Prolactin cooperates with loss of p53 to promote claudin-low mammary carcinomas

Abstract

TP53 is one of the most commonly mutated genes in cancer. In breast cancer, it is mutated in about 40% of primary clinical tumors and is associated with poor survival. The mammotrophic hormone, prolactin (PRL), and/or its receptor are also expressed in many breast cancers, and accumulating epidemiologic data link PRL to breast cancer development and progression. Like TP53 mutations, evidence for PRL activity is evident across several molecular cancer subtypes, and elevated PRL expression and loss of p53 have been observed in some of the same clinical tumors. In order to examine the interaction of these factors, we used genetically modified mouse models of mammary-specific p53 loss and local overexpression of PRL. We demonstrated that mammary PRL decreased the latency of tumors in the absence of p53, and increased the proportion of triple-negative claudin-low carcinomas, which display similarities to human clinical metaplastic carcinomas. Moreover, PRL/p53(-/-) carcinomas displayed higher rates of proliferation and more aggressive behavior. Transcripts associated with cell cycle progression, invasion and stromal reactivity were differentially expressed in carcinomas that developed in the presence of elevated PRL. PRL/p53(-/-) carcinomas also exhibited selectively altered expression of activating protein-1 components, including higher levels of c-Jun and FosL1, which can drive transcription of many of these genes and the epithelial-mesenchymal transition. The ability of PRL to promote claudin-low carcinomas demonstrates that PRL can influence this subset of triple-negative breast cancers, which may have been obscured by the relative infrequency of this cancer subtype. Our findings suggest novel therapeutic approaches, and provide a preclinical model to develop possible agents.

Figure 1

PRL decreases the latency of mammary tumors that develop in the absence of p53 and increases the proportion of claudin low spindle cell carcinomas. NRL-PRL mice (line 1647-13, TgN(Nrl-Prl)23EPS), were generated in the FVB/N strain as described. C57BL/6 mice carrying a deletion for p53 were backcrossed more than ten generations into the FVB/N genetic background. Mammary fragments from 10–13 week old donors of the genotypes of interest were transplanted to mammary fat pads of 3 week old wildtype FVB/N mice which had been cleared of endogenous epithelium as described (5–9 recipients per donor). Nulliparous recipients were examined weekly for tumors until one year of age. (a) Latencies of the tumors that developed from p53^−/− (N=12) and PRL/p53^−/− (N=15) donor cells were compared by Kaplan-Meier analysis. PRL/p53^−/− tumors had a significant shorter latency than p53^−/− tumors (p<0.05). All (100%) of the recipients of wild-type and NRL-PRL cells remained tumor free at 1 year of age. (b) Distribution of carcinoma histotypes that developed from p53^{-/ -} and PRL/p53^−/− donor cells. The proportion of spindle cell carcinomas was significantly higher in the tumors that developed from PRL/p53^−/− donors (*, p=0.024, Fishers exact test), and the proportion of adenocarcinomas tended to be lower (†, p=0.06, Fishers exact test). (c,d) RNA from mammary epithelial cells (MECs) pooled from 12 week old wildtype FVB/N females, individual adenocarcinomas that developed in NRL-PRL females (N=4), and spindle cell carcinomas that developed from PRL/p53^−/− and p53^−/− donor cells in the current study was examined for the transcripts indicated by quantitative RT-PCR (qRT-PCR). Results were analyzed via the delta-delta C(t) method normalized to 18S ribosomal RNA, as described. Primers used for qRT-PCR are listed in Supplementary Table 1. (N=4–6; mean +/− S.E.M.) ***p<0.0001 compared to NRL-PRL adenocarcinomas by the Kruskal-Wallis test for non-parametric data, followed by the Mann-Whitney post test. (e) Heat map of relative levels of the indicated transcripts, normalized to FVB/N MECs. Left, single adenocarcinoma from p53^−/− donor cells; middle, spindle cell carcinoma from p53^−/− donor cells; right, spindle cell carcinoma from PRL/p53^−/− donor cells. Levels of these transcripts in tumors that developed in these genotypes were not significantly different.

Figure 2

Both NRL-PRL/p53^−/− and p53^−/− donor cells give rise to mammary carcinomas of multiple histotypes. (a-d) Hematoxylin/ eosin stained micrographs. (a) Squamous cell carcinoma that developed from p53^−/− donor cells. (b) Adenocarcinoma that developed from p53^−/− donor cells. (c) Spindle cell carcinoma that developed from PRL/p53^−/− donor cells. (d) Giant cells (arrows) in a tumor that developed from p53^−/− donor cells. (e,f) Cytokeratin 8 staining of spindle cell carcinomas that developed from p53^−/− donor cells (e) and PRL/p53^−/− donor cells (f). Note the cytokeratin 8+ giant cells (arrows). (g,h) Picrosirius red-stained spindle cell carcinomas that developed from p53^−/− donor cells (g) and PRL/p53^−/− donor cells (h), visualized with a polarizing microscope (Spot-advanced Olympus BX51 camera, Olympus, Germany). (a-f), Original magnification x100. Bars= 100µ. Insets in (g,h), 200x.

Figure 3

Carcinomas that developed from PRL/p53^−/− donor cells were significantly larger despite the reduced latency and exhibited higher indices of proliferation, and contained different levels of mRNAs for cell cycle inhibitors, proteases associated with invasion, and stromal changes. (a) Tumor wet weight at end stage. (N=9–14; mean +/− S.E.M.) *p<0.05. (b) Proliferation indices were determined by counting PCNA-labeled cells in 1000 total cells from at least 5 randomly chosen microscopic fields in divergent regions of each carcinoma. (N=6–7; mean +/− S.E.M.) **p<0.01, Kruskal-Wallis test for non-parametric data, followed by the Mann-Whitney post test. (c-e) RNA from individual p53^−/− and PRL/p53^−/− spindle cell carcinomas was examined for the transcripts indicated by qRT-PCR as described for Figure 1. (N=4–6; means +/− S.E.M.) (c) Transcripts for p16^Ink4a and p19^Arf . *p<0.05, 2-tailed Student’s t test. (d) Transcripts associated with invasion. *p<0.05 by 2-tailed Student’s t test; ‡p=0.03 by 1-tailed Student’s t test. (e), Transcripts associated with stromal changes. †p=0.08, 1-tailed Student’s t test; §p=0.086, 1-tailed Student’s t test.

Figure 4

PRL/p53^−/− carcinomas express higher levels of c-Jun and Fosl1 compared to p53^−/− tumors. (a) RNA from FVB/N MECs and mammary carcinomas that developed from transplanted p53^−/− and PRL/p53^−/− cells was examined for levels of the transcripts indicated by qRT-PCR as described for Figure 1. (N=6, means +/− S.E.M.) *p<0.05 by Kruskal-Wallis test for non-parametric data, followed by the Mann-Whitney post test. (b) Equal amounts of protein from mammary carcinomas that developed from transplanted p53^−/− and PRL/p53^−/− cells were examined for the indicated proteins by immunoblotting, and signals were quantified by densitometry. (means +/− S.D., N=5; *p<0.05, ***p<0.001 by 2 tailed Student’s t test; †p=0.068 by 1-tailed Student’s t test).