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. 2016 Jan 27;18(1):13.
doi: 10.1186/s13058-016-0673-9.

p53 deficiency linked to B cell translocation gene 2 (BTG2) loss enhances metastatic potential by promoting tumor growth in primary and metastatic sites in patient-derived xenograft (PDX) models of triple-negative breast cancer

Emily Powell  1   2 , Jiansu Shao  3 , Yuan Yuan  4 , Hsiang-Chun Chen  5 , Shirong Cai  6 , Gloria V Echeverria  7 , Nipun Mistry  8 , Keith F Decker  9 , Christopher Schlosberg  10 , Kim-Anh Do  11 , John R Edwards  12 , Han Liang  13 , David Piwnica-Worms  14   15 , Helen Piwnica-Worms  16
Affiliations

p53 deficiency linked to B cell translocation gene 2 (BTG2) loss enhances metastatic potential by promoting tumor growth in primary and metastatic sites in patient-derived xenograft (PDX) models of triple-negative breast cancer

Emily Powell et al. Breast Cancer Res. .

Abstract

Background: Despite advances in early diagnosis and treatment of cancer patients, metastasis remains the major cause of mortality. TP53 is one of the most frequently mutated genes in human cancer, and these alterations can occur during the early stages of oncogenesis or as later events as tumors progress to more aggressive forms. Previous studies have suggested that p53 plays a role in cellular pathways that govern metastasis. To investigate how p53 deficiency contributes to late-stage tumor growth and metastasis, we developed paired isogenic patient-derived xenograft (PDX) models of triple-negative breast cancer (TNBC) differing only in p53 status for longitudinal analysis.

Methods: Patient-derived isogenic human tumor lines differing only in p53 status were implanted into mouse mammary glands. Tumor growth and metastasis were monitored with bioluminescence imaging, and circulating tumor cells (CTCs) were quantified by flow cytometry. RNA-Seq was performed on p53-deficient and p53 wild-type tumors, and functional validation of a lead candidate gene was performed in vivo.

Results: Isogenic p53 wild-type and p53-deficient tumors metastasized out of mammary glands and colonized distant sites with similar frequency. However, p53-deficient tumors metastasized earlier than p53 wild-type tumors and grew faster in both primary and metastatic sites as a result of increased proliferation and decreased apoptosis. In addition, greater numbers of CTCs were detected in the blood of mice engrafted with p53-deficient tumors. However, when normalized to tumor mass, the number of CTCs isolated from mice bearing parental and p53-deficient tumors was not significantly different. Gene expression profiling followed by functional validation identified B cell translocation gene 2 (BTG2), a downstream effector of p53, as a negative regulator of tumor growth both at primary and metastatic sites. BTG2 expression status correlated with survival of TNBC patients.

Conclusions: Using paired isogenic PDX-derived metastatic TNBC cells, loss of p53 promoted tumor growth and consequently increased tumor cell shedding into the blood, thus enhancing metastasis. Loss of BTG2 expression in p53-deficient tumors contributed to this metastatic potential by enhancing tumor growth in primary and metastatic sites. Furthermore, clinical data support conclusions generated from PDX models and indicate that BTG2 expression is a candidate prognostic biomarker for TNBC.

