MARCH 2012 CANCER DISCOVERY | 249The BATTLE Trial: Personalizing Therapy for Lung Cancer RESEARCH ARTICLE efficiently induces EMT. In a clinically relevant orthotopic breast cancer mouse model, Twist1 expression is essential for the formation of lung metastasis (5). In hepatocelluar carcinomas, overexpression of Twist1 correlates with EMTassociated changes and metastasis (6). Twist1 expression can also negatively regulate programmed cell death (7) and overcome oncogene-induced senescence (4).These studies indicate that Twist1 plays multiple roles in the regulation of antiapoptosis and metastasis during tumor progression. Active Akt directly phosphorylates Twist1 on serine 42 (S42) and protects cells from apoptosis induced by DNA damage (8). Twist1 promotes crucial prometastatic roles in tumor development and is frequently associated with aberrant hyperactivation of phosphoinositide 3-kinase (PI3K)/Akt. This prompted us to explore whether phosphorylation of Twist1 by Akt is part of a molecular mechanism contributing to Twist1 activity in cancer and particularly in metastasis.We analyzed Twist1 phosphorylation in human cancer cell lines (with different genetic backgrounds) and invasive human breast tumors and addressed its functional importance in a mouse model of spontaneous breast tumor metastasis. We show that Twist1 is ubiquitously phosphorylated in invasive human breast tumors and is required for breast cancer lung metastasis in vivo. Twist1 phosphorylation supports metastasis by transcriptionally upregulating TGF-β2, leading to enhanced activation of TGF-βR/Smad2 signaling, and these effects could be significantly suppressed by the knockdown of TGF-β2 expression. These data suggest that Twist1 phosphorylation not only mediates a feedback loop allowing cross-talk with TGF-β to maintain hyperactivation of PI3K/Akt but can also potentiate cancer metastasis, at least in part, through enhanced TGF-β signaling in cancer cells. Collectively, our data uncover a novel mechanism Metastatic breast tumor cells display an epithelial-mesenchymal transition (EMT) that increases cell motility, invasion, and dissemination. Although the transcription factor Twist1 has been shown to contribute to EMT and cancer metastasis, the signaling pathways regulating Twist1 activity are poorly understood. Here, we show that Twist1 is ubiquitously phosphorylated in 90% of 1,532 invasive human breast tumors. Akt/protein kinase B (PKB)-mediated Twist1 phosphorylation promotes EMT and breast cancer metastasis by modulating its transcriptional target TGF-β2, leading to enhanced TGF-β receptor signaling, which in turn maintains hyperactive phosphoinositide 3-kinase (PI3K)/Akt signaling. Preventing phosphorylation of Twist1, as well as depletion of TGF-β2, significantly impaired the metastatic potential of cancer cells in vivo, indicating a key role of phosphorylated Twist1 (phospho-Twist1) in mediating cross-talk between the PI3K/Akt and TGF-β/Smad signaling axes that supports metastatic tumor development. Our results describe a novel signalin...

Neoadjuvant and adjuvant chemotherapies provide survival benefits to breast cancer patients, in particular in estrogen receptor negative (ER − ) cancers, by reducing rates of recurrences. It is assumed that the benefits of (neo)adjuvant chemotherapy are due to the killing of disseminated, residual cancer cells, however, there is no formal evidence for it. Here, we provide experimental evidence that ER − breast cancer cells that survived high-dose Doxorubicin and Methotrexate based chemotherapies elicit a state of immunological dormancy. Hallmark of this dormant phenotype is the sustained activation of the IRF7/IFN-β/IFNAR axis subsisting beyond chemotherapy treatment. Upregulation of IRF7 in treated cancer cells promoted resistance to chemotherapy, reduced cell growth and induced switching of the response from a myeloid derived suppressor cell-dominated immune response to a CD4 + /CD8 + T cell-dependent anti-tumor response. IRF7 silencing in tumor cells or systemic blocking of IFNAR reversed the state of dormancy, while spontaneous escape from dormancy was associated with loss of IFN-β production. Presence of IFN-β in the circulation of ER − breast cancer patients treated with neoadjuvant Epirubicin chemotherapy correlated with a significantly longer distant metastasis-free survival. These findings establish chemotherapy-induced immunological dormancy in ER − breast cancer as a novel concept for (neo)adjuvant chemotherapy activity, and implicate sustained activation of the IRF7/IFN-β/IFNAR pathway in this effect. Further, IFN-β emerges as a potential predictive biomarker and therapeutic molecule to improve outcome of ER − breast cancer patients treated with (neo)adjuvant chemotherapy.

