Background
Breast cancer is a highly heterogeneous disease comprising estrogen receptor alpha (ERα)-positive and HER2/ERBB2/NEU-positive subtypes as well as triple-negative breast cancers (TNBCs) that do not express ERα, human epidermal growth factor 2 (HER2) or the progesterone receptor. TNBC can be further divided into several additional groups including basal-like and mesenchymal/claudin-low [
1‐
5]. These tumors contain mutations or deletion in the tumor suppressor p53 in 60–80 % of cases [
4]. In addition, 35 % of TNBC also show loss of expression of the tumor suppressor phosphatase and tensin homolog deleted in chromosome 10 (PTEN). The latter is a phosphatase that converts phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3) into phosphatidylinositol (4, 5)-disphosphate (PIP2), thereby antagonizing phoshotidylinositol-3 kinase (PI3K) pathway activation [
6‐
8]. On the basis of frequency in which each gene is disrupted alone, combined loss of p53 and Pten in TNBC is calculated at about 21–28 %. In addition, using Pten RNA expression and p53 pathway activity it was estimated that 24.4 % of TNBCs are Pten-low, 65.6 % are p53 activity-low, and 18.7 % are both Pten-low and p53 pathway activity-low [
9].
The generation of mice with mutations that occur in breast cancer offers a window into the mechanisms of tumor initiation, progression and spread in immune-competent mice, and provide preclinical models to test potential new therapies. Deletion of p53 in the mouse induces diverse tumors as well as claudin-low-like TNBC [
10]. We recently showed that combined deletion of p53 and Pten via mouse mammary tumor virus (MMTV)-Cre
NLS or whey acidic protein (WAP)-Cre drivers induces almost exclusively claudin-low TNBC-like tumors [
9]. This suggests that p53 deletion in the mouse promotes mesenchymal-like cancer, which is further accelerated by disruption of Pten.
To generate a model for basal-like Pten/p53 mutant TNBC, we here tested the effect of deleting Pten and expressing p53
R270H mutation in the DNA-binding domain (DBD) [
11]. This and similar mutations in p53 DBD were shown to act as dominant-negative or gain-of-function alleles that accelerate metastasis through multiple mechanisms [
12]. Targeted expression of p53
R270H in the mammary epithelium was reported to induce divergent tumors [
13]. Here we show that WAP-Cre:Pten
f/f:p53
lox.stop.lox_R270H composite mice develop spindle-like and poorly differentiated adenocarcinoma (PDA) as well as other subtypes. Using microarray profiling, we found that the spindle and PDA tumors clustered with claudin-low and basal-like breast cancer (BLBC), respectively, and metastasized to the lung following tail vein injection significantly faster than tumors from WAP-Cre:Pten
f/f:p53
f/f double-deletion mice. Thus, WAP-Cre:Pten
f/f:p53
lox.stop.lox_R270H mice provide a preclinical model to study aggressive Pten/p53-mutant basal-like and claudin-low TNBC.
Discussion
We show that targeted deletion of the tumor suppressor Pten together with expression of a p53 DBD mutant, R270H, in mammary epithelium via WAP-Cre induces diverse mammary tumor subtypes including PDA and spindle tumors. Intra- and inter-species cluster analysis classified most PDA and spindle tumors as basal-like and claudin-low like TNBC, respectively. In stark contrast, double-deletion tumors from WAP-Cre:Pten
f/f:p53
f/f mice invariably cluster with human claudin-low breast cancer [
9]. Claudin-low-like TNBC were also found when Pten and p53, or Rb plus p53 were homozygously deleted via MMTV-Cre [
9,
17], or when the MET oncogene was overexpressed in mammary epithelium together with homozygous p53 deletion [
33]. Indeed, these articles showed that homozygous loss of p53 alone induces many, though not exclusively, claudin-low-like tumors, indicating that deletion of this tumor suppressor in the mouse directs tumorigenesis toward this subtype. The Pten
∆f:p53
R270H PDAs expressed the basal keratins K5 and K6, clustered with mouse models of BLBC and with human BLBC samples, exhibited high TIC frequency, and readily formed lung metastases following tail vein injections. Thus, WAP-Cre:Pten
f/f:p53
lsl_R270H/+ mice offer a new model to study basal-like as well as claudin-low Pten/p53-deficient TNBC. Our results also suggest that WAP-Cre:p53
lsl_R270H/+ rather than WAP-Cre:p53
f/f mice should be used to model human breast cancer in conjunction with mutations in other breast cancer drivers.
