Prolactin Interactions in Mammary Gland Development and Tumorigenesis

泌乳素（Prolactin， PRL）通过泌乳素受体（Prolactin receptor， PRLR）作用，通过影响有丝分裂和增殖，在乳腺癌发生过程中发挥重要作用。PRL以内分泌和自分泌/旁分泌方式起作用，大约80%的人类乳腺癌活组织检查中都发现了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人乳腺癌细胞系。非转移细胞系MCF7作为对照。PRL及其受体已在正常和癌变的人类乳腺组织和细胞系中发现；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。关于prl在人类乳腺癌中的作用程度，文献记载较少。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3株乳腺癌细胞系。我们发现PRL在体内和体外都具有更强的侵袭性表型。在体外，它抑制细胞凋亡，促进细胞在塑料和细胞外基质上的有丝分裂，促进软琼脂上的生长和细胞迁移。在体内，在原位异种移植模型中，PRL的过表达增加了血管生成和肿瘤发生。免疫组织化学证实的微阵列分析显示，PRL差异调节Wnt通路的各种成员，导致Wnt信号传导增加。Wnt抑制剂Dkk-1逆转了PRL的促生长作用。这是第一个证明PRL通过激活典型Wnt通路起作用的证据。我们还研究了p调控的含有hox相关同源盒的基因MSX2在肿瘤发生过程中所起的作用，对已发表数据库的挖掘发现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。正常小鼠乳腺上皮细胞系NMuMG细胞经MSX2 cDNA稳定转染后，在血清饥饿条件下生长增强，在软琼脂上生长增强，体外细胞迁移增强。这些功能伴随着cyclin E和cyclin D1的组成性过表达。此外，当将NMuMG-MSX2细胞原位注射到免疫受损小鼠的乳腺脂肪垫中时，肿瘤形成增强。在乳腺中过表达MSX2的转基因小鼠中，25%在15月龄时发生肿瘤；对照小鼠没有产生肿瘤。此外，人浸润性乳腺癌免疫组化仅在浸润性肿瘤细胞中显示MSX2阳性，而非浸润性肿瘤细胞为阴性。这些结果提示MSX2可能在小鼠和人乳腺肿瘤的发展和侵袭中发挥重要作用。上皮细胞一旦分离并置于二维（2D）培养中，就会迅速失去组织特异性功能。除了PRL外，laminin-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，在二维培养中，prlr是基底侧定位的，并且与顶端放置的配体物理分离。细胞脱离使受体暴露于PRL的连接，导致信号转导和转录蛋白5 （STAT5）激活因子的激活，但只是短暂的，不足以诱导乳蛋白的表达。我们发现laminin-111重组乳腺细胞为极化的腺泡，允许暴露于PRLR和STAT5的持续激活。使用组成型活性STAT5结构表明后者对于染色质重组和β -酪蛋白转录是必要和充分的。这些结果强调了连续的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。在非贴壁（3D）培养条件下，正常乳腺细胞或乳腺癌细胞作为球体生长已被用作干细胞或祖细胞存在的替代品。自我更新途径可能被激活，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治疗导致球体形成增加。P与PRL合作，在文化中给予更多、更大和高度无序的领域。参与这种激素激活的自我更新的信号通路和受体异构体目前正在研究中。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。PRL的几种异构体存在于人体循环中，包括全长23kda的PRL的n端切割产物的16kda异构体。16kda PRL在体内和体外均显示出抗血管生成的作用，并能减少前列腺癌和结肠癌细胞系肿瘤的形成。我们利用两种乳腺癌细胞系模型（MCF-7和MDA-MB-231）以及结肠癌细胞系HCT-116，在体外和体内探讨了16kda PRL表达的影响。在体外，与空载体对照相比，在所有三种细胞系中，16kda PRL表达抑制细胞增殖。两种细胞系的体内实验结果有显著差异。表达16kda PRL的HCT-116细胞血管化和肿瘤形成减少，与已发表的结果一致。表达16kda PRL的乳腺癌细胞系在体内也表现出血管生成的抑制作用，但没有减少肿瘤的大小或形成。这些结果表明，16kda PRL对肿瘤形成的影响可能是器官特异性的，乳房细胞对PRL的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，从而超过了16kda PRL的抗血管生成作用。由于PRLR外显子11的选择性剪接，PRLR以多种同工形式存在。我们一直在研究长异构体（LF）和两个短异构体1a （SF1a）和1b (SF1b)，它们缺乏PRLR胞内结构域的成分，但保持相同的胞外结构域。目前的数据表明，短形式的PRLRs在通过LF受体的分化信号传导中起主要的负作用；它们在扩散中的作用仍有待确定。以前对这些异构体的研究只检测了mRNA水平，并通过转染研究确定了潜在的作用。我们已经开发出第一种抗体，可以利用细胞内结构域的差异来区分这三种同种异构体。这些多克隆和单克隆抗体的特异性被western blots和免疫细胞化学证实，这些抗体分别稳定地转染了三个同种异构体的cDNA。随后，我们通过免疫组化对来自合作人体组织网络（CHTN）和包含144例导管癌和24例小叶癌的组织阵列的导管癌和小叶癌样本进行了这些抗体的潜在临床应用。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。我们已经发现了表达上的差异，这表明这些抗体可以作为一种新的临床工具来区分不同亚型的乳腺癌，并有助于诊断和可能的预后。

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.