Volume 42, Issue 4, Pages 524-535 (May 2011) Two Phases of Mitogenic Signaling Unveil Roles for p53 and EGR1 in Elimination of Inconsistent Growth Signals  Yaara Zwang, Aldema Sas-Chen, Yotam Drier, Tal Shay, Roi Avraham, Mattia Lauriola, Efrat Shema, Efrat Lidor-Nili, Jasmine Jacob- Hirsch, Ninette Amariglio, Yiling Lu, Gordon B. Mills, Gideon Rechavi, Moshe Oren, Eytan Domany, Yosef Yarden  Molecular Cell  Volume 42, Issue 4, Pages 524-535 (May 2011) DOI: 10.1016/j.molcel.2011.04.017 Copyright © 2011 Elsevier Inc. Terms and Conditions

Molecular Cell 2011 42, 524-535DOI: (10.1016/j.molcel.2011.04.017) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 Human Mammary Epithelial Cells Commit to Proliferation upon Two Timed Pulses of EGF (A) 184A1 human mammary cells were GF starved for 16 hr. They were then either pulsed with EGF (“1E,” red) for 1 hr or mock pulsed (“1S,” green). Thereafter, cells were washed and incubated in starvation medium for 7 hr, as indicated, either followed by a second, 1 hr pulse of EGF or not. Cells were then washed and incubated for 3 hr with BrdU in starvation medium. Thereafter, the cells were fixed, stained, and counted under a fluorescent microscope. BrdU incorporation into DNA was measured by determining the ratio of BrdU- to DAPI-stained nuclei and was normalized according to the starvation control (1S-7S-1S). Error bars represent standard errors calculated from at least 15 nonoverlapping photomicrograph fields (>500 nuclei). Significant p values of two-tailed student's t test are indicated. The experiment was repeated three times. (B) 184A1 cells were GF starved as in (A) and then treated with two pulses of EGF or continuously stimulated with EGF for 9 hr. Cells were then washed and incubated for 3 hr with BrdU and fixed, and BrdU incorporation analyzed as in (A). Error bars represent standard errors calculated from at least 15 nonoverlapping photomicrograph fields (>500 nuclei). p values of two-tailed student's t test are indicated. The experiment was repeated twice. (C) 184A1 cells were GF starved and treated as in (A). After the second pulse, cells were left in starvation medium for 17 hr, fixed, and stained with methyl violet. Cell-occupied area was then measured from four light photomicrographs of nonoverlapping fields. Error bars represent the standard errors calculated from triplicates. Significant p values of two-tailed student's t test are indicated. Representative light photomicrographs are presented. The experiment was repeated twice. (D) Cells were GF starved and treated as in (A). At the indicated time points, cells were harvested and lysed, and cleared extracts were electroblotted. Phosphorylation of Rb and abundance of c-MYC were determined (left) and quantified by densitometry. Signals were normalized to actin, and fold phosphorylation or expression was calculated (presented under each lane). The right panel presents the corresponding signals determined at the time of BrdU measurement (marked by Roman numbers). The experiment was repeated twice. See also Figure S1. Molecular Cell 2011 42, 524-535DOI: (10.1016/j.molcel.2011.04.017) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Comparative Analyses of Phosphorylation Cascades Stimulated by Each EGF Pulse (A) A scheme presenting phosphorylation cascades activated by EGFR. Light blue-labeled proteins were analyzed with RPPA. (B) 184A1 cells were GF starved for 16 hr. Thereafter they were pulsed for 1 hr with EGF, washed, and incubated in starvation medium for 7 hr, followed by a second pulse with EGF (red) or no treatment (green). At the indicated time points, cells were harvested, and equal amounts of protein were used for RPPA analysis with the indicated antibodies. The mean of phosphorylation signals normalized to the respective total expression level (in triplicates) was calculated. The heat map presents the means in log2 scale, centered to the corresponding mean across all samples. The fold change in phosphorylation between the second pulse peak and the first pulse peak is indicated (right column), if the difference between the peaks was significant. (C) 184A1 cells were GF starved and treated as in (B). Cells were harvested at the indicated time points, lysed, and analyzed by immunoblotting. Quantified and normalized signals are presented under each lane. The experiment was repeated three times. (D) 184A1 cells were treated, and BrdU incorporation measured as in Figure 1A. Cells were treated with U0126 at the indicated concentrations 30 min prior to and throughout the second pulse (shaded area). Error bars represent standard error values (>500 nuclei). ERK activation was calculated according to Figure S2D. The experiment was repeated three times. (E) 184A1 cells were GF starved and treated, and BrdU incorporation measured as in (D). Cells were treated with LY294002 as indicated 30 min prior to and throughout the second pulse (shaded). Error bars represent standard error values. The experiment was repeated three times. See also Figure S2 and Table S1. Molecular Cell 2011 42, 524-535DOI: (10.1016/j.molcel.2011.04.017) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 A Short Pulse of EGF Is Sufficient for the Induction of Metabolic Enzymes Essential for Cell Proliferation (A) A scheme depicting the setup of the microarray experiment. mRNA samples were isolated at the indicated time points (blue triangles). Red and green segments indicate EGF pulses and intervals, respectively. (B) 184A1 cells were treated as in Figure 1A. mRNA abundance was measured at the indicated time points with Affymetrix GeneSet microarrays. Shown are centered and normalized