Abstract Background: Castration-resistant prostate cancer (CRPC) is an incurable disease and a leading cause of cancer death in men worldwide. Olaparib (Lynparza) was among the first PARP inhibitors (PARPi) approved for the treatment of CRPC tumors harboring DNA repair defects. However, clinical resistance to PARPi’s has been documented. The mechanisms underlying resistance to PARPi’s remain elusive. To study acquired resistance, we developed olaparib-resistant LN-OlapR and 2B-OlapR cell lines generated through chronic olaparib treatment of the olaparib-sensitive cell lines LNCaP and C4-2B, respectively. RNA-seq revealed IGFBP3 is overexpressed in both OlapR cell lines. IGFBP3 overexpression is correlated with poor clinical outcome and is thought to participate in DNA repair pathways. IGFBP3 plays a key role in nonhomologous end joining (NHEJ) repair through a ternary complex with EGFR and DNA-PKcs. The IGFBP3/EGFR signaling axis is thought to modulate NHEJ repair and could have implications for PARPi sensitivity. We hypothesize that increased IGFBP3 expression promotes PARPi resistance by enhancing DNA repair capacity. Methods: RNA-sequencing and gene set enrichment analysis were used to determine the expression profile changes in resistant cells compared to parental cells. Real time PCR and western blots confirmed the expression of DNA damage repair genes such as γH2AX, EGFR, and DNA-PKcs. ELISA was used to determine IGFBP3 secretion. RNAi was used to inhibit IGFBP3 and EGFR expression. Gefitinib was used to inhibit EGFR activity. Cell viability assays were used to assess cell growth. Results: Transcriptomic profiling revealed that IGFBP3 is highly expressed in resistant models. We verified increased levels of IGFBP3 RNA and protein in both OlapR models. We found that RNAi inhibition of IGFBP3 increases γH2AX and cleaved-PARP protein levels in the resistant models, which suggests accumulation of DNA double strand breaks (DSBs) leading to genomic instability and cell death. We discovered increased phosphorylation of EGFR and DNA-PKcs in the resistant cells. Furthermore, silencing/inhibiting IGFBP3 and EGFR reduces OlapR cell viability and resensitizes resistant cells to treatment. Conclusions: Our findings demonstrated that inhibiting IGFBP3 and EGFR aids in PARPi sensitivity in the resistant setting. Future work will utilize OlapR models to study how the IGFBP3/EGFR/DNA-PKcs protein complex promotes the development of resistance. Understanding the role of IGFBP3 in PARPi resistance will enhance our ability to re-sensitize resistant CRPC to PARPi therapeutics. Citation Format: Amy R. Leslie, Shu Ning, Leandro S. D'Abronzo, Cameron Armstrong, Masuda Sharifi, Zachary A. Schaaf, Wei Lou, Christopher P. Evans, Hong-Wu Chen, Alan Lombard, Allen C. Gao. IGFBP3 promotes resistance to olaparib via modulating EGFR signaling in advanced prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3862.
<div>Abstract<p>The next-generation antiandrogen drugs such as enzalutamide and abiraterone extend survival times and improve quality of life in patients with advanced prostate cancer. However, resistance to both drugs occurs frequently through mechanisms that are incompletely understood. Wnt signaling, particularly through Wnt5a, plays vital roles in promoting prostate cancer progression and induction of resistance to enzalutamide and abiraterone. Development of novel strategies targeting Wnt5a to overcome resistance is an urgent need. In this study, we demonstrated that Wnt5a/FZD2-mediated noncanonical Wnt pathway is overexpressed in enzalutamide-resistant prostate cancer. In patient databases, both the levels of Wnt5a and FZD2 expression are upregulated upon the development of enzalutamide resistance and correlate with higher Gleason score, biochemical recurrence, and metastatic status, and with shortened disease-free survival duration. Blocking Wnt5a/FZD2 signal transduction not only diminished the activation of noncanonical Wnt signaling pathway, but also suppressed the constitutively activated androgen receptor (AR) and AR variants. Furthermore, we developed a novel bioengineered BERA-Wnt5a siRNA construct and demonstrated that inhibition of Wnt5a expression by the BERA-Wnt5a siRNA significantly suppressed tumor growth and enhanced enzalutamide treatment <i>in vivo</i>. These results indicate that Wnt5a/FZD2 signal pathway plays a critical role in promoting enzalutamide resistance, and targeting this pathway by BERA-Wnt5a siRNA can be developed as a potential therapy to treat advanced prostate cancer.</p></div>
Supplementary Data from Bioengineered BERA-Wnt5a siRNA Targeting Wnt5a/FZD2 Signaling Suppresses Advanced Prostate Cancer Tumor Growth and Enhances Enzalutamide Treatment