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Figures

Fig. 1
Fig. 1
p53 silencing enhances breast tumor growth and metastasis in vivo. BC3-p53WT and BC3-p53KD cells expressing CBR-luc and mCherry were implanted into the humanized mammary fat pads of NOD/SCID mice. Bioluminescence imaging (BLI) was performed to monitor tumor growth and metastasis as a function of time. a Representative images showing growth and metastasis of BC3-p53WT and BC3-p53KD tumors. Due to rapid tumor growth BC3-53KD tumors were resected at approximately 9 weeks post-engraftment. b Total photon flux from each mammary tumor was quantified from BLI, and values were plotted as a function of time. c Tumors were measured with calipers, and values were plotted as a function of time. n = 18, BC3-p53WT; n = 18, BC3-p53KD. p = 0.004, t test (b and c). d and e 5 weeks and 9 weeks post-engraftment, tumors were harvested, formalin-fixed, sectioned, and stained with antibodies against phospho-histone H3 (pHH3) (d) and cleaved caspase 3 (CC3) (e). Positive regions were counted manually, and percentage of positive cells was calculated from DAPI-stained nuclei. Each data point is the average of at least six images from an individual mouse. Paired t tests, p = 0.01 for pHH3 at 5 weeks; p = 0.30 for pHH3 at 9 weeks; p = 0.04 for CC3 at 5 weeks; p = 0.20 for CC3 at 9 weeks. f BLI was performed biweekly to detect the appearance of lymph node metastases. p = 0.001, Wilcoxon rank sum test. Each data point represents one mouse. All error bars represent standard error of the mean (SEM)
Fig. 2
Fig. 2
p53 silencing increases bioluminescence in metastatic sites. BC3-p53WT and BC3-p53KD were implanted into mouse mammary fat pads. Frequency and magnitude of metastasis were assessed with bioluminescence imaging (BLI). a Animals were euthanized when declining health was observed. The time post tumor-engraftment to euthanasia of animals implanted with BC3-p53WT or BC3-p53KD was quantified. Each data point represents one mouse. p = 0.002, Wilcoxon rank sum test. b Lungs, livers, long bones, and brains of mice implanted with BC3-p53WT or BC3-p53KD were imaged with BLI ex vivo at necropsy. Representative images are shown. Scale applies to each image in the panel, and magnitude of scale is indicated below each image. c Mammary tumors and lungs were harvested from mice implanted with BC3-p53KD. Tissues were sectioned and stained with cytokeratin 18 (CK18) to assess regions of human epithelial tumor (positive) and surrounding mouse tissue (negative). Objectives used were: 20 × composite (top panels), 10 × (middle panels), and 20 × (bottom panels). d Frequency of metastasis to the indicated organs was quantified with ex vivo BLI 19–40 weeks post-engraftment. e Lungs were extracted, and total photon flux was assessed with BLI, quantified, and plotted vs. time post-engraftment to study end point. p = 0.03, linear regression analysis of the slopes. f Lungs, livers, bones, and brains were extracted and assessed with BLI. Total photon flux was quantified and normalized to time post tumor-engraftment to euthanasia. n = 21, p53WT; n = 14, p53KD. p <0.001 (lung); p = 0.001 (liver); p = 0.002 (bone); p = 0.01 (brain), Wilcoxon rank sum tests. Each data point represents one organ from one mouse. All error bars represent standard error of the mean (SEM)
Fig. 3
Fig. 3
p53 silencing increases CTCs in the blood as a function of increased tumor growth. Mouse mammary fat pads were engrafted with BC3-p53WT or BC3-p53KD. Whole blood was extracted from mice in a terminal blood draw by cardiac puncture. Red blood cells were lysed, and circulating tumor cells (CTCs) were assessed by flow cytometry for mCherry-positive cells. Bioluminescence imaging (BLI) was performed on mammary tumors and lungs at necropsy. a CTCs were quantified by flow cytometry at the indicated time points. p = 0.71 (5 weeks); p = 0.06 (9 weeks); p = 0.40 (12 weeks); p = 0.10 (15 weeks); p = 0.07 (18 weeks), Wilcoxon rank sum tests. b CTCs were quantified as in (a), and numbers from each time point were combined to increase cohort size. p <0.001, F-test. c CTC number from each mouse was normalized to total photon flux of the corresponding primary tumor. Data are presented as a combination of all time points as in (b). p = 0.072, F-test. d and e BC3-p53WT tumors were implanted to mammary glands 3 weeks prior to implantation of BC3-p53KD, and CTCs were quantified on the same day by flow cytometry (d, p = 0.43, t test) when tumors were equivalent in size (e, p = 0.73, t test). f Spatially distinct regions of bioluminescence in lungs of tumor-bearing mice were quantified manually. p = 0.09, Wilcoxon rank sum test. Each data point represents one mouse. All error bars represent standard error of the mean (SEM)
Fig. 4
Fig. 4
BTG2 negatively regulates growth of BC3-p53KD tumors in primary and metastatic sites. BC3-p53WT or BC3-p53KD were implanted into mouse mammary fat pads, and tumors were harvested when they reached 0.5 cm diameter. Ribonucleic acid (RNA) was extracted from tumors, and gene expression changes were assessed with RNA-sequencing (RNA-Seq). a RNA from BC3-p53WT or BC3-p53KD tumors was reverse transcribed, and quantitative real-time polymerase chain reaction (qRT-PCR) using a probe and primer set for B cell translocation gene 2 (BTG2) was performed. p = 0.0001, t test. Each dot is a biological replicate representing one tumor from one mouse. b BTG2 or green fluorescent protein (GFP) (control) was expressed in BC3-p53KD cells by lentiviral transduction, and cells were implanted to mouse mammary fat pads. Mice were subjected to bioluminescence imaging (BLI) weekly, and total photon flux from mammary tumors was quantified at each time point. n = 5 mice (10 tumors) in each group. p <0.001 for mammary tumors 6 weeks post-engraftment, Wilcoxon rank sum test. p <0.001 using linear mixed-effect model of tumor growth over the time course. c BTG2 or GFP was expressed in BC3-p53KD as in (b), and cells were injected into mouse tail veins to model the final stages of lung metastasis. Mice were subjected to BLI weekly, and total photon flux from lungs was quantified at each time point. n = 4 mice in each group. p = 0.002, linear mixed effect model. d Total photon flux from lungs of mice in (c) was assessed with BLI ex vivo at necropsy and quantified. p = 0.001, t test. Each data point represents one mouse, n = 4 mice per group. e Hematoxylin and eosin (H & E) staining of lungs of mice in (c) and (d). All error bars represent standard error of the mean (SEM)
Fig. 5
Fig. 5
BTG2 is a clinically relevant modulator of tumor progression. Kaplan-Meier curves were generated to assess correlations between B cell translocation gene 2 (BTG2) expression and patient survival. Top and bottom 25 % of patients were used as cutoffs for grouping. a Low BTG2 expression correlates with decreased overall survival of breast cancer patients (all subtypes). b Low BTG2 expression correlates with decreased metastasis-free survival of breast cancer patients (all subtypes). c Low BTG2 expression correlates with decreased overall survival of triple-negative breast cancer (TNBC) patients. Number of patients and p values are indicated in each panel
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