LMW-E renders hMECs tumorigenic, and LMW-E expression is selected with increased <i>in vivo</i> passaging.

<p>(A) Schematic of the generation of <i>in vivo</i> passaged clones with 3 successive injections (T1G2, T1G3, and T1G4). (B) Tumors from <i>in vivo</i> passaging were removed from mice, minced, and cultured on monolayer plates. Lysates were extracted and subjected to Western blot analysis with antibodies against cyclin E, elafin, and β-actin. EL (C), LMW-E (D), and elafin (E) protein levels were quantified by densitometry and compared between different generations of <i>in vivo</i> passaged cells (Student <i>t</i> test, *p<0.05). (F) Paraffin-embedded slides of 4 representative tumors were stained with hematoxylin and eosin (top panel) and cyclin E antibody (bottom panel).</p

LMW-E is tumorigenic.

<p>Athymic mice were injected subcutaneously with 1×10⁷ 76NE6 cells stably transfected with empty vector, EL, LMW-E and MDA-MB-468 cells. After 10 weeks, the tumors were removed for expansion in culture for further <i>in vivo</i> passaging and also for IHC analysis. Tumor incidence rate was estimated with exact 95% confidence intervals and Fisher's exact tests were used to compare tumor incidence rate between/among groups (*p<0.0001: 76NE6-vector vs. 76NE6-LMW-E; 76NE6-EL vs. 76NE6-LMW-E; 76NE6-vector vs. TDCs; 76NE6-EL vs. TDCs).</p

Combination drug treatment prevents induction of aberrant acinar development by LMW-E.

<p>(A) Cells were seeded on Matrigel for 24 hours and then treated with rapamycin, sorafenib, and roscovitine as indicated. Medium containing drugs was replaced every 4 days, and lysates were collected on day 15 for Western blot analysis with the indicated antibodies. (B) On day 15 of Matrigel culture, cells grown as in (A) were fixed and stained with E-cadherin (red) and Ki67 (green), and nuclei were counterstained with DAPI (blue). Scale bar = 50 µm. (C) The diameters of the acini were measured and averaged from three independent experiments. Error bars = SEM (Student <i>t</i> test, *p<0.05). (D) The number of Ki67-positive cells per acinus was counted and averaged from three independent experiments. Error bars = SEM (Student <i>t</i> test, *p<0.05). Kaplan-Meier survival plots demonstrating association between full length and high LMW-E on disease-specific survival. Association of:</p

High LMW-E expression is associated with activated b-Raf-ERK1/2-mTOR pathway <i>in vitro</i> and in patient tissues.

<p>(A) Hierarchal cluster analysis of protein expression in 76NE6, 76NE6-LMW-E and all of the LMW-E-expressing tumor clones grown on 2D (red) and 3D (green) cultures and 276 breast cancer patient samples (blue). (B) Proteins whose expression was associated with high LMW-E levels. Red indicates that high LMW-E along with high protein expression was associated with poor prognosis; grey indicates that high LMW-E along with low protein expression was associated with poor prognosis. (C) The lysates from 3D culture were subjected to Western blot analysis to validate the RPPA data. The cell lines are 76NE6-parental (P) and with stable expression of vector (V), EL, and LMW-E and the tumor-derived cells (TDCs). (D) Linear regression analysis of the RPPA data and the densitometry values of all the proteins analyzed by Western blot analysis in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002538#pgen-1002538-g005" target="_blank">Figure 5C</a>. The same antibodies were used for the two types of analysis.</p

LMW-E induces formation of large and highly proliferative acini.

<p>(A) 76NE6, MCF-10A, HS 578T, and MDA-MB-231 cells were seeded at a density of 70 cells/mm² on 1-mm-thick Matrigel. After 15 days in 3D culture, cells were fixed and immunostained with GM-130 and α6-integrin antibodies. Nuclei were counterstained with DAPI. Scale bar = 50 µm. (B) Lysates from these cells were isolated at days 0 (d0) and 15 (d15) of acinar morphogenesis and subjected to Western blot analysis with the indicated antibodies. (C and D) 76NE6 cells stably transfected with vector, EL, and LMW-E and tumor-derived cells (TDCs) were subjected to analysis similar to that in (A) (V = vector). Scale bar = 50 µm. The diameters of the acini were measured and averaged from 3 independent experiments. Values underneath each figure represents mean (µm) ± SEM. Error bars = SEM (Student <i>t</i> test, *p<0.05). (E) Lysates from these cells were isolated at day 15 and subjected to Western blot analysis with the indicated antibodies. <i>In vitro</i> kinase assay was performed by immunoprecipitation of lysates from 3D culture using polyclonal cyclin E antibody and incubation with (γP32)ATP and GST-Rb. (F and G) Cells cultured on Matrigel for 15 days were fixed and immunostained with E-cadherin and Ki67 antibodies. Nuclei were counterstained with DAPI. Scale bar = 50 µm. The number of Ki67-positive cells per acinus was counted and averaged from 3 independent experiments. Error bars = SEM (Student <i>t</i> test, *p<0.05). (H) Linear regression of the correlation between acinar diameter and percentage of Ki67-positive cells.</p

CDK2-associated kinase activity is required for LMW-E-mediated tumorigenesis and deregulation of acinar morphogenesis.

