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. 2014;10(11):2036-52.
doi: 10.4161/auto.34398. Epub 2014 Oct 30.

Autophagy Regulator BECN1 Suppresses Mammary Tumorigenesis Driven by WNT1 Activation and Following Parity

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Free PMC article

Autophagy Regulator BECN1 Suppresses Mammary Tumorigenesis Driven by WNT1 Activation and Following Parity

Michelle Cicchini et al. Autophagy. .
Free PMC article

Abstract

Earlier studies reported allelic deletion of the essential autophagy regulator BECN1 in breast cancers implicating BECN1 loss, and likely defective autophagy, in tumorigenesis. Recent studies have questioned the tumor suppressive role of autophagy, as autophagy-related gene (Atg) defects generally suppress tumorigenesis in well-characterized mouse tumor models. We now report that, while it delays or does not alter mammary tumorigenesis driven by Palb2 loss or ERBB2 and PyMT overexpression, monoallelic Becn1 loss promotes mammary tumor development in 2 specific contexts, namely following parity and in association with wingless-type MMTV integration site family, member 1 (WNT1) activation. Our studies demonstrate that Becn1 heterozygosity, which results in immature mammary epithelial cell expansion and aberrant TNFRSF11A/TNR11/RANK (tumor necrosis factor receptor superfamily, member 11a, NFKB activator) signaling, promotes mammary tumorigenesis in multiparous FVB/N mice and in cooperation with the progenitor cell-transforming WNT1 oncogene. Similar to our Becn1(+/-);MMTV-Wnt1 mouse model, low BECN1 expression and an activated WNT pathway gene signature correlate with the triple-negative subtype, TNFRSF11A axis activation and poor prognosis in human breast cancers. Our results suggest that BECN1 may have nonautophagy-related roles in mammary development, provide insight in the seemingly paradoxical roles of BECN1 in tumorigenesis, and constitute the basis for further studies on the pathophysiology and treatment of clinically aggressive triple negative breast cancers (TNBCs).

Keywords: 8-O-dG, 8-oxo-7, 8-dihydroguanine; ATG, autophagy-related; BECN1, Beclin 1, autophagy-related; BSA, bovine serum albumin; Beclin 1; CASP3, caspase 3; CD24, cluster of differentiation 24; DAPI, 4′, 6-diamidino-2-phenylindole; DFS, disease-free survival; DMEM, Dulbecco's modified Eagle's medium; E, 17b-estradiol; EGF, epidermal growth factor; EGFP, enhanced green fluorescent protein; EGFR/ERBB1, epidermal growth factor receptor; EM, electron microscopy; EMT, epithelial-to-mesenchymal transition; ERBB2, v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2; ESR1, estrogen receptor 1; FACS, fluorescence activated cell sorting; FGF2/bFGF, fibroblast growth factor 2 (basic); GSEA, gene set enrichment analysis; H&E, hematoxylin &, eosin; HR, hormone receptor; IF, immunofluorescence; IHC, immunohistochemistry; IL, interleukin; ITGB1/CD29, Integrin, beta 1 (fibronectin receptor beta polypeptide, antigen CD29 includes MDF2, MSK12); ITGB3/CD61, integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61); KRT, keratin; Keratin 6; LIN−, lineage negative (CD31− CD45− LY76−); LY76/TER119, lymphocyte antigen 76; MAP1LC3B/LC3B, microtubule-associated protein 1 light chain 3 beta; MEC, mammary epithelial cell; MEGM, mammary epithelial growth medium; MGs, mammary glands; MKI67, marker of proliferation Ki-67; MMTV, mouse mammary tumor virus; MaPC, mammary progenitor cell; MaSC, mammary stem cell; NFKB; NFKB/NFkB, nuclear factor of kappa light polypeptide gene enhancer in B-cells; PBS, phosphate-buffered saline; PECAM1/CD31, platelet/endothelial cell adhesion molecule 1; PGR, progesterone receptor; PI, propidium iodide; PTPRC/CD45, protein tyrosine phosphatase, receptor type, C; RELA/P65, v-rel avian reticuloendotheliosis viral oncogene homolog a; ROS, reactive oxygen species; SD, standard deviation; SNPs, single nucleotide polymorphisms; SQSTM1/p62, sequestosome1; TEBs, terminal end buds; TNBC; TNBCs, triple-negative breast cancers; TNF, tumor necrosis factor; TNF11; TNFRSF11A; TNFRSF11A/TNR11/RANK, tumor necrosis factor receptor superfamily, member 11a, NFKB activator; TNFSF11; TNFSF11/TNF11/RANKL, tumor necrosis factor (ligand) superfamily, member 11; TNR11; TP53 (TRP53 in mice), tumor protein p53 (transformation related protein 53 in mice); WNT1; WNT1, wingless-Type MMTV integration site family, member 1; basal-like breast cancer; iMMECs, immortalized mouse mammary epithelial cells; p-KRT8/p-K8, phosphorylated Keratin 8; parity.

