Oncogenic Potential of CYP24A1 in Lung Adenocarcinoma Hiroe Shiratsuchi, PhD, Zhuwen Wang, MS, Guoan Chen, MD, PhD, Paramita Ray, PhD, Jules Lin, MD, Zhuo Zhang, PhD, Lili Zhao, PhD, David Beer, PhD, Dipankar Ray, PhD, Nithya Ramnath, M.B.B.S.  Journal of Thoracic Oncology  Volume 12, Issue 2, Pages 269-280 (February 2017) DOI: 10.1016/j.jtho.2016.10.010 Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Figure 1 Cytochrome P450 family 24 subfamily A member 1 (CYP24A1) overexpression promotes tumor cell proliferation and invasion in human lung cancer cell lines. (A) Steady-state levels of CYP24A1 mRNA in different human lung adenocarcinoma cell lines as quantified by using RNA sequencing. (B) Human lung cancer cell lines SK-LU-1 and Calu-6 were stably transfected with an empty vector pLX304 or one encoding cytochrome P450 family 24 subfamily A member 1 gene (CYP24A1) and selected in the presence of blasticidin. (B and D) CYP24A1 protein expression in SK-LU-1 (B) and Calu-6 (D) cells transfected with the control vector or CYP24A1-coding vector. CYP24A1-transfected cells expressed a higher amount of CYP24A1 protein (fivefold to 15-fold more) than cells transfected with the control vector. (C and E) Cell growth analyses of SK-LU-1 (C) and Calu-6 (E) cells were assessed by crystal violet staining.16 Data shown in the graph are the mean ± SD of triplicate experiments. CYP24A1-overexpressing SK-LU-1 and Calu-6 cells grew significantly faster than cells transfected with the control vector pLX304 (p < 0.05). As SK-LU-1 failed to form colonies, crystal violet assay was preferred. (F–I) SK-LU-1 and Calu-6 cell invasion measured by using a Boyden chamber. Picture shown is the representative field for each of the cell lines (F and H). Data shown in the graph are the mean ± SD of invaded cell counts in five fields in each experiment (G and I). Invaded cell number was significantly higher in CYP24A1-overexpressing cells than in control vector–transfected cells (p < 0.05). RPKM, reads per kilobase per million; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Cyp, CYP24A1. Journal of Thoracic Oncology 2017 12, 269-280DOI: (10.1016/j.jtho.2016.10.010) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Figure 2 Small interfering RNA (siRNA)-mediated knockdown of cytochrome P450 family 24 subfamily A member 1 gene (CP24A1) reduces survival and mitochondrial DNA content. Cytochrome P450 family 24 subfamily A member 1 (CYP241) protein expression (A) in A549 (high–CYP24A1-expressing human lung cancer cell line) transfected with NT or CYP24A1 siRNA. Transfection with CYP24A1 siRNA decreased CYP24A1 protein expression. A549 cell survival after transfection with NT or CYP24A1 siRNA (B). Transfection with CYP24A1 siRNA significantly reduced A549 cell survival measured by WST-1 assay (p < 0.05) associated with reduced CYP24A1 mRNA expression. (C) Transfection of CYP24A1 siRNA reduced mitochondrial DNA content in A549 cells. Total genomic DNA (mitochondrial and nuclear) was isolated 48 hours after siRNA transfection and subjected to quantitative reverse-transcriptase polymerase chain reaction using mitochondria (D-loop and cytochrome b) and nuclear DNA (ribosomal DNA [rDNA])-specific primers. (D) H441 and H1437 with moderate expression of CYP24A1 mRNA were transfected with siRNA targeting CYP24A1 or NT siRNA. CYP24A1 protein expression after siRNA transfection. CYP24A1 siRNA reduced CYP24A1 protein expression more than 60%. (E) Clonogenic assay (cell survival) of lung cancer cells after transfection with siRNA targeting CYP24A1 or NT. The CYP241 siRNA–transfected cell survival fraction was significantly lower than that in the case of cells transfected with NT siRNA (p < 0.05) in H441 and p < 0.01 in H1437) (F). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Cyp, CYP24A1. Journal of Thoracic Oncology 2017 12, 269-280DOI: (10.1016/j.jtho.2016.10.010) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Figure 3 Short hairpin RNA (shRNA)-mediated stable knockdown of cytochrome P450 family 24 subfamily A member 1 gene (CYP24A1) in A549 cells reduces tumor growth in a mouse xenograft model. Cytochrome P450 family 24 subfamily A member 1 (CYP24A1) protein expression (A) in A549 cells stably transduced with nontargeting (NT) shRNA or CYP24A1 (CYP) shRNA. (B) Cell survival was measured by clonogenic assay. Infection with shCYP24A1 significantly inhibited cell survival (p < 0.001). (C) Bioluminescence intensity (photons/s) was plotted for the corresponding groups showing markedly lower counts in mice injected with shCYP24A1-transduced A549 cells compared with in mice injected with NT shRNA–transduced cells. Bioluminescence intensity (photons/s) was calculated and plotted for the two groups showing significant differences (p < 0.05) between the groups. (D) Tumor size was measured twice a week for up to 4 weeks. Tumor volume was calculated on the basis of tumor width (W) and length (L) (W2L/2, where W < L). Data shown in the graph is mean ± standard effort of the mean of 10 tumors (two injection sites per mouse) in each group. Tumor volume in mice injected with shCYP24A1-transduced A549 was significantly lower than in mice injected with NT shRNA–transduced A549 (**p < 0.0001) (E) Representative micrographs showing immunohistochemistry of Ki67 (upper panel) and cyclin D (lower panel) staining in NT and CYP24A1 shRNA–transduced A549 xenografts (F). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Journal of Thoracic Oncology 2017 12, 269-280DOI: (10.1016/j.jtho.2016.10.010) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Figure 4 Short hairpin RNA (shRNA)-mediated stable knockdown of cytochrome P450 family 24 subfamily A member 1 gene (CYP24A1) in H441 cells reduces tumor growth in a mouse xenograft model. Cytochrome P450 family 24 subfamily A member 1 (CYP24A1) protein (A) expression in H441 stably transfected with nontargeting (NT) or CYP24A1 shRNA. (B) Cell survival measured by clonogenic assay in H441 stably transfected cells. Transfection with CYP24A1 shRNA significantly inhibited cell survival (p < 0.05). (C) 1 × 106 H441 cells were injected into both the flanks of NSG mice. Tumor size was measured once a week and tumor volume was calculated as described in Figure 3. Data shown in the graph is mean ± standard error of the mean. Tumor volume in mice injected with CYP24A1 shRNA–transduced H441 cells was significantly less than that in mice injected with NT shRNA cells (*p < 0.05). (D) Average photonic flux (photons/s) from eight tumors were plotted for the two groups showing significant (*p < 0.05) differences. (E) Representative micrographs showing immunohistochemistry of Ki67 and cyclin D expression in NT and CYP24A1 shRNA–transduced H441 xenografts. Cyp, CYP24A1GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Journal of Thoracic Oncology 2017 12, 269-280DOI: (10.1016/j.jtho.2016.10.010) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

Figure 5 Complex relationship between cytochrome P450 family 24 subfamily A member 1 (CYP24A1) and KRAS. (A) SK-LU-1 and Calu-6 cells were transfected with either empty vector or with CYP24A1 vector DNA. Protein lysates were harvested 48 hours after transfection and subjected to immunoblotting using the indicated antibodies. (B) H441 and H1437 cells were transfected with either scrambled or CYP24A1 small interfering RNA (siRNA), and cell lysates were prepared 48 hours after transfection and subjected to immunoblotting using the indicated antibodies. Band intensity was measured using Image J software and considering control sample as 1 (arbitrary units). (C) Tumor samples with sequencing data isolated from 230 patients with NSCLC were analyzed for KRAS mutation and cytochrome P450 family 24 subfamily A member 1 gene (CYP24A1) amplification (database available from http://www.cbioportal.org). A mutual exclusivity was noted between the two (log OR -1.647, p < 0.01). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Journal of Thoracic Oncology 2017 12, 269-280DOI: (10.1016/j.jtho.2016.10.010) Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions