Journal: eLife

Article Title: Mechanism and preclinical prevention of increased breast cancer risk caused by pregnancy

doi: 10.7554/eLife.00996

Figure Lengend Snippet: ( A and B ) Kaplan–Meier survival curves ( A ) and bar graph showing tumor multiplicity ( B ). Generalized Gehan–Wilcoxon test determined p value for ( A ), and Pearson’s Chi-square test derived p value for ( B ). ( C ) Area and incidence of sub-palpable tumors (indicated by arrows) were calculated at 18-months post infection using ImageJ. n = 24 virgin and 30 parous mice. Student’s t test defined p values. LN, lymph node. ( D ) Immunofluorescence for the HA tag (top panel) located the lesions initiated by RCAS- Wnt1 , and TUNEL assay (bottom panel) performed on the consecutive section identified apoptotic cells in lesions. n = 5 mice. Student’s t test determined p values. ( E ) Immunofluorescence for pSTAT5. Graph indicates the proportion of pSTAT5 + lesions (>5% pSTAT5 + cells). Horizontal bars represent the mean. n ≥3 mice for each group. Student’s t test determined p values. Percentage of pSTAT5 + cells in lesions is shown in associated . For all bar graphs except ( B ), columns represent mean, and error bars represent SEM. All scale bars = 20 μm. DOI: http://dx.doi.org/10.7554/eLife.00996.007

Article Snippet: Primary antibodies used were mouse monoclonal HA (1:1000; Covance), BclXL (1:500; Santa Cruz), cyclinD1 (Cell Signaling, 1:500), and STAT5b (Santa Cruz; 1:500); and rabbit polyclonal Bcl2 (1:500; Santa Cruz), PRLR (1:500; Santa Cruz), pSTAT5 (1:500; Cell Signaling), STAT5a (1:500; Santa Cruz), and GAPDH (1:2000; Santa Cruz).

Techniques: Derivative Assay, Infection, Immunofluorescence, TUNEL Assay

Journal: eLife

Article Title: Mechanism and preclinical prevention of increased breast cancer risk caused by pregnancy

doi: 10.7554/eLife.00996

Figure Lengend Snippet: ( A ) Immunofluorescence for Ki67 detected proliferation in lesions . n = 5 mice. Columns represent mean, and error bars represent SEM. Scale bar = 20 μm. ( B ) Immunofluorescence detected nuclear pSTAT5 in lesions . Graph indicates percentage of pSTAT5 + cells in lesions. Horizontal bars represent the mean. Student's t test determined all p values. DOI: http://dx.doi.org/10.7554/eLife.00996.008

Article Snippet: Primary antibodies used were mouse monoclonal HA (1:1000; Covance), BclXL (1:500; Santa Cruz), cyclinD1 (Cell Signaling, 1:500), and STAT5b (Santa Cruz; 1:500); and rabbit polyclonal Bcl2 (1:500; Santa Cruz), PRLR (1:500; Santa Cruz), pSTAT5 (1:500; Cell Signaling), STAT5a (1:500; Santa Cruz), and GAPDH (1:2000; Santa Cruz).

Techniques: Immunofluorescence

Journal: eLife

Article Title: Mechanism and preclinical prevention of increased breast cancer risk caused by pregnancy

