Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor

Abstract

The biochemical properties of beclin 1 suggest a role in two fundamentally important cell biological pathways: autophagy and apoptosis. We show here that beclin 1-/- mutant mice die early in embryogenesis and beclin 1+/- mutant mice suffer from a high incidence of spontaneous tumors. These tumors continue to express wild-type beclin 1 mRNA and protein, establishing that beclin 1 is a haploinsufficient tumor suppressor gene. Beclin 1-/- embryonic stem cells have a severely altered autophagic response, whereas their apoptotic response to serum withdrawal or UV light is normal. These results demonstrate that beclin 1 is a critical component of mammalian autophagy and establish a role for autophagy in tumor suppression. They both provide a biological explanation for recent evidence implicating beclin 1 in human cancer and suggest that mutations in other genes operating in this pathway may contribute to tumor formation through deregulation of autophagy.

Fig. 1.

Targeted disruption of beclin 1 in embryonic cells. (a) Genomic structure of beclin 1 (showing exons 1–4 only; solid box), targeting vector, and beclin 1 allele after targeted deletion. The start codon is indicated by an asterisk. Arrow ”a” represents a common primer for both the wt and targeted beclin 1 alleles, arrow ”b” represents a specific primer for the wt allele, and arrow ”c” represents a specific primer for the targeted allele. X, XbaI; H, HindIII; R, EcoRI; B, BamHI; neo^r, neomycin resistance cassette; TK, thymidine kinase marker. (b) Southern blot analysis of beclin 1 mutant ES clones after digestion with XbaI. The probe used to distinguish wt and targeted allele is indicated in a. wt allele is detected at a size of 11 kb, and mutant allele is detected at 7 kb. (c) Competitive PCR assay of three different genotypes of ES cells (+/+, +/-, and -/-) with primers a–c described in a. wt allele (250 bp) can be separated from mutated allele (500 bp) on a 1.5% agarose gel. (d) Western blot study of beclin 1 expression in ES cells from three different genotypes with anti-beclin 1 antibody. Anti-RAN antibody was used for protein loading control in each lane.

Fig. 2.

Disruption of beclin 1 causes early embryonic lethality. (a) Genotype distribution of offspring and embryos from beclin 1^+/- intercrosses examined by Southern blotting or PCR. (b) Southern blot analysis of mouse genotype at age of weaning from a litter born by heterozygous parents. (c and d) Immunohistochemical analysis of beclin 1 expression in wt or heterozygous embryo at E6.5 and E7.5, respectively. Sections were counterstained with hematoxylin. (e) Image of wt whole embryo at E7.5. c–e are at the same magnification with a scale bar of 100 μm. (f and g) Sections from the same null mutant embryo (E7.5) were immunostained with anti-beclin 1 antibody and counterstained with (f) or without (g) hematoxylin. (h) Whole embryo of null mutant (E7.5) viewed under a Zeiss Axiovert confocal microscope with differential interference contrast microscopy. (i) The same embryo as in h viewed under serological microscope. (j) Whole embryo of null mutant stained with acridine orange and viewed with fluorescence under a Zeiss confocal microscope. f–j are at the same magnification with a scale bar of 20 μm.

Fig. 3.

Disruption of beclin 1 causes abnormal formation and cell growth in VE of EB. Analysis of EBs (day 14) derived from ES cells of wt (a) and beclin 1^-/- (b) is shown. (a and b, Left) H&E staining of paraffin sections. Note the expanded cystic form for wt and the cystic cavitated form for beclin 1^-/-. (Scale bar, 100 μm.) (a and b, Right) Sections immunostained with anti-amnionless antibody (Upper) and toluidene blue-stained, 1-μm sections (Lower). (Scale bar, 10 μm.)

Fig. 4.

Increase of cancer rate in beclin 1^+/- mice. (a) Summary of cancer incidents and cancer rate in wt and beclin 1^+/- mice (age and gender matched). (b and c) B cell lymphoma. (b) H&E staining of tumor tissue. (c) Immunohistochemical staining of the same tumor in b with antibody against B220 (B cell marker). (d–f) Heptocellular carcinoma. (d) H&E staining of liver tissue containing heptocellular carcinoma. Note the upper portion showing normal tissue and the lower portion showing tumor. (e and f) High magnitude of normal and tumor tissue, respectively, in d.(g) Lung adenocarcinoma; H&E staining showing tumor (left portion) and normal (right portion) tissue of the lung. (Scale bar, 50 μm.)

Fig. 5.

Expression of beclin 1 protein in tumors from beclin 1^+/- mice. (a) Western blot analysis of cell extracts from tumors with anti-beclin 1 antibody or anti-Ran antibody (control). Lane 1, cell lysate from wt spleen; lanes 2–4, B cell lymphomas; lane 5, lung adenocarcinoma; lane 6, heptocellular carcinoma. (Lower) Ran protein level in each lane as loading control. (b) Section from B cell lymphoma stained with (Left) or without (Right) anti-beclin 1 antibody. (c) Section from lung adenocarcinoma stained with (Left) or without (Right) anti-beclin 1 antibody. (Scale bar, 20 μm.)

Fig. 6.

Disruption of beclin 1 causes autophagy deficiency. (a) Apoptotic cell death of wt or beclin 1^-/- ES cells induced by UV irradiation or serum withdrawal. Mean values ± SEM are depicted (n = 3). C1 and C2 represent two independent beclin 1^-/- ES clones. (b) Ultrastructural examination of ES cells after nutrient deprivation from beclin 1 wt (Left) and null mutant (Right) by electronic microscopy. Arrows indicate autophagy vacuoles. N, nucleus. (Scale bar, 1 μm.)