in human breast cancers, we will be able to tell the therapeutic outcomes of breast cancer patients by analyzing how these genes are altered in breast cancer cells. Thus, the results obtained from this study will have significant impact on breast cancer clinics, which will lead to reductions in breast cancer incidence and mortality.