Figures and data
Loss of Frmd8 promotes mammary tumor growth and generates tamoxifen resistance in vivo
(A) A diagram of the Frmd8 targeted alleles. Exons 4, 5 and 6 are flanked by loxp sites. (B) Distinguishing the genotype of littermate mice by mice coat color. Yellow represents MMTV-Cre+; Frmd8fl/fl; PyMT genotype and black represents MMTV-Cre-; Frmd8fl/fl; PyMT genotype. (C) Representative PCR genotyping of mouse tail DNA. (D) Relative mRNA level of Frmd8 in 7-week-old mammary glands from PyMT mice was analyzed by qRT-PCR. Gapdh was used as an internal reference. *p<0.05 by unpaired Student’s t-test. (E) Kaplan-Meier plot showing the appearance of palpable tumors in MMTV-Cre-; Frmd8fl/fl; PyMT (n=9) and MMTV-Cre+; Frmd8fl/fl; PyMT (n=9) mice (Log-rank test). (F) Representative images of tumors from MMTV-Cre-; Frmd8fl/fl; PyMT and MMTV-Cre+; Frmd8fl/fl; PyMT mice. (G-H) Total tumor weight (G) and number of tumors (H) per mice were measured. **p<0.01, ***p<0.001 by unpaired Student’s t-test. (I) Representative H&E staining of tumors from MMTV-Cre-; Frmd8fl/fl; PyMT and MMTV-Cre+; Frmd8fl/fl; PyMTmice. Scale bar, 50 μm. (J) Immunohistochemistry (IHC) staining for Ki67 expression in mammary tumors from PyMT mice. The black boxes represent the magnified typical staining of the original images. Scale bar, 50 μm. (K) Quantification of Ki67-positive cell percentage in (J). *p<0.05 by unpaired Student’s t-test. (L) Kaplan-Meier plot showing the appearance of palpable tumors in MMTV-Cre-; Frmd8fl/fl; PyMT and MMTV-Cre+; Frmd8fl/fl; PyMT mice, with or without tamoxifen treatment (Log-rank test, *p<0.05, ***p<0.001, ****p<0.0001). (M) Representative images of tumors from MMTV-Cre-; Frmd8fl/fl; PyMT and MMTV-Cre+; Frmd8fl/fl; PyMT mice, with or without tamoxifen treatment. (N-O) Total tumor weight (N) and number of tumors (O) per mice from (L) were measured. *p<0.05, **p<0.01, ***p<0.001 by unpaired Student’s t-test or Mann Whitney test.
Frmd8 knockout decreases the proportion of the hormone-sensing differentiated epithelial cells
(A) T-SNE plot showing the distribution of cells from MMTV-Cre-; Frmd8fl/fl; PyMT and MMTV-Cre+; Frmd8fl/fl; PyMT mice. (B) T-SNE plot showing the distribution of epithelial, immune and stromal cells. (C-D) T-SNE plot showing the distribution of main cell linages (C) and their relative percentage (D). (E-F) T-SNE plot showing the distribution of epithelial cell linages (E) and their relative percentage (F). (G) Heatmap showing distinct features of each cell lineages. Rows, genes. Columns, cells. The color key from blue to red indicates low to high gene expression. (H) Boxplot showing CNV score of main cell linages. (I) Dot plot showing the expression of Esr1 and Pgr in epithelial cell linages. (J) T-SNE plot showing the distribution of normal cells and tumor cells. (K) Dot plot showing the expression of Frmd8 in normal and tumor cells from MMTV-Cre-; Frmd8fl/fl; PyMT and MMTV-Cre+; Frmd8fl/fl; PyMT mice. (L) Dot plot showing the expression of Esr1 and Pgr in normal and tumor cells from MMTV-Cre-; Frmd8fl/fl; PyMT and MMTV-Cre+; Frmd8fl/fl; PyMT mice.
FRMD8 promotion of ESR1 expression is mediated by FOXO3A
(A) Representative multiplex immunofluorescence images of tumor tissues and tissues adjacent to tumor from MMTV-Cre-; Frmd8fl/fl; PyMT and MMTV-Cre+; Frmd8fl/fl; PyMT mice. Scale bar, 50 μm. (B) Qualification of Frmd8 and CK8 expression (left panel) and ERα and PR-positive cell percentage in CK8+ cells (right panel) in (A). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by one-way ANOVA (left panel) or unpaired Student’s t-test (right panel). (C) Lysates from MCF7 cells transiently transfected with Flag or Flag-FRMD8 were immunoblotted. (D) Lysates from MCF7 and T47D cells transiently transfected with control or FRMD8 siRNA were immunoblotted. In this and subsequent figures, specific bands are marked with an arrow. (E) Relative mRNA levels of ESR1 and FRMD8 from MCF7 and T47D cells transiently transfected with control or FRMD8 siRNA were analyzed by qRT-PCR. GAPDH was used as an internal reference. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA. (F) Lysates from MCF7 and T47D cells transiently transfected with control or FRMD8 siRNA were immunoblotted. (G) Lysates from MCF7 and T47D cells transiently transfected with HA or HA-FRMD8 were immunoblotted. (H-I) Lysates of MCF7 cells co-transfected with HA-FRMD8 and FOXO3A siRNA as indicated were immunoblotted (H). ERα protein levels were quantified by normalizing to the intensity of the GAPDH band (I). **p<0.01 by unpaired Student’s t-test. (J) Lysates of T47D cells transfected with control or FRMD8 siRNA were subjected to anti-FOXO3A ChIP-qPCR. *p<0.05, ***p<0.001 by one-way ANOVA. (K)Working model for FRMD8 promotes ESR1 transcription via upregulating FOXO3A expression.
