External validation of the Memorial Sloan Kettering Cancer Center preoperative nomogram predicting lymph node invasion in a cohort of high-grade prostate cancer patients.

BACKGROUND: Commonly used preoperative nomograms predicting clinical and pathological outcomes in prostate cancer (PCa) patients have not been yet validated in high-grade only PCa patients. Our objective is to perform an external validation of the Memorial Sloan Kettering Cancer Center (MSKCC) preoperative nomogram as a predictor of lymph node invasion (LNI) in a cohort of high-grade PCa patients. METHODS: We included patients with high-grade PCa (Gleason ≥8) treated at our institution between 2011 and 2020 with radical prostatectomy and pelvic lymph node dissection without receiving neoadjuvant or adjuvant therapy. The area under the curve (AUC) of the receiver operator characteristic (ROC) was used to quantify the accuracy of the model to predict LNI. A calibration plot was used to evaluate the model's precision, and a decision curve analysis was computed to evaluate the net benefit associated with its use. This study was approved by our institution's ethics board. RESULTS: A total of 242 patients with a median age of 66 (60-71) years were included. LNI was observed in 70 (29%) patients with a mean of 16 (median = 15; range = 2-42) resected nodes. The MSKCC nomogram discriminative accuracy, as evaluated by the AUC-ROC was 79.0% (CI: [0.727-0.853]). CONCLUSION: The MSKCC preoperative nomogram is a good predictor of LNI and a useful tool associated with net clinical benefit in this patient population.

Mass spectrometry redefines optimal testosterone thresholds in prostate cancer patients undergoing androgen deprivation therapy.

BACKGROUND: Androgen deprivation therapy (ADT) is the standard of care for prostate cancer treatment. Studies suggest that patients with testosterone levels below 0.7 nM have a longer time to castration resistance. Using the most accurate testosterone measurement method, namely mass spectrometry (MS), we sought to determine if a lower testosterone level under ADT could be associated with longer time to castration resistance. METHODS: This retrospective study included 138 prostate cancer patients undergoing noncurative continuous ADT for which we had access to testosterone measurements assessed by MS. For 108 samples, paired immunoassays (IA) testosterone measurement was available. Primary outcome was time to castration-resistant prostate cancer (CRPC). The Contal and O'Quigley method was used to determine the optimal testosterone castration cut-off point considering the outcome and time-to-event variables. Relationship between testosterone levels assessed either by IA or MS and time to CRPC was evaluated using Cox regression. RESULTS: Mean testosterone level was 0.370 nM by IA and 0.275 nM as assessed by MS. The optimal testosterone cut-off point identified to predict time to CRPC was of 0.705 nM for IA and of 0.270 nM for MS. While no significant difference for time to CRPC was found between patients showing IA testosterone level ≥0.705 nM versus <0.705 nM (hazard ratio [HR]: 1.579; 95% confidence interval [CI]: 0.908-2.745), patients with MS testosterone ≥0.270 nM had an increased risk of progression to CRPC compared to MS testosterone <0.270 nM in univariate (HR: 1.717; 95% CI: 1.160-2.541) and multivariate analysis (HR: 1.662; 95% CI: 1.043-2.648). CONCLUSIONS: The higher sensitivity of MS testosterone measurement methods allows the identification of a lower castration threshold and leads to early identification of patients more likely to progress to CRPC. These patients would likely benefit from treatment intensification by androgen receptor axis-targeted therapies to delay disease progression.

Proximity-dependent Mapping of the Androgen Receptor Identifies Kruppel-like Factor 4 as a Functional Partner.

Prostate cancer (PCa) is the most frequently diagnosed cancer in men and the third cause of cancer mortality. PCa initiation and growth are driven by the androgen receptor (AR). The AR is activated by androgens such as testosterone and controls prostatic cell proliferation and survival. Here, we report an AR signaling network generated using BioID proximity labeling proteomics in androgen-dependent LAPC4 cells. We identified 31 AR-associated proteins in nonstimulated cells. Strikingly, the AR signaling network increased to 182 and 200 proteins, upon 24 h or 72 h of androgenic stimulation, respectively, for a total of 267 nonredundant AR-associated candidates. Among the latter group, we identified 213 proteins that were not previously reported in databases. Many of these new AR-associated proteins are involved in DNA metabolism, RNA processing, and RNA polymerase II transcription. Moreover, we identified 44 transcription factors, including the Kru¨ppel-like factor 4 (KLF4), which were found interacting in androgen-stimulated cells. Interestingly, KLF4 repressed the well-characterized AR-dependent transcription of the KLK3 (PSA) gene; AR and KLF4 also colocalized genome-wide. Taken together, our data report an expanded high-confidence proximity network for AR, which will be instrumental to further dissect the molecular mechanisms underlying androgen signaling in PCa cells.

