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. 2018 Apr 1;78(7):1672-1684.
doi: 10.1158/0008-5472.CAN-17-0985. Epub 2018 Jan 23.

Antiestrogen Therapy Increases Plasticity and Cancer Stemness of Prolactin-Induced ERα+ Mammary Carcinomas

Michael P Shea  1   2 , Kathleen A O'Leary  2 , Saja A Fakhraldeen  3 , Vincent Goffin  4 , Andreas Friedl  5   6 , Kari B Wisinski  6   7 , Caroline M Alexander  3   6 , Linda A Schuler  8   6
Affiliations

Antiestrogen Therapy Increases Plasticity and Cancer Stemness of Prolactin-Induced ERα+ Mammary Carcinomas

Michael P Shea et al. Cancer Res. .

Abstract

Although antiestrogen therapies are successful in many patients with estrogen receptor alpha-positive (ERα+) breast cancer, 25% to 40% fail to respond. Although multiple mechanisms underlie evasion of these treatments, including tumor heterogeneity and drug-resistant cancer stem cells (CSC), further investigations have been limited by the paucity of preclinical ERα+ tumor models. Here, we examined a mouse model of prolactin-induced aggressive ERα+ breast cancer, which mimics the epidemiologic link between prolactin exposure and increased risk for metastatic ERα+ tumors. Like a subset of ERα+ patient cancers, the prolactin-induced adenocarcinomas contained two major tumor subpopulations that expressed markers of normal luminal and basal epithelial cells. CSC activity was distributed equally across these two tumor subpopulations. Treatment with the selective estrogen receptor downregulator (SERD), ICI 182,780 (ICI), did not slow tumor growth, but induced adaptive responses in CSC activity, increased markers of plasticity including target gene reporters of Wnt/Notch signaling and epithelial-mesenchymal transition, and increased double-positive (K8/K5) cells. In primary tumorsphere cultures, ICI stimulated CSC self-renewal and was able to overcome the dependence of self-renewal upon Wnt or Notch signaling individually, but not together. Our findings demonstrate that treatment of aggressive mixed lineage ERα+ breast cancers with a SERD does not inhibit growth, but rather evokes tumor cell plasticity and regenerative CSC activity, predicting likely negative impacts on patient tumors with these characteristics.Significance: This study suggests that treatment of a subset of ERα+ breast cancers with antiestrogen therapies may not only fail to slow growth but also promote aggressive behavior by evoking tumor cell plasticity and regenerative CSC activity. Cancer Res; 78(7); 1672-84. ©2018 AACR.

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Conflict of interest statement

The authors declare no potential conflicts of interest.

