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Activating hotspot L205R mutation in PRKACA and adrenal Cushing’s syndrome

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Science  23 May 2014:
Vol. 344, Issue 6186, pp. 913-917
DOI: 10.1126/science.1249480

Abstract

Adrenal Cushing’s syndrome is caused by excess production of glucocorticoid from adrenocortical tumors and hyperplasias, which leads to metabolic disorders. We performed whole-exome sequencing of 49 blood-tumor pairs and RNA sequencing of 44 tumors from cortisol-producing adrenocortical adenomas (ACAs), adrenocorticotropic hormone–independent macronodular adrenocortical hyperplasias (AIMAHs), and adrenocortical oncocytomas (ADOs). We identified a hotspot in the PRKACA gene with a L205R mutation in 69.2% (27 out of 39) of ACAs and validated in 65.5% of a total of 87 ACAs. Our data revealed that the activating L205R mutation, which locates in the P+1 loop of the protein kinase A (PKA) catalytic subunit, promoted PKA substrate phosphorylation and target gene expression. Moreover, we discovered the recurrently mutated gene DOT1L in AIMAHs and CLASP2 in ADOs. Collectively, these data highlight potentially functional mutated genes in adrenal Cushing’s syndrome.

Candidate Cushing's culprit identified

Cushing's syndrome is a rare condition resulting from the excess production of cortisol. About 15% of Cushing's syndrome cases are associated with an adrenocortical tumor. However, the genetic etiology of these adrenocortical tumors is ill defined (see the Perspective by Kirschner). Cao et al. and Sato et al. both performed whole-exome sequencing of tumors from individuals with adrenal Cushing's syndrome and compared it with the patient's own matched non-tumor DNA and identified recurrent mutations in the protein kinase A catalytic subunit alpha (PRKACA) gene, as well as less frequent mutations in other putative pathological genes. The most common recurrent mutation activated the kinase, which may suggest a potential therapeutic target.

Science, this issue p. 913, p. 917; see also p. 804

Cushing’s syndrome is caused by excessive glucocorticoid production, which may lead to a series of metabolic disorders such as obesity, glucose intolerance, and hypertension. Adrenal Cushing’s syndrome results from autonomous production of cortisol from adrenocortical tumors (ACTs), which are most common in adult females. Cortisol-producing ACTs include benign adrenocortical adenoma (ACA), malignant adrenocortical carcinoma (ACC), and rare forms of bilateral adrenal hyperplasia (BAH) and adrenocortical oncocytoma (ADO) (1). BAHs consist of macronodular and micronodular hyperplasias, such as adrenocorticotropin-independent macronodular adrenocortical hyperplasia (AIMAH) and primary pigmented nodular adrenocortical disease (PPNAD).

Several genetic alterations have been described in inherited or sporadic BAHs. The inactivating mutations in PRKAR1A [cyclic adenosine monophosphate (cAMP)–dependent protein kinase regulatory subunit type Iα] are dominant in patients with Carney complex and PPNAD. Germline or somatic mutations in phosphodiesterase genes (PDE11A and PDE8B) and in GNAS are associated with adrenal hyperplasia (2, 3). However, the genetic architecture of adrenal Cushing’s syndrome remains largely uncharacterized, hampering the development of diagnostic and therapeutic approaches for Cushing’s syndrome.

To investigate the genetic lesions in adrenal Cushing’s syndrome, we performed whole-exome sequencing of DNA from 49 cortisol-producing ACTs and matched blood pairs, including 39 ACAs, 7 AIMAHs, and 3 ADOs (table S1). The histological subtypes of all tumors were confirmed by pathological examination. The average sequencing depth was 169× (82× to 277×), and 95.61% (91.20 to 99.50%) of the target regions were covered by at least 10× (table S2 and fig. S1). Meanwhile, we conducted RNA sequencing (RNA-seq) on 44 ACTs, including 36 whole-exome sequenced tumors, and obtained an average of 12.6 (9.4 to 17.4) Gb of sequenced data per sample (table S3).

