IGFBP3 promotes resistance to Olaparib via modulating EGFR signaling in advanced prostate cancer.

Olaparib is a pioneering PARP inhibitor (PARPi) approved for treating castration-resistant prostate cancer (CRPC) tumors harboring DNA repair defects, but clinical resistance has been documented. To study acquired resistance, we developed Olaparib-resistant (OlapR) cell lines through chronic Olaparib treatment of LNCaP and C4-2B cell lines. Here, we found that IGFBP3 is highly expressed in acquired (OlapR) and intrinsic (Rv1) models of Olaparib resistance. We show that IGFBP3 expression promotes Olaparib resistance by enhancing DNA repair capacity through activation of EGFR and DNA-PKcs. IGFBP3 depletion enhances efficacy of Olaparib by promoting DNA damage accumulation and subsequently, cell death in resistant models. Mechanistically, we show that silencing IGFBP3 or EGFR expression reduces cell viability and resensitizes OlapR cells to Olaparib treatment. Inhibition of EGFR by Gefitinib suppressed growth of OlapR cells and improved Olaparib sensitivity, thereby phenocopying IGFBP3 inhibition. Collectively, our results highlight IGFBP3 and EGFR as critical mediators of Olaparib resistance.

Therapeutic Resistance Models and Treatment Sequencing in Advanced Prostate Cancer.

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.

Wntless expression promotes lineage plasticity and is associated with neuroendocrine prostate cancer.

Resistance to androgen receptor (AR) targeted therapies remains as the main reason for most prostate cancer related deaths. Lineage plasticity resulting in altered, treatment insensitive prostate tumor cell phenotypes such neuroendocrine differentiated prostate cancer is a common manifestation within resistant tumors upon AR-targeted therapies. The mechanisms responsible for lineage plasticity in prostate cancer remain incompletely understood. Here we demonstrate that the enzalutamide resistant MDVR cell line possesses lineage plastic characteristics associated with overexpression of the Wnt transporter Wntless (WLS). Furthermore, we present evidence that overexpression of WLS is common in varying cell line models of lineage plastic prostate cancer, is higher in neuroendocrine patient samples, and positively correlates with the neuroendocrine marker SYP in clinical data. Targeting WLS in lineage plastic cellular models reduces viability and represses lineage plasticity associated gene expression. Our study provides insight into the importance of WLS to the development of lethal resistant prostate cancer and provides a potential target for the treatment of advanced disease.

Activation of neural lineage networks and ARHGEF2 in enzalutamide-resistant and neuroendocrine prostate cancer and association with patient outcomes.

Background: Treatment-emergent neuroendocrine prostate cancer (NEPC) after androgen receptor (AR) targeted therapies is an aggressive variant of prostate cancer with an unfavorable prognosis. The underlying mechanisms for early neuroendocrine differentiation are poorly defined and diagnostic and prognostic biomarkers are needed. Methods: We performed transcriptomic analysis on the enzalutamide-resistant prostate cancer cell line C4-2B MDVR and NEPC patient databases to identify neural lineage signature (NLS) genes. Correlation of NLS genes with clinicopathologic features was determined. Cell viability was determined in C4-2B MDVR and H660 cells after knocking down ARHGEF2 using siRNA. Organoid viability of patient-derived xenografts was measured after knocking down ARHGEF2. Results: We identify a 95-gene NLS representing the molecular landscape of neural precursor cell proliferation, embryonic stem cell pluripotency, and neural stem cell differentiation, which may indicate an early or intermediate stage of neuroendocrine differentiation. These NLS genes positively correlate with conventional neuroendocrine markers such as chromogranin and synaptophysin, and negatively correlate with AR and AR target genes in advanced prostate cancer. Differentially expressed NLS genes stratify small-cell NEPC from prostate adenocarcinoma, which are closely associated with clinicopathologic features such as Gleason Score and metastasis status. Higher ARGHEF2, LHX2, and EPHB2 levels among the 95 NLS genes correlate with a shortened survival time in NEPC patients. Furthermore, downregulation of ARHGEF2 gene expression suppresses cell viability and markers of neuroendocrine differentiation in enzalutamide-resistant and neuroendocrine cells. Conclusions: The 95 neural lineage gene signatures capture an early molecular shift toward neuroendocrine differentiation, which could stratify advanced prostate cancer patients to optimize clinical treatment and serve as a source of potential therapeutic targets in advanced prostate cancer.

Bioengineered BERA-Wnt5a siRNA Targeting Wnt5a/FZD2 Signaling Suppresses Advanced Prostate Cancer Tumor Growth and Enhances Enzalutamide Treatment.

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 in vivo. 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.

Olaparib-Induced Senescence Is Bypassed through G2-M Checkpoint Override in Olaparib-Resistant Prostate Cancer.

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.