Extensive Alteration of Androgen Precursor Levels After Castration in Prostate Cancer Patients and Their Association With Active Androgen Level.

PURPOSE: Dihydrotestosterone and testosterone are thought to be major contributors of prostate cancer progression and resistance. We studied the modulation of 15 circulating steroids by castration and their association with dihydrotestosterone and testosterone levels. MATERIALS METHODS: A total of 116 serum samples were collected from 99 prostate cancer patients and categorized as eugonadal, castration-sensitive prostate cancer, castration-resistant prostate cancer, or castration-resistant prostate cancer under abiraterone acetate. Serum levels of 15 steroids were measured using mass spectrometry and compared between groups using analysis of variance. Intrapatient association of steroid levels and the androgens testosterone and dihydrotestosterone were assessed using Pearson correlation and linear regression. RESULTS: Testosterone, dihydrotestosterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone-sulfate, androsterone, androstenediol, estrone, estrone-sulfate, estradiol, and androsterone/3α-diol-3/3α-diol-17-glucuronide levels were significantly decreased in castration-sensitive prostate cancer (castrated) compared to eugonadal patients. Testosterone levels were strongly associated with multiple steroids under eugonadal conditions, whereas they were weakly affected by precursor steroids in castrated patients. By contrast, dihydrotestosterone levels under androgen deprivation therapy were associated with testosterone and the backdoor pathway metabolite androsterone. In castration-resistant prostate cancer patients, levels of androstenedione were significantly associated with testosterone level, while testosterone was the only steroid associated with dihydrotestosterone levels. CONCLUSIONS: Androgen deprivation therapy significantly reduces the levels of 13 circulating steroids. Upon androgen deprivation therapy initiation, the backdoor pathway metabolite androsterone are strongly associated with dihydrotestosterone levels. Under castration-resistant prostate cancer conditions, androstenedione was significantly associated with testosterone levels, suggesting the presence of tumor-related circulating androgens in these patients. These results provide further rationale to intensify treatments with androgen receptor axis signaling pathway inhibitors in patients with prostate cancer.

The Triple-Tracer strategy against Metastatic PrOstate cancer (3TMPO) study protocol.

OBJECTIVE: To determine the prevalence of intra-patient inter-metastatic heterogeneity based on positron emission tomography (PET)/computed tomography (CT) in patients with metastatic castration-resistant prostate cancer (mCRPC) and to determine the prevalence of neuroendocrine disease in these patients and their eligibility for radioligand therapies (RLTs). PATIENTS AND METHODS: This multicentre observational prospective clinical study will include 100 patients with mCRPC from five Canadian academic centres. Patients with radiological or biochemical progression and harbouring at least three metastases by conventional imaging will be accrued. Intra-patient inter-metastatic heterogeneity will be determined with triple-tracer imaging using fluorine-18 fluorodeoxyglucose (18 F-FDG), gallium-68-(68 Ga)-prostate-specific membrane antigen (PSMA)-617 and 68 Ga-DOTATATE, which are a glucose analogue, a PSMA receptor ligand and a somatostatin receptor ligand, respectively. The 68 Ga-PSMA-617 and 18 F-FDG PET/CT scans will be performed first. If at least one PSMA-negative/FDG-positive lesion is observed, an additional PET/CT scan with 68 Ga-DOTATATE will be performed. The tracer uptake of individual lesions will be assessed for each PET tracer and patients with lesions presenting discordant uptake profiles will be considered as having inter-metastatic heterogeneous disease and may be offered a biopsy. EXPECTED RESULTS: The proposed triple-tracer approach will allow whole-body mCRPC characterisation, investigating the inter-metastatic heterogeneity in order to better understand the phenotypic plasticity of prostate cancer, including the neuroendocrine transdifferentiation that occurs during mCRPC progression. Based on 68 Ga-PSMA-617 or 68 Ga-DOTATATE PET positivity, the potential eligibility of patients for PSMA and DOTATATE-based RLT will be assessed. Non-invasive whole-body determination of mCRPC heterogeneity and transdifferentiation is highly innovative and might establish the basis for new therapeutic strategies. Comparison of molecular imaging findings with biopsies will also link metastasis biology to radiomic features. CONCLUSION: This study will add novel, biologically relevant dimensions to molecular imaging: the non-invasive detection of inter-metastatic heterogeneity and transdifferentiation to neuroendocrine prostate cancer by using a multi-tracer PET/CT strategy to further personalise the care of patients with mCRPC.

