DNA damage signals through differentially modified E2F1 molecules to induce apoptosis

E2F transcription can lead to cell proliferation or apoptosis, indicating that E2Fs control opposing functions. In a similar manner, DNA double-strand breaks can signal to induce cell cycle arrest or apoptosis. Specifically, pRB is activated following DNA damage, allowing it to bind to E2Fs and block transcription at cell cycle promoters; however, E2F1 is simultaneously activated, leading to transcription at proapoptotic promoters. We examined this paradoxical control of E2F transcription by studying how E2F1\u27s interaction with pRB is regulated following DNA damage. Our work reveals that DNA damage signals create multiple forms of E2F1 that contain mutually exclusive posttranslational modifications. Specifically, E2F1 phospho-serine 364 is found only in complex with pRB, while E2F1 phosphorylation at serine 31 and acetylation function to create a pRB-free form of E2F1. Both pRB-bound and pRB-free modifications on E2F1 are essential for the activation of TA-p73 and the maximal induction of apoptosis. Chromatin immunoprecipitation demonstrated that E2F1 phosphorylated on serine 364 is also present at proapoptotic gene promoters during the induction of apoptosis. This indicates that distinct populations of E2F1 are organized in response to DNA damage signaling. Surprisingly, these complexes act in parallel to activate transcription of proapoptotic genes. Our data suggest that DNA damage signals alter pRB and E2F1 to engage them in functions leading to apoptotic induction that are distinct from pRB-E2F regulation in cell cycle control. © 2012, American Society for Microbiology

Dysregulation of Cell Polarity Proteins Synergize with Oncogenes or the Microenvironment to Induce Invasive Behavior in Epithelial Cells

Changes in expression and localization of proteins that regulate cell and tissue polarity are frequently observed in carcinoma. However, the mechanisms by which changes in cell polarity proteins regulate carcinoma progression are not well understood. Here, we report that loss of polarity protein expression in epithelial cells primes them for cooperation with oncogenes or changes in tissue microenvironment to promote invasive behavior. Activation of ErbB2 in cells lacking the polarity regulators Scribble, Dlg1 or AF-6, induced invasive properties. This cooperation required the ability of ErbB2 to regulate the Par6/aPKC polarity complex. Inhibition of the ErbB2-Par6 pathway was sufficient to block ErbB2-induced invasion suggesting that two polarity hits may be needed for ErbB2 to promote invasion. Interestingly, in the absence of ErbB2 activation, either a combined loss of two polarity proteins, or exposure of cells lacking one polarity protein to cytokines IL-6 or TNFα induced invasive behavior in epithelial cells. We observed the invasive behavior only when cells were plated on a stiff matrix (Matrigel/Collagen-1) and not when plated on a soft matrix (Matrigel alone). Cells lacking two polarity proteins upregulated expression of EGFR and activated Akt. Inhibition of Akt activity blocked the invasive behavior identifying a mechanism by which loss of polarity promotes invasion of epithelial cells. Thus, we demonstrate that loss of polarity proteins confers phenotypic plasticity to epithelial cells such that they display normal behavior under normal culture conditions but display aggressive behavior in response to activation of oncogenes or exposure to cytokines

SCRIBBLE Interactome and Proliferation in Breast Cancer

Disruption of ductal epithelial organization in the mammary gland during carcinogenesis of the breast is a hallmark and frequently utilized clinical marker of invasive ductal breast cancer. The Scribble (SCRIB) polarity protein is required to regulate organization and distribution of proteins in epithelial cells. Specifically, in the normal mammary gland, SCRIB maintains ductal organization, by acting as a molecular scaffold to assemble regulatory components of signaling pathways. During breast cancer, SCRIB is frequently mislocalized and overexpressed. Mouse models have suggested that overexpressed mislocalized SCRIB is sufficient to cause mammary tumourigenesis, with the development of tumours with basal characteristics and hyperactivation of S6 kinase. With the aim of defining SCRIB function, studies have identified individual interactors for SCRIB, however, the SCRIB interactome in the mammary epithelium remains uncharacterized. My research has validated and characterized a unique interaction between SCRIB and the sub-apical AF6 polarity protein, that arose from our unbiased proteomic screen for SCRIB interactors in mammary epithelial cells. I have shown that this interaction can regulate proliferation/survival of three-dimensional acini under nutrient stress, and loss of this interaction in mammary epithelial cells can compromise p85-S6K phosphorylation and RHEB-GTP hydrolysis. Second, in a mouse model of mammary-specific Tp53 loss, overexpression of mislocalized SCRIB did not accelerate Tp53-loss driven mammary tumourigenesis, and in fact, may increase the latency of mammary tumourigenesis and lung metastases, suggesting that mislocalized SCRIB may retain some tumour suppressive function. Finally, I have screened and identified several cancer-associated mutations in the SCRIB leucine rich repeat region (LRR) that result in mislocalization of a SCRIB-RFP reporter construct, suggesting that acquisition of point mutations in the LRR of SCRIB is a physiologically relevant means of SCRIB mislocalization in tumourigenesis. These studies suggest that novel SCRIB interactions may modulate cell survival under certain conditions, such as nutrient stress and growth factor stimulation, while SCRIB mislocalization is a cancer-associated event and may be attributed to tumourigenesis in specific genetic contexts.Ph.D.2020-11-19 00:00:0

