Chitinase-like proteins promoting tumorigenesis through disruption of cell polarity via enlarged endosomal vesicles

Introduction: Chitinase-like proteins (CLPs) are associated with tissue-remodeling and inflammation but also with several disorders, including fibrosis, atherosclerosis, allergies, and cancer. However, CLP’s role in tumors is far from clear.

Methods: Here, we utilize Drosophila melanogaster and molecular genetics to investigate the function of CLPs (imaginal disc growth factors; Idgf’s) in Ras^V12 dysplastic salivary glands.

Results and discussion: We find one of the Idgf’s members, Idgf3, is transcriptionally induced in a JNK-dependent manner via a positive feedback loop mediated by reactive oxygen species (ROS). Moreover, Idgf3 accumulates in enlarged endosomal vesicles (EnVs) that promote tumor progression by disrupting cytoskeletal organization. The process is mediated via the downstream component, aSpectrin, which localizes to the EnVs. Our data provide new insight into CLP function in tumors and identifies specific targets for tumor control.

1 Introduction

Chitinase-like protein (CLPs), including human YKL-39 and YKL-40 are synthesized and secreted under various conditions, including tissue injury, inflammatory and regenerative responses. Under pathological conditions they may contribute to asthma, sepsis, fibrosis and tumor progression (1, 2) including ductal tumors, such as the lung, breast, and pancreas (3, 4). CLPs are regulated by growth factors, cytokines, stress and the extracellular matrix (ECM). However, the causal connection between CLPs’ function and disease progression is only partially elucidated (5).

Animal models have been increasingly used in molecular oncology. This includes the fruitfly Drosophila melanogaster, where overexpression of dominant-active Ras (Ras^V12) in proliferating tissue leads to benign tumors and simultaneous reduction of cell polarity genes to progression towards an invasive stage. (6–9). Central to this switch towards increasing malignancy is the C-Jun N-terminal kinase (JNK)-signaling pathway, which becomes activated via loss of cell polarity and promotes tumor growth (10). However, the outcome of activated JNK is mediated in a context-dependent manner due to downstream effects several of which are yet to be elucidated (11, 12). Among potential JNK regulators, spectrin family members belong to cytoskeletal proteins which form a spectrin-based membrane skeleton (SBMS) (13). Through the Rac family of small GTPases, cell polarity and SBMS organization are maintained (14, 15). Although the exact relationship between Spectrin and JNK in tumors remains to be established, Rac1 under physiological conditions cooperates with JNK in tissue growth (16–18).

To explore CLPs’ tissue autonomous function in a ductal tumor, we utilize the Drosophila melanogaster salivary glands (SGs). Generally, Drosophila CLPs are endogenously expressed in the larvae and include six members, termed Idgf 1-6 (Imaginal disc growth factors), that are involved in development, establishment of the cuticle, wound healing and restoration of cell organization (19–23). The SGs’ epithelial luminal organization and the conserved activation of the tumor-promoting signaling factors make them suitable for dissecting CLP function. Moreover, the lumen separating a single layer of cells can be disrupted by constitutive active Drosophila Ras (Ras^V12) (24) leading to the loss of ECM integrity, the formation of fibrotic lesions and of the loss of secretory activity (25).

Here we investigated the role of Drosophila Idgf’s in Ras^V12-expressing SGs. We show that one of the CLP’s members, Idgf3, is induced in tumor glands, leading to a partial loss of epithelial polarity and promoting a reduction of lumen size. The mechanism is driven through JNK signaling upstream of Idgf3. In line with previous work, ROS production via JNK mediates induction of Idgf3, creating a tumor-promoting signaling loop. Idgf3 further promotes the formation of enlarged endosomal vesicles (EnVs) via αSpectrin. Inhibiting EnVs formation by individually knocking-down Idgf3 and αSpectrin, restores cell organization. Similar effects are observed upon expression of human CLP members in Ras^V12 SGs. Thus, our work identifies a phylogenetically conserved contribution of tumor-induced CLP’s towards the dysplasia of ductal organs and supports a role for spectrins as tumor modifiers.

2 Materials and methods

2.1 Drosophila maintenance and larvae staining

Stocks were reared on standard potato meal supplemented with propionic acid and nipagin in a 25°C room with a 12 h light/dark cycle. Female virgins were collected for five days and crossed to the respective males (see supplementary cross-list) after two days. Eggs were collected for six hours and further incubated for 18 h at 29°C. 24 h after egg deposition (AED), larvae were transferred to a vial containing 3 mL food supplemented with antibiotics (see Supplementary Table S1). 96 h and 120 h after egg deposition (AED), larvae were washed out with tap water before being dissected.

