No abstract

To address the role of transforming growth factor (TGF) ␤ in the progression of established tumors while avoiding the confounding inhibitory effects of TGF-␤ on early transformation, we generated doxycycline (DOX)-inducible triple transgenic mice in which active TGF-␤1 expression could be conditionally regulated in mouse mammary tumor cells transformed by the polyomavirus middle T antigen. DOX-mediated induction of TGF-␤1 for as little as 2 weeks increased lung metastases >10-fold without a detectable effect on primary tumor cell proliferation or tumor size. DOX-induced active TGF-␤1 protein and nuclear Smad2 were restricted to cancer cells, suggesting a causal association between autocrine TGF-␤ and increased metastases. Antisense-mediated inhibition of TGF-␤1 in polyomavirus middle T antigen-expressing tumor cells also reduced basal cell motility, survival, anchorage-independent growth, tumorigenicity, and metastases. Therefore, induction and/or activation of TGF-␤ in hosts with established TGF-␤-responsive cancers can rapidly accelerate metastatic progression.

Biomarkers that predict therapeutic response are essential for the development of anticancer therapies. We have used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to directly analyze protein profiles in mouse mammary tumor virus/HER2 transgenic mouse frozen tumor sections after treatment with the erbB receptor inhibitors OSI-774 and Herceptin. Inhibition of tumor cell proliferation and induction of apoptosis and tumor reduction were predicted by a >80% reduction in thymosin ␤4 and ubiquitin levels that were detectable after 16 hours of a single drug dose before any evidence of in situ cellular activity. These effects were time-and dose-dependent, and their spatial distribution in the tumor correlated with that of the small-molecule inhibitor OSI-774. In addition, they predicted for therapeutic synergy of OSI-774 and Herceptin as well as for drug resistance. These results suggest that drug-induced early proteomic changes as measured by MALDI-MS can be used to predict the therapeutic response to established and novel therapies.

Purpose: A significant fraction of HER2-overexpressing breast cancers exhibit resistance to the HER2 antibody trastuzumab. Hyperactivity of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway confers trastuzumab resistance, and mammalian target of rapamycin (mTOR) is a major downstream effector of PI3K/AKT. Therefore, we examined whether mTOR inhibitors synergize with trastuzumab. Experimental Design: Immunocompetent mice bearing HER2 + mammary tumors were treated with trastuzumab, the mTOR inhibitor rapamycin, or the combination. Mice were imaged for tumor cell death using an optical Annexin-V probe and with [18 F] FDG positron emission tomography. The signaling and growth effects of the mTOR inhibitor RAD001 on HER2 + cells treated with trastuzumab or lapatinib were evaluated. Results: Treatment of mice with trastuzumab plus rapamycin was more effective than single-agent treatments, inducing complete regression of 26 of 26 tumors. The combination induced tumor cell death (Annexin-V binding) and inhibited FDG uptake. Rapamycin inhibited mTOR and tumor cell proliferation as determined by phosphorylated S6 and Ki-67 immunohistochemistry, respectively. In culture, the combination of RAD001 plus trastuzumab inhibited cell growth more effectively than either drug alone. Trastuzumab partially decreased PI3K but not mTOR activity. Knockdown of TSC2 resulted in HER2-independent activation of mTOR and dampened the response to trastuzumab and lapatinib. Treatment with the HER2 inhibitor lapatinib decreased phosphorylated S6 and growth in TSC2-expressing cells but not in TSC2-knockdown cells.

