Effects of E2 stimulation on EGF− and FGF-dependent branching morphogenesis.

<p>(A) Brightfield images of representative wt and p130Tg organoids, cultured for 5 days in the absence or presence of E2, EGF, or the combination of both. (B) Quantification of primary and secondary branching of wt vs. p130Tg organoids upon treatment with either E2, EGF or EGF+E2 for 5 days. Experiments were repeated at least five times. Two-way ANOVA analysis show a significant effect of both treatment (EGF vs. EGF+E) and genotype (wild type vs. transgenic) for Acinar Structures, secondary branching and enlarged spheroids (P<0.0001 for both treatment and genotype). For primary branching only treatment has a significant effect (P<0.0001). Interaction between treatment and genotype is significant for all variables (Acinar: P<0.0065; primary branching: P<0.0001; secondary branching: P<0.0001; enlarged spheroids: P<0.0001). (C) Quantification of spherical organoids size in untreated and EGF+E2 treated wt and p130Tg organoids was performed by using ImageJ software. Statistical significance is indicated (** p<0.001).</p

Effects of p130Cas expression on branching morphogenesis in primary mouse mammary gland organoids grown in 3D Matrigel cultures.

<p>(A) Let panel: qRT-PCR expression analysis of p130Cas using total RNA isolated from the 4th inguinal mammary gland of at least three FVB wt mice during different stages of puberty. The amount of p130Cas mRNA was normalized to 18S rRNA. Right panel: Analysis of p130Cas protein expression from wt mice (three mice/stage) at the same stages of puberty as in the left panel. (B) Analysis of p130Cas protein expression from 5 day untreated wt and p130Tg organoids (upper panel) and quantification analysis expressed as the mean of p130Cas/tubulin ± standard deviation (SD) (*p<0.05) (lower panel). (C) Brightfield images of representative wt and p130Tg organoids, after 1, 4 and 5 days of culture in presence of EGF (35 ng/ml) or FGF-2 (35 ng/ml) in the culture medium. Images were captured by using the Axio Observer Z1 microscope. (D) Quantitative analyses of branching morphogenesis after treatment of wt and p130Tg organoids with EGF and FGF-2 at 5 days. The plot represents the mean number of overall branching response in at least three wells from 4 independent experiments. Similar results were obtained with a second p130Cas transgenic line (data not shown). Quantification of the mean number of branched organoids buds was significantly higher in EGF and FGF-2 treated p130Tg organoids compared to wt organoids (*p<0.002).</p

EGF+E2 treatment leads to Erk1/2 MAPKs and Akt hyperactivation in p130Tg organoids.

<p>(A) Western blot analysis of phospho-Erk1/2 MAPKs in wt or p130Tg organoids stimulated with EGF and EGF+E2 at the indicated times. Vinculin and total Erk1/2 MAPKs blots are provided as loading controls. Blots are representative of five independent experiments. (B) Quantification analysis of Erk1/2 MAPKs activation following stimulation for 5, 30 minutes and 1 hour. Data are represented as the mean of phospho-Erk1/2 MAPK levels/total Erk1/2 MAPK ± standard deviation (SD) of five independent experiments (*p<0.05). (C) Western blot analysis of phospho-Akt in wt or p130Tg organoids stimulated as in (A). Total Akt blots are provided as loading controls. Blots are representative of three independent experiments. (D) Quantification analysis of Akt activation following stimulation as in (A). Data are represented as the mean of phospho-Akt levels/total Akt ± standard deviation (SD) of three independent experiments (*p<0.05).</p

E2 treatment of p130Tg organoids alters myoepithelial-luminal architecture and lumen clearance.

