Integrins Cooperate With the EGFR/Ras Pathway to Preserve Epithelia Survival and Architecture in Development and Oncogenesis

Integrins Cooperate With the EGFR/ Ras Pathway to Preserve Epithelia Survival and Architecture in Development and Oncogenesis Andrea Valencia-Expósito 1 † , M. Jesús Gómez-Lamarca 1 , 2 † , Thomas J. Widmann 3 and María D. Martín-Bermudo 1 * 1 Centro Andaluz de Biología del Desarrollo CSIC-Universidad Pablo de Olavide, Sevilla, Spain, 2 Departamento de Biología Celular, Universidad de Sevilla, Sevilla, Spain, 3 GENYO, Granada, Spain Adhesion to the extracellular matrix (ECM) is required for normal epithelial cell survival. Disruption of this interaction leads to a specific type of apoptosis known as anoikis. Yet, there are physiological and pathological situations in which cells not connected to the ECM are protected from anoikis, such as during cell migration or metastasis. The main receptors transmitting signals from the ECM are members of the integrin family. However, although integrin-mediated cell-ECM anchorage has been long recognized as crucial for epithelial cell survival, the in vivo significance of this interaction remains to be weighed. In this work, we have used the Drosophila wing imaginal disc epithelium to analyze the importance of integrins as survival factors during epithelia morphogenesis. We show that reducing integrin expression in the wing disc induces caspase-dependent cell death and basal extrusion of the dead cells. In this case, anoikis is mediated by the activation of the JNK pathway, which in turn triggers expression of the proapoptotic protein Hid. In addition, our results strongly suggest that, during wing disc morphogenesis, the EGFR pathway protects cells undergoing cell shape changes upon ECM detachment from anoikis. Furthermore, we show that oncogenic activation of the EGFR/Ras pathway in integrin mutant cells rescues them from apoptosis while promoting their extrusion from the epithelium. Altogether, our results support the idea that integrins promote cell survival during normal tissue morphogenesis and prevent the extrusion of transformed cells. Keywords: integrins, survival, JNK, EGFR, oncogenesis INTRODUCTION To survive or die is a cellular decision controlled by environmental cues and physical stimuli. This life and death decision is deeply influenced by the extracellular matrix (ECM) (Meredith et al., 1993). When cells lose their normal interactions with the ECM, the cell cycle is arrested and a specific form of apoptosis, knownasanoikis,isinitiated(Frisch and Ruoslahti, 1997). During development, aberrant anoikis alters tissue architecture and function, compromising embryonic viability (Mole et al., 2021). Conversely, programmed anoikis contributes to physiological developmental processes and tissue renewal (Gilmore, 2005;Chiarugi and Giannoni, 2008). Anoikis is also an important surveillance mechanism, ensuring that any cell that loses its appropriate position within a tissue is targeted for death. For this reason, impaired anoikis contributes to the malignancy of many cancer cells, allowing them to survive without ECM Edited by: Claudia Tanja Mierke, Leipzig University, Germany Reviewed by: Susan LaFlamme, Albany Medical College, United States Dan Bergstralh, University of Rochester, United States *Correspondence: María D. Martín-Bermudo [email protected] † These authors have contributed equally to this work Specialty section: This article was submitted to Cell Adhesion and Migration, a section of the journal Frontiers in Cell and Developmental Biology Received: 09 March 2022 Accepted: 09 May 2022 Published: 13 June 2022 Citation: Valencia-Expósito A, Gómez-Lamarca MJ, Widmann TJ and Martín-Bermudo MDD (2022) Integrins Cooperate With the EGFR/Ras Pathway to Preserve Epithelia Survival and Architecture in Development and Oncogenesis. Front. Cell Dev. Biol. 10:892691. doi: 10.3389/fcell.2022.892691 Frontiers in Cell and Developmental Biology | www.frontiersin.org June 2022 | Volume 10 | Article 8926911 ORIGINAL RESEARCH published: 13 June 2022 doi: 10.3389/fcell.2022.892691 anchorage and facilitating their dispersion [reviewed in (Zhong and Rescorla, 2012)]. Unraveling the mechanisms regulating anoikis is therefore crucial to understand both normal morphogenesis and cancer progression. Although the concept of anchorage-dependent cell survival has been recognized for many years, it was in the early 1990’swhenitwas demonstrated that cells deprived of ECM attachment