ACLY alternative splicing correlates with cancer phenotypes.

ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism and provides nucleocytosolic acetyl-CoA necessary for protein acetylation. ACLY has two major splice isoforms: the full-length canonical "long" isoform and an uncharacterized "short" isoform in which exon 14 is spliced out. Exon 14 encodes 10 amino acids within a disordered region of the protein and includes at least 1 site that is dynamically phosphorylated. Both isoforms are expressed in healthy tissues to varying degrees. Analysis of human transcriptomic data revealed that the Percent Spliced In (PSI) of exon 14, i.e., the proportion of long isoform, is increased in several cancers and correlated with poorer overall survival in a pan-cancer analysis, though not in individual tumor types, which prompted us to explore potential biochemical and functional differences between ACLY isoforms. Here, we show that there are no discernible differences in enzymatic activity or stability between isoforms or phosphomutants of ACLY in vitro. Similarly, both isoforms and phosphomutants were able to rescue ACLY functions, including fatty acid synthesis and bulk histone acetylation, when re-expressed in Acly knockout cells. Deletion of Acly exon 14 in mice did not overtly impact development or metabolic physiology, nor did it attenuate tumor burden in a genetic model of intestinal cancer. Notably, expression of epithelial splicing regulatory protein 1 (ESRP1) is highly correlated with ACLY PSI. We report that ACLY splicing is regulated by ESRP1. In turn, both ESRP1 expression and ACLY PSI are correlated with specific immune signatures in tumors. Despite these intriguing patterns of ACLY splicing in healthy and cancer tissues, functional differences between the isoforms remain elusive.

Acetylcarnitine shuttling links mitochondrial metabolism to histone acetylation and lipogenesis.

The metabolite acetyl-CoA is necessary for both lipid synthesis in the cytosol and histone acetylation in the nucleus. The two canonical precursors to acetyl-CoA in the nuclear-cytoplasmic compartment are citrate and acetate, which are processed to acetyl-CoA by ATP-citrate lyase (ACLY) and acyl-CoA synthetase short-chain 2 (ACSS2), respectively. It is unclear whether other substantial routes to nuclear-cytosolic acetyl-CoA exist. To investigate this, we generated cancer cell lines lacking both ACLY and ACSS2 [double knockout (DKO) cells]. Using stable isotope tracing, we show that both glucose and fatty acids contribute to acetyl-CoA pools and histone acetylation in DKO cells and that acetylcarnitine shuttling can transfer two-carbon units from mitochondria to cytosol. Further, in the absence of ACLY, glucose can feed fatty acid synthesis in a carnitine responsive and carnitine acetyltransferase (CrAT)-dependent manner. The data define acetylcarnitine as an ACLY- and ACSS2-independent precursor to nuclear-cytosolic acetyl-CoA that can support acetylation, fatty acid synthesis, and cell growth.

JAZF1: A Metabolic Regulator of Sensitivity to a Polyamine-Targeted Therapy.

Identifying and leveraging unique points of metabolic dysregulation in different disease settings is vital for safe and effective incorporation of metabolism-targeted therapies in the clinic. In addition, it has been shown identification of master metabolic transcriptional regulators (MMTR) of individual metabolic pathways, and how they relate to the disease in question, may offer the key to understanding therapeutic response. In prostate cancer, we have previously demonstrated polyamine biosynthesis and the methionine cycle were targetable metabolic vulnerabilities. However, the MMTRs of these pathways, and how they affect treatment, have yet to be explored. We sought to characterize differential sensitivity of prostate cancer to polyamine- and methionine-targeted therapies by identifying novel MMTRs. We began by developing a gene signature from patient samples, which can predict response to metabolic therapy, and further uncovered a MMTR, JAZF1. We characterized the effects of JAZF1 overexpression on prostate cancer cells, basally and in the context of treatment, by assessing mRNA levels, proliferation, colony formation capability, and key metabolic processes. Lastly, we confirmed the relevance of our findings in large publicly available cohorts of prostate cancer patient samples. We demonstrated differential sensitivity to polyamine and methionine therapies and identified JAZF1 as a MMTR of this response. IMPLICATIONS: We have shown JAZF1 can alter sensitivity of cells and its expression can segregate patient populations into those that do, or do not highly express polyamine genes, leading to better prediction of response to a polyamine targeting therapy.

