Exon-skipping strategy by ratio modulation between cytoprotective versus pro-apoptotic clusterin forms increased sensitivity of LNCaP to cell death

Abstract

Background: In prostate cancer the secreted form of clusterin (sCLU) has been described as an anti-apoptotic protein whose expression is increased after therapeutic intervention, whereas, the nuclear protein form nCLU was reported to have pro-apoptotic properties.

Methodology: In order to provide new therapeutic approaches targeting CLU, we developed a strategy based on exon skipping by using a lentiviral construct to preferentially induce the nuclear spliced form of the protein. The molecular construct was transduced in LNCaP cells for testing the modulation of sensitivity of the transduced cells to pro-apoptotic stress.

Results and conclusions: We showed an increase of nCLU/sCLU expression ratio in the prostate cancer cell line "LNCaP" after lentiviral vector-U7 nCLU transduction. Moreover, we showed a significant inhibition of cell proliferation in nCLU-U7 LNCaP cells after treatment with cisplatin and after exposure to ionizing radiation compared to control cells. Finally, we showed that nCLU-U7 LNCaP cells exposure to UV-C significantly reduced an increase of cell death compared to control. Finally, we showed that modulating nCLU expression had profound impact on Ku70/Bax interaction as well as Rad17 expression which could be a key mechanism in sensitizing cells to cell death. In conclusion, this is the first report showing that increasing of nCLU/sCLU expression ratio by using an "on demand alternative splicing" strategy successfully increased sensitivity to radiotherapy and chemotherapy of prostate cancer cells.

Figure 1. U7-nCLU vector construct and CLU mRNA expression in LNCaP.

(A) (Top) Antisens sequence position to induce exon 2 skipping. (Bottom) Structure of the pHIV-1-U7-nCLU. U7snRNA including antisens sequence is placed under the control of its own promoter and its regulating domains 3′. SmOPT: sequence indicates the Sm protein binding site. LTR : long terminal repeats. (B) U7 vector integration in transduced LNCaP cell genome. Curves represent qPCR amplification of U7-nCLU genomic DNA in different transduction conditions (middle curves). Human β-actin amplification corresponded to positive control (Top curves) and genomic DNA of non transduced cells was used as negative control. (C) (Top) mRNA from transduced LNCaP cells with U7-nCLU vector was analyzed by RT-PCR at 3 weeks posttransduction. The 344 bp and 218 bp bands correspond to sCLU and skipped form nCLU mRNA respectively without alteration of the sequence. Controls were from RNA of LNCaP alone or LNCaP transduced with U7-control. (Bottom) Relative mRNA expression levels determined by densitometry. βactin used to normalize changes in specific gene expressions. (D) DNA sequence of the 218 bp band showed skipping of exon 2.

Figure 2. Effect of cisplatin drug on nCLU-U7 LNCaP cells.

(A) LNCaP cells were treated at the indicated doses for 24 hours and proliferation was assessed. (*): P value <0.05 (Student’s t-test) vs control cells. (B) Effect of cisplatin treatment on DNA fragmentation of LNCaP cells. LNCaP cells were treated with 50 µM cisplatin for 0, 4 and 24 hours. Cellular DNA was isolated and subjected to agarose gel electrophoresis.

Figure 3. Increase sensitivity of LNCaP cells to irradiation following U7-nCLU LNCaP transduction.

IR dose exposure effect at 48 hours on LNCaP cells proliferation. Data are expressed as % inhibition of the mean counts per minute (CPM) from 3 individual experiments. (*): P value <0.05 (Student’s t-test) vs control cells.

Figure 4. Increased UV-C induced death following U7-nCLU vector expression in LNCaP cells.

FACS analysis was performed at 0, 2 and 4 hours after 10 min UV-C cell exposure. Cell death was assessed using propidium iodide (PI)/annexin V staining. The red rectangle surrounds PI−/Annexin V+cells. Histogram represent % of dead cells in each condition. Results are means +/− s.e.m. from 3 individual experiments. (*): P value <0.05 (Student’s t-test).

Figure 5. nClu expression led to defective interaction between Bax and Ku70.

(A) Coimmunoprecipitation of Bax and Ku70 following or not irradiation. No Bax- Ku70 coimmunoprecipitation was found at time 0 in not irradiated U7-nClu cells compare to U7 control cells. No coimmunoprecipitation of Bax and Ku70 was observed in all irradiated cells both at 4 hrs (not shown) and 24 hrs. (B) Defective expression of KU70 in nCLU-transduced cells. Note the upregulation of Rad17 protein following irradiation of nCLU-LNCap.