权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Prolactin Interactions in Mammary Gland Development and Tumorigenesis

催乳素在乳腺发育和肿瘤发生中的相互作用

基本信息

批准号：
8175288
负责人：
Barbara Vonderhaar
金额：
$ 52.86万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8175288
关键词：
Acinus organ component Agar Age-Months Alternative Splicing Angiogenesis Inhibition Antibodies Apoptosis Architecture Biological Process Biopsy Blood Circulation Breast Breast Cancer Cell Breast Carcinoma Cancer cell line Cancerous Cell Line Cell Proliferation Cells Chinese Hamster Ovary Cell Chromatin Cleaved cell Clinical Complementary DNA Complex Cooperative Human Tissue Network Cyclin D1 Cyclin E Data Development Diagnosis Dimerization Dominant-Negative Mutation Ductal Ductal Carcinoma Endocrine Epithelial Cells Estrogen receptor positive Etiology Exhibits Exons Extracellular Domain Extracellular Matrix Fatty acid glycerol esters Genes Genetic Transcription Goals Growth Growth Factor HCT116 Cells Homeobox Hormones Human Immune Immunohistochemistry In Vitro Investigation Laminin Length Ligands Ligation Lobular Carcinoma MCF7 cell Maintenance Mammary Gland Parenchyma Mammary Neoplasms Mammary Tumorigenesis Mammary gland Messenger RNA Milk Proteins Mining Modeling Monoclonal Antibodies Mus N-terminal Neoplasm Metastasis Organ Pathway interactions Pattern Phenotype Plasma Plastics Play Polymerase Chain Reaction Postmenopause Progesterone Prolactin Prolactin Receptor Prostatic Neoplasms Protein Isoforms Proteins Published Database Publishing Regulation Research Risk Rodent Model Role STAT protein Sampling Serum Signal Pathway Signal Transduction Signaling Pathway Gene Specificity Staining method Stains Stem cells Stimulation of Cell Proliferation System Tissue Microarray Tissue Sample Tissue-Specific Gene Expression Tissues Transfection Transgenic Mice Vascularization Western Blotting Woman Work Xenograft Model angiogenesis autocrine beta-Casein carcinogenesis cell growth cell motility colon cancer cell line immunocytochemistry in vivo inhibitor/antagonist malignant breast neoplasm mammary epithelium mammary gland development member neoplastic cell outcome forecast paracrine protein expression receptor self-renewal stem tool trafficking transcription factor tumor tumor growth tumorigenesis two-dimensional vector control