Reduced NCOR2 expression accelerates androgen deprivation therapy failure in prostate cancer.

This study addresses the roles of nuclear receptor corepressor 2 (NCOR2) in prostate cancer (PC) progression in response to androgen deprivation therapy (ADT). Reduced NCOR2 expression significantly associates with shorter disease-free survival in patients with PC receiving adjuvant ADT. Utilizing the CWR22 xenograft model, we demonstrate that stably reduced NCOR2 expression accelerates disease recurrence following ADT, associates with gene expression patterns that include neuroendocrine features, and induces DNA hypermethylation. Stably reduced NCOR2 expression in isogenic LNCaP (androgen-sensitive) and LNCaP-C4-2 (androgen-independent) cells revealed that NCOR2 reduction phenocopies the impact of androgen treatment and induces global DNA hypermethylation patterns. NCOR2 genomic binding is greatest in LNCaP-C4-2 cells and most clearly associates with forkhead box (FOX) transcription factor FOXA1 binding. NCOR2 binding significantly associates with transcriptional regulation most when in active enhancer regions. These studies reveal robust roles for NCOR2 in regulating the PC transcriptome and epigenome and underscore recent mutational studies linking NCOR2 loss of function to PC disease progression.

Lactate supports a metabolic-epigenetic link in macrophage polarization.

Lactate accumulation is a hallmark of solid cancers and is linked to the immune suppressive phenotypes of tumor-infiltrating immune cells. We report herein that interleukin-4 (IL-4)­induced M0 → M2 macrophage polarization is accompanied by interchangeable glucose- or lactate-dependent tricarboxylic acid (TCA) cycle metabolism that directly drives histone acetylation, M2 gene transcription, and functional immune suppression. Lactate-dependent M0 → M2 polarization requires both mitochondrial pyruvate uptake and adenosine triphosphate­citrate lyase (ACLY) enzymatic activity. Notably, exogenous acetate rescues defective M2 polarization and histone acetylation following mitochondrial pyruvate carrier 1 (MPC1) inhibition or ACLY deficiency. Lastly, M2 macrophage­dependent tumor progression is impaired by conditional macrophage ACLY deficiency, further supporting a dominant role for glucose/lactate mitochondrial metabolism and histone acetylation in driving immune evasion. This work adds to our understanding of how mitochondrial metabolism affects macrophage functional phenotypes and identifies a unique tumor microenvironment (TME)­driven metabolic-epigenetic link in M2 macrophages.

Glutamine deprivation triggers NAGK-dependent hexosamine salvage.

Tumors frequently exhibit aberrant glycosylation, which can impact cancer progression and therapeutic responses. The hexosamine biosynthesis pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a major substrate for glycosylation in the cell. Prior studies have identified the HBP as a promising therapeutic target in pancreatic ductal adenocarcinoma (PDA). The HBP requires both glucose and glutamine for its initiation. The PDA tumor microenvironment is nutrient poor, however, prompting us to investigate how nutrient limitation impacts hexosamine synthesis. Here, we identify that glutamine limitation in PDA cells suppresses de novo hexosamine synthesis but results in increased free GlcNAc abundance. GlcNAc salvage via N-acetylglucosamine kinase (NAGK) is engaged to feed UDP-GlcNAc pools. NAGK expression is elevated in human PDA, and NAGK deletion from PDA cells impairs tumor growth in mice. Together, these data identify an important role for NAGK-dependent hexosamine salvage in supporting PDA tumor growth.