Extragonadal Steroids Contribute Significantly to Androgen Receptor Activity and Development of Castration Resistance in Recurrent Prostate Cancer after Primary Therapy.

PURPOSE: Beyond testosterone, several steroids contribute to the activation of the androgen receptor pathway, but their relative contributions to the activation of the androgen receptor signaling axis in patients with castrated prostate cancer remain unknown. MATERIALS AND METHODS: Serum levels of 9 steroids were measured by mass spectrometry from continuously castrated patients of the PR.7 study (219) and from the PCA24 cohort (116). For each steroid standard curves for dose dependent prostate specific antigen promoter activation were built in castration sensitive (LAPC4) and resistant (VCaP) prostate cancer models. Standard curves were used to determine the androgen receptor activation potency for each steroid measurement from patients in these trials. RESULTS: In LAPC4 and VCaP cells testosterone, dihydrotestosterone and androstenedione induced androgen receptor transcriptional activity, while dehydroepiandrosterone, 5alpha-androstan-3beta,17beta-diol, androstenediol and androsterone stimulated androgen receptor only in VCaP cells. Extragonadal steroids were responsible for 34% (LAPC4) and 88% (VCaP) of the serum total androgen receptor transcriptional activity found in castrated cases. The total androgen receptor transcriptional activity secondary to testosterone, dihydrotestosterone and androstenedione was associated with time to castration resistance in patients from the PR.7 study (HR 2.17, 95% CI 1.12-4.23, p=0.02) in multivariate analysis using the castration sensitive model (LAPC4). Androgen receptor transcriptional activity of extragonadal androstenedione was the only steroid statistically associated with time to castration resistance in univariate analysis (HR 1.89, 95% CI 1.04-3.44, p=0.036). CONCLUSIONS: Extragonadal steroids contribute significantly to the androgen receptor axis activation at castration levels of testosterone in recurrent nonmetastatic prostate cancer and these sustain the development of castration resistance after primary local treatment.

Development of a Transcriptional Amplification System Based on the PEG3 Promoter to Target Androgen Receptor-Positive and -Negative Prostate Cancer Cells.

Localized prostate cancer (PCa) is often curable, whereas metastatic disease treated by castration inevitably progresses toward castration-resistant PCa (CRPC). Most CRPC treatments target androgen receptor (AR) signaling. However, not all CRPC cells rely on AR activity for survival and proliferation. With advances in immunotherapy and fluid biopsies for cancer management, expression systems specific for both AR-positive and -negative PCa are required for virus-based vaccines and cell imaging. To target both AR-responsive and non-responsive cells, we developed a three-step transcriptional amplification (3STA) system based on the progression elevated gene-3 (PEG3) promoter named PEG3AP1-3STA. Notably, we report on different genetic modifications that significantly improved PEG3 promoter's strength in PCa cells. Adenoviruses incorporating PEG3 promoter with and without transcriptional amplification systems were generated. The potential of PEG3AP1-3STA to target PCa cells was then evaluated in vitro and in vivo in androgen-responsive and non-responsive PCa cell lines. PEG3AP1-3STA was shown to be active in all PCa cell lines and not regulated by androgens, and its activity was amplified 97-fold compared to that of a non-amplified promoter. The PEG3AP1-3STA system can thus be used to target advanced AR+ and AR- cells for imaging or immunovirotherapy in advanced PCa.

An Extracellular Matrix Overlay Model for Bioluminescence Microscopy to Measure Single-Cell Heterogeneous Responses to Antiandrogens in Prostate Cancer Cells.