We identified a total of 425 candidate somatic mutations among the 49 samples, including 97 synonymous, 298 missense, 15 nonsense, 3 splicing, 4 frameshift, and 8 in-frame, resulting in an average of 8.7 (0 to 28) somatic mutations in exonic regions (tables S4 and S5). We validated 55 of 57 predicted somatic mutations (96.5%) by Sanger sequencing and also verified a subset of highly expressed mutations using RNA-seq data (table S4). The predominant substitution in these mutations was the C:G > T:A transversion, consistent with most cancer types (fig. S2).

We defined 12 potentially functional mutated genes, including 8 recurrently mutated genes, 3 consensus cancer genes with potentially damaging mutations, and the only mutated gene in AIMAH3 (4). PRKACA (cAMP-dependent protein kinase catalytic subunit α) was the only significantly mutated gene in our study (false discovery rate q = 3.77 × 10–11) (4). Strikingly, a hotspot somatic c.T617G/p.L205R mutation in PRKACA was discovered in 55.1% of sequenced ACTs (27 of 49) and exclusively in 69.2% of ACAs (27 of 39). Further screening revealed this somatic c.T617G/p.L205R mutation in 65.5% of ACAs (57 of 87) and none in AIMAHs (n = 13), ACCs (n = 16), and ADOs (n = 3) by Sanger sequencing (fig. S3 and table S6). The L205R (Lys205 → Arg) mutations were mainly detected in adult females (Fisher’s exact test, P = 0.017), and there were no significant differences in serum cortisol, ACTH, and urinary free cortisol levels between ACA patients with and without the PRKACA mutation (tables S7 and S8). In addition, only one somatic PRKAR1A mutation was observed in an ACA. Thus, the PRKACA L205R mutation was believed to be the dominant genetic alteration in sporadic ACAs.

Activating CTNNB1 mutations are frequently observed in benign and malignant ACTs (5). Germline APC mutations in patients with familial adenomatous polyposis could result in the emergence of ACT (6). We identified activating CTNNB1 mutations (Ser33 → Phe, Ser45 → Ala) in ACA and ADO, and an APC truncating mutation in ACA (fig. S4 and table S4). In addition, activating mutations in Arg201 and Gln227 of GNAS (encodes stimulatory G protein α subunit), resulting in PKA activation, have been reported in McCune-Albright syndrome and in adrenal and pituitary tumors (7, 8). We found two activating GNAS mutations (Arg201 → His, Arg201 → Cys) in two PRKACA wild-type ACAs (Fig. 1 and table S4).

Fig. 1 PRKACA L205R mutations in ACAs and landscape of somatic mutations in cortisol-producing ACTs.

Candidate cancer genes (4) are listed vertically on the left. Columns represent samples, with the average mutation rate in exon regions at the top. Samples are arranged from left to right by subtypes and mutation numbers. Histological subtypes and gender of each patient are reported at the bottom (green, ACA; red, AIMAH; orange, ADO; purple, female; blue, male). Colored rectangles indicate mutation category observed in the cohort. Asterisks indicate significantly mutated genes in tumor subtypes (PRKACA, 28/39 in ACA; DOT1L, 2/7 in AIMAH; CLASP2, 2/3 in ADO).

We also uncovered novel mutated genes in cortisol-producing ACTs. In the ACAs, we identified mutations in cancer-related genes, including ARID1A and STAT3. We also detected recurrently mutated GPR98, colocalizing with PRKACA mutations in ACAs. In the AIMAHs, we identified two mutations in the highly conserved methyltransferase domain of DOT1L, a histone H3 Lys79 methyltransferase (Fig. 1 and fig. S5). DOT1L regulates gene transcription and cell proliferation and mediates leukemic transformation in MLL (mixed lineage leukemia)–rearranged leukemias (9, 10). Moreover, the histone deacetylase HDAC9 was mutated in one AIMAH3 (table S4). These data suggest that deregulated chromatin modification might contribute to tumorigenesis of AIMAH. A recent study revealed recurrent ARMC5 mutations in AIMAHs (11), whereas DOT1L and HDAC9 mutations have not been reported. Functional adrenocortical oncocytoma causing Cushing’s syndrome is exceptionally rare. We found CLASP2 mutated in two of three ADOs, in which we predict the CLIP-associating proteins (CLASPs) to serve a functional role in cell division (12). In addition, we also observed PRKAR1A and CTNNB1 mutations in ADOs and PRUNE2 mutations in both ACA and AIMAH (Fig. 1). Taken together, our findings indicate that multiple genes may be responsible for adrenal Cushing’s syndrome.