undergo anoikis (Meredith et al., 1993;Frisch and Francis, 1994). Anoikis can be considered a multistep process [reviewed in (Zhong and Rescorla, 2012)].Firstofall,lossofanchorageresultsininactivationof focal complex signalling molecules, including FAK and Src (Giancotti, 2000;Parsons, 2003). As a consequence, several prosurvival pathways such as PI3K/Akt and Raf/MEK/ERK are disrupted [reviewed in (Vachon, 2011)].Thesecondstepin anoikis is the simultaneous disassembly of focal adhesions and the destabilization of the cytoskeleton, which leads to the release of proapoptotic Bcl-2 proteins (Martin and Vuori, 2004). The final step is the activation of apoptotic kinases, including JNK, which results in an increase in the expression and phosphorylation of the pro-apoptotic BH3-only proteins BIM an BMF (Girnius and Davis, 2017). However,theroleofJNKinanoikisremainscontroversial,as, depending on the cell type, kinase isoform and stimulus, it can perform pro- or anti-apoptotic functions (Liu and Lin, 2005). Analysing JNK function in different cellular contexts shall help resolving its debated role in anoikis. The major transmitters of survival signals from the ECM are members of the integrin family (Frisch and Ruoslahti, 1997;Meredith et al., 1993;Vachon, 2011). Integrins are a widely expressed family of heterodimeric transmembrane glycoproteins, composed of an aand a βsubunit, that link the ECM to the actin cytoskeleton (Hynes, 1992). The vertebrate integrin family is comprised of 18 aand 8 β transmembrane subunits enabling about 24 different heterodimeric receptors for a diverse array of ECM proteins. However, only some integrins are capable of regulating cell viability and these include the β1integrinsubfamily(Stupack and Cheresh, 2002). The regulation of cell survival mediated by integrins implicates an increasing complexity of players depending on the tissue, cell type, specie and the cell differentiation state, emphasizing the need to study anoikis in a given tissue within its physiological context [reviewed in (Ignotz and Massague, 1987;Frisch and Screaton, 2001;Vachon, 2011)]. Nonetheless, the early embryonic lethality observed in some vertebrate models lacking β1 integrins has long precluded a direct evidence for a role of β1 integrins as survival factors in vivo (Stephens et al., 1995). The use of cell lineage-specific gene deletion approaches and transplantation experiments to circumvent early embryonic lethality has revealed that the role of integrin function in cell survival in vivo is multifaceted and sometimes contradictory. Thus, while depletion of β1 integrins from either the developing mouse lens, the epidermis or the endothelia results in apoptosis (DiPersio et al., 2000;Simirskii et al., 2007;Carlson et al., 2008), conditional deletion in the intestinal epithelium causes anoikis resistance, increased cell proliferation and defective differentiation (Jones et al., 2006). Furthermore, in some situations, cells are protected from anoikis, such as during cell migration or metastasis. In these cases, cells escape death using a variety of strategies, including hyperactivation of receptor tyrosine kinases (Guadamillas et al., 2011;Paolietal.,2013). Since the main components of the cell death machinery are evolutionary conserved,Drosophila, due to its unique genetic and cell biological advantages, has become an ideal model system to study apoptosis in the context of a developing organism. As in mammals, the caspase family of cysteine proteases is essential for the regulation of apoptosis also in Drosophila. These proteins are constitutively expressed as procaspases that have low but significant protease activity. To prevent unwanted consequences of basal procaspase activity, living cells express high levels of the Inhibitor of Apoptosis (IAP) proteins, Diap1 and Diap2, which act as E3- ubiquitin ligases and target activator and effector caspases for proteasome degradation [reviewed in (Hay, 2000)]. Upon an apoptotic stimulus, Diap1 antagonists, including hid,reaper (rpr), grim,sickle (skl), jafrac2,anddOmi/HtrA2 are transcriptionally activated and physically interact with Diap1 [reviewed in (Xu et al., 2009)]. This leads to the blockage of its caspase inhibitory function and the degradation of Diap1 by auto-inhibitory ubiquitinization. As a consequence, the caspase cascade is activated and the apoptosome is formed. First of all, initiator procaspases, including DRONC, the casp9 ortholog, DREDD and DCP- 2, are activated by