Interaction between Ras(V12) and scribbled clones induces tumour growth and invasion

Abstract

Human tumours have a large degree of cellular and genetic heterogeneity. Complex cell interactions in the tumour and its microenvironment are thought to have an important role in tumorigenesis and cancer progression. Furthermore, cooperation between oncogenic genetic lesions is required for tumour development; however, it is not known how cell interactions contribute to oncogenic cooperation. The genetic techniques available in the fruitfly Drosophila melanogaster allow analysis of the behaviour of cells with distinct mutations, making this the ideal model organism with which to study cell interactions and oncogenic cooperation. In Drosophila eye-antennal discs, cooperation between the oncogenic protein Ras(V12) (ref. 5) and loss-of-function mutations in the conserved tumour suppressor scribbled (scrib) gives rise to metastatic tumours that display many characteristics observed in human cancers. Here we show that clones of cells bearing different mutations can cooperate to promote tumour growth and invasion in Drosophila. We found that the Ras(V12) and scrib(-) mutations can also cause tumours when they affect different adjacent epithelial cells. We show that this interaction between Ras(V12) and scrib(-) clones involves JNK signalling propagation and JNK-induced upregulation of JAK/STAT-activating cytokines, a compensatory growth mechanism for tissue homeostasis. The development of Ras(V12) tumours can also be triggered by tissue damage, a stress condition that activates JNK signalling. Given the conservation of the pathways examined here, similar cooperative mechanisms could have a role in the development of human cancers.

Figure 1. Interclonal cooperation between Ras^V12 and scrib⁻ causes tumours

a-e, Clones of cells marked with GFP in the eye-antennal discs of third-instar larvae. Upper subpanels show the cephalic complex (CC), consisting of eye-antennal discs (EA), brain (B) and ventral nerve cord (VNC). Lower subpanels show the dissected VNC. Compared to wild-type clones (a), Ras^V12 clones overgrow moderately (b). scrib⁻ clones are eliminated from the tissue (c). Double mutant Ras^V12scrib⁻ clones (d, intraclonal cooperation), as well as Ras^V12 clones confronted with scrib⁻ clones (e, interclonal cooperation), cause tumours that overgrow and invade the VNC. f-i, Confocal sections of the inner tumour mass in Ras^V12scrib⁻ (f, g) and Ras^V12//scrib⁻ tumours (h, i) at day 6 and 14 after egg laying (AEL). Non-Ras^V12 cells (absence of GFP) are progressively eliminated from the tissue. Cell nuclei labeled with DAPI. Yellow arrowheads point to scattered remaining GFP-negative cells (insets in g and i).

Figure 2. Synergy between Ras and JAK/STAT signaling promotes growth and invasion in Ras^V12scrib⁻ and Ras^V12//scrib⁻ tumours

a, Quantification by real-time RT-PCR of expression of the upd genes, encoding the JAK/STAT-activating cytokines Upd, Upd2 and Upd3, in eye-antennal discs containing wild-type clones, Ras^V12-expressing clones, scrib⁻ clones (day 6 AEL), Ras^V12scrib− tumours and Ras^V12//scrib⁻ tumours (day 10 AEL). Expression is normalized to the housekeeping gene rp49. Error bars depict 95% confidence intervals (1.96 × s.e.m., n=3). b-e, Expression of the JAK/STAT reporter STAT-GFP (green) in day 6 AEL eye-antennal discs containing wild-type clones (b, red), Ras^V12 clones (c), Ras^V12scrib⁻ (d) and Ras^V12//scrib⁻ tumours (e). f, g, Suppression of Ras^V12scrib⁻ (f) and Ras^V12//scrib⁻ (g) tumours by expression of a dominant negative form of the JAK/STAT receptor Domeless (Dome^DN). h, Clones overexpressing Upd. i-l, Tumours caused by Ras^V12 clones co-overexpressing Upd (i), Upd2 (j), Upd3 (k) and both Upd and Upd2 (l). m, Ras^V12Upd tumours are suppressed by Dome^DN.

Figure 3. JNK signaling drives oncogenic cooperation upstream of JAK/STAT

a-c, STAT-GFP expression in wing discs of wild-type larvae (a) and scrib⁻ larvae heterozygous (b) or hemizygous (male, c) for the JNK-kinase loss-of-function mutation hep^r75. Overgrowth and STAT-GFP upregulation are suppressed by hep^r75. Arrowheads point to normal STAT-GFP expression in the wing hinge. Discs stained with phalloidin (red). d-g, Expression of a dominant negative form of the Jun-kinase Basket (Bsk^DN) suppresses Ras^V12scrib⁻ tumours (d, e), but not Ras^V12Upd tumours (f, g). h, i, Expression of Bsk^DN in Ras^V12 cells partially suppresses Ras^V12//scrib⁻ tumours. j, Quantification by real-time RT-PCR of expression of the upd genes in Ras^V12//scrib⁻ and Ras^V12Bsk^DN//scrib⁻ tumours (day 6 AEL). Error bars represent 95% confidence intervals (n=3). k, Propagation of JNK activity (puc-lacZ reporter, green) into the posterior (P) compartment (arrowhead) in wing discs wounded in the anterior (A) compartment (asterisk) 24 hours after wounding. l, puc-lacZ expression in discs expressing the JNK-phosphatase Puc under control of ptc-GAL4 (red cells, expressing RFP), wounded as in k. Puc is both a downstream target and a negative regulator of JNK. Propagation of puc-lacZ expression into the P compartment is not observed (hollow arrowhead).

Figure 4. Tissue damage, compensatory growth and a model for interclonal oncogenic cooperation

a-d, Cooperation between Ras^V12 and tissue damage. Right (wounded) and left (unwounded) wing discs of a wild-type larva (a, b) and a larva expressing Ras^V12 under control of nub-GAL4 (c, d). Discs were wounded by repeated pinching and dissected 48h later. Expression of RFP driven by nub-GAL4 marks the wing blade region (red). Cell nuclei stained with DAPI (blue). e-h, Requirement of JAK/STAT signaling in compensatory proliferation. Wild-type (e) and stat92E⁻ (g) clones in adult eyes, marked by absence of red pigment. In eyes containing scrib⁻ clones confronted with wild-type cells (f), scrib⁻ cells (red) are mostly absent and size of the eye is largely normal. In eyes containing scrib⁻ clones (red) confronted with stat92E⁻ cells (h), the size of the eye is reduced. i, Model for the involvement of JNK and JAK/STAT signaling in intraclonal and interclonal cooperation between Ras^V12 and scrib⁻. See text for details.