<p>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. <b>A,</b> Transcriptomic data reveal increased expression of PINK1 in the 2b-OlapR subline. <b>B,</b> qRT-PCR verification of increased <i>PINK1</i> gene expression in 2b-OlapR cells. <b>C,</b> Western immunoblot analysis showing increased cellular PINK1 protein in 2b-OlapR cells. <b>D,</b> Deceased patients with prostate cancer have higher amounts of tumor PINK1 expression (Abida 2019). <b>E,</b> High-PINK1–expressing striated patients with prostate cancer show lower survival over time (GSE21032). <b>F,</b> 2B-OlapR cells treated with siPINK1 show lower mRNA expression through qRT-PCR (left) and decrease in cellular protein (right). <b>G,</b> siPINK1 treatment decreases growth of 2B-OlapR cells and increases relative olaparib efficacy at 5 μmol/L. <math><mo>*</mo><mo>,</mo><mo> </mo><mi>P</mi><mo>≤</mo><mn>0</mn><mo>.</mo><mn>05</mn><mo>;</mo><mo> </mo><mo>*</mo><mo>*</mo><mo>,</mo><mo> </mo><mi>P</mi><mo>≤</mo><mn>0</mn><mo>.</mo><mn>01</mn><mo>;</mo><mo> </mo><mo>*</mo><mo>*</mo><mo>*</mo><mo>,</mo><mo> </mo><mi>P</mi><mo>≤</mo><mn>0</mn><mo>.</mo><mn>001</mn><mo>;</mo><mo> </mo><mo>*</mo><mo>*</mo><mo>*</mo><mo>*</mo><mo>,</mo><mo> </mo><mi>P</mi><mo>≤</mo><mn>0</mn><mo>.</mo><mn>0001</mn></math>.</p>
Abstract Meiosis produces haploid gametes that will give rise to the next diploid generation. Chromosome segregation errors occurring at one or both meiotic divisions result in aneuploidy, which can lead to miscarriages or birth defects in humans. During meiosis I, ring-shaped cohesin complexes play important roles to aid in the proper segregation of homologous chromosomes. While REC8 is a specialized meiosis-specific cohesin that functions to hold sister chromatids together, the role of its vertebrate-specific paralog, RAD21L, is poorly understood. Here we tested if Rad21l1, the zebrafish homolog of human and mouse RAD21L, is required for meiotic chromosome dynamics during oogenesis and spermatogenesis. We found that Rad21l1 is an abundant component of meiotic chromosomes where it localizes to both the chromosome axes and the transverse filament of the synaptonemal complex (SC). Knocking out rad21l1 causes nearly the entire mutant population to develop as fertile males, suggesting the mutation triggers a sex reversal from female to male due to a failure in oocyte production. The rad21l1 −/− mutant males display normal fertility at sexual maturity. Sex reversal was partially suppressed in the absence of tp53, suggesting that the rad21l1 −/− mutation causes defects leading to a Tp53 dependent response, specifically in females. The rad21l1 −/− ;tp53 −/− double mutant females produced elevated rates of decomposing eggs and deformed offspring compared to tp53 −/− controls. This response, however, is not linked to a defect in repairing Spo11-induced double-strand breaks since deletion of Spo11 does not suppress the sex reversal phenotype. Overall, our data highlight an exceptional sexually dimorphic phenotype caused by knocking out a meiotic-specific cohesin subunit. We propose that Rad21l1 is required for maintaining the integrity of meiotic chromatin architecture during oogenesis. Author Summary A prominent symptom of age-linked reproductive decline in women is the increased rate of miscarriage and birth defects due to aneuploidy. Aneuploidy can arise when chromosomes fail to segregate properly during meiosis, the process of creating haploid gametes from a diploid germ cell. Oocyte progression normally arrests prior to anaphase I, after homologous chromosomes have formed crossovers, but before ovulation, which triggers the first round of segregation. This prolonged arrest makes oocytes especially vulnerable to degradation of meiotic chromosome structure and homolog connections over time. Cohesin complexes play a major role in maintaining the meiotic chromosome architecture. Here we assess the role of the vertebrate-specific Rad21l1 cohesin subunit in zebrafish. We find that while males appear mostly unaffected by loss of Rad21l1, oocyte production is massively compromised, leading to sex reversion to males. Sex reversion can be partially prevented in the absence of Tp53, demonstrating that loss or Rad21l1 leads to a Tp53-dependent response in oocytes. Strikingly, double mutant rad21l1 tp53 females produce large numbers of poor quality eggs and malformed offspring. This demonstrates a cohesin-linked vulnerability in female meiosis not present in males and sheds light on a potential mechanism associated with the decline in female reproductive health.
