A tumor-suppressing mechanism in Drosophila involving cell competition and the Hippo pathway

Abstract

Mutant larvae for the Drosophila gene lethal giant larva (lgl) develop neoplastic tumors in imaginal discs. However, lgl mutant clones do not form tumors when surrounded by wild-type tissue, suggesting the existence of a tumor-suppressing mechanism. We have investigated the tumorigenic potential of lgl mutant cells by generating wing compartments that are entirely mutant for lgl and also inducing clones of various genetic combinations of lgl(-) cells. We find that lgl(-) compartments can grow indefinitely but lgl(-) clones are eliminated by cell competition. lgl mutant cells may form tumors if they acquire constitutive activity of the Ras pathway (lgl(-) UAS-ras(V12)), which confers proliferation advantage through inhibition of the Hippo pathway. Yet, the majority of lgl(-) UAS-ras(V12) clones are eliminated in spite of their high proliferation rate. The formation of a tumor requires in addition the formation of a microenvironment that allows mutant cells to evade cell competition.

Fig. 1.

Behavior of mutant lgl clones developing in a normal (lgl⁻/+) disc. (A and A′) lgl⁻ clones labeled green with GFP. Several clones in the wing pouch contain caspase activity (red). The A′ image shows the green channel to illustrate that the cells undergoing apoptosis are mutant for lgl. Note that clones in the hinge and the thorax do not show caspase activity. (B and B′) Several lgl clones (green) in the process of being eliminated. Caspase activity is in red. The dying cells are in the periphery of the top clone. (C–C′′) Dying clone (green) labeled with caspase (red) and puc-LacZ (blue). The latter is an indicator of JNK activity. (D–G) Results of an experiment in which lgl mutant cells are labeled by the loss of GFP (see main text). The twin control clones are labeled by 2x GFP. By 48 h after clone initiation the size of lgl and control twin clones is the same (D), but by 72 h (E) lgl⁻ clones are smaller and also contain cells in apoptosis, normally at the borders (red). (F) By 96 h most of the lgl⁻ clones have disappeared and only the control twins remain. (G) Comparison of the clone size between control and lgl⁻ clones 48 and 72 h after clone initiation. Note that up to 48 h, control and lgl⁻ clones are of the same size, but after 72 h, lgl⁻ clones are significantly smaller than controls. Error bars indicate SE. *P < 0.0001.

Fig. 2.

Proliferation rate of lgl⁻ ras^V12 cells and down-regulation of the Hpo pathway. (A and A′) Cell proliferation rate in lgl⁻ ras^V12 clones is higher than in surrounding tissue, as indicated by BrdU incorporation. In contrast, ras^V12 clones (B and B′) grow normally. (C and C′) Disc with a large tumor showing elevated levels of diap1 (red) in the tumor cells. (D and D′) Disc containing several small lgl⁻ ras^V12 clones. The disc is triply stained for GFP, Yki (red), and TOPRO (blue), the latter labeling cell nuclei. The panels in E–E′′′ are high magnifications of the clones in the Inset in D. Note in E′′ and E′′′ that Yki and TOPRO are coextensive in the clone cells. The small nuclear regions with very high TOPRO label (E′–E′′′) correspond to heterochromatin and Yki is not present there (E′′).

Fig. 3.

Behavior of lgl⁻ ras^V12 and scrib⁻ ras^V12 clones. (A, A′, B, and B′) Typical discs from larvae given a 7-min heat shock (HS) and fixed 72 (A) or 96 h (B) after HS. Note the small size of the lgl⁻ ras^V12 clones and also that one of them is almost entirely apoptotic (caspase in red) and will be eliminated. The disc in B also shows small clones. One of them contains apoptotic cells in the border (arrowhead). The larger clone at the Bottom (yellow arrowhead) is surrounded by a group of apoptotic cells, but in this case they do not belong to the clone. (C, C′, D, and D′) Discs from the 15-min HS experiment. (C and C′). The 72-h after-heat-shock (AHS) disc contains several lgl⁻ ras^V12 clones, some of which appear to be close to fuse. Nevertheless some of their cells are in apoptosis (arrowheads, caspase in red) and probably some clones, like the one in the Top, will be eliminated. (D and D′) 96-h AHS disc containing a large tumor covering the disc almost entirely. Even though the lgl⁻ ras^V12 tumor is overgrowing, some of its cells undergo apoptosis (arrowhead, caspase in red). There are also lgl⁺ cells close to the border of the tumor that are in apoptosis (yellow arrowhead). (E–E′′, F, and F′) Two discs containing several scrib⁻ ras^V12 clones in which numerous cells in the border are dying. In some cases (E′) the apoptotic cells form a near complete ring. In other cases (F, Top Left, arrows) the entire clone is in apoptosis.

Fig. 4.

Behavior of lgl⁻ UAS-Yki clones. (A and A′) Wing disc almost filled with lgl⁻ UAS-Yki clones. Note the areas of apoptosis (red) close to the borders of the tumors. (B and B′). High magnification of a region of the disc in A. Note (arrowheads) the incidence of apoptosis in lgl⁻ UAS-Yki cells located in the tumor borders or in clones that are isolated.

Fig. 5.

Merging of lgl⁻ ras^V12 clones in tumors. The expression of engrailed (en, in red) marks the anterior or posterior compartment origin of the clones. The Top row shows two discs from the 7-min HS experiment. The disc in A and A′ is the typical case fixed 72 h after clone initiation, in which there are very few clones and these are small. The disc in B and B′ was fixed after 96 h and represents the unusual case in which the disc develops a tumor after 7 min of HS. Note that the tumor contains cells of anterior and posterior origin and therefore must have been formed by at least two clones. The Bottom row shows typical cases from the 15-min HS experiment. The 72-h AHS disc (C and C′) contains several clones, some anterior and some posterior. Note the closeness of some clones, suggesting they are about to merge. The 96-h AHS disc (D and D′) has a large tumor that contains cells of anterior and posterior origin.