Understanding and Targeting Fatty Acid-CoA Ligase ACSL4 in Advanced Human Prostate Cancer

Abstract

Prostate cancer (PCa) ranks as the most prevalent non-cutaneous malignancy among men. While localized prostate cancer can often be cured, the recurrence of metastatic castration-resistant prostate cancer (mCRPC) remains a formidable challenge and is the leading cause of mortality in PCa patients. Recent findings from our lab and others highlights the pivotal role of RB1 loss in driving lineage plasticity, metastasis, and lethality of prostate tumors. One downstream target of RB1 loss/E2F activation is acyl-CoA synthetase long-chain family member 4 (ACSL4), a fatty acid ligase crucial for the utilization of long-chain fatty acids. Dysregulated fatty acid metabolism fuels oncogenic processes and tumor progression through excessive energy production and lipogenesis, and reprogramming fatty acid metabolism shows promise in advanced cancer therapy. ACSL4 has been reported to be upregulated in cancers of various histological origins, including prostate cancer, and its elevated expression is correlated with cancer aggressiveness. However, the metabolic regulation and clinical significance of ACSL4 in prostate tumorigenesis and progression remains elusive. Through both gain-of-function and loss-of-function approach aiming to examine the outcome of high ACSL4 level in PCa patients, we revealed ACSL4 as an oncogenic driver that promoted prostate cancer progression via enhanced cell proliferation and ATP production. Further characterization via untargeted lipidomic profiling and RNA sequencing has unveiled a distinct metabolic profile and transcriptomic landscape resulting from ACSL4 overexpression, characterized by altered organelle membrane composition, extracellular matrix component and increased neuroendocrine markers that support cancer progression. These findings underscore the potential of ACSL4 as a novel target for advanced cancer therapy. By employing a structure-based virtual screening approach targeting ATP binding domains of ACSL4, followed by in vitro drug testing, we successfully identified four candidate ACSL4 inhibitors. These inhibitors demonstrated significant efficacy in suppressing prostate cancer (PCa) cell proliferation in a dose-response manner. Notably, we showed that these candidate inhibitors exhibit enhanced effectiveness in inhibiting cell proliferation in isogenic ACSL4-high PCa cells. Moreover, we assessed inhibition selectivity through an isotope-labeled ACSL4 activity assay for these inhibitors. Moving forward, our focus will shift towards analyzing pharmacodynamics/pharmacokinetics (PD/PK) for these inhibitors and modifying drug structures to facilitate in vivo application.The first three chapters of this dissertation are introductory information that reviews the role of lipid metabolism in cancer progression and therapy resistance through the regulation of energy production, phospholipid composition and lipid storage, and the clinical significance of targeting lipid metabolism and fatty acid activation as a cancer therapy. Chapter 1 provides a comprehensive review of the current knowledge of how altered lipid metabolism contributes to aberrant cellular activities and oncogenic events in cancer cells, particularly in prostate cancer. Fatty acid activation is key to lipid utilization, and upregulation of multiple Acyl-CoA synthetase, including ACSL4, is frequently observed in cancer patients. The dissertation aims to bridge the existing gap in knowledge by unraveling the biological mechanisms underlying the positive correlation between prostate tumorigenesis and ACSL4 as a long-chain fatty acid activator. Furthermore, it seeks to explore the therapeutic potential of targeting ACSL4 as a viable option for managing advanced prostate cancer. Chapter 2 provided the background of lipid metabolism, and the impact of dysregulated lipid metabolism on cancer cell activities as a result of oncogenic signaling. In this chapter, we highlight the current understanding of the intricate interplay between cancer lipid metabolism and well-known oncogenic signaling. By elucidating these connections, we illuminate how altered lipid metabolic pathways contribute to the reshaping of cellular components, modulation of energy production levels, and perturbation of lipid storage mechanisms, all of which drive cancer progression. Chapter 3 discusses the knowledge and considerations in the current field on the potential of targeting various pathways in lipid metabolism as effective cancer therapies. Given the pivotal role of dysregulated lipid metabolism during tumorigenesis, there has been significant clinical interest in developing pharmacological inhibitors to target altered lipid metabolic activities in cancer. Recent studies have discovered multiple targets as key fatty acid metabolic regulators during oncogenesis with several inhibitors demonstrating effectiveness in both preclinical studies and clinical trials. Most of these drugs exert their effects on tumorigenesis and tumor expansion by targeting enzymes in different lipid metabolic pathways, including de novo fatty acid synthesis, metabolic substrates, fatty acid degradation and lipid storage. The major obstacle in this field has been related to target specificity, as many enzymes within the same family share high structural similarities, leading to off-target issues for many lipogenesis inhibitors. In addition, inhibition of some crucial steps in fatty acid metabolism can cause severe side effects due to accumulation of unexpected side products and toxicity to normal tissues. In many cases, single treatment with lipid synthesis inhibitors shows