ONCOSTATIN M INDUCES VEGF IN HUMAN BREAST CARCINOMA CELLS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 OSM是一种多效性细胞因子，由多种细胞类型产生，包括中性粒细胞和肿瘤相关巨噬细胞。OSM在体外抑制乳腺癌细胞的增殖，因此正在研究其在癌症治疗中的潜在用途。我们实验室的初步研究结果表明，OSM可以促进肿瘤细胞中的促血管生成因子，就像在血管内皮细胞中观察到的那样，这一努力对这一努力很重要。其他研究表明，OSM由肿瘤相关的中性粒细胞和乳腺癌上皮细胞表达，但不在正常乳腺组织中表达。此外，从全血中分离的中性粒细胞在与乳腺癌细胞共同培养之前不会表达OSM。我们的数据表明，OSM可以刺激乳腺癌细胞产生血管生成相关的基质金属蛋白酶(MMPs)和血管内皮生长因子-A(VEGF)，这是一种非常有效的促血管生成因子。后一项发现的含义是，虽然OSM在体外可能导致乳腺癌细胞生长停滞，但它也可能通过产生血管内皮生长因子而诱导肿瘤中的血管生成。这种血管生成的诱导将通过促进血管生成依赖型乳腺癌的进展来对抗OSM的生长抑制特性。鉴于我们的发现，重要的是要更好地理解血管内皮生长因子诱导的含义，以正确评估OSM作为临床癌症治疗的潜力。 这项建议的具体目标是： 特异性目的1.证实中性粒细胞来源的OSM以旁分泌方式诱导乳腺癌细胞产生血管内皮生长因子。中性粒细胞与乳腺癌细胞共培养时会分泌OSM，但其病理生理学意义尚不清楚。将测试中性粒细胞与乳腺癌细胞的共培养，以确定中性粒细胞分泌的OSM是否会以旁分泌的方式诱导乳腺癌细胞产生血管内皮生长因子。采用酶联免疫吸附试验检测OSM和VEGF水平。 特定目的2.识别和表征刺激中性粒细胞释放OSM的信号。我们将通过对经GM-CSF等特定细胞因子中和抗体处理的共培养细胞的细胞裂解物/条件培养液进行Western blots/ELISA检测，确定乳腺癌细胞产生的刺激中性粒细胞释放OSM的信号。此外，我们将确定中性粒细胞在共培养系统(细胞接触)和Transwell培养系统(无细胞接触)中是否需要中性粒细胞-乳腺癌细胞接触来释放OSM。 特异性目的3.研究OSM诱导的血管内皮生长因子在体内刺激血管生成和促进乳腺癌进展的能力。我们将使用Matrigel Plug实验来研究OSM诱导的血管生成是否会刺激体内的血管生成，并评估OSM转基因的乳腺癌细胞注射到裸鼠清除的乳腺脂肪垫中是否会通过刺激依赖于VEGF的血管生成而促进肿瘤进展。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Oncostatin M (OSM) is a pleiotropic cytokine produced by many cell types, including neutrophils and tumor-associated macrophages. OSM inhibits the proliferation of breast cancer cells in vitro, and for this reason is being examined for its potential use in cancer treatment. Important to this endeavor are the results of preliminary studies done in our laboratory that show that OSM may promote pro-angiogenic factors in tumor cells, as is observed for endothelial cells. Additional studies show that OSM is expressed by tumor-associated neutrophils and breast cancer epithelial cells, but not by normal breast tissue. Furthermore, neutrophils isolated from whole blood do not express OSM until they are co-cultured with breast cancer cells. Our data demonstrates that OSM will stimulate breast cancer cells to produce angiogenesis-related matrix metalloproteinases (MMPs) and vascular endothelial growth factor-A (VEGF), which is an extremely potent pro-angiogenic factor. The implication of the latter finding is that while OSM may cause growth-arrest in breast cancer cells in vitro, it may also lead to the induction of angiogenesis in the tumor through production of VEGF. Such an induction of angiogenesis would counter the growth arrest properties of OSM by promoting angiogenesis-dependent breast cancer progression. In light of our findings, it is important to better understand the implications of VEGF induction to correctly evaluate the potential of OSM as a clinical cancer treatment. The Specific Aims of the proposal are: SPECIFIC AIM 1. DEMONSTRATE THAT NEUTROPHIL-DERIVED OSM WILL INDUCE VEGF FROM BREAST CANCER CELLS IN A PARACRINE FASHION. Neutrophils will secret OSM when they are co-cultured with breast cancer cells, but the pathophysiologic significance of this is unknown. Neutrophil-breast cancer cell co-cultures will be tested to determine whether neutrophil-secreted OSM will induce VEGF from breast cancer cells in a paracrine fashion. OSM and VEGF levels will be analyzed by ELISA. SPECIFIC AIM 2. IDENTIFY AND CHARACTERIZE THE SIGNAL THAT STIMULATES NEUTROPHILS TO RELEASE OSM. We will identify the signal generated by breast cancer cells that stimulates neutrophils to release OSM by performing Western blots/ELISAs on cell lysates/conditioned media of co-cultured cells treated with neutralizing antibodies to specific cytokines such as GM-CSF. In addition, we will determine whether neutrophil-breast cancer cell contact is needed for release of OSM by neutrophils in both a co-culture system (cell-contact) and a Transwell-culture (no cell contact). SPECIFIC AIM 3. INVESTIGATE THE ABILITY OF OSM-INDUCED VEGF TO STIMULATE ANGIOGENESIS AND PROMOTE BREAST CARCINOMA PROGRESSION IN VIVO. We will investigate whether OSM-induced VEGF will stimulate angiogenesis in vivo using the Matrigel plug assay, and assess whether OSM-transfected breast cancer cells injected into the cleared mammary fat pads of nude mice will promote tumor progression by stimulating VEGF-dependent angiogenesis in an autocrine fashion.