项目摘要

Prolactin (PRL) acting through the prolactin receptor (PRLR) has an important role in breast carcinogenesis through its effects on mitogenesis and proliferation. PRL acts in an endocrine and an autocrine/paracrine manner and both PRL and PRLR are found in about 80% of human breast cancer biopsies. We are examining the role of the autocrine PRL system in metastasis. For these studies we used the highly metastatic MDA-MB-435 and MDA-MB-231 human breast cancer cell lines. The non-metastatic cell line MCF7 was used as a control. PRL and its receptors have been identified in normal and cancerous human breast tissues and cell lines; however, much of the evidence implicating a role for PRL in mammary tumorigenesis is from rodent models. The extent of PRLs involvement in human breast cancer is less well documented. To evaluate the role autocrine PRL plays in tumor growth, progression, and metastasis, we stably transfected the three breast cancer cell lines with a human prolactin cDNA. We found that PRL confers a more aggressive phenotype in vitro and in vivo. In vitro, it inhibited apoptosis and increased mitogenesis on plastic and on extracellular matrices, enhanced growth on soft agar and increased cell migration. In vivo, over-expression of PRL increased angiogenesis and tumorigenesis in an orthotopic xenograft model. Microarray analyses, confirmed by immunohistochemistry, revealed that PRL differentially regulated various members of the Wnt pathway resulting in increased Wnt signaling. The Wnt inhibitor Dkk-1 reversed the growth promoting effects of PRL. This is the first evidence demonstrating that PRL acts through activation of the canonical Wnt pathway. We also examined the role that the P-regulated Hox-related homeobox-containing gene, MSX2, plays during tumorigenesis where mining of published databases found a strong correlation between elevated MSX2 expression and estrogen receptor (ER) positive breast cancer. NMuMG cells, a normal mouse mammary epithelial cell line, stably-transfected with a MSX2 cDNA showed increased growth in serum-starved conditions, enhanced growth on soft agar and increased cell migration in vitro. These functions were accompanied by constitutive over-expression of cyclin E and cyclin D1. Additionally, the NMuMG-MSX2 cells demonstrated enhanced tumor formation when injected orthotopically into the mammary fat pad of immune compromised mice. 25% of transgenic mice over-expressing MSX2 in the mammary glands developed tumors by 15 months of age; control mice failed to develop tumors. Additionally, immunohistochemistry of human infiltrating breast carcinomas showed positive staining for MSX2 only in the infiltrating tumor cells while the non-infiltrating tumor cells were negative. These results suggest that MSX2 may play a significant role in mouse and human mammary tumor development and invasion. Epithelial cells, once dissociated and placed in two-dimensional (2D) cultures, rapidly lose tissue-specific functions. In addition to PRL, signaling by laminin-111 is necessary to restore functional differentiation of mammary epithelia. We showed that in 2D cultures, PRLRs are basolaterally localized and physically segregated from the apically placed ligand. Detachment of the cells exposes the receptor to ligation by PRL leading to signal transducers and activators of transcription protein 5 (STAT5) activation, but only transiently and not sufficiently for induction of milk protein expression. We showed that laminin-111 reorganizes mammary cells into polarized acini, allowing both the exposure of the PRLR and sustained activation of STAT5. The use of constitutively active STAT5 constructs showed that the latter is necessary and sufficient for chromatin reorganization and beta-casein transcription. These results underscore the crucial role of continuous laminin signaling and polarized tissue architecture in maintenance of transcription factor activation, chromatin organization, and tissue-specific gene expression. Growth of normal breast cells or breast cancer cells as spheres in non-adherent (3D) culture conditions has been used as a surrogate for the presence of stem or progenitor cells. Self-renewal pathways may be activated to increase the number of stem cells contributing to the formation of the spheres. For both primary normal breast cells, cell lines and breast cancer cells, we found that only PRL treatment results in increased sphere formation. P acts in cooperation with PRL to give more, larger and highly disorganized spheres in culture. The signaling pathways and receptor isoforms involved in this hormonally activated self-renewal is currently under investigation. High levels of PRL in plasma correlate with increased risk of breast cancer, especially among postmenopausal women. Several isoforms of PRL exist in human circulation, including a 16 kDa isoform that is an N-terminal cleavage product of the full-length 23 kDa PRL. 16 kDa PRL has been shown to be anti-angiogenic in vitro and in vivo, and to reduce formation of tumors from prostate and colon cancer cell lines. We explored the effect of 16 kDa PRL expression in vitro and in vivo using two breast cancer cell line models (MCF-7 and MDA-MB-231) and also the HCT-116 colon cancer cell line. In vitro, in all three cell lines, 16 kDa PRL expression inhibited cell proliferation compared to empty vector controls. In vivo results were markedly different between the two types of cell lines. HCT-116 cells expressing 16 kDa PRL exhibited reduced vascularization and tumor formation, consistent with published results. The breast cancer cell lines expressing 16 kDa PRL also exhibited inhibition of angiogenesis in vivo but no reduction in tumor size or formation. These results suggest that the effects of 16 kDa PRL on tumor formation may be organ specific and that the unique sensitivity of breast cells to the mitogenic effects of PRL may play a dominant role in tumor formation, thus outweighing the anti-angiogenic effects of 16 kDa PRL. PRLRs exist in multiple isoforms resulting from alternative splicing of PRLR exon 11. We have been studying the long form (LF) and two short isoforms, 1a (SF1a) and 1b (SF1b) that lack components of the intracellular domain of PRLR but maintain the same extracellular domain. Current data suggests that the short form PRLRs act as dominant negatives for differentiation signaling through the LF receptor; their roles in proliferation remain to be determined. Previous work on these isoforms has only examined mRNA levels and established potential roles through transfection studies. We have developed the first antibodies that can distinguish between these three isoforms using differences in the intracellular domains. The specificity of these polyclonal and monoclonal antibodies was demonstrated by western blots and immunocytochemistry on CHO cells stably transfected with the cDNA independently for each of the three isoforms. Subsequently we examined potential clinical uses for these antibodies by immunohistochemistry on ductal and lobular carcinoma samples obtained from the Cooperative Human Tissue Network (CHTN) and a tissue array containing 144 ductal carcinoma and 24 lobular carcinomas. We were able to correlate these results with quantitative polymerase chain reaction (qPCR) on whole tissue samples. We have found differences in expression that demonstrate these antibodies could be used as a new clinical tool to distinguish between different subclasses of breast cancers and aid in diagnosis and possibly prognosis.