Inside tumors, cancer cells often have to compete with each other for food and other resources they need to survive. This is a key factor driving the growth and progression of cancer. One of the resources cells need is a molecule called UDP-GlcNAc, which they use to modify many proteins so they can work properly. Because cancer cells grow quickly, they likely need much more UDP-GlcNAc than healthy cells. Many tumors, including those derived from pancreatic cancers, have very poor blood supplies, so their cells cannot get the nutrients and other resources they need to grow from the bloodstream. This means that tumor cells have to find new ways to use what they already have. One example of this is developing alternative ways to obtain UDP-GlcNAc. Cells require a nutrient called glutamine to produce UDP-GlcNAc. Limiting the supply of glutamine to cells allows researchers to study how cells are producing UDP-GlcNAc in the lab. Campbell et al. used this approach to study how pancreatic cancer cells obtain UDP-GlcNAc when their access to glutamine is limited. They used a technique called isotope tracing, which allows researchers to track how a specific chemical is processed inside the cell, and what it turns into. The results showed that the pancreatic cancer cells do not make new UDP-GlcNAc but use a protein called NAGK to salvage GlcNAc (another precursor of UDP-GlcNAc), which may be obtained from cellular proteins. Cancer cells that lacked NAGK formed smaller tumors, suggesting that the cells grow more slowly because they cannot recycle UDP-GlcNAc fast enough. Pancreatic cancer is one of the most common causes of cancer deaths and is notable for being difficult to detect and treat. Campbell et al. have identified one of the changes that allows pancreatic cancers to survive and grow quickly. Next steps will include examining the role of NAGK in healthy cells and testing whether it could be targeted for cancer treatment.

The Bidirectional Relationship Between Cancer Epigenetics and Metabolism.

Metabolic and epigenetic reprogramming are characteristics of cancer cells that, in many cases, are linked. Oncogenic signaling, diet, and tumor microenvironment each influence the availability of metabolites that are substrates or inhibitors of epigenetic enzymes. Reciprocally, altered expression or activity of chromatin-modifying enzymes can exert direct and indirect effects on cellular metabolism. In this article, we discuss the bidirectional relationship between epigenetics and metabolism in cancer. First, we focus on epigenetic control of metabolism, highlighting evidence that alterations in histone modifications, chromatin remodeling, or the enhancer landscape can drive metabolic features that support growth and proliferation. We then discuss metabolic regulation of chromatin-modifying enzymes and roles in tumor growth and progression. Throughout, we highlight proposed therapeutic and dietary interventions that leverage metabolic-epigenetic cross talk and have the potential to improve cancer therapy.

Dynamic patterns of DNA methylation in the normal prostate epithelial differentiation program are targets of aberrant methylation in prostate cancer.

Understanding the epigenetic control of normal differentiation programs might yield principal information about critical regulatory states that are disturbed in cancer. We utilized the established non-malignant HPr1-AR prostate epithelial cell model that upon androgen exposure commits to a luminal cell differentiation trajectory from that of a basal-like state. We profile the dynamic transcriptome associated with this transition at multiple time points (0 h, 1 h, 24 h, 96 h), and confirm that expression patterns are strongly indicative of a progressive basal to luminal cell differentiation program based on human expression signatures. Furthermore, we establish dynamic patterns of DNA methylation associated with this program by use of whole genome bisulfite sequencing (WGBS). Expression patterns associated with androgen induced luminal cell differentiation were found to have significantly elevated DNA methylation dynamics. Shifts in methylation profiles were strongly associated with Polycomb repressed regions and to promoters associated with bivalency, and strongly enriched for binding motifs of AR and MYC. Importantly, we found that dynamic DNA methylation patterns observed in the normal luminal cell differentiation program were significant targets of aberrant methylation in prostate cancer. These findings suggest that the normal dynamics of DNA methylation in luminal differentiation contribute to the aberrant methylation patterns in prostate cancer.

Pharmacological polyamine catabolism upregulation with methionine salvage pathway inhibition as an effective prostate cancer therapy.

Prostatic luminal epithelial cells secrete high levels of acetylated polyamines into the prostatic lumen, sensitizing them to perturbations of connected metabolic pathways. Enhanced flux is driven by spermidine/spermine N1-acetyltransferase (SSAT) activity, which acetylates polyamines leading to their secretion and drives biosynthetic demand. The methionine salvage pathway recycles one-carbon units lost to polyamine biosynthesis to the methionine cycle to overcome stress. Prostate cancer (CaP) relies on methylthioadenosine phosphorylase (MTAP), the rate-limiting enzyme, to relieve strain. Here, we show that inhibition of MTAP alongside SSAT upregulation is synergistic in androgen sensitive and castration recurrent CaP models in vitro and in vivo. The combination treatment increases apoptosis in radical prostatectomy ex vivo explant samples. This unique high metabolic flux through polyamine biosynthesis and connected one carbon metabolism in CaP creates a metabolic dependency. Enhancing this flux while simultaneously targeting this dependency in prostate cancer results in an effective therapeutic approach potentially translatable to the clinic.

