MYC-driven increases in mitochondrial DNA copy number occur early and persist throughout prostatic cancer progression

Increased mitochondrial function may render some cancers vulnerable to mitochondrial inhibitors. Since mitochondrial function is regulated partly by mitochondrial DNA copy number (mtDNAcn), accurate measurements of mtDNAcn could help reveal which cancers are driven by increased mitochondrial function and may be candidates for mitochondrial inhibition. However, prior studies have employed bulk macrodissections that fail to account for cell type–specific or tumor cell heterogeneity in mtDNAcn. These studies have often produced unclear results, particularly in prostate cancer. Herein, we developed a multiplex in situ method to spatially quantify cell type–specific mtDNAcn. We show that mtDNAcn is increased in luminal cells of high-grade prostatic intraepithelial neoplasia (HGPIN), is increased in prostatic adenocarcinomas (PCa), and is further elevated in metastatic castration-resistant prostate cancer. Increased PCa mtDNAcn was validated by 2 orthogonal methods and is accompanied by increases in mtRNAs and enzymatic activity. Mechanistically, MYC inhibition in prostate cancer cells decreases mtDNA replication and expression of several mtDNA replication genes, and MYC activation in the mouse prostate leads to increased mtDNA levels in the neoplastic prostate cells. Our in situ approach also revealed elevated mtDNAcn in precancerous lesions of the pancreas and colon/rectum, demonstrating generalization across cancer types using clinical tissue samples.

gene expression measures for known mtDNA replication genes.Full details and reporting of the WGS and RNA-seq data will be reported as part of another study.

Differential gene expression analysis and gene set enrichment analysis
Differential gene expression analysis and pathway analysis on RNAseq data from laser capture microdissected prostate samples were performed using DESeq2 (103) and GSVA with zscore method (104).Gene set enrichment analysis on microarray data from cell lines with MYC knockdown (76) was performed using the fgsea R package (105).The mtDNA replication related gene set was obtained from the Mitocarta 3.0 database (106).

Quantitative PCR
Quantitative PCR to measure mtDNAcn from DNA extracted from LCM prostate samples was conducted as described previously (48).Probes used in this study were Hs-MT-CO1 (Thermo Fisher, Hs4331182) as the mitochondrial gene and BGLT3 (Thermo Fisher, Hs01629437) as the nuclear gene reference.
COX and SDH enzyme histochemistry COX and SDH enzyme histochemistry was performed on fresh frozen human prostate tissues.Briefly, fresh prostate tissues were collected, frozen, cryosectioned into 5 μm (SDH) or 10 μm (COX) sections.The frozen slides were air dried at room temperature for 1 hour.COX reaction mix containing 25 µL of 4 mM Cytochrome c, 67 µL of 10 mg/mL 3,3′-diaminobenzidine tetrahydrochloride (DAB) in 1 mL 1X PBS was freshly prepared, followed by addition of 2 µg bovine catalase and vortexing, and added to slides for 40 min at 37 °C in a moist chamber.SDH reaction mix was prepared using 1.5 mM NBT, 130 mM sodium succinate, 0.2 mM PMS, and 1.0 mM sodium azide in 1X PBS.The mixture was applied to tissue slides for 40 min at 37 °C in a moist chamber.The slides were washed in PBS for 4 x 10 min, dehydrated in an ethanol gradient and xylene, mounted and coverslipped in Cytoseal 60.

