Amy Leslie

All Publications

• IGFBP3-SphK1/S1P Signaling Axis Drives Enzalutamide Resistance in Advanced Prostate Cancer. Molecular cancer therapeutics Leslie, A. R., Ning, S., Sharifi, M., Schaaf, Z. A., Maine, J. P., Lou, W., Leslie, K. D., Liu, C., Xu, H., Lombard, A. P., Chen, H. W., Parikh, M., Dall'Era, M., Gao, A. C. 2025

  Abstract

  Enzalutamide resistance remains a significant challenge in the treatment of advanced prostate cancer. Identifying molecular drivers of enzalutamide resistance is crucial for developing effective therapeutic strategies. In this study, we identify insulin-like growth factor binding protein 3 (IGFBP3) as a key driver of enzalutamide resistance in castration-resistant prostate cancer (CRPC). We demonstrate that IGFBP3 expression is significantly upregulated in enzalutamide-resistant C4-2B MDVR cells compared to parental C4-2B cells. This upregulation was consistently observed across multiple enzalutamide-resistant CRPC models, including LNCaP-derived 42D and 42F cells, as well as long-term enzalutamide-resistant cell lines derived from LNCaP, VCaP, LAPC-4, and CWR-R1 cells. Additionally, Enzalutamide treatment directly induced IGFBP3 expression in sensitive cells. Elevated IGFBP3 expression was also observed in CRPC patient samples post-enzalutamide treatment and was associated with higher Gleason scores and reduced disease-free survival. Mechanistically, IGFBP3 activates the sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P) signaling pathway, which promotes cell survival and resistance to enzalutamide. IGFBP3 knockdown decreased SphK1 expression, reduced S1P secretion, and enhanced enzalutamide sensitivity, whereas IGFBP3 overexpression induced SphK1 expression and S1P production, conferring enzalutamide resistance. Inhibition of IGFBP3 via siRNA reduced cell viability, induced apoptosis, and re-sensitized resistant models to enzalutamide. Similarly, targeting SphK1 with the inhibitor SKI-II suppressed SphK1 activity, reduced S1P production, enhanced enzalutamide sensitivity, and significantly inhibited resistant tumor growth while enhancing enzalutamide sensitivity. Collectively, these findings highlight IGFBP3-mediated SphK1 signaling as a critical mediator of enzalutamide resistance and suggest that targeting the IGFBP3/SphK1/S1P axis represents a promising therapeutic strategy to overcome resistance in advanced prostate cancer.

  View details for DOI 10.1158/1535-7163.MCT-25-0644

  View details for PubMedID 41347288

  View details for PubMedCentralID PMC12795166

• Steroid Sulfatase Regulates Metabolic Reprogramming in Advanced Prostate Cancer. Cancers Sharifi, M., Armstrong, C. M., Ning, S., Leslie, A. R., Schaaf, Z. A., Maine, J. P., Lou, W., Li, P. K., Xu, H., Liu, C., Gao, A. C. 2025; 17 (12)