Prostate cancer (PCa) displays diverse intra-tumoral traits, impacting its progression and treatment outcomes. This study aimed to refine PCa cell culture conditions for dynamic monitoring of androgen receptor (AR) activity at the single-cell level. We introduced an extracellular matrix-Matrigel (ECM-M) culture model, enhancing cellular tracking during bioluminescence single-cell imaging while improving cell viability. ECM-M notably tripled the traceability of poorly adherent PCa cells, facilitating robust single-cell tracking, without impeding substrate permeability or AR response. This model effectively monitored AR modulation by antiandrogens across various PCa cell lines. Single-cell imaging unveiled heterogeneous antiandrogen responses within populations, correlating non-responsive cell proportions with drug IC50 values. Integrating ECM-M culture with the PSEBC-TSTA biosensor enabled precise characterization of ARi responsiveness within diverse cell populations. Our ECM-M model stands as a promising tool to assess heterogeneous single-cell treatment responses in cancer, offering insights to link drug responses to intracellular signaling dynamics. This approach enhances our comprehension of the nuanced and dynamic nature of PCa treatment responses.

Preclinical models of prostate cancer - modelling androgen dependency and castration resistance in vitro, ex vivo and in vivo.

Prostate cancer is well known to be dependent on the androgen receptor (AR) for growth and survival. Thus, AR is the main pharmacological target to treat this disease. However, after an initially positive response to AR-targeting therapies, prostate cancer will eventually evolve to castration-resistant prostate cancer, which is often lethal. Tumour growth was initially thought to become androgen-independent following treatments; however, results from molecular studies have shown that most resistance mechanisms involve the reactivation of AR. Consequently, tumour cells become resistant to castration - the blockade of testicular androgens - and not independent of AR per se. However, confusion still remains on how to properly define preclinical models of prostate cancer, including cell lines. Most cell lines were isolated from patients for cell culture after evolution of the tumour to castration-resistant prostate cancer, but not all of these cell lines are described as castration resistant. Moreover, castration refers to the blockade of testosterone production by the testes; thus, even the concept of "castration" in vitro is questionable. To ensure maximal transfer of knowledge from scientific research to the clinic, understanding the limitations and advantages of preclinical models, as well as how these models recapitulate cancer cell androgen dependency and can be used to study castration resistance mechanisms, is essential.

Isocitrate dehydrogenase 1 sustains a hybrid cytoplasmic-mitochondrial tricarboxylic acid cycle that can be targeted for therapeutic purposes in prostate cancer.

The androgen receptor (AR) is an established orchestrator of cell metabolism in prostate cancer (PCa), notably by inducing an oxidative mitochondrial program. Intriguingly, AR regulates cytoplasmic isocitrate dehydrogenase 1 (IDH1), but not its mitochondrial counterparts IDH2 and IDH3. Here, we aimed to understand the functional role of IDH1 in PCa. Mouse models, in vitro human PCa cell lines, and human patient-derived organoids (PDOs) were used to study the expression and activity of IDH enzymes in the normal prostate and PCa. Genetic and pharmacological inhibition of IDH1 was then combined with extracellular flux analyses and gas chromatography-mass spectrometry for metabolomic analyses and cancer cell proliferation in vitro and in vivo. In PCa cells, more than 90% of the total IDH activity is mediated through IDH1 rather than its mitochondrial counterparts. This profile seems to originate from the specialized prostate metabolic program, as observed using mouse prostate and PDOs. Pharmacological and genetic inhibition of IDH1 impaired mitochondrial respiration, suggesting that this cytoplasmic enzyme contributes to the mitochondrial tricarboxylic acid cycle (TCA) in PCa. Mass spectrometry-based metabolomics confirmed this hypothesis, showing that inhibition of IDH1 impairs carbon flux into the TCA cycle. Consequently, inhibition of IDH1 decreased PCa cell proliferation in vitro and in vivo. These results demonstrate that PCa cells have a hybrid cytoplasmic-mitochondrial TCA cycle that depends on IDH1. This metabolic enzyme represents a metabolic vulnerability of PCa cells and a potential new therapeutic target.

A transcriptional biosensor to monitor single cancer cell therapeutic responses by bioluminescence microscopy.