Overexpression of ErbB-2͞Neu has been causally associated with mammary epithelial transformation. Here we report that blockade of the epidermal growth factor receptor (EGFR) kinase with AG-1478 markedly delays breast tumor formation in mouse mammary tumor virus (MMTV)͞Neu ؉ MMTV͞transforming growth factor ␣ bigenic mice. This delay was associated with inhibition of EGFR and Neu signaling, reduction of cyclin-dependent kinase 2 (Cdk2) and mitogenactivated protein kinase (MAPK) activities and cyclin D1, and an increase in the levels of the Cdk inhibitor p27 Kip1 . In addition, BrdUrd incorporation into tumor cell nuclei was prevented with no signs of tumor cell apoptosis. These observations prompted us to investigate the stability of p27. Recombinant p27 was degraded rapidly in vitro by untreated but not by AG-1478-treated tumor lysates. Proteasome depletion of the tumor lysates, addition of the specific MEK1͞2 inhibitor U-0126, or a T187A mutation in recombinant p27 all prevented p27 degradation. Cdk2 and MAPK precipitates from untreated tumor lysates phosphorylated recombinant wild-type p27 but not the T187A mutant in vitro. Cdk2 and MAPK precipitates from AG-1478-treated tumors were unable to phosphorylate p27 in vitro. These data suggest that increased signaling by ErbB receptors up-regulates MAPK activity, which, in turn, phosphorylates and destabilizes p27, thus contributing to dysregulated cell cycle progression.T he ErbB family of receptors includes the epidermal growth factor receptor (EGFR) ErbB-1, the orphan ErbB-2 (Neu), and the Neuregulin receptors ErbB-3 and ErbB-4 (1-4). Binding of ligands to the ectodomain of these receptors results in the formation of homodimeric and heterodimeric complexes (5), which is followed rapidly by the activation of the receptors' intrinsic tyrosine kinase. Consequently, phosphorylation of specific C-terminal tyrosine residues and the recruitment of specific second messengers activate a plethora of intracellular signaling pathways that play central roles in cell proliferation, development, differentiation, migration, and oncogenesis (6).Many studies support a pivotal role for the orphan ErbB-2͞ Neu in ErbB signaling and tumorigenesis (7-9), suggesting that this receptor plays a critical role in the cellular responses mediated by the ligand-dependent activation of other ErbB receptors (10, 11). Indeed, overexpression of ErbB-2͞Neu alone or in combination with EGFR (ErbB-1) or ErbB-3 in vitro can transform mammary epithelial cells (8) and fibroblasts (12, 13), respectively. A central role for ErbB-2͞Neu in transformation has been shown by using transgenic mice overexpressing the protooncogene under the control of the mouse mammary tumor virus (MMTV) promoter (14, 15). In these tumors, DNA sequence analysis revealed the presence of a 16-aa in-frame deletion in the extracellular domain of ErbB-2͞Neu, resulting in a constitutively activated receptor capable of transforming Rat-1 fibroblasts (16). So far, no such activating mutations have been found in human tumors, where the m...

No abstract

Expansion of conifer woodlands into sagebrush (Artemisia spp.) ecosystems is a primary threat to the greater sage‐grouse (Centrocercus urophasianus) in the Great Basin, southeast Oregon, USA. Conifer removal to restore sage‐grouse habitats has been widely implemented, yet limited information exists on the effects of conifer expansion on sage‐grouse habitat selection. Our objective was to evaluate the landscape‐scale effects of conifer expansion on selection of sites for nesting and to estimate direction, magnitude, and scale of effects. We classified 160 nests and 167 available sites with random forests during 2010–2011 to assess effects of conifer cover and tree clustering at multiple scales along with other vegetation and topographic covariates on selection of nesting habitat. Relative probability of nesting was negatively associated with >3% conifer cover within 800 m of nests. When trees were present within 800 m of nests, sage‐grouse nested where trees were clustered rather than dispersed, suggesting selection for more open habitat. Results further indicated that sage‐grouse are nesting in landscapes susceptible to conifer expansion that have yet to be invaded. Sage‐grouse are expected to lose nesting habitat as conifer expansion continues, but management intervention may be a possible solution to increase habitat availability where open space for nesting is a limiting factor. © 2016 The Wildlife Society.

Sagebrush (Artemisia spp.) obligate wildlife species such as the imperiled greater sage-grouse (Centrocercus urophasianus) face numerous threats including altered ecosystem processes that have led to conifer expansion into shrub-steppe. Conifer removal is accelerating despite a lack of empirical evidence on grouse population response. Using a before-after-control-impact design at the landscape scale, we evaluated effects of conifer removal on two important demographic parameters, annual survival of females and nest survival, by monitoring 219 female sage-grouse and 225 nests in the northern Great Basin from 2010 to 2014. Estimates from the best treatment models showed positive trends in the treatment area relative to the control area resulting in an increase of 6.6% annual female survival and 18.8% nest survival relative to the control area by 2014. Using stochastic simulations of our estimates and published demographics, we estimated a 25% increase in the population growth rate in the treatment area relative to the control area. This is the first study to link sage-grouse demographics with conifer removal and supports recommendations to actively manage conifer expansion for sage-grouse conservation. Sage-grouse have become a primary catalyst for conservation funding to address conifer expansion in the West, and these findings have important implications for other ecosystem services being generated on the wings of species conservation.