E-cigarettes can Induce α7 Expression at the Transcriptional, Translational, and Protein Levels.

<p>(A) Transient transfection showed that 1.5μM of three different brands of e-cigarettes could induce the α7 promoter after 48 hours of stimulation in A549 cells, to a similar extent as nicotine. (B and C) Immunofluorescent staining and western blot analysis showed that 1.5μM of e-cigarettes could induce α7 at the protein level after 48 hours in A549 cells, to a similar extent as nicotine. For western blot, arrow indicates the α7 protein band and quantification of the data is depicted in the corresponding graphs. (D) qRT-PCR analysis showed that 1.5μM of e-cigarettes resulted in increased α7 mRNA levels after 48 hours, decreased α3 mRNA levels after 24 hours, and increased α5 mRNA levels after 48 hours.</p

Schematic Depicting Signaling Cascades Initiated by Nicotine-Mediated Activation of the α7 nAChR and Inhibitors Used to Target these Pathways in this Study.

<p>Upon nicotine binding to α7 nAChR, oligomeric complexes form including the receptor, β-arrestin-1 scaffolding protein, and Src kinase. This activates Src, resulting in activation of Raf-1 kinase which acts along with activated CDK/cyclins to hyperphosphorylate the Rb tumor suppressor, resulting in its dissociation from E2F transcription factors, allowing them to activate their target genes including a number of genes involved in multiple aspects of tumor progression. The PI3K-AKT and MAPK signaling pathways are also known to be activated by nicotine-mediated α7 nAChR activation subsequent to Src phosphorylation; and activation of MEK-ERK is known to result in activation of CDK/cyclin complexes. AKT1 activation downstream of PI3KC in response to nicotine has additionally been shown to result in activation of NFκB transcription factors. Further, Src activates STAT proteins up nicotine-mediated activation of α7 nAChR. Inhibitors used in this study are depicted in black and white boxes, and their targets are indicated accordingly.</p

E2F and STAT Transcription Factors Have Predicted Binding Sites and can Bind to the α7 Promoter.

<p>(A) Schematic representation of 1000bp regions of the α3 nAChR, α5 nAChR, and α7nAChR gene promoters showing potential E2F binding sites as black ovals. (B) Schematic representation of 1100bp region of nAChR α7 gene promoter showing potential E2F binding sites as black ovals and potential STAT binding sites as green ovals. Arrows represent the position of primers spanning E2F and STAT binding sites used for ChIP assays. Sequence data of overlapping E2F and STAT binding sites is detailed. (C) ChIP assays showed binding of E2F1-5 to the α7 nAChR promoter region -177 through +13 in A549 cells and binding of E2F1-4 in H460 cells. E2F1 and E2F4, and to some extent E2F2 and E2F3 could also bind region -294 through -463 in A549 cells while E2F1-5 could bind this region in H460cells. STAT1 and STAT3 additionally could bind region -294 through -463 in A549 cells, while STAT1 alone could bind in H460 cells. Sonicated DNA was used as input control, and there was no detectable amplification from irrelevant IgG, used as negative control. Quantification of the data is depicted as percent of input, in the corresponding graphs.</p

Nicotine-Mediated Induction of α7 Could be Abrogated by Inhibitors of Src, PI3K, MEK, CDK4/6, Rb/Raf, or α-BT; or by siRNA Depletion of E2F1.

<p>(A, B, C) Western blot, Li-Cor in-cell western blot, and qRT-PCR analysis showed that treatment with indicated inhibitors for 30 minutes prior to nicotine stimulation could abrogate the nicotine-mediated induction of α7 levels in A549 cells. For western blots, arrows indicate the α7 protein band. (D) Transient transfection showed that nicotine-mediated induction of the α7 promoter was decreased when E2F1 was depleted using siRNA, but was increased when STAT1 was depleted using siRNA in A549 and H460 cells. (E) Similar results were seen by qRT-PCR analysis which showed that nicotine-mediated induction of α7 mRNA was abrogated when E2F1 was depleted using siRNA, but enhanced when STAT1 was depleted using siRNA.</p

E2F and STAT Have Overlapping Binding Sites on the α7 Promoter, and Differentially Act to Regulate its Expression.

