Patient derived tumoroids of high grade neuroendocrine neoplasms for more personalized therapies.

There are no therapeutic predictive biomarkers or representative preclinical models for high-grade gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN), a highly aggressive, fatal, and heterogeneous malignancy. We established patient-derived (PD) tumoroids from biobanked tissue samples of advanced high-grade GEP-NEN patients and applied this model for targeted rapid ex vivo pharmacotyping, next-generation sequencing, and perturbational profiling. We used tissue-matched PD tumoroids to profile individual patients, compared ex vivo drug response to patients' clinical response to chemotherapy, and investigated treatment-induced adaptive stress responses.PD tumoroids recapitulated biological key features of high-grade GEP-NEN and mimicked clinical response to cisplatin and temozolomide ex vivo. When we investigated treatment-induced adaptive stress responses in PD tumoroids in silico, we discovered and functionally validated Lysine demethylase 5 A and interferon-beta, which act synergistically in combination with cisplatin. Since ex vivo drug response in PD tumoroids matched clinical patient responses to standard-of-care chemotherapeutics for GEP-NEN, our rapid and functional precision oncology approach could expand personalized therapeutic options for patients with advanced high-grade GEP-NEN.

Hexokinase 3 enhances myeloid cell survival via non-glycolytic functions.

The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed, the less well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly upregulated during terminal differentiation of some acute myeloid leukemia (AML) cell line models. Here we show that expression of HK3 is predominantly originating from myeloid cells and that the upregulation of this glycolytic enzyme is not restricted to differentiation of leukemic cells but also occurs during ex vivo myeloid differentiation of healthy CD34+ hematopoietic stem and progenitor cells. Within the hematopoietic system, we show that HK3 is predominantly expressed in cells of myeloid origin. CRISPR/Cas9 mediated gene disruption revealed that loss of HK3 has no effect on glycolytic activity in AML cell lines while knocking out HK2 significantly reduced basal glycolysis and glycolytic capacity. Instead, loss of HK3 but not HK2 led to increased sensitivity to ATRA-induced cell death in AML cell lines. We found that HK3 knockout (HK3-null) AML cells showed an accumulation of reactive oxygen species (ROS) as well as DNA damage during ATRA-induced differentiation. RNA sequencing analysis confirmed pathway enrichment for programmed cell death, oxidative stress, and DNA damage response in HK3-null AML cells. These signatures were confirmed in ATAC sequencing, showing that loss of HK3 leads to changes in chromatin configuration and increases the accessibility of genes involved in apoptosis and stress response. Through isoform-specific pulldowns, we furthermore identified a direct interaction between HK3 and the proapoptotic BCL-2 family member BIM, which has previously been shown to shorten myeloid life span. Our findings provide evidence that HK3 is dispensable for glycolytic activity in AML cells while promoting cell survival, possibly through direct interaction with the BH3-only protein BIM during ATRA-induced neutrophil differentiation.

Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer.

The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular FAs and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto, all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs. Structural analysis of the resulting glycerophospholipids revealed that labeled FAs from uptake were largely incorporated to canonical (sn-) positions on the glycerol backbone. Surprisingly, labeled FA uptake also disrupted canonical isomer patterns of the unlabeled lipidome and induced repartitioning of n-3 and n-6 PUFAs into glycerophospholipid classes. These structural changes support the existence of differences in the metabolic fates of FAs derived from uptake or de novo sources and demonstrate unique signaling and remodeling behaviors usually hidden from conventional lipidomics.

Aquaporin 9 induction in human iPSC-derived hepatocytes facilitates modeling of ornithine transcarbamylase deficiency.

