Inflammatory breast cancer (IBC) is the most lethal form of locally advanced breast cancer with unique clinical and pathologic features. Clinically, patients present with skin erythema and nodularity; pathologically, IBC is highly angiogenic and angioinvasive with multiple tumor emboli that plug the dermal lymphatics and are responsible for the striking clinical picture. Recently, a novel gene, WISP3 (LIBC) whose expression is lost in 80% of IBC and in 20% of non-IBC tumors has been identified in our laboratory. WISP3 gene belongs to the CCN family of proteins together with connective tissue growth factor, Cyr61 and NOV. Based on amino acid sequence analysis, WISP3 encodes for a putative IGF-binding protein related protein. We demonstrated that WISP3 has growth, invasion and angiogenesis inhibitory functions in breast cancer, both in vivo and in vitro. We hypothesize that loss of WISP3 expression results in unregulated breast cancer growth and angiogenesis by two possible mechanisms: 1. increasing the availability of IGF to the IGF receptor on epithelial and mesenchymal cells, and 2. directly through IGF-independent effects. The broad, long-term objectives of this proposal are to elucidate the role of WISP3 in breast cancer development and progression. In order to test this hypothesis and address the broad objective, we propose the following aims: 1) To elucidate the structure-function relationship of the WISP3 protein, and its relationship to the IGF family, 2) To investigate the role of WISP3 in breast cancer growth and in the in vivo angiogenic switch, and 3) To investigate the usefulness of detecting WISP3 protein as a potential prognostic marker in patients with breast cancer. The research design to study the structure/function relationship of the WISP3 protein involves immunoprecipitation and binding assays utilizing WISP3 protein and recombinant IGF-I and IGF-II. The relevance of the IGFBP domain in WISP3 function will be studied using mutagenesis assays of the IGFBP domain of the WISP3 protein, followed by transfection experiments and functional biological assays, including growth in soft agar, invasion, and angiogenesis assays. The in vivo role of WISP3 in the angiogenic switch involves the development of mammary tumors in athymic nude mice derived from transfected cells with different WISP3 protein expression and detection of angiogenic factors in the mice serum using ELISA assays. Immunohistochemistry of the resected mice tumors will be performed using antibodies for VEGF, bFGF and CD31. The translational study of patients' breast cancers and normal breast tissues will complete these experiments and involves immunohistochemistry for WISP3, CD31 and apoptosis, as well as development of a high-density tissue microarray. The experiments proposed may provide the foundation for the design of new therapeutic interventions that target WISP3. Also, understanding the role of WISP3 will shed light on the mechanisms of cancer development in general and serve as a model for other related CCN genes involved in carcinogenesis.
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