催乳素（PRL）通过催乳素受体（PRLR）发挥作用，通过影响有丝分裂和增殖，在乳腺癌发生中发挥重要作用。 PRL 以内分泌和自分泌/旁分泌方式发挥作用，约 80% 的人类乳腺癌活检中都发现了 PRL 和 PRLR。我们正在研究自分泌 PRL 系统在转移中的作用。在这些研究中，我们使用了高度转移的 MDA-MB-435 和 MDA-MB-231 人类乳腺癌细胞系。使用非转移细胞系MCF7作为对照。 PRL 及其受体已在正常和癌性人类乳腺组织和细胞系中得到鉴定；然而，许多表明 PRL 在乳腺肿瘤发生中的作用的证据来自啮齿动物模型。 PRL 参与人类乳腺癌的程度尚不清楚。为了评估自分泌 PRL 在肿瘤生长、进展和转移中的作用，我们用人催乳素 cDNA 稳定转染了三种乳腺癌细胞系。我们发现 PRL 在体外和体内赋予更具攻击性的表型。在体外，它抑制细胞凋亡并增加塑料和细胞外基质的有丝分裂，促进软琼脂上的生长并增加细胞迁移。在体内，PRL 的过度表达增加了原位异种移植模型中的血管生成和肿瘤发生。经免疫组织化学证实的微阵列分析表明，PRL 差异调节 Wnt 通路的各个成员，导致 Wnt 信号传导增强。 Wnt 抑制剂 Dkk-1 逆转了 PRL 的生长促进作用。这是证明 PRL 通过激活经典 Wnt 通路发挥作用的第一个证据。我们还研究了 P 调节的 Hox 相关同源盒基因 MSX2 在肿瘤发生过程中的作用，对已发表的数据库的挖掘发现 MSX2 表达升高与雌激素受体 (ER) 阳性乳腺癌之间存在很强的相关性。 NMuMG 细胞是一种正常小鼠乳腺上皮细胞系，用 MSX2 cDNA 稳定转染后，在血清饥饿条件下显示出生长加快、在软琼脂上生长增强以及体外细胞迁移增加。这些功能伴随着细胞周期蛋白 E 和细胞周期蛋白 D1 的组成型过度表达。此外，当原位注射到免疫受损小鼠的乳腺脂肪垫中时，NMuMG-MSX2 细胞显示出增强的肿瘤形成。乳腺中过度表达 MSX2 的转基因小鼠中有 25% 在 15 个月大时出现肿瘤；对照小鼠没有出现肿瘤。此外，人浸润性乳腺癌的免疫组织化学显示，仅在浸润性肿瘤细胞中 MSX2 染色呈阳性，而非浸润性肿瘤细胞呈阴性。这些结果表明MSX2可能在小鼠和人类乳腺肿瘤的发展和侵袭中发挥重要作用。上皮细胞一旦解离并置于二维 (2D) 培养物中，就会迅速丧失组织特异性功能。除了 PRL 之外，层粘连蛋白 111 的信号传导对于恢复乳腺上皮的功能分化也是必需的。我们发现，在 2D 培养物中，PRLR 位于基底外侧，并与顶部放置的配体物理分离。细胞的分离使受体暴露于 PRL 连接，导致信号转导器和转录蛋白 5 (STAT5) 激活剂激活，但只是短暂的，不足以诱导乳蛋白表达。我们发现，laminin-111 将乳腺细胞重组为极化腺泡，从而使 PRLR 暴露并持续激活 STAT5。使用组成型活性 STAT5 构建体表明后者对于染色质重组和 β-酪蛋白转录是必要且充分的。这些结果强调了连续层粘连蛋白信号传导和极化组织结构在维持转录因子激活、染色质组织和组织特异性基因表达中的关键作用。正常乳腺细胞或乳腺癌细胞在非贴壁 (3D) 培养条件下以球体形式生长已被用作干细胞或祖细胞存在的替代物。自我更新途径可以被激活以增加有助于球体形成的干细胞的数量。对于原代正常乳腺细胞、细胞系和乳腺癌细胞，我们发现只有 PRL 治疗才会导致球体形成增加。 P 与 PRL 合作，提供更多、更大且高度无组织的文化领域。目前正在研究参与这种激素激活的自我更新的信号通路和受体亚型。血浆中高水平的 PRL 与乳腺癌风险增加相关，尤其是绝经后妇女。人体循环中存在多种 PRL 亚型，包括 16 kDa 亚型，它是全长 23 kDa PRL 的 N 末端裂解产物。 16 kDa PRL 已被证明在体外和体内具有抗血管生成作用，并能减少前列腺癌细胞系和结肠癌细胞系肿瘤的形成。