BACKGROUND AND AIMS: Patient-derived human-induced pluripotent stem cells (hiPSCs) differentiated into hepatocytes (hiPSC-Heps) have facilitated the study of rare genetic liver diseases. Here, we aimed to establish an in vitro liver disease model of the urea cycle disorder ornithine transcarbamylase deficiency (OTCD) using patient-derived hiPSC-Heps. APPROACH AND RESULTS: Before modeling OTCD, we addressed the question of why hiPSC-Heps generally secrete less urea than adult primary human hepatocytes (PHHs). Because hiPSC-Heps are not completely differentiated and maintain some characteristics of fetal PHHs, we compared gene-expression levels in human fetal and adult liver tissue to identify genes responsible for reduced urea secretion in hiPSC-Heps. We found lack of aquaporin 9 (AQP9) expression in fetal liver tissue as well as in hiPSC-Heps, and showed that forced expression of AQP9 in hiPSC-Heps restores urea secretion and normalizes the response to ammonia challenge by increasing ureagenesis. Furthermore, we proved functional ureagenesis by challenging AQP9-expressing hiPSC-Heps with ammonium chloride labeled with the stable isotope [15 N] (15 NH4 Cl) and by assessing enrichment of [15 N]-labeled urea. Finally, using hiPSC-Heps derived from patients with OTCD, we generated a liver disease model that recapitulates the hepatic manifestation of the human disease. Restoring OTC expression-together with AQP9-was effective in fully correcting OTC activity and normalizing ureagenesis as assessed by 15 NH4 Cl stable-isotope challenge. CONCLUSION: Our results identify a critical role for AQP9 in functional urea metabolism and establish the feasibility of in vitro modeling of OTCD with hiPSC-Heps. By facilitating studies of OTCD genotype/phenotype correlation and drug screens, our model has potential for improving the therapy of OTCD.

Correction to 'Genome instability and pressure on non-homologous end joining drives chemotherapy resistance via a DNA repair crisis switch in triple negative breast cancer'.

[This corrects the article DOI: 10.1093/nar/zcab022.].

Isomer-Resolved Imaging of Prostate Cancer Tissues Reveals Specific Lipid Unsaturation Profiles Associated With Lymphocytes and Abnormal Prostate Epithelia.

Prostate cancer is the fourth most common cancer worldwide with definitive diagnosis reliant on biopsy and human-graded histopathology. As with other pathologies, grading based on classical haematoxylin and eosin (H&E) staining of formalin fixed paraffin-embedded material can be prone to variation between pathologists, prompting investigation of biomolecular markers. Comprising around 50% of cellular mass, and with known metabolic variations in cancer, lipids provide a promising target for molecular pathology. Here we apply isomer-resolved lipidomics in combination with imaging mass spectrometry to interrogate tissue sections from radical prostatectomy specimens. Guided by the histopathological assessment of adjacent tissue sections, regions of interest are investigated for molecular signatures associated with lipid metabolism, especially desaturation and elongation pathways. Monitoring one of the most abundant cellular membrane lipids within these tissues, phosphatidylcholine (PC) 34:1, high positive correlation was observed between the n-9 isomer (site of unsaturation 9-carbons from the methyl terminus) and epithelial cells from potential pre-malignant lesions, while the n-7 isomer abundance was observed to correlate with immune cell infiltration and inflammation. The correlation of lipid isomer signatures with human disease states in tissue suggests a future role for isomer-resolved mass spectrometry imaging in assisting pathologists with prostate cancer diagnoses and patient stratification.

Genome instability and pressure on non-homologous end joining drives chemotherapy resistance via a DNA repair crisis switch in triple negative breast cancer.

Chemotherapy is used as a standard-of-care against cancers that display high levels of inherent genome instability. Chemotherapy induces DNA damage and intensifies pressure on the DNA repair pathways that can lead to deregulation. There is an urgent clinical need to be able to track the emergence of DNA repair driven chemotherapy resistance and tailor patient staging appropriately. There have been numerous studies into chemoresistance but to date no study has elucidated in detail the roles of the key DNA repair components in resistance associated with the frontline clinical combination of anthracyclines and taxanes together. In this study, we hypothesized that the emergence of chemotherapy resistance in triple negative breast cancer was driven by changes in functional signaling in the DNA repair pathways. We identified that consistent pressure on the non-homologous end joining pathway in the presence of genome instability causes failure of the key kinase DNA-PK, loss of p53 and compensation by p73. In-turn a switch to reliance on the homologous recombination pathway and RAD51 recombinase occurred to repair residual double strand DNA breaks. Further we demonstrate that RAD51 is an actionable target for resensitization to chemotherapy in resistant cells with a matched gene expression profile of resistance highlighted by homologous recombination in clinical samples.

Leptin antagonism inhibits prostate cancer xenograft growth and progression.