<p>(A) 76NE6-TetR cells were cultured with or without doxycycline induction, and the lysates were extracted and subjected to Western blot analysis with antibodies against cyclin E, CDK2, and β-actin. <i>In vitro</i> kinase assay was performed by immunoprecipitation with FLAG antibody and histone H1 and GST-Rb were added as substrates. Doxycycline was administered to achieve approximately 1× and 2× cyclin E protein levels. (The doxycycline concentrations for the 76NE6-TetR-V and wild-type EL and LMW-E cells were 0, 0.2, and 0.4 ng/ml, and the doxycycline concentrations for the EL^R130A and LMW-E^R130A cells were 0, 1, and 2 ng/ml.) (B) 76NE6-TetR cells were cultured on Matrigel for 15 days with or without doxycycline induction. Bright-field images were taken at day 15. Values underneath each figure represent mean diameter (µm) ± SEM. (C) The diameters of at least 100 acini from 3 different experiments were measured. Error bars = SEM (Wilcoxon rank-sum test, *p<0.05). (D) Multi-acinar complexes were counted. Error bars = SEM (Wilcoxon rank-sum test, *p<0.05). Multi-acinar complexes were defined as complexes with more than 2 acini growing on top of each other. Logistic regression models were used to compare the rate of formation of multi-acinar complexes between/among groups (*p<0.05).</p

Patient protein expression based on low and high LMW-E and EL levels by RPPA analysis.

*<p>Wilcoxon ranksum test.</p

The tumorigenicity of LMW-E requires CDK2–associated kinase activity.

<p>Athymic mice were injected with 1×10⁷ 76NE6-TetR cells with inducible expression for empty vector, EL, LMW-E, EL^R130A, and LMW-E^R130A. Doxycycline was added to drinking water containing 1% sucrose 24 hours after injection and replaced twice weekly. The diameter of the tumors were measured and recorded weekly. Tumor incidence rate was estimated with exact 95% confidence intervals and Fisher's exact tests were used to compare tumor incidence rate between/among groups (*p<0.0001: LMW-E 0 vs. LMW-E 500; vector 500 vs. LMW-E 500; EL 500 vs. LMW-E 500; LMW-E 500 vs. EL^R130A 500; LMW-E 500 vs. LMW-E^R130A).</p

LMW-E induces ductal hyperplasia in vivo and invasion in Boyden chamber assays.

<p>(A) Doxycycline-dependent expression of LMW-E in MTB/TLMW mice induces ductal hyperplasias. Bi-transgenic mice carrying both the MMTV-rtTA-pA (MTB) transgene and the TetO-LMW-E (TLMW) transgene express the rtTA transactivator in the mammary epithelium but do not express LMW-E unless doxycycline is added. (B) Virgin female mice (6 weeks old) of the indicated genotypes were either left untreated or administered 2 mg/ml doxycycline in drinking water for 4 days. Mammary gland extracts were prepared and subjected to luciferase activity assay and Western blot analysis of LMW-E expression. (C) Whole-mount staining of mammary glands from 8-week-old mice either left untreated (upper panels) or administered 2 mg/ml doxycycline in drinking water for 7 days (lower panels). TEB, terminal end buds. (D) Representative BrdU and cyclin E analyses performed on mammary sections from MTB/TLMW 6-week-old females administered doxycycline for 4 days (left panels, magnification: ×200). BrdU incorporation and percentage of cyclin E-positive cells were quantified (right panels) by counting 1,000 cells per section in 3 mice. (E) Boyden Chamber assays. 76NE6 parental and different EL, LMW-E and TDC variants were seeded on Matrigel-coated transwell chamber and incubated on top of fibronectin-containing media for 24 hours. The cells on top of the membrane were removed and the cells remaining on the bottom were stained with crystal violet and images were taken with a light microscope. (F) After 24 hours incubation, the cells on the bottom of the transwell were collected and counted. Statistical analysis used was unpaired student's <i>t</i>-test. MDA-MB-231 cells were used as positive control for the invasion assay.</p