Paraquat (PQ) is a potential human neurotoxicant and is used in models of oxidative stress. We determined the toxicokinetics (TK) and toxicodynamics (TD) of PQ in adult mouse brain following repeated or prolonged PQ exposure. PQ accumulated in different brain regions and reached a plateau after ~18 i.p. (10 mg/kg) doses and resulted in modest morbidity and mortality unpredictably associated with dose interval and number. PQ had divergent effects on horizontal locomotor behavior depending on the number of doses. PQ decreased striatal dopamine levels after the 18th to 36th i.p. dose (10 mg/kg) and reduced the striatal level of tyrosine hydroxylase. Drinking water exposure to PQ (0.03– 0.05 mg/ml) did not result in any mortality and resulted in concentration and time dependent levels in the brain. The brain half-life of PQ varied with mouse strain. PQ accumulates and may saturate a site in mouse brain resulting in complex PQ level and duration-related consequences. These findings should alter our risk assessment of this compound and demonstrate a useful, but complex dynamic model for understanding the consequences of PQ in the brain.

Keywords: CDK, cyclin, paclitaxel, Smad3, triple negative breast cancer Abbreviations: BCSC, breast cancer stem cells; CDK, cyclin dependent kinase; CDKi, cyclin dependent kinase inhibitor; CK, cytokeratin; EGFR, epidermal growth factor receptor; EMT, epithelial-mesenchymal transition; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; Pin1, peptidyL-prolyl cis-trans isomerase NIMA-interacting 1; PR, progesterone receptor; TNBC, triple negative breast cancerBreast cancer onset and disease progression have been linked to members of the TGFb superfamily and their downstream signaling components, the Smads. Alterations in Smad3 signaling are associated with the dichotomous role of TGFb in malignancy, mediating both tumor suppressant and pro-metastatic behaviors. Overexpression of cell cycle regulators, cyclins D and E, renders cyclin-dependent kinases (CDKs) 4/2 hyperactive. Noncanonical phosphorylation of Smad3 by CDK4/2 inhibits tumor suppressant actions of Smad3. We hypothesized that CDK inhibition (CDKi) would restore Smad3 action and help promote cancer cell regression. Treatment of triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, MDA-MB-436, Hs578T) with CDK2i or CDK4i resulted in increased Smad3 activity and decreased cell migration. Transfection with a 5M Smad3 construct containing inhibitory mutations in 5 CDK phosphorylation sites also resulted in decreased TNBC cell migration and invasion. MDA-MB-231 cells treated with CDK2i or CDK4i resulted in decreased Smad3 protein phosphorylation at the CDK phosphorylation T179 site, decreased MMP2 and c-myc expression, and increased p15 and p21 expression. Using a novel transfected cell array, we found that CDK2i treatment decreased activity of the epithelial-to-mesenchymal transition related transcription factors Snail and Twist. In vivo studies in an MDA-MB-231 tumor model showed that individual and combination treatment with paclitaxel and CDK2i resulted in decreased tumor volume and Ki67 staining. Collectively, these data support further investigation of targeted CDK inhibitors as a promising therapeutic strategy for TNBC, a breast cancer subtype with limited treatment options.

No abstract

Cyclin D1/CDK4 activity is upregulated in up to 50% of breast cancers and CDK4-mediated phosphorylation negatively regulates the TGFβ superfamily member Smad3. We sought to determine if CDK4 inhibition and doxorubicin chemotherapy could impact Smad3-mediated cell/colony growth and apoptosis in breast cancer cells. Parental and cyclin D1-overexpressing MCF7 cells were treated with CDK4 inhibitor, doxorubicin, or combination therapy and cell proliferation, apoptosis, colony formation, and expression of apoptotic proteins were evaluated using an MTS assay, TUNEL staining, 3D Matrigel assay, and apoptosis array/immunoblotting. Study cells were also transduced with WT Smad3 or a Smad3 construct resistant to CDK4 phosphorylation (5M) and colony formation and expression of apoptotic proteins were assessed. Treatment with CDK4 inhibitor/doxorubicin combination therapy, or transduction with 5M Smad3, resulted in a similar decrease in colony formation. Treating cyclin D overexpressing breast cancer cells with combination therapy also resulted in the greatest increase in apoptosis, resulted in decreased expression of anti-apoptotic proteins survivin and XIAP, and impacted subcellular localization of pro-apoptotic Smac/DIABLO. Additionally, transduction of 5M Smad3 and doxorubicin treatment resulted in the greatest change in apoptotic protein expression. Collectively, this work showed the impact of CDK4 inhibitor-mediated, Smad3-regulated tumor suppression, which was augmented in doxorubicin-treated cyclin D-overexpressing study cells.