When several life-prolonging drugs are indicated for cancer treatment, predictive drug-response tumor biomarkers are essential to guide management. Most conventional biomarkers are based on bulk tissue analysis, which cannot address the complexity of single-cell heterogeneity responsible for drug resistance. Therefore, there is a need to develop alternative drug response predictive biomarker approaches that could directly interrogate single-cell and whole population cancer cell drug sensitivity. In this study, we report a novel method exploiting bioluminescence microscopy to detect single prostate cancer (PCa) cell response to androgen receptor (AR)-axis-targeted therapies (ARAT) and predict cell population sensitivity. Methods: We have generated a new adenovirus-delivered biosensor, PCA3-Cre-PSEBC-ITSTA, which combines an integrated two-step transcriptional amplification system (ITSTA) and the activities of the prostate cancer antigen 3 (PCA3) and modified prostate-specific antigen (PSEBC) gene promoters as a single output driving the firefly luciferase reporter gene. This system was tested on PCa cell lines and on primary PCa cells. Single cells, exposed or not to ARAT, were dynamically imaged by bioluminescence microscopy. A linear discriminant analysis (LDA)-based method was used to determine cell population sensitivities to ARAT. Results: We show that the PCA3-Cre-PSEBC-ITSTA biosensor is PCa-specific and can dynamically monitor single-cell AR transcriptional activity before and after ARAT by bioluminescence microscopy. After biosensor transduction and bioluminescence microscopy single-cell luminescence dynamic quantification, LDA analysis could discriminate the cell populations overall ARAT sensitivity despite heterogeneous single-cell responses. Indeed, the biosensor could detect a significant decrease in AR activity following exposure to conventional ARAT in hormone-naive primary PCa cells, while in castration-resistant PCa patients, treatment response correlated with the observed clinical ARAT resistance. Conclusion: The exploitation of bioluminescence microscopy and multi-promoter transcriptionally-regulated biosensors can aptly define the overall treatment response of patients by monitoring live single cell drug response from primary cancer tissue. This approach can be used to develop predictive biomarkers for drug response in order to help clinicians select the best drug combinations or sequences for each patient.

Subversion of infiltrating prostate macrophages to a mixed immunosuppressive tumor-associated macrophage phenotype.

Tumor-associated macrophages (TAMs) support tumor progression within the tumor microenvironment (TME). Many questions remain as to the origin, development, and function of TAMs within the prostate TME. Evaluation of TAMs in prostate cancer (PCa) patients identified the immunosuppressive TAM marker CD163 in adjacent normal epithelium as an independent predictor of metastases or PCa death. Flow cytometry analyses identified prostate TAMs as frequently expressing both proinflammatory M1 (CCR7+) and immunosuppressive M2 (CD163+) markers. In vitro, we demonstrate PCa cells similarly subvert human M1 macrophages toward a mixed M1/M2 macrophage phenotype favoring tumor growth. Further the cytokine milieu-induced transition between immunosuppressive M2 to proinflammatory M1 (M2→M1) macrophages is abrogated by the presence of PCa cells. RNA sequencing suggests alterations in chemokine expression in prostate TAMs due to the presence of PCa cells. Together, our results suggest that prostate TAMs originate from inflammatory infiltrating macrophages, which are then reprogrammed mainly by PCa cells, but also the cytokine milieu. A better understanding of this subversion of macrophages within the prostate may lead to novel treatment strategies.

Multiple metabolic pathways fuel the truncated tricarboxylic acid cycle of the prostate to sustain constant citrate production and secretion.

OBJECTIVE: The prostate is metabolically unique: it produces high levels of citrate for secretion via a truncated tricarboxylic acid (TCA) cycle to maintain male fertility. In prostate cancer (PCa), this phenotype is reprogrammed, making it an interesting therapeutic target. However, how the truncated prostate TCA cycle works is still not completely understood. METHODS: We optimized targeted metabolomics in mouse and human organoid models in ex vivo primary culture. We then used stable isotope tracer analyses to identify the pathways that fuel citrate synthesis. RESULTS: First, mouse and human organoids were shown to recapitulate the unique citrate-secretory program of the prostate, thus representing a novel model that reproduces this unusual metabolic profile. Using stable isotope tracer analysis, several key nutrients were shown to allow the completion of the prostate TCA cycle, revealing a much more complex metabolic profile than originally anticipated. Indeed, along with the known pathway of aspartate replenishing oxaloacetate, glutamine was shown to fuel citrate synthesis through both glutaminolysis and reductive carboxylation in a GLS1-dependent manner. In human organoids, aspartate entered the TCA cycle at the malate entry point, upstream of oxaloacetate. Our results demonstrate that the citrate-secretory phenotype of prostate organoids is supported by the known aspartate-oxaloacetate-citrate pathway, but also by at least three additional pathways: glutaminolysis, reductive carboxylation, and aspartate-malate conversion. CONCLUSIONS: Our results add a significant new dimension to the prostate citrate-secretory phenotype, with at least four distinct pathways being involved in citrate synthesis. Better understanding this distinctive citrate metabolic program will have applications in both male fertility as well as in the development of novel targeted anti-metabolic therapies for PCa.

