Par-4 inhibits Akt and suppresses Ras-induced lung tumorigenesis

Abstract

The atypical PKC-interacting protein, Par-4, inhibits cell survival and tumorigenesis in vitro, and its genetic inactivation in mice leads to reduced lifespan, enhanced benign tumour development and low-frequency carcinogenesis. Here, we demonstrate that Par-4 is highly expressed in normal lung but reduced in human lung cancer samples. We show, in a mouse model of lung tumours, that the lack of Par-4 dramatically enhances Ras-induced lung carcinoma formation in vivo, acting as a negative regulator of Akt activation. We also demonstrate in cell culture, in vivo, and in biochemical experiments that Akt regulation by Par-4 is mediated by PKCzeta, establishing a new paradigm for Akt regulation and, likely, for Ras-induced lung carcinogenesis, wherein Par-4 is a novel tumour suppressor.

Figure 1

Par-4 is highly expressed in the lung and is downregulated in human lung tumours. (A) Par-4 protein expression in normal mouse tissues. (B) Par-4 mRNA levels in normal mouse tissues. (C) Par-4 distribution in human and mouse tissues, determined by IHC. (D) Commercial tissue microarrays of NSCLC human samples (n=133) were stained with anti-Par-4 antibody. Par-4 expression was analysed in adenocarcinoma and squamous cell carcinoma tumours compared with normal lung tissue. A representative example of positive and negative tumour samples and normal control for Par-4 staining is shown at two magnifications ( × 2.5 and × 20). Scale bar=50 μm.

Figure 2

Par-4 cooperates with Ras-induced tumorigenesis in the lung. (A) H&E staining of lungs from WT and Par-4 KO mice crossed with K-ras^+/V12, RERT2^T/T mice and analysed 5 months after activation of the K-rasV12 allele by injection of 4-hydroxytamoxifen (n=5 per genotype). Overall tumour burden was determined by quantification of the tumour area as a percentage of total area of H&E-stained tissue (right panel). (B) Number of total tumours per mouse in WT and Par-4 KO mice. (C) Survival of mice of different genotypes represented as percentage of total; the cause of death was asphyxiation. (D) Loss of Par-4 leads to increased Ras-induced high-grade lung adenocarcinomas. (E) Representative sections and quantification of proliferation index measured as percentage of positive nuclear staining for Ki67 in lung sections from normal alveolar or tumour tissue from WT and Par-4 KO Ras-expressing mice. (F) Par-4 expression levels in the lung from different mouse genotypes. ^**, P<0.01; ^****, P<0.0001.

Figure 3

Increased levels of XIAP and activation of RelA and Akt in lungs of Par-4 KO mice. (A) The levels of XIAP, phospho-Akt-S473, Akt, Par-4 and actin were determined in lung extracts from WT and Par-4 KO mice. These are representative experiments where there were at least two others with similar results. (B, C) Sections of alveolar lung tissue from WT and Par-4 KO mice were stained by IHC with anti-RelA (B) or anti-phospho-Akt-S473 (C) antibody and scored for the number of cells with nuclear staining. Results are the mean±s.d. of 10 different fields per mouse, with a total of five mice for each condition. Scale bar=50 μm. ^**, P<0.01; ^****, P<0.003.

Figure 4

Increased activation of Akt but not RelA in Ras-expressing Par-4 KO lung tumours. Sections of lung tumours from WT and Par-4 KO mice expressing Ras were stained by IHC with anti-phospho-Akt-S473 (A) or anti-RelA (C) antibody. (B) Quantitation of cells showing positive nuclear staining for phospho-Akt-S473. Results are the mean±s.d. of 10 different fields per mouse, with a total of five mice for each condition. Scale bar=50 μm. ^***, P<0.001.

Figure 5

Par-4 deficiency induces increased nuclear phospho-Akt in vivo. (A) Confocal immunofluorescence on WT and Par-4 KO EFs seeded on the same coverslip, double-stained for Par-4 and XIAP (upper panels), or XIAP and phospho-Akt-S473 (lower panels). WT and Par-4 KO EFs stimulated with serum (FCS) for different durations or a dose–response for 15 min (C) in total extracts (C, D) or in nuclear and cytosol extracts (B). Reconstitution of Par-4 KO EFs with Par-4 restored phospho-Akt-S473 levels to basal levels (D). (E, F) A549 or 293 cells treated with a control siRNA or with Par-4-specific siRNA were stimulated with serum for different times and the levels of phospho-Akt-S473 were determined. Knockdown of Par-4 was confirmed by immunoblot. (G) WT and Par-4 KO EFs stimulated with serum were analysed by immunoblot for phospho-Akt-S473 and phospho-Akt-T308 levels as well as the Akt substrates, Gsk3β-S9 and Foxo3-T32. (H) Knockdown of Akt blocks the increased cell proliferation induced by knockdown of Par-4 in A549 cells. Cell number was determined by trypan blue exclusion. Knockdown of Par-4 and Akt was analysed by immunoblot. These are representative experiments where there were at least two others with similar results.

Figure 6

PKCζ directly interacts and phosphorylates Akt. (A) Overexpression of Par-4 in 293 cells inhibited serum-induced phospho-Akt-S473 phosphorylation and was reversed by PKCζ co-expression. (B) Serum-stimulated 293 cells extracts were immunoprecipitated with anti-Akt or IgG control antibody and the immunoprecipitates were analysed by immunoblot with anti-PKCζ or anti-Akt, as a loading control. (C) In vitro phosphorylation of recombinant Akt by recombinant PKCζ with γP³²-ATP. Part of the assay reaction was also immunoblotted with anti-phospho-Akt-Ser473, anti-phospho-Akt-T308 and anti-Akt antibodies. (D) Immunoprecipitates of WT or KD Akt were phosphorylated in vitro by PKCζ and phospho-Akt-Ser473 levels were determined. (E) Phosphopeptide map of in vitro PKCζ phosphorylated-Akt (left panel). Phospho-amino-acid analysis of peptides A, B, C and the total reaction. (F) MS/MS spectra of the identified phosphopeptides corresponding to sites S124, S473 and T308 phosphorylated in Akt by PKCζ. (G) A549 cells treated with a control siRNA, Par-4–siRNA, Rictor-siRNA or both siRNAs were stimulated with serum and the levels of phospho-Akt-S473 were determined. Knockdown of Par-4 and Rictor was confirmed by immunoblot. These are representative experiments where there were at least two others with similar results.

Figure 7

Activation of Akt by Par-4 deficiency is dependent on PKCζ in vivo. (A) WT and PKCζ KO EFs stimulated with serum were analysed by immunoblot for phospho-Akt-S473 and phospho-Akt-T308 levels. (B) Lung extracts from WT and PKCζ KO were immunoblotted with XIAP, phospho-Akt-S473 and phospho-Akt-T308 antibodies. (C–E) Phosphorylation of Akt in Par-4 KO is reverted in DKO (Par-4/PKCζ). Phosphorylation of Akt and its substrate FOXO3 were determined in lung extracts of the different KO mice (C). Lung sections were stained for phospho-Akt-S473. Quantitation of cells showing positive nuclear staining for phospho-Akt-S473 (D). Results are the mean±s.d. of 10 different fields per mouse with a total of five mice for each condition. (E) Lung sections were stained for phospho-Akt-S124. Negative control was performed on Par-4 KO sample with no primary antibody. These are representative experiments where there were at least two others with similar results. Scale=50 μm.