Research ArticleCELL BIOLOGY

The RNA binding protein CPEB2 regulates hormone sensing in mammary gland development and luminal breast cancer

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Science Advances  15 May 2020:
Vol. 6, no. 20, eaax3868
DOI: 10.1126/sciadv.aax3868
  • Fig. 1 CPEB2 regulates mammary gland postnatal development.

    (A) mRNA levels of Cpeb1 to Cpeb4 normalized to Gapdh in whole tissue mammary gland (n = 2; n = 7 for adult nulliparous). Tissue was obtained from mice at puberty (5 weeks old), adult nulliparous (10 weeks old), midpregnancy (day 12 of gestation), lactation (2 weeks of lactation), or involution (6 days after weaning). Gapdh expression is also shown. Statistics were determined using two-way analysis of variance (ANOVA), **P < 0.01, ***P < 0.001, and ****P < 0.0001. (B) mRNA levels of Cpeb1 to Cpeb4 normalized to Gapdh in sorted cells from adult virgin mammary gland (n = 3). Statistics using two-way ANOVA, ****P < 0.0001. Myo, myoepithelial. (C) Representative carmine-stained mammary gland whole mounts and automatic quantification of the number junctions in virgin 10- to 12-week-old WT (n = 11) and constitutive CPEB1 KO (n = 4), CPEB2 KO (n = 10), CPEB3 KO (n = 5), and CPEB4 KO (n = 4) mice. Statistics were determined using the Mann-Whitney test, *P < 0.05 and **P < 0.01. (D) Representative mammary whole mounts and automatic quantification of the number of junctions in virgin 10- to 12-week-old epithelial-specific WTCK14 (n = 4), CPEB1 KOCK14 (n = 6), and CPEB2 KO CK14 (n = 8) mice. Statistics were determined using the Mann-Whitney test, *P < 0.05. (E) Representative mammary whole mounts and quantification of the area of the fat pad filled with epithelial ducts at puberty in WT and CPEB2 KO females (5 weeks old) (n = 5). Statistics were determined using the Mann-Whitney test, *P < 0.05. (F) Ratio between the percentage of luminal and myoepithelial cells gated on lineage-negative (WT, n = 7; CPEB1 KO, n = 4; CPEB2 KO, n = 6; CPEB3 KO, n = 4; and CPEB4 KO, n = 4). Statistics were determined using the Mann-Whitney test, *P < 0.05.

  • Fig. 2 CPEB2 KO females display aberrant Sca1+ ductal luminal progenitors.

    (A) Representative fluorescence-activated cell sorting (FACS) plots gated on luminal cells depicting luminal subpopulations: ductal differentiated (DD; Sca1+CD49b), ductal progenitors (DPs; Sca1+CD49b+), and alveolar progenitors (APs; Sca1CD49b+). (B) Representative FACS plots for Sca1 gated on luminal cells. FSC-W, forward scatter width. (C) Ratio of the percentage of Sca1high and Sca1low populations in luminal cells (n = 10). Statistics were determined using the Mann-Whitney test, **P < 0.01. (D) Quantification of the percentage of CD49b+ cells gated on luminal cells (n = 17). Statistics were determined using the Mann-Whitney test, *P < 0.05. (E) Quantification of the luminal subpopulations as in (A). Statistics were determined using two-way ANOVA, ***P < 0.001 (n = 17). (F) Preranked GSEA graphical output for the enrichment in DPKO versus DPWT cells of the up-regulated genes in the “WT progenitor signature” generated by P value (n = 181, see Methods). FDR q ≤ 0.0001. FDR, false discovery rate; NES, normalized enrichment score. (G) Expression of luminal progenitor markers in DPWT and DPKO cells. (H) Expression of luminal progenitor markers in APWT and APKO cells. (I) Representative images of organoids from sorted DPWT and DPKO cells and automatic quantification of the number of organoids from sorted DD or DP cells. Scale bars, 100 μm. Statistics were determined using two-way ANOVA, **P < 0.01.

  • Fig. 3 CPEB2 regulates proliferation in the mammary gland.

    (A) Representative images and automatic quantification of Ki67+ cells by immunohistochemistry in adult virgin mammary gland in WT and CPEB2 KO (n = 7) mice. Statistics were determined using the Mann-Whitney test, *P < 0.05. Scale bars, 50 μm. (B) Representative FACS plots (gated on MECs) and quantification of percentage of EdU incorporation. FSC-A, FSC area. Statistics were determined using two-tailed unpaired Student’s t test, *P < 0.05. MECs, mammary epithelial cells. (C) Representative images and automatic quantification of ER+ cells by immunohistochemistry in adult virgin mammary gland in WT and CPEB2 KO (n = 5). Statistics were determined using the Mann-Whitney test, *P < 0.05. Scale bars, 25 μm. (D) Representative images and automatic quantification of PR+ cells by immunohistochemistry in adult virgin mammary gland in WT and CPEB2 KO (n = 5). Statistics were determined using the Mann-Whitney test, *P < 0.05. Scale bars, 25 μm. (E) Preranked GSEA graphical output for the enrichment in Sca1+KO cells (DPKO + DDKO) of the gene set estrogen response early from the Molecular Signatures Database Hallmarks collection (see Methods). FDR q = 0.0139. (F) Heat map representing the log2FC expression of hormone-driven genes in DPKO compared to DPWT. (G) Cpeb2 expression levels normalized by Gapdh in epithelial subpopulations.

  • Fig. 4 CPEB2 regulates the synthesis of key effectors in the hormonal signaling pathways.

