Aneuploidy-induced delaminating cells drive tumorigenesis in Drosophila epithelia

Abstract

Genomic instability has been observed in essentially all sporadic carcinomas. Here we use Drosophila epithelial cells to address the role of chromosomal instability in cancer development as they have proved useful for elucidating the molecular mechanisms underlying tumorigenic growth. We first show that chromosomal instability leads to an apoptotic response. Interestingly, this response is p53 independent, as opposed to mammalian cells, and depends on the activation of the c-Jun N-terminal kinase (JNK) signaling cascade. When prevented from undergoing programmed cell death (PCD), chromosomal instability induces neoplasic overgrowth. These tumor-like tissues are able to grow extensively and metastasize when transplanted into the abdomen of adult hosts. Detailed analysis of the tumors allows us to identify a delaminating cell population as the critical one in driving tumorigenesis. Cells loose their apical-basal polarity, mislocalize DE-cadherin, and delaminate from the main epithelium. A JNK-dependent transcriptional program is activated specifically in delaminating cells and drives nonautonomous tissue overgrowth, basement membrane degradation, and invasiveness. These findings unravel a general and rapid tumorigenic potential of genomic instability, as opposed to its proposed role as a source of mutability to select specific tumor-prone aneuploid cells, and open unique avenues toward the understanding of the role of genomic instability in human cancer.

Fig. 1.

Dp53-independent CIN-induced apoptosis in epithelial cells. (A and B) Loss of heterozygocity analyses of the Minute (A) and yellow (B) genes. Adult thorax with Minute thin bristles (A, red arrow) and magnification of an anterior adult wing margin with yellow bristles (B, red arrows) upon expression of asp^RNAi (A) or rod^RNAi (B) under the control of the ci-gal4 driver, and histogram plotting the frequencies of Minute (A) and yellow (B) bristles of flies expressing GFP^RNAi, asp^RNAi, rod^RNAi, bub3^RNAi, or orc2^RNAi under the control of the ci-gal4 driver. ci-gal4 drives expression in anterior (A) cells in the wing and in the whole adult thorax. Genotypes: (A) +/+; ci-gal4 (II)/+; UAS-dsRNA (III) and (B) yellow/+ (I); ci-gal4 (II)/+; UAS-dsRNA (III)/+. Only females were scored. (C–J) Larval wing primordia, adult wings, and eyes from individuals expressing GFP (C and D) and the indicated transgenes (D–J) under the control of the en-gal4 (wings) or ey-gal4 (eyes) drivers, and stained for GFP (green), DAPI (blue), and TUNEL staining (red). The wing primordium shown in I is also mutant for dp53. X-Y sections of the basal side of the wing epithelia are shown. H′ shows a magnification of the squared region in H. H′′ shows a cross-section of the posterior compartment. Note in H′′ that TUNEL positive cells are located on the basal side of the epithelium. Vertical line in E–J depicts the boundary between anterior (A) and posterior (P) cells. (K–O′) Larval wing primordia from individuals expressing the indicated transgenes and GFP under the control of the en-gal4 (K and M–O′) or ci-gal4 (L) drivers, and stained for GFP (green), DAPI (blue), and MMP1 (red in K–M) or TUNEL staining (red in N–O′). X-Y sections of the basal side of the wing epithelia are shown. O shows a magnification of the squared region in N. O′ shows a cross-section of the posterior compartment. Note in O′ that TUNEL negative cells are located in the basal side of the epithelium. en-gal4 drives expression in posterior (P) cells and is depicted in green in the adult wings. ci-gal4 drives expression in anterior (A) cells. ap, apical; bs, basal.

Fig. 2.

CIN-induced tumor growth and tissue invasiveness upon additional blockade of programmed cell death. (A–F and I–K) Larval wing primordia from wild-type individuals (A) or from individuals expressing the indicated transgenes and GFP in posterior (P) cells under the control of the en-gal4 (wings) driver and stained for GFP (green or white, A and F), P35 (B–E), DAPI (blue or white, A and I–K), and Ci (red, I and J) protein expression. Ci labels anterior (A) cells in I and J. Wing primordia shown in F and K are also mutant for dronc (F) or heterozygous for Df(H99) (K). (G) Histogram plotting the P/A size ratio of wing primordia expressing p35 and dsRNA for the indicated genes under the control of the en-gal4 driver. Error bars represent SEM. *P < 0.001. Larvae were 4 d old in A–G and 8 d old in I–K. (H) DNA content analysis by fluorescence associated cell sorter (FACS) of wing cells expressing the indicated transgenes under the control of the ap-gal4 driver. Percentage of cells with DNA content higher than 4n is indicated. (L) Adult fly micrographs taken 20 d after implantation (a.i.) of GFP-labeled larval wing tissue expressing the indicated transgenes. Ratios showing the reproducibility of the phenotype are shown. (M) Larval wing tissue 0 and 20 d a.i. (N–P) GFP (green) and P35 (blue) positive cells are observed inside the host ovary stained for DAPI (gray) and actin (red). O is a cross-section of N along the white line, and P is a magnification of N.

