We are examining the role of the autocrine PRL system in metastasis. For these studies we used the highly metastatic MDA-MB-435 human breast cancer cell line. 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 435 cells 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, overexpression of PRL increased 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 have examined the role that the Hox-related homeobox-containing gene, Msx2, plays during branching morphogenesis and tumorigenesis where our studies in vivo and in vitro showed that P in the presence of E regulates its expression. Mining of published databases found a strong correlation between elevated MSX2 expression and estrogen receptor (ER) positive breast cancer. Epithelial-Mesenchymal Transition (EMT) is a process occurring during both embryogenesis and early stages of invasive cancer. Epithelial cells that undergo EMT become more migratory and invasive with a mesenchymal morphology. We assessed EMT induction in a mouse mammary epithelial cell line driven by Msx2. NMuMG cells, a normal mouse mammary epithelial cell line, stably-transfected with a Msx2 cDNA showed downregulation of an epithelial marker E-cadherin and upregulation of the mesenchymal markers vimentin and N-cadherin. Furthermore, overexpression of Cripto-1, a member of the epidermal growth factor-CFC protein family already known to be involved in EMT, was detected in Msx2-transfected cells. The expression of Cripto-1 was accompanied by activation of the tyrosine kinase c-Src pathway and an increase in the invasive ability of the cells. Functional assays also demonstrated inhibition of the invasive behavior of the Msx2-transfected cells by a c-Src specific inhibitor. Moreover, 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 promoting EMT in epithelial cells that acquire properties involved in tumor invasion. MSX2 is also an important downstream component of the ras gene signaling pathway. Most pancreatic cancers harbor a K-ras gene mutation. In collaboration with Dr. Satoh in Sendai, Japan, we also showed that MSX-2 was expressed in four human pancreatic cancer cell lines and in 23 of 32 (71.8 %) of human pancreatic carcinoma tissues as determined by RT-PCR and immunohistochemistry, respectively. Increased expression of MSX2 was significantly correlated with higher tumor grade. BxPC3 cells stably expressing MSX2 showed a flattened and scattered morphology accompanied by a change in localization of E-cadherin and beta-catenin from membrane to cytoplasm. Cell proliferation rate, cell migration and anchorage independent cell growth were enhanced in MSX2 expressing cells. MSX2 expressing cells also show significantly more frequent liver metastases and disseminations in nude mice than did control cells when cells were injected into the pancreas. In addition, microarray analysis revealed a significant induction of Twist 1 by MSX2. Twist 1 expression was significantly associated with MSX2 expression in human pancreatic carcinomas and was down-regulated in MSX2 pancreatic cancer cells by transfected with a small interfering RNA expressing vector to inactivate MSX2. Taken together these data indicate that MSX2 plays a crucial role in pancreatic cancer cell development by leading pancreatic cancer cells to a state consistent with EMT through enhanced expression of Twist 1. Utilizing our knowledge of the MSX homeobox genes, we examined the role of the homeobox-containing gene, BP-1, in human breast cancer. In collaboration with Dr. Pat Berg of George Washington University who showed this gene to be more prevalent in breast cancer from African American women vs. Caucasians, and over-expressed in all ER negative breast cancers examined, We examined the role of BP1 in aggressiveness using MCF-7 breast cancer cell lines stably overexpressing BP1 (MCF-BP1). Four assays were used to predict aggressiveness: serum independent growth, anchorage independent growth, invasion potential, and tumorigenesis in mice. MCF-BP1 cells were more aggressive by all four assays; tumors were larger in mice injected with BP1 cells than in mice injected with control cells. MCF-BP1 cells developed tumors in mice in the absence of estrogen, while control cells only produced tumors in the presence of estrogen supplementation, indicating that high-level BP1 expression can confer estrogen independence. One mechanism of estrogen independence is repression of the estrogen receptor alpha (ER) gene. We show that pBP1 binds to the first intron of the ER gene and that MCF-BP1 cells are more frequently ER negative by immunostaining than control cells, suggesting that BP1 may repress the ER. Moreover, in the presence of increased BP1 levels, tumor size was increased in human breast carcinoma as well as in mice. Microarray analysis demonstrated increased expression of genes involved in angiogenesis, invasion and metastasis, including c-FOS, MET, TWIST and MMP9. Thus, by all measures we examined, overexpression of BP1 confers a more aggressive phenotype on breast cancer cells, making BP1 a strong potential therapeutic target. Expression of BP-1 in breast cancer cells is regulated by PRL.
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