A knotty turnabout?: Akt1 as a metastasis suppressor.

Akt is well known to enhance malignancy and is recognized as a key target for antineoplastic therapies. However, intriguing findings reported by Yoeli-Lerner et al. in the November 23, 2005 issue of Molecular Cell, suggest a novel, antimetastasis function of Akt: activation of Akt1 inhibited invasion in some cancer cells. One possible mechanism for this surprising phenotype was that Akt activated the E3 ubiquitin ligase HDM2, causing ubiquitination and degradation of NFAT, an invasion-promoting factor. These findings clearly justify further investigations and, if validated in vivo, call for reevaluation of some Akt-targeting therapeutic strategies currently under development.

Activation of p21(CIP1/WAF1) in mammary epithelium accelerates mammary tumorigenesis and promotes lung metastasis.

While p21 is well known to inhibit cyclin-CDK activity in the nucleus and it has also been demonstrated to have oncogenic properties in different types of human cancers. In vitro studies showed that the oncogenic function of p21is closely related to its cytoplasmic localization. However, it is unclear whether cytoplasmic p21 contributes to tumorigenesis in vivo. To address this question, we generated transgenic mice expressing the Akt-phosphorylated form of p21 (p21T145D) in the mammary epithelium. The results showed that Akt-activated p21 was expressed in the cytoplasm of mammary epithelium. Overexpression of Akt-activated p21 accelerated tumor onset and promoted lung metastasis in MMTV/neu mice, providing evidence that p21, especially cytoplasmic phosphorylated p21, has an oncogenic role in promoting mammary tumorigenesis and metastasis.

Preclinical testing of clinically applicable strategies for overcoming trastuzumab resistance caused by PTEN deficiency.

PURPOSE: We have previously shown that PTEN loss confers trastuzumab resistance in ErbB2-overexpressing breast cancer using cell culture, xenograft models, and patient samples. This is a critical clinical problem because trastuzumab is used in a variety of therapeutic regimens, and at the current time, there are no established clinical strategies to overcome trastuzumab resistance. Here, we did preclinical studies on the efficacy of clinically applicable inhibitors of the Akt/mammalian target of rapamycin (mTOR) pathway to restore trastuzumab sensitivity to PTEN-deficient cells. EXPERIMENTAL DESIGN: Cell culture and xenograft models were used to test a panel of clinically applicable, small-molecule inhibitors of the Akt/mTOR signal transduction pathway, a critical pathway downstream of ErbB2, and identify compounds with the ability to restore trastuzumab sensitivity to PTEN-deficient cells. RESULTS: When trastuzumab was combined with the Akt inhibitor triciribine, breast cancer cell growth was inhibited and apoptosis was induced. In a xenograft model, combination therapy with trastuzumab and triciribine dramatically inhibited tumor growth. The combination of trastuzumab and the mTOR inhibitor RAD001 also slowed breast cancer cell growth in vitro and in vivo. CONCLUSIONS: Combining trastuzumab with inhibitors of the Akt/mTOR pathway is a clinically applicable strategy and combinations of trastuzumab with triciribine or RAD001 are promising regimens for rescue of trastuzumab resistance caused by PTEN loss.

Progesterone receptor repression of prolactin/signal transducer and activator of transcription 5-mediated transcription of the beta-casein gene in mammary epithelial cells.

Prolactin (PRL) and glucocorticoids act synergistically to stimulate transcription of the beta-casein milk protein gene. Signal transducer and activator of transcription 5 (Stat5) mediates PRL-dependent trans-activation, and glucocorticoid potentiation occurs through cross talk between glucocorticoid receptor (GR) and Stat5 at the beta-casein promoter. In the mouse, progesterone withdrawal leads to terminal differentiation and secretory activation of the mammary gland at parturition, indicating progesterone's role in repressing milk protein gene expression during pregnancy. To investigate the mechanism of the inhibitory action of progesterone, experiments were performed with cell culture systems reconstituted to express progesterone receptor (PR), the PRL receptor/Stat5 signaling pathway, and GR, enabling evaluation of PR, GR, and Stat5 interactions at the beta-casein promoter. With COS-1, normal murine mammary gland, HC-11, and primary mammary epithelial cells, progestin-PR directly repressed the PRL receptor/Stat5a signaling pathway's mediation of PRL-induced beta-casein transcription. Progestin-PR also inhibited glucocorticoid-GR enhancement of PRL induced trans-activation of beta-casein. Inhibition depended on a functional PR DNA binding domain and specific PR-DNA interactions at the beta-casein promoter. Chromatin immunoprecipitation assays in HC-11 cells revealed recruitment of PR and Stat5a to the beta-casein promoter by progestin or PRL, respectively. Recruitment was disrupted by cotreatment with progestin and PRL, suggesting a mutual interference between activated PR and Stat5a. Without PRL, progestin-PR also recruited Stat5a to the beta-casein promoter, suggesting that recruitment of an unactivated form of Stat5a may contribute to inhibition of beta-casein by progesterone. These results define a negative cross talk between PR and Stat5a/GR that may contribute to the physiological role of progesterone to repress lactogenic hormone induction of the beta-casein gene in the mammary gland during pregnancy.

ErbB2 increases vascular endothelial growth factor protein synthesis via activation of mammalian target of rapamycin/p70S6K leading to increased angiogenesis and spontaneous metastasis of human breast cancer cells.

ErbB2 overexpression in breast tumors results in increased metastasis and angiogenesis and reduced survival. To study ErbB2 signaling mechanisms in metastasis and angiogenesis, we did a spontaneous metastasis assay using MDA-MB-435 human breast cancer cells stably transfected with constitutively active ErbB2 kinase (V659E), a kinase-dead mutant of ErbB2 (K753M), or vector control (neo). Mice injected with V659E had increased metastasis incidence and tumor microvessel density than mice injected with K753M or control. Increased angiogenesis in vivo from the V659E transfectants paralleled increased angiogenic potential in vitro. V659E produced increased vascular endothelial growth factor (VEGF) through increased VEGF protein synthesis. This was mediated through signaling events involving extracellular signal-regulated kinase, phosphatidylinositol 3-kinase/Akt, mammalian target of rapamycin (mTOR), and p70S6K. The V659E xenografts also had significantly increased phosphorylated Akt, phosphorylated p70S6K, and VEGF compared with controls. To validate the clinical relevance of these findings, we examined 155 human breast tumor samples. Human tumors that overexpressed ErbB2, which have been previously shown to have higher VEGF expression, showed significantly higher p70S6K phosphorylation as well. Increased VEGF expression also significantly correlated with higher levels of Akt and mTOR phosphorylation. Additionally, patients with tumors having increased p70S6K phosphorylation showed a trend for worse disease-free survival and increased metastasis. Our findings show that ErbB2 increases VEGF protein production by activating p70S6K in cell lines, xenografts, and in human cancers and suggest that these signaling molecules may serve as targets for antiangiogenic and antimetastatic therapies.

The mineralocorticoid receptor may compensate for the loss of the glucocorticoid receptor at specific stages of mammary gland development.

To study the role of glucocorticoid receptor (GR) at different stages of mammary gland development, mammary anlage were rescued from GR-/- mice by transplantation into the cleared fat pad of wild-type mice. In virgin mice, GR-/- outgrowths displayed abnormal ductal morphogenesis characterized by distended lumena, multiple layers of luminal epithelial cells in some regions along the ducts, and increased periductal stroma. In contrast, the loss of GR did not result in overt phenotypic changes in mammary gland development during pregnancy, lactation, and involution. Surprisingly, despite the known synergism between glucocorticoids and prolactin in the regulation of milk protein gene expression, whey acidic protein and beta-casein mRNA levels were unaffected in GR-/- transplants as compared with wild-type transplants. That mineralocorticoid receptor (MR) might compensate for the loss of GR was suggested by the detection of MR in the mammary gland at d 1 of lactation. This hypothesis was tested using explant cultures derived from the GR-/- transplants in which the mineralocorticoid fludrocortisone was able to synergistically induce beta-casein gene expression in the presence of prolactin and insulin. These studies suggest that MR may compensate for the absence of GR at some, but not at all stages of mammary gland development.

14-3-3ζ orchestrates mammary tumor onset and progression via miR-221-mediated cell proliferation.

14-3-3ζ is overexpressed in more than 40% of breast cancers, but its pathophysiologic relevance to tumorigenesis has not been established. Here, we show that 14-3-3ζ overexpression is sufficient to induce tumorigenesis in a transgenic mouse model of breast cancer. MMTV-LTR promoter-driven HA-14-3-3ζ transgenic mice (MMTV-HA-14-3-3ζ) developed mammary tumors, whereas control mice did not. Whey acidic protein promoter-driven HA-14-3-3ζ transgenic mice (WAP-HA-14-3-3ζ) developed hyperplastic lesions and showed increased susceptibility to carcinogen-induced tumorigenesis. When crossed with MMTV-neu transgenic mice, 14-3-3ζ.neu transgenic mice exhibited accelerated mammary tumorigenesis and metastasis compared with MMTV-neu mice. Mechanistically, 14-3-3ζ overexpression enhanced MAPK/c-Jun signaling, leading to increased miR-221 transcription, which inhibited p27 CDKI translation and, consequently, promoted cell proliferation. Importantly, this 14-3-3ζ-miR-221-p27 proliferation axis is also functioning in breast tumors in patients and is associated with high-grade cancers. Taken together, our findings show that overexpression of 14-3-3ζ has a causal role in mammary tumorigenesis and progression, acting through miR-221 in cooperation with known oncogenic events to drive neoplastic cell proliferation.

Activation of murine double minute 2 by Akt in mammary epithelium delays mammary involution and accelerates mammary tumorigenesis.

Amplification or overexpression of murine double minute 2 (MDM2) promotes a variety of human tumors by degrading tumor suppressor proteins such as p53. Phosphorylation of MDM2 on Ser(166) and Ser(186) by the survival kinase Akt inhibits p53-mediated apoptosis. However, it is unclear whether this pathway contributes to normal or malignant pathophysiology in vivo. To address these questions, we generated transgenic mice expressing the Akt-phosphorylated form of MDM2 (MDM2DDS166D/S186D) in the mammary epithelium. Activation of MDM2 delayed mammary gland involution and accelerated tumor progression in mouse mammary tumor virus/neu transgenic mice by inhibiting apoptosis in a manner associated with decreased p53 expression. Our findings offer in vivo evidence that activation of MDM2 by Akt contributes to mammary development and tumorigenesis.

14-3-3 zeta down-regulates p53 in mammary epithelial cells and confers luminal filling.

Recent progress in diagnostic tools allows many breast cancers to be detected at an early preinvasive stage. Thus, a better understanding of the molecular basis of early breast cancer progression is essential. Previously, we discovered that 14-3-3 zeta is overexpressed in >40% of advanced breast cancers, and this overexpression predicts poor patient survival. Here, we examined at what stage of breast disease 14-3-3 zeta overexpression occurs, and we found that increased expression of 14-3-3 zeta begins at atypical ductal hyperplasia, an early stage of breast disease. To determine whether 14-3-3 zeta overexpression is a decisive early event in breast cancer, we overexpressed 14-3-3 zeta in MCF10A cells and examined its effect in a three-dimensional culture model. We discovered that 14-3-3 zeta overexpression severely disrupted the acini architecture resulting in luminal filling. Proper lumen formation is a result of anoikis, apoptosis due to detachment from the basement membrane. We found that 14-3-3 zeta overexpression conferred resistance to anoikis. Additionally, 14-3-3 zeta overexpression in MCF10A cells and in mammary epithelial cells (MEC) from 14-3-3 zeta transgenic mice reduced expression of p53, which is known to mediate anoikis. Mechanistically, 14-3-3 zeta induced hyperactivation of the phosphoinositide 3-kinase/Akt pathway which led to phosphorylation and translocation of the MDM2 E3 ligase resulting in increased p53 degradation. Ectopic expression of p53 restored luminal apoptosis in 14-3-3 zeta-overexpressing MCF10A acini in three-dimensional cultures. These data suggest that 14-3-3 zeta overexpression is a critical event in early breast disease, and down-regulation of p53 is one of the mechanisms by which 14-3-3 zeta alters MEC acini structure and increases the risk of breast cancer.