expression patterns of genes associated with cholesterol biosynthetic processes included in the “persistently induced” profile (see Figure S3B). (C) 184A1 cells were grown and processed as in Figure 1A except that cells were treated with Metformin (0.1 mM), Mevastatin (1 μM), or AICAR (0.5 mM) during the interval (shaded). Error bars represent standard errors calculated from at least 15 nonoverlapping photomicrograph fields (>500 nuclei). A significant p value of two-tailed student's t test is indicated. The experiment was repeated three times. See also Figure S3 and Tables S2 and S3. Molecular Cell 2011 42, 524-535DOI: (10.1016/j.molcel.2011.04.017) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Differential Induction of Immediate-Early Transcription Factors by the Two Pulses (A) 184A1 cells were treated with two pulses of EGF, as in Figure 3B. Shown are centered and normalized expression levels of the indicated immediate-early induced TFs from the profile denoted 1st < 2nd (Figure S3D). (B) 184A1 cells were treated with two pulses of EGF. At the end of each pulse, mRNA was isolated, followed by cDNA synthesis and RT-qPCR with primers for either c-FOS or EGR1. Presented are the average ratios of expression, calculated from four biological repeats. Error bars represent standard error values. (C) GF-starved 184A1 cells were pulsed for 1 hr with EGF, washed, and incubated for 7 hr in starvation medium, followed by a second EGF pulse. Cells were treated with U0126 at the indicated concentrations, 30 min prior to and throughout the second pulse. Immunoblotting was used to quantify EGR1 levels of induction relative to the level at 1E-7S-1E, normalized to actin. (D) GF-starved 184A1 cells were pulsed for 1 hr with EGF (“1E,” red), IGF-1 (“1IGF,” purple), or left untreated (“1S,” green). After GF removal, cells were incubated in starvation medium for 7 hr, followed by a second pulse of EGF or IGF-1, as indicated. For BrdU incorporation analysis, see Figure S4D. For determination of EGR1 fold induction, mRNA was isolated at the end of each pulse (blue triangles), and cDNA was analyzed by RT-qPCR. The right column schematically presents time profiles of EGR1's patterns of expression. (E) GF-starved 184A1 cells were treated and analyzed as in Figure 1A. The shaded rectangle indicates transfection with control or EGR1-specific siRNA oligonucleotides. Error bars represent standard error values. p values of two-tailed Student's t test are indicated. See also Figure S4. Molecular Cell 2011 42, 524-535DOI: (10.1016/j.molcel.2011.04.017) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 The Second Pulse Downregulates Antiproliferative Genes Induced by the First Pulse (A) 184A1 cells were treated with two pulses of EGF. Shown are centered and normalized levels of antiproliferation genes (marked in black are known p53 targets) from the profile “downregulated by a second pulse” (see Figure S3F). (B) 184A1 cells were treated as in Figure 4D. Where indicated, cells were treated for 30 min with LY294002. BrdU incorporation results are presented in Figure 2E and Figure S4D. For determination of fold change, mRNA was isolated before the second pulse and 60 min after completion of the pulse (blue triangles), and cDNA was analyzed by RT-qPCR. Listed are the ratios of expression levels after and before the second pulse. (C) GF-starved cells were pulsed for 60 min with EGF, washed, and incubated in starvation medium for 7 hr, followed by a second pulse. At the indicated time points, cells were harvested for a chromatin association assay, and DNA-bound proteins were isolated and analyzed by immunoblotting. (D) Cells were transfected with control or p53-specific siRNAs. Twenty-four hours later, the cells were replated on coverslips, and another 24 hr later, they were GF starved for 16 hr and treated with EGF, and BrdU incorporation was measured. Error bars represent standard errors calculated from 15 nonoverlapping photomicrograph fields. p values of two-tailed student's t test are indicated. (E) NIH-3T3 cells were transfected with control or p53-specific siRNAs. Twenty-four hours later, the cells were replated, and another 24 hr later they were GF starved for 24 hr, treated for 1 hr without (“1S,” green) or with (“1P,” blue) PDGF, washed, and incubated in starvation medium for 7 hr, followed by a second PDGF pulse. Cells were left in starvation medium or PDGF-containing medium for additional 18 hr and were then fixed and stained with methyl violet. Representative photomicrographs are shown (right). The cell-occupied area was measured from five photomicrographs (left). Error bars represent standard errors of triplicates. The experiment was repeated twice. p values of two-tailed Student's t test are indicated. See also Figure S5. Molecular Cell 2011 42, 524-535DOI: (10.1016/j.molcel.2011.04.017) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 Schematic Representations of the Proposed Biochemical Events Elicited by a Single and a Dual EGF Pulse The first pulse of EGF induces expression of lipid biosynthesis-associated genes, along with activation of p53. The latter propels expression of antiproliferation genes, such as BTG2 and SESN1. When cells are treated with a second pulse of EGF, enhanced activation of ERK and subsequent induction of EGR1 exceed a critical threshold. In parallel, signaling through PI3K and the resulting suppressed expression of antiproliferative genes permit cells to cross the restriction point (R) and enter the S phase. See also Figure S6. Molecular Cell 2011 42, 524-535DOI: (10.1016/j.molcel.2011.04.017) Copyright © 2011 Elsevier Inc. Terms and Conditions