DNA-PKcs-Mediated Transcriptional Regulation Drives Prostate Cancer Progression and Metastasis

Abstract

Emerging evidence demonstrates that the DNA repair kinase DNA-PKcs exerts divergent roles in transcriptional regulation of unsolved consequence. Here, in vitro and in vivo interrogation demonstrate that DNA-PKcs functions as a selective modulator of transcriptional networks that induce cell migration, invasion, and metastasis. Accordingly, suppression of DNA-PKcs inhibits tumor metastases. Clinical assessment revealed that DNA-PKcs is significantly elevated in advanced disease and independently predicts for metastases, recurrence, and reduced overall survival. Further investigation demonstrated that DNA-PKcs in advanced tumors is highly activated, independent of DNA damage indicators. Combined, these findings reveal unexpected DNA-PKcs functions, identify DNA-PKcs as a potent driver of tumor progression and metastases, and nominate DNA-PKcs as a therapeutic target for advanced malignancies.

Figure 1. DNA-PKcs binds AR and is recruited to sites of AR action

(A) C4-2 cells were treated with ADT (CSS) for 24 hrs and immunoblot analysis for phospho-S2056 DNA-PKcs, total DNA-PKcs, and Ku70, performed. (B,C) C4-2 cells in hormone proficient media were (B) harvested for ChIP-qPCR analysis and percent (input) occupancy of AR (left) or DNA-PKcs (right) reported or (C) treated with 10nM DHT and harvested for ChIP-qPCR analysis with percent (input) occupancy of AR, DNA-PKcs, p300, or RNPII set relative to control at each time point. (D) C4-2 cells were treated with 10nM DHT and relative transcript expression analyzed as normalized to GAPDH mRNA at each timepoint. (E,F) C4-2 cells were treated with 10nM DHT for 6 hrs and co-immunoprecipitation performed in the absence (E) or presence (F) of 50μg/mL ethidium bromide. Data are reported as mean +/− SD. *p<0.05 **p<0.01. See also Fig S1.

Figure 2. DNA-PKcs selectively impacts gene expression in CRPC

(A) RNA harvested from C4-2 cells depleted of DNA-PKcs or treated with 1μM NU7441 (DNA-PKcsi) for 24 hrs was analyzed by microarray analysis (left). Immunoblot of phospho-S2056 DNA-PKcs, total DNA-PKcs, and Ku70 after knockdown or NU7441 treatment (right). (B) Genes identified as upregulated (left) or downregulated (right) by ≥1.5 fold compared to untreated. (C,D) GSEA motif (left) or gene ontology (right) analyses of all genes altered at least 1.5-fold after DNA-PKcs knockdown.

Figure 3. DNA-PKcs and AR cooperate to suppress UGT enzyme expression in CRPC

(A) GSEA KEGG pathway analysis of genes upregulated by ≥1.5 fold compared to control after DNA-PKcs knockdown. (B) Heat map of transcript change of UGT enzymes in the DNA-PKcs knockdown groups. (C,D) C4-2 cells depleted of DNA-PKcs were harvested for ChIP-qPCR analysis and percent (input) occupancy of AR (D, left) or DNA-PKcs (D, right) at indicated loci reported, TSS= transcriptional start site. (E,F) CRPC cells depleted of DNA-PKcs were subject to either qPCR (E, C4-2 left, 22Rv1 right) or immunoblot (F, C4-2) analysis. (G) Free (left) and G-DHT (right) levels in C4-2 cells depleted of DNA-PKcs were determined by HPLC. (H) Tumor samples were profiled for mRNA expression of DNA-PKcs, UGT2B15, and UGT2B17 and correlation coefficients determined. Data are reported as mean +/− SD. *p<0.05 **p<0.01. See also Fig S2.

