SIDT2 RNA Transporter Promotes Lung and Gastrointestinal Tumor Development

Abstract

RNautophagy is a newly described type of selective autophagy whereby cellular RNAs are transported into lysosomes for degradation. This process involves the transmembrane protein SIDT2, which transports double-stranded RNA (dsRNA) across the endolysosomal membrane. We previously demonstrated that SIDT2 is a transcriptional target of p53, but its role in tumorigenesis, if any, is unclear. Unexpectedly, we show here that Sidt2^-/- mice with concurrent oncogenic Kras^G12D activation develop significantly fewer tumors than littermate controls in a mouse model of lung adenocarcinoma. Consistent with this observation, loss of SIDT2 also leads to enhanced survival and delayed tumor development in an Apc^min/+ mouse model of intestinal cancer. Within the intestine, Apc^min/+;Sidt2^-/- mice display accumulation of dsRNA in association with increased phosphorylation of eIF2α and JNK as well as elevated rates of apoptosis. Taken together, our data demonstrate a role for SIDT2, and by extension RNautophagy, in promoting tumor development.

Keywords: Biological Sciences; Cancer; Cell Biology; Molecular Biology.

Graphical abstract

Figure 1

Kras^LSL-G12D/+;Sidt2^−/− Mice Have Increased Tumor Burden Compared with Controls (A) Representative images of H&E-stained lung sections from Kras^LSL-G12D/+;Sidt2^+/+ and Kras^LSL-G12D/+;Sidt2^−/− mice 18 weeks following inoculation with 4 × 10⁶ plaque-forming unit adenovirus containing Cre recombinase by intratracheal intubation. (B and C) (B) Average tumor number and (C) tumor burden as a percentage of total lung area was assessed per lung section (n = 19–23 mice per genotype). (D) Representative images of Ki67-stained lung sections of Kras^LSL-G12D/+;Sidt2^+/+ and Kras^LSL-G12D/+;Sidt2^−/− mice. (E) Quantification of average number of Ki67-positive cells per mm² tumor tissue. Analysis was performed on >25 tumors per mouse (n = mice per genotype). Error bars represent ±SEM. *p < 0.05, as calculated by unpaired Student's t test.

Figure 2

- Apc^min/+;Sidt2^−/− Mice Have Enhanced Survival and Smaller Tumors Compared with Controls (A) Kaplan-Meier survival curve of Apc^min/+;Sidt2^+/+, Apc^min/+;Sidt2^+/−, and Apc^min/+;Sidt2^−/− mice. Median survival rates of Apc^min/+;Sidt2^+/+, Apc^min/+;Sidt2^+/−, and Apc^min/+;Sidt2^−/− mice are 93, 99, and 131 days respectively. (B–I) (B) Representative image of distal small intestine of 100-day-old Apc^min/+;Sidt2^+/+ and Apc^min/+;Sidt2^−/− mouse. Total number (C–E) and area (F–H) of visible tumors in the colon and small intestine (distal, medial, and proximal) was quantified in 100-day-old Apc^min/+;Sidt2^+/+ (n = 12) and Apc^min/+;Sidt2^−/− (n = 12) mice. Error bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 as calculated by unpaired Student's t test. See also Figures S1 and S2.

Figure 3

Loss of SIDT2 Impairs Tumor Proliferation (A–C) (A) Representative H&E- and Ki67-stained histological sections of distal small intestine of Apc^min/+;Sidt2^+/+ and Apc^min/+;Sidt2^−/− mice at 100 days of age. Quantification of (B) Ki67-positive staining area as a percentage of total tumor area and (C) average number of Ki67-positive cells per tumor. Analysis was performed on >20 high-power fields across three mice. Error bars represent mean ± SEM. ***p < 0.001 as calculated by unpaired Student's t test.

Figure 4

- Loss of SIDT2 Leads to dsRNA Accumulation within Intestinal Crypts and Increased Phosphorylation of eIF2α (A) Representative image of dsRNA staining of distal small intestine of 100-day-old Sidt2^+/+;Apc^min/+ and Sidt2^−/−;Apc^min/+ mice. Arrowheads indicate cells with positive dsRNA staining. (B–D) (B) Quantification of dsRNA-positive stained area per crypt (n = 3–4 mice per genotype). Western blot and densitometry analysis of eIF2α phosphorylation in non-tumor (C) and tumor (D) tissue from distal small intestine of 100-day-old Sidt2^+/+;Apc^min/+ and Sidt2^−/−;Apc^min/+ mice normalized to total eIF2α. Each lane corresponds to an individual mouse (n = 4 mice per genotype). Error bars represent mean ± SEM. *p < 0.05, **p < 0.01 as calculated by unpaired Student's t test. See also Figures S3 and S4.

Figure 5

- Sidt2^−/−;Apc^min/+ Mice Have Increased Apoptosis in Intestinal Tissue (A–C) (A) Western blot analysis. Phosphorylation of JNK and cleavage of caspase 8 was assessed in normal intestinal tissue of 100-day-old Sidt2^+/+;Apc^min/+ and Sidt2^−/−;Apc^min/+ mice. Each lane corresponds to an individual mouse (n = 4 mice per genotype). Densitometry quantification of (B) p-JNK and (C) cleaved caspase 8 normalized to HSP70. (D) Representative images of cleaved caspase 3 staining of distal small intestine of 100-day-old Sidt2^+/+;Apc^min/+ and Sidt2^−/−;Apc^min/+ mice. (E) Quantification of dsRNA-positive stained area per crypt (n = 7 mice per genotype). (F) Representative images of TUNEL staining of positive cells from distal small intestine of 100-day-old Sidt2^+/+;Apc^min/+ and Sidt2^−/−;Apc^min/+ mice, n = 3 animals per genotype. Arrowheads indicate TUNEL⁺ cells. Error bars represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01 as calculated by unpaired Student's t test. See also Figure S4.

Figure 6

– Lower SIDT2 Expression Is Associated with Improved Survival in Some Cancers (A–E) Kaplan-Meier curves of overall survival of patients with (A) renal, (B) thyroid, (C) gastric, and (D) urothelial cancer and (E) glioma stratified against SIDT2 expression from publicly available RNA-seq data. The results shown are in whole based upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/. See also Figure S5.

Figure 7

Loss of SIDT2-Mediated RNAutophagy Leads to Increased Apoptosis and Cell Proliferation SIDT2 is a dsRNA transporter that mediates the lysosomal degradation of cellular RNAs via RNAutophagy. Loss of SIDT2 leads to accumulation of cellular RNA within the cytosol. Binding of these RNAs by protein kinase RNA-activated (PKR) leads to its activation and subsequent phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). Phosphorylation of eIF2α leads to inhibition of protein translation and cell proliferation, as well as induction of apoptosis.