The Retinoblastoma Protein Regulates Pericentric Heterochromatin

The retinoblastoma protein (pRb) has been proposed to regulate cell cycle progression in part through its ability to interact with enzymes that modify histone tails and create a repressed chromatin structure. We created a mutation in the murine Rb1 gene that disrupted pRb's ability to interact with these enzymes to determine if it affected cell cycle control. Here, we show that loss of this interaction slows progression through mitosis and causes aneuploidy. Our experiments reveal that while the LXCXE binding site mutation does not disrupt pRb's interaction with the Suv4-20h histone methyltransferases, it dramatically reduces H4-K20 trimethylation in pericentric heterochromatin. Disruption of heterochromatin structure in this chromosomal region leads to centromere fusions, chromosome missegregation, and genomic instability. These results demonstrate the surprising finding that pRb uses the LXCXE binding cleft to control chromatin structure for the regulation of events beyond the G(1)-to-S-phase transition

Combined loss of two regulators from apical and basal polarity complexes is sufficient to induce invasive behavior.

<p>(A) Lysates were immunoblotted to test knockdown for indicated proteins (B) Phase morphology of cells grown on plastic dishes (left panels) or M/Col-1 matrix (right panels). Also refer to <i>SI </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034343#pone.0034343.s002" target="_blank">Fig. S2</a>B. Arrows indicate area of the image magnified in the inset. Scale bars, 100 µm. (C) Quantification of cell invasion plotted as mean ± S.E.M. from at least three independent experiments. *, p<0.005, **, p<0.0001 obtained in an unpaired t-test comparing indicated cell lines with parental MCF10A cells (10A). See Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034343#s4" target="_blank">Methods</a> for details.</p

Loss of Scribble, Dlg1 or AF6 cooperates with ErbB2 activation to promote migration and invasion in MCF-10A cells.

<p>(A) Immunoblot of lysates from 10A.B2 expressing control (Luciferace, 10A.B2.Luc) or Scribble (10A.B2.Scrib) or Dlg1 (10A.B2.Dlg1) or AF6 (10B2.AF6) shRNAs. (B) Transwell cell migration assay in of cell lines in Panel A in the presence (+) or absence (−) of the ErbB2 activator. The graph represents mean of three independent experiments ± S.E.M. *, p<0.05, **, p<0.005. calculated using an unpaired t-test comparing ErbB2-activated polarity-gene knockdown cells with Luc control cells. (Ci-iv) Morphology of 3D acini derived from 10A.B2.Luc,10A.B2.Scrib, 10A.B2.Dlg1 and 10A.B2.AF6 cells grown in M/Col-I in absence (ErbB2−) or presence (ErbB2+) of ErbB2 activator. Scale bars, 100 µm. (Di-ii) M/Col-I grown 10A.B2.Luc or 10A.B2.Scrib acini treated or untreated with ErbB2 activator fixed and immunostained for Laminin (Red) and DAPI-stained for nuclei (Blue). Scale bars, 50 µm. Arrows indicate area of the image magnified in the inset. (E) Percentage of acini showing invasive protrusions were quantified and mean (± S.E.M.) plotted from at least three independent experiments. *, p<0.05 based on an unpaired t-test comparing ErbB2-activated polarity-gene knockdown B2 cells and control Luc.B2 cells. (F) Lysates from 10A.B2 and 10A.B2.Scrib transfected with Par6K19A-Flag (K19 and K19+Scrib) or untransfected (Scrib) cells were immunoblotted for Flag to show Par6.K19A overexpression. ErbB2 blot shows expression levels of ErbB2 in transfected and untransfected lines. (G) Percentage of invasive acini quantified and mean ± S.E.M. plotted for K19, Scrib and K19+Scrib cells. Note the suppression of invasion in K19+Scrib cells compared to Scrib knockdown cells. **, p<0.005 in an unpaired t-test comparing ErbB2− and ErbB2+ in Scrib knockdown cells. See Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034343#s4" target="_blank">Methods</a> for details.</p

Loss of one polarity gene and cooperation with pro-tumorigenic cytokines.

<p>(A) Phase images showing induction of invasion in 4-day old acini of parental MCF10A cells or cells expressing shRNAi for indicated polarity genes growing in M/Col-I matrix and treated with 1∶6 diluted supernatant from CpG-treated dendritic cells (DC Sup.), or pro-inflammatory cytokines TNF-α (2 ng/ml) and IL-6 (25 ng/ml) (refer to <i>SI </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034343#pone.0034343.s003" target="_blank">Fig. S3</a>A). Arrows indicate region of the image magnified in the inset. Scale bars, 100 µm (B) Quantification of invasion of DC Sup-treated acini plotted as mean ± S.E.M. from at least three independent experiments. (C–D) Quantification of invasion in MCF10A acini after treatment with recombinant TNF-α (C) or IL-6 (D). Also refer to SI <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034343#pone.0034343.s003" target="_blank">Fig. S3</a>B,C. *, p<0.05, **, p<0.005. p values are based on an unpaired t-test comparing cytokine-treated to the untreated values for the same knockdown.</p

Loss of two polarity genes upregulates EGFR and activate Akt.