auto-cleavage. Then, initiator caspases catalyse the activation of the effector pro-caspases drICE and DCP-1, and this in turn cleave their substrates, including cytoskeleton proteins and DNA metabolic proteins (Cooper et al., 2009). However, despite the conservation of the cell death response between flies and vertebrates, little is known about the process of anoikis in Drosophila. Using the primordium of the Drosophila wing, the wing imaginal disc, as model system, we have studied the implication of integrins in the regulation of epithelial cell survival in the context of a developing tissue. The formation of the wing starts during early embryonic development when about 30 cells are allocated to form the wing imaginal disc primordium. During larval life, disc cells divide and form an epithelial sac comprised of a folded columnar epithelium on one side and a squamous epithelium on the other (Garcia-Bellido and Merrian, 1971).Thematurediscwillgiverisetotheadultwing and notum. At the onset of metamorphosis, local cell shape changes promote the folding of the single-layered columnar epithelium into a bi-layered sheet in which epithelial cells face each other along their basal sides (Fristrom et al., 1993). Integrins perform two distinct functions in the wing disc, to hold the two layers together and to regulate the cell shape changes underlying folding (Brabant et al., 1996;Dominguez-Gimenez et al., 2007). However, despite the known role of integrins as survival factors in both benign and malignant epithelia [reviewed in (Gilcrease, 2007)], integrins have not yet been implicated in cell survival in wing disc epithelial cells. Here, we show that low integrin levels induces caspase-dependent wing disc cell death. This anoikis process is mediated by the activation of the JNK pathway, which in turn induces expression of the proapoptotic gene hid.Wealsofind that integrins cooperate with the EGFR pathway to maintain wing disc epithelial cell survival. Finally, our results demonstrate that an oncogenic version of Ras, Ras V12 , blocks anoikis in the wing disc and that elimination of integrins from transformed Ras V12 cells stimulates their basal extrusion from the wing disc epithelium. Our results unravel new roles for integrins in epithelial cells in vivo, promoting cell survival during tissue morphogenesis and preventing the extrusion of transformed cells. Frontiers in Cell and Developmental Biology | www.frontiersin.org June 2022 | Volume 10 | Article 8926912 Valencia-Expósito et al. Integrins: Essential Developmental Survival Factors MATERIALS AND METHODS Fly Strains The following stocks were used: UAS-Ras V12 (Lee et al., 1996); nub- Gal4 (Calleja et al., 1996); hid 5′F-GFP (Tanaka-Matakatsu et al., 2009); UAS-DER DN ,UAS-Diap1,UAS-puc,ap-Gal4,puc-LacZ,rpr- LacZ,andbrk-LacZ (Bloomington Drosophila Stock Center); mys RNAi ,UAS-diβ(Martin-Bermudo and Brown, 1999)and hid RNAi (Vienna Drosophila RNA-i Center). To generate mutant clonesinthewingdiscweusedtheFRT/FLPtechnique(Chou and Perrimon, 1992). Mutant clones were marked by the absence of GFP. The following mutant alleles and chromosomes were used: mys 11 [also known as mys XG43 (Bunch et al., 1992)] and e22c-Gal4 UAS- flipase (Duffy et al., 1998). The e22c-Gal4 driver is expressed in some posterior wing disc cells and was therefore used in combination with UAS- flpto generate large mys clones. Flies were raised at 25°C. Immunohistochemistry and Imaging Wing imaginal discs were stained using standard procedures and mounted in Vectashield (Vector Laboratories, Burlingame, CA, United States). The following primary antibodies were used: goat anti-GFP FICT (Abcam, 1:500), rabbit anti-caspase Dcp1 (Cell Signaling; 1:100), rabbit anti-pJNK (Promega, 1:200), mouse antiβGal (Promega, 1:1000), mouse anti-βPS (Developmental Studies Hybridoma Bank, DSHB, University of Iowa, United States, 1:50) and mouse anti-myc (Promega, 1:100). The secondary antibodies used were Alexa fluor488,(MolecularProbes TM ) and Cy3 and Cy5 (Jackson ImmunoReseach Laboratories, Inc.) at 1: 200. DNA was labelled using Hoechst (Molecular Probes, 1:1000). Confocal images were obtained using a Leica SP5-MP-AOBS, equipped with a Plan- Apochromat 40X oil objective (NA 1.4). Quantification of Fluorescence Intensity To quantify fluorescent intensity of the different markers, fluorescent signaling was measured from maximum projections of 30 Z-confocal sections taken with 1 μm of interval per wing disc and genotype. Quantifications