FRMD8 stabilizes ERα via prevention of its degradation
(A) Lysates of HEK293T cells co-transfected with Flag-ERα and FRMD8 siRNA as indicated were immunoblotted. (B-C) Lysates from T47D cells transiently transfected with control or FRMD8 siRNA were subjected to immunoblotting. Cells were treated with 100 μg/ml CHX for the indicated times (B). ERα protein levels were quantified by normalizing to the intensity of the Actin band (C). (D-E) Immunoblot analysis of ERα in MCF7 (D) and T47D (E) cells transiently transfected with control or FRMD8 siRNA. The cells were treated with MG132 (25 μM) or chloroquine (50 μM) for 6 h.
FRMD8 inhibits ERα degradation by blocking UBE3A binding with ERα
(A-B) Lysates of MCF7 (A) and T47D (B) cells transfected with Flag or Flag-FRMD8 were anti-Flag immunoprecipitated and immunoblotted for ERα and Flag. (C) Lysates from MCF7 cells were immunoprecipitated with IgG or anti-ERα, then immunoblotted for FRMD8 and ERα. (D) HEK293T cells were transiently transfected with Flag-ERα. ERα proteins in HEK293T whole cell lysates (WCL) pulled down by GST or GST-FRMD8 recombinant proteins were subjected to Western blot. Asterisks indicate proteins at the expected molecular weight. (E) Venn diagram showing overlap of E3 ligases related to ERα and proteins interacting with FRMD8. (F) Lysates of HEK293T cells transfected with Flag or Flag-FRMD8 were anti-Flag immunoprecipitated and immunoblotted. (G) Lysates of HEK293T cells transfected with Flag or Flag-UBE3A were anti-Flag immunoprecipitated and immunoblotted. (H-I) HEK293T (H) and T47D (I) cells were co-transfected with Flag-ERα, HA-Ub and FRMD8 siRNA as indicated. Cells were treated with MG132 (25 μM) for 6 h. WCL were immunoprecipitated with anti-Flag and then immunoblotted for ubiquitinated ERα. (J-K) Lysates of T47D cells co-transfected with FRMD8 and UBE3A siRNA as indicated were immunoblotted (J). ERα protein levels were quantified by normalizing to the intensity of the Actin band (K). ***p<0.001, ****p<0.0001 by one-way ANOVA. (L) Working model for FRMD8 disrupts the interaction between ERα and UBE3A, and protects ERα from UBE3A-mediated degradation.
FRMD8 promoter is methylated and low FRMD8 level predicts poor prognosis in breast cancer patients
(A) Lysates from human mammary epithelial cell (MEC) and breast cancer cells were subjected to immunoblotting. (B) Dot plot showing the expression of Frmd8 in epithelial cell linages from PyMT mice. (C) Methylation of FRMD8 promoter region in breast cancer according to the University of California Santa Cruz (UCSC) database (http://xena.ucsc.edu/). (D) SUM159 and MDA-MB-231 cells were treated with 5-Aza-dC (10 μM) for 48 h. Protein expression of FRMD8 was examined by Western blot. (E) SUM159 and MDA-MB-231 cells were treated with 5-Aza-dC (10 μM) for 48 h. FRMD8 mRNA levels was examined by quantitative reverse transcription PCR (qRT-PCR) GAPDH was used as an internal reference. **p<0.01 by unpaired Student’s t-test. (F) IHC analysis of FRMD8 expression in human breast carcinoma TMA was performed. Representative examples (scale bar, 100 μm) of normal tissue adjacent to tumor and breast cancer with different levels of FRMD8 expression are shown, with the magnification of selected areas inserted. (G) Kaplan-Meier analysis for the overall survival of breast cancer patients according to FRMD8 expression (Log-rank test). (H-J) Recurrence free survival of breast cancer patients according to FRMD8 expression were analyzed according to Kaplan-Meier plotter (http://kmplot.com/analysis/).
