Prostate Cancer Viability Research Articles

Abstract IA001: Exploiting pathway activation as a new form of synthetic lethality

Abstract Targeting activated oncogenes is an effective treatment strategy across many cancers now including therapeutics targeting KRAS. Genetic events including DNA damage deficiencies and tumor suppressor mutations require alternative strategies and the concept of synthetic lethality has been applied to these alterations. PARPi inhibitors were a founding member of this class of therapeutics demonstrating enhanced activity in the setting of BRCA-deficiency. By applying large-scale loss-of-function approaches1 we and other discovered the vulnerability to PRMT5 and WRN inhibition imposed by co-deletions of MTAP and CDKN2A, and the MSI+ genotype respectively2,3. Such inhibitors are now in clinical trials. To explore the potential for paralogous genes to act as synthetic lethal gene pairs we enacted dual knockout screens. These early efforts led to the discovery of DUSP4/6 paralog dependence in the setting of BRAF and NRAS mutations in melanoma4. Intriguingly the loss of DUSP4/6 impaired cancer cell viability through the increased activation of ERK highlighting the susceptibility of cancers to pathway activation in addition to the more common sensitivity to pathway inhibition. This inappropriate activation of the ERK signaling pathway, the conflict between EGFR and KRAS activation, the synthetic lethality enacted by TRIM8 knockouts, and the effects of AR agonists on prostate cancer viability, points to a wider than expected vulnerability of cancer to inappropriate gene activation. To systematically identify context-specific gene activation induced lethalities in cancer, we developed methods for enacting gain-of-function perturbations across ∼500 barcoded cancer cell lines. With this approach, we queried the pan-cancer vulnerability landscape upon activating 10 key cancer pathway revealing activation dependencies in MAPK and PI3K pathways. Notably, we discovered novel pathway hyperactivation dependencies in subsets of APC-mutant colorectal cancers where further activation of the WNT pathway by APC knockdown or direct β-catenin overexpression led to robust antitumor effects in xenograft and patient-derived organoid models. These latter discoveries paradoxically point to the residual activity of the APC ubiquitin-ligase complex as a target in APC-mutant CRC5. 1. McDonald, E. R., 3rd et al. Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening. Cell 170, 577-592.e10 (2017). 2. Mavrakis, K. J. et al. Disordered methionine metabolism in MTAP/CDKN2A-deleted cancers leads to dependence on PRMT5. Science 351, 1208–1213 (2016). 3. Chan, E. M. et al. WRN helicase is a synthetic lethal target in microsatellite unstable cancers. Nature 568, 551–556 (2019). 4. Ito, T. et al. Paralog knockout profiling identifies DUSP4 and DUSP6 as a digenic dependence in MAPK pathway-driven cancers. Nat. Genet. 53, 1664–1672 (2021). 5. Chang, L. et al. Systematic profiling of conditional pathway activation identifies contextdependent synthetic lethalities. Nat. Genet. 55, 1709–1720 (2023). Citation Format: William R. Sellers. Exploiting pathway activation as a new form of synthetic lethality [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA001.

Abstract 995: Integrative genomic characterization and CRISPR-mediated gene editing studies identify IRAKs as novel therapeutic targets for inflammation-driven prostate tumorigenesis