doi: 10.7554/eLife.00996

Figure Lengend Snippet: ( A and B ) Immunofluorescence for pSTAT5 in lesions ( A ) and accompanying quantification ( B ). Insets show staining of normal ducts ( A ). pSTAT5 + lesions are those that have >5% pSTAT5 + cells ( B ). n ≥ 3 mice. Quantification of percentage of pSTAT5 + cells in lesions is presented in associated and analysis of pSTAT1, pSTAT3 and pSTAT6 is presented in . ( C ) Generalized linear regression analysis for correlation between the square root of the number of pSTAT5 + cells in individual I10 lesions and percentage of CC3 + cells in their corresponding lesions. Dots represent individual lesions. Colors represent individual mice (n = 4). ( D ) Immunofluorescence for Bcl XL . Positive staining in cytoplasmic (arrow) and perinuclear (arrowhead) regions was observed. Bar graph shows the percentage of Bcl XL + cells in early lesions. n = 4. Supporting data in . ( E ) Immunohistochemical staining for GSK3-β phosphorylated at Ser9. n = 4 mice. ( F ) Immunohistochemical staining for PRLR. Black arrows indicate cells with membrane PRLR. Bar graph shows the percentage of PRLR + cells in early lesions. n = 4 mice. Supporting data in . Scale bars = 20 μm. All columns indicate the mean, and error bars represent SEM. p value for the P7.5 time point was derived by a Wilcoxon Rank Sum test. All other p values were generated by Student’s t test. Supporting Western blotting data are presented in associated figure supplements. DOI: http://dx.doi.org/10.7554/eLife.00996.010

Article Snippet: Primary antibodies used were mouse monoclonal HA (1:1000; Covance), BclXL (1:500; Santa Cruz), cyclinD1 (Cell Signaling, 1:500), and STAT5b (Santa Cruz; 1:500); and rabbit polyclonal Bcl2 (1:500; Santa Cruz), PRLR (1:500; Santa Cruz), pSTAT5 (1:500; Cell Signaling), STAT5a (1:500; Santa Cruz), and GAPDH (1:2000; Santa Cruz).

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Derivative Assay, Generated, Western Blot

Journal: eLife

Article Title: Mechanism and preclinical prevention of increased breast cancer risk caused by pregnancy

doi: 10.7554/eLife.00996

Figure Lengend Snippet: ( A ) The percentage of pSTAT5 + and total STAT5a + cells in 15 early lesions per mouse was quantified. Representative photomicrographs and additional quantification are shown in . n ≥ 3 mice. For pSTAT5 + cell frequency, Wilcoxon rank-sum test derived p values for P7.5 and L12, and Student's t test measured p values for I10. For total STAT5a + cell frequency, Student's t test measured all p values. ( B ) Western blotting quantified pSTAT5 and total STAT5a and b isoforms in protein extracts from whole mammary glands of virgin and parous mice injected with RCAS- caErbb2 . Student's t test measured p value for pSTAT5. DOI: http://dx.doi.org/10.7554/eLife.00996.011

Article Snippet: Primary antibodies used were mouse monoclonal HA (1:1000; Covance), BclXL (1:500; Santa Cruz), cyclinD1 (Cell Signaling, 1:500), and STAT5b (Santa Cruz; 1:500); and rabbit polyclonal Bcl2 (1:500; Santa Cruz), PRLR (1:500; Santa Cruz), pSTAT5 (1:500; Cell Signaling), STAT5a (1:500; Santa Cruz), and GAPDH (1:2000; Santa Cruz).

Techniques: Derivative Assay, Western Blot, Injection

Journal: eLife

Article Title: Mechanism and preclinical prevention of increased breast cancer risk caused by pregnancy

doi: 10.7554/eLife.00996

Figure Lengend Snippet: Representative photomicrographs depicting immunohistochemical detection of nuclear pSTAT5 in mammary glands of age-matched virgin (V) and parous (I10) transgenic MMTV- Erbb2 mice. n = 5 mice. Scale bar represents 50 µm. DOI: http://dx.doi.org/10.7554/eLife.00996.009

Article Snippet: Primary antibodies used were mouse monoclonal HA (1:1000; Covance), BclXL (1:500; Santa Cruz), cyclinD1 (Cell Signaling, 1:500), and STAT5b (Santa Cruz; 1:500); and rabbit polyclonal Bcl2 (1:500; Santa Cruz), PRLR (1:500; Santa Cruz), pSTAT5 (1:500; Cell Signaling), STAT5a (1:500; Santa Cruz), and GAPDH (1:2000; Santa Cruz).

