An SH3PX1-Dependent Endocytosis-Autophagy Network Restrains Intestinal Stem Cell Proliferation by Counteracting EGFR-ERK Signaling

Abstract

The effect of intracellular vesicle trafficking on stem-cell behavior is largely unexplored. We screened the Drosophila sorting nexins (SNXs) and discovered that one, SH3PX1, profoundly affects gut homeostasis and lifespan. SH3PX1 restrains intestinal stem cell (ISC) division through an endocytosis-autophagy network that includes Dynamin, Rab5, Rab7, Atg1, 5, 6, 7, 8a, 9, 12, 16, and Syx17. Blockages in this network stabilize ligand-activated EGFRs, recycling them via Rab11-dependent endosomes to the plasma membrane. This hyperactivated ERK, calcium signaling, and ER stress, autonomously stimulating ISC proliferation. The excess divisions induced epithelial stress, Yki activity, and Upd3 and Rhomboid production in enterocytes, catalyzing feedforward ISC hyperplasia. Similarly, blocking autophagy increased ERK activity in human cells. Many endocytosis-autophagy genes are mutated in cancers, most notably those enriched in microsatellite instable-high and KRAS-wild-type colorectal cancers. Disruptions in endocytosis and autophagy may provide an alternative route to RAS-ERK activation, resulting in EGFR-dependent cancers.

Keywords: Drosophila sorting nexins; EGFR-Ras-MAPK; Keren; Rab GTPases; SH3PX1; autophagy-related proteins; calcium signaling; colorectal cancer; endocytosis-autophagy network; intestinal stem-cell proliferation.

Conflict of interest statement

Declaration of Interests

The authors declare no competing interests.

Copyright © 2019 Elsevier Inc. All rights reserved.

Figures

Figure 1.. Drosophila SH3PX1 restrains ISC proliferation

(A, C, F, G) Midguts were stained with anti-pH3 antibody. ISC mitoses were quantified by pH3+ cells. Quantification data shown in A, C, F, and G represent the mean±SD (t-test, nsP>0.05, **P<0.01, ****P<0.0001). Each dot represents one sample. (B, E) Midguts were stained with anti-GFP and anti-pH3 antibodies, and DAPI. (D) 2–3 day-old esg-Gal4,UAS-GFP;tubGal80ts (esgts) flies were shifted from 18°C to 29°C and raised for 2d on normal fly food before infection. Flies were then transferred to empty vials containing Whatman discs soaked with 5% sucrose and yeast paste +/− P.e. for 16hr at 29°C. Midguts were dissected and stained with anti-SH3PX1 antibody. (E-F) Two independent RNAi lines against SH3PX1 were driven using an ISC-specific driver esg-Gal4,UAS-2xEYFP;Su(H)GBE-Gal80,tub-Gal80ts (esgts;Su(H)GBE-Gal80). 2–3 day-old adult females were shifted from 18°C to 29°C for 7d before dissection. The SH3PX1RNAi lines’ knockdown efficiencies are shown in Supplementary Figures 2A–2C’. (G) UAS-GFP-SH3PX1 was overexpressed using esg-Gal4 at 25°C for 3d in the SH3PX1d1/d1 mutant background. (H) Flies were raised at 25°C on normal fly food. Survival curves are shown for each genotype as indicated (n=30, per genotype). Scale bars in B and E, 40 μm; D, 10 μm.

Figure 2.. SH3PX1-dependent autophagy restricts ISC proliferation

(A) Flies were raised at 18°C. 3-day-old esg-Gal4/UASp-GFP-mCherry-atg8a;SH3PX1d1FRT2A/TM6B or esg-Gal4/UASp-GFP-mCherry-atg8a;SH3PX1d1FRT2A/SH3PX1d1FRT2A flies were starved for 6hr in the vials containing Whatman paper wetted with 5% sucrose, then were stained with anti-GFP and anti-RFP antibodies, and DAPI. esgGal4>UASp-GFP-mCherry-atg8a marked autophagosomes in progenitor cells. The red boxes in the left panels were enlarged and visualized in the middle/right panels. Arrowheads indicate autophagosome formation. (B-G) Different genetic manipulations in progenitor cells driven by esgts. 2~3-day-old adult females were shifted from 18°C to 29°C for 5–6d (as indicated in panels) before dissection. Midguts were stained with anti-GFP and anti-pH3 antibodies, and DAPI. ISC mitoses were quantified by pH3+ cells. Quantification data shown in B, D-G represent the mean±SD (t-test, nsP>0.05, **P<0.01, ***P<0.001, ****P<0.0001). Each dot represents one sample. Scale bars in A, 5 μm; C, 40 μm.