were made in the wing pouch region, which was manually selected using the FIJI-Image J line selection tool. Microscope settings were maintained between imaging sessions in each experiment. The background value taken from cell-free region was subtracted from all data series. Measurements of whole fluorescence intensity were done dividing the mean of all included pixels intensity by the outlined cell area, using the FIJI-Image J measure tool. Student’s t tests were used for statistical comparisons of fluorescence intensity values. RESULTS Integrins are Required for the Survival of Wing Disc Epithelial Cells To deepen our understanding of the role of integrins as survival factors during epithelia morphogenesis, we tested whether removing integrins in Drosophila wing disc epithelial cells affected their survival. The Drosophila genome contains two integrin βsubunits, βPS and βν [reviewed in (Brown, 1993;Yee and Hynes, 1993). βPS, encoded by the gene myospheroid (mys), is the only βchain present in the wing imaginal disc (Brabant et al., 1996); Supplementary Figure S1]. First, we generated mosaic wing disc epithelia containing clones of cells homozygous for the null allele mys XG43 (from now on mys cells, white arrow in Figures 1A–B”). To detect apoptosis, we used an antibody to cleaved effector caspase Dcp-1 (Yu et al., 2002). In control wing imaginal discs, Dcp-1 activity is very low, being detected only occasionally in a few cells scattered throughout the disc (Figure 1A,n= 20, where n represents the number of wing discs analyzed). In contrast, all wing discs containing mysclonesshowed apoptotic cells within the clone area (Figure 1B,n=18).Second,we reduced integrin levels in larger areas of the disc using the nubbin (nub) Gal4 line to express a mys RNAi (nub>mys RNAi)inthewing pouch (inset in Figure 1C) region in the center of the disc that gives rise to the adult wing, indicated with a dotted white circle in all figures (Brand and Perrimon, 1993;Calleja et al., 1996). Immunostaining of control (n= 30) and experimental nub>mys RNAi wing imaginal discs (n= 36) with antibodies against the βPS protein and cleaved Dcp-1 showed that the expression of the mys specificRNAiinthewing pouch caused a strong reduction in βPS protein levels (Figures 1C,C’) and a robust increase in apoptosis (Figures 1C,C’’,E). Thus, integrin expression in wing disc epithelial cells prevent them from undergoing cell death. It is known that embryonic epithelial cells undergoing apoptosis are removed from the epithelium by extrusion rather than phagocytosis (Rosenblatt et al., 2001). Similarly, integrin-defective cells were extruded (Figures 1C–C’’) and accumulated between the basal surface of the disc epithelium and the ECM, as seen with an antibody against the ECM component Perlecan (Supplementary Figure S2). Integrins can mediate both signaling and adhesion [reviewed in (Adams and Watt, 1993)]. To address which integrin function is required to facilitate cell survival in the wing imaginal disc, we used a chimeric integrin (diβ) that lacks integrin-adhesive function but maintains its ability to signal (Martin-Bermudo and Brown, 1999;Dominguez-Gimenez et al., 2007). We had previously shown that expression of diβin the wing disc inhibited the basal localization of endogenous integrin [(Supplementary Figures S3B,B’;(Dominguez-Gimenez et al., 2007)]. Here, we found that it caused an increase in apoptosis (Supplementary Figures S3B,B”), strongly suggesting that integrin signaling is not sufficient to prevent cell death in detached wing disc cells. Altogether, these results propose that integrin-mediated adhesion to the ECM promotes cell survival in the wing disc epithelium by regulating caspase activity. To further support this hypothesis, we tested whether overexpression of one of the Drosophila IAP (inhibitor of apoptosis) proteins, Diap1, suppressed the cell death caused by expression of mys RNAi and found that it did (Figures 1D-D’’,E,n= 35). Integrins DownRegulate Caspase Activity by Inhibiting Hid Expression Previous experiments have shown that overexpression of hid or rpr induces downregulation of Diap1 levels and apoptosis in Drosophila wing imaginal discs (Milan et al., 1997;Bergantinos et al., 2010;Yoo et al., 2002). As Diap1 overexpression rescues cell death due to integrin downregulation, integrins could promote cell survival through the regulation of hid and/or rpr expression. To test this, we analysed hid and rpr expression in control and Frontiers