only moderate efficacy. Therefore, it is imperative for future studies to discover novel targets more specific to oncogenesis and explore the potential of combinatorial treatment with other therapeutic options such as chemotherapy and androgen deprivation therapy to achieve best efficacy with minimized toxicity. Chapter 4 provides the basis for this thesis project based on our previous findings and preliminary data on the key modulators of advanced prostate cancer progression and metastasis. The Retinoblastoma Transcriptional Corepressor 1 (RB) is a key cell cycle regulator and tumor suppressor through regulating essential transcriptional activities via the RB1/E2F signaling axis. Loss of RB1 activity is known to drive cancer cell proliferation and correlates with cancer aggressiveness. Consistently, our preliminary data also showed that co-deletion of RB1 and PTEN in a mouse model led to a highly aggressive and lethal metastatic prostate cancer compared with PTEN deletion only. In addition to its well-established role in mitotic cell cycle regulation, RB1 has been reported to execute tumor-suppressing function through controlling important oncogenic transcriptional activities. Importantly, our previous study identified an RB1 downstream effector called ACSL4, also known as Acyl-CoA Synthetase Long Chain Family Member 4. We showed that RB1/E2F negatively regulates ACSL4 transcription through multiple binding sites on its promoter region, and ACSL4 levels are found to be significantly upregulated in RB-loss PCa cells and patient samples. ACSL4 catalyzes the activation and membrane incorporation of fatty acids by promoting fatty acid utilization and subsequently regulates cell lipid metabolism. Moreover, ACSL4 has been reported to be overexpressed in advanced cancers, including metastatic castration-resistant prostate cancer (mCRPC) and neuroendocrine prostate cancer (NEPC), and its upregulation is often associated with therapy resistance and poor prognosis. In addition to RB1, multiple genes have been reported to regulate ACSL4 level in cancer cells, including the well-known oncogenic signaling AR. However, the role of ACSL4 in reshaping cancer fatty acid metabolism thus promoting tumorigenesis remains elusive. Understanding the biological function of ACSL4 in the context of cancer metabolic landscape will provide great insight into novel targeted therapy by inhibiting ACSL4 activity. Chapter 5 discusses the findings from the first part of this thesis project, which focuses on characterizing the impact of ACSL4 on prostate tumorigenesis through its regulation on cancer cell activities. To explore the role of ACSL4 in cancer cell proliferation and to understand the underlying mechanism, we examined ACSL4 levels in common human PCa cell lines, and generated ACSL4 overexpression cell lines to represent the clinical observation of ACSL4 upregulation. Examination of cell proliferation through crystal violet assay showed that ACSL4 overexpression led to significantly increased cell growth in human PCa cell lines. In addition, ACSL4-overexpressing xenograft tumors had increased growth rate in vivo. Further characterization through the seahorse assay revealed that these cells showed significantly higher oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), indicating boosted ATP-dependent cell viability that accounts for the increased cell proliferation. ACSL4 activates intracellular fatty acids, preferably long-chain fatty acids by esterification of CoA to free fatty acids and generate Acyl-CoA. One metabolic fate of Acyl-CoA is to be diverted into mitochondria, where they are further catalyzed into Acetyl-CoA and participate in tricarboxylic acid (TCA) cycle. Acceleration of TCA cycle due to increased Acetyl-CoA production can cause excessive ATP production supporting cancer cell growth. This result is also consistent with the previous finding that cancer cells tend to shift fatty acid utilization towards de novo synthesis. To confirm these results through loss-of-function studies, we sought to generate ACSL4 knockout and knockdown cell lines. Interestingly, shRNA knockdown of ACSL4 could not be stabilized due to impaired cell viability in parental DU145 and PC-3 cell lines. Alternatively, we found that cells with double knockdown of RB1 and ACSL4 can be stably passed with the knockdown effects well maintained. Our previous study found that RB1 loss granted PCa cells growth advantage and supported cancer progression, and that RB1 deficient PCa is highly lethal and lacks effective therapy options. Examination of the double knockdown PCa cells revealed that the proliferation of these cells was significantly impaired compared. Consistently, we found that ACSL4 knockout PCa cells had a significantly decreased cell proliferation rate and ATP production level. This result provides strong rationale for targeting ACSL4 activity as an efficient way to treat advanced prostate cancers with RB1 deficiency. To further investigate the role of ACSL4 in reshaping cancer cell fatty acid metabolic landscape, in chapter 6 we aim to dissect the mechanism behind the effect of ACSL4 on PCa tumor growth. To this end, we performed untargeted lipidomic profiling analysis and RNA sequencing analysis on ACSL4 overexpression cells and control cells. Here we emphasize the role of ACSL4 in reshaping cancer cell lipid metabolic landscape to support cancer cell growth and drug resistance. Our lipidomic profiling analysis revealed upregulated lipid species in ACSL4 overexpressing PCa cells including triglyceride (TG), bismethyl phosphatidic acid (BisMePA), cholesteryl ester (ChE) and phosphatidylethanolamine (PE). Notably, upregulation