Smg1 haploinsufficiency predisposes to tumor formation and inflammation

Abstract

SMG1 is a member of the phosphoinositide kinase-like kinase family of proteins that includes ATM, ATR, and DNA-PK, proteins with known roles in DNA damage and cellular stress responses. SMG1 has a well-characterized role in nonsense-mediated decay as well as suggested roles in the DNA damage response, resistance to oxidative stress, regulation of hypoxic responses, and apoptosis. To understand the roles of SMG1 further, we generated a Genetrap Smg1 mouse model. Smg1 homozygous KO mice were early embryonic lethal, but Smg1 heterozygous mice showed a predisposition to a range of cancers, particularly lung and hematopoietic malignancies, as well as development of chronic inflammation. These mice did not display deficiencies in known roles of SMG1, including nonsense-mediated decay. However, they showed elevated basal tissue and serum cytokine levels, indicating low-level inflammation before the development of tumors. Smg1 heterozygous mice also showed evidence of oxidative damage in tissues. These data suggest that the inflammation observed in Smg1 haploinsufficiency contributes to susceptibility to cancer and that Smg1-deficient animals represent a model of inflammation-enhanced cancer development.

Fig. 1.

Generation of Smg1 mice. (A) Schematic shows targeting of the Smg1 gene by insertion of a Genetrap cassette. Arrows indicate the primers used for PCR and sequencing to map the insertion site. (B) cDNA was isolated from Smg1^+/gt and Smg1^+/+ mice. 5′ RACE was performed on these samples, and products were visualized on an agarose gel. Lane 1: positive control for PCR, lane 2: negative control, lane 3: 5′ RACE sample, and lane 4: negative control for 5′ RACE. (C) Genomic DNA from Smg1^+/gt and Smg1^+/+ mice was used for Southern blotting. Multiple restriction digests, as indicated, were run for each sample and then separated on an agarose gel. DNA was transferred to a nylon membrane and incubated with a probe targeting either the neomycin gene in the Genetrap cassette (Upper; NEO) or intron4-exon 4 of the Smg1 gene (Lower; Exon 4). (D) Smg1^gt/gt mice die around E8.5. Embryos were harvested and genotyped by real-time PCR assay at the indicated days after timed mating. Three litters were analyzed at each time point. Numbers in brackets indicate the expected number of embryos for each genotype.

Fig. 2.

Smg1 heterozygous mice have decreased viability compared with WT littermates. (A) Kaplan–Meier survival plot shows the significantly decreased viability of Smg1^+/gt mice compared with Smg1^+/+ littermates. Statistical significance was determined using Prism 5 software (GraphPad Software). H&E staining of paraffin-embedded sections shows key pathological findings observed in Smg1^+/gt mice [lymphoma (B), chronic inflammation (C), papillary adenocarcinoma (D), and steatosis (E)] and WT tissues for comparison [lung (F), spleen (G), and liver (H)].

Fig. 3.

Smg1^+/gt mice do not show a deficiency in NMD. (A) SMG1 Western blotting shows reduced levels of SMG1 protein expression in the lung, heart, spleen, thymus, and brain compared with Smg1^+/+ levels. Tissues were harvested, total protein was extracted, and SMG1 expression was measured by Western blotting. GAPDH was used to show loading. (B) Real-time PCR quantification of endogenous NMD target transcript Gas5 in the indicated tissues. Gas5 expression is shown relative to control gene rpl13a. Data are pooled from five independent experiments and expressed as mean ± SEM. (C) Quantification of NMD-regulated alternative splicing in MEFs. Transcripts for luc7l, sfrs10, and alkbh3 were amplified by PCR, and samples were separated by electrophoresis. Products from the PTC-containing transcripts are a different size from the major non-PTC transcript. The amount of product for each splice variant was determined by ethidium bromide incorporation. Data shown are pooled from three independent pairs of MEFs and expressed as mean ± SEM. (D) Smg1^+/gt and Smg1^+/+ MEFs were transfected with plasmid containing normal β-globin (normal) or β-globin with an introduced PTC and a control for transfection efficiency, murine urinary tract protein (MUP). Twenty-four hours after transfection, samples were analyzed for β-globin mRNA expression by PCR. The amount of PCR product was determined by ethidium bromide incorporation. Data are normalized to Smg1^+/+ normal MEFs and are pooled from analysis of three pairs of MEFs and expressed as mean ± SEM. (E) Indicated T-cell receptor β-chains (1, 5, 13.3, and 13.1) were amplified from thymocytes from Smg1^+/gt and Smg1^+/+ mice. PCR products were cloned, sequenced, and analyzed for inclusion of a PTC in the transcript. No difference in the frequency of PTC inclusion was observed between transcripts from Smg1^+/gt and Smg1^+/+ mice.

Fig. 4.

Smg1^+/gt mice do not show defects in DNA damage responses. (A) Immortalized MEF survival after the indicated dose of IR was determined by colony formation assay. Data points show the average of two independent experiments, and the error bars, which may fall within the size of the symbol, indicate the standard error. (B) γH2AX foci formation and resolution were measured by immunofluorescence at the indicated time points after 10-Gy irradiation in Smg1^+/gt and Smg1^+/+ MEFs. The number of foci per cell was counted for a minimum of 150 cells at each time point. Data shown are pooled from three independent experiments and expressed as mean ± SEM. (C) Smg1^+/gt and Smg1^+/+ littermates were exposed to two 3-Gy doses of IR 4 mo apart, followed by 5 Gy of IR another 3 mo later, and survival was monitored. An ATM^−/− mouse was included to ensure irradiation was effective. Smg1^+/gt mice did not show sensitivity to IR. (D) Genomic DNA was isolated from the indicated tissues from Smg1^+/gt and Smg1^+/+ mice at 9 mo of age. Telomere length was measured by real-time PCR. Data shown are pooled from the indicated number of replicates for each genotype. Data are expressed as mean ± SEM. No significant difference was observed between genotypes.

Fig. 5.

Smg1^+/gt mice have elevated basal levels of cancer-related cytokines. (A) RNA was isolated from the indicated tissues, cDNA synthesis was performed, and cytokine mRNA levels were measured by real-time PCR. Expression levels are expressed relative to the control gene rpl13a. Data presented are pooled from a minimum of five pairs of Smg1^+/gt and Smg1^+/+ littermates analyzed in independent experiments. Data are expressed as mean ± SEM. A Student t test was performed to determine statistical significance: *P < 0.05; **P < 0.01. (B) Serum cytokine levels were measured by cytokine bead array in Smg1^+/gt and Smg1^+/+ mice. (C) Serum cytokine levels for mice not showing extreme cytokine production (as in B) were pooled and analyzed. Data shown are from a minimum of seven Smg1^+/gt or Smg1^+/+ mice. Data are expressed as mean ± SEM. A Student t test was performed to determine statistical significance: *P < 0.05. (D) Oxidative damage to tissues was measured by immunofluorescent staining for 8oxodG or 4-hydroxynonenal (4HNE; green), and tissues were counterstained with DAPI (blue) to show nuclei. Tissues from Smg1^+/gt spleens of mice with hyperplasia were compared with those of Smg1^+/+ mice of a similar age. (Scale bar = 100 μM.)

Fig. P1.

Smg1 heterozygous mice show elevated levels of tissue cytokines and oxidative damage despite showing no defect in the characterized roles of Smg1. The enhanced basal inflammation can then lead to either chronic inflammatory disease or cancer development.