Signal transducer and activator of transcription (Stat)5a is a well-established regulator of mammary gland development. Several pathways for activating Stat5a have been identified, but little is known about the mechanisms that regulate its expression in this tissue. In this report, we used immunofluorescent staining to examine Stat5a expression in mammary epithelial cells during normal development and in response to treatment with the ovarian hormones estrogen (E) and progesterone (P). Stat5a was present at very low levels in the prepubertal gland and was highly induced in a subset of luminal epithelial cells during puberty. The percentage of positive cells increased in adult virgin, pregnant, and lactating animals, dropped dramatically during involution, and then increased again after weaning. Ovariectomy ablated Stat5a expression in virgin animals, and treatment with both E and P was necessary to restore it. Double-labeling experiments in animals treated with E plus P for 3 d demonstrated that Stat5a was localized exclusively to cells containing both E and P receptors. Together, these results identify a novel role for E and P in inducing Stat5a expression in the virgin mammary gland and suggest that these hormones act at the cellular level through their cognate receptors.

Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase (MAPK) kinase kinase that activates MAPK pathways, including the c-Jun NH 2 -terminal kinase (JNK) and p38 pathways. MLK3 and its family members have been implicated in JNK-mediated apoptosis. A survey of human cell lines revealed high levels of MLK3 in breast cancer cells. To learn more about MLK3 regulation and its signaling pathways in breast cancer cells, we engineered the estrogen-responsive human breast cancer cell line, MCF-7, to stably, inducibly express FLAG epitope-tagged MLK3. FLAG⅐MLK3 complexes were isolated by affinity purification, and associated proteins were identified by in-gel trypsin digestion followed by liquid chromatography/tandem mass spectrometry. Among the proteins identified were heat shock protein 90␣,␤ (Hsp90) and its kinase-specific cochaperone p50 cdc37 . We show that endogenous MLK3 complexes with Hsp90 and p50 cdc37 . Further experiments demonstrate that MLK3 associates with Hsp90/ p50 cdc37 through its catalytic domain in an activity-independent manner. Upon treatment of MCF-7 cells with geldanamycin, an ansamycin antibiotic that inhibits Hsp90 function, MLK3 levels decrease dramatically. Furthermore, tumor necrosis factor ␣-induced activation of MLK3 and JNK in MCF-7 cells is blocked by geldanamycin treatment. Our finding that geldanamycin treatment does not affect the cellular levels of the downstream signaling components, MAPK kinase 4, MAPK kinase 7, and JNK, suggests that Hsp90/p50 cdc37 regulates JNK signaling at the MAPK kinase kinase level. Previously identified Hsp90/p50 cdc37 clients include oncoprotein kinases and protein kinases that promote cellular proliferation and survival. Our findings reveal that Hsp90/p50 cdc37 also regulates protein kinases involved in apoptotic signaling.

Signal transducer and activator of transcription (Stat)5a is a critical regulator of mammary gland development. Previous studies have focused on Stat5a's role in the late pregnant and lactating gland, and although active Stat5a is detectable in mammary epithelial cells in virgin mice, little is known about its role during early mammary gland development. In this report, we compare mammary gland morphology in pubertal and adult nulliparous wild-type and Stat5aϪ/Ϫ mice. The Stat5a-null mammary glands exhibited defects in secondary and side branching, providing evidence that Stat5a regulates these processes. In addition, Stat5aϪ/Ϫ mammary glands displayed an attenuated proliferative response to pregnancy levels of estrogen plus progesterone (EϩP), suggesting that it plays an important role in early pregnancy. Finally, we examined one potential mediator of Stat5a's effects, receptor activator of nuclear factor-B ligand (RANKL). Stat5aϪ/Ϫ mammary glands were defective in inducing RANKL in response to EϩP treatment. In addition, regulation of several reported RANKL targets, including inhibitor of DNA binding 2 (Id2), cyclin D1, and the cyclin-dependent kinase inhibitor p21 Waf1/Cip1 , was altered in Stat5aϪ/Ϫ mammary cells, suggesting that one or more of these proteins mediate the effects of Stat5a in EϩP-treated mammary epithelial cells. (Endocrinology 151: 2876 -2885, 2010)

Progesterone, through the progesterone receptor (PR), promotes development of the normal mammary gland and is implicated in the etiology of breast cancer. We identified PRA-regulated genes by microarray analysis of cultured epithelial organoids derived from pubertal and adult mouse mammary glands, developmental stages with differing progesterone responsiveness. Microarray analysis showed significant progestin (R5020)-regulation of 162 genes in pubertal organoids and 104 genes in adult organoids, with 68 genes regulated at both developmental stages. Greater induction of receptor activator of NFκB ligand and calcitonin expression was observed in adult organoids, suggesting possible roles in the differential progesterone responsiveness of the adult and pubertal mammary glands. Analysis of the R5020-responsive transcriptome revealed several enriched biological processes including cell proliferation, adhesion, and survival. R5020 both induced Agtr1 and potentiated angiotensin II-stimulated proliferation, highlighting the functional significance of these processes. Striking up-regulation of genes involved in innate immunity processes included the leukocyte chemoattractants serum amyloid A1, 2 and 3 (Saa1, 2, 3). In vivo analysis revealed that progesterone treatment increased SAA1 protein expression and leukocyte density in mammary gland regions undergoing epithelial expansion. These studies reveal novel targets of PRA in mammary epithelial cells and novel linkages of progesterone action during mammary gland development.