GLUT1 expression in high-risk prostate cancer: correlation with 18F-FDG-PET/CT and clinical outcome.

BACKGROUND: Tumour 18F-FDG-uptake is of prognostic value in high-risk and metastatic prostate cancer (PCa). The aim of this study is to investigate the underlying glucose metabolism mechanisms of 18F-FDG-uptake on PET/CT imaging in PCa. METHODS: Retrospective analysis was conducted for 94 patients diagnosed with a Gleason sum ≥8 adenocarcinoma of the prostate at biopsy between July 2011 and July 2014 who underwent 18F-FDG-PET/CT imaging before radical prostatectomy (RP). 18F-FDG-uptake in primary lesion was measured by a blinded reader using maximum standardised uptake value (SUVmax). GLUT1, GLUT12 and HK2 expression were blindly scored after immunohistochemistry on specimens RP by three pathologists. Correlations between GLUT1, GLUT12 and HK2, and SUVmax were assessed using Spearman's rank correlation test. Survival probabilities were based on the Kaplan-Meier method. RESULTS: With a median follow-up of 4.5 years, 56% (n = 53) of patients had biochemical recurrence (BCR), 7% (n = 7) progressed to castration-resistant prostate cancer (CRPC) disease, 13% (n = 12) developed metastasis and 6% (n = 6) died. Correlation was found between GLUT1 expression and SUVmax level (r = 0.25, p = 0.02). In addition, SUVmax was significantly higher in tumours with high GLUT1 expression (n = 17, 5.74 ± 1.67) than tumours with low GLUT1 expression (n = 71, 2.68 ± 0.31, p = 0.004). Moreover, a significant association was found between GLUT1 expression levels and SUVmax level (p = 0.005), lymph node status (p = 0.05), volume of cancer (p = 0.01), CRPC disease progression (p = 0.02) and metastasis development (p = 0.04). No significant difference between GLUT12 and HEX2 expression and SUVmax have been found. CONCLUSIONS: GLUT1 expression in PCa tumours correlates with 18F-FDG-uptake and poor prognostic factors. These results suggest that this transporter is involved in the molecular mechanism of 18F-FDG-uptake in high-risk PCa and raise interest in targeting metabolic dependencies of PCa cells as a selective anticancer strategy.

Usefulness of heterologous promoters in the Pseudozyma flocculosa gene expression system.

The basidiomycetous fungus Pseudozyma flocculosa represents a promising new host for the expression of complex recombinant proteins. Two novel heterologous promoter sequences, the Ustilago maydis glyceraldehyde-3-phosphate dehydrogenase (GPD) and Pseudozyma tsukubaensis alpha-glucosidase promoters, were tested for their ability to provide expression in P. flocculosa. In liquid medium, these two promoters produced lower levels of intracellular green fluorescent protein (GFP) as compared to the U. maydis hsp70 promoter. However, GPD and alpha-glucosidase sequences behaved as constitutive promoters whereas the hsp70 promoter appeared to be morphology-dependent. When using the hsp70 promoter, the expression of GFP increased proportionally to the concentration of hygromycin in the culture medium, indicating possible induction of the promoter by the antibiotic. Optimal solid-state culture conditions were designed for high throughput screening of hygromycin-resistant transformants with the hsp70 promoter in P. flocculosa.

Recombinant protein secretion in Pseudozyma flocculosa and Pseudozyma antarctica with a novel signal peptide.

Secretion of recombinant proteins aims to reproduce the correct posttranslational modifications of the expressed protein while simplifying its recovery. In this study, secretion signal sequences from an abundantly secreted 34-kDa protein (P34) from Pseudozyma flocculosa were cloned. The efficiency of these sequences in the secretion of recombinant green fluorescent protein (GFP) was investigated in two Pseudozyma species and compared with other secretion signal sequences, from S. cerevisiae and Pseudozyma spp. The results indicate that various secretion signal sequences were functional and that the P34 signal peptide was the most effective secretion signal sequence in both P. flocculosa and P. antarctica. The cells correctly processed the secretion signal sequences, including P34 signal peptide, and mature GFP was recovered from the culture medium. This is the first report of functional secretion signal sequences in P. flocculosa. These sequences can be used to test the secretion of other recombinant proteins and for studying the secretion pathway in P. flocculosa and P. antarctica.

