The mutational landscape of lethal castration-resistant prostate cancer

Abstract

Characterization of the prostate cancer transcriptome and genome has identified chromosomal rearrangements and copy number gains and losses, including ETS gene family fusions, PTEN loss and androgen receptor (AR) amplification, which drive prostate cancer development and progression to lethal, metastatic castration-resistant prostate cancer (CRPC). However, less is known about the role of mutations. Here we sequenced the exomes of 50 lethal, heavily pre-treated metastatic CRPCs obtained at rapid autopsy (including three different foci from the same patient) and 11 treatment-naive, high-grade localized prostate cancers. We identified low overall mutation rates even in heavily treated CRPCs (2.00 per megabase) and confirmed the monoclonal origin of lethal CRPC. Integrating exome copy number analysis identified disruptions of CHD1 that define a subtype of ETS gene family fusion-negative prostate cancer. Similarly, we demonstrate that ETS2, which is deleted in approximately one-third of CRPCs (commonly through TMPRSS2:ERG fusions), is also deregulated through mutation. Furthermore, we identified recurrent mutations in multiple chromatin- and histone-modifying genes, including MLL2 (mutated in 8.6% of prostate cancers), and demonstrate interaction of the MLL complex with the AR, which is required for AR-mediated signalling. We also identified novel recurrent mutations in the AR collaborating factor FOXA1, which is mutated in 5 of 147 (3.4%) prostate cancers (both untreated localized prostate cancer and CRPC), and showed that mutated FOXA1 represses androgen signalling and increases tumour growth. Proteins that physically interact with the AR, such as the ERG gene fusion product, FOXA1, MLL2, UTX (also known as KDM6A) and ASXL1 were found to be mutated in CRPC. In summary, we describe the mutational landscape of a heavily treated metastatic cancer, identify novel mechanisms of AR signalling deregulated in prostate cancer, and prioritize candidates for future study.

Figure 1. Integrated mutational landscape of lethal metastatic castrate resistant prostate cancer (CRPC)

Exomes of 50 CRPC (WA3-WA60; three foci from WA43) and 11 high-grade untreated localized prostate cancers (T8-T97) were sequenced to identify somatic mutations and copy number alterations. Heatmap of high-level copy number alterations and non-synonymous mutations. Samples are stratified by ETS status in localized prostate cancer and CRPCs, and ordered by the total number of aberrations in shown genes. ETS gene fusions, RAF/RAS family aberrations, and SPINK1 outlier expression is indicated for all samples (black is present). For each gene, aberrations as indicated are shown (two aberrations in the same gene are indicated by divided boxes). Significantly mutated genes have white names. Mutations in the hypermutated sample WA16 are not shown.

Figure 2. Integrated exome sequencing and copy number analysis highlights novel aspects of ETS genes in prostate cancer biology: deregulation of CHD1 and ETS2

a–b. CHD1 deregulation through deletion or mutation in ETS fusion negative (ETS⁻) prostate cancer. a. Genome wide copy number analysis identified a peak of copy number loss on chr5q21 centered on CHD1 (upper panel, blue bar). The expanded view shows individual samples as rows, with indicated genes represented by boxes. The area and size of each box indicates the copy number call (see legend). Only samples with at least one gene in the region with a called copy number gain/loss are shown, and missing boxes indicate that gene has no called copy number gain/loss. Mutations in CHD1 are indicated according to the legend and samples with focal deletions or mutation of CHD1 are bolded. b. Co-occurrence of CHD1 deregulation (CHD1⁻) and ETS⁺ from the current exome study and 3 aCGH studies (Exome/aCGH), 9 gene expression profiling studies (Gene Expr.), and all studies (All). The total number of samples in each set (n) is shown, and two sided Fisher’s exact tests were performed. c–d. ETS2 is a prostate cancer tumour suppressor deregulated through deletion and mutation. c. WA30 (yellow) harbored a R437C mutation that disrupts a residue conserved in class I ETS transcription factors (red), but not in class IIb or III factors (blue and black, respectively). R437 (yellow) contacts DNA (blue and magenta) in the ETS domain (brown), as shown by the structure of the homologous residue in ETS1 (R409, PDB:3MFK). d. VCaP prostate cancer cells (ERG⁺) stably expressing ETS2 (wild type [wt] or R437C) or LACZ were evaluated for migration (left panel, n=8), invasion (middle panel, n=12) and proliferation (right panel, n=4). For migration and invasion, fold change relative to VCaP LACZ was plotted. For each experiment, mean ± S.E. is plotted; * and ** indicate p<0.05 or <0.001 from two tailed t-tests.

Figure 3. Castrate resistant prostate cancer (CRPC) harbors mutational aberrations in chromatin/histone modifiers that physically interact with AR

Deregulation through mutation or high-level copy aberrations of multiple chromatin/histone modifying genes was identified (see Fig. 1). a. Interaction of deregulated chromatin/histone modifiers with AR. AR (or IgG as control) was immunoprecipitated from VCaP cells and Western blotting for the indicated chromatin/histone modifier was performed. 1% lysate was used as control. EZH2 and FOXA1 were used as negative and positive controls, respectively. b. VCaP cells were treated with siRNAs against MLL or ASH2L (or non-targeting as control), starved, stimulated with vehicle or 1nm R1881 for the indicated times and harvested. qPCR for KLK3 (PSA) expression (relative to vehicle) is plotted (n=3, mean + S.E.). c. Summary of genes interacting with AR that are deregulated in CRPC. Frequency of high copy alterations, somatic mutations, and both aberration types according to the color scales, are shown for chromatin/histone modifiers, the AR collaborating factor FOXA1 and ERG. MLL aberration frequency includes MLL, MLL2, MLL3 and MLL5. Genes encoding AR interactors identified in this and previous studies are indicated by bold and regular arrows, respectively.

Figure 4. Recurrent mutations in the androgen receptor (AR) collaborating factor FOXA1 promote tumour growth and affect AR signaling

a. Exome sequencing and subsequent screening of 147 prostate cancers (101 treatment naïve localized and 46 CRPCs) identified 5 samples with FOXA1 mutations, and transcriptome sequencing of 11 prostate cancer cell lines identified indels in LAPC-4 and DU-145 (shown in black). Locations of mutations are indicated on the domain structure of FOXA1 (TA= transactivation domains). b. Wild type FOXA1 (wt, black) and FOXA1 mutants observed in clinical samples were stably expressed in LNCaP cells as N-terminal FLAG fusions (empty vector, purple, as control). Western blotting with anti-FLAG antibody confirmed expression. c. Cell proliferation in 1% charcoal-dextran stripped serum with 10 nM DHT was measured by WST-1 colorimetric assay. Mean + S.E. (n=4) is plotted. d. FOXA1 wild-type and mutations identified in prostate cancer repress androgen signaling. Indicated LNCaP cells were treated with vehicle or 10 nM DHT for 48 hrs prior to expression profiling. The heatmap shows probes with >2 fold change after DHT stimulation in LNCaP vector DHT/vehicle cells. Probes were clustered using centroid linkage. For each FOXA1 mutant (or wild-type) DHT/vehicle condition, the percentage of filtered probes showing <1.5 or >-1.5 fold change (indicating repression) is indicated. e. Subcutaneous xenografts were generated from LNCaP cells stably expressing LACZ (control, purple), or N-terminally HA-tagged FOXA1 (wild type [wt] or S453fs). Tumour volume is plotted and representative tumours are shown. Mean + S.E. (n=3) is plotted; * indicates p < 0.05 from two tailed t-test.