Techniques: Immunohistochemical staining, Transgenic Assay

Journal: eLife

Article Title: Mechanism and preclinical prevention of increased breast cancer risk caused by pregnancy

doi: 10.7554/eLife.00996

Figure Lengend Snippet: ( A ) Kaplan–Meier tumor-free survival curves of virgin mice injected with RCAS- caErbb2 alone (black) or with both RCAS- caErbb2 and RCAS- caStat5a (blue). The parous group from is shown for comparison (red). Comparison of lesion apoptosis and proliferation between the two virgin groups is presented in associated . ( B ) Kaplan–Meier tumor-free survival curves of the virgin cohort from that was stratified into pSTAT5 hi (blue) and pSTAT5 lo (black) groups based on baseline pSTAT5 levels in the normal mammary glands of each of these mice . The parous group from is shown for comparison (red). ( C and D ) Immunofluorescence detected pSTAT5 + cells ( C ) and apoptotic cells ( D ) in lesions induced by RCAS- caErbb2 in MMTV- tva and WAP- tva mice. n = 6 ( C ) and 4 ( D ) mice. Scale bar=20 μm. Columns represent the mean, and error bars indicate SEM. Student’s t test determined p values. Proliferation in lesions is shown in , and relevant baseline characteristics of these two mouse lines are delineated in . ( E ) Kaplan–Meier tumor-free survival curves comparing MMTV- tva and WAP- tva virgin mice injected with RCAS- caErbb2 . Tumor multiplicity is presented in . ( F ) Kaplan-Meier tumor-free survival curves comparing WAP- tva mice injected with RCAS- caErbB2 and then either kept as virgin or impregnated. The p value for all tumor-free survival comparisons was generated using generalized Gehan-Wilcoxon test. DOI: http://dx.doi.org/10.7554/eLife.00996.015 10.7554/eLife.00996.016 Figure 4—source data 1. Comparison of uninfected and infected cells in MMTV- tva vs WAP- tva mice. Table indicates percentages of infected mammary epithelial cells (identified by the HA tag in RCAS- β-actin ) that are positive for pSTAT5 in MMTV- tva and WAP- tva virgin mice that were injected with RCAS- β-actin (HA) (n = 4 mice) 4 days earlier, percentages of pSTAT5 + cells in uninfected control ducts (n = 5 mice), and percentages of infected cells in mammary glands of MMTV- tva and WAP- tva virgin mice that were injected with RCAS- GFP and analyzed by FACS 4 days later (n = 10 mice). Student's t test derived the p values. Associated histological and graphical data are presented in and associated figure supplements. DOI: http://dx.doi.org/10.7554/eLife.00996.016

Article Snippet: Primary antibodies used were mouse monoclonal HA (1:1000; Covance), BclXL (1:500; Santa Cruz), cyclinD1 (Cell Signaling, 1:500), and STAT5b (Santa Cruz; 1:500); and rabbit polyclonal Bcl2 (1:500; Santa Cruz), PRLR (1:500; Santa Cruz), pSTAT5 (1:500; Cell Signaling), STAT5a (1:500; Santa Cruz), and GAPDH (1:2000; Santa Cruz).

Techniques: Injection, Immunofluorescence, Generated, Infection, Derivative Assay

Journal: eLife

Article Title: Mechanism and preclinical prevention of increased breast cancer risk caused by pregnancy

doi: 10.7554/eLife.00996

Figure Lengend Snippet: Linear correlation between the percentage of pSTAT5 + cells in early lesions vs percentage of pSTAT5 in normal ducts ( A ) of virgin mice, and between the percentage of pSTAT5 + cells in early lesions and percentage of apoptotic cells (as identified by CC3) in early lesions ( B ). Each dot represents a mouse. Statistical significance and R 2 were derived using a generalized linear regression model. Associated tumor latency data are presented in . DOI: http://dx.doi.org/10.7554/eLife.00996.018

Article Snippet: Primary antibodies used were mouse monoclonal HA (1:1000; Covance), BclXL (1:500; Santa Cruz), cyclinD1 (Cell Signaling, 1:500), and STAT5b (Santa Cruz; 1:500); and rabbit polyclonal Bcl2 (1:500; Santa Cruz), PRLR (1:500; Santa Cruz), pSTAT5 (1:500; Cell Signaling), STAT5a (1:500; Santa Cruz), and GAPDH (1:2000; Santa Cruz).

