Endogenous ether lipids differentially promote tumor aggressiveness by regulating the SK3 channel.

SK3 channels are potassium channels found to promote tumor aggressiveness. We have previously demonstrated that SK3 is regulated by synthetic ether lipids, but the role of endogenous ether lipids is unknown. Here, we have studied the role of endogenous alkyl- and alkenyl-ether lipids on SK3 channels and on the biology of cancer cells. Experiments revealed that the suppression of alkylglycerone phosphate synthase or plasmanylethanolamine desaturase 1, which are key enzymes for alkyl- and alkenyl-ether-lipid synthesis, respectively, decreased SK3 expression by increasing micro RNA (miR)-499 and miR-208 expression, leading to a decrease in SK3-dependent calcium entry, cell migration, and matrix metalloproteinase 9-dependent cell adhesion and invasion. We identified several ether lipids that promoted SK3 expression and found a differential role of alkyl- and alkenyl-ether lipids on SK3 activity. The expressions of alkylglycerone phosphate synthase, SK3, and miR were associated in clinical samples emphasizing the clinical consistency of our observations. To our knowledge, this is the first report showing that ether lipids differentially control tumor aggressiveness by regulating an ion channel. This insight provides new possibilities for therapeutic interventions, offering clinicians an opportunity to manipulate ion channel dysfunction by adjusting the composition of ether lipids.

Spatial transcriptomic analysis of virtual prostate biopsy reveals confounding effect of tissue heterogeneity on genomic signatures.

Genetic signatures have added a molecular dimension to prognostics and therapeutic decision-making. However, tumour heterogeneity in prostate cancer and current sampling methods could confound accurate assessment. Based on previously published spatial transcriptomic data from multifocal prostate cancer, we created virtual biopsy models that mimic conventional biopsy placement and core size. We then analysed the gene expression of different prognostic signatures (OncotypeDx®, Decipher®, Prostadiag®) using a step-wise approach with increasing resolution from pseudo-bulk analysis of the whole biopsy, to differentiation by tissue subtype (benign, stroma, tumour), followed by distinct tumour grade and finally clonal resolution. The gene expression profile of virtual tumour biopsies revealed clear differences between grade groups and tumour clones, compared to a benign control, which were not reflected in bulk analyses. This suggests that bulk analyses of whole biopsies or tumour-only areas, as used in clinical practice, may provide an inaccurate assessment of gene profiles. The type of tissue, the grade of the tumour and the clonal composition all influence the gene expression in a biopsy. Clinical decision making based on biopsy genomics should be made with caution while we await more precise targeting and cost-effective spatial analyses.

Persistent organochlorine pesticides in periprostatic adipose tissue from men with prostate cancer: Ethno-geographic variations, association with disease aggressiveness.

Although several studies have examined the relationship between organochlorine pesticides (OCPs) and prostate cancer (PCa) risk, no data are available concerning the association between OCPs concentrations in periprostatic adipose tissue (PPAT), which reflects cumulative exposure, and PCa aggressiveness. Moreover, no previous study has compared OCPs exposure in two distinct ethno-geographical populations. The objectives were to analyze OCPs in PPAT of PCa patients from either Mainland France or French West Indies in correlation with features of tumor aggressiveness, after adjusting for potential confounders such age, BMI, and polyunsaturated fatty acid (PUFA) content of PPAT. PPAT was analyzed in 160 patients (110 Caucasians and 50 African-Caribbeans), 80 with an indolent tumor (ISUP group 1 + pT2), and 80 with an aggressive tumor (ISUP group more than 3 + pT3). The concentrations of 29 OCPs were measured in PPAT concomitantly with the characterization of PUFA content. Exposure patterns of OCPs differed according to the ethno-geographical origin. Most OCPs were found at higher concentration in Caucasian patients, whereas pp'-DDE content was twice as high in African-Caribbeans. Chlordecone was only detected in PPAT from African-Caribbean patients. Most OCP concentrations were positively correlated with age, and some with BMI. After adjusting for age, BMI, and PUFA composition of PPAT, no significant association was found between OCPs content and risk of aggressive disease, except of mirex which appeared inversely associated with aggressive features of PCa in Caucasian patients. These results highlight a significant ethno-geographic variation in internal exposure to OCPs, which likely reflects differences in consumption patterns. The inverse relationship observed between mirex concentration and markers of PCa aggressiveness need to be further investigated.

Roles of endogenous ether lipids and associated PUFAs in the regulation of ion channels and their relevance for disease.