Figures

Figure 1
Figure 1. NRL-PRL mice develop ERα+ adenocarcinomas composed of luminal and basal epithelial subpopulations
(A, i,ii) Adenocarcinomas of varying histotypes (“Tumor 1” (i) and “Tumor 2” (ii)) develop spontaneously in nulliparous female NRL-PRL mice (hematoxylin and eosin stain). (A, iii,iv) ERα expression in Tumor 1 (iii) and Tumor 2 (iv). (A, v,vi) Undetectable progesterone receptor (PR) expression in both Tumor 1 (v) and Tumor 2 (vi). Insets, ERα and PR staining in luminal epithelium; cross sections of mammary ducts. Scale bars, 50 μm. (B) Representative flow cytometric plots showing differences in tumor subpopulations among NRL-PRL, MMTV-neu, and p53−/− tumors. Bar graphs represent percentages of luminal (EPCAMhiCD49f+) and basal (EPCAMmedCD49f+) cells relative to total Lin-tumor cells (N=3; mean±S.D.). (C) Immunofluorescent staining of Tumors 1 and 2 using lineage-specific markers, cytokeratin-8 (K8) (red) and cytokeratin-5 (K5) (green). Inset shows a cross section from a normal mammary duct. Scale bars, 50 μm. (D) Relative Krt8 and Krt5 mRNA levels in FACS sorted luminal and basal subpopulations from the two tumors. (E) ERα+ patient tumor cores from a clinical breast tumor microarray showing pseudocolored K8 (red) and K5 (green) immunostaining (left panel) and ERα immunostaining with hematoxylin counterstain of a nearby section from the same core (right panel). Insets show staining of a normal mammary duct.
Figure 2
Figure 2. Transplantation of limiting numbers of cells from the luminal and basal tumor subpopulations regenerates carcinomas with characteristics of the parental tumor
Representative photomicrographs of secondary tumors derived from sorted luminal and basal subpopulations demonstrating similar morphology, ERα expression and heterogeneity of the parent tumor. (A)H&E, (B) ERα IHC, and (C) flow cytometric analysis (Tumor 1: N=3, mean±S.D.; Tumor 2: N=1; others were too small for analysis). Scale bars, 50 μm.
Figure 3
Figure 3. Treatment with the estrogen receptor antagonist, ICI 182,780, does not alter growth of passaged tumors, but transiently depletes the tumor subpopulation with basal characteristics
(A) Schema of the in vivo treatment methodology. When transplanted tumor cells developed into tumors of 7mm in diameter, mice were treated with or without ICI 182,780 (ICI) as described in the Methods, and tumors were harvested after 2 or 14 days of treatment. (B) Uterine wet-weights of treated and untreated mice confirm the systemic anti-estrogenic activity of ICI 182,780 (N=3; mean±S.D.). (C) ICI did not alter the rate of growth of established tumors (Tumor 1 shown, Tumor 2 in Supplementary Fig. 2A). (N=3; mean±S.D.). (D) ICI did not alter the rate of proliferation in established tumors measured by Ki-67 staining. (N=3; mean±S.D.). (E) Flow cytometric analysis in response to in vivo ICI 182,780, 2 or 14 days after initiation of treatment (N=3; mean±S.D.). (F) Representative K5 and K8 immunofluorescence at 2 and 14 days after initiation of treatment. (G) Quantification of K8/K5 staining was performed as described in the Methods. N= 3 independent tumors. (Means±S.D. and statistical evaluations are shown in Supplementary Table 2).
Figure 4
Figure 4. ICI 182,780 antagonizes cancer stem cells and alters markers for EMT, Notch, and Wnt activity
(A) Primary tumorsphere forming ability of tumor cells from control or ICI 182,780 treated mice (see Methods; N= 3 independent tumors; mean±S.D.). (B) Relative mRNA levels of Esr1, Wnt4, and Axin2 from FACS-sorted luminal and basal subpopulations from Tumor 1 without treatment (N=3; mean±S.D.). Light bars, luminal cells (L); dark bars, basal cells (B). Asterisks indicate significant differences determined by Student’s t test, p<0.05. (C,D) Relative mRNA levels of Wnt4, Axin2, Hes1 (C), and Sox2, Bmi1, Twist1 (D) from control, 2 day and 14 day ICI-treated tumor-burdened mice (N=3-5; mean±S.D.). Asterisks indicate significant differences determined by one-way ANOVA followed by Tukey’s multiple comparison tests, p<0.05. (E,F) Relative levels of Wnt4, Axin2, Hes1 mRNAs (E), and Sox2, Bmi1, Twist1 mRNAs (F) in FACS-sorted luminal and basal tumor subpopulations harvested from control or 14 day ICI-treated mice (N=3; mean±S.D.). Light bars, luminal cells (L); dark bars, basal cells (B). Different letters indicate significant differences (one-way ANOVA followed by Tukey’s multiple comparison tests, p<0.05).
Figure 5
Figure 5. In vitro ICI 182,780 treatment of tumor cells increases CSC self-renewal independent of Wnt and Notch pathways
(A) Primary (left panel) and secondary (self-renewal, right panel) tumorsphere formation from Tumor 1 cells treated with 17β-estradiol (E2) or ICI during primary tumorsphere formation, relative to ethanol vehicle (See Methods). (B) Size (left panel) and frequency of double-positive (K8+/K5+) cells (right panel) of primary tumorspheres that developed with ethanol or ICI treatment (see Methods). Asterisks indicate significant differences (unpaired Student’s t-test, p<0.05). (C) Relative secondary tumorsphere generation (self-renewal) from primary tumorspheres treated with inhibitors of the Wnt (iCRT14) or Notch (GSI) pathways, with or without ICI co-treatment. Different letters indicate significant differences (one-way ANOVA followed by Tukey’s multiple comparison tests, p<0.05). A-C, N=3 independent experiments; mean±S.D.
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