2.2 Sample preparation and immunohistochemistry

SGs were dissected in 1 x phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA) for 20 min. For extracellular protein staining, the samples were washed three times for 10 min in PBS and with PBST (1% TritonX-100) for intracellular proteins. Subsequently, samples stained for H2 were blocked with 0.1% bovine serum albumin (BSA) in PBS, and SG stained for pJNK, Idgf3, Spectrin, Dlg, p62 (ref(2)P), and GFP were blocked with 5% BSA for 20 min. After that, samples were incubated with the respective primary antibodies. Anti-pJNK (1:250), anti-Idgf3 (0.0134 µg/ml), anti-Spectrin (0.135 µg/ml) diluted in PBST were incubated overnight 4°C. anti-GFP (1 µg/ml) in PBST, H2 (1:5), and anti-SPARC (1:3000) in PBS were incubated for one hour at room temperature (RT). Samples were washed three times with PBS or PBST for 10 min and incubated with secondary antibody anti-mouse (4 µg/ml, Thermofisher #A11030) or anti-rabbit (4 µg/ml, Thermofisher #A21069) for one hour at RT. Subsequently, samples were washed three times in PBS or PBST for 10 min and mounted in FluoromountG.

2.3 Salivary gland size imaging and analysis

SG samples were imaged with Axioscope II (Objective 4x) (Zeiss, Germany) using AxioVision LE (Version 4.8.2.0). The images were exported as TIF and analyzed in FIJI (ImageJ: Version 1.53j). Representative confocal pictures were selected for figure panels and the complete set of replicate figures processed further for quantification (see below). Region of Interest (ROI) were drawn with the Polygon selection tool, and the scale was set to pixels (Px). The SG area was summarized as a boxplot with whisker length min to max. The bar represents the median. Statistical analysis was done with Prism software (GraphPad Software, 9.1.2, USA), the population was analyzed for normality with D’Agostino-Pearson and p-value quantified with Student’s t-test.

2.4 Nuclear volume imaging and quantification

Nuclei were stained with DAPI (1 µg/ml, Sigma-Aldrich D9542) in PBST for 1 h at RT. Mounted glands were imaged with Zeiss LSM780 (Zeiss, Germany) using a plan-apochromat 10x/0.45 objective with a pixel dwell 3.15 µs and 27 µm pinhole in z-stack and tile scan mode. Zeiss images were imported into ImageJ and viewed in Hyperstack. The selection threshold was set individually for each sample, and the analysis was performed with 3D objects counter. The nuclei volume was presented in boxplot, whisker length min to max and bar represent median. P-value quantified with Student’s t-test and the scale bar represent µm³.

2.5 Intensity and hemocyte quantification

The images for quantifying pJNK, TRE, Idgf3, and SPARC intensity and hemocyte recruitment were captured with AxioscopeII (Objective 4x) (Zeiss, Germany). The images were exported as TIF and analyzed in ImageJ. ROI was drawn with the Polygon selection tool, and subsequently, the total intensity was measured (pixel scale). The intensity was quantified according to the equation: Integrated Density – (SG area*Mean gray value). Hemocyte area was selected with Threshold Color and quantified by using the following equation: Ln (Hemocyte area + 1)/Ln (SG size + 1). Representative images were taken with Zeiss LSM780 (Zeiss, Germany). The images were then processed using Affinity Designer (Serif, United Kingdom). Graphs and statistical analysis were generated with Prism software (GraphPad Software, 9.1.2, USA). The population was analyzed for normality with D’Agostino-Pearson. Statistical significance was determined with Student’s t-test, One-way ANOVA with Tukey’s multiple comparison, and two-way ANOVA with Dunnett’s multiple comparison.

2.6 Enlarged endosomal vesicles penetrance quantification

The penetrance of the enlarged vesicles was subjectively quantified based on positive actin staining. Samples were analyzed in Axioscope II (Objective 20x) (Zeiss, Germany). At least 15 samples were analyzed with three independent replicates.

2.7 Humanized transgenic Drosophila lines

Plasmids were generated and transformed at VectorBuilder (https://en.vectorbuilder.com/). Human CH3L1 and CH3L2 genes were inserted into Drosophila Gene Expression Vector pUASTattB vector generating VB200527-1248haw and VB200518-1121xyy, respectively and transformed into E. coli. The bacteria were cultured in 3 ml LB supplemented with ampicillin (AMP: 100 ug/ml) for 15 h, at 37°C. The plasmid was extracted according to the GeneJetTm Plasmid Miniprep Kit #K0503 standard procedure. Plasmids were validated through sequencing at Eurofins (https://www.eurofins.se/: For primer details see Supplementary Table S1). Drosophila transgenic lines were generated at thebestgene (https://www.thebestgene.com/). Plasmids were extracted with QIAGEN Plasmid Maxi Kit according to the standard procedure and injected into w¹¹¹⁸ strains. Expression of the human CLPs was validated with qPCR.

2.8 In situ hybridization

The Idgf3 (GH07453: DGRC) probe was generated according to (26). The staining procedure is described elsewhere with the following changes (26). The procedure was conducted in 200 µl transwells containing four salivary glands. The procedure included three technical replicates per genotype. Images were aqured with Leica MZ16 (Leica, Germany) microscope and Leica DFC300x FX digital color camera (Leica, Germany). Representative images were taken, and figures were generated in Affinity Designer (Serif, United Kingdom).