Ether lipids (ELs) are lipids characterized by the presence of either an ether linkage (alkyl lipids) or a vinyl ether linkage [i.e., plasmalogens (Pls)] at the sn1 position of the glycerol backbone, and they are enriched in PUFAs at the sn2 position. In this review, we highlight that ELs have various biological functions, act as a reservoir for second messengers (such as PUFAs) and have roles in many diseases. Some of the biological effects of ELs may be associated with their ability to regulate ion channels that control excitation-contraction/secretion/mobility coupling and therefore cell physiology. These channels are embedded in lipid membranes, and lipids can regulate their activities directly or indirectly as second messengers or by incorporating into membranes. Interestingly, ELs and EL-derived PUFAs have been reported to play a key role in several pathologies, including neurological disorders, cardiovascular diseases, and cancers. Investigations leading to a better understanding of their mechanisms of action in pathologies have opened a new field in cancer research. In summary, newly identified lipid regulators of ion channels, such as ELs and PUFAs, may represent valuable targets to improve disease diagnosis and advance the development of new therapeutic strategies for managing a range of diseases and conditions.

Functional Organotypic Cultures of Prostate Tissues: A Relevant Preclinical Model that Preserves Hypoxia Sensitivity and Calcium Signaling.

In prostate cancer research, there is a lack of valuable preclinical models. Tumor cell heterogeneity and sensitivity to microenvironment signals, such as hypoxia or extracellular calcium concentration, are difficult to reproduce. Here, we developed and characterized an ex vivo tissue culture model preserving these properties. Prostate tissue slices from 26 patients were maintained ex vivo under optimized culture conditions. The expression of markers associated with proliferation, androgen-receptor signaling, and hypoxia was assessed by immunostaining. A macroscope was used to achieve real-time calcium fluorescence optical imaging. Tissue morphology was maintained successfully without necrosis for 5 days. Compared with native tumors and tissue cultured with androgens, androgen deprivation in the medium led to decreased expression of both androgen receptor and its target gene products, prostate specific antigen (PSA) and ETS-related gene (ERG). Ex vivo cultured slices also were sensitive to hypoxia because carbonic anhydrase IX and zinc finger E-box binding homeobox 1 (Zeb1) protein levels increased in 1% oxygen. Exposure of slices to supraphysiological extracellular Ca2+ concentration induced a robust and rapid Ca2+ entry, with a greater response in tumor compared with nontumor tissue. This ex vivo model reproduces the morphologic and functional characteristics of human prostate cancer, including sensitivity to androgen deprivation and induced response to hypoxia and extracellular Ca2+. It therefore could become an attractive tool for drug response prediction studies.

Hypoxia Promotes Prostate Cancer Aggressiveness by Upregulating EMT-Activator Zeb1 and SK3 Channel Expression.

Hypoxia is a well-established feature of prostate cancer (PCa) and is associated with disease aggressiveness. The hypoxic microenvironment initiates multiple adaptive responses including epithelial-to-mesenchymal transition (EMT) and a remodeling of calcium homeostasis involved in cancer progression. In the present study, we identified a new hypoxia signaling pathway with a positive feedback loop between the EMT transcription factor Zeb1 and SK3, a Ca2+-activated K+ channel, which leads to amplifying store-operated Ca2+ entry. Zeb1 and SK3 channel were strongly upregulated by hypoxia both in vitro and ex vivo in organotypic cultures of human PCa. Taking into account the sensitivity of the SK3 channel to the membrane lipid composition, we identified lipids such as Ohmline (an alkyl ether lipid and SK3 inhibitor), linoleic acid (LA) and eicosapentaenoic acid (EPA) (fatty acids associated with indolent PCa), which were able to completely abrogate the hypoxia-induced changes in Zeb1 expression. Ultimately, better understanding of this new hypoxia-induced EMT pathway may allow to develop adjuvant therapeutic strategies, in order to control PCa aggressiveness and improve treatment outcomes.

Identifying proteomic risk factors for overall, aggressive, and early onset prostate cancer using Mendelian Randomisation and tumour spatial transcriptomics.