    (A) Western blot image for CPEB2 and vinculin (as a control) from unbound, input, and immunoprecipitated fractions with anti-CPEB2 antibody in WT and CPEB2 KO MECs. (B) Percentage of genes with (+CPEs, red) or without (–CPEs, gray) CPEs in the 3′UTR, comparing RIP targets to the mouse transcriptome (all). Statistics were determined using Fisher’s exact test, ****P < 0.0001. (C) Significantly enriched KEGG pathways (adjusted P < 0.05) in the analyzed RIP targets. cGMP-PKG, cyclic guanosine monophosphate (cGMP)–cGMP-dependent Protein Kinase G (PKG). mTOR, mammalian target of rapamycin. (D) RIP-qPCR results showing the RIP values normalized by each input in WT (n = 4) and KO (n = 3) MECs. Gapdh mRNA and RIP in CPEB2 KO MECs are used as negative controls for enrichment in RIP as compared to input. Statistics were determined using the Mann-Whitney test, *P < 0.05. IP, immunoprecipitation. (E) Western blot image for CPEB2, CREB1, and α-tubulin (loading control) and normalized quantification of CREB1 protein levels in WT and KO MECs (n = 6). Statistics were determined using the Mann-Whitney test, **P < 0.01. (F) Representative images and manual quantification of RANKL+ cells by immunohistochemistry in adult virgin mammary gland in WT and CPEB2 KO animals (n = 6). Scale bar, 50 μm. Statistics were determined using the Mann-Whitney test, **P < 0.01. (G) Western blot image for CPEB2, CyclinD1, and α-tubulin (loading control) and normalized quantification of CyclinD1 protein levels in WT and KO MECs (n = 6). Statistics were determined using the Mann-Whitney test, *P < 0.05. (H) mRNA levels of Rankl, Ccnd1, and Creb1 normalized to Gapdh and to WT in MECs (WT, n = 6; KO, n = 4). Statistics were determined using the Mann-Whitney test.

  • Fig. 5 Absence of CPEB2 protects against luminal breast cancer.

    (A) Violin plots for CPEB2 RNA expression depending on ER status; METABRIC cohort (n = 1974). Statistics were determined using the Wald test, P < 10 × 2.22−16. (B) Violin plots for CPEB2 RNA expression in the PAM50 subtypes; METABRIC cohort (n = 1974). Statistics were determined using the Wald test compared to the luminal A subtype: basal-like, P < 10 × 2.22−16; HER2, P < 10 × 2.22−16; and luminal B, P = 0.99003. (C) Quantification of CPEB2 expression levels by RT-qPCR in the indicated breast cancer cell lines. B2M was used as endogenous control. (D) Kaplan-Meier survival curves for patients with luminal A breast cancer [HR (<10 years) = 1.89; P = 0.021; multivariate using tumor size and lymph node as other risk factors n = 550]. (E) Schematic representation of the chemical-induced breast cancer model and kinetics of mammary tumor onset in mice treated with medroxyprogesterone acetate (MPA) and 7,12-dimethylbenz(a)anthracene (DMBA) as indicated. Statistics were determined using the log-rank test, *P < 0.05. (F) Number of macroscopic tumors per animal at time of sacrifice (16 weeks after MPA administration) in WTCK14 (n = 11) and CPEB2 KOCK14 (n = 11) animals. Statistics were determined using the Mann-Whitney test, *P < 0.05. (G) Tumor incidence in WTCK14 (n = 11) and CPEB2 KOCK14 (n = 11) mice. Statistics were determined using chi-square test, *P < 0.05. (H) Western blot image for CPEB2 and vinculin (loading control) in ZR75 cells after KD of CPEB2 using sh_CPEB2 #28 or #78 or in control cells (sh_Control). (I) Relative growth curve of ZR75 cells sh_Control or KD of CPEB2. Cell numbers were quantified relative to day 0 at the indicated time points. Statistics were determined using a two-tailed unpaired Student’s t test, ***P < 0.001. (J) Surviving fraction of CPEB2 KD ZR75 cells (using sh_CPEB2 #28 and #78) or control ZR75 cells treated with vehicle (0 μM), 0.5 μM 4-OHT, or 1 μM 4-OHT. Number of viable cells was quantified 6 days after 4-OHT treatment. Surviving fraction refers to the fraction of cells present after 4-OHT treatment. Statistics were determined using a two-tailed unpaired Student’s t test, *P < 0.05 and ***P < 0.001. n.s., not significant. (K) RT-qPCR quantification of MYC expression levels in CPEB2 KD ZR75 cells (sh_CPEB2 #28 or #78) or control ZR75 cells (sh_Control) treated with vehicle (0 μM) or 1 μM 4-OHT for 48 hours. B2M was used as an endogenous control. Statistics were determined using a two-tailed unpaired Student’s t test, **P < 0.01 and ***P < 0.001. (L) Quantification of CCND1 expression levels by RT-qPCR in CPEB2 KD ZR75 cells (sh_CPEB2 #28 and #78) or control cells (sh_Control) treated with vehicle (0 μM) or 1 μM 4-OHT for 48 hours. B2M was used as an endogenous control. Statistics were determined using a two-tailed unpaired Student’s t test, *P < 0.05, **P < 0.01, and ***P < 0.001.

Supplementary Materials

  • Supplementary Materials

    The RNA binding protein CPEB2 regulates hormone sensing in mammary gland development and luminal breast cancer

    Rosa Pascual, Judit Martín, Fernando Salvador, Oscar Reina, Veronica Chanes, Alba Millanes-Romero, Clara Suñer, Gonzalo Fernández-Miranda, Anna Bartomeu, Yi-Shuian Huang, Roger R. Gomis, Raúl Méndez

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