Bertrand Neveu

The potential of Pseudozyma yeastlike epiphytes for the production of heterologous recombinant proteins

Although Basidiomycetes represent the most evolved class of fungi, they have been neglected with regard to recombinant gene expression. In this work, basidiomycetous yeasts belonging to Pseudozyma spp. were studied with respect to their amenability to heterologous protein production. Single plasmid or cotransformation experiments routinely afforded 100 to 200 independent transformants for the two tested species of Pseudozyma. Green fluorescent protein (GFP) was expressed in the correctly folded conformation, as demonstrated by fluorescence microscopy, and hen egg white lysozyme (HEWL) was expressed in its active form, as revealed by its lytic activity on Micrococcus lysodeikticus cells. Protease analysis established that Pseudozyma spp. contained equivalent or less extracellular protease activity than yeasts and far less protease activity than ascomycetous filamentous fungi in similar culture conditions. This proteolytic activity was inhibited by over 97% with a combination of PMSF and Pepstatin A. N-glycosylation patterns of native Pseudozyma flocculosa secreted proteins were comprised of one or a few short glycan chains that possess a classic eukaryotic structure typical of higher fungi and animal cells. This is the first report of a Basidiomycete that possesses multiple intrinsic characteristics necessary for use as a heterologous gene expression system.

Cloning of the glyceraldehyde-3-phosphate dehydrogenase gene from Pseudozyma flocculosa and functionality of its promoter in two Pseudozyma species.

Pseudozyma flocculosa is a yeast-like epiphyte recently classified as a basidiomycete related to the Ustilaginales. In this study, we report the cloning of its gene coding for a putative glyceraldehyde-3-phosphate dehydrogenase (GPD). This gene was selected on the premise that its transcripts are abundant during the growth phase of P. flocculosa. The complete sequence of this gene was found to contain two introns in the coding region and one in the 3′-untranslated region. This gene was present in a single copy in the genome of P. flocculosa. By comparing its deduced amino acid sequence with various sequences from basidiomycetous and ascomycetous fungi, we observed a stronger homology with the former group as predicted by the new classification of P. flocculosa. The promoter region lacked a typical TATA or CAAT box but contained a CT-rich region including the transcription start site. Although the GPD promoter showed a stronger affinity within P. flocculosa, it remained active across species as shown by expressing the green fluorescent protein in Pseudozyma antarctica. The cloning of this gene and its promoter brings new and versatile options to the limited genetic tools currently available for the study of the recently defined Pseudozyma genus.

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 α-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 α-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.

IL-8 secretion in primary cultures of prostate cells is associated with prostate cancer aggressiveness

Background Chronic inflammation is believed to be a major factor in prostate cancer initiation and promotion and has been studied using prostate cancer cells and immortalized cell lines. However, little is known about the contribution of normal cells to the prostatic microenvironment and inflammation. We aim to study the contribution of normal prostate epithelial cells to prostate inflammation and to link the inflammatory status of normal cells to prostate cancer aggressiveness. Materials and methods Short-term primary cell cultures of normal epithelial prostate cells were derived from prostate biopsies from 25 men undergoing radical prostatectomy, cystoprostatectomy, or organ donation. Cells were treated with polyinosinic:polycytidylic acid, a mimic of double-stranded viral RNA and a potent inducer of the inflammatory response. Secretion of interleukin (IL)-8 in the cell culture medium by untreated and treated cells was measured and we determined the association between IL-8 levels in these primary cell cultures and prostate cancer characteristics. The Fligner–Policello test was used to compare the groups. Results Baseline and induced IL-8 secretion were highly variable between cultured cells from different patients. This variation was not related to drug use, past medical history, age, or preoperative prostate-specific antigen value. Nonetheless, an elevated secretion of IL-8 from normal cultured epithelial cells was associated with prostate cancer aggressiveness (P=0.0005). Conclusion The baseline secretion of IL-8 from normal prostate epithelial cells in culture is strongly correlated with cancer aggressiveness and may drive prostate cancer carcinogenesis. A better characterization of individual prostate microenvironment may provide a basis for personalized treatment and for monitoring the effects of strategies aimed at preventing aggressive prostate cancer.

A PCA3 gene-based transcriptional amplification system targeting primary prostate cancer

Targeting specifically primary prostate cancer (PCa) cells for immune therapy, gene therapy or molecular imaging is of high importance. The PCA3 long non-coding RNA is a unique PCa biomarker and oncogene that has been widely studied. This gene has been mainly exploited as an accurate diagnostic urine biomarker for PCa detection. In this study, the PCA3 promoter was introduced into a new transcriptional amplification system named the 3-Step Transcriptional Amplification System (PCA3-3STA) and cloned into type 5 adenovirus. PCA3-3STA activity was highly specific for PCa cells, ranging between 98.7- and 108.0-fold higher than that for benign primary prostate epithelial or non-PCa cells, respectively. In human PCa xenografts, PCA3-3STA displayed robust bioluminescent signals at levels that are sufficient to translate to positron emission tomography (PET)-based reporter imaging. Remarkably, when freshly isolated benign or cancerous prostate biopsies were infected with PCA3-3STA, the optical signal produced from primary PCa biopsies was significantly higher than from benign prostate biopsies (4.4-fold, p < 0.0001). PCA3-3STA therefore represents a PCa-specific expression system with the potential to target, with high accuracy, primary or metastatic PCa epithelial cells for imaging, vaccines, or gene therapy.

