Role of LIBC in Inflammatory Breast Cancer Phenotype

描述（由申请人提供）： 炎性乳腺癌（IBC）是局部晚期乳腺癌中最致命的形式，具有独特的临床和病理特征。 临床上，患者表现为皮肤红斑和结节;病理学上，IBC具有高度血管生成性和血管侵入性，具有多个肿瘤栓塞，这些肿瘤栓塞堵塞了真皮血管，并导致了引人注目的临床表现。 最近，一个新的基因，WISP 3（LIBC），其表达在80%的IBC和20%的非IBC肿瘤中丢失，已经在我们的实验室中鉴定。 WISP 3基因与结缔组织生长因子（CTGF）、Cyr 61和NOV同属CCN蛋白家族。 我们证明WISP 3在乳腺癌中具有生长、侵袭和血管生成抑制功能，无论是在体内还是体外。 我们假设WISP 3表达的缺失通过两种可能的机制导致乳腺癌生长和血管生成不受调控：1.增加IGF对上皮细胞和间充质细胞上的IGF受体的可用性，和2.直接通过胰岛素样生长因子独立的影响。 该提案的广泛，长期目标是阐明WISP 3在乳腺癌发展和进展中的作用。 为了检验这一假设并解决广泛的目标，我们提出以下目标：1）阐明WISP 3蛋白的结构-功能关系及其与IGF家族的关系，2）研究WISP 3在乳腺癌生长和体内血管生成开关中的作用，3）探讨WISP 3蛋白作为乳腺癌患者预后指标的价值。 研究WISP 3蛋白的结构/功能关系的研究设计包括利用WISP 3蛋白和重组IGF-I和IGF-II的免疫沉淀和结合测定。 IGFBP结构域在WISP 3功能中的相关性将使用WISP 3蛋白的IGFBP结构域的诱变测定进行研究，然后进行转染实验和功能生物学测定，包括在软琼脂中的生长、侵袭和血管生成测定。 WISP 3在血管生成开关中的体内作用涉及在无胸腺裸鼠中乳腺肿瘤的发展，所述无胸腺裸鼠源自具有不同WISP 3蛋白表达的转染细胞，并且使用ELISA测定法检测小鼠血清中的血管生成因子。 将使用VEGF、bFGF和CD 31的抗体进行切除的小鼠肿瘤的免疫组织化学。 对患者乳腺癌和正常乳腺组织的转化研究将完成这些实验，并涉及WISP 3，CD 31和细胞凋亡的免疫组织化学，以及高密度组织微阵列的开发。 提出的实验可能为设计针对WISP 3的新治疗干预措施提供基础。 此外，了解WISP 3的作用将揭示癌症发展的一般机制，并作为参与致癌作用的其他相关CCN基因的模型。

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.