Matricellular proteins play multiple roles in primary tumor growth, local invasion and tumor angiogenesis. However, their contribution to metastasis and the putative mechanisms involved are less well characterized. In ER-negative human breast cancer, elevated expression levels of the matricellular protein Cysteine-rich angiogenic inducer 61 (CYR61) are associated with more aggressive progression. Here, we investigated the role of CYR61 in breast cancer lung metastasis using the triple negative human breast cancer cell lines MDA-MB-231 and SUM159. Silencing of CYR61 significantly decreased lung metastasis from tumors orthotopically implanted in pre-irradiated or naive mammary tissue and upon tail vein injection. Constitutive CYR61 silencing impaired cancer cell extravasation to the lung during the first 24 hours after tail vein injection. In contrast, CYR61 inducible silencing starting 24 hours after cancer cell injection had no impact on lung metastasis formation. In vitro experiments revealed that CYR61 silencing decreased cancer cell transendothelial migration and motility, reduced CYR61 levels present at the cell surface and sensitized cancer cells to anoikis. Furthermore, we demonstrate that CYR61-dependent cell survival under non-adhesive conditions relied, at least partially, on β1 integrin ligation and AMPKα signaling while it was independent of AKT, FAK and ERK1/2 activation. Our data provide the first evidence that CYR61 promotes breast cancer lung metastasis by facilitating tumor cell extravasation and protecting from anoikis during initial seeding to the lung. The uncovered CYR61-β1 integrin-AMPKα axis may serve as a potential therapeutic target to prevent breast cancer metastasis to the lung.

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The significance of matricellular proteins during development and cancer progression is widely recognized. However, how these proteins actively contribute to physiological development and pathological cancer progression is only partially elucidated. In this study, we investigated the role of the matricellular protein Cysteine-rich 61 (CYR61) in pancreatic islet development and carcinogenesis. Transgenic expression of CYR61 in β cells (Rip1CYR mice) caused irregular islets morphology and distorted sorting of α cells, but did not alter islets size, number or vascularization. To investigate the function of CYR61 during carcinogenesis, we crossed Rip1CYR mice with Rip1Tag2 mice, a well-established model of β cell carcinogenesis. Beta tumors in Rip1Tag2CYR mice were larger, more invasive and more vascularized compared to tumors in Rip1Tag2 mice. The effect of CYR61 on angiogenesis was fully abrogated by treating mice with the anti-VEGFR2 mAb DC101. Results from in vitro assays demonstrated that CYR61 modulated integrin α6β1-dependent invasion and adhesion without altering its expression. Taken together, these results show that CYR61 expression in pancreatic β cells interferes with normal islet architecture, promotes islet tumor growth, invasion and VEGF/VERGFR-2-dependent tumor angiogenesis. Taken together, these observations demonstrate that CYR61 acts as a tumor-promoting gene in pancreatic neuroendocrine tumors.

Branching morphogenesis is a characteristic feature of many essential organs such as the lung, kidney, and most glands, and the net result of two tissue behaviors: branch point initiation and elongation. Each branched organ has a distinct architecture customized to its physiological function, but how patterning occurs in these ramified tubular structures is a fundamental problem of development. The conserved molecular pathways that control branching morphogenesis are well defined, but their contribution to growth versus branch patterning is often challenging to discern. Here we use quantitative 3D morphometrics, time-lapse imaging and manipulation of ex vivo cultured embryonic organs, and genetically modified mice to address these questions in the developing mammary gland. Our results show that the embryonic epithelial trees are highly complex in topology owing to the flexible use of two distinct modes of branch point initiation: lateral branching and tip bifurcation. This non-stereotypy was contrasted by the remarkably constant average branch frequency indicating a time-invariant, yet stochastic propensity to branch. The probability to branch was malleable, and could be increased or decreased by exogenous Fgf10 and Tgf-β1, respectively. Our in vivo and ex vivo time-lapse imaging also suggest involvement of convergent extension in mammary branch elongation, a conclusion supported by the phenotype analysis of mice deficient in the PCP component Vangl2.

Mammary gland development begins with the appearance of epithelial placodes that invaginate, sprout, and branch to form small arborized trees by birth. The second phase of ductal growth and branching is driven by the highly invasive structures called terminal end buds (TEBs) that form at ductal tips at the onset of puberty. Ectodysplasin (Eda), a tumor necrosis factor-like ligand, is essential for the development of skin appendages including the breast. In mice, Eda regulates mammary placode formation and branching morphogenesis, but the underlying molecular mechanisms are poorly understood. Fibroblast growth factor (Fgf) receptors have a recognized role in mammary ductal development and stem cell maintenance, but the ligands involved are ill-defined. Here we report that Fgf20 is expressed in embryonic mammary glands and is regulated by the Eda pathway. Fgf20 deficiency does not impede mammary gland induction, but compromises mammary bud growth, as well as TEB formation, ductal outgrowth and branching during puberty. We further show that loss of Fgf20 delays formation of Eda-induced supernumerary mammary buds and normalizes the embryonic and postnatal hyperbranching phenotype of Eda overexpressing mice. These findings identify a hitherto unknown function for Fgf20 in mammary budding and branching morphogenesis.

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