Hyperleptinaemia is a well-established therapeutic side effect of drugs inhibiting the androgen axis in prostate cancer (PCa), including main stay androgen deprivation therapy (ADT) and androgen targeted therapies (ATT). Given significant crossover between the adipokine hormone signalling of leptin and multiple cancer-promoting hallmark pathways, including growth, proliferation, migration, angiogenesis, metabolism and inflammation, targeting the leptin axis is therapeutically appealing, especially in advanced PCa where current therapies fail to be curative. In this study, we uncover leptin as a novel universal target in PCa and are the first to highlight increased intratumoural leptin and leptin receptor (LEPR) expression in PCa cells and patients' tumours exposed to androgen deprivation, as is observed in patients' tumours of metastatic and castrate resistant (CRPC) PCa. We also reveal the world-first preclinical evidence that demonstrates marked efficacy of targeted leptin-signalling blockade, using Allo-aca, a potent, specific, and safe LEPR peptide antagonist. Allo-aca-suppressed tumour growth and delayed progression to CRPC in mice bearing LNCaP xenografts, with reduced tumour vascularity and altered pathways of apoptosis, transcription/translation, and energetics in tumours determined as potential mechanisms underpinning anti-tumour efficacy. We highlight LEPR blockade in combination with androgen axis inhibition represents a promising new therapeutic strategy vital in advanced PCa treatment.

Apocryphal FADS2 activity promotes fatty acid diversification in cancer.

Canonical fatty acid metabolism describes specific enzyme-substrate interactions that result in products with well-defined chain lengths, degree(s), and positions of unsaturation. Deep profiling of lipids across a range of prostate cancer cell lines reveals a variety of fatty acids with unusual site(s) of unsaturation that are not described by canonical pathways. The structure and abundance of these unusual lipids correlate with changes in desaturase expression and are strong indicators of cellular phenotype. Gene silencing and stable isotope tracing demonstrate that direct Δ6 and Δ8 desaturation of 14:0 (myristic), 16:0 (palmitic), and 18:0 (stearic) acids by FADS2 generate new families of unsaturated fatty acids (including n-8, n-10, and n-12) that have rarely-if ever-been reported in human-derived cells. Isomer-resolved lipidomics reveals the selective incorporation of these unusual fatty acids into complex structural lipids and identifies their presence in cancer tissues, indicating functional roles in membrane structure and signaling.

Adiponectin receptor activation inhibits prostate cancer xenograft growth.

Adiponectin is an adipokine originally identified as dysregulated in obesity, with a key role in insulin sensitisation and in maintaining systemic energy balance. However, adiponectin is progressively emerging as having aberrant signalling in multiple disease states, including prostate cancer (PCa). Circulating adiponectin is lower in patients with PCa than in non-malignant disease, and inversely correlates with cancer severity. More severe hypoadiponectinemia is observed in advanced PCa than in organ-confined disease. Given the crossover between adiponectin signalling and several cancer hallmark pathways that influence PCa growth and progression, we hypothesised that targeting dysregulated adiponectin signalling may inhibit tumour growth and progression. We, therefore, aimed to test the efficacy of correcting the hypoadiponectinemia and dysregulated adiponectin signalling observed in PCa, a world-first PCa therapeutic approach, using peptide adiponectin receptor (ADIPOR) agonist ADP355 in mice bearing subcutaneous LNCaP xenografts. We demonstrate significant evidence for PCa growth inhibition by ADP355, which slowed tumour growth and delayed progression of serum PCa biomarker, prostate-specific antigen (PSA), compared to vehicle. ADP355 conferred a significant advantage by increasing time on treatment with a delayed ethical endpoint. mRNA sequencing and protein expression analyses of tumours revealed ADP355 PCa growth inhibition may be through altered cellular energetics, cellular stress and protein synthesis, which may culminate in apoptosis, as evidenced by the increased apoptotic marker in ADP355-treated tumours. Our findings highlight the efficacy of ADP355 in targeting classical adiponectin-associated signalling pathways in vivo and provide insights into the promising future for modulating adiponectin signalling through ADIPOR agonism as a novel anti-tumour treatment modality.

Fatty Acid Oxidation Is an Adaptive Survival Pathway Induced in Prostate Tumors by HSP90 Inhibition.

