The PI3K/AKT pathway regulates growth in C4-HI but not in C4-HD tumors.

<p>C4-HD tumors were transplanted into BALB/c females carrying a MPA depot while C4-HI tumors were transplanted in the absence of MPA. When tumors reached a 150 mm² of size, they were removed and processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010786#s4" target="_blank">Materials and Methods</a> for western blot or immunohistochemistry. <b>A</b>. Left. Western blots of protein extracts from C4-HD and C4-HI tumors using specific antibodies against phosphorylated Ser473 AKT (p-AKT), total AKT, phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2 and β-actin (loading control). Three representative samples of a total of six of each tumor type are shown. Right. Quantification of AKT activation (p-AKT relative to total AKT levels) and ERK activation (p-ERK relative to total ERK levels). n = 6 for each tumor type; *: p<0.05 C4-HI vs. C4-HD. <b>B</b>. Immunohistochemistry of slices of paraffin embedded C4-HD or C4-HI tumors using antibodies against phosphorylated Ser473 AKT, showing a higher number of p-AKT-positive cells in C4-HI than in C4-HD tumors. Brown corresponds to the antibody signal and blue marks nuclei stained with hematoxylin. Scale bar: 60 µm. <b>C</b>. 3.6 mg/kg PD98059 (MEK inhibitor), 4 mg/kg LY294002 (PI3K inhibitor) or 100 µl of saline solution (c, control) were administrated i.p. every other day for 12 days to animals carrying C4-HD or C4-HI tumors. Length and width of mammary tumors (mm²) were measured every 2 days. Treatments with the inhibitors started once the tumors reached a size of approximately 30 mm². Treatment with LY294002 causes C4-HI tumors to have a decreased growth rate. n = 6 for each group; **: p<0.01 vs. control. <b>D</b>. Left. At the end of 12 days of treatment, tumors were removed to evaluate by western blot the inhibitory effect of PD98059 and LY294002 on p-ERK1/2 and p-AKT, respectively. Two representative samples of a total of six are shown in the gel (left). Right. Quantification of AKT and ERK activation as in A. n = 6 for each group; a,b: p<0.01 C4-HI vs. C4-HD; *:p<0.05 vs. control. <b>E</b>. Quantification of the percentage of apoptosis at the end of treatment. Data corresponds to the mean±SEM percentage of apoptotic cells in ten high-power fields corresponding to six independent experiments. Apoptosis is higher in C4-HI tumors treated with LY294002. **: p<0.01 vs. control. <b>F</b>. C4-HI tumors are more differentiated than C4-HD tumors. After 12 days of treatment with LY294002, there is an increase in the number of ductal-like structures only in the C4-HI tumors, as indicated by the black arrows. Scale bar: 30 µm.</p

C4-HD tumor cells are more sensitive to steroid receptor inhibitors.

<p>Apoptosis induced by inhibitors of ER (ICI182780) and PR (ZK230211) is higher in C4-HD than C4-HI cells. Top: Phase contrast microscopy showing representative C4-HD (left) and C4-HI (right) cell clusters in the presence of 1 µM ICI182780, 0.01 µM ZK230211 or vehicle as control. Confocal images from a fluorescence microscope of AO/EB staining show a higher number of apoptotic cells (red cells) in C4-HD cultures treated with ICI182780 or ZK230211 than in C4-HD untreated cultures. C4-HI cultures exhibit no change in cell death after endocrine treatments. Bottom. Quantification of the percentage of apoptotic C4-HD (left) and C4-HI (right) cells per cluster, of four independent experiments with ten clusters in each. Data corresponds to the mean +/− SEM. ***:p<0.001 vs. control. Scale bar: 30 µm.</p

Cell death induced by LY294002 in C4-HI cancer cells involves intrinsic BAX/mitochondrial/caspase-9 apoptotic pathway.