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.

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.

Abstract 4764: Development of a multigenic bioluminescence imaging system to detect prostate cancer cells and assess their response to therapy

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA BACKGROUND Currently, liquid biposies for imaging single cancer cell to provide personalised medicine is gaining importance. In the last years, molecular imaging techniques using transcriptional amplification systems have been developed but a system enabling both PCa cell detection and treatment response assessment is lacking. PCA3 RNA is a unique PCa biomarker that has been widely studied for PCa screening and detection while the PSA gene is another biomarker of high clinical significance as it gives an account of response to androgen deprivation treatments (ADT). In this study, we have developed and studied an imaging system based on the combined transcriptional activities of the PCA3 and PSA gene promoters for single PCa cell detection and ADT response assessment from patients body fluids. METHODS Adenoviruses (Ad) were constructed utilizing the ability of site-specific recombination of the Cre-Lox system. The PCA3 and PSA promoters were integrated into a single Ad backbone with one promoter driving the expression of CRE recombinase and the other driving the Two Step Transcriptional Amplification system and the Firefly luciferase gene (fl) to generate a new system that we named the Multigenic Integrative Transcriptional Amplification System (MP-ITSTA). PCa cells specificity and ADT response was tested by transient infection. To detect cells in body fluid, 22Rv1-GFP cells were spiked in urine or blood of healthy control, infected with MP-ITSTA after purification and single cell imaging was done using the LV200 bioluminescence microscope. RESULTS We show that the PCA3-TSTA driven fl expression is specific to PCa cells (22Rv1, LAPC4, PC3, DU145) giving 8.5-108.4 fold higher expression when compared to SW780 bladder cancer cells. Contrary to PCA3-TSTA, the PSA-TSTA activity is regulated by androgen treatment but is not prostate cancer-specific as it is active in AR responsive breast cancer cells (CAMA-1 and ZR-75). We show that MP-ITSTA reporter expression is dependent on the combined activation of two promoters (PCA3 and PSA promoter) in a DHT dependent manner. MP-ITSTA could therefore also give an account of responsiveness to bicalutamide or enzalutamide treatments PCa cells. The signal obtained by MP-ITSTA system is 2.3 and 1.6 times higher than PCA3-TSTA in 22Rv1 and LAPC4, respectively proving that MP-ITSTA has the ability to enhance the reporter gene expression from a weak but PCa specific PCA3 promoter. Finally, MP-ITSTA could specifically target spiked 22Rv1-mcherry cells isolated from urine while no signal was found in non-spiked samples. CONCLUSIONS MP-ITSTA therefore represents a prostate cancer specific and non-invasive tool to target with high accuracy PCa cells and to detect their response to ADT cell per cell from body fluids. Citation Format: Pallavi Jain, Bertrand Neveu, Yves Fradet, Frederic Pouliot. Development of a multigenic bioluminescence imaging system to detect prostate cancer cells and assess their response to therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4764.

Abstract 206: Development of a molecular imaging system based on the transcriptional activity of the DD3/PCA3 non-coding RNA for imaging specifically the prostate cancer cells

Development of a molecular imaging system based on the transcriptional activity of the DD3/PCA3 non-coding RNA for imaging specifically the prostate cancer cells OBJECTIVE: Molecular imaging plays an important role in oncology for staging of tumors. Unfortunately, the specificity and sensitivity of current techniques remains low. This study aims to improve the existing imaging system based on transcriptional activation, named as “Two -Step- Transcriptional Amplification (TSTA) system”, with the goal to precisely image in vivo prostate cancer cells (PCa) by Positron Emission Tomography (PET). We have studied the potential of DD3/PCA3 promoter, a gene specifically expressed in PCa, to achieve this goal. METHODS: Various adenovirus constructs incorporating the DD3 promoter, the TSTA system and the Firefly luciferase reporter gene were generated and their specificity for PCa cells was tested in transient infection. By molecular engineering, we have improved the TSTA system and generated the 3STA. The luciferase activities of DD3-TSTA, DD3-3STA and PSA-TSTA (Prostate Specific Antigen) were compared in vivo by bioluminescence in xenograft mouse models. Ex vivo prostate biopsy from RP (radical prostatectomy) specimen were exposed to DD3-3STA and luciferase expression was evaluated using bioluminescence and Immunohistochemistry. RESULTS: The DD3 promoter activity is both specific to prostate and cancer cells. When DD3 promoter is incorporated in the amplification systems TSTA and 3STA, it is specific to PCa cells and its activity is amplified 32 and 95 times, respectively, when compared to the activity of the DD3 promoter alone. Moreover, activity of DD3-TSTA and DD3-3STA is androgen-independent. In vivo, DD3-3STA activity is comparable to that obtained with the PSA-TSTA whose activity can be imaged by PET. DD3-3STA could detect primary prostate cancer cells ex vivo in prostate biopsy obtained from RP specimen (Figure 1). CONCLUSIONS: The new system DD3-3STA allows specific and sensitive imaging of PCa cells. The new amplification system 3STA allows the DD3 promoter to produce reporter signal high enough to be detected by PET, a technology available in the clinic. Citation Format: Pallavi Jain, Bertrand Neveu, Yves Fradet, Frederic Pouliot. Development of a molecular imaging system based on the transcriptional activity of the DD3/PCA3 non-coding RNA for imaging specifically the prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 206. doi:10.1158/1538-7445.AM2015-206