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. 2020 Jul 10;12(7):1861.
doi: 10.3390/cancers12071861.

NCL Inhibition Exerts Antineoplastic Effects against Prostate Cancer Cells by Modulating Oncogenic MicroRNAs

Tyler Sheetz  1   2   3 , Joseph Mills  1   2 , Anna Tessari  1   2 , Megan Pawlikowski  1   2 , Ashley E Braddom  1   2 , Tasha Posid  3 , Debra L Zynger  4 , Cindy James  5 , Valerio Embrione  1   2 , Kareesma Parbhoo  1   2 , Claudia Foray  1 , Vincenzo Coppola  1   2 , Carlo M Croce  1   2 , Dario Palmieri  1   2
Affiliations

NCL Inhibition Exerts Antineoplastic Effects against Prostate Cancer Cells by Modulating Oncogenic MicroRNAs

Tyler Sheetz et al. Cancers (Basel). .

Abstract

Prostate cancer (PCa) is the most frequently diagnosed cancer in men and second most common cause of cancer-related deaths in the United States. Androgen deprivation therapy (ADT) is only temporarily effective for advanced-stage PCa, as the disease inevitably progresses to castration-resistant prostate cancer (CRPC). The protein nucleolin (NCL) is overexpressed in several types of human tumors where it is also mislocalized to the cell surface. We previously reported the identification of a single-chain fragment variable (scFv) immuno-agent that is able to bind NCL on the surface of breast cancer cells and inhibit proliferation both in vitro and in vivo. In the present study, we evaluated whether NCL could be a valid therapeutic target for PCa, utilizing DU145, PC3 (CRPC), and LNCaP (androgen-sensitive) cell lines. First, we interrogated the publicly available databases and noted that higher NCL mRNA levels are associated with higher Gleason Scores as well as with recurrent and metastatic tumors. Then, using our anti-NCL scFv, we demonstrated that NCL is expressed on the surface of all three tested cell lines and that NCL inhibition results in reduced proliferation and migration. We also measured the inhibitory effect of NCL targeting on the biogenesis of oncogenic microRNAs such as miR-21, -221 and -222, which was cell context dependent. Taken together, our data provide evidence that NCL targeting inhibits the key hallmarks of malignancy in PCa cells and may provide a novel therapeutic option for patients with advanced-stage PCa.

Keywords: castration-resistant prostate cancer; microRNAs; nucleolin; prostate cancer.

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Conflict of interest statement

D.P. and C.M.C. are inventors of the patent application WO2017011411A1 (methods and compositions relating to anti-nucleolin recombinant immunoagents). At the time of submission, Koru Biopharma had an option to license this patent. Other authors declare no conflict of interests.