The cancer stem cell hypothesis posits that many cancers, including breast cancer, are driven by a subpopulation of cells displaying stem cell properties. These cancer stem cells may also be responsible for mediating tumor metastasis and therapeutic resistance. Recently, a number of therapeutic agents designed to target tumor angiogenesis have been developed and have entered clinical trials in breast and other malignancies. Although these agents may delay tumor progression, once these tumors progress, they display accelerated growth and, as a result, these agents have had only limited effects on prolonging patient survival. Since cancer stem cells may mediate tumor invasion and metastasis, we hypothesize that antiangiogenic agents might increase cancer stem cell populations through the generation of tumor hypoxia. We examined the effect of the multikinase VEGFR inhibitor sunitinib on mammary cancer stem cells in mouse xenografts. Sunitinib treatment increased the percentage of cells expressing the stem cell marker Aldehyde dehydrogenase as assessed by the Aldefluor assay, in SUM159 and MDA-MB-231 breast cancer xenografts in NOD/SCID mice. Tumor cells obtained from sunitinib treated mice exhibit enhanced tumor initiating capacity when transplanted in secondary NOD/SCID mice. In order to determine the relationship between generation of tumor hypoxia and increase cancer stem cell frequency, we utilized hypoxyprobe staining to identify areas of tumor hypoxia and ALDH-1 staining to identify cancer stem cells. Tumors from sunitinib-treated mice displayed regions of tumor hypoxia containing enriched populations of ALDH1-expressing cells which also displayed increased nuclear β-catenin staining suggesting activation of the Wnt pathway in these cells. The effect of hypoxia on the cancer stem cell population was recapitulated in vitro by the demonstration that culture of SUM159 and MDA-MB-231 cells under moderate hypoxia (1-2% oxygen) increased the Aldefluor population by over two-fold. Knockdown of HIF1α utilizing an siRNA completely inhibits the increase in cancer stem cells following hypoxia treatment while knockdown of HIF2α exhibited a significant but lesser effect. Cells grown under hypoxic conditions also exhibited increased expression of phospho-Akt and activated β-catenin. Together these studies suggest that antiangiogenic agents increase the cancer stem cell populations through generation of tissue hypoxia, which, in turn, activates Wnt signaling mediated by HIF1α and HIF2α, Akt and β-catenin. The increase in cancer stem cell populations may account for the limited effectiveness of angiogenesis inhibitors and suggests that the use of these agents should be combined with agents capable of targeting the cancer stem cell population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3336. doi:10.1158/1538-7445.AM2011-3336

There is increasing evidence from our lab and others supporting the “cancer stem cell” hypothesis in breast cancer, which proposes that tumors are driven a small population of cells exhibiting stem cell properties. These cancer stem/progenitor cells may also be responsible for mediating tumor metastasis and resistance to cancer treatments. Recently, several new cancer therapies targeting tumor angiogenesis have been developed and validated in clinical trials. Specifically, the VEGFR and multikinase inhibitors sunitinib and sorafenib have been approved for treatment of some tumor types. However, clinical trials in breast cancer and other malignancies have demonstrated that these therapies ultimately result in drug resistance and only modestly prolong patient survival. Recent reports have suggested that these angiogenesis inhibitors may promote tumor invasiveness and metastasis in rodent breast cancer models. We hypothesize that the tumor stem/progenitor cell population increases in response to hypoxia in anti-angiogenic treated cancers, leading to increased tumor aggressiveness. We have used in vitro and in vivo assays to study the possible role of hypoxia in regulating mammary cancer stem/progenitor cells. We demonstrate that moderate hypoxia (2% oxygen) enriches for stem/progenitor cells in several breast cancer cell lines grown in vitro. The percentage of cells expressing ALDH as assessed by the Aldefluor assay approximately doubles in response to hypoxia. In vivo, sunitinib treatment increases the proportion of Aldefluor positive cells in SUM-159 and MDA-MB-231 breast cancer xenografts in NOD/SCID mice. In addition, tumor cells obtained from sunitinib treated mice exhibit enhanced tumor initiation and growth upon transplantation into secondary NOD/SCID mice. These studies indicate that tissue hypoxia results in expansion of the breast cancer stem/progenitor cell population. This may contribute to the increased aggressiveness and metastasis of cancers that evolve during therapy with angiogenesis inhibitors. If this is the case, then the addition of cancer stem cell targeting agents to antiangiogenic agents may reduce the development of treatment resistance and enhance treatment efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4215.