我们使用两种乳腺癌细胞系模型（MCF-7 和 MDA-MB-231）以及 HCT-116 结肠癌细胞系探索了 16 kDa PRL 表达在体外和体内的影响。在体外，在所有三种细胞系中，与空载体对照相比，16 kDa PRL 表达抑制细胞增殖。两种类型的细胞系之间的体内结果明显不同。表达 16 kDa PRL 的 HCT-116 细胞表现出血管化和肿瘤形成减少，与已发表的结果一致。表达 16 kDa PRL 的乳腺癌细胞系也表现出体内血管生成的抑制作用，但肿瘤大小或形成没有减少。这些结果表明，16 kDa PRL 对肿瘤形成的影响可能是器官特异性的，并且乳腺细胞对 PRL 促有丝分裂作用的独特敏感性可能在肿瘤形成中起主导作用，从而超过 16 kDa PRL 的抗血管生成作用。 PRLR 存在于 PRLR 外显子 11 选择性剪接产生的多种亚型中。我们一直在研究长亚型 (LF) 和两种短亚型 1a (SF1a) 和 1b (SF1b)，它们缺乏 PRLR 胞内结构域的成分，但保留相同的胞外结构域。目前的数据表明，短型 PRLR 作为通过 LF 受体的分化信号传导的显性失活；它们在扩散中的作用仍有待确定。之前对这些亚型的研究仅检查了 mRNA 水平并通过转染研究确定了潜在的作用。我们开发了第一个可以利用细胞内结构域的差异来区分这三种亚型的抗体。这些多克隆和单克隆抗体的特异性通过对稳定转染三种亚型的 cDNA 的 CHO 细胞进行蛋白质印迹和免疫细胞化学来证明。随后，我们通过免疫组织化学对从合作人体组织网络 (CHTN) 获得的导管癌和小叶癌样本以及包含 144 个导管癌和 24 个小叶癌的组织阵列检查了这些抗体的潜在临床用途。我们能够将这些结果与整个组织样本的定量聚合酶链反应 (qPCR) 相关联。我们发现表达差异表明这些抗体可以用作新的临床工具来区分乳腺癌的不同亚类，并有助于诊断和可能的预后。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Msx2 induces epithelial-mesenchymal transition in mouse mammary epithelial cells through upregulation of Cripto-1.

DOI：
10.1002/jcp.21712
发表时间：
2009-06
期刊：
JOURNAL OF CELLULAR PHYSIOLOGY
影响因子：
5.6
作者：
Di Bari, M. G.;Ginsburg, E.;Plant, J.;Strizzi, L.;Salomon, D. S.;Vonderhaar, B. K.
通讯作者：
Vonderhaar, B. K.

Identification of a regulatory loop for the synthesis of neurosteroids: a steroidogenic acute regulatory protein-dependent mechanism involving hypothalamic-pituitary-gonadal axis receptors.

DOI：
10.1111/j.1471-4159.2009.06192.x
发表时间：
2009-08
期刊：
Journal of neurochemistry
影响因子：
4.7
作者：
Meethal SV;Liu T;Chan HW;Ginsburg E;Wilson AC;Gray DN;Bowen RL;Vonderhaar BK;Atwood CS
通讯作者：
Atwood CS

Up-regulation of MSX2 enhances the malignant phenotype and is associated with Twist 1 expression in human pancreatic cancer cells