2.9 qPCR

mRNA isolation and cDNA synthesis were performed according to manufacture instructions (AM1931). qPCR procedures were performed as described earlier (24) with an adjusted Kappa concentration to 0.5x. At least three replicates and two technical replicates were performed for each qPCR. See Supplementary Table S1 for primer list.

3 Results

3.1 Idgf3 promotes a dysplastic phenotype

Obstruction of SG lumen by the constitutive-active oncogene, Ras^V12, under Beadex-Gal4 driver (Ras^V12) disrupts organ function between 96 h and 120 h after egg deposition (AED) (25). Being that CLPs have been implicated in the loss of cell polarity (27), we investigated whether Drosophila CLPs contribute to the observed phenotype. First, to find out whether CLPs were induced in the Ras^V12 glands, we assessed relative mRNA levels at two different time points, 96 h and 120 h AED. Only one of the CLP members, namely Idgf3, was significantly upregulated at both time points (Figures 1A, S1A). Therefore, we decided to focus on Idgf3’s effects on dysplastic glands.

FIGURE 1

Figure 1 Idgf3 promotes growth and disrupts tissue architecture (A) qPCR data showing induction of Idgf3 in 120 h AED Ras^V12 glands. (B, C) Idgf3 tagged with GFP was localized in the dysplastic glands. (D-G) Knock-down of Idgf3 in Ras^V12 glands confirmed reduced mRNA levels as shown by in situ hybridization. (H) SG size quantification showing a reduction in tissue size in Idgf3^KD;Ras^V12 SG compared to Ras^V12 alone. (I-L) F-actin (Phalloidin) staining revealed partial restoration of the lumen in Idgf3^KD;Ras^V12 glands, in comparison to Ras^V12 alone. (M-P) Nuclei in DAPI stained SG displayed a reduced size in Idgf3^KD;Ras^V12; quantified in (Q). Scale bars in (B, C) represent 100 µm, (D-G) represent 0.3 mm and (I-L) represent 20 µm (M-P) represents 10 µm. Data in (A) represent 3 independent replicates summarized as mean ± SD. Boxplot in (H, Q) represent at least 20 SG pairs. Whisker length min to max, bar represent median. P-value quantified with Student’s t-test.

Idgf3 contains an N-terminal signal peptide and has been detected in hemolymph (28). To analyze its subcellular tissue distribution in SGs, we used a C-terminally GFP tagged version of Idgf3 (21). At first we used in situ hybridization to show distribution of Idgf3 in salivary glands (Figures S1B-E). At 96 h we could not detect Idgf3 in the whole WT or Ras^V12 animals (Figures S1F-G’), possibly due to limited sensitivity. Likewise, 120 h old WT larvae did not show any detectable signal (Figure S1H-H’) while a strong Idgf3 signal was detected in Ras^V12 SGs (Figure S1I-I’). To better understand Idgf3 distribution at a higher resolution, we dissected 120 h AED glands. WT glands had a weaker Idgf3::GFP signal in comparison to the Ras^V12 (Figures 1B, C). Moreover, Idgf3 was unevenly distributed throughout Ras^V12 SGs (Figure 1C).

The increased level of Idgf3 between 96 h and 120 h strongly correlated with loss of tissue- and cell-organization and an increased nuclear volume (24). In order to characterize the role of Idgf3 in Ras^V12 glands, we used a specific Idgf3 RNA-interference line (Idgf^KD). Moreover, we focused on 120 h larvae, unless otherwise stated, since they showed the most robust and developed dysplastic phenotype. Efficient knockdown of Idgf3 was confirmed using in situ hybridization and at the protein level (Figures 1D-G, S1J-M; quantified in N, (21)). Macroscopic inspection showed that Idgf^KD;Ras^V12 SGs were smaller than Ras^V12 SGs (Figure 1H), resembling WT controls. To gain insight into the cellular organization, we stained the glands for F-actin (Phalloidin: Ph) and DNA using DAPI. In Idgf^KD the cells retained their cuboidal structure, and the lumen was visible as in WT, indicating that Idgf3 on its own does not affect apicobasal polarity (Figures 1I, J). In contrast, in Ras^V12 glands apicobasal polarity was lost, and the lumen was absent (Figures 1K, (25)). In Idgf^KD;Ras^V12 SGs a reversal to the normal distribution of F-actin and partial restoration of the lumen was observed (Figure 1L). Similarly, the nuclear volume, which increased in Ras^V12 SGs returned to near wild type levels upon Idgf^KD (Figure 1M-P, quantified in Figure 1Q). This indicates that Idgf^KD can rescue Ras^V12-induced dysplasia.

In order to unravel the specific effects mediated by Idgf3 we further investigated Ras^V12 associated phenotypes, including fibrosis and the cellular immune response. As recently reported, Ras^V12 SGs displayed increased levels of the extracellular matrix components (ECM), including collagen IV and SPARC (BM40, (25)). Idgf^KD did not affect SPARC levels in comparison to the WT (Figures S1O-P) but Idgf-KD;Ras^V12 SGs displayed significantly reduced SPARC levels in comparison to Ras^V12 (Figures S1Q-R, quantified in S). To assess whether this led to a reduced inflammatory response, we investigated the recruitment of plasmatocytes, macrophage-like cells previously reported to be recruited towards tumors (9). We found that both control and Idgf^KD glands did not show recruitment of hemocytes (Figures S1T-U). In contrast to the effects on ECM components, Idgf^KD in Ras^V12 glands did not lead to any changes in hemocyte attachment (Figures S1V-W, quantified in X). Taken together, upon Ras^V12 overexpression, Idgf3 promotes SG overgrowth, loss of cell organization, and fibrotic-like accumulation of the ECM, but not immune cell recruitment.