Figure 4. DNA-PKcs promotes pro-metastatic signaling

(A) GSEA KEGG pathway analysis of genes downregulated by ≥1.5 fold compared to control after DNA-PKcs knockdown. (B,C) Heat map of transcript change of pathways in cancer (B) or focal adhesion (C) pathway genes in the DNA-PKcs knockdown groups. (D) C4-2 and PC3-ML cells in hormone proficient or LNCaP cells in hormone deficient media treated with siDNA-PKcs or siControl were subject to qPCR analysis with control data set to 1 for each cell line. (E) Immunoblot analyses of C4-2 cells depleted of DNA-PKcs or treated with 1μM NU7441. (F) C4-2 cells depleted of ATM were harvested for qPCR analysis with relative expression of indicated transcripts analyzed and normalized to GAPDH. (G) C4-2 cells harvested for ChIP-qPCR analysis and percent (input) occupancy of DNA-PKcs at the indicated regulatory regions. (H) C4-2 cells depleted of DNA-PKcs or treated with 1μM NU7441 for 48 hrs were subject to immunoblot analysis. (I) C4-2 cells depleted of DNA-PKcs or treated with 1μM NU7441 for 48 hrs were analyzed for activated (GTP-bound) Rho and Rac1 by column binding followed by immunoblot. Data are reported as mean +/− SD. *p<0.05, **p<0.01. See also Fig S3.

Figure 5. DNA-PKcs induces metastatic phenotypes

(A) Cells depleted of DNA-PKcs were seeded into hormone deficient media and allowed to migrate (left) or invade through matrigel (right) towards hormone proficient media. (B) Cells pretreated with 1μM NU7441, SLx-2119 or combination of both for 24 hrs were seeded into hormone deficient media and allowed to migrate for 24 hrs (top) or invade through matrigel for 72 hrs (bottom) towards hormone proficient media. Data are reported as mean +/− SD. *p<0.05, **p<0.01 compared to control unless otherwise indicated. See also Fig S4.

Figure 6. DNA-PKcs inhibitors delay formation of metastases in vivo

(A) Mice were injected with luciferin 31 days post tail vein injection of PC3-ML cells and imaged using the IVIS imaging system with total luciferase signal reported (left) and representative images shown (right). Indicated mice were selected for crossover studies. (B,C) Mice were injected with luciferin and imaged for 2 weeks after initiation of crossover studies with total luciferase signal reported (left), representative images shown (middle), and average doubling times pre and post crossover calculated. (D) CASP-NPK-YFP tumors were measured twice weekly for 30 days after initiation of treatment (end point for survival was the predefined tumor volume of 1.5cm³) with volumes calculated using the formula volume=(width)²xlength/2. (E) At time of sacrifice, metastases were documented ex vivo in the lungs and livers by visualizing fluorescence with the total number of metastatic nodules for the lungs and livers assessed. (F) CASPNPK-YFP tumors were harvested for qPCR analysis with the indicated transcripts set relative to Gapdh mRNA. Data are reported as mean +/− SD. **p<0.01. See also Fig S5.

Figure 7. DNA-PKcs inhibition modulates expression of pro-metastatic factors in primary human disease

(A) Schematic of explant assay, adapted from (Schiewer et al., 2012). (B) Representative images of explant tissues treated with control or 1μM NU7441 and stained with hematoxylin & eosin. (C) Explant tissues were harvested on day 6 for qPCR analysis with indicated transcripts set relative to GAPDH. Data are reported as mean +/− SD. *p<0.05.

Figure 8. DNA-PKcs is associated clinically with disease recurrence and metastases

(A-C) Tumor samples were profiled for DNA-PKcs mRNA, which was split into high vs. low by the 80^th percentile for Kaplan Meier analysis. (D-G) GSEA analyses showed enrichment of the AR pathway (D), MAZ (E) and SP1 (F) targets, and the focal adhesion pathway (G) in genes correlated to DNA-PKcs. (H) DNA-PKcs and histone H2AX phosphorylation were measured by mass spectrometry in organ confined, treatment naïve PCa and metastatic CRPC tissues. (I) DNA-PKcs modulates cancer-associated transcriptional networks, inducing expression of AR targets and genes that regulate pro-metastatic Rho/Rac signaling pathways and suppressing expression of UGT enzymes known to impact DHT metabolism, identifying DNA-PKcs as a clinically actionable driver of metastatic CRPC. ***p<0.001, *p<0.05. See also Fig S6, Table S1, S2.