HSP90 is a molecular chaperone required for stabilization and activation of hundreds of client proteins, including many known oncoproteins. AUY922 (luminespib), a new-generation HSP90 inhibitor, exhibits potent preclinical efficacy against several cancer types including prostate cancer. However, clinical use of HSP90 inhibitors for prostate cancer has been limited by toxicity and treatment resistance. Here, we aimed to design an effective combinatorial therapeutic regimen that utilizes subtoxic doses of AUY922, by identifying potential survival pathways induced by AUY922 in clinical prostate tumors. We conducted a proteomic analysis of 30 patient-derived explants (PDE) cultured in the absence and presence of AUY922, using quantitative mass spectrometry. AUY922 significantly increased the abundance of proteins involved in oxidative phosphorylation and fatty acid metabolism in the PDEs. Consistent with these findings, AUY922-treated prostate cancer cell lines exhibited increased mitochondrial mass and activated fatty acid metabolism processes. We hypothesized that activation of fatty acid oxidation is a potential adaptive response to AUY922 treatment and that cotargeting this process will sensitize prostate cancer cells to HSP90 inhibition. Combination treatment of AUY922 with a clinical inhibitor of fatty acid oxidation, perhexiline, synergistically decreased viability of several prostate cancer cell lines, and had significant efficacy in PDEs. The novel drug combination treatment induced cell-cycle arrest and apoptosis, and attenuated the heat shock response, a known mediator of HSP90 treatment resistance. This combination warrants further preclinical and clinical investigation as a novel strategy to overcome resistance to HSP90 inhibition. IMPLICATIONS: Metabolic pathways induced in tumor cells by therapeutic agents may be critical, but targetable, mediators of treatment resistance.

Synthesis of a Unique Psammaplysin F Library and Functional Evaluation in Prostate Cancer Cells by Multiparametric Quantitative Single Cell Imaging.

The spirooxepinisoxazoline alkaloid psammaplysin F (1) was selected as a scaffold for the generation of a unique screening library for both drug discovery and chemical biology research. Large-scale extraction and isolation chemistry was performed on a marine sponge (Hyattella sp.) collected from the Great Barrier Reef in order to acquire >200 mg of the desired bromotyrosine-derived alkaloidal scaffold. Parallel solution-phase semisynthesis was employed to generate a series of psammaplysin-based urea (2-9) and amide analogues (10-11) in low to moderate yields. The chemical structures of all analogues were characterized using NMR and MS data. The absolute configuration of psammaplysin F and all semisynthetic analogues was determined as 6R, 7R by comparison of ECD data with literature values. All compounds (1-11) were evaluated for their effect on cell cycle distribution and changes to cancer metabolism in LNCaP prostate cancer cells using a multiparametric quantitative single-cell imaging approach. These investigations identified that in LNCaP cells psammaplysin F and some urea analogues caused loss of mitochondrial membrane potential, fragmentation of the mitochondrial tubular network, chromosome misalignment, and cell cycle arrest in mitosis.

Therapy-induced lipid uptake and remodeling underpin ferroptosis hypersensitivity in prostate cancer.

BACKGROUND: Metabolic reprograming, non-mutational epigenetic changes, increased cell plasticity, and multidrug tolerance are early hallmarks of therapy resistance in cancer. In this temporary, therapy-tolerant state, cancer cells are highly sensitive to ferroptosis, a form of regulated cell death that is caused by oxidative stress through excess levels of iron-dependent peroxidation of polyunsaturated fatty acids (PUFA). However, mechanisms underpinning therapy-induced ferroptosis hypersensitivity remain to be elucidated. METHODS: We used quantitative single-cell imaging of fluorescent metabolic probes, transcriptomics, proteomics, and lipidomics to perform a longitudinal analysis of the adaptive response to androgen receptor-targeted therapies (androgen deprivation and enzalutamide) in prostate cancer (PCa). RESULTS: We discovered that cessation of cell proliferation and a robust reduction in bioenergetic processes were associated with multidrug tolerance and a strong accumulation of lipids. The gain in lipid biomass was fueled by enhanced lipid uptake through cargo non-selective (macropinocytosis, tunneling nanotubes) and cargo-selective mechanisms (lipid transporters), whereas de novo lipid synthesis was strongly reduced. Enzalutamide induced extensive lipid remodeling of all major phospholipid classes at the expense of storage lipids, leading to increased desaturation and acyl chain length of membrane lipids. The rise in membrane PUFA levels enhanced membrane fluidity and lipid peroxidation, causing hypersensitivity to glutathione peroxidase (GPX4) inhibition and ferroptosis. Combination treatments against AR and fatty acid desaturation, lipase activities, or growth medium supplementation with antioxidants or PUFAs altered GPX4 dependence. CONCLUSIONS: Our work provides mechanistic insight into processes of lipid metabolism that underpin the acquisition of therapy-induced GPX4 dependence and ferroptosis hypersensitivity to standard of care therapies in PCa. It demonstrates novel strategies to suppress the therapy-tolerant state that may have potential to delay and combat resistance to androgen receptor-targeted therapies, a currently unmet clinical challenge of advanced PCa. Since enhanced GPX4 dependence is an adaptive phenotype shared by several types of cancer in response to different therapies, our work might have universal implications for our understanding of metabolic events that underpin resistance to cancer therapies.

