Role of LIBC in Inflammatory Breast Cancer Phenotype

描述（申请人提供）：炎症性乳腺癌（IBC）是局部晚期乳腺癌中最致命的一种，具有独特的临床和病理特征。临床表现为皮肤红斑和结节性；病理上，IBC是高度血管生成和血管侵入性的，有多个肿瘤栓子堵塞真皮淋巴管，这是引起显著临床表现的原因。最近，我们实验室发现了一个新的基因WISP3 (LIBC)，它在80%的IBC和20%的非IBC肿瘤中表达缺失。WISP3基因与结缔组织生长因子、Cyr61和11一起属于CCN蛋白家族。根据氨基酸序列分析，WISP3编码一种推定的igf结合蛋白相关蛋白。我们在体内和体外均证明了WISP3在乳腺癌中具有生长、侵袭和血管生成抑制功能。我们假设WISP3表达的缺失通过两种可能的机制导致不受调节的乳腺癌生长和血管生成：1。1 .增加上皮细胞和间充质细胞上IGF受体的可用性；直接通过与igf无关的作用本研究的长期目标是阐明WISP3在乳腺癌发生和发展中的作用。为了验证这一假设并解决广泛的目标，我们提出以下目标：1)阐明WISP3蛋白的结构-功能关系及其与IGF家族的关系；2)研究WISP3在乳腺癌生长和体内血管生成开关中的作用；3)研究检测WISP3蛋白作为乳腺癌患者潜在预后标志物的有效性。为了研究WISP3蛋白的结构/功能关系，本研究设计了利用WISP3蛋白和重组IGF-I和IGF-II进行免疫沉淀和结合试验。IGFBP结构域与WISP3功能的相关性将通过WISP3蛋白IGFBP结构域的诱变试验进行研究，随后进行转染实验和功能生物学试验，包括软琼脂生长、侵袭和血管生成试验。在体内，WISP3在血管生成开关中的作用涉及由转染不同WISP3蛋白表达的细胞衍生的胸腺裸鼠乳腺肿瘤的发展，以及用ELISA法检测小鼠血清中的血管生成因子。使用VEGF、bFGF和CD31抗体对切除的小鼠肿瘤进行免疫组化。乳腺癌患者和正常乳腺组织的转化研究将完成这些实验，涉及WISP3、CD31和细胞凋亡的免疫组化，以及高密度组织芯片的开发。所提出的实验可能为设计针对WISP3的新治疗干预提供基础。此外，了解WISP3的作用将揭示癌症发展的一般机制，并作为其他相关CCN基因参与癌变的模型。

DESCRIPTION (provided by applicant): 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.