GFP technology for the study of biocontrol agents in tritrophic interactions: a case study with Pseudozyma flocculosa.

GFP technology was applied to the biocontrol agent (BCA) Pseudozyma flocculosa to study its development and interactions at the tritrophic level plant-powdery mildew-BCA. Transformation experiments with GFP led to the production of a strongly fluorescent strain, Act-4, that displayed biocontrol traits typical of P. flocculosa WT. Following inundative applications, growth of P. flocculosa Act-4 was closely and almost exclusively associated with the colonies of the pathogen regardless of the powdery mildew species or the host plant tested. Development of P. flocculosa Act-4 on control leaves alone was extremely limited 24 h after its application and was typical of the epiphytic growth characterizing this type of yeast-like fungus. Based on the strong correlation between the colonization pattern of the different powdery mildew species tested and the presence of P. flocculosa Act-4, as determined by its fluorescence, it seems that growth of the BCA is dependant on the presence of powdery mildews. These results demonstrate that the GFP technology can be used to study plant-pathogen-BCA interactions and fulfill a wide array of purposes ranging from fundamental observations of the biocontrol behavior of a BCA to very applied ones serving some of the requirements for the registration of BCA's such as defining their environmental fate.

Investigation of the In Vitro and In Vivo efficiency of RM-532-105, a 17ß-hydroxysteroid dehydrogenase type 3 inhibitor, in LAPC-4 prostate cancer cell and tumor models.

In the fight against androgen-sensitive prostate cancer, the enzyme 17ß-hydroxysteroid dehydrogenase type 3 (17ß-HSD3) is an attractive therapeutic target considering its key role in the formation of androgenic steroids. In this study, we attempted to assess the in vivo efficacy of the compound RM-532-105, an androsterone derivative developed as an inhibitor of 17ß-HSD3, in the prostate cancer model of androgen-sensitive LAPC-4 cells xenografted in nude mice. RM-532-105 did not inhibit the tumor growth induced by 4-androstene-3,17-dione (4-dione); rather, the levels of the androgens testosterone (T) and dihydrotestosterone (DHT) increased within the tumors. In plasma, however, DHT levels increased but T levels did not. In troubleshooting experiments, the non-androgenic potential of RM-532-105 was confirmed by two different assays (LAPC-4 proliferation and androgen receptor transcriptional activity assays). The enzyme 5α-reductase was also revealed to be the predominant enzyme metabolizing 4-dione in LAPC-4 cells, yielding 5α-androstane-3,17-dione and not T. Other 17ß-HSDs than 17ß-HSD3 seem responsible in the androgen synthesis. From experiments with LAPC-4 cells, we fortuitously came across the interesting finding that 17ß-HSD3 inhibitor RM-532-105 is concentrated inside tumors.

Tissue-engineered human 3D model of bladder cancer for invasion study and drug discovery.

The tumour microenvironment is critical to both the initiation and maintenance of tumorigenesis. Reconstitution of the microenvironment is a major challenge for in vitro cancer models. Indeed, conventional 2D culture systems cannot replicate the complexity, diversity and dynamic nature of the tumour microenvironment. In this study, we have developed a 3D endotheliazed vesical equivalent by using tissue engineering from primary human cells in which non-invasive or invasive bladder cancer (BCa) cell lines, cultured as compact spheroids, were incorporated. Invasive BCa cells cross the basement membrane and invade the stromal compartment whereas non-invasive BCa cells are confined to the urothelium. Our 3D BCa model could be used as a reliable model for assessing drug responses, potentially reducing or partially replacing animal experiments, and thus should have applications in the identification of novel targets as well as toxicological evaluation of anti-cancer therapies.

Bioluminescence Microscopy as a Method to Measure Single Cell Androgen Receptor Activity Heterogeneous Responses to Antiandrogens.

Cancer cell heterogeneity is well-documented. Therefore, techniques to monitor single cell heterogeneous responses to treatment are needed. We developed a highly translational and quantitative bioluminescence microscopy method to measure single cell androgen receptor (AR) activity modulation by antiandrogens from fluid biopsies. We showed that this assay can detect heterogeneous cellular response to drug treatment and that the sum of single cell AR activity can mirror the response in the whole cell population. This method may thus be used to monitor heterogeneous dynamic treatment responses in cancer cells.