ILC-CORE (Collaboration, Openness, REproducibility)

Rigor, Reproducibility, Transparency, and Collaboration Network for ILC Researc

Peroral Estradiol Is Sufficient to Induce Carcinogen-Induced Mammary Tumorigenesis in Ovariectomized Rats without Progesterone.

A role for estrogens in breast cancer is widely accepted, however, recent evidence highlights that timing and exposure levels are important in determining whether they elicit harmful versus beneficial effects. The rat chemical carcinogen model has been widely used to study the effects of estrogens but conclusions on the levels that lead to tumor development and an absolute requirement for progesterone (P4) are lacking. A newer method of hormone administration mixes hormones with nut butter for peroral consumption allowing for a less stressful method of long-term administration with lower spikes in serum estradiol (E2) levels. The present study was designed to determine if estrogens alone at a physiological dose can drive carcinogen-induced tumors in ovariectomized (OVX) rats or if P4 is also required using this method of hormone administration. Short-term studies were conducted to determine the dose of estrogen (E) that would lead to increased uterine weight following OVX. Subsequently, rats were OVX on postnatal day (PND) 40 then treated daily with E (600 μg/kg/day), P4 (15 mg/kg/day), or the combination. On PND 50, all rats were injected with nitrosomethylurea to induce mammary tumors. Uterine weights, body weights, and serum E2 levels were measured to demonstrate the efficacy of the method for increasing E2 levels during long-term treatment. After 26 weeks, tumor incidence was similar in Sham, E, and E + P4 animals indicating that E was sufficient to induce tumorigenesis when hormone levels were normalized by this method. This study demonstrates peroral administration can be used in long-term studies to elucidate relationships between different types and levels of steroid hormones

ROS is a master regulator of in vitro matriptase activation.

Matriptase is a type II transmembrane serine protease that is widely expressed in normal epithelial cells and epithelial cancers. Studies have shown that regulation of matriptase expression and activation becomes deranged in several cancers and is associated with poor disease-free survival. Although the central mechanism of its activation has remained unknown, our lab has previously demonstrated that inflammatory conditions such as intracellular pH decrease strongly induces matriptase activation. In this investigation, we first demonstrate clear matriptase activation following Fulvestrant (ICI) and Tykerb (Lapatinib) treatment in HER2-amplified, estrogen receptor (ER)-positive BT474, MDA-MB-361 and ZR-75-30 or single ER-positive MCF7 cells, respectively. This activation modestly involved Phosphoinositide 3-kinase (PI3K) activation and occurred as quickly as six hours post treatment. We also demonstrate that matriptase activation is not a universal hallmark of stress, with Etoposide treated cells showing a larger degree of matriptase activation than Lapatinib and ICI-treated cells. While etoposide toxicity has been shown to be mediated through reactive oxygen species (ROS) and MAPK/ERK kinase (MEK) activity, MEK activity showed no correlation with matriptase activation. Novelly, we demonstrate that endogenous and exogenous matriptase activation are ROS-mediated in vitro and inhibited by N-acetylcysteine (NAC). Lastly, we demonstrate matriptase-directed NAC treatment results in apoptosis of several breast cancer cell lines either alone or in combination with clinically used therapeutics. These data demonstrate the contribution of ROS-mediated survival, its independence of kinase-mediated survival, and the plausibility of using matriptase activation to indicate the potential success of antioxidant therapy

Peroral treatment with E and E + P4 after OVX results in similar tumor incidence but more adenocarcinomas following NMU relative to Sham.

<p>(a) Rats were OVX on PND 40, injected with NMU on PND 50, and palpated biweekly for the appearance of tumors. There was no difference in tumor incidence between E, E + P4 and Sham but all three groups were different from P only and OVX, which were not different from one another (p < 0.05, Mantel-Cox Log-rank test). (b) Tumor type was classified by a histological pathologist as adenoma or adenocarcinoma based on the most malignant part of each section. The effect of treatment on tumor type was analyzed using a Chi-square analysis (* p < 0.05). (c) Tumors were measured with a Vernier caliper the morning of sacrifice. To calculate tumor burden, the volume of each tumor was added together per animal. There were no differences in tumor burden among treatment groups.</p

Serum E2 levels are elevated with peroral hormone treatment.

<p>Blood samples were drawn from the lateral tail vein 5, 3, and 5 hours after treatments were presented 4, 8, and 12 weeks after NMU injection, respectively. Blood draws from all animals were completed within 2 hours on each of the three days. Serum was analyzed using an E2 ELISA (Calbiotech). Blood from one animal in the 12-week E only group and one animal in the 8-week E + P4 group hemolyzed, thus these samples were not included in the analysis. All values from E and E + P4 animals are presented to demonstrate the range of serum E2 across animals and treatments while a subset of Sham animals and serum pools from OVX animals were included for comparison. Horizontal lines indicate mean values for each time point.</p

Treatment of OVX animals with E or E + P4 results in tumors with similar proliferation indices and receptor status relative to tumors from Sham controls.