Fig. 3.

CIN-induced cell delamination and BM degradation. (A) Cartoon depicting the columnar epithelium (ce) of the wing primordium. (B–J) Cross-sections of the posterior compartment of wing primordia expressing the indicated transgenes under the control of the en-gal4 driver and stained for DAPI (blue), fasciclin III (red, B, C, and I), laminin-γ (labels the BM in green, B, C, and I), Dlg (red, D and E), aPKC (green, D and E), E-cad (red, F–H), and viking-GFP (collagen IV, J). ap, apical, and bs, basal. (C) Magnification of the squared regions shown in B. (D′–H′) X-Y sections of the apical and basal sides of the wing epithelia shown in D–H. Yellow arrows indicate delaminating cells. (K and K′) FACS sorted delaminated and nondelaminated cells from wing primordia expressing p35 and a dsRNA form for rod under the control of the en-gal4 driver were subject to DNA content profile analysis (K) and chromosome labeling with chromosome-specific probes (K′). In K, percentage of cells with DNA content higher than 4n is indicated. In K′, yellow arrows indicate aberrant number of chromosomes in delaminated cells. Ratios showing the number of nuclei presenting an aberrant number of chromosomes (more than two chromosomes 2 or 3 and loss of chromosome 4) are shown below each panel.

Fig. 4.

CIN-induced JNK activation in delaminating cells. (A and C) Wing primordia expressing the indicated transgenes in posterior (P) cells under the control of the en-gal4 driver, and stained for MMP1 (red, A), Wingless (red, C), and Ci (green) protein expression. Ci labels anterior (A) cells. (B, D, and E–G) Cross-sections of the posterior compartment of wing primordia expressing the indicated transgenes under the control of the en-gal4 driver and stained in red for MMP1 (B and E), Wingless (D and F), or E-cadherin (G), in green for laminin-γ (B, E, and F), and in blue for DAPI. Yellow arrows indicate delaminating cells. (H) Wing primordium with clones of cells mutant for Df(H99) and expressing a dsRNA form of asp and GFP (green) and stained in red for Wingless and in blue for DAPI. (Right) Cross-section of the clone expressing Wingless. Note in this clone expression of Wg in delaminating cells (yellow arrow). (I) Scheme showing the genomic region of the wingless gene, the location of the BRV118 enhancer, and the deletion in the wingless¹ mutation. (J and K) Wing primordia expressing the indicated transgenes in the posterior (P) compartment under the control of the en-gal4 driver and stained in red for Wg, in green for BRV118-lacZ expression (J) or Ci (K), and in blue for DAPI (K). In K, Ci labels the anterior (A) compartment. Wing primordium shown in K is also mutant for wingless¹. Asterisks represent endogenous Wg expression. ap, apical; bs, basal.

Fig. 5.

A central role of the delaminating cell population in inducing tumorigenesis. (A and C) Histograms plotting the P/A size ratio of wing primordia expressing, under the control of the en-gal4 driver, p35 alone (red bars), or p35 and dsRNA forms for asp, orc2, bub3, or rod (green and blue bars). Blue bars express also basket-DN (A) or a dsRNA form for wingless (C). Error bars represent SEM. *P < 0.001. (B and D) Adult fly micrographs taken 20 d after implantation (a.i.) of GFP-labeled larval wing tissue expressing the indicated transgenes. Ratios showing the reproducibility of the phenotype are shown. (E) Cartoon depicting CIN-induced tumorigenesis. CIN induces cell delamination of epithelial cells, and delaminating cells activate a JNK-dependent transcriptional response (dark blue nuclei) that leads to the expression of MMP1 (green) and Wingless (red) protein expression. MMP1 has an active role in degrading the basement membrane (purple), a critical step in tissue invasiveness, and Wingless contributes to the hyperplastic growth of the monolayered epithelium.