Techniques: Derivative Assay

Journal: eLife

Article Title: Mechanism and preclinical prevention of increased breast cancer risk caused by pregnancy

doi: 10.7554/eLife.00996

Figure Lengend Snippet: ( A ) Immunofluorescent staining for pSTAT5 in early lesions of mice with the indicated genotype. n = 5. Scale bar = 20 µm. ( B ) Levels of apoptosis in early lesions were quantified via immunostaining for CC3. n = 5 mice. ( C and D ) Average lesion number ( C ) and area ( D ) were quantified using images of immunostaining for RCAS- caErbb2 -HA. n = 5 mice. ( E ) Tumor incidence at day 66 post injection with RCAS- caErbb2 . Black region indicates percentage of mice with palpable tumors. n = 18 parous mice and 19 virgin mice. ( F ) Kaplan–Meier tumor-free survival curves comparing Stat5a −/− parous and virgin mice. p values were determined by Student’s t test ( B – D ), Fisher’s exact test ( E ), and generalized Gehan–Wilcoxon test ( F ). Columns represent means and error bars SEM except for ( E ). DOI: http://dx.doi.org/10.7554/eLife.00996.021

Article Snippet: Primary antibodies used were mouse monoclonal HA (1:1000; Covance), BclXL (1:500; Santa Cruz), cyclinD1 (Cell Signaling, 1:500), and STAT5b (Santa Cruz; 1:500); and rabbit polyclonal Bcl2 (1:500; Santa Cruz), PRLR (1:500; Santa Cruz), pSTAT5 (1:500; Cell Signaling), STAT5a (1:500; Santa Cruz), and GAPDH (1:2000; Santa Cruz).

Techniques: Staining, Immunostaining, Injection

Journal: eLife

Article Title: Mechanism and preclinical prevention of increased breast cancer risk caused by pregnancy

doi: 10.7554/eLife.00996

Figure Lengend Snippet: ( A – F ) Bar graphs showing the percentage of pSTAT5 + RCAS- caErbb2 -induced lesions ( A and D ), apoptotic cells in lesions ( B and E ), and average lesion area ( C and F ) of I10 or virgin mice that were treated with either AG490 ( A – C ) at the indicated doses or Ruxolitinib ( D – F ). n ≥ 3 mice. Statistical significance was determined by Student’s t test ( A and B , D – F ) and ANOVA ( C ). The impact of AG490 on Bcl XL in these lesions is presented in . ( G ) Bar graphs showing percentage of CC3 + cells in lesions of mice treated with DMSO or C188-9. n = 4 mice. Student’s t test derived p value. The potency of C188-9 on STAT5 is shown in . The impact of C188-9 on Bcl XL and cell proliferation is shown in . ( H ) Early lesions in parous mice treated either with DMSO or C188-9 were identified by immunohistochemical staining for the HA tag of RCAS- caErbb2 , and their areas were then quantified using ImageJ. n = 4 mice. Scale bar = 50 μm. Student’s t test derived p value. ( I and J ) Kaplan–Meier tumor-free survival curves ( I ) and tumor incidence plot ( J ) of parous and age-matched virgin controls treated with either C188-9 or DMSO. Generalized Gehan–Wilcoxon test determined p-value for ( I ), and Fisher’s Exact test determined p-value for ( J ). Tumor latency and incidence for virgin mice treated with C188-9 or DMSO are presented in . For all bar graphs, columns represent the mean, and error bars indicate SEM. DOI: http://dx.doi.org/10.7554/eLife.00996.024

Article Snippet: Primary antibodies used were mouse monoclonal HA (1:1000; Covance), BclXL (1:500; Santa Cruz), cyclinD1 (Cell Signaling, 1:500), and STAT5b (Santa Cruz; 1:500); and rabbit polyclonal Bcl2 (1:500; Santa Cruz), PRLR (1:500; Santa Cruz), pSTAT5 (1:500; Cell Signaling), STAT5a (1:500; Santa Cruz), and GAPDH (1:2000; Santa Cruz).

