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1.
Figure 4

Figure 4. ΔNp63-mediated MaSC function is dependent on Fzd7. From: ΔNp63 promotes stem cell activity in mammary gland development and basal-like breast cancer by enhancing Fzd7 expression and Wnt signaling.

(a) Quantification of colonies formed by P4 cells transduced with indicated lentiviruses (n = 3 samples; data represents mean ± s.d.). (b) P4 cells were transduced with control or Fzd7 lentiviruses and limiting numbers of cells were transplanted into cleared fat pads. Table shows take rate quantifications. (c) Quantification of colonies formed by P4 cells from WT and ΔNp63gfp/+ mice with or without Fzd7 overexpression (n = 5 samples; data represents mean ± s.d.). (d) Representative images of colonies from c. (e) Table showing reconstitution efficiency at limiting dilution of P4 cells from WT and ΔNp63gfp/+ mice with Fzd7 overexpression. (f) Representative images and pie graph summary of outgrowths from e. (g) Table showing reconstitution efficiency at limiting dilution of P4 cells from control and ΔNp63 overexpressing cells with or without Fzd7 KD. (h) Representative images and pie graph summary of outgrowths from g. ● 80–100%, 30–80%, ◔ 0–30% and ○ No reconstitution. *p < 0.05 by Student’s t test in a and c. In b, e, and g, n= number of mammary fat pad injections as indicated in tables. p value was obtained by Pearson’s Chi-squared test using ELDA software. Size bar, 1 mm in d and f, and 2 mm in h, respectively.

Rumela Chakrabarti, et al. Nat Cell Biol. ;16(10):1004-13.
2.
Figure 8

Figure 8. Loss of ΔNp63 attenuates Wnt1-driven hyperplasia and tumorigenesis. From: ΔNp63 promotes stem cell activity in mammary gland development and basal-like breast cancer by enhancing Fzd7 expression and Wnt signaling.

(a) Representative mammary gland whole mounts from 5- and 8-week-old WT; ΔNp63+/+, MMTV-Wnt1; ΔNp63 +/+ and MMTV-Wnt1; ΔNp63gfp/+ mice. (b) Representative FACS profiles of mammary epithelial cells from the indicated. (c) Quantification of mammospheres formed by P4 cells (10,000 cells) from 8-week-old mice of indicated genotypes (n = 3 samples, data represents mean ± s.d.).*p < 0.05 by Student’s t test. (d) Table showing reconstitution efficiency at limiting dilution of P4 cells from mice of the indicated genotypes (n = number of mammary fat pad injections as indicated in the table). P value was obtained by Pearson’s Chi-squared test using ELDA software. (e) Representative images of outgrowths from d. (f) Kaplan-Meier curve analysis of tumor-free mammary glands at the indicated ages (n = 12 mice for MMTV-Wnt1; ΔNp63 +/+ and n = 10 mice for MMTV-Wnt1; ΔNp63 gfp/+). Log rank test was used for statistical analysis. (g) Schematic model for function of the ΔNp63-Fzd7 axis in mammary cell fate regulation and basal breast cancer initiation. High expression of ΔNp63 in mammary stem cells and TICs maintain their self renewal abilities through activating Fzd7 expression and Wnt signaling. High expression of ΔNp63 can also confer luminal differentiated/progenitor cells with MaSC-like properties. While TICs in basal-like breast cancer may arise from the oncogenic transformation of MaSCs with intrinsic elevated expression of ΔNp63, progenitor cells may also acquire high ΔNp63 expression during their transformation to become TICs. Size bar, 3 mm and 2 mm respectively in a and e.

Rumela Chakrabarti, et al. Nat Cell Biol. ;16(10):1004-13.
3.
Figure 6

Figure 6. Loss of ΔNp63 attenuates tumor initiation in basal-like MMTV-Wnt1 tumors. From: ΔNp63 promotes stem cell activity in mammary gland development and basal-like breast cancer by enhancing Fzd7 expression and Wnt signaling.