Abstract A major drawback to accurate diagnosis and treatment of inflammation-driven prostate cancer (PCa) progression is our limited understanding of the molecular mechanisms underlying aberrant inflammation signaling in PCa patients. Since asymptomatic chronic inflammation is hard to diagnose, we do not have adequate knowledge of how specific genes within the inflammatory cascade drive PCa aggressiveness. Hence, an integrative genomic approach may provide us with effective prognostic and diagnostic tools to manage inflammation-driven PCa progression. The aim of this study was to characterize inflammation-associated hub genes as predictors of PCa progression and investigate the effect of aberrant signaling of interleukin-1 receptor-associated kinases (IRAKs) on prostate tumorigenesis. WES and RNA-seq datasets from a total of 726 PCa patients, stratified into subtypes: primary PCa (PPC; n=493), metastatic PCa (MPC; n=150) and the castration-resistant PCa (CRPC; n=52) and neuroendocrine PCa (NEPC; n=30) from 3 cohort studies were acquired from cBioPortal. A bioinformatics pipeline was designed to analyze the datasets. Weighted gene co-expression network analysis (WGCNA) was used to construct gene co-expression networks and identify inflammation hub genes associated with PCa progression. The top inflammation hub genes, such as ILR1, TLRs, IRAK1-4, and NF-kB were validated. IRAK1-4 hub genes were found to be differentially expressed and genetically altered in NEPC and CRPC (>40%) compared to PPC and MPC (<20%) patients. GISTIC and MutSigCV were used to identify somatic mutations and copy-number alterations (CNAs). IRAK1 was found to have the highest CNA in CRPC and NEPC compared to MPC and PPC patients. Interestingly, no mutation was identified in CRPC and NEPC patients. The co-occurrence of IRAK1/4 CNVs and mRNA with AR, PI3K and AURKA signaling pathways suggest a role in PCa progression. To mechanistically investigate the role of IRAK1/4 signaling on PCa progression, CRISPR-mediated knockdown of IRAK1/4 was performed in PCa cell lines (LNCaP, PC3, and NCI-H660). mRNA and protein expression analyses were performed to determine changes in the levels of AR, PI3K/AKT, and AURKA between IRAK1/4-/- and IRAK1/4+/+ cell lines. Inhibition of IRAK1/4 significantly altered AR, PI3K, and AURKA signaling, leading to a reduction in PCa viability and metastatic potential. To investigate the effect of aberrant IRAK1/4 signaling on chemoresistance induction, IRAK+/+ PCa cells were treated with either TLR agonists or IRAK1/4 inhibitors and then treated with different anticancer drugs, such as Enzalutamide, Docetaxel, and Alisertib (AURKA inhibitor) dose-dependently. IRAK1/4-/- cells were more susceptible to anticancer drug treatment compared to IRAK1+/+ cells. The combination treatments were shown to be synergistic, thereby suppressing inflammation-induced chemoresistance. In conclusion, we have identified IRAK1/4 as a potential target for treating inflammation-driven prostate cancer progression. Citation Format: Saheed Oluwasina Oseni, Mirjana Pavlovic, James Hartmann, James Kumi-Diaka. Integrative genomic characterization and CRISPR-mediated gene editing studies identify IRAKs as novel therapeutic targets for inflammation-driven prostate tumorigenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 995.

Reduction of circular RNA Foxo3 promotes prostate cancer progression and chemoresistance to docetaxel.

Dysregulation of circular RNA Foxo3 (circFoxo3) has been reported to be involved in breast cancer and non-small lung cancer progression. However, little is known about the role of circFoxo3 in prostate cancer, which the present study seeks to investigate. CircFoxo3 expression was analyzed in 22 low-grade prostate cancer samples, 24 high-graded prostate cancer samples, and 18 normal prostate tissues, finding that its quantity was significantly decreased in high-graded compared to low-grade prostate cancer and normal prostate tissues. CircFoxo3 inhibited prostate cancer cell survival, migration, invasion and chemoresistance to docetaxel, which was related to circFoxo3's repression of Foxo3 and EMT. Silencing circFoxo3 expression promoted prostate cancer cell survival, migration, invasion and chemoresistance to docetaxel, as well as the positive effects of androgen on prostate cancer viability. Delivery of circfoxo3 enhanced chemosensitivity to docetaxel of prostate tumor-bearing mice and prolonged the life span of mice, while reduction with siRNAs promoted chemoresistance to docetaxel and shorted the life span of the tumor-bearing mice. Targeting circFoxo3/Foxo3/EMT may provide an applicable strategy for exploring potential prognostic and therapeutic approaches for prostate cancer.

Abstract 3799: Heparan sulfate modifiers and signaling between tumor-associated macrophages and cancer-associated fibroblasts in a biomimetic hydrogel model of bone-metastatic prostate cancer

Abstract The establishment of a new tumor niche for most carcinomas is orchestrated primarily by the bidirectional engagement of cancer cells and fibroblasts. Cancer cells reprogram and activate fibroblasts into a wound healing-like state and, in turn, these cancer-associated fibroblasts (CAFs) react by remodeling the extracellular matrix (ECM) and secreting chemokines and growth factors. Macrophages are rapidly recruited to the tumor and represent the primary immune cell in the microenvironment. Tumor-associated macrophages (TAMs) enter a wound healing-like loop, displaying the full spectrum of macrophage phenotypes and behaviors as the disease progresses. Among these activities is the ability to modulate growth factor bioavailability. Deposition of the heparan sulfate (HS) proteoglycan perlecan/HSPG2 in the ECM and secretion of HS modulators are increased as result of the cancer-CAF-TAM trialogue and play critical roles in the availability and signaling of growth factors, cytokines, and chemokines. In this work, we developed a novel, biomimetic 3D hydrogel system composed of hyaluronic acid and collagen I (1:1) to study prostate cancer bone metastases. Our goal was to explore the molecular signaling between TAMs and CAFs to learn how this interaction affects the expression of the enzymes that modulate HS as a growth factor depot. Essential modulators of HS include heparanase (HPSE) and sulfatases 1 and 2 (SULF1, 2), all of which selectively release growth factors to influence signaling in the tumor microenvironment. We found that alternatively activated macrophages (M2) and TAMs (M2-like) increase levels of HPSE and SULFs in CAFs. We next examined the impact of TAM-CAF co-signaling on prostate cancer viability, growth, and dependence on HS modulators. Our data show that as a consequence of the TAM-CAF interaction, the elevated levels of HS-modifying enzymes are sufficient to trigger substantial changes in prostate cancer cell behavior, including viability, proliferation, and response to growth factors. We currently are assessing if these outcomes are seen in a series of patient-derived prostate xenograft lines representing different prostate cancer subtypes. We conclude that our 3D hydrogel model is a physiologically relevant in vitro system to discover fundamental aspects of HS signaling in the tumor microenvironment and thus uncover new ways to reduce the bioavailability of growth factors in the prostate cancer microenvironment and interfere with cancer progression. (Supported by NIH P01 and CAPES.) Citation Format: Fabio Henrique Brasil da Costa, Nora M. Navone, Mary C. Farach-Carson, Daniel D. Carson. Heparan sulfate modifiers and signaling between tumor-associated macrophages and cancer-associated fibroblasts in a biomimetic hydrogel model of bone-metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3799.