BACKGROUND: Understanding the role of circulating proteins in prostate cancer risk can reveal key biological pathways and identify novel targets for cancer prevention. METHODS: We investigated the association of 2002 genetically predicted circulating protein levels with risk of prostate cancer overall, and of aggressive and early onset disease, using cis-pQTL Mendelian randomisation (MR) and colocalisation. Findings for proteins with support from both MR, after correction for multiple-testing, and colocalisation were replicated using two independent cancer GWAS, one of European and one of African ancestry. Proteins with evidence of prostate-specific tissue expression were additionally investigated using spatial transcriptomic data in prostate tumour tissue to assess their role in tumour aggressiveness. Finally, we mapped risk proteins to drug and ongoing clinical trials targets. FINDINGS: We identified 20 proteins genetically linked to prostate cancer risk (14 for overall [8 specific], 7 for aggressive [3 specific], and 8 for early onset disease [2 specific]), of which the majority replicated where data were available. Among these were proteins associated with aggressive disease, such as PPA2 [Odds Ratio (OR) per 1 SD increment = 2.13, 95% CI: 1.54-2.93], PYY [OR = 1.87, 95% CI: 1.43-2.44] and PRSS3 [OR = 0.80, 95% CI: 0.73-0.89], and those associated with early onset disease, including EHPB1 [OR = 2.89, 95% CI: 1.99-4.21], POGLUT3 [OR = 0.76, 95% CI: 0.67-0.86] and TPM3 [OR = 0.47, 95% CI: 0.34-0.64]. We confirmed an inverse association of MSMB with prostate cancer overall [OR = 0.81, 95% CI: 0.80-0.82], and also found an inverse association with both aggressive [OR = 0.84, 95% CI: 0.82-0.86] and early onset disease [OR = 0.71, 95% CI: 0.68-0.74]. Using spatial transcriptomics data, we identified MSMB as the genome-wide top-most predictive gene to distinguish benign regions from high grade cancer regions that comparatively had five-fold lower MSMB expression. Additionally, ten proteins that were associated with prostate cancer risk also mapped to existing therapeutic interventions. INTERPRETATION: Our findings emphasise the importance of proteomics for improving our understanding of prostate cancer aetiology and of opportunities for novel therapeutic interventions. Additionally, we demonstrate the added benefit of in-depth functional analyses to triangulate the role of risk proteins in the clinical aggressiveness of prostate tumours. Using these integrated methods, we identify a subset of risk proteins associated with aggressive and early onset disease as priorities for investigation for the future prevention and treatment of prostate cancer. FUNDING: This work was supported by Cancer Research UK (grant no. C8221/A29017).

Identifying proteomic risk factors for overall, aggressive and early onset prostate cancer using Mendelian randomization and tumor spatial transcriptomics.

Background: Understanding the role of circulating proteins in prostate cancer risk can reveal key biological pathways and identify novel targets for cancer prevention. Methods: We investigated the association of 2,002 genetically predicted circulating protein levels with risk of prostate cancer overall, and of aggressive and early onset disease, using cis-pQTL Mendelian randomization (MR) and colocalization. Findings for proteins with support from both MR, after correction for multiple-testing, and colocalization were replicated using two independent cancer GWAS, one of European and one of African ancestry. Proteins with evidence of prostate-specific tissue expression were additionally investigated using spatial transcriptomic data in prostate tumor tissue to assess their role in tumor aggressiveness. Finally, we mapped risk proteins to drug and ongoing clinical trials targets. Results: We identified 20 proteins genetically linked to prostate cancer risk (14 for overall [8 specific], 7 for aggressive [3 specific], and 8 for early onset disease [2 specific]), of which a majority were novel and replicated. Among these were proteins associated with aggressive disease, such as PPA2 [Odds Ratio (OR) per 1 SD increment = 2.13, 95% CI: 1.54-2.93], PYY [OR = 1.87, 95% CI: 1.43-2.44] and PRSS3 [OR = 0.80, 95% CI: 0.73-0.89], and those associated with early onset disease, including EHPB1 [OR = 2.89, 95% CI: 1.99-4.21], POGLUT3 [OR = 0.76, 95% CI: 0.67-0.86] and TPM3 [OR = 0.47, 95% CI: 0.34-0.64]. We confirm an inverse association of MSMB with prostate cancer overall [OR = 0.81, 95% CI: 0.80-0.82], and also find an inverse association with both aggressive [OR = 0.84, 95% CI: 0.82-0.86] and early onset disease [OR = 0.71, 95% CI: 0.68-0.74]. Using spatial transcriptomics data, we identified MSMB as the genome-wide top-most predictive gene to distinguish benign regions from high grade cancer regions that had five-fold lower MSMB expression. Additionally, ten proteins that were associated with prostate cancer risk mapped to existing therapeutic interventions. Conclusion: Our findings emphasize the importance of proteomics for improving our understanding of prostate cancer etiology and of opportunities for novel therapeutic interventions. Additionally, we demonstrate the added benefit of in-depth functional analyses to triangulate the role of risk proteins in the clinical aggressiveness of prostate tumors. Using these integrated methods, we identify a subset of risk proteins associated with aggressive and early onset disease as priorities for investigation for the future prevention and treatment of prostate cancer.