Techniques: Derivative Assay, Immunohistochemical staining, Staining

Journal: eLife

Article Title: Mechanism and preclinical prevention of increased breast cancer risk caused by pregnancy

doi: 10.7554/eLife.00996

Figure Lengend Snippet: ( A ) Bar graphs showing the percentage of Bcl XL + ( A ; n = 5 mice) cells in lesions of parous mice treated with AG490 at the indicated doses. ( B ) Bar graphs showing the percentage of pSTAT1 + , pSTAT3 + , and pSTAT6 + cells in lesions of virgin and parous mice treated with AG490 at indicated doses ( B ; n = 3 mice). Effects of AG490 on pSTAT5, cell survival, and mean lesion area are presented in . ( C and D ) Bar graphs showing the percentage of pSTAT5 + lesions ( C ; defined as early lesions with >5% pSTAT5 + cells) and apoptotic cells ( D ) assayed based on TUNEL immunofluorescence in WAP- tva virgin mice treated with AG490 at the indicated dose. n = 4 mice. For all graphs, columns represent the mean, and error bars the SEM. Student's t test determined all p values. DOI: http://dx.doi.org/10.7554/eLife.00996.025

Article Snippet: Primary antibodies used were mouse monoclonal HA (1:1000; Covance), BclXL (1:500; Santa Cruz), cyclinD1 (Cell Signaling, 1:500), and STAT5b (Santa Cruz; 1:500); and rabbit polyclonal Bcl2 (1:500; Santa Cruz), PRLR (1:500; Santa Cruz), pSTAT5 (1:500; Cell Signaling), STAT5a (1:500; Santa Cruz), and GAPDH (1:2000; Santa Cruz).

Techniques: TUNEL Assay, Immunofluorescence

Journal: eLife

Article Title: Mechanism and preclinical prevention of increased breast cancer risk caused by pregnancy

doi: 10.7554/eLife.00996

Figure Lengend Snippet: ( A ) Flow histograms for pSTAT5 and pSTAT3 in Kasumi-1 cells pre-treated with C188-9 at the concentrations indicated or with DMSO. Where indicated, G-CSF (30 ng/ml) or media was added, and the cells were incubated for 15 min. Data were analyzed using Cytobank. ( B ) The percentage of positive cells was normalized to the G-CSF-stimulated and C188-9-untreated control, and then plotted. The IC 50 for G-CSF-induced pSTAT5 (2.8 μM) and for pSTAT3 (7.4 μM) was determined using GraphPad Prism. DOI: http://dx.doi.org/10.7554/eLife.00996.026

Article Snippet: Primary antibodies used were mouse monoclonal HA (1:1000; Covance), BclXL (1:500; Santa Cruz), cyclinD1 (Cell Signaling, 1:500), and STAT5b (Santa Cruz; 1:500); and rabbit polyclonal Bcl2 (1:500; Santa Cruz), PRLR (1:500; Santa Cruz), pSTAT5 (1:500; Cell Signaling), STAT5a (1:500; Santa Cruz), and GAPDH (1:2000; Santa Cruz).