Figure 3.. SH3PX1-mediated autophagy restrains ISC mitoses by damping EGFR/ERK activity

(A-D) Different genetic manipulations in progenitor cells driven by esgts. 2~3-day-old adult females were shifted from 18°C to 29°C for 6d or 12d (as indicated in panels) before dissection. Midguts were stained with anti-GFP and anti-pH3 antibodies, and DAPI. ISC mitoses were quantified by pH3+ cells. Quantification data shown in A, C and D represent the mean±SD (t-test, nsP>0.05, ****P<0.0001). Each dot represents one sample. (E-F) Flies with indicated genetic manipulations were shifted from 18°C to 29°C for 6d or 7d (as indicated in panels) before dissection. Midguts were stained with anti-GFP and anti-dpERK antibodies, and DAPI. Scale bars in B, E and F, 40 μm.

Figure 4.. EC-localized stress signaling is dispensable for proliferation of SH3PX1 mutant ISCs

(A, C, F) UAS-SH3PX1RNAi−2 was overexpressed using esgts at 29°C for 3d or 4d, as indicated in panels before dissection. Ex-lacZ (A, red) expression indicated Yki activity. Upd3 expression levels were indicated by the upd3-lacZ reporter (C, red). Rho induction was indicated by the rho-lacZ reporter (F, red). Progenitor cells were marked by GFP. (B, D, G and H) Midguts were stained with anti-pH3 antibody. ISC mitoses were quantified by pH3+ cells. (B) UAS-ykiRNAi was overexpressed using myo1A-Gal4 at 25°C for 8d in the SH3PX1d1/d1 mutant background. (D) Adult males of +/Y;mira-GFP;SH3PX1d1/d1 (control) and upd2Δ,3Δ/Y;mira-GFP;SH3PX1d1/d1 were incubated at 25°C for 4 or 8d after eclosion. (E) The same genotype flies as described in panel D were stained with anti-GFP and anti-dpERK antibodies and DAPI at day 7. (G-H) UAS-rhoRNAi (G) or UAS-KrnRNAi (H) was overexpressed using Myo1A-Gal4 at 25°C for 3d or 6d (as indicated in panels) in the SH3PX1d1/d1 mutant background. Quantification data shown in C, D, G and H represent the mean±SD (t-test, nsP>0.05, ****P<0.0001). Each dot represents one sample. Scale bars in A, 30 μm; C, E and F, 20 μm.

Figure 5.. Cell autonomous EGFR/MAPK activation triggers SH3PX1--ISC division

(A, D) Midguts were stained with anti-pH3 antibody. ISC mitoses were quantified by pH3+ cells. (A) SH3PX1 knockdown or SH3PX1/stg double knockdown was driven by esgts. esgts>w1118 was used as control. Flies were raised at 18°C and then shifted to 29°C for 5d before dissection. (B-C) SH3PX1/stg or SH3PX1/MEK double knockdown was driven by esgts at 29°C for 4d. Induction of upd3 or rho was indicated by upd3-lacZ (upper panels of B, and C) or rho-lacZ (lower panels of B, and C), respectively. Progenitor cells were marked by GFP. (D-E) UAS-MEKRNAi was overexpressed using myo1A-Gal4 at 25°C for 3d in the SH3PX1d1/d1 mutant background. Myo1A-Gal4/+;SH3PX1d1/d1 flies were selected as positive control (D and left panels of E). Myo1A-Gal4/MEKRNAi; SH3PX1d1/+ flies were selected as negative control (E, right panels). (E-F) Midguts were stained with anti-GFP antibody and anti-dpERK antibodies, and DAPI. White asterisk in (E) indicates the EC cell. (F) SH3PX1 knockdown (control) or SH3PX1/stg double knockdown was driven by esgts at 29°C for 5d. Quantification data shown in A and D represent the mean ± SD (t-test, nsP>0.05, ***P<0.001, ****P<0.0001). Each dot represents one sample. Scale bars in B and C, 20 μm; E, 30 μm; F, 40 μm.