3.2 Idgf3 induces dysplasia via JNK-signaling

Dysplasia is driven by internal and external factors that either work in concert or independently. Similar to what we observed in Idgf^KD;Ras^V12 glands blocking the sole Drosophila JNK member basket reverts many tumor phenotypes (24). Moreover, the dysplastic loss of apical and basolateral polarity between 96 h and 120 h is driven by the JNK-pathway (24). The time frame when we observed upregulation of Idgf3 (Figures 1A, S1A) coincides with the period during which blocking JNK restores tissue organization and homeostasis, similar to what occurs in Idgf^KD;Ras^V12 tissues (Figures 1L, S1R). Therefore, we decided to test a possible involvement of JNK-signaling in the regulation of Idgf3.

First, we performed a targeted JNK RNAi-screen using Idgf3::GFP intensity in the glands as readout upon KD of JNK signaling components. We first confirmed the sensitivity of the Idgf3::GFP construct by Idgf3-KD in Ras^V12 SGs compared to Ras^V12 glands (Figures S2A, B, quantified in Figure S2C). Knockdown of the two classical TNF receptors upstream of JNK, Grnd (Grindelwald) and Wgn (Wengen) (Figure S2D) similarly reduced Idgf3::GFP intensity (Figures 2A–C, quantified in Figure 2E (29). Similar effects were observed with Bsk^KD (Figure 2D, quantified in E). Altogether this suggests that Idgf3 protein levels are regulated downstream of JNK and the TNF members Grnd and Wgn.

FIGURE 2

Figure 2 Idgf3 dysplasia is mediated through JNK activity (A-D) Representative images of Idgf3::GFP in a JNK targeted screen. (E) Quantification showing Idgf3::GFP intensity was reduced by Grnd^KD, Wgn^KD and Bsk^KD in Ras^V12 SG. Scale bars in (A-D) represent 100 µm. Boxplot in (E) represents at least 20 SG pairs. Whisker length min to max, bar represent median. P-value quantified with Student’s t-test.

3.3 ROS promotes Idgf3 induction via JNK

To further dissect Idgf3 regulation, we focused on the positive JNK regulators, reactive oxygen species (ROS) both intra- and extracellularly (9, 30). We previously reported that ROS production in Ras^V12 SGs increases via JNK (24). To inhibit ROS intra- and extracellularly, we separately overexpressed the H₂O₂ scavengers Catalase (Cat) and a secreted form of Catalase, IRC (immune-regulated Catalase), and ${\text{O}}_2^{-}$ scavenger SOD (Superoxide dismutase A), in the Ras^V12 background and quantified Idgf3::GFP intensity. Reducing levels of intracellular H₂O₂ (Cat^OE), but not ${\text{O}}_2^{-}$(SOD^OE) lowered Idgf3::GFP intensity (Figures S3A-D quantified in E). Similarly, reduction of extracellular H₂O₂ by the secreted version of Catalase (Irc^OE) lowered Idgf3::GFP levels (Figures 3A-D, quantified in Figure 3E) as well as JNK signaling (Figures 3J, K-N’, quantified in Figure 3O). We used detection of pJNK and TRE-GFP1b reporter construct, which recapitulates JNK-activation by expressing GFP under control of biding sites for JNK-specific AP-1 transcription factors (31). Confirming JNK-activation, three known JNK targets (puckered, puc, a negative feedback regulator of JNK; metalloproteinase 1, MMP1 and head involution defective, hid (32–34)) as well as Idgf3 itself showed the same dependence on Irc^OE. In line with the reduced tissue size and improved tissue integrity in Idgf3^KD;Ras^V12, overexpression of IRC in Ras^V12 SGs also reduced SG size (Figure 3T), improved tissue integrity and restored the SG lumen (Figure 3F-I, P-S).

FIGURE 3

Figure 3 Antioxidants overexpression reduce Idgf3 and JNK signaling activity (A-D) Reduction of H₂O₂ by overexpression of secreted catalase (immune regulated catalase; IRC) lowered Idgf3::GFP levels, quantified in (E). (F-I) ISH showing reduced expression of Idgf3 in IRC-OE;Ras^V12glands. (J) qPCR data showing reduction of Idgf3, puc, MMP1 and Hid in IRC^OE;Ras^V12glands. Selection of JNK targets with the most significant induction was partially based on our previous experiments ((24), Figure, S3A). (K-N’) pJNK staining and TRE reporter constructs showing reduced intensity in IRC^OE;Ras^V12 in comparison to Ras^V12 glands, quantified in (O). (P-S) Phalloidin staining showing partially restored lumen in IRC^OE;Ras^V12 glands, quantified in (T). Scale bars in (A-D, K-S) represent 100 µm and (F-I) represent 0.3 mm. Data in (J) represent 3 independent replicates summarized as mean ± SD. Boxplot in (E, O, T) represent at least 20 SG pairs. Whisker length min to max, bar represent median. P-value quantified with Student’s t-test.

