Research Grants Awarded
Role Of Ikk/Nf-Kappab/Bmi-1 In Mammary Carcinogenesis And Metastasis
RATIONALE: Dr. Karin?s laboratory was the first to identify and clone IKKbeta, a key enzyme required for NF-kappaB activation and also the first laboratory to discover IKKalpha-mediated alternative NF-kappaB pathway. Deletion of Ikkbeta in hepatocytes enhanced cancer development while it inhibited tumorigenesis when deleted in intestinal epithelial cells. In both cases, IKKbeta deficiency led to the enhanced apoptosis, a kind of cell suicide. Cao et al. in our lab found IKKalpha is critical in mammary gland development and tumorigenesis, likely through mediating self-renewal of mammary cancer stem cells. IKKalpha is also involved in metastasis through repression of Maspin expression in prostate cancer model. IKKbeta is known to regulate bone metastasis of breast cancer cells. My preliminary data indicates a role of IKK/NF-kappaB in transcriptional regulation of Bmi-1 expression in mouse embryonic fibroblasts (MEFs). Bmi-1 was originally identified as a proto-oncogene and upregulated in a variety of human tumors; further studies revealed the function of Bmi-1 as a self-renewal factor of normal and cancer stem cell.
HYPOTHESIS: Through Bmi-1 gene regulation, IKK-dependent NF-kappaB pathway plays crucial roles in self-maintaining of breast cancer stem cells, thus may contribute to tumorigenesis, especially metastasis by supporting the growth in distant organ.
RESEARCH AIMS AND DESIGN: In the proposed study, I will explore the mechanism of how Bmi-1 gene is regulated through IKK/NF-kappaB pathway and the role of this specific IKK?/NF-kappaB/Bmi-1 axis in the regulation of mammary tumorigenesis and metastasis, likely through maintaining self-renewal ability of cancer stem cells. To this end, different methods including promoter assay, EMSA, and CHIP assays will be performed to map out the minimal promoter region that is regulated by IKK/NF-kappaB pathway. Ikkalpha and Ikkbeta will be also specifically deleted in mammary epithelial from different mammary tumor mouse models, including MMTV-c-Neu, MMTV-PyV-mT transgenic mice. In vitro mammosphere formation and in vivo serial transplantations will be used to evaluate the importance of IKK in stem cell self-renewal. Metastasis will be specifically examined in MMTV-PyV-mT mouse model. 4TO7 and 4T1 xenograph model will be utilized to examine the role of Bmi-1, IKKalpha and IKKbeta in colonization, the last and least-understood step of metastasis. IKKbeta inhibitor-VI (CalBiochem Cat# 401483) and ML120B (Millenium) will be administrated to the above mouse models in which IKK? deficiency leads to altered tumor growth, especially metastasis. I will also analyze the clinical breast cancer samples and evaluate the clinical significance of this signaling pathway in metastasis.
EXPECTED RESULTS: I anticipate that IKK-regulated Bmi-1 expression is through NF-kappaB activation, especially p65/RelA and c-Rel, two members from NF-kappaB family, given that there are consensus binding sites at the promoter and intron region of Bmi-1 gene. I will be able to identify the specific region where NF-kappaB binds to. Decreased tumor growth and reduced metastasis may be caused by loss of IKKbeta and Bmi-1 likely through loss of self-renewal ability of cancer stem cells, owing to the facts that IKK/NF-kappaB pathways is critically involved in proliferation and inhibition of apoptosis.
ADVANCES AND DISEASE RELEVANCE: The importance of cancer stem cells in tumor-initiation has been firmly established in leukemia and recently reported in a variety of solid tumors. However, the signaling pathways involved in self-renewal of cancer stem cells are poorly understood. Cancer stem cells are assumed to play a central role in both tumorigenesis and metastasis. Better understanding of cancer stem cells as a fundamental component of the metastatic cascade will lead to novel therapeutic strategies against metastatic cancers. The proposed study will shed light on the role of signaling pathway that allows inflammation and chronic infections to affect stem cell renewal, tumorigenesis and metastasis, intervention of which might be a potential target for cancer therapy. Use of selective kinase inhibitors, if succeed, may result in novel drug validation for breast cancer therapy. The proposed studies are therefore of great translational and clinical relevance. Although I will focus on mammary tumorigenesis and metastasis, the lesson to be learned from the proposed study is likely relevant to all solid cancers.
STUDY HYPOTHESIS AND EXPERIMENTAL DESIGN:
My hypothesis is that cancer stem cell plays a critical role in metastasis to establish the tumor cell growth in metastatic organ. One of the relevant genes that might govern cancer stem property is Bmi-1. My initial study and results from other scientists suggest that inhibitor of kappaB kinase (IKK)-dependent NF-kappaB pathway regulates Bmi-1 gene expression, thus may contribute to tumorigenesis, especially metastasis by supporting the growth in distant organ.
To test the hypothesis, I will explore the mechanism of how Bmi-1 gene is regulated through IKK/NF-kappaB pathway and the role of this specific IKK?/NF-kappaB/Bmi-1 axis in the regulation of mammary tumorigenesis and metastasis, likely through maintaining self-renewal ability of cancer stem cells. To this end, different molecular and biochemical methods including luciferase promoter assay, eletrophoresis mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) assays will be performed to map out the minimal promoter region that is regulated by IKK/NF-kappaB pathway.
My preliminary data suggest this IKK-Bmi-1 pathway is critical for metastasis, not primary tumor growth. I will consolidate the data will other methods. Ikkalpha and Ikkbeta will be specifically deleted in mammary epithelial from different mammary tumor mouse models, including MMTV-c-Neu and MMTV-PyV-mT transgenic mice. Those models mimic human breast cancer development and progression. I will use these mouse models to study the role of IKK and Bmi-1 in mammary tumorigenesis, metastasis and cancer stem cells renewal. In vitro culturing method and flow cytometry based on cell surface markers will be used to isolate cancer stem cell population. Novel inhibitors of IKK will be used to examine if the blockage of IKK pathway will decrease the metastatic ability of cancer cells. 4TO7 and 4T1 xenograph model will be utilized to examine the role of Bmi-1, IKKalpha and IKKbeta in colonization, the last and least-understood step of metastasis. I will also analyze the clinical breast cancer samples and evaluate the clinical significance of this signaling pathway in metastasis.
ADVANCES IN BREAST CANCER AND IMPORTANCE TO CACNER PATIENTS
The mechanistic study will allow us to understand the molecular detail of gene regulation and provide the targets for designing or screening novel therapeutic drugs. Early stage cancer patients are relatively easy to cure. My proposed study is trying to understand the reason why invasive cancer cells will form metastasis in other organs in human, the critical fatal step for majority of cancer patients. Inhibitors of this pathway will be used for a pre-clinical study, which, if succeed, will be potential drug for late stage cancer patients. Although this study is based on breast cancers, it is likely true for other cancers. The proposed studies are therefore of great scientific, translational and clinical relevance. The proposed study, if succeed, will advance our knowledge of how cancer re-colonizes in other organs, provide targets for novel drug design and benefit especially late stage cancer patients.