<p>(A) Confocal images representing immunofluorescence staining for K14 (red; myoepithelium) and K18 (green; luminal epithelium) of wt and p130Tg organoids stimulated with EGF and E2 at day 5 of culture. Scale bar = 0,50 micron. (B) Confocal z-stacks of DAPI staining of wt and p130Tg organoids at day 5 of culture to visualize lumen clearance. Images in A and B were acquired using a Leica TCS-SP5 II confocal microscope. Images in A and B are representative of four independent experiments.</p

p130Cas Over-Expression Impairs Mammary Branching Morphogenesis in Response to Estrogen and EGF

<div><p>p130Cas adaptor protein regulates basic processes such as cell cycle control, survival and migration. p130Cas over-expression has been related to mammary gland transformation, however the <em>in vivo</em> consequences of p130Cas over-expression during mammary gland morphogenesis are not known. In ex vivo mammary explants from MMTV-p130Cas transgenic mice, we show that p130Cas impairs the functional interplay between Epidermal Growth Factor Receptor (EGFR) and Estrogen Receptor (ER) during mammary gland development. Indeed, we demonstrate that p130Cas over-expression upon the concomitant stimulation with EGF and estrogen (E2) severely impairs mammary morphogenesis giving rise to enlarged multicellular spherical structures with altered architecture and absence of the central lumen. These filled acinar structures are characterized by increased cell survival and proliferation and by a strong activation of Erk1/2 MAPKs and Akt. Interestingly, antagonizing the ER activity is sufficient to re-establish branching morphogenesis and normal Erk1/2 MAPK activity. Overall, these results indicate that high levels of p130Cas expression profoundly affect mammary morphogenesis by altering epithelial architecture, survival and unbalancing Erk1/2 MAPKs activation in response to growth factors and hormones. These results suggest that alteration of morphogenetic pathways due to p130Cas over-expression might prime mammary epithelium to tumorigenesis.</p> </div

p130Cas over-expression enhances cell proliferation and survival in EGF and EGF+E2-treated primary organoids.

<p>(A) Quantification of nuclear Ki67 staining from total nuclei count of each organoid at day 2. Quantification analysis of Ki67 staining as mean of Ki67 positive cells/total number of cells ±SD and significance are reported (*p<0.05, **p<0.001). More than 20 organoids from each condition were counted in three independent experiments. (B) Western blot analysis of cleaved caspase-3 from 2 and 5 day of EGF, E2 and EGF+E2-treated wt and p130Tg cultured organoids. Quantification analysis at day 2 and day 5 of cleaved-caspase 3 are reported on the right (*p<0.05). (C) TUNEL analysis from 2 day treated wt and p130Cas cultured organoids. TUNEL positive cells in the organoids are stained in red. The plot represents the quantification of the results of three independent experiments in which apoptotic cells are counted and normalized to the total number of nuclei/organoid for at least 10 organoids from each condition ± standard deviation (*p<0.05).</p

Inhibition of ER activity in EGF+E2-treated p130Tg mammary organoids prevents Erk1/2 MAPK hyper-activation and rescues branching morphogenesis.

<p>(A) Western blot analysis of phospho-Erk1/2 MAPKs in wt or p130Tg organoids pre-treated for 1 hour with ICI 182,780 or AG1478 followed by 5 minutes of EGF or EGF+E2 stimulation. Vinculin and total Erk1/2 MAPKs blots are provided as loading controls. Blots are representative of three independent experiments. (B) Quantification analysis of Erk1/2 MAPKs activation of wt and p130Tg organoids pre-treated for 1 hour with ICI 182,780 or AG1478 followed by 5 minutes of EGF or EGF+E2 stimulation is shown. Data are represented as the mean of the ratio of phospho-Erk1/2 MAPK and total Erk1/2 MAPK levels ± standard deviation (SD) (*p<0.05) of three independent experiments. (C) Brightfield images of representative wt and p130Tg organoids at 5 days of culture treated with ICI 182,780 and stimulated with EGF or EGF+E2 every other day. Images are representative of three independent experiments. (D) Quantification of primary and secondary branching of wt vs. p130Tg organoids at 5 days of culture treated with ICI 182,780 and stimulated with EGF or EGF+E2 of three independent analyses. Two-way ANOVA analysis show a significant effect of both treatment (EGF vs. EGF+E) and genotype (wild type vs. transgenic) for Acinar Structures, primary and secondary branching (P<0.0001 for both treatment and genotype). Interaction between treatment and genotype is significant for all variables (all P<0.0001).</p

Dbl oncogene expression in MCF-10 A epithelial cells disrupts mammary acinar architecture, induces EMT and angiogenic factor secretion