in Cell and Developmental Biology | www.frontiersin.org June 2022 | Volume 10 | Article 8926913 Valencia-Expósito et al. Integrins: Essential Developmental Survival Factors nub>mys RNAi wing discs, using the hid 5′F-GFP [from now on hidGFP,(Tanaka-Matakatsu et al., 2009)] and rprLacZ (Nordstrom et al., 1996) transcriptional reporters. hidGFP levels in control discs (hidGFP;nubGal4) are hardly detectable, consistent with a low occurrence of cell death in this tissue (Tanaka-Matakatsu et al., 2009); Figures 2A-A’’’,E]. This expression was dramatically upregulated in nub >mys RNAi discs (n= 25, Figures 2B-B’’’). In contrast, the normal expression pattern of rprLacZ, confined to a small region of the notum and two stripes in the D-V and A-P boundaries [(Nordstrom et al., 1996), Supplementary Figures S4A,A’], was not altered in nub >mys RNAi discs (n= 28, Supplementary Figures S4B,B’). These results suggest that integrins block hid, but not rpr expression in normal wing disc epithelial cells to promote cell survival. Furthermore, consistent with the idea that Drosophila hid acts upstream of Diap1 (Wang et al., 1999;Goyal et al., 2000), we found that expression of Diap1 was not able to rescue the increase in hidGFP levels observed in mys RNAi cells (n= 24, Figures 2C–C’’’). To further confirm hid involvement in integrin loss of functiondependent cell death, we tested whether reducing hid levels could rescue apoptosis in nub >mys RNAi discs. We found that expression FIGURE 1 | Integrins promote cell survival in the wing imaginal disc. (A–D99 )Maximal projection of confocal views of wing imaginal discs from third-instar larvae stained with anti-βPS [green in (A–D), white in (A9–D9)], anti-Dcp1 [red in (A–D), white in (A99 –D99 )] and the nuclear marker Hoechst [DNA, blue in (A–D)]. (A) Control nubGal4 wing disc. (B) Wing disc carrying mys mutant clones (white arrow), marked by the absence of βPS (green). (C) Wing disc expressing GFP or a mys RNAi under the control of nubGal4,nub >GFP (inset) and nub >mys RNAi , respectively. (yz) Confocal yz section along the white dotted line shown in (C).(D) Wing disc coexpressing a mys RNAi and Diap-1 under the control of nubGal4,nub >mys RNAi ;Diap1.(E) Quantification of Dcp1 levels in wing discs of the designated genotypes. The dotted white circles indicate the wing pouch area in all figures. The statistical significance of differences was assessed with a t-test, ***, ** and * pvalues <0.001, <0.01, and <0.05, respectively. Scale bar in all panels, 30 μm. Frontiers in Cell and Developmental Biology | www.frontiersin.org June 2022 | Volume 10 | Article 8926914 Valencia-Expósito et al. Integrins: Essential Developmental Survival Factors of an RNAi against hid (hidGFP;nub >mys RNAi ;hid RNAi )resulted in a significant reduction of mys RNAi-induced apoptosis (n=20, Figures 2A-A’’’,B-B’’’,D-D’’’,E). These findings demonstrate that integrins promote cell survival in wing imaginal discs through the negative regulation of hid expression. Altering the levels of Decapentaplegic (Dpp), a Drosophila transforming growth factor βhomologue), in wing discs results in upregulation of brinker (brk), a transcriptional repressor that prevents apoptosis (Adachi-Yamada et al., 1999;Moreno et al., 2002). However, reducing integrin function does not seem to affect Dpp signalling, as the expression pattern of the brk reporter brkLacZ was not altered in nub >mys RNAi wing discs (n=26,Supplementary Figure S5B). Downregulation of Integrin Expression in Wing Discs Induces JNK Activation As mentioned in the introduction, the role of JNK in anoikis is controversial, as, depending on the cell type and cellular context, it FIGURE 2 | Integrins stimulate cell survival through inhibition of hid expression. (A–D-)Maximal projection of confocal views of third-instar wing imaginal discs stained with anti-βPS [blue in (A–D), white in (A9–D9)], anti-Dcp1 [red in (A–D), white in (A99 –D99 )] and anti-GFP [green in (A–D), white in (A-–D-)].(A–A-)Control nubGal4 wing disc carrying a reporter of hid expression, hidGFP,hidGFP; nubGal4.(B–B-)hidGFP wing disc expressing a mys RNAi under the control of nubGal4, hidGFP;nub >mys RNAi .(C–D-)hidGFP wing disc co-expressing a mys RNAi under the control of nubGal4 with either Diap1, hidGFP; nub >mys RNAi ;Diap1 (C–C-), or a hid RNAi, hidGFP;nub >mys RNAi ;hid RNAi (D–D-).