<p>The 3D Matrigel culture system of primary C4-HI cancer cells reproduces the increased pro-apoptotic effect of the PI3K inhibitor observed <i>in vivo</i>. <b>A</b>. Top. Phase contrast microscopy showing a representative C4-HD (left) and C4-HI (right) cell cluster cultured for 96 hrs on Matrigel and treated for the last 48 hrs with 10 µM PD98059, LY294002, or vehicle as control. Confocal images from a fluorescence microscope of acridine orange/ethidium bromide (AO/EB) staining was used to discriminate live from apoptotic cells. AO fluoresces green in live cells and EB fluoresces orange/red when intercalated with DNA in dead cells. Most C4-HI cell clusters on Matrigel exhibit a central lumen. In contrast, no C4-HD cell clusters possess a central lumen. A higher number of apoptotic cells in and around the central lumen of LY294002-treated C4-HI cells was also noted. Scale bar: 30 µm. Bottom. Quantification of the percentage of apoptotic cells per cluster, of four independent experiments with ten clusters in each. Data corresponds to the mean +/− SEM. LY294002 induces cell death in C4-HI cells; **:p<0.01 vs. control. <b>B</b>. Confocal images showing higher BAX and activated caspase-9 staining, and lower Bcl-XL staining in C4-HI cells treated with 10 µM LY294002. Nuclei were stained red with propidium iodide. Scale bar: 30 µm.</p

Differential sensitivity to PI3K/AKT pathway regulation is lost in isolated cancer cells.

<p><b>A</b>. Phase contrast microscopy of primary cultures isolated from C4-HD and C4-HI tumors showing flattened monolayers on tissue culture plastic. Scale bar: 30 µm. <b>B</b>. Top. Western blots showing p-AKT, total AKT, p-ERK1/2, ERK1/2 and β-actin in protein extracts from primary cells cultured for 96 hrs. Three representative samples of a total of six are shown in the gel. Bottom. Quantification of p-AKT/total AKT and p-ERK/total ERK levels, n = 6 for each cell type. <b>C</b>. Proliferation assay (³H-thymidine incorporation) in primary C4-HD (left) or C4-HI (right) cells incubated during the last 48 hrs with 0.01 µM MPA and 5, 10 and 20 µM PD98059 or LY294002. For the combined treatment, the inhibitor was used at a dose of 10 µM. ³H-thymidine was added in the last 18 hrs before harvesting the cells. MPA has a stimulatory effect while PD98059 and LY294002 have an inhibitory effect on MPA-treated and untreated cells. n = 8 for each treatment. ***:p<0.001; **:p<0.01; *: p<0.05 vs. control (untreated cells); different letters indicate significant differences between treatments in the presence of MPA with p<0.01. A representative experiment of a total of three is shown here. <b>D</b>. Left. Western blots showing that phosphorylation levels of AKT and ERK1/2 decrease in primary C4-HI cultures incubated for 48 hrs with 10 µM of specific inhibitors. β-actin levels in the blot serve as a loading control. Two representative samples of a total of six are shown (left). Right. Quantification of p-AKT/total AKT and p-ERK/total ERK levels. n = 6 for each group; *p<0.05; **p<0.01 vs. control.</p

AKT regulates ERα protein expression.

<p><b>A</b>. ERα levels determined by western blot are reduced in C4-HI tumors treated with PD98059 or LY294002 administrated as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010786#pone-0010786-g001" target="_blank">Figure 1</a>. However, ERα levels are more variable between tumors and not regulated by either inhibitor in C4-HD tumor samples. Top. Two representative samples of a total of six are shown in the gel. Bottom. Quantification of ERα relative to β-actin (used as a loading control) levels. n = 6 for each treatment; **:p<0.01 vs. control. <b>B</b>. A similar pattern of regulation of ERα protein is observed in primary cells cultured on top of Matrigel and incubated for 48 hrs with 10 µM of either of the inhibitors. Top. Two representative samples of a total of six of each group are shown in two independent gels. Bottom. Quantificaton of ERα relative to β-actin in each cell type. n = 6 for each treatment; **p<0.01 vs. control. <b>C</b>. Representative confocal images showing ERα determined by immunofluoresence (green). Lower levels of the protein are seen in PD98059-treated and in LY294002-treated C4-HI cells growing on Matrigel, whereas similar levels of ERα are seen in C4-HD treated and un-treated cells. Nuclei were stained red with propidium iodide. Scale bar: 30 µm. <b>D</b>. Top. A representative sample for each treatment is shown in a western blot. Scp2, a mouse mammary cell line, transfected with a constitutively active form of AKT1, myristoylated AKT1-Δ4-129 (Scp2Akt), displays in the same gel AKT with a typical molecular weight of 59 kDa and the myristoylated deleted variant of AKT1 (p-myrAKT) with a molecular weight of 45 kDa. The antibody used to detect total AKT recognizes only wild type AKT. E-cadherin was used as a loading control for Scp2 cells. Scp2Akt cells exhibit higher levels of ERα than Scp2 cells transfected with the vector control (vc) or Scp2 control cells. In Scp2 and Scp2vc cells, but not in Scp2Akt cells, 2 and 5 µM LY294002 downregulate p-AKT and ERα levels, whereas total AKT levels remains invariable. Bottom. Quantification of ERα relative to E-cadherin levels. n = 4 for each treatment; a,b p<0.05 Scp2Akt vs. Scp2 and Scp2vc; *p<0.05 LY294002 vs. control.</p