Pten
∆f:p53
∆f and Pten
∆f:p53
R270H tumors provide the means to compare histologically indistinguishable tumors with either mutation of deletion of p53. We therefore performed FDA-approved drug screens to identify compounds that can kill both tumors or preferentially target tumors driven by p53 deletion or mutation. We found that 8-azaguanine, a purine analog, efficiently killed both Pten
∆f:p53
∆f and Pten
∆f:p53
R270H tumors. Sensitivity to 8-azaguanine may reflect high cell proliferation or low levels of guanine deaminase, which can convert this purine analog to a noncytotoxic metabolite (8-azaxanthine) [
34]. 8-azaguanine incorporation via hypoxanthine guanine phosphoribosyl transferase (HGPRTase) leads to inhibition of purine nucleotide synthesis. It may also be toxic due to its incorporation into RNA [
35]. Despite some benefits to leukemia patients, adverse dermatological reactions, nausea and vomiting limit its use [
36]. Additional analysis is required to assess possible benefits, if any, of 8-azaguanine either alone or in combination with PI3K antagonists or other inhibitors for Pten/p53-deficient TNBC. Notably, in a kinome screen of Pten
∆f:p53
∆f tumors we identified eEF2K antagonists as better inhibitors than AKT and PI3K inhibitors [
9]. Such pathway-specific drugs are much more likely to have a large therapeutic window as compared to cytotoxic drugs like 8-azaguanine. Additional screens with both Pten
∆f:p53
∆f and Pten
∆f:p53
R270H tumors as well as with human Pten/p53-deficient breast cancer lines using large libraries of divergent compounds may uncover new potent drugs for this aggressive cancer subtype.
Contrary to published results on increased sensitivity of mutant p53 to statins [
31], we observed consistent but mild and statistically insignificant increase in sensitivity of Pten
∆f:p53
∆f tumor cells to HMG-CoA inhibitors relative to Pten
∆f:p53
R270H tumor cells. One explanation for this discrepancy may be that although p53 mutation alone increases sensitivity to statins by inducing the mevalonate pathway, tumors that develop in the absence of p53 (p53 deletion) may increase sensitivity to statins through other mechanisms. Indeed, we found that a signature that predicts sensitivity to statins [
37] is elevated in Pten
∆f:p53
∆f relative to Pten
∆f:p53
R270H tumor cells (not shown). We note that the FDA-approved drug library we used here does not include the HSP90 inhibitor geldanamycin or its more potent derivatives, which destabilize mutant p53. However, we found that knockdown of p53
R270H protein impeded cell growth (not shown) indicating that Pten
∆f:p53
R270H tumor cells are addicted to continuous expression of mutant p53.
Despite – and perhaps because of – the inter- and intra-tumor heterogeneity seen in WAP-Cre:Ptenf/f:p53lsl_R270H/+ mice, they offer certain advantages over WAP-Cre:Ptenf/f:p53f/f mice. First, they develop PDA/basal-like tumors in approximately 10 % of mice and spindle/claudin-low-like tumors in approximately 30 % of mice. Tumor-bearing animals can be biopsied to determine histology and then treated with candidate drugs, allowing their effect to be tested on diverse tumor types with similar oncogenic initiation events (Pten loss; p53 mutation). Alternatively, as described herein, tumors can be harvested, frozen, classified and then transplanted to obtain multiple xenografts with uniform tumor subtype. Together, the WAP-Cre:Ptenf/f:p53lsl_R270H/+ and WAP-Cre:Ptenf/f:p53f/f mice model the spectrum of p53 gene aberrations and Pten loss seen in human TNBC.
Using GSEA and functional analysis we showed that Pten
∆f:p53
R270H tumor cells are more metastatic than Pten
∆f:p53
∆f tumors and this was correlated with induction of E-cadherin in lung metastases from Pten
∆f:p53
R270H tumor cells. P53 mutations in the DBD were shown to increase metastasis through induction of EMT via several routes including sequestration of p63, stabilization/activation of SLUG/SNAIL, induction of the mir130b-Zeb1 axis, TWIST and SLUG transcription factors and other mechanisms [
12,
38‐
42]. Our study suggests that once disseminating mutant p53 tumors home at distal sites, they more readily undergo MET, which enhances colonization, the dominant rate-limiting step of the metastatic cascade [
32,
43,
44].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SW designed and performed experiments, analyzed results, and wrote the manuscript. JCL performed the bioinformatic analysis. DK helped with histology and genotype analysis. AD supervised the HTP screen and analysis. EZ designed and supervised the study, analyzed the results, and wrote the manuscript. All authors read and approved the final manuscript.