(a) Fold change of ΔNp63 mRNA level in primary MMTV-Wnt1 tumor MECs after transduction with indicated shRNAs (n = 3 tumors). (b) Quantification of tumorsphere numbers from MMTV-Wnt1 tumor cells transduced with indicated shRNAs (n = 3 tumors). (c) Kaplan-Meier curve of tumor-free mammary gland incidence using MMTV-Wnt1 tumor MECs (10,000 cells) transudced with indicated shRNAs. Log rank test was used for statistical analysis (n = 8 mice). (d) Average tumor volume at end point control (n = 8 tumors) and p63 KD1 (n = 7 tumors). (e) Tumor incidence of MMTV-Wnt1 tumor cells expressing the indicated shRNAs (). (f) Representative FACS profile of primary tumors. (g) Quantification of percentage of CD45CD24+Thy-1+ cells in control and p63-KD Wnt1 tumor cells as shown in f (n = 10 tumors). (h) qRT-PCR analysis of Fzd7 and p63 expression in control or p63-KD Wnt1 tumor MECs (n = 3 samples). (i) Western blot of ΔNp63 in control and ΔNp63-KD MMTV-Wnt1 tumor cells. (j) Fold change of TAp63 mRNA level in MMTV-Wnt1 tumor cells after TAp63 KD (n = 3 tumors). (k and l) Quantification of tumorspheres formed by 10,000 MMTV-Wnt1 tumor cells transduced with indicated shRNAs (n = 3 tumors in k and n = 4 tumors in l). (m) Kaplan-Meier curve analysis of tumor-free mammary glands after injection of mice using control or Fzd7-KD MMTV-Wnt1 tumor MECs (10,000 cells). n = 8 mice. Log rank test was used for statistical analysis. (n) Average tumor volume at end point (n = 6 tumors). (o) Tumor incidence of MMTV-Wnt1 tumor cells expressing the indicated shRNAs. In this figure, bar graph data represent mean ± s.d. *p < 0.05 by Student’s t test in a, b, h, j, k and l. *p < 0.05 by Mann Whitney test in d, g and n. In e and o, n= number of mammary fat pad injections as indicated in the table. p value was obtained by Pearson’s Chi-squared test using ELDA software. Uncropped images of blots are shown in .

Rumela Chakrabarti, et al. Nat Cell Biol. ;16(10):1004-13.
4.
Figure 5

Figure 5. ΔNp63 expression positively correlates with FZD7 expression and Wnt signaling in human breast cancer. From: ΔNp63 promotes stem cell activity in mammary gland development and basal-like breast cancer by enhancing Fzd7 expression and Wnt signaling.

(a) ΔNp63 expression in clinical samples of basal and luminal type human tumors. Box represents 75th, 50th and 25th percentile of the values. Top and bottom lines represent maximum and minimal data points within 1.5x IQ (inter quarter) range, respectively (n = 286 tumors). (b) Scatter plot showing correlation between ΔNp63 and FZD7 expression in breast tumors from the TCGA-BRCA-RNAseqV2 dataset (n = 1000 tumors). (c) IHC analysis of ΔNp63 and FZD7 expression. (d) Relative expression of Wnt luciferase reporter in MDA-MB-231 cells after lentiviral transduction of a Wnt reporter (7TFC), together with indicated shRNAs. (e) Western blot of ΔNp63, TAp63 and FZD7 expression in MDA-MB-231 and SUM-1315 cells. (f, g) Western blot of ΔNp63 and FZD7 expression in MDA-MB-231 and SUM-1315 cells after transduction with indicated shRNAs. (h) Fold change of TAp63 mRNA level in MDA-MB-231 cells and SUM-1315 cells respectively, after transduction with control and TAp63 shRNA. (i) Relative expression of Wnt luciferase reporter in MDA-MB-231 cells after lentiviral transduction of a Wnt reporter (7TFC), together with indicated shRNAs. In d, h and i, n = 3 samples, data represents mean ± s.d. (j) Tumor incidence of MDA-MB-231 cells expressing indicated shRNAs. (k) Tumor incidence of SUM-1315 cells expressing indicated shRNAs. In j and k, n= number of mammary fat pad injections as indicated in the table. P value was obtained by Pearson’s Chi-squared test using ELDA software. (l) Quantification of tumorsphere formed by control, p63-KD and Fzd7-KD tumors (10,000 cells) from PDX-2 (HCI002). (m) Representative images of tumorspheres from l. (n–p) Quantification of tumorsphere formed by control, ΔNp63 KD and TAp63 KD cells (10,000 cells) from PDX-2 tumors (HCI002) (n–o) and PDX-3 tumors (HCI009) (p). In l, n–p, n = 4 tumors; data represents mean ± s.d. *p < 0.05 by Student’s t test. Size bar, 40 μm in c and m respectively. Uncropped images of blots are shown in .

Rumela Chakrabarti, et al. Nat Cell Biol. ;16(10):1004-13.
5.
Figure 3

Figure 3. ΔNp63 but not TAp63 increases Wnt signaling via direct activation of Fzd7 expression. From: ΔNp63 promotes stem cell activity in mammary gland development and basal-like breast cancer by enhancing Fzd7 expression and Wnt signaling.