Abstract 5195: Modulating the UPR using the impridone ONC201 to change the impact of radiation on prostate cancer cells

Abstract Prostate cancer is the highest incidence non-cutaneous cancer in men, and a significant cause of cancer mortality. Localized disease is treated with surgery or radiotherapy and whilst androgen deprivation therapy can extend survival in the subset of cases that progress, metastatic disease is incurable. We have found that oncogenic drivers of prostate cancer, as well as therapy, lead to changes in the activation of prostate cancer cell stress signaling pathways and in particular the unfolded protein response (UPR). We have previously reported that the androgen receptor sustains the cytoprotective activity of the IRE1-XBP1 arm of the UPR and represses the activation of the PERK-ATF4 arm. We showed that treating prostate cancer cells with radiation leads to the activation of all three arms of the UPR, culminating in treatment resistance in surviving cells. In this study we assess whether the activation of the UPR in response to radiation mirrors the immunogenic/inflammatory response, which is known to occur in an initially acute apoptotic phase before resolving to a chronic sustainable level. Further we have evaluated the impact of combining radiation with ONC201, a novel anti-cancer small molecule in clinical trials that is an activator of the UPR, on the viability of prostate cancer. We find that ONC201 has a delayed cytotoxic effect as a single agent, with the impact on viability occurring from 72 hours onwards at low microMolar concentrations. However activation of multiple arms of the UPR occurs earlier and is detectable at 24 hours for ATF4, ATF6 and IRE1-XBP1 at the protein/mRNA levels. This time difference creates a window in which to introduce sequential administration of radiation to test ONC201 as a primer for cytotoxic response radiation. We have undertaken this work in a panel of cell-lines (PC3, DU145 and AR-expressing cell-lines) in which we have also knocked down key regulators (PERK, IRE1, CHOP) of separate arms of the UPR. This represents the first in vitro study to characterise the impact of ONC201 on the radiation-responsiveness of prostate cancer cells. Citation Format: Francesca Amoroso, Adam Pickard, Alice McNaney, Rohinton Tarapore, Joshua E. Allen, Ian G. Mills. Modulating the UPR using the impridone ONC201 to change the impact of radiation on prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5195. doi:10.1158/1538-7445.AM2017-5195

Chemosensitization of carmustine with maitake beta-glucan on androgen-independent prostatic cancer cells: involvement of glyoxalase I.

To improve the poor efficacy (< 10%) of chemotherapy for patients with hormone-refractory prostate cancer, we investigated a possible cytotoxic effect of carmustine/beta-glucan combination on prostatic cancer PC-3 cells, focusing on a glutathione-dependent detoxifying enzyme, glyoxalase I (Gly-I). Carmustine (BCNU) is an anticancer agent and a putative inhibitor of Gly-I, while beta-glucan is a unique, nontoxic polysaccharide extracted from maitake mushrooms. The cytotoxic effects of BCNU or other anticancer agents with beta-glucan on PC-3 cells were assessed by cell-viability testing and Gly-I activity was measured using the spectrophotometric method. BCNU, 5-fluorouracil (5-FU), and methotrexate (MTX) were capable of inducing approximately a 50% reduction in cell viability at 72 hours, while etoposide, cisplatin, and mitomycin C were all ineffective. Only the combination of BCNU (50 micro ;mol) and beta-glucan (60 micro g/mL) exhibited an enhanced cytotoxicity with an approximate 90% cell viability reduction, but little improvement was seen with any combinations of 5-FU, MTX, or beta-glucon. Gly-I assays revealed that such a profound (approximately 90%) cell death was accompanied by an approximate 80% reduction in Gly-I activity by 6 hours. This study demonstrates a sensitized cytotoxic effect of BCNU with beta-glucan in PC-3 cells, which was associated with a drastic (approximately 80%) inactivation of Gly-I. Therefore, the BCNU/beta-glucan combination may help to improve current treatment efficacy by targeting Gly-I, which appears to be critically involved in prostate cancer viability.