<p>Ki67, ERα, and PR IHC was performed and measured as described. Images depicting low (a, d, g) and high (b, e, h) expression of Ki67, ERα, and PR are presented with corresponding scores in the bottom left corner of each image. There were no differences in Ki67 or ERα expression between treatment groups. Animals treated with E + P4 had more PR expression than animals treated with E alone (Mann-Whitney test comparing E only to E + P4, p < 0.05).</p

Long-term daily peroral E treatment is physiologically active.

<p>At sacrifice, uteri were excised and weighed. Images of uteri were taken before storage in 10% NBF. (a) Representative images of uteri from each treatment group. (b) Uterine weights were reduced in OVX rats treated with vehicle but increased with E or E + P4 treatment. P4 alone was unable to restore uterine weights. Uterine weights are expressed as mean ± SEM; n = 8, 7 for P4 only; p < 0.05, one-way ANOVA; Newman-Keul’s posttest; different letters denote significant difference. (c) Rats were weighed biweekly for the duration of the study. Long-term exposure to E or E+ P4 resulted in body weights that were similar to those of Sham controls. Daily treatment with P4 did not prevent weight gain following OVX. Body weights are expressed as mean ± SEM, n = 8, 7 for P4 only; repeated measures ANOVA through the first animal sacrifice at 13 weeks post NMU (PND 148) (treatment: p < 0.01) with Bonferroni-Dunn multiple comparisons test (* start of p < 0.05 for OVX, # start of p < 0.05 for P4 only; compared to Sham). The pattern persisted in the remaining animals at the end of the study (p < 0.05, one-way ANOVA, Newman-Keul’s posttest).</p

Investigating ICI and Lapatinib induced matriptase determined that only modest activation resulted from PI3K inhibition with no activation from MAPK inhibition and occurs as early as six hours post treatment.

A) BT474 and ZR-75-30 cells were seeded into 35mm dishes and when ~90% confluent, washed twice with sterile 1X PBS and incubated for 24 hours in serum-replete media with 1uM ICI. Cells were then treated with 5.4uM Lapatinib, 10uM Trametinib, 10uM Pictilisib, or 0.01% DMSO. Conditioned media was collected with the appropriate lysis buffer (see methods). B) BT474 cells were seeded into 35mm dishes and when ~90% confluent, washed twice with sterile 1X PBS and incubated for 24 hours in serum-replete media with 1uM ICI. Cells were then spiked with 5.4uM Lapatinib for increasing amounts of time. Cell lysates and conditioned media were collected with the appropriate lysis buffer. The levels of phospho-AKT, total and activated matriptase were determined by immunoblotting with p-S473 AKT and total matriptase (M24) monoclonal antibodies. C) BT474 cells were seeded into 35mm dishes and when ~90% confluent, washed twice with sterile 1X PBS and incubated for increasing amounts of time in serum-free media with 1uM ICI. After each time point, cells were spiked with 5.4uM Lapatinib for 1 hour. Cell lysates were collected with the appropriate lysis buffer and the levels of total and activated matriptase were determined by immunoblotting with the total matriptase (M24) monoclonal antibody. GAPDH was used as a loading control. Panels were the highest quality representative of 4 biological replicates.</p

Treatment with E leads to increased uterine metaplasia in treated animals.

<p>Cross sections of uteri were stained with hematoxylin and eosin. A toxicological pathologist staged and graded the slides from 0–3 for each category leading to scores of stage + grade, specifically focusing on the uterine lumen (arrows) and not on dilated ducts (arrow heads). Images are representative of 0+0 (relatively normal), 1+1, 2+2, and 3+3 respectively. The larger images were taken at 20X magnification and the inset at 4X magnification. Scores were derived by adding stage and grade to get a final score of 0–6. P4 only and OVX animals were not included in analysis as all uteri were atrophied and not metaplastic. Uterine scores are expressed as mean ± SEM; n = 8; p < 0.05, Kruskal-Wallis test with Dunn’s Multiple Comparison’s posttest; different letters denote significant difference.</p

Mammary gland development of E and E + P4 treated animals is similar to Sham controls.

<p>Representative images of whole mounts (4X magnification) demonstrated ductal thickness was similar in Sham (a), E only (b), and E + P4 (c) while the overall morphology of glands was similar between OVX (d) and P4 only (e) with thinner ducts relative to the other groups. The length of the mammary gland parenchyma was measured from the lymph node to the most distal edge of the ducts using FIJI as shown in (f). The parenchyma from Sham, E only, and E + P4 glands exhibited similar ductal lengths while OVX and P4 only were shorter (g). Mammary gland length is expressed as mean ± SEM; p < 0.05, one-way ANOVA; Newman-Keul’s posttest.</p