Lipid Uptake Is an Androgen-Enhanced Lipid Supply Pathway Associated with Prostate Cancer Disease Progression and Bone Metastasis.

De novo lipogenesis is a well-described androgen receptor (AR)-regulated metabolic pathway that supports prostate cancer tumor growth by providing fuel, membrane material, and steroid hormone precursor. In contrast, our current understanding of lipid supply from uptake of exogenous lipids and its regulation by AR is limited, and exogenous lipids may play a much more significant role in prostate cancer and disease progression than previously thought. By applying advanced automated quantitative fluorescence microscopy, we provide the most comprehensive functional analysis of lipid uptake in cancer cells to date and demonstrate that treatment of AR-positive prostate cancer cell lines with androgens results in significantly increased cellular uptake of fatty acids, cholesterol, and low-density lipoprotein particles. Consistent with a direct, regulatory role of AR in this process, androgen-enhanced lipid uptake can be blocked by the AR-antagonist enzalutamide, but is independent of proliferation and cell-cycle progression. This work for the first time comprehensively delineates the lipid transporter landscape in prostate cancer cell lines and patient samples by analysis of transcriptomics and proteomics data, including the plasma membrane proteome. We show that androgen exposure or deprivation regulates the expression of multiple lipid transporters in prostate cancer cell lines and tumor xenografts and that mRNA and protein expression of lipid transporters is enhanced in bone metastatic disease when compared with primary, localized prostate cancer. Our findings provide a strong rationale to investigate lipid uptake as a therapeutic cotarget in the fight against advanced prostate cancer in combination with inhibitors of lipogenesis to delay disease progression and metastasis. IMPLICATIONS: Prostate cancer exhibits metabolic plasticity in acquiring lipids from uptake and lipogenesis at different disease stages, indicating potential therapeutic benefit by cotargeting lipid supply.

Correction: A molecular portrait of epithelial-mesenchymal plasticity in prostate cancer associated with clinical outcome.

Following the publication of the above article, the authors noted an error in Figure 4, panel B. The colours of the localized and mCRPC samples were accidentally switched. The authors have corrected the colour scheme and added a key to the figure. They have also updated the colour scheme of panel C, both bars are now red instead of one red and one blue. The authors wish to apologize for any inconvenience caused.

A molecular portrait of epithelial-mesenchymal plasticity in prostate cancer associated with clinical outcome.

The propensity of cancer cells to transition between epithelial and mesenchymal phenotypic states via the epithelial-mesenchymal transition (EMT) program can regulate metastatic processes, cancer progression, and treatment resistance. Transcriptional investigations using reversible models of EMT, revealed the mesenchymal-to-epithelial reverting transition (MErT) to be enriched in clinical samples of metastatic castrate resistant prostate cancer (mCRPC). From this enrichment, a metastasis-derived gene signature was identified that predicted more rapid cancer relapse and reduced survival across multiple human carcinoma types. Additionally, the transcriptional profile of MErT is not a simple mirror image of EMT as tumour cells retain a transcriptional "memory" following a reversible EMT. This memory was also enriched in mCRPC samples. Cumulatively, our studies reveal the transcriptional profile of epithelial-mesenchymal plasticity and highlight the unique transcriptional properties of MErT. Furthermore, our findings provide evidence to support the association of epithelial plasticity with poor clinical outcomes in multiple human carcinoma types.

Dysregulated fibronectin trafficking by Hsp90 inhibition restricts prostate cancer cell invasion.

The molecular chaperone Hsp90 is overexpressed in prostate cancer (PCa) and is responsible for the folding, stabilization and maturation of multiple oncoproteins, which are implicated in PCa progression. Compared to first-in-class Hsp90 inhibitors such as 17-allylamino-demethoxygeldanamycin (17-AAG) that were clinically ineffective, second generation inhibitor AUY922 has greater solubility and efficacy. Here, transcriptomic and proteomic analyses of patient-derived PCa explants identified cytoskeletal organization as highly enriched with AUY922 treatment. Validation in PCa cell lines revealed that AUY922 caused marked alterations to cell morphology, and suppressed cell motility and invasion compared to vehicle or 17-AAG, concomitant with dysregulation of key extracellular matrix proteins such as fibronectin (FN1). Interestingly, while the expression of FN1 was increased by AUY922, FN1 secretion was significantly decreased. This resulted in cytosolic accumulation of FN1 protein within late endosomes, suggesting that AUY922 disrupts vesicular secretory trafficking pathways. Depletion of FN1 by siRNA knockdown markedly reduced the invasive capacity of PCa cells, phenocopying AUY922. These results highlight a novel mechanism of action for AUY922 beyond its established effects on cellular mitosis and survival and, furthermore, identifies extracellular matrix cargo delivery as a potential therapeutic target for the treatment of aggressive PCa.

