PINK1-Mediated Mitochondrial Activity Confers Olaparib Resistance in Prostate Cancer Cells

Abstract

Olaparib, a PARP inhibitor, is effective against various cancers, including prostate cancer. However, resistance to olaparib poses a significant challenge. This study uncovers that mitochondrial alterations and PINK1 gene overexpression contribute to this resistance in prostate cancer cells. Enhanced mitochondrial functionality and increased PINK1 expression in olaparib-resistant cells underscore the importance of targeting mitochondrial dynamics and PINK1 to develop more effective treatments for overcoming olaparib resistance in prostate cancer.

Figure 1

Gene enrichment in olaparib-resistant derived C4-2B cells (2B-OlapR) related to OxPhos. A, Hallmark collection gene sets in 2B-OlapR cells, with the OxPhos gene set the most enriched. B–E, Gene set expression heatmap for full set of the genes (left) in parental and resistant 2B cells, with the top 15 genes listed (right), and GSEA plots with summary statistics for the following: GOBP OxPhos (B), GOBP ATP synthesis coupled electron transport (C), GOBP Mitochondrial Electron Transport NADH to Ubiquinone (D), and the Hallmark OxPhos (E) from the MSigDB. FWER, family-wise error rate; NES, normalized enrichment score.

Figure 2

Seahorse Mito Stress Test assay results comparing C4-2B and 2B-OlapR cells at basal state and with mitochondrial inhibitor supplementation to deduce relative function. A and B, Mito Stress Test summary graphic representing differential OCR and extracellular acidification rate between naïve and resistant cell lines under normal conditions and with the addition of mitochondrial inhibitors. C, Cellular OCR at basal state is increased in the olaparib-resistant cell line. D, Amount of oxygen consumption utilized for ATP production, derived through OCR difference before and after oligomycin (ETC complex V inhibitor) is added. 2B-OlapR displays increased oxygen consumption related to ATP production. E, Change in the OCR between basal readings and maximal readings after FCCP (mitochondrial membrane uncoupler) is added. 2B-OlapR cells exhibit greater ability to increase respiration under mitochondrial insult. F, Difference in cellular OCR reading after rotenone and antimycin A (complex I and III inhibitors) are added, eliminating all ETC-based oxygen consumption. 2B-OlapR cells show amplified maximal respiration compared with parental C4-2B. G, Difference between readings after oligomycin addition and the readings after antimycin A/rotenone addition, inferring oxygen consumption not ultimately utilized for ATP production. The rate in 2B-OlapR cells is almost twice that in C4-2B. ECAR, extracellular acidification rate. $**, P\le 0.01; ***, P\le 0.001; ****, P\le 0.0001$.

Figure 3

Mitochondrial live stain and ETC functional test demonstrate higher function in 2B-OlapR cells. A, Left, MitoTracker staining counterstained with nuclear-specific DAPI reveals higher amounts of relative mitochondrial mass in 2B-OlapR cells over C42B. 10× images shown, with 20× crops within. Right, TCF ratio of MitoTracker to DAPI displaying quantification of relative increased active mitochondria in the resistant cell subline. B, Left, ETCC1 kit assay results express a higher rate of ETCC1 activity in 2B-OlapR cells. Right, Reported rate of change in NAD+ associated absorbance between 300 and 2,000 seconds show significant increased ETCC1 rate in 2B-OlapR cells. $***, P\le 0.001; ****, P\le 0.0001$.

Figure 4

PINK1 expression is increased in 2B-OlapR cells and associated with negative patient prognosis. Treating 2B-OlapR cells with siPINK1 ASOs decreases PINK1 expression and protein level, as well as cell growth, and improves olaparib efficacy. A, Transcriptomic data reveal increased expression of PINK1 in the 2b-OlapR subline. B, qRT-PCR verification of increased PINK1 gene expression in 2b-OlapR cells. C, Western immunoblot analysis showing increased cellular PINK1 protein in 2b-OlapR cells. D, Deceased patients with prostate cancer have higher amounts of tumor PINK1 expression (Abida 2019). E, High-PINK1–expressing striated patients with prostate cancer show lower survival over time (GSE21032). F, 2B-OlapR cells treated with siPINK1 show lower mRNA expression through qRT-PCR (left) and decrease in cellular protein (right). G, siPINK1 treatment decreases growth of 2B-OlapR cells and increases relative olaparib efficacy at 5 μmol/L. $*, P\le 0.05; **, P\le 0.01; ***, P\le 0.001; ****, P\le 0.0001$.

Figure 5

Seahorse Mito Stress Test assay results comparing 2B-OlapR cells with and without siPINK treatment at basal state and with mitochondrially relevant pharmacologic supplementation to deduce relative function convey decreased function after PINK1 expression ablation. A and B, Mito Stress Test summary graphic representing differential OCR and extracellular acidification rate between nonspecific and siPINK1-targeted OlapR cells. C, Cellular OCR at basal state is greatly reduced with PINK1 excision. D, ATP production–associated oxygen consumption is markedly reduced in 2B-OlapR cells treated with siPINK1. E and F, PINK1 expression reduction reveals consequential decreased spare capacity for respiration, as well as lessened maximal rate of ETC-based oxygen consumption. G, Decreasing PINK1 levels leads to less proton leakage out of the inter-mitochondrial membrane space inferred by OCR reading differences after oligomycin addition and after rotenone and antimycin addition. ECAR, extracellular acidification rate. $**, P\le 0.01; ***, P\le 0.001$.

Figure 6

Inhibiting PINK1 expression decreases relative active mitochondria and lowers ETCC1 activity. A, Left, 2B-OlapR cells treated with siPINK1 show lower relative positive MitoTracker staining. 10× images shown, with 20× crops within. Right, MitoTracker to DAPI TCF ratio is reduced in the siPINK1 group. B, Left, Decreased PINK1 expression results in lower ETCC1 activity. Right, siPINK1-dosed 2B-OlapR cells report decreased the ETCC1 activity rate. $**, P\le 0.01; ****, P\le 0.0001$.