<p>Mitochondrial live stain and ETC functional test demonstrate higher function in 2B-OlapR cells. <b>A,</b> 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>B,</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. <math><mo>*</mo><mo>*</mo><mo>*</mo><mo>,</mo><mo> </mo><mi>P</mi><mo>≤</mo><mn>0</mn><mo>.</mo><mn>001</mn><mo>;</mo><mo> </mo><mo>*</mo><mo>*</mo><mo>*</mo><mo>*</mo><mo>,</mo><mo> </mo><mi>P</mi><mo>≤</mo><mn>0</mn><mo>.</mo><mn>0001</mn></math>.</p>
<p>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. <b>A</b> and <b>B,</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. <b>C,</b> Cellular OCR at basal state is increased in the olaparib-resistant cell line. <b>D,</b> 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. <b>E,</b> 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. <b>F,</b> 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. <b>G,</b> 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. <math><mo>*</mo><mo>*</mo><mo>,</mo><mo> </mo><mi>P</mi><mo>≤</mo><mn>0</mn><mo>.</mo><mn>01</mn><mo>;</mo><mo> </mo><mo>*</mo><mo>*</mo><mo>*</mo><mo>,</mo><mo> </mo><mi>P</mi><mo>≤</mo><mn>0</mn><mo>.</mo><mn>001</mn><mo>;</mo><mo> </mo><mo>*</mo><mo>*</mo><mo>*</mo><mo>*</mo><mo>,</mo><mo> </mo><mi>P</mi><mo>≤</mo><mn>0</mn><mo>.</mo><mn>0001</mn></math>.</p>
Abstract PARP inhibition represents the dawn of precision medicine for treating prostate cancer. Despite this advance, questions remain regarding the use of PARP inhibitors (PARPi) for the treatment of this disease, including (i) how specifically do PARPi-sensitive tumor cells respond to treatment, and (ii) how does PARPi resistance develop? To address these questions, we characterized response to olaparib in sensitive LNCaP and C4-2B cells and developed two olaparib-resistant derivative cell line models from each, termed LN-OlapR and 2B-OlapR, respectively. OlapR cells possess distinct morphology from parental cells and display robust resistance to olaparib and other clinically relevant PARPis, including rucaparib, niraparib, and talazoparib. In LNCaP and C4-2B cells, we found that olaparib induces massive DNA damage, leading to activation of the G2–M checkpoint, activation of p53, and cell-cycle arrest. Furthermore, our data suggest that G2–M checkpoint activation leads to both cell death and senescence associated with p21 activity. In contrast, both LN-OlapR and 2B-OlapR cells do not arrest at G2–M and display a markedly blunted response to olaparib treatment. Interestingly, both OlapR cell lines harbor increased DNA damage relative to parental cells, suggesting that OlapR cells accumulate and manage persistent DNA damage during acquisition of resistance, likely through augmenting DNA repair capacity. Further impairing DNA repair through CDK1 inhibition enhances DNA damage, induces cell death, and sensitizes OlapR cells to olaparib treatment. Our data together further our understanding of PARPi treatment and provide a cellular platform system for the study of response and resistance to PARP inhibition.
Current common treatments for castration-resistant prostate cancer (CRPC) typically belong to one of three major categories: next-generation anti-androgen therapies (NGAT) including enzalutamide, abiraterone acetate, apalutamide, and darolutamide; taxane therapy represented by docetaxel; and PARP inhibitors (PARPi) like olaparib. Although these treatments have shown efficacy and have improved outcomes for many patients, some do not survive due to the emergence of therapeutic resistance. The clinical landscape is further complicated by limited knowledge about how the sequence of treatments impacts the development of therapeutic cross-resistance in CRPC. We have developed multiple CRPC models of acquired therapeutic resistance cell sublines from C4-2B cells. These include C4-2B MDVR, C4-2B AbiR, C4-2B ApaR, C4-2B DaroR, TaxR, and 2B-olapR, which are resistant to enzalutamide, abiraterone, apalutamide, darolutamide, docetaxel, and olaparib, respectively. These models are instrumental for analyzing gene expression and assessing responses to various treatments. Our findings reveal distinct cross-resistance characteristics among NGAT-resistant cell sublines. Specifically, resistance to enzalutamide induces resistance to abiraterone and vice versa, while maintaining sensitivity to taxanes and olaparib. Conversely, cells with acquired resistance to docetaxel exhibit cross-resistance to both cabazitaxel and olaparib but retain sensitivity to NGATs like enzalutamide and abiraterone. OlapR cells, significantly resistant to olaparib compared to parental cells, are still responsive to NGATs and docetaxel. Moreover, OlapR models display cross-resistance to other clinically relevant PARP inhibitors, including rucaparib, niraparib, and talazoparib. RNA-sequencing analyses have revealed a complex network of altered gene expressions that influence signaling pathways, energy metabolism, and apoptotic signaling, pivotal to cancer’s evolution and progression. The data indicate that resistance mechanisms are distinct among different drug classes. Notably, NGAT-resistant sublines exhibited a significant downregulation of androgen-regulated genes, contrasting to the stable expression noted in olaparib and docetaxel-resistant sublines. These results may have clinical implications by showing that treatments of one class can be sequenced with those from another class, but caution should be taken when sequencing drugs of the same class.
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