(E) Quantification of Dcp1 levels in wing discs of the indicated genotypes. The statistical significance of differences was assessed with a t-test, ***, ** and * pvalues <0.001, <0.01, and <0.05, respectively. NS, non-statistically significant. Scale bar in all panels, 30 μm. Frontiers in Cell and Developmental Biology | www.frontiersin.org June 2022 | Volume 10 | Article 8926915 Valencia-Expósito et al. Integrins: Essential Developmental Survival Factors can be either essential (Frisch et al., 1996)ordispensable(Khwaja and Downward, 1997). In Drosophila wing discs, JNK activation triggers apoptosis in response to several stimuli, including distortion of positional information (Adachi-Yamada et al., 1999), TNF signalling (Igakietal.,2002;Moreno et al., 2002)orirradiation (Luo et al., 2007). To study the role of the JNK pathway in apoptosis induced by integrin loss of function, we analyzed JNK activation using an antibody against phosphorylated JNK (pJNK, Figure 3). Compared to controls (n=16,Figures 3A,A’,D), pJNK levels were upregulated in nub >mys RNAi discs (n=18,Figures 3B,B’,D), suggesting that integrins control JNK activity in the wing disc. This was further confirmed using a reporter that monitors the transcription of a target of the JNK pathway, the JNK inhibitor puckered (pucLacZ)(Adachi-Yamada et al., 1999). We found that while in wild type wing discs, pucLacZ expression was restricted to a small proximal region (Adachi-Yamada et al., 1999), Supplementary Figures S6A,A’), in nub >mys RNAi wing discs, pucLacZ levels were strongly upregulated in the pouch (n= 28,Supplementary Figures S6B,B’). In some contexts, JNK signalling can be activated as a consequence of apoptosis (Kuranaga et al., 2002). To test whether activation of JNK lies upstream or downstream of loss of integrin function, JNK activation was assessed in nub >mys RNAi ;Diap1 wing discs (Figure 3). We found that pJNK (n=20,Figures 3C,D)and pucLacZ (n= 25, Supplementary Figures S6C,C’) levels were still upregulated despite rescue of apoptosis (Figure 1D), suggesting that JNK activation is upstream of cell death due to integrin loss of function in wing disc cells. Stress induced cell death in the wing disc activates JNK upstream of caspase activator proteins, which, in turn, reinforce JNK activation in a positive feedback loop to amplify the apoptotic response (Shlevkov and Morata, 2012). Here, we found that, in fact, the increase in JNK activity of nub >mys RNAi ;Diap1 FIGURE 3 | Elimination of integrins activates the JNK pathway. (A–C9)Maximal projection of confocal views of wing imaginal discs from third-instar larvae stained with anti-pJNK [green in (A–C), white in (A9–C9)] and the nuclear marker Hoechst [DNA, blue in (A–C)]. (A,A9)Control nubGal4 wing disc. (B,B9)Wing disc expressing a mys RNAi under the control of nubGal4,nub >mys RNAi .(C,C9)Wing disc co-expressing RNAi against mys and Diap1 under the control of nubGal4,nub >mys RNAi ; Diap1.(D) Quantification of pJNK levels in wing discs of the indicated genotypes. The statistical significance of differences was assessed with a t-test, *** and ** p values <0.001 and <0.01, respectively. Scale bar in all panels, 30 μm. Frontiers in Cell and Developmental Biology | www.frontiersin.org June 2022 | Volume 10 | Article 8926916 Valencia-Expósito et al. Integrins: Essential Developmental Survival Factors discs was lower than the one observed in nub >mys RNAi discs (Figures 3B–D). This result suggests that the feedback loop between JNK activity and apoptosis observed in stress-induced apoptosis may also operate in integrin loss of function-dependent apoptosis. JNK Mediates Apoptosis due to Loss of Integrin Function in Wing Disc Cells To test whether the JNK pathway mediates cell death due to integrin loss of function in wing imaginal discs, we analyzed if overexpression of the JNK inhibitor puc suppressed the cell death caused by mys RNAi (nub >mys RNAi ;puc) and found that it did (Figures 4A–D,n= 24). The relationship between JNK signalling, pro-apoptotic proteins and caspase activation is context-dependent. Thus, the JNK pathway can act upstream or downstream of pro-apoptotic genes such as rpr or hid. For instance, experiments in Drosophila S2 cells suggested that Rpr could stimulate JNK activation through DIAP1 degradation (Kuranaga et al., 2002). In contrast, JNK signalling promotes hid expression (and apoptosis) in eye discs, and it is required for rpr-reporter induction in response to radiation in wing discs (Kuranaga et al., 2002;Moreno et al., 2002). In our system, inhibition of JNK signalling via puc overexpression was able to