<p>The proteins of the Dbl family are guanine nucleotide exchange factors (GEFs) of Rho GTPases and are known to be involved in cell growth regulation. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders, neoplastic transformation, and tumor metastasis. We have previously demonstrated that expression of Dbl oncogene in lens epithelial cells modulates genes encoding proteins involved in epithelial-mesenchymal-transition (EMT) and induces angiogenesis in the lens. Our present study was undertaken to investigate the role of Dbl oncogene in epithelial cells transformation, providing new insights into carcinoma progression.To assess how Dbl oncogene can modulate EMT, cell migration, morphogenesis, and expression of pro-apoptotic and angiogenic factors we utilized bi- and 3-dimensional cultures of MCF-10 A cells. We show that upon Dbl expression MCF-10 A cells undergo EMT. In addition, we found that Dbl overexpression sustains Cdc42 and Rac activation inducing morphological alterations, characterized by the presence of lamellipodia and conferring a high migratory capacity to the cells. Moreover, Dbl expressing MCF-10 A cells form altered 3D structures and can induce angiogenesis by producing proangiogenic factors such as CCL2. These results support a role for Dbl oncogene in epithelial cell differentiation and transformation and suggest the relevance of GEF deregulation in tumor onset and progression.</p

Identification of Abl as the tyrosine kinase responsible of p140Cap tyrosine phosphorylation on EPLYA and EGLYA sequences. A–B.

<p>For each recombinant kinase, bar charts of the mean values of the triplicate activity raw counts of kinase activity and the means of the corresponding background values of the synthetic peptides with (black bars) or without (white bars) enzyme are indicated. The synthetic peptides contain respectively EPLYA (A) and EGLYA (B) sequences. A. cDNAs encoding GFP and GFP-p140Cap full length (p140 WT) were used to transfect HEK-293 cells. After 24 hours, cells were starved and treated with 10 micromolar Src inhibitor SU6656 or Abl inhibitor Imatinib for 16 hours. Cell extracts were immunoprecipitated with a specific antibody to p140Cap and analysed by western blotting using monoclonal antibodies for phosphotyrosine and p140Cap. <b>B.</b> Left panel. cDNAs encoding GFP, GFP-p140Cap full length (p140 WT) and its double mutant (p140 EPLY/FA, EGLY/FA) were used to transfect HEK-293 cells together with cDNA encoding for active BCR-Abl. Extracts were immunoprecipitated with a specific antibody to p140Cap and analysed by western blotting using monoclonal antibodies to phosphotyrosines (PY99) and p140Cap. Right panel. cDNAs encoding GFP, GFP-p140Cap full length (p140 WT) and its double mutant (p140 EPLY/FA, EGLY/FA) were used to transfect HEK-293 cells. Cells were treated with 100 micromolar pervanadate solution for five minutes and extracts were processed as in the left panel.</p

p140Cap tyrosine phosphorylation depends mainly on two tyrosine residues. A.

<p>Schematic representation of p140Cap structure and localization of FYELE, EPLYA, EGLYA, and ADPYG sequences into the tyrosine rich region. These four tyrosine residues have been mutated to phenylalanine. <b>B.</b> cDNAs encoding GFP, GFP-p140Cap full length (p140 WT) and its single (p140 EPL<b>Y/F</b>A, p140 EGL<b>Y/F</b>A), double (p140 EPL<b>Y/F</b>A, EGL<b>Y/F</b>A; p140 F<b>Y/F</b>ELE, ADP<b>Y</b>/<b>F</b>G) and triple (p140 EPL<b>Y/F</b>A, EGL<b>Y/F</b>A, ADP<b>Y</b>/<b>F</b>G) mutants were used to transfect HEK-293 cells. 48 hours after transfection cells were treated for 5 minutes with 100 micromolar pervanadate solution. Cell extracts were immunoprecipitated with a specific antibody to p140Cap and immunocomplexes were analysed by western blotting using monoclonal antibodies to phosphotyrosine (PY99), p140Cap and Tubulin respectively. The results are representative of six independent experiments.</p