Embryonic stem cell secreted factors decrease invasiveness of triple-negative breast cancer cells through regulome modulation.

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.

Inhibition of CDK-mediated phosphorylation of Smad3 results in decreased oncogenesis in triple negative breast cancer cells.

Breast cancer onset and disease progression have been linked to members of the TGFß superfamily and their downstream signaling components, the Smads. Alterations in Smad3 signaling are associated with the dichotomous role of TGFß 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.

CDK4 inhibition and doxorubicin mediate breast cancer cell apoptosis through Smad3 and survivin.

Cyclin D1/CDK4 activity is upregulated in up to 50% of breast cancers and CDK4-mediated phosphorylation negatively regulates the TGFß 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.

Phospho-specific Smad3 signaling: impact on breast oncogenesis.

Members of the TGFß superfamily are known to exert a myriad of physiologic and pathologic growth controlling influences on mammary development and oncogenesis. In epithelial cells, TGFß signaling inhibits cell growth through cytostatic and pro-apoptotic activities but can also induce cancer cell EMT and, thus, has a dichotomous role in breast cancer biology. Mechanisms governing this switch are the subject of active investigation. Smad3 is a critical intracellular mediator of TGFß signaling regulated through phosphorylation by the TGFß receptor complex at the C terminus. Smad3 is also a substrate for several other kinases that phosphorylate additional sites within the Smad protein. This discovery has expanded the understanding of the significance and complexity of TGFß signaling through Smads. This review highlights recent advances revealing the critical role of phospho-specific Smad3 in malignancy and illustrates the potential prognostic and therapeutic impact of Smad3 phospho-isoforms in breast cancer.

Biochemical and morphological consequences of human α-synuclein expression in a mouse α-synuclein null background.

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 (hm(2) αSYN) 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 with 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 9 months of age. Human αSYN was predominantly located in neuronal cell bodies, neurites, synapses, and in intraneuronal/intraneuritic aggregates. The hm(2) αSYN 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 with the levels of the hm(2) αSYN transgene. These observations suggest that mutations in αSYN may impair specific functional domains, leaving others intact. These lines may be useful for exploring interactions between hαSYN and environmental or genetic risk factors in dopamine-related disorders using a mouse model.

Toxicokinetics and toxicodynamics of paraquat accumulation in mouse brain.

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 approximately 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.