WP 1: Assessment of the gene and protein expression of “up-regulated” metabolic gene candidates in invasive and non-invasive PCa.
Milestone: Establishment of PCa cellular models.
Results: A battery of cell models was tested to identify suitable for genetic manipulation in the project. Various prostate cancer (PCa) invasive/non-invasive cell lines (DU145, LNCaP, PC3, VCAP, 22RV1, C4-2, BPH-1, PWR1E and RWPE1) were tested. Protein and RNA expression of the metabolic genes CIT, HES6 and TCF19 was consistently identified and shown in DU145, LNCaP, PC3.
Deliverable: Three PCa cell lines were characterized and confirmed as a model system for subsequent studies.
WP 2: Inducible genetic modulation of “up-regulated” metabolic gene candidates, to investigate causal contribution to the metastatic potential of PCa.
Milestone: Modulation of metabolic gene candidates by CRISPR/Cas9/short-hairpin:
Results: Next, CIT, HES6 and TCF19 roles as potential metabolic oncogenes were explored. To this we used a gain- and loss-of-function approach. To this, we used a combination of experimental techniques. This included the use of sequence-specific control of transcription via CRISPR-CAS9-based gene editing (CRISPRi) as well as short-hairpin RNA 19–21. In the latter case we benefit from the IRB Barcelona access to the mission shorthairpin RNA library. We used both approaches in parallel, with several sequences targeting each gene in each of the approaches. Whereas the use of CRISPR/Cas9 knockout seem to cause major limitations for the cells to grow in vitro, shorthairpin and its partial downregulation seemed a better approach. The complete depletion of the various gene causes a cellular catastrophe impeding the subsequent use of the cells. Genetic depletion of gene candidates was used to investigation of targets and effectors of the perturbed genes, the metabolic reprogramming, and stromal impact during metastatic cascades in PCa. In addition, we engineered PCa cells with a pTRIPZ lentiviral vector with doxycycline-inducible shRNA targeting CIT, HES6 and TCF19 and TurboRFP fluorescent protein (tRFP) expression (10.1038/ncomms15618). CIT, HES6 and TCF19 expression was abrogated in DU145 PCa cells expressing this inducible shRNA targeting CIT, HES6 and TCF19 genes by treating the cells with doxycycline (2ug/ml for 72 hours).
Deliverable: DU145, LNCaP, PC3 genetically modified cells lines with two independent shorthairpins against CIT, HES6 and TCF19 were generated. DU145 cells with an inducible pTRIPZ_shortharipin against CIT, HES6 and TCF19 were completed.
WP 3: In vitro characterization of genetically modulated PCa cell lines.
Milestone: Characterization of PCa perturbed cells.
Results: In this aim we seek to fully characterize the loss-of-function of CIT, HES6 and TCF19 in invasive PCa. First, we validated the loss-of-expression of the candidates by quantitative real-time PCR (rtPCR) and Western-botting (WB). This will demonstrate the quality and strength of the loss of function of the genes of interest. Similarly, we also used immunofluorescence to confirme the inducibility of pTRIPZ_shorthairpin in DU145 cells. We used specific staining for HES6 and TCF19 (no antibodies were identified that were suitable for CIT IF). Upon treating the cells with doxycycline (2ug/ml for 72 hours) we confirmed the expression of RFP (readout of the pTRIPZ_shorthairpin expression) and loss of our gene of interest. Also, we confirmed that upon doxycycline removal (7 days later) the expression of the genes of interest returned and RFP expression disappeared.
Deliverable: A battery of qPCR, WB, IFs analyses were completed, and TCF9 and HES6 genetic perturbations confirmed.
WP 4: In vitro characterization of aggressiveness and metabolic function of modulated cell lines for “up-regulated” metabolic gene candidates.
Milestone: Functional assays to test aggressive PCa phenotypes after genetic modulations of metabolic gene candidates:
Results: Herein, we characterize the functional role of our genes of interest. To this we used control and target depleted cells from objective 3 (WP3) to assess invasive/metastatic PCa behaviour in experimental in vitro functional assays. Similarly, we assessed the contribution to metastatic aggressiveness of “up-regulated” metabolic gene candidates (CIT, HES6 and TCF19). We explored proliferation by BrdU incorporation, growth and metabolic dependencies. A subset of this were completed, whereas others did not. The termination of the contract earlier than expected did not allow to complete the battery of experiments.
Deliverable: Only a subset of the experiments planned were completed due to an early termination of the postdoctoral contract. Proliferation, growth and a subset of metabolic analyses were completed for DU145 cells with TCF19 perturbation).
WP 5: In vivo characterization of tumor outgrowth and metabolic capability of genetically modulated PCa cell lines.
Milestone: Analyses of tumor growth, systemic metastasis or bone colonization
Results: We tested in vivo the functional relevance of the candidates CIT, HES6 and TCF19 and their impact on metastatic PCa progression. We used the genetically modulated DU145 PCa cell lines (WP4). The cells were also transduced with a non-invasive imaging reporter (Luciferase), implanted intra tibiae or via left ventricle and generate metastasis xenografted experimental mouse models. We confirmed the role of TCF19 for metastasis whereas neither CIT nor HES6 significantly altered the capacity of the DU145 cells to colonize the bones.
Deliverable: Only a small subset of the experiments planned were completed due to an early termination of the postdoctoral contract. Analyses were completed for DU145 cells with TCF19 perturbation, but experimental replicates, the expansion to other experimental cell systems and the used of the inducible system are pending.