Figures

Figure 1.
Figure 1.
Monoallelic Becn1 loss results in KRT6 upregulation in mammary epithelial cells and tissues. (A) Microarray analysis was performed on samples (three each) of Becn1+/+ and Becn1+/− apoptosis-competent and apoptosis-defective iMMECS, as well as mammary tumors derived from orthotopic implantation of apoptosis-defective iMMECs in nude mice. KRT6 expression relative to that in apoptosis-competent Becn1+/+ iMMECs is presented as means ± SDs (n = 3). (B–D) Becn1+/+ (left) and Becn1+/− (right) with DAPI (blue) by IF: (B) More KRT6-expressing cells are seen in Becn1+/− apoptosis-competent iMMECs (top) and Becn1+/− apoptosis-defective iMMECs (bottom) under normal growth conditions. (C) More KRT6-expressing (green, top) and KRT14-expressing (red, bottom) cells are seen in allograft mammary tumors generated by Becn1+/− apoptosis-defective iMMECs. (D) MGs from 5-wk-old (top) and 9-mo-old (bottom) Becn1+/− mice reveals increased number of KRT6-positive (green) cells that are not KRT8-positive (red). (E) Costaining for KRT6 (purple), CD24 (red), CD29 (green) in MGs from 9-mo-old Becn1+/− mice reveals that KRT6-positve cells colocalize with mammary progenitor cell markers. Scale bar: (B and D) 30 μm; (C and E) 37.5 μm.
Figure 2.
Figure 2.
Monoallelic Becn1 loss results in accelerated mammary fat pad filling and excessive ductal side-branching. (A–D) Analysis of mouse mammary epithelium from Becn1+/+ (left) and Becn1+/− (right) mice (n = 3 to 5): (A) MG whole mounts from 6.5-wk-old mice, with circle identifying an unfilled (left) vs. filled (right) fat pad and (B) (top) quantification of number of side-branches per primary branch per field and (bottom) quantification of percentage of mammary fat pad 4 filled with epithelium. Three MG specimen per genotype from cohoused Becn1+/+ and Becn1+/− littermates were evaluated. Results are presented as means ± SDs. (C) MG whole mounts (top) and hematoxylin and eosin staining of MG sections (bottom) from 6-mo-old mice, with mammary hyperplasia in separate insert. (D) MG whole mounts from 12-mo-old mice. Scale bar: (A, C,and D) 5 mm for whole mounts; (A, C, and D) 2 mm for whole mount enlargements. Scale bar: (C) 100 μm for H&Es; 200 μm for hyperplasia. *P < 0.05 by a 2-tailed Student t test.
Figure 3.
Figure 3.
Monoallelic Becn1 loss results in functionally enriched mammary stem and progenitor cell populations. (A–C) Freshly isolated Becn1+/+ (blue) and Becn1+/− (red) MECs were used, experiments were performed 3 independent times and outcomes are presented as means ± SDs. (A) Primary mammosphere assays performed using Pi LIN(CD31,CD45,LY76) CD24+ CD29hi MECs plated at 20,000 cells per well (left) and in limiting dilution of 500 to 25 CD24+ CD29hi MECs plated (right) reveal increased MaSC activity in Becn1+/− MECs. (B) 3D colony-formation by CD24+ CD29hi Becn1+/− compared with Becn1+/+ MECs plated at 20,000 cells per well shows increased MaSC activity. (C) Colony formation by CD24+ CD29lo Becn1+/− compared with Becn1+/+ MECs plated at 1,000 cells per well reveals increased colony forming ability in 2D-matrigel conditions. (D) Increased repopulation frequency and mammary fat pad filling are seen following transplantation of 250 CD24+ CD29hi Becn1+/− MECs isolated from 5-wk-old mice. Becn1+/+ (left) and Becn1+/− (right), contralateral transplantations in wild-type recipient mouse. Circles represent fat pads and the black color represents the percentage of the mammary fat pad that is filled in. (E) Representative whole mount images of outgrowths from contralateral CD24+ CD29hi Becn1+/+ and Becn1+/− MEC transplantation are shown and reveal increased ductal side branching in Becn1+/− samples. *P < 0.05 by a 2-tailed Student t test. Scale bar: (E) 5 mm for whole mounts; 2 mm for whole mount enlargements.