In summary, ROSs contribute to pJNK signaling. In addition, overexpression of extracellular and intracellular Catalase but not SOD reduces Idgf3 induction via JNK, similar to the feedback loop that has been identified in other tumor models (9).

3.4 Idgf3 accumulates in large vesicles, which display markers for endocytosis and macropinocytosis

We previously noted the uneven distribution of Idgf3 in Ras^V12 SGs (Figure 1C). To further understand how Idgf3 promotes dysplasia, we dissected its subcellular localization (Figure 4A). We stained the glands for F-actin (Phalloidin) and addressed Idgf3::GFP localization at high resolution (Figures 4B, C’). Interestingly, we observed Idgf3::GFP clusters surrounded by F-actin (Figures 4C-C’: arrow). Using a different salivary gland driver (AB-Gal4) to drive expression of Ras^V12, we also observed increased expression of Idgf3::GFP and its localization within vesicular structures (Figure S4A-B’: arrow). The size of the vesicle-like structures was between 10-43 µm in comparison to secretory Drosophila vesicles (3-8µm, Figure 4D) (35). We refer to these as enlarged vesicles (EnVs). Based on the increased Idgf3 levels, we wondered whether the protein was aggregating in EnVs. The aggregation marker p62, which is autophagic adaptor marking cytoplasmic protein aggregates prepared for clearance (36) was strongly bound to the cytoplasm of Ras^V12 SGs unlike from WT glands. However, the EnVs did not contain any aggregated proteins (Figure S4C-D’). This may imply that Idgf3 is even taken up from the SG lumen in a soluble state.

FIGURE 4

Figure 4 Idgf3 promotes formation of enlarged endosomes in the Ras^V12 background (A) The overall figure shows Ras^V12 salivary gland with EnVs marked by red color. The inlet depict Idgf3 enclosed by enlarged vesicles (EnVs) coated by cytoskeletal and cell polarity proteins. (B-C’) Idgf3::GFP clusters coated with Phalloidin. (D) Vesicle size quantification showing Ras^V12 enlarged vesicles in comparison to prepupae SG vesicles. (E-J’) Non secreted MFGE8 localizes to the surface of EnVs, co-stained with phalloidin. The secreted MFGE8 is packaged into EnVs in Ras^V12glands. (K-M) ISH showing no induction of Idgf3 by constitutively active Pak. (K’-M’’) Idgf3::GFP is not detectable in the Pak^CA glands and the lumen is detectable (Phalloidin). (N-R’’) Restoration of the lumen packed with Idgf3::GFP, no formation of EnVs and Idgf3 in Pak^KD;Ras^V12 glands. Scale bars in (B-C’, E-J’, K’-M’’, N’-R’’) represent 20 µm, (K-M) represents 0.3 mm and (N-R) represents 100 µm. Boxplot in (D) represents 20 EnVs and (S) represents at least 20 SG pairs. Whisker length min to max, bar represent median. P-value quantified with Student’s t-test.

Since we had observed a loss of secretion in RasV12 SGs we next addressed the presence of EnVs within the secretory pathway. We overexpressed two versions of human phosphatidylserine binding protein, MFG-E8 (Milk fat globule-EGF factor), without (referred as non-secreted: Figures 4F, F’, I, I’) and with a signal peptide (referred as secreted: Figures 4G, G’,J, J’37). In controls, the non-secreted form was found in the cytoplasm, whereas the secreted version was detected in the cytoplasm and in the lumen (Figures 4E–E’, F–G’). In Ras^V12 SGs, the non-secreted form was surrounding the EnVs (arrow), indicating the presence of phosphatidylserine on their membrane (Figures 4H–H’, I, I’). In contrast, the secreted form localized to the inside of the EnVs (Figures 4J, J’: arrow). These data suggest that EnVs are surrounded by a lipid membrane and probably derive from the secretory pathway.