suppress hid upregulation in nub > mys RNAi wing discs to a large extent (n=17,Figures 4C-C’’’,E). Altogether, these results allow us to conclude that reduction of integrin function in wing discs leads to apoptosis by stimulating hid transcription through JNK activation. Integrins Cooperate With the EGFR Pathway to Promote Cell Survival The EGFR activates a network of signaling pathways promoting survival in many different cellular contexts [reviewed in (Henson FIGURE 4 | Integrins promote cell survival by inhibiting the JNK pathway. (A–C-)Maximal projection of confocal views of third-instar wing imaginal discs carrying the reporter hidGFP, stained with anti-βPS [blue in (A–C), white in (A9–C9)], anti-Dcp1 [red in (A–C), white in (A99 –C99 )] and anti-GFP [green in (A–C), white in (A-–C-)]. (A–A-)Control hidGFP;nubGal4 wing disc. (B–B-)hidGFP wing disc expressing a mys RNAi under the control of nubGal4,hidGFP;nub >mys RNAi .(C–C-)hidGFP wing disc co-expressing a mys RNAi and the negative regulator of the JNK pathway puc, under the control of nubGal4 hidGFP;nub >mys RNAi ;puc.(D,E) Quantification of Dcp1 (D) and hidGFP (E) levels in wing discs of the indicated genotypes. The statistical significance of differences was assessed with a t-test, *** and * pvalues < 0.001, <0.01, and <0.05, respectively. NS, non-statistically significant. Scale bar in all panels, 30 μm. Frontiers in Cell and Developmental Biology | www.frontiersin.org June 2022 | Volume 10 | Article 8926917 Valencia-Expósito et al. Integrins: Essential Developmental Survival Factors and Gibson, 2006)]. In addition, experiments mainly from cell culture have revealed synergistic cooperation between growth factors and integrins in cell survival (Reginato et al., 2003;Miranti and Brugge, 2002).For instance, EGFR promotes survival through regulation of the Ras/MAPK pathway and consequent inhibition of the proapoptotic gene hid in the eye disc (Bergmann et al., 1998;Kurada and White, 1998;Yu et al., 2002). Moreover, wing disc mutant clones for EGFR or for some of their dowmstream effectors, such as Ras, are smaller than their twin spot, suggesting a role for the EGFR pathway in cell survival (Diaz-Benjumea and Garcia-Bellido, 1990; Diaz-Benjumea and Hafen, 1994; Zecca and Struhl, 2002). To directly address a role for EGFR in cell survival in the wing disc, we expressed an EGFR dominant negative construct (UAS-DER DN ) under the control of the nub-Gal4 line and found it led to increase cell death in the pouch region (nub >DER DN ,n= 21, Figures 5A, A’,C,C’,E). However, in contrast to what happens in nub >mys RNAi wing discs (Figures 5B, B’,E), where cell death was found distributed all over the pouch, expression of DER DN restricted apoptosis to regions of high EGFR activity (Gabay et al., 1997), such as the wing margin and the presumptive vein territories (yellow arrow and asterisks in Figures 5A, A’,C,C, respectively). In addition, and contrary to what happens in the eye, EGFR did not seem to exert its pro-survival function through regulation of hid or rpr transcription, as hidGFP or rpr-lacZ expression were not altered in nub >DER DN wing discs (Figures 5A, A’,C,C”;Supplementary Figure S7). We thus conclude that the EGFR pathway has a limited role in the control of cell survival in the wing disc that is independent of the transcriptional regulation of the proapoptotic genes hid and rpr. Finally, we examined a possible cooperation between integrins and the EGFR pathway to mediate cell survival. We found that the simultaneous coexpression of mys RNAi and DER DN in wing discs (nub > mys RNAi ;DER DN ) enhanced the cell death phenotype caused by the expression of any of them on their own (Figures 5B–E), strongly suggesting that integrins and the EGFR pathway act in paralell to promote cell survival in wing imaginal discs. Oncogenic Ras Suppresses Anoikis in Wing Discs Suppression of anoikis after detachment of cancer cells from the ECM is a key step in tumor metastasis [reviewed in (Simpson et al., 2008)]. In fact, the ability of oncogenic Ras to suppress anoikis has long been considered critical to Ras transformation. However, recent evidence suggests that Ras and other oncogenes can induce both pro- and anti-apoptotic signals depending on the cell type and context [reviewed in (Cox and Der, 2003)]. In Drosophila, Ras overactivation renders cells refractory to stress- and irradiation-induced apoptosis (Bergmann et al., 1998; Kurada and White, 1998;Pinal et al., 2018). To test