Figure 4.
Figure 4.
Outgrowths from CD24+ CD29hi Becn1+/− MEC transplantation display increased proliferation and TNFSF-NFKB pathway activation. (A–D, and F) Examination of outgrowths from contralateral transplantations of CD24+ CD29hi Becn1+/+ and Becn1+/− MECs, and (E) native MGs from 6.5-wk-old cohoused Becn1+/+ and Becn1+/− littermates. (A) Representative images show more MKI67+ cells in ducts (top) and TEBs (bottom) of outgrowths from CD24+ CD29hi Becn1+/− MEC transplantation; (B) quantification of MKI67+ cells in mammary ducts and TEBs is shown as means ± SDs (n = 3 mammary gland specimens per genotype). (C) Representative images show fewer cleaved CASP3-staining cells in ducts (top) and TEBs (bottom) of outgrowths from CD24+ CD29hi Becn1+/− MEC transplantation; (D) quantification of cleaved CASP3+ cells in mammary ducts and TEBs is shown as means ± SDs (n = 3 mammary gland specimens per genotype). Increased TNFSF11 (red) expression is seen in (E) native MGs from 6.5-wk-old Becn1+/− mice and (F) outgrowths from contralateral CD24+ CD29hi Becn1+/− MEC transplantation (top panel). Increased TNFRSF11A (middle panel) and RELA (bottom panel) levels are also seen in outgrowths from CD24+ CD29hi Becn1+/− MEC transplantation. Scale bar: (A, C, E, and F, middle and bottom panels) 30 μm ; (F, top panel) 40 μm.
Figure 5.
Figure 5.
Augmented progesterone signaling, cytokines, and oxidative stress detected upon monoallelic Becn1 loss. (A and C) Nuclear PGR (red) and KRT6 (green) cell staining in native MGs from cohoused Becn1+/+ and Becn1+/− MEC and outgrowths from contralateral CD24+ CD29hi Becn1+/+ and Becn1+/− MEC transplantation. (A) Representative images of PGR+ and KRT6+ cells and (B) quantification of nuclear PGR+ cells (left) and KRT6+ cells (right) in MGs from 5-wk old mice reveals increased numbers of PGR+ and KRT6+ cells in Becn1+/− mice. (C) Representative images of nuclear PGR+ cells (top panel) and KRT6+ cells (bottom panel) in outgrowths from contralateral CD24+ CD29hi Becn1+/+ and Becn1+/− MEC transplantation with (D) quantification of PGR+ cells (top panel) and KRT6+ cells (bottom panel). Results are presented as means ± SDs (n = 3 mammary gland specimens per genotype). (E) Quantification of ductal side-branching in MGs from hormonally stimulated following ovariectomization, where Sham = ovariectomized, Vehicle = ovariectomized + 100 μL sesame oil daily, and E + P = ovariectomized + 5 μg 17B-estradiol + 5 mg progesterone in 100 μL of sesame oil daily. (F–H) Examination of outgrowths from contralateral transplantations of CD24+ CD29hi Becn1+/+ and Becn1+/− MECs. (F) Increased IL1B (top panel) and TNF (bottom panel) seen in outgrowths from Becn1+/− MEC transplantation. (G) Similar F4/80 staining detected in outgrowths from Becn1+/+ and Becn1+/− MEC transplantation. (H) Increased 8-O-dG is seen in outgrowths from Becn1+/− MEC transplantation. *P < 0.05, **P < 0.01, and ***P < 0.001 by a 2-tailed Student t test. Scale bar: (A, C, F, G, and H) 30 μm; (E) 50 μm.
Figure 6.
Figure 6.