In order to further characterize Idgf3-containing EnVs we co-expressed vesicle-specific Rab’s coupled with a GFP fluorophore, a lysosomal marker (Atg8), an autophagy marker (Vps35), and a marker for phosphatidylinositol-3-phosphate-(PtdIns3P: FYVE)-positive endosomes in Ras^V12 glands (For a complete set, see Figure S4E-M’’) which marks macroautophagy vesicles. To increase sensitivity and to identify EnVs, we stained with anti-GFP and co-stained with Phalloidin. Localization of Rabs and phalloidin to the same vesicles was observed with endosomal marker (Rab5) and recycling endosomal marker (Rab11) but not endosomal marker (Rab7) (Figure S4E-H’’, S4M-M’’). Moreover, EnVs were also positive for PtdIns3 (Figure S4L-L’’). In line with their dependence on secretion, this potentially identifies EnVs as enlarged recycling endosomes. EnV accumulation in Ras^V12 glands between 96 h and 120 h implies that (i) endosome formation is either increased compared to WT or (ii) that endosomes are not normally recycled leading to their accumulation. The latter hypothesis correlates with the loss of apico-basolateral polarity and the disruption of secretion due to a lack of a luminal structure in Ras^V12 glands (25). To test the first hypothesis, we blocked the formation of early endosomes with Rab5^DN. Apico-basolateral polarity, detected by a visible lumen, was not affected by Rab5^DN. Moreover, Rab5^DN;Ras^V12 did not block EnV formation and restoration of apicobasal polarity (Figure S4N-Q). Halting the recycling endosome pathway via Rab11^DN increases the endosomes’ accumulation without affecting cell polarity (Figure S4R). In contrast, in Rab11^DN;Ras^V12 SGs, endosomes were not accumulating, and EnVs were still detected (Figure S4S). Taken together, EnV formation is independent of the classical recycling pathway, suggesting other candidates are involved in their generation.

In SGs, overexpression of Rac generates enlarged vesicles with similarity to the EnVs described here (14). Supporting a role in dysplasia in our system, Ras^V12 SGs showed stronger Rac1 expression in comparison to the control. Due to the pleiotropic effects of the Rac1^DN construct, we addressed Rac1 function by modulating the expression of the Rac1 effector molecule, Pak (14). Overexpression of Pak^CA did not increase Idgf3 levels and had no detectable effect on F-actin distribution (Figures 4K-M’’). In contrast, Rac1 activity via Pak does affect Ras^V12 SG integrity: Idgf3::GFP levels were increased in Rac1-OE SGs and F-actin was disorganized (Figures 4N-P’’ quantified in S). However, the Rac1-OE glands did not grow larger compared to Ras^V12,indicating additional signals are necessary for gland overgrowth. Also, Pak^KD;Ras^V12 SGs displayed a more regular F-actin distribution leading to restoration of the lumen and proper secretion of Idgf3 (Figures 4Q-R’’ quantified in S). Moreover, we observed Rac1 also localized to EnVs (Figure S4T-U’). Decoration with Rac1 and actin as well as their dependence on Ras activation potentially identifies EnVs as macropinocytotic vesicles ((38), see also discussion). The enlarged vesicles that form upon Rac overexpression in SGs (14) also stain positive for Spectrins identifying them as additional candidates for EnVs formation. Of note, Spectrins under physiological settings are involved in the maintenance of cellular integrity including epithelial organization, which is lost in Ras^V12 SGs.

3.5 JNK promotes EnVs formation via Idgf3 upstream of αSpectrin

To analyze Spectrin contribution to EnVs formation, we stained for αSpectrin, one of the three members in flies (39) and found it to be induced in Ras^V12 SGs and to localize to the EnVs (Figure S5A-B’’). Knockdown of Idfg3 in Ras^V12 SGs reduced both αSpectrin levels and EnVs formation (Figures 5A-D’). Despite efficient Idfg3^KD, transcript levels for both α- and β_HeavySpectrin as well as for Rac1 were not affected indicating regulation at the posttranscriptional level (Figure 5E). Moreover, we found markers for cell polarity including Dlg, and Myosin II also decorate the EnVs (Figure S4V-Y’: arrow). In contrast, αSpectrin^KD (Figure S5C-F quantified G) reduced Idgf3 levels (Figures 5F-I’ quantified Figure 5J) as well as JNK signaling upstream of Idgf3 (Figures 5U, V). Further supporting a role for Spectrins in SG dysplasia, knockdown of αSpectrin in Ras^V12 glands abolished EnVs formation and partially restored the SG lumen (Figure 5I’’).

FIGURE 5

Figure 5 JNK promotes EnVs formation via Idgf3 upstream of αSpectrin (A-D’) αSpectrin staining showing restoration of normal distribution in Idgf3^KD;Ras^V12glands. (E) qPCR data showing no reduction of αSpectrin and β_HeavySpectrin in Idgf3^KD;Ras^V12. (F-I’’) Reduced levels of αSpectrin (αSpectrin^KD/Ras^V12) reduces Idgf3::GFP levels quantified in (J), prevents formation of EnVs and largely restores the SG lumen (arrows indicate EnVs). (K-S) Phalloidin staining showing epistasis of EnVs formation in which Idgf3 acts downstream of JNK. (T) EnVs penetrance quantification showing a strong induction of EnVs in JNK^DN;Idgf3^OE/Ras^V12 glands. (U) pJNK intensity quantification showing reduced levels in aSpectrin^KD/Ras^V12. (V) TRE intensity quantification showing reduced levels in aSpectrin^KD/Ras^V12. (W) Idgf3 promotes formation of EnVs, upstream of Rac1. Scale bars in (A-D’, F’-I’’, K-S’) represent 20 µm, (F-I) represents 100 µm. Data in (E) represent 3 independent replicates summarized as mean ± SD. Barplot in (T) represent 3 independent replicates with at least 10 SG pairs, summarized as mean ± SD. Boxplot in (J, U, V) represent at least 20 SG pairs. Whisker length min to max, bar represent median. P-value quantified with Student’s t-test.