Supplemental Figure 1 .
Image analysis results showing higher mtDNAcn in normal basal cells compared to normal luminal cells in organ donor prostates.The center line in the box shows the median %mtDNA area in each group.N = 4 regions from 3 organ donors for each cell type.Wilcoxon test, ***P = 0.0005.Supplemental Figure 2. Heterogeneity of mtDNAcn in human prostate.Quantitative image analysis results from individual regions of normal, HGPIN and primary prostate carcinoma.Each symbol represents an individual region of interest examined.N = 40, 24, 45 for normal, HGPIN, and Primary PCa lesions.Kruskal-Wallis test followed by Dunn's test for nonparametric pairwise multiple comparisons, ****P < 0.0001.%mtDNA ISH area Supplemental Figure 3. Intratumoral heterogeneity in a single prostatic tumor nodule.(A) Low power view of a prostatectomy specimen with tumor nodule that is homogeneously positive for ERG protein, indicative of an ERG gene fusion.Original magnification, x10.(B) Marked heterogeneity of mtDNA in situ hybridization signals from different subregions of this tumor using an adjacent slide to that shown in A and corresponding quantitative image analysis results supporting statistically significant heterogeneity among different tumor regions.Kruskal-Wallis test, P = 0.0003.Original magnification, x10 and x70 (boxed regions).Supplemental Figure 4. Increased mtDNAcn occurs in prostatic adenocarcinoma tissues from both Black and White men.(A) Similar increases in mtDNAcn were found in tumors compared to matched benign prostate samples from Black and White men after WGS.N = 43 and 72 for Black and White Patients, respectively.Wilcoxon test, ***P = 0.0003 for Black patients, and **** P < 0.0001 for White patients.(B) Scatter plot shows a strong correlation of mtDNA levels when comparing the results from WGS to qPCR using the same prostate samples from LCM. (C) Relative mtDNAcn by qPCR shows a similar pattern of increased levels in tumor tissue to the results found by in situ hybridization (Fig. 2) and WGS (Fig. 3) (n = 10 patients).Wilcoxon test, **P = 0.0098.(D) Correlation between age and mtDNAcn was observed in prostate cancer samples.Supplemental Figure 5. Increased COX and SDH enzyme activity is observed in prostate cancer compared to normal adjacent glands.(A and C) SDH histochemistry and (B and D) COX histochemistry both reveal increased and heterogenous signals in the cancer regions, similar to the results of mtDNA in situ hybridization.E and F are representative images showing higher SDH and COX enzymatic activities in normal basal cells compared to the normal luminal cells in a different area from the same samples as in A-D.Original magnification, x20 (A and B), x70 (C and D) and x200 (E and F).N = 11 patients.Supplemental Figure 6.MYC regulates a number of genes encoding factors involved in mtDNA replication.(A) Microarray analysis in cell lines after siRNA-based MYC knockdown (59) reveals down regulation of mtDNA replication-related genes from 3 different human prostate cancer cell lines.(B) Publicly available ChIP-seq and RNA-seq data using 22Rv1 prostate cancer cells shows MYC protein binds to the promoter region of genes encoding proteins for mtDNA replication, and MYC occupancy and expression levels were decreased after the cells were treated with a MYC inhibitor.(C) Correlation between MYC and TWNK (encoding TWINKLE) in mCRPC cases in the COMBAT-CRPC study.Each point represents the Log2 mRNA TPM from RNAseq from laser captured frozen tissue biopsy samples.(D) Positive correlations between MYC and many other mtDNA replication related genes were observed by RNAseq using the U01 study samples.Supplemental Figure 9. HGPIN luminal cells exhibit higher mtDNA in situ hybridization signals than normal luminal cells in the human prostate.Tissues were subjected to multiplex CISH-IHC as in Fig. 1.Quantitative image analysis results from individual regions of normal and HGPIN glands in which luminal and basal cells were assessed separately.Nonparametric Mann-Whitney tests were done on luminal cells between HGPIN and normal as well as basal cells in normal vs. luminal cells in HGPIN.n = 2 regions per tissue type from 3 patients.**P = 0.0087.

Supplemental Table 1. Pathology of Cases used in Chromogenic in situ hybridization for mtDNAcn.
* Race is self identified.B = Black patients, W = White patients.** P Stage is the pathological stage at radical prostatectomy using the American Joint Committee on Cancer Staging 2007.

Supplemental Table 2. Demographic and pathological features of 115 patients with the laser captured microdissected prostate samples for whole genome sequencing and RNAseq.
Supplemental Table3.mtDNAcn in matched benign and cancer samples from 115 patients with the laser captured microdissected prostate samples for whole genome sequencing.
* Race is self identified.B = Black patients, W = White patients.** P Stage is the pathological stage at radical prostatectomy using the American Joint Committee on Cancer Staging 2007.