Epigenetic Control of Fatty-Acid Metabolism Sustains Glioma Stem Cells.

In this issue of Cancer Discovery, Gimple and colleagues examine superenhancers in glioblastoma and glioma stem cells (GSC), identifying one which promotes expression of ELOVL2, an enzyme in polyunsaturated fatty acid (PUFA) synthesis. They find that ELOVL2 products help maintain cell membrane organization and EGFR signaling in GSCs, and that targeting PUFA metabolism along with EGFR offers a potential novel therapeutic strategy for glioblastoma.See related article by Gimple et al., p. 1248.

Adipocyte ACLY Facilitates Dietary Carbohydrate Handling to Maintain Metabolic Homeostasis in Females.

Sugars and refined carbohydrates are major components of the modern diet. ATP-citrate lyase (ACLY) is upregulated in adipocytes in response to carbohydrate consumption and generates acetyl-coenzyme A (CoA) for both lipid synthesis and acetylation reactions. Here, we investigate the role of ACLY in the metabolic and transcriptional responses to carbohydrates in adipocytes and unexpectedly uncover a sexually dimorphic function in maintaining systemic metabolic homeostasis. When fed a high-sucrose diet, AclyFAT-/- females exhibit a lipodystrophy-like phenotype, with minimal fat accumulation, insulin resistance, and hepatic lipid accumulation, whereas AclyFAT-/- males have only mild metabolic phenotypes. We find that ACLY is crucial for nutrient-dependent carbohydrate response element-binding protein (ChREBP) activation in adipocytes and plays a key role, particularly in females, in the storage of newly synthesized fatty acids in adipose tissue. The data indicate that adipocyte ACLY is important in females for the systemic handling of dietary carbohydrates and for the preservation of metabolic homeostasis.

ATP-citrate lyase multimerization is required for coenzyme-A substrate binding and catalysis.

ATP-citrate lyase (ACLY) is a major source of nucleocytosolic acetyl-CoA, a fundamental building block of carbon metabolism in eukaryotes. ACLY is aberrantly regulated in many cancers, cardiovascular disease, and metabolic disorders. However, the molecular mechanisms determining ACLY activity and function are unclear. To this end, we investigated the role of the uncharacterized ACLY C-terminal citrate synthase homology domain in the mechanism of acetyl-CoA formation. Using recombinant, purified ACLY and a suite of biochemical and biophysical approaches, including analytical ultracentrifugation, dynamic light scattering, and thermal stability assays, we demonstrated that the C terminus maintains ACLY tetramerization, a conserved and essential quaternary structure in vitro and likely also in vivo Furthermore, we show that the C terminus, only in the context of the full-length enzyme, is necessary for full ACLY binding to CoA. Together, we demonstrate that ACLY forms a homotetramer through the C terminus to facilitate CoA binding and acetyl-CoA production. Our findings highlight a novel and unique role of the C-terminal citrate synthase homology domain in ACLY function and catalysis, adding to the understanding of the molecular basis for acetyl-CoA synthesis by ACLY. This newly discovered means of ACLY regulation has implications for the development of novel ACLY modulators to target acetyl-CoA-dependent cellular processes for potential therapeutic use.

Acetyl-CoA Metabolism Supports Multistep Pancreatic Tumorigenesis.

Pancreatic ductal adenocarcinoma (PDA) has a poor prognosis, and new strategies for prevention and treatment are urgently needed. We previously reported that histone H4 acetylation is elevated in pancreatic acinar cells harboring Kras mutations prior to the appearance of premalignant lesions. Because acetyl-CoA abundance regulates global histone acetylation, we hypothesized that altered acetyl-CoA metabolism might contribute to metabolic or epigenetic alterations that promote tumorigenesis. We found that acetyl-CoA abundance is elevated in KRAS-mutant acinar cells and that its use in the mevalonate pathway supports acinar-to-ductal metaplasia (ADM). Pancreas-specific loss of the acetyl-CoA-producing enzyme ATP-citrate lyase (ACLY) accordingly suppresses ADM and tumor formation. In PDA cells, growth factors promote AKT-ACLY signaling and histone acetylation, and both cell proliferation and tumor growth can be suppressed by concurrent BET inhibition and statin treatment. Thus, KRAS-driven metabolic alterations promote acinar cell plasticity and tumor development, and targeting acetyl-CoA-dependent processes exerts anticancer effects. SIGNIFICANCE: Pancreatic cancer is among the deadliest of human malignancies. We identify a key role for the metabolic enzyme ACLY, which produces acetyl-CoA, in pancreatic carcinogenesis. The data suggest that acetyl-CoA use for histone acetylation and in the mevalonate pathway facilitates cell plasticity and proliferation, suggesting potential to target these pathways.See related commentary by Halbrook et al., p. 326.This article is highlighted in the In This Issue feature, p. 305.