Taken together this suggests that Idgf3 promotes EnVs formation (Figures 5C, C’) most likely post-transcriptionally (Figure 5E). In line, overexpression of Idgf3 throughout the whole gland, at 96 h, as shown by ISH (Figure S5I-L), led to an increase in the number of glands with endosomes (Figure S5M-P’’’, quantified in Q). To address epistasis between Idgf3 and JNK we calculated the penetrance of EnVs formation. In Ras^V12 SGs we observed EnVs in 100% of the glands, an effect that was strongly blocked in Bsk^DN;Ras^V12 (Figures 5O, S, quantified in Figure 5T). Blocking JNK and overexpressing Idgf3 in Ras^V12 strongly reverted the Bsk^DN;Ras^V12 phenotype, a lumen could not be detected, and around 98% of the glands contained enlarged endosomes (Figures 5O–S quantified in Figure 5T) while control SGs using RFP-overexpression retained the Bsk^DN;Ras^V12 phenotype. Overexpression of Idgf3 alone did not result in EnVs formation (Figures 5K–N). In conclusion, the data suggest that Idgf3 acts downstream of JNK and - through formation of EnV’s - disrupts luminal integrity. The proposed activity of Idgf3 in EnVs formation is summarized in Figure 5W.

3.6 Human CLP members enhance dysplasia in Drosophila SGs

Finally, we wished to determine whether the tumor-modulating effects we had observed for Drosophila Idgf3 also applies to human CLP members. For this we expressed two human CLPs (Ch3L1 or Ykl-40; 29% amino acid identity to Idgf3 and Ch3L2 or Ykl-39; 26% amino acid identity, Figure 6A) in SGs, both on their own and in combination with Ras^V12. Overexpression of CLPs in salivary glands was confirmed by qPCR (Figure 6B). Similar to Idgf3, both CLPs enhanced the hypertrophy observed in Ras ^V12 SGs (Figures 6C-H quantified in Figure 6I). The lumen integrity stayed highly disturbed when CLPs were overexpressed in Ras^V12 background (Figures 6J–O). Additionally, Ch3L1 enhanced the prevalence of EnVs in the Ras mutant background (Figure 6P). Taken together this means that the tumor-promoting effect of CLPs is conserved between Drosophila and humans and may affect different phenotypes of dysplasia depending on the CLP under study.

FIGURE 6

Figure 6 Human Chitinase-like proteins similarly to Idgf3 promotes EnVs formation (A) Comparison of Idgf3, CH3L1 and CH3L2 protein motifs (https://prosite.expasy.org). (B) qPCR confirmation of CH3L1 and CH3L2 expression in SG. (C-H) Representative images of phalloidin staining used for size quantification. (I) SG size quantification showing an increase in tissue size in CH3L1^OE;Ras^V12 and CH3L2^OE;Ras^V12 SG compared to Ras^V12 alone. (J-O) Phalloidin staining depicting disrupted lumen integrity in Ras^V12 glands. (P) EnVs penetrance quantification showing an induction of EnVs in CH3L1^OE;Ras^V12 glands. Barplot in (B) represent 4 independent replicates with at least 10 SG pairs, summarized as mean ± SD. Scale bars in (C-H) represent 100 µm and (J-O) 20 µm. Boxplot in (I) represent at least 20 SG pairs. Whisker length min to max, bar represent median. P-value quantified with Student’s t-test. Barplot in (P) represent 3 independent replicates with at least 10 SG pairs, summarized as mean ± SD.

4 Discussion

The levels of Chitinase-like proteins (CLPs) are elevated during a wide range of inflammatory processes as well as neoplastic disorders. Their physiological function has been more elusive but includes the formation of extracellular assemblages (40) including the insect cuticle (22), wound healing and in both mammals (40) and insects (41) and the restoration of cell integrity after oxidative damage (42). Conversely, induction of CLPs has been associated with the development of fibrotic lesions and cancer development with poor prognosis (reviewed in (40)). We used Drosophila as a tumor model to dissect CLP (Idgf3) function genetically in a secretory ductal organ, the salivary glands. We show that Idgf3 promotes tumor overgrowth through the disruption of cell polarity. The induction of Idgf3 disrupts cell organization and leads to the formation of enlarged endosome vesicles (EnVs) which accumulate in the cytoplasm. Genetically, Idgf3 is induced via a pro-tumorigenic JNK and ROS signaling feedback loop. Consequently, Idgf3 recruits the spectrin-based membrane skeleton (SBMS) for the formation of EnVs. Significantly, KD of Idgf3 inhibits overgrowth, restores cell polarity, reduces ECM size and blocks EnV formation.