  Abstract

  The expression of human steroid sulfatase (STS) is upregulated in castration-resistant prostate cancer (CRPC) and is associated with resistance to anti-androgen drugs, such as enzalutamide (Enza) and abiraterone (Abi). Despite the known link between STS overexpression and therapeutic unresponsiveness, the mechanism by which STS confers this phenotype remains incompletely understood. In this study, we sought to understand how STS induces treatment resistance in advanced prostate cancer (PCa) cells by exploring its role in altering mitochondrial activity.To examine the effects of increased STS expression on mitochondrial respiration and programming, we performed RNA sequencing (RNA-seq) analysis, the Seahorse XF Mito Stress Test, and a mitochondrial Complex I enzyme activity assay in STS-overexpressing cells (C4-2B STS) and in enzalutamide-resistant CPRC cells (C4-2B MDVR). We employed SI-2, the specific chemical inhibitor of STS, on C4-2B STS and C4-2B MDVR cells and evaluated STS activity inhibition on mitochondrial molecular pathways and mitochondrial respiration. Lastly, we examined the effects of dehydroepiandrosterone sulfate (DHEAS) supplementation on C4-2B STS organoids.We present evidence from the transcriptomic profiling of C4-2B STS cells that there are enriched metabolic pathway signatures involved in oxidative phosphorylation, the electron transport chain, and mitochondrial organization. Moreover, upon STS inhibition, signaling in the electron transport chain and mitochondrial organization pathways is markedly attenuated. Findings from the Seahorse XF Mito Stress Test and mitochondrial Complex I enzyme activity assay demonstrate that STS overexpression increases mitochondrial respiration, whereas the inhibition of STS by SI-2 significantly reduces the oxygen consumption rate (OCR) and Complex I enzyme activity in C4-2B STS cells. Similarly, an increased OCR and electron transport chain Complex I enzymatic activity are observed in C4-2B MDVR cells and a decreased OCR upon SI-2 inhibition. Lastly, we show that STS overexpression promotes organoid growth upon DHEAS treatment.Our study demonstrates STS as a key driver of metabolic reprogramming and flexibility in advanced prostate cancer. Disrupting enhanced mitochondrial respiration via STS presents a promising strategy in improving CRPC treatment.

  View details for DOI 10.3390/cancers17121959

  View details for PubMedID 40563609

  View details for PubMedCentralID PMC12190948

• LX1 Dual Targets AR Variants and AKR1C3 in Advanced Prostate Cancer Therapy. Cancer research Ning, S., Armstrong, C. M., Xing, E., Leslie, A. R., Gao, R. Y., Sharifi, M., Schaaf, Z. A., Lou, W., Han, X., Xu, D. H., Yang, R., Cheng, J., Mohammed, S., Mitsiades, N., Liu, C., Lombard, A. P., Wu, C. Y., Cheng, X., Li, P. K., Gao, A. C. 2024; 84 (21): 3617-3628

  Abstract

  The development of resistance to current standard-of-care treatments, such as androgen receptor (AR) targeting therapies, remains a major challenge in the management of advanced prostate cancer. There is an urgent need for new therapeutic strategies targeting key resistant drivers, such as AR variants like AR-V7, and steroidogenic enzymes, such as aldo-keto reductase 1C3 (AKR1C3), to overcome drug resistance and improve outcomes for patients with advanced prostate cancer. Here, we have designed, synthesized, and characterized a novel class of LX compounds targeting both the AR/AR variants and AKR1C3 pathways. Molecular docking and in vitro studies demonstrated that LX compounds bind to the AKR1C3 active sites and inhibit AKR1C3 enzymatic activity. LX compounds were also shown to reduce AR/AR-V7 expression and to inhibit their target gene signaling. LX1 inhibited the conversion of androstenedione into testosterone in tumor-based ex vivo enzyme assays. In addition, LX1 inhibited the growth of cells resistant to antiandrogens including enzalutamide (Enza), abiraterone, apalutamide, and darolutamide in vitro. A synergistic effect was observed when LX1 was combined with antiandrogens and taxanes, indicating the potential for this combination in treating resistant prostate cancer. Treatment with LX1 significantly decreased tumor volume, serum PSA levels, as well as reduced intratumoral testosterone levels, without affecting mouse body weight. Furthermore, LX1 was found to overcome resistance to Enza treatment, and its combination with Enza further suppressed tumor growth in both the CWR22Rv1 xenograft and LuCaP35CR patient-derived xenograft models. Collectively, the dual effect of LX1 in reducing AR signaling and intratumoral testosterone, along with its synergy with standard therapies in resistant models, underscores its potential as a valuable treatment option for advanced prostate cancer. Significance: LX1 simultaneously targets androgen receptor variants and the steroidogenic enzyme AKR1C3, offering a promising approach to combat drug resistance and enhancing therapeutic efficacy in conjunction with standard treatments for advanced prostate cancer.