Inhibition of the aryl hydrocarbon receptor/polyamine biosynthesis axis suppresses multiple myeloma.

Polyamine inhibition for cancer therapy is, conceptually, an attractive approach but has yet to meet success in the clinical setting. The aryl hydrocarbon receptor (AHR) is the central transcriptional regulator of the xenobiotic response. Our study revealed that AHR also positively regulates intracellular polyamine production via direct transcriptional activation of 2 genes, ODC1 and AZIN1, which are involved in polyamine biosynthesis and control, respectively. In patients with multiple myeloma (MM), AHR levels were inversely correlated with survival, suggesting that AHR inhibition may be beneficial for the treatment of this disease. We identified clofazimine (CLF), an FDA-approved anti-leprosy drug, as a potent AHR antagonist and a suppressor of polyamine biosynthesis. Experiments in a transgenic model of MM (Vk*Myc mice) and in immunocompromised mice bearing MM cell xenografts revealed high efficacy of CLF comparable to that of bortezomib, a first-in-class proteasome inhibitor used for the treatment of MM. This study identifies a previously unrecognized regulatory axis between AHR and polyamine metabolism and reveals CLF as an inhibitor of AHR and a potentially clinically relevant anti-MM agent.

Dietary Protein Restriction Reprograms Tumor-Associated Macrophages and Enhances Immunotherapy.

PURPOSE: Diet and healthy weight are established means of reducing cancer incidence and mortality. However, the impact of diet modifications on the tumor microenvironment and antitumor immunity is not well defined. Immunosuppressive tumor-associated macrophages (TAMs) are associated with poor clinical outcomes and are potentially modifiable through dietary interventions. We tested the hypothesis that dietary protein restriction modifies macrophage function toward antitumor phenotypes. EXPERIMENTAL DESIGN: Macrophage functional status under different tissue culture conditions and in vivo was assessed by Western blot, immunofluorescence, qRT-PCR, and cytokine array analyses. Tumor growth in the context of protein or amino acid (AA) restriction and immunotherapy, namely, a survivin peptide-based vaccine or a PD-1 inhibitor, was examined in animal models of prostate (RP-B6Myc) and renal (RENCA) cell carcinoma. All tests were two-sided. RESULTS: Protein or AA-restricted macrophages exhibited enhanced tumoricidal, proinflammatory phenotypes, and in two syngeneic tumor models, protein or AA-restricted diets elicited reduced TAM infiltration, tumor growth, and increased response to immunotherapies. Further, we identified a distinct molecular mechanism by which AA-restriction reprograms macrophage function via a ROS/mTOR-centric cascade. CONCLUSIONS: Dietary protein restriction alters TAM activity and enhances the tumoricidal capacity of this critical innate immune cell type, providing the rationale for clinical testing of this supportive tool in patients receiving cancer immunotherapies.

Dietary folate levels alter the kinetics and molecular mechanism of prostate cancer recurrence in the CWR22 model.

Folate impacts the genome and epigenome by feeding into one-carbon metabolism to produce critical metabolites, deoxythymidine monophosphate and s-adenosylmethionine. The impact of folate exposure and intervention timing on cancer progression remains controversial. Due to polyamine metabolism's extraordinary biosynthetic flux in prostate cancer (CaP) we demonstrated androgen stimulated CaP is susceptible to dietary folate deficiency. We hypothesized dietary folate levels may also affect castration recurrent CaP. We used the CWR22 human xenograft model which recurs following androgen withdrawal. Engrafted mice were fed a folate depleted or supplemented diet beginning at androgen withdrawal, or prior to xenograft implantation. Both folate depletion and supplementation at the time of withdrawal significantly decreased recurrence incidence. Folate supplementation prior to xenograft implantation increased time to recurrence, suggesting a protective role. By contrast, folate depleted recurrent tumors exhibited transcriptional adaptive responses that maintained high polyamine levels at the expense of increased DNA damage and DNA methylation alterations. Mining of publically available data demonstrated folate related pathways are exceptionally dysregulated in human CaP, which correlated with decreased time to biochemical recurrence. These findings highlight the potential for novel therapeutic interventions that target these metabolic pathways in CaP and provide a rationale to apply such strategies alongside androgen withdrawal.