Our identification of a contribution of JNK signaling and both extra- and intracellular ROS to dysplasia is in line with previous findings from other Drosophila tumor models (43). Similarly, like others (43) we observe an amplification loop between ROS and JNK signaling, which augments the dysplastic phenotype (24). Several studies have demonstrated that activation of JNK signaling in mammals promotes the progression of ductal tumors (44–46). Here we identify Idgf3 as an additional component that feeds into JNK signaling. Ultimately in Ras^V12-expressing SGs this leads to the formation of EnVs involving Spectrins. Under physiological conditions, members of the Spectrin family have a supporting role in maintaining cellular architecture through interaction with phospholipids and actively promoting polymerization of F-actin (47–49). Moreover, the secretory activity of ductal organs has been shown to be facilitated by Spectrins (50).

During Drosophila development and under physiological conditions, the pathway that involves Spectrins, Rac1 and Pak1 has been shown to be required for the maintenance of cell polarity while when deregulated it leads to the formation of enlarged vesicles similar to the EnVs (14). Thus, our results provide a possible link between the observed induction of CLPs in a range of tumors and the effects of Spectrins and their deregulation in tumors (51, 52). In addition to the genetic interaction we find, previous work suggests an additional mechanical link via a Spectrin binding protein (Human spectrin Src homology domain binding protein1; Hssh3bp1, (53)) the loss of which has been associated with prostatic tumors (54). Hhh3bp1 may influence tumor progression possibly through interaction with tyrosine kinases such as Abelson kinase (54). Interestingly Hhh3bp1 is a marker and possible regulator of macropinocytosis (55), a recycling pathway that is known to be hijacked by Ras-transformed tumor cells to acquire nutrients (38) and also leads to the formation of large intracellular vesicles (56). In favor of this hypothesis macropinocytosis is known to depend on Rac1/Pak1 signaling although the resulting vesicles are usually smaller (0.2-5 micrometers) than EnVs (57). We find that - like macropinocytosis - EnV-formation depends on the activity of growth factors (38), in this case Idgf3, much in line with its original description as an in vitro mediator of insulin signaling (20). In vivo, under normal conditions Idgf3 is required for proper formation of chitin-containing structures, wound healing and cellular integrity (22). Thus, under these circumstances Idgf3 acts to preserve cellular integrity including the epithelial character of SG cells upstream of spectrins. We propose that in a non-physiological setting such as upon overexpression of Ras^V12, this mechanism is overwhelmed leading to the breakdown of homeostasis, loss of cell polarity and the gland lumen, loss of secretory activity and the formation of EnVs larger than macropinocytotic vesicles. Large vesicles accompany several scenarios of non-apoptotic programed cell death, which occurs a.o. in apoptosis-resistant tumors (58, 59). Such modes of cell death include methuosis, a deregulated form of macropinocytosis (56, 58). Of note, apoptotic cell death is inhibited in Drosophila polytenic SGs to account for the increased number of DNA breaks that occur during endoreplication, which in mitotic cells induce apoptosis in both a p53-dependent and independent manner (60, 61). In line, despite the activation of caspase activity and nuclear fragmentation, which are considered hallmarks of apoptosis, Ras^V12 SG cells don’t disintegrate to produce apoptotic bodies (24). This may also explain the difference to mitotically cycling tumor models, which also activate JNK – yet with apoptosis as an outcome (32, 62, 63). Thus, SGs provide a suitable model for apoptosis-resistant tumors. In a mammalian setting, the phenotypes that are associated with non-apoptotic cell death such as disruption of cellular polarity and reorganization of the ECM provide potential targets for therapeutic treatments (46). Our work adds CLPs and spectrins to this list. Depending on the tissue environment and similar to JNK signaling, CLP’s may have varying roles in a context-dependent manner. Overexpression of Idgf3 alone is not sufficient for the loss of cell polarity, overgrowth, and fibrosis. Collectively, this suggests a tumor-specific phenotype for Idgf3 (Figures 6C-H), in line with mammalian CLPs (reviewed in (40)). Due to their pleiotropic effects, further investigation of CLPs role will be required to dissect their molecular function in a given tissue and to ultimately design tumor-specific treatments (64).

Taken together our findings provide new insight into the loss of tissue integrity in a neoplastic tumor model including the contribution of CLPs, Spectrins and alternative forms of cell death. This may provide further ways to test how developmentally and physiologically important conserved mechanisms that maintain cellular hemostasis - when deregulated - contribute to tumor progression.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Author contributions

DK, UT and MK conceived the research and designed the experiments. DK, MK, SH and AM performed experiments and data analysation. DK, UT and MK wrote the paper and participated in the revisions. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by Swedish Cancer Foundation (CAN 2015-546), Wenner-Gren Foundation (UPD2020-0094 and UPD2021-0095 to MK), Swedish Research Council (VR 2016-04077 and VR 2021-04841).

Acknowledgments

We would like to thank Chris Molenaar, Roger Karlsson, Stina Höglund and the Imaging facility at Stockholm University for support with all aspects of microscopy. We would also like to thank Vasilios Tsarouhas for his critical feedback. This work was supported by grants from the Swedish Cancer Foundation (CAN 2015-546), the Wenner-Gren Foundation (UPD2020-0094 and UPD2021-0095 to MK) and the Swedish Research Council (VR 2016-04077 and VR 2021-04841).

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2023.1170122/full#supplementary-material

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