of TG and PE has been associated with increased risk of tumor metastasis and progression. ACSL4 preferably consumes long-chain fatty acids including arachidonic acid (AA), eicosatetraenoic acid (EA), stearic acid (SA) and palmitic acid (PA) as substrates. Particularly, BisMePA and ChE with AA, EA and SA acyl tails are also enriched in ACSL4 overexpressing PCa cells. Interestingly, upregulation of BisMePA was observed for both ACSL4-specific acyl chains and in total amount. BisMePA is a major component of lysosomal and endosomal membranes, regulating endo-lysosomal capacity and cell response to stress, and elevated BisMePA could promote cancer cell survival and drug resistance. Interestingly, RNA-seq analysis on ACSL4-overexpressing cells revealed significant change of a set of genes that regulates lysosome stability and activity, consistent with our lipidomic data. Moreover, we found that ACSL4 OE may play a role in remodeling cancer cell extracellular matrix (ECM) to favor cancer cell migration and drug resistance as indicated by significant changes in the ADAMTS family and A2M. Intriguingly, we also noticed that ACSL4 OE cells had a distinct pattern of neuroendocrine prostate carcinomas (NEPC) signature genes expression independent of AR signaling compared with the wildtype cells, which warrants further study of how ACSL4 is involved in lineage plasticity during the progression of NEPC. Given these results, it is imperative to explore the feasibility of pharmacological inhibition of ACSL4 for clinical application. Chapter 7 focuses on the discovery of novel ACSL4 inhibitors as potential treatment for advanced prostate cancer. Structure-based virtual screening (SBVS) utilizes computational docking approach based on the protein 3D structures, which has been widely applied in the discovery of novel small molecule ligands in early-stage drug discovery. To identify ACSL4 inhibitors, we performed SBVS by rendering and docking ACSL4 structure against 100,000 ligands obtained from three Maybridge compound libraries. These ligands were docked against ATP binding pocket and K572 acyl group accommodating channel on ACSL4 structure, respectively to optimize target selectivity. Standard docking followed by precision docking of the top hits generated each round was performed to maximize targeting accuracy. As a result, we identified 158 hits in total on ACSL4 structure, from which we selected top 15 hit compounds for subsequent in vitro screening. SBVS complements high-throughput screening (HTS) through computational approach and provides valuable insights into novel compound discovery, however, downstream in vitro validation is required to examine the pharmacological effects of these candidate molecules. Therefore, we established an in vitro drug testing system using human PCa cell lines to test the potency of the 15 top candidates. We found that 4 out of 15 compounds effectively inhibited PCa cell proliferation in a dose-response manner and their inhibitory effects positively correlated with cellular levels of ACSL4. Notably, we highlight the potency of our candidate inhibitors on suppressing PCa cell growth compared with the only previously reported ACSL4 inhibitor PRGL493, particularly in aggressive ACSL4-high PCa cells such as RB1-deficient PC-3 and neuroendocrine prostate cancer (NEPC) cell line H660. Achieving inhibitor selectivity for desired targets in the development of new drugs is often challenging and can lead to severe side effects due to off targeting. Here we performed an isotope-labeled enzyme activity assay to assess the inhibitor selectivity of our candidate inhibitors on ACSL4 activity. Through measuring the consumption and conversion of arachidonic acid (AA) to AA-CoA by ACSL4 activity, we confirmed that 2 of our candidate inhibitors showed high selectivity towards ACSL4 inhibition. Chapter 8 highlights the potential of ACSL4 inhibitors in the context of clinical application. ACSL4 is known to regulate cancer cell sensitivity to ferroptosis through increased lipid peroxidation. Chemotherapeutic agents inducing cancer cell apoptosis such as docetaxel have been widely used to treat patients with metastatic prostate cancer. It has been debated that the effect of chemotherapy in certain contexts also involves other types of cell death including ferroptosis, arguing that ACSL4 inhibitor could weaken the potency of chemotherapy. Therefore, we investigated the mechanism of action of docetaxel and ACSL4 inhibitors in the context of prostate cancer. We showed that docetaxel treatment mainly induced apoptosis in PCa cell lines PC-3 and DU145, given that the inhibition of cell growth was only rescued by apoptosis inhibitors Z-VAD, not ferroptosis inhibitors ferrostatin and deferoxamine (DFO). This result indicates that inhibition of ACSL4 will not attenuate the effect of chemotherapy. Consistently, ACSL4 overexpressing PCa cells are more resistant to docetaxel treatment. Therefore, we further sought to maximize the therapeutic efficacy for ACSL4 inhibitors, and we found that combinational treatment of ACSL4 inhibitors and docetaxel displayed a synergistic effect on PCa cells, which was partially rescued by apoptosis inhibitor Z-VAD. Our data provides evidence for a novel precision therapeutic option for advanced PCa patients with high ACSL4 expression. Chapter 9 concludes the findings of this study and offers directions for future research. It underscores the significance of targeting fatty acid metabolism and ACSL4 as a pivotal metabolic regulator and oncogenic driver in tumorigenesis and progression, providing valuable insights into treatment options for targeted therapy in advanced prostate cancer.