  View details for DOI 10.1158/0008-5472.CAN-24-0440

  View details for PubMedID 39088701

  View details for PubMedCentralID PMC11534543

• PINK1-Mediated Mitochondrial Activity Confers Olaparib Resistance in Prostate Cancer Cells. Cancer research communications Schaaf, Z. A., Ning, S., Leslie, A. R., Sharifi, M., Gao, R. Y., Maine, J. P., Lou, W., Lombard, A. P., Liu, C., Yu, A. M., Mitsiades, N., Gao, A. C. 2024; 4 (11): 2976-2985

  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.

  View details for DOI 10.1158/2767-9764.CRC-24-0339

  View details for PubMedID 39440945

  View details for PubMedCentralID PMC11577557

• IGFBP3 promotes resistance to Olaparib via modulating EGFR signaling in advanced prostate cancer. iScience Leslie, A. R., Ning, S., Armstrong, C. M., D'Abronzo, L. S., Sharifi, M., Schaaf, Z. A., Lou, W., Liu, C., Evans, C. P., Lombard, A. P., Gao, A. C. 2024; 27 (2): 108984

  Abstract

  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.

  View details for DOI 10.1016/j.isci.2024.108984

  View details for PubMedID 38327800

  View details for PubMedCentralID PMC10847745

• Therapeutic Resistance Models and Treatment Sequencing in Advanced Prostate Cancer. Cancers Schaaf, Z. A., Ning, S., Leslie, A. R., Sharifi, M., Han, X., Armstrong, C., Lou, W., Lombard, A. P., Liu, C., Gao, A. C. 2023; 15 (21)

  Abstract

  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.

  View details for DOI 10.3390/cancers15215273

  View details for PubMedID 37958444

  View details for PubMedCentralID PMC10650051

• Wntless expression promotes lineage plasticity and is associated with neuroendocrine prostate cancer. American journal of clinical and experimental urology D'Abronzo, L. S., Lombard, A. P., Ning, S., Armstong, C. M., Leslie, A. R., Sharifi, M., Schaaf, Z. A., Lou, W., Gao, A. C. 2022; 10 (5): 299-310

  Abstract

  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.

  View details for PubMedID 36313205

  View details for PubMedCentralID PMC9605943

• Bioengineered BERA-Wnt5a siRNA Targeting Wnt5a/FZD2 Signaling Suppresses Advanced Prostate Cancer Tumor Growth and Enhances Enzalutamide Treatment. Molecular cancer therapeutics Ning, S., Liu, C., Lou, W., Yang, J. C., Lombard, A. P., D'Abronzo, L. S., Batra, N., Yu, A. M., Leslie, A. R., Sharifi, M., Evans, C. P., Gao, A. C. 2022; 21 (10): 1594-1607

  Abstract

  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.

  View details for DOI 10.1158/1535-7163.MCT-22-0216

  View details for PubMedID 35930737

  View details for PubMedCentralID PMC9547958

• Activation of neural lineage networks and ARHGEF2 in enzalutamide-resistant and neuroendocrine prostate cancer and association with patient outcomes. Communications medicine Ning, S., Zhao, J., Lombard, A. P., D'Abronzo, L. S., Leslie, A. R., Sharifi, M., Lou, W., Liu, C., Yang, J. C., Evans, C. P., Corey, E., Chen, H. W., Yu, A., Ghosh, P. M., Gao, A. C. 2022; 2: 118

  Abstract

  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.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.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.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.

  View details for DOI 10.1038/s43856-022-00182-9

  View details for PubMedID 36159187

  View details for PubMedCentralID PMC9492734

• Olaparib-Induced Senescence Is Bypassed through G2-M Checkpoint Override in Olaparib-Resistant Prostate Cancer. Molecular cancer therapeutics Lombard, A. P., Armstrong, C. M., D'Abronzo, L. S., Ning, S., Leslie, A. R., Sharifi, M., Lou, W., Evans, C. P., Dall'Era, M., Chen, H. W., Chen, X., Gao, A. C. 2022; 21 (4): 677-685

  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.

  View details for DOI 10.1158/1535-7163.MCT-21-0604

  View details for PubMedID 35086956

  View details for PubMedCentralID PMC8983570