Human cancer-associated mutations in the Aα subunit of protein phosphatase 2A increase lung cancer incidence in Aα knock-in and knockout mice

Abstract

Strong evidence has indicated that protein phosphatase 2A (PP2A) is a tumor suppressor, but a mouse model for testing the tumor suppressor activity was missing. The most abundant forms of trimeric PP2A holoenzyme consist of the scaffolding Aα subunit, one of several regulatory B subunits, and the catalytic Cα subunit. Aα mutations were discovered in a variety of human carcinomas. All carcinoma-associated mutant Aα subunits are defective in binding the B or B and C subunits. Here we describe two knock-in mice expressing cancer-associated Aα point mutants defective in binding B' subunits, one knockout mouse expressing truncated Aα defective in B and C subunit binding, and a floxed mouse for generating conditional Aα knockouts. We found that the cancer-associated Aα mutations increased the incidence of cancer by 50 to 60% in lungs of FVB mice treated with benzopyrene, demonstrating that PP2A acts as a tumor suppressor. We show that the effect of Aα mutation on cancer incidence is dependent on the tumor suppressor p53. The finding that the Aα mutation E64D, which was detected in a human lung carcinoma, increases the lung cancer incidence in mice suggests that this mutation also played a role in the development of the carcinoma in which it was discovered.

Fig. 1.

Diagram of PP2A holoenzyme containing B′γ. The B′γ subunit binds to intrarepeat loops 2 to 8, and the Cα subunit binds to intrarepeat loops 11 to 15. Amino acid E64 of Aα binds to L309 of the C-terminal tail of Cα. E64 mutants are found in human lung and mammary carcinomas and are defective in B′ binding. R183 of Aα interacts with E214 of B′. R183 mutants were found in ovary carcinomas and are predicted to be defective in B′ binding.

Fig. 2.

Generation of E64D mice. (A) The Aα allele spans nearly 20 kb from exon 1 to exon 15 (first row, scale). The region targeted reaches from before exon 2 into exon 9 (black horizontal line, second row). Amino acid E64 of wild-type (WT) Aα is encoded in exon 3 (second row). The targeting construct (i) replaces codon E64 with codon D64, (ii) introduces an NheI site due to silent mutations, and (iii) places a neomycin (neo) cassette flanked by loxP sites (open triangles) into intron 3 (third row). ES cells were screened for the E64D_neo allele by using forward primer A (A>) and reverse primer D (<D) (fourth row). A> binds 5′ of the targeted region, while <D binds specifically to D64/NheI but not WT E64 (fourth row). E64D mice were identified with primers D> and <A (fifth row). D> binds to D64/NheI but not WT E64, while <A binds past neo. (B) PCR screening with primer pair A> and <D yields a product of 3,625 bp from the E64D_neo (D_neo) and E64D (D) alleles. (C) Primer pair D> and <A distinguishes E64D_neo (2,950 bp) from E64D mice (1,050 bp).

Fig. 3.

Correct sequence of E64D and E64G mRNAs. RNA from ES cell clones that yielded E64D and E64G mice was reverse transcribed and amplified by PCR with primers based on the reference sequence for mouse Aα mRNA, NM_016891.3 (top line, wild type [WT]). Nucleotide (nt) numbers are given in the right column. Only relevant sequence stretches are shown. The forward primer (nt 24 to 44) binds 5′ of the ATG start codon (nt 53 to 55), the reverse primer (nt 2047 to 2026) 3′ of the TGA stop codon (nt 1820 to 1822). The PCR products were cloned, and clones sensitive to NheI (GCTAGC at nt 250 to 255) were sequenced. The obtained sequences showed codon 64 as GAC for E64D and as GGC for E64G (nt 242 to 244). Stars indicate identity. Silent substitutions (nt 247 to 259) 3′ of E64D and E64G permitted design of primers that distinguish WT (primer

Fig. 4.

Generation of Aα-floxed (F5-6) and Aα knockout (Δ5-6) mice. (A) The region targeted reaches from before exon 3 past exon 11 (black horizontal line, second row, Aα wild type [WT]). The targeting construct contains (i) a loxP site (open triangle) 5′ of exon 5, (ii) a neo cassette flanked by FRT sites (black triangles) 3′ of exon 6, and (iii) a second loxP site 3′ of neo (third row). F5-6_neo ES cells and mice were identified by Southern blotting of EcoRV-digested DNA (Eco) with the 3′ probe (black box) (second and fourth rows). F5-6 and Δ5-6 mice were identified by PCR using primer P>, binding to the 5′ loxP site, and primer <I, binding to intron 6 past neo (fifth and sixth rows). (B) Southern blotting of EcoRV-digested DNA with the 3′ probe identifies WT (24,000 bp) and F5-6_neo (F_neo) alleles (8,200 bp). (C) PCR screening with primer pair P> and <I identifies F5-6 (F) (1,836 bp), Δ5-6 (Δ) (1,032 bp), and Δ5-6/F5-6 (1,032 and 1,836 bp) mice. (D) F5-6 and Δ5-6 mice were routinely screened with primer U> binding 5′ of the targeted region and primer <P binding to the 5′ loxP site (4,507 bp).

Fig. 5.

Viability of E64D/E64D, E64G/E64G, Δ5-6/E64D, and Δ5-6/E64G mice. (A) Inbreeding of E64D/+ mice yields +/+, E64D/+, and E64D/E64D progeny. Tail DNA was screened for the E64D (D) allele with primer pair A> and and and and and and and and

and and

Fig. 6.

