<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>
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.
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 Introduction and Objectives: Androgen receptor (AR) blockade using antiandrogens is a mainstay for the treatment of castration-resistant prostate cancer (CRPC). However, resistance to antiandrogens occurs unavoidably due to the escaping mechanisms of tumor cells that are not completely understood. Upregulation of non-canonical Wnt ligand Wnt5a is identified from circulating tumor cells from CRPC patients after being treated with enzalutamide. FZD2, the cognate frizzled receptor for Wnt5a, is the most commonly co-upregulated non-canonical Wnt signaling molecule in prostate cancer. In this study, we determined the role of non-canonical Wnt5a/FZD2 to enzalutamide resistance and neural lineage plasticity, and explore the potential of targeting Wnt5a/FZD2 to overcome resistance in advanced prostate cancer. Methods: Wnt5a and FZD2 expression was determined in enzalutamide-resistant C4-2B MDVR cells. Wnt5a and FZD2 expressions were modulated using specific siRNAs. Cell growth, colony formation, and migration were studied in vitro. Transcriptomic analysis was performed on C4-2B MDVR cells treated with FZD2 knocked down; gene program of non-canonical Wnt signaling, hallmark androgen response and AR-V7 associated genes, and neural lineage pathways were analyzed. A novel tRNA bioengineered Wnt5a siRNA was developed to target Wnt5a/FZD2 signaling. The effect of tRNA-siWnt5a on tumor growth, sensitivity to enzalutamide treatment, and neural lineage plasticity was evaluated in vitro and in vivo. Results: Wnt5a and FZD2 are highly upregulated in castration-resistant prostate cancer patients, which is verified overexpression in enzalutamide-resistant C4-2B MDVR cells. Knocking down Wnt5a and FZD2 diminishes the enrichment of the non-canonical Wnt signaling pathway. Downregulating Wnt5a and FZD2 expression by specific siRNAs suppresses prostate cancer cell proliferation and resensitized C4-2B MDVR cells to enzalutamide treatment. Suppressing Wnt5a and FZD2 abrogated the enrichment of gene programs regulating cancer cell survival/proliferation, and neural lineage pathways including neuron differentiation and embryonic stem cell markers. Using the bioengineered tRNA-Wnt5a siRNA effectively inhibited the growth of enzalutamide-resistant prostate cancer cells and resensitized tumor cells to enzalutamide treatment in vitro, and resistant CRPC PDX LuCaP35CR tumor growth in vivo. Conclusions: Our results suggest that activation of noncanonical Wnt5a/FZD2 signaling confers to enzalutamide resistance and neural lineage plasticity. Targeting the Wnt5a signal could provide benefits for CRPC patients with tumors expressing a high level of Wnt5a, FZD2, and lineage markers, not only enhancing the anti-tumor effects of enzalutamide but suppressing the potential of neuroendocrine differentiation in advanced prostate cancer patients. Citation Format: Shu Ning, Wei Lou, Chengfei Liu, Joy C. Yang, Alan P. Lombard, Leandro S. Abronzo, Neelu Batra, Aiming Yu, Christopher P. Evans, Allen C. Gao. Targeting noncanonical Wnt5a signaling suppresses the neural lineage network and overcomes enzalutamide resistance 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 3069.
<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>
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