Loss of endocrine resistance in isolated C4-HIR tumor cells is not prevented by 3D Matrigel.

<p>Isolated C4-HI and C4-HIR tumor cells are sensitive to cell death in response to RU486 when cultured on plastic, and the 3D Matrigel culture system is not sufficient to revert this phenotype. <b>A</b>. C4-HI and C4-HIR tumors were transplanted in syngeneic mice and measured every 2 days (length and width). RU486 was inoculated s.c. at a dose of 12 mg/kg/day once the tumors were 60 mm². C4-HI tumors (left) regress following treatment with antiprogestin, while C4-HIR tumors (right) are unresponsive. <b>B</b>. Fluorescent confocal images of AO/EB staining show similar number of apoptotic cells (red cells) after 48 hrs of treatment with 0.01 µM MPA and a higher number of apoptotic cells with 0.01 µM RU486 treatment. Right. Quantification of the percentage of apoptotic C4-HI (upper) and C4-HIR (lower) cells per cluster, of three independent experiments with ten clusters in each. Data corresponds to the mean +/− SEM; ***:p<0.001 vs. control. Scale bar: 100 µm.</p

Effects of asPR or RU 486 on MAPK phosphorylation and on ER-α and PR expression

<p><b>Copyright information:</b></p><p>Taken from "Antisense oligonucleotides targeting the progesterone receptor inhibit hormone-independent breast cancer growth in mice"</p><p>Breast Cancer Research 2005;7(6):R1111-R1121.</p><p>Published online 9 Nov 2005</p><p>PMCID:PMC1410760.</p><p>Copyright © 2005 Lamb et al.; licensee BioMed Central Ltd.</p> Immunoblots of ER-α (MC-20; Santa Cruz Biotechnology), total ERK (K-23; Santa Cruz Biotechnology), and pERK (E-4; Santa Cruz Biotechnology) in whole extracts of tumors obtained from animals treated with saline, asPR, or scPR for 5 days. Tumor samples were obtained after day 5 of treatment; tumor growth kinetics is shown in Fig. 2d. Arrows show ERK1 (42 kDa) and ERK2 (44 kDa). PR immunoblots were performed using extracts obtained from mice treated with asPR over 10 days (Ab1; Dr Shyamala). Arrows show the classical PRof 115 kDa and the classical PRof 83 kDa. Immunoblots of ER-α, PR (Ab7; Neomarkers), E-cadherin (E cad; BD Transduction Lab), total ERK, and pERK using wholeextracts of tumors obtained from animals treated with saline or RU 486 for 24 hours. Tumor kinetics are shown in Fig. 2c. A representative Western blot of three is shown. Immunohistochemistry of ER-α and PR (C-20 Santa Cruz) of the same tumor samples used in Western blot studies shown in panel b (125×). Experimental details are described in Materials and method. asPR, antisense oligodeoxynucleotides to progesterone receptors; ER, estrogen receptor; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PR, progesterone receptor; scPR, scrambled oligodeoxynucleotides to progesterone receptors

Tumor growth and hormone receptor regulation in endocrine treated murine mammary carcinomas tumors.