whether oncogenic Ras could protect cells from anoikis in the wing imaginal disc, we overexpressed an activated form of DRas, Ras V12 (Karim and Rubin, 1998) and found it substantially suppressed the cell death phenotype caused by integrin depletion (nub >mys RNAi ,ras V12 ;Figures 6A-A’’,6B–B’’, C–C’’,D,n= 18). These results demonstrate that oncogenic Ras is able to suppress anoikis in wing imaginal discs. In epithelial cell lines, oncogenic Ras confers resistance to anoikis partly by downregulating the expression of proapoptotic Bcl-2 members FIGURE 5 | Integrins and EGFR cooperate to promote cell survival. (A–D”)Maximal projection of confocal views of wing imaginal discs from thirdinstar larvae carrying the reporter hidGFP, stained with anti-Dcp1 [red in (A–D), white in (A9–D9)], anti-GFP [green in (A–D), white in (A’’–D’’)] and the nuclear marker Hoechst [DNA, blue in (A–D)]. (A–D’’)Control hidGFP; nubGal4 wing disc. (B–B’’)hidGFP wing disc expressing a mys RNAi under the control of nubGal4,hidGFP;nub >mys RNAi .(C–C’’)hidGFP wing disc expressing a dominant negative form of the EGFR, DER DN , under the control of nubGal4,hidGFP;nub >DER DN .(C–D’’)hidGFP wing disc co-expressing a mys RNAi and DER DN under the control of nubGal4 hidGFP;nub >mys RNAi ; DER DN .(E) Quantification of Dcp1 levels in wing discs of the indicated genotypes. The statistical significance of differences was assessed with a t-test, *** and ** pvalues <0.001 and <0.01, respectively. NS, non-statistically significant. Scale bar in all panels, 30 μm. Frontiers in Cell and Developmental Biology | www.frontiersin.org June 2022 | Volume 10 | Article 8926918 Valencia-Expósito et al. Integrins: Essential Developmental Survival Factors (Rak et al., 1995) and by preventing downregulation of antiapoptotic proteins (Rosen et al., 2000). In Drosophila,it appears that the Ras pathway regulates Hid activity at the level of both, protein phosphorylation and gene expression, in hidinduced apoptosis (Bergmann et al., 1998;Kurada and White, 1998). However, we found that, in the Drosophila wing disc epithelium, the ability of oncogenic Ras to confer resistance to anoikis does not involve transcriptional regulation of hid,as hidGFP levels were not altered in nub >mys RNAi ; Ras V12 (n= 21, Figures 6A’’’,C’’’) discs compared to nub >mys RNAi (n= 20, Figure 6B’’’). Ectopic expression of Ras V12 in the dorsal compartment of wing imaginal discs, by means of the apterous Gal4 (ap) Gal4 line, causes tissue overgrowth and cell shape changes, which results in the formation of ectopic folds [ap >Ras V12 ,n= 20, Figures 7A,A’,C,C’,(Prober and Edgar, 2000;Soler Beatty et al., 2021). Reducing integrin function also alters cell shape causing a mild folding phenotype (Dominguez-Gimenez et al., 2007), Figures 7B, B’E, E’]. Here, we found that co-expression of mys RNAi and Ras V12 enhanced the cell shape and folding phenotype caused by the sole expression of any of them (ap >mys RNAi ; Ras V12 ,n= 20, Figures 7D,D’). In this work, we show that reducing integrin expression ensued basal extrusion of the dead cells (Figures 1C–C’’, Figures 7F,G). However, even though oncogenic K-Ras MCDK cells survive and extrude basally, a mechanism proposed to initiate invasion into surrounding tissues (Slattum et al., 2014), we found that expression of Ras V12 on its own in wing disc cells (ap >Ras V12 ) did not result in cell extrusion (Figure 7H). In striking contrast, the expression of Ras V12 in FIGURE 6 | Ectopic activation of the Ras pathway rescues anoikis. (A–C’’’)Maximal projection of confocal views of third-instar wing imaginal discs carrying the reporter hidGFP, stained with anti-βPS [blue in (A–C), white in (A9–C9)], anti-Dcp1 [red in (A–C), white in (A’’–C’’)] and anti-GFP [green in (A–C), white in (A-–C-)]. (A–A-)Control hidGFP;nubGal4 wing disc. (B–B-)hidGFP wing disc expressing a mys RNAi under the control of nubGal4,hidGFP;nub >mys RNAi .(C–C-)hidGFP wing disc co-expressing a mys RNAi and an activated form of Ras, Ras V12 , under the control of nubGal4 hidGFP;nub >mys RNAi ; Ras V12 .(D) Quantification of Dcp1 levels in wing discs of the indicated genotypes. The statistical significance of differences was assessed with a t-test, *** and * pvalues <0.001 and <0.05, respectively. Scale bar in all panels, 30 μm. Frontiers in Cell and Developmental Biology | www.frontiersin.org June 2022 | Volume 10 | Article 8926919 Valencia-Expósito et al. Integrins: Essential Developmental Survival Factors