(a) GSEA of Wnt gene signatures in unsorted (upper panels) or P4 (lower panels) WT versus ΔNp63gfp/+ MECs. (b) Relative expression of Wnt luciferase reporter in P4 cells after lentiviral transduction of vector control, ΔNp63 or TAp63 lentiviruses (n = 3 samples; data represents mean ± s.d.). (c, d) Heat map of microarray data (c) and qRT-PCR (d) showing the expression of Wnt pathway genes in wild type and ΔNp63gfp/+ P4 cells from 7 week-old virgin mice. (e) qRT-PCR analysis of the Wnt signaling genes in control or p63-KD primary MECs. (f) qRT-PCR analysis of the Wnt signaling genes in control or ΔNp63 KD MECs. In d–f, n = 3 samples; data represents mean ± s.d. (g) Western blot analysis of ΔNp63 and Fzd7 in control, ΔNp63 and TAp63 KD MECs. (h) qRT-PCR analysis of the expression of FZD7 and WNT5A in control, ΔNp63, or TAp63 overexpressing HMLE cells at 48 h (n = 3 samples) or 10 days (n = 6 technical replicates pooled from 2 independent experiments) post-infection. Real time values were normalized to the housekeeping gene GAPDH. Data represents mean ± s.d. (i) ChIP-seq data from human keratinocytes show ΔNp63 binding region to a putative FZD7 enhancer. Dnase: DNAse I hypersensitive sites. Mamm cons: mammalian conserved areas. (j) ChIP analysis of p63 (using H129 and 4A4 antibody) binding to the FZD7 enhancer in HMEC (n = 3 samples; data represents mean ± s.d.). (k) ChIP analysis of p63 binding to Fzd7 enhancer in MMTV-Wnt1 primary mammary tumor cells (n = 6 technical repeats pooled from 2 independent experiments; data represents mean ± s.d.). (l, m) Relative expression of FZD7 enhancer-driven luciferase reporter in HMLE (l) and iMMEC (m) cells transiently transfected with indicated expression plasmids (n = 3 samples, data represents mean ± s.d.). *p < 0.05 by Student’s t test. Uncropped images of blots are shown in .

Rumela Chakrabarti, et al. Nat Cell Biol. ;16(10):1004-13.
6.
Figure 1

Figure 1. ΔNp63 is enriched in MaSCs and promote MaSC activity. From: ΔNp63 promotes stem cell activity in mammary gland development and basal-like breast cancer by enhancing Fzd7 expression and Wnt signaling.

(a) Heat map of differential expression of 26 transcriptional regulators, including p63 (arrow), in different mouse MEC subpopulations (upper panel). qRT-PCR analysis of ΔNp63 and TAp63 mRNAs in MECs (lower panel) (n = 3 samples; data represents mean ± s.d.). qRT-PCR values were normalized to Gapdh. (b) Immunoblot of ΔNp63 expression in different mouse MEC subpopulations. (c) Immunohistochemical analysis of ΔNp63 and TAp63 expression in the terminal end buds (TEBs) and ducts of mammary gland serial sections. (d) qRT-PCR analysis of ΔNp63 and TAp63 mRNAs in normal human mammary epithelium (left panel, n = 3 samples; data represents mean ± s.d.) and CD49fhiEpCAMneg/low MaSC-enriched population (right panel, n = 3 samples; data represents mean ± s.d.). qRT-PCR values were normalized to GAPDH. *p < 0.05 by Student’s t test. (e) Strategy of lentiviral transduction of different MEC subpopulations. (f) MaSC-enriched LinCD24+CD29hi (P4) population was isolated and transduced with indicated lentivirus constructs and transplanted into cleared fat pads (1000 cells). (g) 1000 luminal cells (LinCD24+CD29lo) (P5) were transduced with indicated lentivirus constructs and injected to cleared fat pad. Representative mammary outgrowths shown in f and g. In pie graph below representative images, each circle represents one mammary gland, with blackened area representing the degree of gland filling with outgrowth. ● 80–100%, 30–80%, ◔ 0–30% and no reconstitution. (h, i) Pie graph (h) and representative fluorescence images (i) of the mammary fat pads after competitive reconstitution assay. (j) Representative images of 3-D matrigel colonies of P4 population from GFP and dsRED MECs mixed equally. (k) Number of colonies formed in three generations in 3-D matrigel assay (n = 3 samples; data represents mean ± s.d.). (l) Table showing reconstitution rate of the mammary gland over successive generations after transplantation of 10,000 unsorted MECs transduced with control or ΔNp63 expressing lentiviruses. *p < 0.05, **p < 0.01 by Student’s t test. Size bar, 40 μm in c, 1 mm in f and g, 2mm in i, and 3mm in j. Uncropped images of blots are shown in .

