Research Grants Awarded
Inflammation And Maspin In Breast Cancer Metastasis
Investigator Initiated Research
Breast cancer has extremely high morbidity with bone metastasis. Clinical studies show that about 70% of cancer patients have bone tumors. It appears that breast tumor cells, the ?seeds?, prefer to metastasize to bone which serves as an especially fertile ?soil?. Bone metastasis is an extremely complicated process involving local stromal invasion, intravasation and extravasation into and out of the capillary vascular system, tumor growth in the bone marrow, and angiogenesis. The process of breast cancer metastasis ultimately depends on the interactions between primary tumors and the bone host environment. Understanding why certain tumor cells prefer homing to the bone is of key importance to breast cancer research.
Breast cancer cells that are capable of metastasizing to bone must possess some unique features that make them suitable to colonize and grow in bone environment. These unique features are likely represented by gene expression patterns, which serve as the molecular signatures of bone metastasis. We have identified a list of candidate signature genes through microarray analysis. Among 86 genes that were identified as differentially expressed candidates, signal transducer and activator of transcription 1 (STAT1) was consistently up regulated about 4 fold at mRNA level in highly metastatic TM40D-MB cells (p value < 0.0001). The result was confirmed by repeated quantitative RT-PCR analyses. Preliminary study in my laboratory showed that the level of phosphorylated STAT1, an indicator of STAT1 activity, was highly elevated in TM40D-MB cells compared to TM40D cells. Several other interferon gamma response genes were also up regulated in TM40D-MB cells, and interferon gamma is known to induce STAT1 expression and activity. Since transcription factors generally control an array of downstream target genes, we hypothesize that STAT1 may act as a master switch that controls many signal pathways involved in tumor-bone interaction and breast cancer bone metastasis. In addition, STAT1 may act synergistically with other candidate genes (such as Fgf-7) to control bone metastasis. The long-term objective of this study is to study the function of STAT1 and its regulated genes in bone metastasis and the tumor-bone host interaction during the process of bone metastasis. Three specific aims are proposed in this application.
Specific Aim 1. To analyze the effect of STAT1 overexpression on tumor cell behavior and the rate of bone metastasis. Stable STAT1 clones will be established, and the effect of STAT1 overexpression on tumor invasion, migration, and enzymatic activities will be investigated. STAT1 overexpression tumor cells will be tested in the mouse model of bone metastasis.
Specific Aim 2. To analyze the effect of STAT1 overexpressing tumor cells on the interaction with osteoclast and osteoblast cells. We will study the interaction between STAT1 overexpressing tumor cells with osteoclasts and osteoblasts. Osteoclast formation, osteoblast differentiation and proliferation will be analyzed. These analyses will help us understand how tumor cells within bone microenvironment can affect bone remodeling through its interaction with osteoclasts and osteoblasts.
Specific Aim 3. To identify STAT1 targets that mediate tumor metastasis and tumor-bone cell interaction. Chromatin immunoprecipitation (ChIP) assay will be performed to identify STAT1 target genes. Identified target genes will be cloned in expression vector and transfected into TM40D cells. Stale clones will be analyzed in in vitro cell culture assays and in vivo bone metastasis assay.
Bone metastasis is the most frequently found metastasis in breast cancer patients. Despite its importance, limited progress has been made in this research field. Breast cancer patients face the threat of metastasis, and most of them do not die from the primary tumors but rather die due to aggressive metastases. Therefore, studying cancer metastasis and finding means to interfere with the process should be considered a high priority of cancer research, since these studies directly benefit patients with metastatic spread. In that sense, our proposal is designed to study gene functions involved in controlling breast cancer bone metastasis, with a goal that these studies may have therapeutic applications in the near future.
Breast cancer has extremely high morbidity with bone metastasis. Clinical studies show that about 70% of cancer patients have bone tumors. It appears that breast tumor cells, the ?seeds?, prefer to metastasize to bone which serves as a fertile ?soil?. To study bone tumor metastasis, one must establish an appropriate animal model. Our laboratory has recently developed another model system using mouse mammary tumor TM40D-MB cells in syngeneic mice. We showed that mice that had been implanted with TM40D-MB cells developed bone metastasis with high potency, suggesting TM40D-MB tumor cells have the features for preferential metastasis to the bone. Further studies by my laboratory identified a list of candidate genes, which we believe may represent the signatures of breast cancer bone metastasis.
The significance of this study is obvious. For years, there has been a debate whether tumor metastasis is achieved after tumor cells acquire multiple alterations that render them more competent to establish metastatic lesions in distant organs or that a small portion of primary tumor cells may have existing molecular signatures for metastasis. Our recent study, described above, suggests that sub-population(s) of cells with a bone metastasis signature may be present in parental mouse mammary tumor TM40D cells, and possibly are enriched in TM40D-MB tumor cells when they were in the bone environment. Through microarray analysis using Affymetrix array chips, candidate molecular signature genes of breast cancer cells with a high potential for bone metastasis have been identified. One of the key genes, STAT1, may act as a master controller for breast cacner bone metastasis. The object of this proposal is to study the functions of STAT1 and STAT1 regulated target signature genes, using both in vitro and in vivo approaches. Identifying the molecular signature of metastasis is of key importance to our understanding of the processes of breast cancer metastasis, which will help us design therapeutic intervention against breast cancer.
The impact of our study on breast cancer research is clear. As the most common malignancy, breast cancer accounts for nearly one out of every three cancers diagnosed in the United States. Currently, chemotherapy, radiation therapy, and surgery are used to treat breast cancer patients, with removal of the primary tumor by surgery as one of the most effective therapies for those diagnosed with breast cancer. However, residual tumor cells that metastasize to secondary sites, such as the bone and lung, become the ultimate threat to every patient following surgery. It is clear that most breast cancer patients do not die from the primary tumor but rather die due to aggressive metastases. Therefore, studying cancer metastasis and finding means to interfere with the process should be considered as the priority of breast cancer research, since they directly benefit patients with metastatic spread. In that sense, our proposal is designed to study gene functions involved in controlling breast cancer bone metastasis, with a goal that these studies may have therapeutic applications in the near future.