Figures

Figure 1
Figure 1
NCL is Upregulated in Aggressive Forms of PCa Violin plots displaying nucleolin (NCL) mRNA expression levels in patient tumor samples stratified by clinical characteristics from (A) TCGA provisional database, queried through cBioPortal (Gleason 6–7 n = 291; Gleason 8–10 n = 206; Pearson: r = 0.134; Spearman: rs = 0.127), and (B) Sun et al. (non-recurrent n = 39; biochemical recurrence n = 38), (C) Yu et al., and Chandran et al. queried through NCBI GEO (normal tissue n = 81; localized prostate cancer (PCa) n = 65; metastatic PCa n = 25 sample locations from four patients). A Student’s t-test was used for group analyses. ** p < 0.01, **** p < 0.0001.
Figure 2
Figure 2
4LB5 Binds to PCa Cells and Inhibits Cell Proliferation (A) Basal expression levels of whole-cell NCL protein was measured in LNCaP, PC3, and DU145 PCa cell lines and compared to MDA-MB-231 expression levels via Western blot. Uncropped blots of Figure 2A are shown in Figure S4. (B) 4LB5 binding cell surfaces was assessed by an ELISA performed after incubating cells with serial dilutions of 4LB5. ELISA data shown are representative of two independent experiments performed in quadruplicate. (C) Relative cell counts of DU145, PC3, and LNCaP cells treated with 50 nM 4LB5 or control solution for 48 h. Cell survival data are the average of five biological replicates. (D) MTS assay performed after 72 h-treatment with increasing concentrations of 4LB5. MTS data are average of two assays performed in biological triplicate. (E) Light microscopy images of PCa cells taken at 48 h post-treatment with 50 nM 4LB5 or control solution. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 3
Figure 3
Treatment with 4LB5 Reduces Mature Forms of Oncogenic MicroRNAs and alters cell survival (A) microRNA expression levels were assessed via qRT-PCR in DU145, PC3, and LNCaP cells after 48 h treatment with 50 nM 4LB5 or control solution. qRT-PCR data are the average of three independent experiments performed in at least technical duplicate. (B) Caspase 3/7 activity was assessed after 48 and 72 h of 4LB5 treatment. Caspase assay was performed in biological triplicate for each time point and averaged. (C) PARP cleavage was assessed by Western blot after PCa cell treatment with 4LB5 or control solution for 48 and 72 h. PARP Western blots are representative of at least two independent experiments. Student’s t-test (2-tailed, homoscedastic) was utilized for statistical analysis. Uncropped blots of Figure 3C are shown in Figure S4. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 4
Figure 4
4LB5 Impairs PCa Cell Migration (A) Wound healing assays performed with DU145 and PC3 cells after 48h of pretreatment with 50 nM 4LB5 or control solution. Representative photos were taken at t = 0h and t = 24h. (B) Trans-well migration experiments performed with DU145 and PC3 cells after 24h pretreatment with 100 nM 4LB5 or control solution and 12h migration time. Migrated cells were stained with crystal violet and photographed with phase contrast microscopy. (C) Images of control- and 4LB5-treated migrated cells for experiments shown in A and B were processed and analyzed with ImageJ software in parallel. Quantification of images from five independent wound healing experiments performed in technical triplicate is reported. Trans-well migration data represents average of two independent experiments performed in biological triplicate and was generated by calculating percent area of migrated cells. ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 5
Figure 5
4LB5 Reduces AR Expression in Hormone-Sensitive LNCaP Cells (A) Basal androgen receptor (AR) expression in PCa cell lines. (B) LNCaP cells were treated with the indicated increasing concentration of 4LB5 for 48h and AR expression was assessed by Western blot. Additional loading control using Ponceau S staining is included in Figure S3A. (C) Levels of AR were normalized to GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), plotted, and subjected to linear correlation testing (Pearson (displayed): r = −0.621, p = 0.006, Spearman: rs = −0.650, p = 0.003). Plotted data are from the experiment shown in Figure 5B as well as two additional independent experimental replicates.
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References

    1. Siegel R.L., Miller K.D., Jemal A. Cancer statistics, 2019. CA Cancer J. Clin. 2019;69:7–34. doi: 10.3322/caac.21551. - DOI - PubMed
    1. James N.D., Spears M.R., Clarke N.W., Dearnaley D.P., De Bono J.S., Gale J., Hetherington J., Hoskin P.J., Jones R.J., Laing R., et al. Survival with Newly Diagnosed Metastatic Prostate Cancer in the “Docetaxel Era”: Data from 917 Patients in the Control Arm of the STAMPEDE Trial ( MRC PR08, CRUK/06/019 ) Eur. Urol. 2015;67:1028–1038. doi: 10.1016/j.eururo.2014.09.032. - DOI - PubMed
    1. Kirby M., Hirst C., Crawford E.D. Characterising the castration-resistant prostate cancer population: A systematic review. Int. J. Clin. Pract. 2011;65:1180–1192. doi: 10.1111/j.1742-1241.2011.02799.x. - DOI - PubMed
    1. Shevrin D. Genomic predictors for treatment of late stage prostate cancer. Asian J. Androl. 2016;18:586. doi: 10.4103/1008-682X.177121. - DOI - PMC - PubMed
    1. Shiota M., Yokomizo A., Naito S. Increased androgen receptor transcription: A cause of castration-resistant prostate cancer and a possible therapeutic target. J. Mol. Endocrinol. 2011;47:R25–R41. doi: 10.1530/JME-11-0018. - DOI - PubMed

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