<p>(A–B) Parental or knockdown cell lysates were analyzed for EGFR mRNA expression (A) and immunoblotted for EGFR or ErbB2 or ErbB3. Note the increase in EGFR expression without any change in expression of ErbB2 or ErbB3 in polarity knockdown cells. (B) Parental or polarity knockdown cells were grown overnight in low-serum medium without growth factor supplements and next day replenished with Assay medium with 5 ng/ml EGF for indicated times (see Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034343#s4" target="_blank">Methods</a> for details) and immunoblotted for phospho-Akt (Ser-473) first and then stripped and reblotted for pan-Akt. (D) One-day old parental or knockdown MCF10A acini were left untreated or treated with 1nM perifosine and phase images obtained and quantified after 72–96 hr post-perifosine treatment. Data represents mean ± S.E.M. from at least three independent experiments. p<0.05 obtained in an unpaired t-test comparing perifosine-treated to the untreated values for the same knockdown.</p

K14+ cells secrete more Col6a1 and express higher levels of Amigo2 than K14− cells.

<p>(A) Anti-Col6a1 immunoblots of secreted proteins from K14.GFP+ or K14.GFP− reporter cell lines. CM were concentrated and analyzed for levels of secreted Col6a1. ITGB1, also present in the secretome, was used for loading control. (B) RT-PCR for <i>Amigo2</i> mRNA level on K14+ and K14− cells. Results show the mean ± SD of 3 independent experiments, <i>p</i> = 0.0126 by paired <i>t</i> test. (C) Amigo2 protein level was detected in K14+ and K14− cell lysates by western blot. Quantification of 3 independent experiments, <i>p</i> < 0.0001 by paired <i>t</i> test; mean ± SD is shown. (D) Chip for H3K27Ac shows the magnitude of the peaks for K14+ and K14− replicates at the <i>Amigo2</i> locus. (E) Cartoon of self-inactivating lentiviral K14.tRPT (upper cartoon) and K8.tGPD reporters (lower cartoon). (F) RT-PCR analysis of <i>Amigo2</i> mRNA expression in K14+ or K14− human breast cancer cell line HCC1143. Quantification of independent experiments in triplicates <i>p</i> = 0.0148 by paired <i>t</i> test; mean ± SD is shown. (G) Kaplan-Meier plot in TP53 mutant and TP53 WT breast cancer show relationship between <i>Amigo2</i> expression and relapse-free survival. Amigo2, amphoterin-induced protein 2; Chip, chromatin immunoprecipitation; CM, conditioned medium; Col6a1, Collagen VI subunit A; DTR, diphtheria toxin receptor; <i>EF-1α</i>, <i>elongation factor 1α</i>; GFP, green fluorescent protein; H3K27Ac, histone 3 lysine 27; ITGB1, integrin β-1; K, cytokeratin; K8.tGPD, keratin-8 promoter followed by turbo green fluorescent protein and diphtheria toxin receptor; K14.tRPT, keratin-14 promoter followed by a turbo red fluorescent protein and herpes simplex virus thymidine kinase; LTR, long terminal repeat; RT-PCR, real-time PCR; tBFP, turbo blue fluorescent protein; tGFP, turbo green fluorescent protein; TK, thymidine kinase; tRFP, turbo red fluorescent protein; WT, wild-type.</p

Generation and characterization of transgenic mice expressing K14 and K8 reporters.

<p>(A) Flow cytometry analysis for stromal, basal, and luminal compartments of cells from mammary glands of control or K14.tRPT/K8.tGPD double-positive transgenic mouse. For each type of mouse, the first dot plot shows the total population in each cell compartment, whereas the second plot shows only the cells that are positive for the reporters. Gates were set based on the negative control and dots pseudocolored to represent the reporter-positive cells. (B) Fluorescent IHC showing colocalization of tGFP with endogenous K8 in the lung of a K8.tGPD-positive mouse (upper panels). Lower panel shows the control staining on a WT mouse in which no tGFP was detected; scale bar 10 μm. (C) Positive and negative mice were injected i.p. with either high (“H”; GCV = 100 μg/g; DT = 50 ng/g) or low (“L”; GCV = 20 μg/g; DT = 10 ng/g) doses at indicated time points (days). DAPI, 4’,6-diamidino-2-phenylindole; DT, diphtheria toxin; GCV, ganciclovir; IHC, immunohistochemistry; i.p., intraperitoneally; K8.tGPD, keratin-8 promoter followed by turbo green fluorescent protein and diphtheria toxin receptor; K14.tRPT, keratin-14 promoter followed by a turbo red fluorescent protein and herpes simplex virus thymidine kinase; tGFP, turbo green fluorescent protein; WT, wild-type</p