A consensus about the functions of human wild-type or mutated α-synuclein (αSYN) is lacking. Both forms of αSYN are implicated in Parkinson’s disease, whereas the wild-type form is implicated in substance abuse. Interactions with other cellular proteins and organelles may meditate its functions. We developed a series of congenic mouse lines containing various allele doses or combinations of the human wild type αSYN (hwαSYN) or a doubly mutated (A30P*A53T) αSYN (hm2αSYN) in a C57Bl/6J line spontaneously deleted in mouse αSYN (C57BL/6JOla). Both transgenes had a functional role in the nigrostriatal system, demonstrated by significant elevations in striatal catecholamines, metabolites, and the enzyme tyrosine hydroxylase compared to null-mice without a transgene. Consequences occurred when the transgenes were expressed at a fraction of the endogenous level. Hemizygous congenic mice did not exhibit any change in the number or size of dopaminergic neurons in the ventral midbrain at nine months of age. Human αSYN was predominantly located in neuronal cell bodies, neurites, synapses, and in intraneuronal/intraneuritic aggregates. The hm2αSYN transgene resulted in more aggregates and dystrophic neurites than did the hw5 transgene. The hwαSYN transgene resulted in higher expression of two striatal proteins, synaptogamin 7 and UCHL1, compared to the levels of the hm2αSYN transgene. These observations suggest that mutations in αSYN may impair specific functional domains, leaving others intact. These lines may also be useful for exploring interactions between hαSYN and environmental or genetic risk factors in dopamine-related disorders using a mouse model.

Stem cell microenvironments decrease the invasiveness of cancer cells, and elucidating the mechanisms associated with disease regression could further the development of targeted therapies for aggressive cancer subtypes. To this end, we applied an emerging technology, TRanscriptional Activity CEll aRray (TRACER), to investigate the reprogramming of triple-negative breast cancer (TNBC) cells in conditions that promoted a less aggressive phenotype. The repressive environment was established through exposure to mouse embryonic stem cell conditioned media (mESC CM). Assessment of carcinogenic phenotypes indicated that mESC CM exposure decreased proliferation, invasion, migration, and stemness in TNBC cells. Protein expression analysis revealed that mESC CM exposure increased expression of the epithelial protein E-cadherin and decreased the mesenchymal protein MMP9. Gene expression analysis showed that mESC CM decreased epithelial to mesenchymal transition (EMT) markers fibronectin, vimentin, and Snail. Over a period of 6 d, TRACER quantified changes in activity of 11 transcription factors (TFs) associated with oncogenic progression. The EMT profile was decreased in association with the activity of 7 TFs (Smad3, NF-κΒ, MEF2, GATA, Hif1, Sp1, and RXR). Further examination of Smad3 and GATA expression and phosphorylation revealed that mESC CM exposure decreased noncanonical Smad3 phosphorylation and Smad3-mediated gene expression, increased GATA3 expression and phosphorylation, and resulted in a synergistic decrease in migration of GATA3 overexpressing MDA-MB-231 cells. Collectively, the application of TRACER to examine TF activity associated with the transition of cancer cells to a less aggressive phenotype, as directed by mESC CM, identified novel mechanistic events linking the embryonic microenvironment to both favorable changes and cellular plasticity in TNBC cell phenotypes.

No abstract

Introduction: Breast cancer onset and disease progression has been linked to members of the TGFb superfamily and their downstream signaling components, the Smads. Changes in Smad signaling have been associated with the dichotomous role of TGFβ in malignancy, mediating both tumor suppressant and tumor promoting behaviors in breast cancer. Our previous work showed that cyclin/CDK mediated phosphorylation of Smad3 resulted in the inhibition of canonical tumor suppressant Smad3 action. We thus hypothesized that activation of CDKs leads to phosphorylation and inhibition of Smad3, releasing cell cycle arrest and promoting cell proliferation and metastasis. Using triple negative breast cancer cell lines, we examined the impact of specific CDK inhibitors (CDKis) on signaling patterns, metastatic phenotypes, and protein expression profiles. Methods: Triple negative cell lines Hs578T, MDA-MB-231 and MDA-MB-436 were treated with CDK2i or CDK4i and Smad3 activity was measured using a luciferase assay. Transwell migration and Matrigel invasion assays were used to show the effect of transfection with Smad3 CDK phosphorylation site mutants, resistant to inhibitory CDK phosphorylation, or treatment with CDK2i or CDK4i on the study cells. Immunoblotting was performed to measure protein expression levels of Smad3, pSmad3 T179, and MMP2. A novel live cell array was implemented to quantify changes in activity of 14 cancer-related transcription factors over a period of 3 days. TUNEL and Ki67 staining analysis and a xenograft mouse model were used to determine impact of CDKis, in combination with paclitaxel chemotherapy, on apoptosis and tumor growth respectively. Results: Treatment of study cells with CDK2i or CDK4i resulted in increased Smad3 activity and decreased migration and invasion in the study cells. Transfection with a 5M Smad3 mutant construct, containing mutations in all 5 CDK phosphorylation sites, resulted in the greatest decrease in cell migration, when compared with cells transfected with both vector control or WT Smad3. CDK2i or CDK4i therapy resulted in decreased Smad3 protein phosphorylation at the T179 site, while total Smad3 levels were unaffected. CDK2i/4i treatment also resulted in decreased MMP2 expression. The array studies revealed decreased activity of EMT transcription factors B-catenin, Snail, Twist, in MDA-MB-231 cells following CDK2i treatment. CDK2i and paclitaxel combination treatment resulted in increased apoptosis and decreased tumor volume and Ki67 staining compared to control treatment. Conclusions: We have shown for the first time that cyclin/CDK activity, in part, mediates the metastasis-associated role of Smad3 in triple negative breast cancer cells. Importantly, both CDK2i and CDK4i treatment resulted in decreased cell migration/invasion and also corresponded to decreased MMP2 expression. Lastly, the array studies revealed CDK2i mediated effects on key transcription factors associated with the promotion of EMT. Future studies will explore the significance of Smad3 interaction with these EMT factors and will pursue the clinical application of CDK2i for patients with triple negative/cyclin overexpressing breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-12.