Lethality of the Δ5-6/Δ5-6 genotype. Inbreeding of Δ5-6/+ mice yielded +/+ and Δ5-6/+ (Δ/+) offspring (lanes 1 and 2) but no Δ5-6/Δ5-6 pups. Δ5-6/+ inbreeding yielded Δ5-6/+ embryos and corresponding yolk sacs at E10.5 (lanes 3 and 4) but no Δ5-6/Δ5-6 embryos. Several unusually small implantation sites contained no embryos but contained Δ5-6/Δ5-6 (Δ/Δ) yolk sacs at E10.5 (lane 5). The Δ5-6 allele was identified with primer pair U> and

and has no target in the Δ5-6 allele, the Δ5-6/Δ5-6 genotype yields no product (lane 5).

Fig. 7.

Lethality of knockout of Aα in adult mice. Mice were generated containing one Δ5-6 (Δ) knockout allele, one F5-6 (F) floxed allele, and a transgene (CreER^TM) expressing Cre ubiquitously and inducible by TM. (A) Nine control mice, Δ5-6/+; CreER^TM, and four floxed littermates, Δ5-6/F5-6; CreER^TM, ages 2 to 4 months, were injected with TM on days 1 through 5 and weighed daily. All four Δ5-6/F5-6; CreER^TM mice died on day 6, while the nine control mice survived. The error bars represent the 95% confidence intervals. The weight losses of the floxed mice were significant, with P values of 0.02 (day 1 versus 2), 0.0002 (day 4 versus 5), and 0.006 (day 5 versus 6). (B) One control and one floxed mouse were treated as described for panel A and sacrificed on day 6, at which time the floxed mouse had a hunched back and difficulty walking. The weights of both mice were similar to the averages shown in panel A. Several organs were Western blotted for Aα (6F9). GAPDH was used as a loading control.

Fig. 8.

Reduced Aα levels in Δ5-6/+ mice. Various organs of Δ5-6/+ (Δ/+) mice and +/+ control littermates were Western blotted for Aα using 6F9 antibody. All organs except brain showed approximately 50% Aα in the heterozygous knockout mice compared to +/+ controls. GAPDH and β-actin were used as loading controls. MG, mammary gland; Cereb, cerebellum. The results are representative of three experiments.

Fig. 9.

PP2A subunit levels in wild-type mouse organs. Various organs were Western blotted for Aα, B′δ, B′α, and Bα. GAPDH and β-actin were used as loading controls. Cereb, cerebellum. The same results were obtained in two separate experiments.

Fig. 10.

Reduced binding of B′δ and B′α to E64D and E64G. Extracts of lung (A) and cortex (B) of +/+, E64D/E64D (D/D), and E64G/E64G (G/G) mice were used for immunodepletion of Aα using 6F9. Both the depleted supernatants (Sup) and the immunoprecipitates (IP) were Western blotted for Aα, B′δ, B′α (A), and Bα (B). Controls (Con) are immunodepletions for which we used beads without antibody. The first rows of percentages (below the blots) compare subunit amounts between supernatants and IP from the same organ. The second rows of percentages (in bold italics) compare input amounts of subunits between wild-type and mutant organs. GAPDH was used as a loading control. Coimmunoprecipitation of B′α and B′δ with E64G from extracts of lung, cortex, and liver (liver not shown) was carried out twice in independent experiments using different mice. Coimmunoprecipitation of B′α and B′δ with E64D from extracts of lung, cortex, and kidney (kidney not shown) was carried out once. In addition, quantitatively similar binding defects of B′α and B′δ to E64D were observed when we used extracts from +/+ and E64D/E64D mouse embryo fibroblasts from embryonic day 13.5 (data not shown).

Fig. 11.

Increased lung cancer risk in Aα mutant mice. E64D/+;dnp53 and Δ5-6/+ mice were crossed to obtain the eight genotypes indicated. dnp53 is a dominant-negative fragment of p53. All mice received BP at 5 weeks of age. Mice without dnp53 were harvested 10 months later (black columns), and mice with dnp53 were harvested 6 months after BP application (grey columns). The table on the bottom shows the total number of mice, the number of mice with tumors, lung tumor incidences, and the average numbers of tumors (Tu) per tumor-bearing mouse. The higher tumor numbers in Δ/D (2.5) and Δ/D;dnp53 (2.8) have P values of 0.05 and 0.01, respectively, compared to the wild type. The P values above columns 2 to 4 are for the incidences in the mutant mice compared to the wild-type mice. D, E64D; Δ, Δ5-6. Note that neither tumor numbers nor sizes were comparable between mice with and without dnp53, since they were harvested at different times.

Fig. 12.

Lung adenomas and adenocarcinomas in Aα mutant mice. (A and C) An adenoma at low magnification (40×; A) appears as a small parenchymal nodule (n →). At high magnification (400×; C), there is a focus of neoplastic cells, each with abundant cytoplasm and uniform monomorphic, small round nuclei. (B and D) An adenocarcinoma presented on gross evaluation as a large nodule that protruded above the pleural surface. Low magnification (40×; B) showed that the ill-defined capsule (c →) was eroded and that small foci of neoplastic cells infiltrated (i →) into the surrounding parenchyma. At high magnification (400×; D), the tumor mass shows anaplastic pleomorphic cells with tubular (t →) differentiation within solid (s →) areas. The majority of neoplastic cells contains large pleomorphic nuclei with an altered nuclear-to-cytoplasmic ratio, prominent nucleoli (o →), and occasional mitotic (m →) figures, all indicative of malignancy.