A Novel Calcium-Mediated EMT Pathway Controlled by Lipids: An Opportunity for Prostate Cancer Adjuvant Therapy.

The composition of periprostatic adipose tissue (PPAT) has been shown to play a role in prostate cancer (PCa) progression. We recently reported an inverse association between PCa aggressiveness and elevated PPAT linoleic acid (LA) and eicosapentaenoic acid (EPA) content. In the present study, we identified a new signaling pathway with a positive feedback loop between the epithelial-to-mesenchymal transition (EMT) transcription factor Zeb1 and the Ca2+-activated K+ channel SK3, which leads to an amplification of Ca2+ entry and cellular migration. Using in vitro experiments and ex vivo cultures of human PCa slices, we demonstrated that LA and EPA exert anticancer effects, by modulating Ca2+ entry, which was involved in Zeb1 regulation and cancer cellular migration. This functional approach using human prostate tumors highlights the clinical relevance of our observations, and may allow us to consider the possibility of targeting cancer spread by altering the lipid microenvironment.

Fatty acid profile in peri-prostatic adipose tissue and prostate cancer aggressiveness in African-Caribbean and Caucasian patients.

BACKGROUND: Genetic and nutritional factors have been linked to the risk of aggressive prostate cancer (PCa). The fatty acid (FA) composition of peri-prostatic adipose tissue (PPAT), which reflects the past FA intake, is potentially involved in PCa progression. We analysed the FA composition of PPAT, in correlation with the ethno-geographical origin of the patients and markers of tumour aggressiveness. METHODS: From a cohort of 1000 men treated for PCa by radical prostatectomy, FA composition of PPAT was analysed in 156 patients (106 Caucasians and 50 African-Caribbeans), 78 with an indolent tumour (ISUP group 1 + pT2 + PSA <10 ng/mL) and 78 with an aggressive tumour (ISUP group 4-5 + pT3). The effect of FA extracted from PPAT on in-vitro migration of PCa cells DU145 was studied in 72 patients, 36 Caucasians, and 36 African-Caribbeans. RESULTS: FA composition differed according to the ethno-geographical origin. Linoleic acid, an essential n-6 FA, was 2-fold higher in African-Caribbeans compared with Caucasian patients, regardless of disease aggressiveness. In African-Caribbeans, the FA profile associated with PCa aggressiveness was characterised by low level of linoleic acid along with high levels of saturates. In Caucasians, a weak and negative association was observed between eicosapentaenoic acid level (an n-3 FA) and disease aggressiveness. In-vitro migration of PCa cells using PPAT from African-Caribbean patients was associated with lower content of linoleic acid. CONCLUSION: These results highlight an important ethno-geographical variation of PPAT, in both their FA content and association with tumour aggressiveness.

Clinical significance of epithelial-mesenchymal transition markers in prostate cancer.

The process of epithelial-to-mesenchymal transition (EMT) contributes to cancer progression, with activation of transcription factors leading to loss of epithelial characteristics and acquirement of mesenchymal properties. We analyzed in human prostate cancer (PCa) the expression of EMT markers at the different stages of PCa natural history, and evaluated its clinical significance. The expression of the key EMT transcription factor Zeb1, together with E-cadherin, vimentin, and N-cadherin, was evaluated by immunohistochemistry on tissue microarrays containing samples of normal prostate (n = 58), clinically localized cancer (CLC) (n = 242), castration-resistant PCa (CRPC) (n = 48), and metastases (n = 43). Zeb 1 expression was not found in normal tissues, and significantly increased with disease progression from pT2 (20% of cases) to pT3 tumors (34%), and then from CLC to metastases and CRPC (62% and 92%). The expression of EMT target genes was more fluctuant according to disease stages, although in CLC N-cadherin was closely associated with Zeb1 staining. In CLC, after adjusting for classical prognostic markers, only vimentin expression was significantly predictive of shorter recurrence-free survival. In CRPC, preserved E-cadherin staining was associated with longer overall survival, and Zeb1 expression in metastases was predictive of decreased survival. Although Zeb1 expression increased according to the different steps of PCa progression, the expression of its target genes does not seem to follow the same kinetics. However, the potential clinical interest of these EMT markers at several stages of the disease is strongly suggested by their predictive value on both recurrence-free and overall survival.