Internally ratiometric fluorescent sensors for evaluation of intracellular GTP levels and distribution.

GTP is a major regulator of multiple cellular processes, but tools for quantitative evaluation of GTP levels in live cells have not been available. We report the development and characterization of genetically encoded GTP sensors, which we constructed by inserting a circularly permuted yellow fluorescent protein (cpYFP) into a region of the bacterial G protein FeoB that undergoes a GTP-driven conformational change. GTP binding to these sensors results in a ratiometric change in their fluorescence, thereby providing an internally normalized response to changes in GTP levels while minimally perturbing those levels. Mutations introduced into FeoB to alter its affinity for GTP created a series of sensors with a wide dynamic range. Critically, in mammalian cells the sensors showed consistent changes in ratiometric signal upon depletion or restoration of GTP pools. We show that these GTP evaluators (GEVALs) are suitable for detection of spatiotemporal changes in GTP levels in living cells and for high-throughput screening of molecules that modulate GTP levels.

The essential role of methylthioadenosine phosphorylase in prostate cancer.

Prostatic epithelial cells secrete high levels of acetylated polyamines into the prostatic lumen. This distinctive characteristic places added strain on the connected pathways, which are forced to increase metabolite production to maintain pools. The methionine salvage pathway recycles the one-carbon unit lost to polyamine biosynthesis back to the methionine cycle, allowing for replenishment of SAM pools providing a mechanism to help mitigate metabolic stress associated with high flux through these pathways. The rate-limiting enzyme involved in this process is methylthioadenosine phosphorylase (MTAP), which, although commonly deleted in many cancers, is protected in prostate cancer. We report near universal retention of MTAP expression in a panel of human prostate cancer cell lines as well as patient samples. Upon metabolic perturbation, prostate cancer cell lines upregulate MTAP and this correlates with recovery of SAM levels. Furthermore, in a mouse model of prostate cancer we find that both normal prostate and diseased prostate maintain higher SAM levels than other tissues, even under increased metabolic stress. Finally, we show that knockdown of MTAP, both genetically and pharmacologically, blocks androgen sensitive prostate cancer growth in vivo. Our findings strongly suggest that the methionine salvage pathway is a major player in homeostatic regulation of metabolite pools in prostate cancer due to their high level of flux through the polyamine biosynthetic pathway. Therefore, this pathway, and specifically the MTAP enzyme, is an attractive therapeutic target for prostate cancer.

Metastatic phenotype in CWR22 prostate cancer xenograft following castration.

BACKGROUND: CWR22 is a human xenograft model of primary prostate cancer (PCa) that is often utilized to study castration recurrent (CR) PCa. CWR22 recapitulates clinical response to androgen deprivation therapy (ADT), in that tumors regress in response to castration, but can recur after a period of time. METHODS: Two cohorts of mice, totaling 117 mice were implanted with CWR22, allowed to develop tumors, castrated by pellet removal and followed for a period of 32 and 50 weeks. Mice presenting with tumors >2.0 cm(3) at the primary site, moribund appearance, or palpable masses other than the primary tumor were sacrificed prior to the endpoint of the study. Tumor tissue, serum, and abnormal lesions were collected upon necropsy and analyzed by IHC, H&E, and PCR for presence of metastatic lesions arising from CWR22. RESULTS: Herein, we report that CWR22 progresses after castration from a primary, hormonal therapy-naïve tumor to metastatic disease in 20% of castrated nude mice. Histological examination of CWR22 primary tumors revealed distinct pathologies that correlated with metastatic outcome after castration. CONCLUSION: This is the first report and characterization of spontaneous metastasis in the CWR22 model, thus, CWR22 is a bona-fide model of clinical PCa representing the full progression from androgen-sensitive, primary PCa to metastatic CR-PCa.