Rumela Chakrabarti, et al. Nat Cell Biol. ;16(10):1004-13.
7.
Figure 7

Figure 7. Loss of ΔNp63 and Fzd7 attenuates tumor initiation in MMTV-Myc tumors. From: ΔNp63 promotes stem cell activity in mammary gland development and basal-like breast cancer by enhancing Fzd7 expression and Wnt signaling.

(a) Western blot analysis of Fzd7 expression in primary MMTV-Myc tumor cells transduced with indicated shRNAs. (b, c) Reduced level of ΔNp63 mRNA (b) and protein (c) expression in primary MMTV-Myc tumor cells with or without p63 KD. *p < 0.05 by Mann Whitney U test and n = 5 tumors per group in b. Box represents 75th, 50th and 25th percentile of the values. The top and bottom lines represent the maximum and minimal data points within 1.5x IQ (inter quarter) range, respectively. (d, e) Freshly isolated tumor MECs with or without p63 KD were used for tumorsphere assays in low adherent plates. Bar graphs show quantification of tumorsphere numbers in control and p63-KD MECs (10,000 cells) (n = 4 tumors; data represents mean ± s.d.). (f, g) Kaplan-Meier curves showing mammary gland tumor-free survival using MMTV-Myc tumor MECs transduced with control and p63 shRNAs (p63 KD1 and p63 KD2). 10,000 cells (n = 6 mice per group) and 5000 cells (n = 10 mice per group) were injected in f and g, respectively. Log rank test was used for statistical analysis and p values computed for both plots are < 0.001. (h, i) Tumor incidence (h) and volume (i) was calculated at end point. P value was obtained by Pearson’s Chi-squared test using ELDA software in h (n= number of mammary fat pad injections as indicated in table). *p < 0.05 by Mann Whitney U test in i (n = 6 tumors per group; data represents mean ± s.d.). (j, k) Bar graphs show quantification of tumorsphere numbers in control, p63-KD2 and Fzd7-KD tumor MECs (10,000 cells) from Blg-Cre-Brca1f/fp53+/− and MMTV-Myc tumors respectively (n = 3 tumors per genotype; data represents mean ± s.d.). P value was computed by Student’s t test. *p < 0.05. Size bar, 40 μm in c. Uncropped images of blots are shown in .

Rumela Chakrabarti, et al. Nat Cell Biol. ;16(10):1004-13.
8.
Figure 2

Figure 2. ΔNp63 deficiency reduces MaSC activity. From: ΔNp63 promotes stem cell activity in mammary gland development and basal-like breast cancer by enhancing Fzd7 expression and Wnt signaling.

(a, b) Box plot (a) and western blot (b) of ΔNp63 expression in P4 cells from WT and ΔNp63gfp/+ mice. Boxes in a represents 75th, 50th and 25th percentile of the values. Top and bottom lines represent maximum and minimal data points within 1.5x IQ (inter quarter) range, respectively. P value computed by Mann Whitney U test (n = 5 samples per genotype). (c) Representative mammary outgrowths from WT and ΔNp63gfp/+ mice. (d) Representative FACS profiles of MECs from WT and ΔNp63gfp/+ mouse. (e) Representative mammary outgrowths from f. Each circle below the images represents one mammary gland, with blackened area representing the degree of gland filling with outgrowth. 80–100%, 30–80%, ◔ 0–30% and no reconstitution. (f) Table showing transplantation of limiting numbers of P4 cells into cleared mammary fat pads from indicated mice (n= number of mammary fat pad injections as indicated in the table). P value was obtained by Pearson’s Chi-squared test using ELDA software. (g) Fold change (FC) of ΔNp63 mRNA level in primary MECs transduced with control and two ΔNp63 shRNA lentiviral constructs (ΔN KD1 and ΔN KD2) (left panel). Fold change of TAp63 mRNA level in control or ΔNp63-KD MECs (right panel). (h) Western blot of ΔNp63 in control and ΔNp63 KD MECs. (i) Fold change of TAp63 mRNA level in primary MECs transduced with control and two TAp63 shRNA lentiviral constructs. TA isoform-specific KD of TAp63 was confirmed by qRT-PCR since TAp63 protein is undetectable in MECs. (j) Quantification of mammosphere formed by control, ΔNp63-KD and TAp63-KD P4 cells (20,000 cells) from WT mice mammary gland. In g, i and j, n = 3 samples; data represents mean ± s.d. *p < 0.05 by Student’s t test. (k) GSEA demonstrating enrichment of MaSC gene signatures in WT versus ΔNp63gfp/+ MECs using MaSC signatures derived from the current study (left panel) or a previously reported MaSC gene signature (right panel). Size bar, 2 mm and 4 mm in c and e respectively. Uncropped images of blots are shown in .

Rumela Chakrabarti, et al. Nat Cell Biol. ;16(10):1004-13.

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