<p>(<b>A</b>) Effects of MPA or MFP on tumor growth. <b>(Left)</b> BALB/c mice were sc transplanted in the inguinal flank with C4-HD mammary carcinomas and simultaneously MPA- (40 mg) HMSP or Hormone Pellet Press pellets were sc implanted into the back of the mice (N = 4–6/group). Treatments began when tumors were transplanted. <b>(Right)</b> Mice bearing C4-HI mammary carcinomas were treated with 6 mg MFP-HMSP (N = 3), MFP solution (12 mg/kg/day; N = 3) or left untreated (N = 6). Treatments began when tumors reached approximately 50 mm² (arrow). Tumor sizes are expressed as mean ± SD. (<b>B</b>) Immunohistochemical expression of ERα (<b>right</b>) and PR (<b>left</b>) in C4-HD (<b>upper panel</b>) and C4-HI (<b>lower panel</b>) mammary carcinomas. C4-HD carcinomas were treated with MPA and when tumors reached approximately 50 mm², the pellet was removed for 48 h in untreated tumors. C4-HI-bearing mice were treated for 72 h with E2 (5 mg) once the tumors reached 50 mm². Bar: 50 µm.</p

Effect of E2- or Pg-HMSP in uterine and mammary gland morphology.

<p>(<b>A</b>) Histological features of 5- µm-thick sections of uteri and mammary glands from mice treated for 14 days with control, E2- (20 µg), E2- (5 mg) and Pg- (5 mg) HMSP. Mice were ovariectomized followed by a 7 day washout period and treated for 14 days. <b>Uterus</b>. <b>Control:</b> The endometrium is lined by a low cylindrical epithelium (arrow head, inset); the endometrial stroma is densely packed (star, inset), with embedded small endometrial glands (arrow heads). The star in the main picture shows the thinned myometrial wall. <b>E2 20 µg:</b> Well-developed endometrial glands with proteinaceous material inside (arrow heads). The surface epithelium is locally stratified, composed of more than one layer of cells (arrow heads, inset). <b>E2 5 mg:</b> The surface epithelium is irregular and composed of one or more cell layers (inset, white arrow head) with focal apoptosis, the lumen discloses plugs of coagulated secretion (inset, arrow head) and mononuclear cells. <b>Pg 5 mg:</b> The cells lining the glandular structures are cylindrical and the nuclei are basally located (inset, arrow head). <b>Mammary gland</b>. <b>E2 20 µg:</b> Well-developed ductal structure lined by a stratified cuboidal epithelium; a thin stromal layer is evident in this picture (arrow head). <b>Pg 5 mg:</b> Ductal structure showing signs of initial lobular development (arrow heads). Magnification bar: 50 µm. (<b>B</b>) Effect of E2- and Pg-HMSP on luminal epithelium height following 14 days of pellet implantation to ovariectomized mice. Bar: 10 µm. ***: p<0.001; **: p<0.01. (<b>C</b>) Effect of co-administration of E2- and Pg-HMSP on luminal epithelium height following 7 days of pellet implantation to ovariectomized mice. ***: p<0.001; a <i>vs</i>. b: p<0.001.</p

E2, Pg, LH and Prl serum levels after E2- or Pg-HMSP implantation.

<p>Effect of E2- or Pg-HMSP on serum levels of E2 (<b>A</b>), Pg (<b>B</b>), LH (<b>C</b>) or Prl (<b>D</b>) in ovariectomized adult mice treated for up to 28 days with the HMSP (n = 4–6/group). Values represent means ± S.E.M. Values with different letters are significantly different (a <i>vs.</i> b or a′ <i>vs.</i> b′: p<0.05; a <i>vs.</i> c or a′ <i>vs.</i> c′: p<0.01; and a <i>vs.</i> d or a′ <i>vs.</i> d′ or a′′ <i>vs.</i> d′′: p<0.001). Two-way ANOVA (treatment by time) with <i>post-hoc</i> Fisher’s tests was performed for LH (p interaction = 0.0089), Prl (p interaction = 0.011) and E2 (p interaction = 0.00018); and one-way ANOVA with <i>post-hoc</i> Fisher’s tests in Pg (p time <0.0001). Inset: Student t test (p<0.01).</p