<p>(A) Schematic representation of the overlapping E2F1 and STAT1/3 binding sites on the α7 promoter at region -294 through -463. (B and C) Transient transfection showing increasing concentrations of STAT1 can repress E2F1-mediated induction of the α7 promoter in A549 cells; reciprocally increasing concentrations of E2F1 can alleviate STAT1-mediated repression of the α7 promoter in A549 cells. (D) Schematic representation of DNA sequence of overlapping E2F and STAT binding sites. Nucleotides which were mutated to disrupt STAT binding on α7 promoter are depicted in red on the left and the resulting mutated sequence is depicted in red on the right. (E) Transient transfections showed that STAT1 could repress the α7 promoter but not the α7-STAT-site-mutant promoter in A549 and H460 cells. (F) Transient transfection showed that STAT1 could repress the 1115bp α7 promoter, but not 235bp α7 promoter lacking predicted STAT binding sites.</p

E2F and STAT Transcription Factors Regulate α7 nAChR Expression.

<p>(A and B) Transient transfection experiments showing that E2F1-3 can induce the α7 promoter, E2F4 has no effect, and E2F5, STAT1, and STAT3 act repress it in A549 and H460 cells. (C and D) Depletion of E2F1 by siRNA results in reduced expression of α7 mRNA, while depletion of STAT1 results in increased expression in A549 and H460 cells.</p

A model depicting KDM2A function in a mammalian cell.

<p>It can be implied that KDM2A regulates Rb-E2F1 function in the progression of the cell cycle. In a quiescent state, KDM2A represses E2F1 functions on various promoters. Addition of VEGF or serum dissociates KDM2A from these promoters facilitating various cellular processes by transcriptional activation leading to enhanced angiogenesis, invasion and migration of cells.</p

KDM2A represses transcriptional activity of MMPs and VEGF receptors by inhibiting E2F1-mediated transcription.

<p>(A) ChIP assay demonstrates occupancy of KDM2A on MMP9, -14, -15, KDR and FLT-1 similar to E2F1. (B, C, D, E) Transient transfection experiments in MCF-7 cells showed that E2F1 induces MMP2 (B), MMP-9 (C), MMP-14 (D) and MMP-15 (E) promoters, and this was repressed by co-transfection of KDM2A or Rb large pocket. (F, G) Transient transfection experiments in MDA-MB-231 cells demonstrate that KDM2A represses the transcriptional activity of E2F1 on KDR (F) and FLT-1 (G) promoters.</p

KDM2A interacts with Rb and E2F1 in a cell cycle specific manner.

<p>(A) GST pull down assay showing KDM2A binding to Rb and E2F1 <i>in vitro</i>. ³⁵S-lysate lane has 1/10^th protein loaded. (B) MCF-7 cells were serum starved for 48 hr and serum stimulated for 2 hr, 4 hr, 6 hr, 8 hr and 18 hr. Western blotting showing protein expression of KDM2A, Rb, E2F1 and actin. There is no significant change in the expression of KDM2A and E2F1 at different time points when normalized to actin levels. Rb shows increasing hyperphosphorylation from 6 hr to 18 hr of serum stimulation. (C) KDM2A interacts with Rb and E2F1 <i>in vivo</i> in MCF-7 cells as seen by immunoprecipitation-western blot experiment. Interaction of KDM2A with E2F1 decreases at 8 hr of serum stimulation. (D) Chromatin Immunoprecipitation (ChIP) assays showing KDM2A occupancy on E2F1-regulated proliferative promoters, CDC25A and TS. KDM2A occupies the promoters at all time points from 0 hr to 6 hr, reduction is seen at 8 hr and complete absence of KDM2A from the promoters is observed at 18 hr of serum stimulation. (E) KDM2A significantly represses E2F1-mediated E2.Luc transcription in a dose-dependent manner in Renilla luciferase assay.</p

Myoepithelial cells of the breast express KDM2A.

<p>(A) Immunohistochemical staining of KDM2A on human breast cancer tissue microarray. Immunostaining was performed using rabbit anti-human KDM2A antibody and representative images of KDM2A expression in normal breast, Ductal Carcinoma in situ, Infiltrating Ductal Carcinoma with and without metastasis, lymph node metastasis are shown. Magnification is 200X, scale bar  = 50 µm. (B) Enlarged images of normal and cancer breast tissue sections. Arrows indicate positively stained myoepithelial cells. Scale bar  = 50 µm. (C) Quantitative analysis of KDM2A in breast tissue microarray. The Immunostaining of KDM2A was quantified by using semi quantitative scoring method based on cellularity and intensity of expression. The means of two independent arrays are shown. All <i>p</i>-values were calculated using a two-sided Student <i>t-</i>test.</p