The L205R mutation in PKA catalytic subunit (PKA C) appears to be a dominant genetic characteristic of ACAs, although PKA C mutation has not been associated with a human disease. Structure analyses of PKA C shows that Lys205 is a component of the P+1 loop (Lys198-Lys205), which controls the specific binding between the kinase and its substrates and mediates the communication between catalytic residues and their substrates (1320). The highly conserved P+1 loop and activation loop constitute a conserved protein kinase core and contain the activation segment [DFG (Asp-Phe-Gly) to APE (Ala-Pro-Glu) motif] (fig. S6). Previous studies have demonstrated that the Tyr204 → Ala mutation in the P+1 loop altered the enzyme catalysis and phosphoryl transfer of substrate (1618). Lys198, Pro202, and Lys205 in the P+1 loop form a hydrophobic binding pocket for accommodating the hydrophobic side chain of the P+1 residue in the substrate consensus (17). Crystal structure analyses of the complex between PKA C and regulatory subunit (PKA R) and of PKA C in complex with substrate peptide (Fig. 2) suggested that L205R changed the surface for substrate binding (15, 19, 20). Together, the structure-based analyses indicate that the L205R mutation might alter the PKA activity by affecting the substrate contact dynamics and catalytic efficiency of PKA C in a manner that contributes to ACA formation.

Fig. 2 L205R mutation occurs in the catalytic subunit of PKA (PKA C).

Crystal structures of the interaction between catalytic and regulatory subunits of PKA (left, PDB: 2QCS) and a binding substrate peptide (right, PDB: 1L3R) are shown. The L205R mutation in the P+1 loop is shown in pink; the substrate peptide derived from inhibitor PKI is in red.

We analyzed RNA-seq data and obtained 232 genes differentially expressed between PRKACA mutant and wild-type ACAs (Fig. 3A and table S9) (4). Of note, we did not observe a significantly expressed change of PRKACA, which was further supported by quantitative polymerase chain reaction (qPCR) quantification (Fig. 3B). Pathway analysis of these 232 genes using DAVID and WebGestalt (tables S10 and S11) nominated the associated Gene Ontology terms of “biosynthesis and metabolism of steroid and cholesterol” and “response to chemical stimulus.” The genes StAR, MC2R, GSTA1, CXCL2, and S100A8/9 involved in these two terms were significantly up-regulated in the PRKACA L205R mutant ACAs (fig. S7). These genes may participate in tumor growth, survival, and adrenal steroidogenesis (2124). Especially, the mRNA and protein levels of StAR were markedly elevated in the PRKACA L205R mutant ACAs relative to normal adrenocortical tissues and PRKACA wild-type tumors (Fig. 3, B and C). Furthermore, Western blot analysis showed significantly increased phosphorylation of PKA substrates in ACAs with the L205R mutation (Fig. 3D). Together with the findings that PKA activation can promote StAR expression as well as phosphorylate and activate StAR (25), our results indicate that the L205R mutation triggers the activation of PKA, which promotes tumorigenesis and steroidogenesis through phosphorylation of substrates.

Fig. 3 PRKACA L205R mutation activates PKA signaling in ACAs.