Spontaneously arising mammary tumors are detected in multiparous Becn1+/− FVB/N mice. (A and B) Hematoxylin and eosin staining (H&E) of MG sections from Becn1+/+ (left) and Becn1+/− (right) FVB/N mice: (A) Representative images from MGs of nulliparous 21- to 22-mo-old mice reveals increased epithelial cell density and inflammatory cell infiltration (brown, identified with arrow) in Becn1+/− mice. (B) Representative H&E images of MGs from parous 14- to 17-mo-old mice reveals increased epithelial cell density and disorganized cell layering in MGs from Becn1+/− mice. (C) Representative images from 4 spontaneously arising mammary tumors in parous Becn1+/− FVB/N mice (Tumors #1 to 4). Left panels are representative H&Es; middle panels, KRT6 (green) and nuclear PGR (red) staining; and right panels, KRT8 (green) and KRT14 (red) staining. (*) and (*) denote keratin pearls and basement membrane/eosinophilic hyaline materials, respectively. Tumor type denotations are identified by: (i) for glandular components, (ii) for poorly to moderately differentiated squamous cell carcinoma, (iii) for well-differentiated squamous cell carcinoma, and (iv) for basaloid squamous cell carcinoma. Scale bar: (A and B) 30 μm and KRT8 and KRT14 (C); (C) 200 μm for H&Es; (C) 50 μm for KRT6 and PGR.
Figure 7.
Figure 7.
Monoallelic Becn1 loss accelerates WNT1-driven tumorigenesis and gives rise to mammary tumors with TNFRSF11A-NFKB1 pathway activation and basal-like characteristics. Basal-like characteristics and TNFRSF11A-NFKB1 signaling activation are detected in spontaneous mammary tumors that arise faster in Becn1+/−;MMTV-Wnt1 (right) compared with Becn1+/+;MMTV-Wnt1 mice (left). (A) Kaplan-Meier curves for mammary tumor-free survival in Becn1+/+;MMTV-Wnt1 (n = 25) and Becn1+/−;MMTV-Wnt1 (n = 20) mice demonstrates decreased mammary tumor-free survival in Becn1+/−;MMTV-Wnt1 mice. (B–E) Examination of mammary tumors and premalignant MGs from Becn1+/+;MMTV-Wnt1 (left) and Becn1+/−;MMTV-Wnt1 (right) mice. (B) Representative images of H&E staining (top panel), KRT6 (green) and nuclear PGR (red) cell staining (second panel), KRT8 (green) and KRT14 (red) cell staining (third panel), and nuclear ESR1 staining (bottom panel) on tumor sections. (C) Heat map representation of microarray analysis confirms basal-like tumor characteristics along with higher CTNNB1 expression in tumors from Becn1+/−;MMTV-Wnt1 mice. Statistical significance of upregulation of a given signature on a group of samples was determined using a Fischer exact test for enrichment of samples with significant (P < 0.05) signature expression in the group relative to samples outside the group. (D) Representative images of TNFSF11 (top panel) and RELA (bottom panel) expression in mammary tumors, and (E) TNFSF11 (top panel), TNFRSF11A (center panel), and RELA (bottom panel) expression in premalignant MGs from 6- to 10-wk-old cohoused Becn1+/+;MMTV-Wnt1 and Becn1+/−;MMTV-Wnt1 littermates. P < 0.01 determined by a Mantel-Cox test for Kaplan-Meier curves. Scale bar: (B) 200 μm for H&Es; (B, D, and E) 30 μm for all other panels.
Figure 8.
Figure 8.
Human breast cancers with low BECN1 expression and an activated WNT pathway gene signature have poor prognosis. (A–D) Kaplan-Meier curves and GSEA for breast tumors displaying an activated WNT pathway gene signature and stratified for BECN1 expression (see Methods). (A) Disease-free survival for patients with breast cancers annotated in the Sabatier cohort (P 7.2 × 10−5). (B) Disease-free survival for patients with ERBB2-negative breast cancers annotated in the Hatzis cohort (P 2.7 × 10−3). Gene expression analysis comparison in (C) the Sabatier cohort and (D) the Hatzis cohort reveals that breast cancers with low BECN1 and an activated WNT pathway gene signature are primarily triple-negative, have basal-like characteristics, and display TNFRSF11A pathway activation and higher CTNNB1. P values were calculated by the Fischer exact test (see Materials and Methods). (E) Model for the cooperation between monoallelic BECN1 loss and WNT pathway activation in mammary tumorigenesis. *P < 0.05, **P < 0.01, ***P < 0.001.

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