Figure 6.. SH3PX1 controls the EGFR pathway via multiple routes

(A) UAS-EGFRwt or UAS-SH3PX1RNAi−1+UAS-EGFRwt was overexpressed by the esgtsF/O system. EsgtsF/O>w1118 was used as control. Flies were raised at 18°C and then shifted to 29°C for 3d before dissection. Midguts were stained with anti-EGFR and anti-GFP antibodies, and DAPI. White asterisks indicate ISC or EB cells. Yellow asterisks indicate EC cells. (B) w1118, UAS-SH3PX1RNAi−1 and UAS-EGFRwt were overexpressed by esgts. Flies were raised at 18°C and then shifted to 29°C for 1–6d before dissection. Samples were dissected every day from d1 to d6 and stained with anti-pH3 antibody. ISC mitoses were quantified by pH3+ cells. Values represent mean±SEM. (C-F, I) Midguts were stained with anti-pH3 antibody. ISC mitoses were quantified by pH3+ cells. (C-F) Different genetic manipulations in progenitor cells driven by esgts. 2~3-day-old adult females were shifted from 18°C to 29°C for 6d before dissection. (G-H) UAS-GCaMP6s or together with UAS-SH3PX1RNAi−1 was overexpressed using esgts. (G) Representative Ca2+ plots of single esg+ midgut cells from wild-type (blue) and SH3PX1 knockdown (red) flies. Normalized fluorescence intensity is plotted over time, highlighting differences in intracellular Ca2+ peak durations. (H) Peak durations as determined from Ca2+ plots of wild-type (n=155 peaks) and SH3PX1 knockdown (n=80 peaks) esg+ midgut cells; P-value calculated by Mann-Whitney two-tailed U test. (I) SH3PX1 knockdown, SH3PX1/TrpA1 double knockdown, and SH3PX1/RyR double knockdown were driven by esg-Gal4 at 25°C for 6d. Quantification data shown in C-F and I represent the mean±SD (t-test, nsP>0.05, ****P<0.0001). Each dot represents one sample. Scale bars in A, 10 μm.

Figure 7.. Autophagy mutations are associated with MSI-H and CIMP-H colorectal cancers

(A) UAS-hSNX9, hSNX18 or hSNX33 were overexpressed using esg-Gal4 at 25°C for 3d in the SH3PX1d1/d1 mutant background. (B-E) RPE-1 cells were serum starved for 18hr, then treated with the autophagy inhibitor, 3-MA (4mM), for the times indicated. Cell lysates were subjected to western blot analysis with the indicated antibodies. (C) The induction (fold change) of dpERK samples in (B) was quantified after normalization against total-ERK. Values represent mean±SEM (n=4 independent experiments, t-test, *P<0.05). (E) The relative levels of EGFR shown in (D) were quantified after normalization against Vinculin. Values represent mean±SEM (n=2 independent experiments, t-test, *P<0.05). (F) Summary of SH3PX1-dependent endocytosis/autophagy mutations and deep deletions by cancer type in human TCGA datasets (n=14 datasets with ≥10% mutation and deep deletion alteration frequency and n³30 cases). The network gene list and corresponding human orthologs are shown in Table S1. (G-J) Analysis of somatic mutations in human colorectal adenocarcinoma (DFCI, n=619 cases). (G) Summary of somatic mutations by gene. (H-J) Proportion of wild-type (black) versus mutated (red) samples with the MSI-H genomic phenotype (H) and the CIMP-H epigenomic phenotype (I) (n=5 genes with ≥1% altered cases). (J) Proportion of SH3PX1 human homologs SNX9/18/33 wild-type versus mutated samples by KRAS wild-type status.