Techniques: Incubation

Journal: eLife

Article Title: Mechanism and preclinical prevention of increased breast cancer risk caused by pregnancy

doi: 10.7554/eLife.00996

Figure Lengend Snippet: ( A ) Immunofluorescence detected a decrease in pSTAT5 + and Bcl XL + cells in the lesions of parous mice treated with C188-9. n = 4 mice. Scale bars = 20 μm. Student's t test determined p value. ( B – D ) Bar graphs showing insignificant effects of C188-9 on pSTAT1 + , pSTAT3 + , and pSTAT6 + cells in early lesions ( B ), cell proliferation in early lesions ( C ), and on the number of early lesions per section per mouse ( D ). n ≥ 4. Student's t test derived p values. For all bar graphs, columns represent the mean, and error bars the SEM. DOI: http://dx.doi.org/10.7554/eLife.00996.027

Article Snippet: Primary antibodies used were mouse monoclonal HA (1:1000; Covance), BclXL (1:500; Santa Cruz), cyclinD1 (Cell Signaling, 1:500), and STAT5b (Santa Cruz; 1:500); and rabbit polyclonal Bcl2 (1:500; Santa Cruz), PRLR (1:500; Santa Cruz), pSTAT5 (1:500; Cell Signaling), STAT5a (1:500; Santa Cruz), and GAPDH (1:2000; Santa Cruz).

Techniques: Immunofluorescence, Derivative Assay

Journal: eLife

Article Title: Mechanism and preclinical prevention of increased breast cancer risk caused by pregnancy

doi: 10.7554/eLife.00996

Figure Lengend Snippet: Western blotting ( A ) and densitometric quantification ( B ) of both pSTAT5 and total STAT5a protein levels in mammary glands at lactation day 12 from mice at16 weeks (O) or 6 weeks (Y) of age. Columns represent mean and errors bars the SEM. Student's t test determined p values. DOI: http://dx.doi.org/10.7554/eLife.00996.032

Article Snippet: Primary antibodies used were mouse monoclonal HA (1:1000; Covance), BclXL (1:500; Santa Cruz), cyclinD1 (Cell Signaling, 1:500), and STAT5b (Santa Cruz; 1:500); and rabbit polyclonal Bcl2 (1:500; Santa Cruz), PRLR (1:500; Santa Cruz), pSTAT5 (1:500; Cell Signaling), STAT5a (1:500; Santa Cruz), and GAPDH (1:2000; Santa Cruz).

Techniques: Western Blot

Journal: eLife

Article Title: Mechanism and preclinical prevention of increased breast cancer risk caused by pregnancy

doi: 10.7554/eLife.00996

Figure Lengend Snippet: Mice that had completed a pregnancy, 3 weeks of lactation, and 2 months of involution were injected with RCAS- caErbb2 . 3 weeks later, the resulting lesions were compared with those in age-matched virgin controls for pSTAT5 ( A ), apoptosis (via TUNEL) ( B ), and Ki67 ( C and E ). Levels of apoptosis ( B ) and proliferation in normal ducts ( D ) of uninfected mice are shown for comparison. Columns represent the mean, and error bars the SEM. Student’s t test measured p values. n = 5 mice. ( F ) Schematic Model. Breast cells with oncogenic activation (red) progress to cancer slowly due to the apoptosis anticancer barrier. However, with a pregnancy, these preexisting precancerous cells activate PRLR-Jak2-STAT5 signaling (becoming pink), and maintain the activated state of this pathway even at involution likely through oncoprotein-initiated phosphorylation and inactivation of GSK3β. pSTAT5 overcomes both the apoptosis anticancer barrier and the apoptotic force unleashed by involution, consequently accelerating progression to malignancy. DOI: http://dx.doi.org/10.7554/eLife.00996.031

Article Snippet: Primary antibodies used were mouse monoclonal HA (1:1000; Covance), BclXL (1:500; Santa Cruz), cyclinD1 (Cell Signaling, 1:500), and STAT5b (Santa Cruz; 1:500); and rabbit polyclonal Bcl2 (1:500; Santa Cruz), PRLR (1:500; Santa Cruz), pSTAT5 (1:500; Cell Signaling), STAT5a (1:500; Santa Cruz), and GAPDH (1:2000; Santa Cruz).

Techniques: Injection, TUNEL Assay, Activation Assay