(A) Heat map of significantly differentially expressed genes identified by RNA-seq in ACAs. Columns represent samples; genes are in rows. The mutation status of PRKACA for each sample is reported at the top (blue, wild type; red, L205R). Colors of rectangles represent gene expression levels (red, up-regulation; green, down-regulation; black, no change). Samples are arranged by the clustering of gene expression patterns. (B) Quantitative reverse transcription PCR analysis of PRKACA and StAR in normal human adrenocortical tissues (green), L205R mutant ACAs (red), and wild-type ACAs (blue) (normal, n = 10; L205R, n = 20; wild type, n = 9). (C) Elevated protein level of StAR in L205R mutant ACAs. (D) The phosphorylation of PKA substrates is enhanced in L205R mutant ACAs. (E) The PKA activity is significantly higher in L205R mutant ACAs (n = 5). Data are means ± SD; t test, *P < 0.05, **P < 0.01, ***P < 0.001.

To further investigate the function and characteristic features of the PRKACA L205R mutation, we performed gain-of-function experiments in 293T cells. Overexpression of L205R mutants significantly enhanced the phosphorylation of PKA substrates relative to the wild type (Fig. 4A). The activity of wild-type and mutated PKA C could be suppressed by the selective inhibitor H89 (fig. S8). L205R mutants induced more phosphorylation of CREB (cAMP response element binding protein), which initiates transcription of target genes such as StAR and MC2R (Fig. 4B) (25). In addition, the phosphorylation of PKA C at Thr197, which is critical for catalytic activity of PKA (26), was facilitated by the L205R mutation (Fig. 4C). Coimmunoprecipitation assays showed that mutated PKA C was pulled down by PKA R, indicating that the interaction was not interrupted by the L205R mutation (Fig. 4D).

Fig. 4 PRKACA L205R mutation promotes PKA activation and substrate phosphorylation.

(A) Overexpression of L205R-mutated PKA C increases phosphorylation of PKA substrates relative to the wild type in 293T cells. (B) Overexpression of L205R-mutated PKA C promotes phosphorylation of CREB in 293T cells. (C) The phosphorylation of Thr197 is elevated in L205R-mutated PKA C. (D) Coimmunoprecipitation of wild type, L205R-mutated PKA C, and Flag-tagged regulatory subunit of PKA (PKA R) in 293T cells. Protein lysates were prepared and immunoprecipitated with antibody to Flag or control immunoglobulin G, respectively. (E and F) Coexpression of PKA R and PKA C in 293T cells. L205R-mutated PKA C leads to more phosphorylation of PKA substrates (E) and CREB (F). (G and H) L205R-mutated and wild-type PKA C were overexpressed in 293T cells for PKA activity assay. The activity of L205R-mutated PKA C is significantly higher (G). The peptide inhibitor PKI inhibits the activity of L205R-mutated PKA C (H).

Furthermore, coexpression of L205R mutants and PKA R exhibited more activity than the wild type upon phosphorylation of PKA substrates and CREB in 293T cells, although protein levels of phosphorylated PKA C were comparable (Fig. 4, E and F). PKA activity assays demonstrated that the L205R mutants induced a significant increase in PKA activity, whereas the substrate competitive peptide inhibitor (PKI) could block the catalytic activity of mutated PKA C (Fig. 4, G and H). Taken together, these results indicated that the L205R mutation enhanced the phosphorylation catalysis of PKA.

The identification of activating mutations in oncogenes is particularly valuable in developing targeted therapies for cancer. PKA signaling plays a pivotal role in various physiological and pathological processes, including development, metabolism, and tumorigenesis. Although inactivating mutations in PRKAR1A and phosphodiesterase genes are known in adrenal hyperplasia, we revealed the activating PRKACA L205R mutation and several potential functional mutated genes in ACTs. Future investigations on mutagenesis and hormones or environmental initiators of the L205R mutation may provide clues for prevention or treatment of Cushing’s syndrome.

Supplementary Materials

www.sciencemag.org/content/344/6186/913/suppl/DC1

Materials and Methods

Figs. S1 to S8

Tables S1 to S11

References (2740)

References and Notes

  1. See supplementary materials on Science Online.
  2. Acknowledgments: Supported by Natural Science Foundation of China grants 30900702, 81130016, and 81270859, and by Guangdong Innovative Research Team Program grant 2009010016. The sequencing data have been deposited in the European Genome-phenome Archive (accession no. EGAS00001000712).
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