Recurrent activating mutation in PRKACA in cortisol-producing adrenal tumors

Abstract

Adrenal tumors autonomously producing cortisol cause Cushing's syndrome. We performed exome sequencing of 25 tumor-normal pairs and identified 2 subgroups. Eight tumors (including three carcinomas) had many somatic copy number variants (CNVs) with frequent deletion of CDC42 and CDKN2A, amplification of 5q31.2 and protein-altering mutations in TP53 and RB1. Seventeen tumors (all adenomas) had no somatic CNVs or TP53 or RB1 mutations. Six of these had known gain-of-function mutations in CTNNB1 (β-catenin) or GNAS (Gαs). Six others had somatic mutations in PRKACA (protein kinase A (PKA) catalytic subunit) resulting in a p.Leu206Arg substitution. Further sequencing identified this mutation in 13 of 63 tumors (35% of adenomas with overt Cushing's syndrome). PRKACA, GNAS and CTNNB1 mutations were mutually exclusive. Leu206 directly interacts with the regulatory subunit of PKA, PRKAR1A. Leu206Arg PRKACA loses PRKAR1A binding, increasing the phosphorylation of downstream targets. PKA activity induces cortisol production and cell proliferation, providing a mechanism for tumor development. These findings define distinct mechanisms underlying adrenal cortisol-producing tumors.

Figure 1

Somatic mutations in cortisol-producing adrenal tumors. (a) The number of somatic mutations in each tumor is shown. Tumors are either adrenocortical carcinomas (ACC, in orange), CNV+ (in off-white) or CNV− (in white). (b) Recurrent copy number variants and GISTIC peaks identified in tumors. Likely driver genes are listed in parenthesis. Red, amplification; blue, deletion. (c) Somatic protein-altering mutations in genes frequently mutated in tumors. Each track describes presence (colored) or absence (off-white, white) of mutation in tumor for gene specified.

Figure 2

PRKACA Leu206 interacts with PRKAR1A. (a) Representative chromatograms of Sanger DNA sequence of normal and tumor DNA for PRKACA codons 205–207; the tumor shows a heterozygous somatic p.Leu206Arg mutation. (b) Multiple sequence alignment of the activation loop of PRKACA. This segment is highly conserved from yeast to humans, with leucine at the position orthologous to Leu206 through invertebrates, and isoleucine in yeast. (c) Crystal structure of PRKACA and inhibitor (PDB: 1ATP). The inhibitory peptide (Arg⁹⁴-Arg-Asn-Ala-Ile⁹⁸) is shown in cyan while the P+1 loop of PRKACA is shown in yellow. Leu206 and Leu199 of PRKACA make van der Waals contacts with Ile98 of the inhibitory peptide, while arginines of the inhibitory peptide form salt bridges with glutamates of the catalytic cleft^,,. (d) Schematic illustration of the key interactions between the regulatory and catalytic subunit (adapted from ref. 10). (e) Close-up view of the interaction between Ile98 and Leu206, and (f) likely rotamer with Arg206 substitution.

Figure 3

PRKACA^L206R does not bind regulatory subunit and shows increased phosphorylation of CREB and ATF1. (a) FLAG-tagged constructs of wild type (Wt) or mutant (M) catalytic subunits (PRKACA^WT and PRKACA^L206R) and the regulatory subunit (PRKAR1A) were expressed in HEK293 cells in indicated combinations. Cell lysates were immunoprecipitated with anti-FLAG and the products of IP were analyzed by Western blotting with antibodies specific for FLAG and PRKAR1A. While IP of PRKACA^WT pulls down the regulatory subunit, IP of PRKACA^L206R does not. Duplicates shown are from independent transfections. (b) Western blots with antibodies specific for CREB phosphorylated at Ser133 (pCREB) and ATF1 phosphorylated at Ser63 (pATF), CREB, ATF1, and FLAG in cells expressing FLAG-tagged PRKAR1A and either wildtype or mutant PRKACA(both tagged with FLAG). Representative results of triplicate experiments are shown. Bar graph shows results of quantitation (mean ± SEM; n = 3). Cells expressing PRKACA^L206R have 3 to 4-fold higher levels of pCREB and pATF1 than cells expressing PRKACA^WT (**P<0.05). neg, no transfection of PRKACA or PRKAR1A.