Identification of Gibberellic Acid Derivatives That Deregulate Cholesterol Metabolism in Prostate Cancer Cells.

The naturally occurring pentacyclic diterpenoid gibberellic acid (1) was used in the generation of a drug-like amide library using parallel-solution-phase synthesis. Prior to the synthesis, a virtual library was generated and prioritized based on drug-like physicochemical parameters such as log P, hydrogen bond donor/acceptor counts, and molecular weight. The structures of the synthesized analogues (2-13) were elucidated following analysis of the NMR, MS, UV, and IR data. Compound 12 afforded crystalline material, and its structure was confirmed by X-ray crystallographic analysis. All compounds were evaluated in vitro for cytotoxicity and deregulation of lipid metabolism in LNCaP prostate cancer cells. While no cytotoxic activity was identified at the concentrations tested, synthesized analogues 3, 5, 7, 10, and 11 substantially reduced cellular uptake of free cholesterol in prostate cancer cells, suggesting a novel role of gibberellic acid derivatives in deregulating cholesterol metabolism.

Discovery of thalicthuberine as a novel antimitotic agent from nature that disrupts microtubule dynamics and induces apoptosis in prostate cancer cells.

We report for the first time the mechanism of action of the natural product thalicthuberine (TH) in prostate and cervical cancer cells. TH induced a strong accumulation of LNCaP cells in mitosis, severe mitotic spindle defects, and asymmetric cell divisions, ultimately leading to mitotic catastrophe accompanied by cell death through apoptosis. However, unlike microtubule-binding drugs (vinblastine and paclitaxel), TH did not directly inhibit tubulin polymerization when tested in a cell-free system, whereas it reduced cellular microtubule polymer mass in LNCaP cells. This suggests that TH indirectly targets microtubule dynamics through inhibition of a critical regulator or tubulin-associated protein. Furthermore, TH is not a major substrate for P-glycoprotein (Pgp), which is responsible for multidrug resistance in numerous cancers, providing a rationale to further study TH in cancers with Pgp-mediated treatment resistance. The identification of TH's molecular target in future studies will be of great value to the development of TH as potential treatment of multidrug-resistant tumors.

6α-Acetoxyanopterine: A Novel Structure Class of Mitotic Inhibitor Disrupting Microtubule Dynamics in Prostate Cancer Cells.

The lack of a cure for metastatic castrate-resistant prostate cancer (mCRPC) highlights the urgent need for more efficient drugs to fight this disease. Here, we report the mechanism of action of the natural product 6α-acetoxyanopterine (6-AA) in prostate cancer cells. At low nanomolar doses, this potent cytotoxic alkaloid from the Australian endemic tree Anopterus macleayanus induced a strong accumulation of LNCaP and PC-3 (prostate cancer) cells as well as HeLa (cervical cancer) cells in mitosis, severe mitotic spindle defects, and asymmetric cell divisions, ultimately leading to mitotic catastrophe accompanied by cell death through apoptosis. DNA microarray of 6-AA-treated LNCaP cells combined with pathway analysis identified very similar transcriptional changes when compared with the anticancer drug vinblastine, which included pathways involved in mitosis, microtubule spindle organization, and microtubule binding. Like vinblastine, 6-AA inhibited microtubule polymerization in a cell-free system and reduced cellular microtubule polymer mass. Yet, microtubule alterations that are associated with resistance to microtubule-destabilizing drugs like vinca alkaloids (vinblastine/vincristine) or 2-methoxyestradiol did not confer resistance to 6-AA, suggesting a different mechanism of microtubule interaction. 6-AA is a first-in-class microtubule inhibitor that features the unique anopterine scaffold. This study provides a strong rationale to further develop this novel structure class of microtubule inhibitor for the treatment of malignant disease. Mol Cancer Ther; 16(1); 3-15. ©2016 AACR.