Hiroe Shiratsuchi1, Zhuwen Wang2, Guoan Chen2, Paramita Ray3, Jules Lin2, Zhuo Zhang4, Lili Zhao5, David Beer2, Dipankar Ray3, Nithya Ramnath6. 1. Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan. 2. Section of Thoracic Surgery, Department of Surgery, University of Michigan, Ann Arbor, Michigan. 3. Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan. 4. Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan. 5. Department of Biostatistics, University of Michigan, Ann Arbor, Michigan. 6. Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan; Ann Arbor Veterans Administration Medical Center, Ann Arbor, Michigan. Electronic address: nithyar@umich.edu.
Abstract
INTRODUCTION: We have previously demonstrated that a subset of lung cancer cells express higher CYP24A1 mRNA, a metabolizing enzyme for 1,25-D3, compared to benign tumors or surrounding normal lung and that high CYP24A1 mRNA expression is associated with poor prognosis in resected lung adenocarcinoma (AC). We hypothesized that CYP24A1 has oncogenic potential and increased CYP24A1 expression may contribute to tumor growth, whereas, CYP24A1 targeting may reduce tumor burden. METHODS: Two low CYP24A1 expressing human lung cancer cell lines (SK-LU-1 and Calu-6) were stably transfected either with an empty lentiviral vector or with the CYP24A1 expressing vector. Over-expression of mRNA and protein levels of CYP24A1 in SK-LU-1 and Calu-6 were confirmed using qRT-PCR and immunoblotting respectively. Next, effects of targeting CYP24A1 were examined in lung cancer cells (A549 and H441), which express higher basal levels of CYP24A1. Finally, we studied the effects of stable knockdown of CYP24A1 in xenograft models. RESULTS: Over-expression of CYP24A1 correlated with accelerated cell growth and invasion compared to control vector-transfected cells. CYP24A1 over-expression also increased RAS protein expression. Knockdown of CYP24A1 using either si- or shRNA reduced CYP24A1 mRNA and protein expression and significantly decreased cell proliferation (30-60%) and reduced mitochondrial DNA content compared to non-targeting (NT) si-/shRNA transfected/transduced cells. Transfection with CYP24A1 siRNA also decreased total RAS protein, thus reducing phosphorylated AKT. Importantly, stable knockdown of CYP24A1 in A549 and H441 lung tumor xenograft models resulted in tumor growth delay and smaller tumor size as evident from tumor bioluminescence and tumor volume measurement studies. Such observations were correlated with decreased tumor cell proliferation as evidenced by reduced Ki67 and Cyclin D staining. CONCLUSIONS: Our data suggest that CYP24A1 has oncogenic properties mediated by increasing RAS signaling, targeting of which may provide an alternate strategy to treat a subset of lung AC.
INTRODUCTION: We have previously demonstrated that a subset of lung cancer cells express higher CYP24A1 mRNA, a metabolizing enzyme for 1,25-D3, compared to benign tumors or surrounding normal lung and that high CYP24A1 mRNA expression is associated with poor prognosis in resected lung adenocarcinoma (AC). We hypothesized that CYP24A1 has oncogenic potential and increased CYP24A1 expression may contribute to tumor growth, whereas, CYP24A1 targeting may reduce tumor burden. METHODS: Two low CYP24A1 expressing humanlung cancer cell lines (SK-LU-1 and Calu-6) were stably transfected either with an empty lentiviral vector or with the CYP24A1 expressing vector. Over-expression of mRNA and protein levels of CYP24A1 in SK-LU-1 and Calu-6 were confirmed using qRT-PCR and immunoblotting respectively. Next, effects of targeting CYP24A1 were examined in lung cancer cells (A549 and H441), which express higher basal levels of CYP24A1. Finally, we studied the effects of stable knockdown of CYP24A1 in xenograft models. RESULTS: Over-expression of CYP24A1 correlated with accelerated cell growth and invasion compared to control vector-transfected cells. CYP24A1 over-expression also increased RAS protein expression. Knockdown of CYP24A1 using either si- or shRNA reduced CYP24A1 mRNA and protein expression and significantly decreased cell proliferation (30-60%) and reduced mitochondrial DNA content compared to non-targeting (NT) si-/shRNA transfected/transduced cells. Transfection with CYP24A1 siRNA also decreased total RAS protein, thus reducing phosphorylated AKT. Importantly, stable knockdown of CYP24A1 in A549 and H441 lung tumor xenograft models resulted in tumor growth delay and smaller tumor size as evident from tumor bioluminescence and tumor volume measurement studies. Such observations were correlated with decreased tumor cell proliferation as evidenced by reduced Ki67 and Cyclin D staining. CONCLUSIONS: Our data suggest that CYP24A1 has oncogenic properties mediated by increasing RAS signaling, targeting of which may provide an alternate strategy to treat a subset of lung AC.
Authors: Miller Huang; Jignesh Tailor; Qiqi Zhen; Aaron H Gillmor; Matthew L Miller; Holger Weishaupt; Justin Chen; Tina Zheng; Emily K Nash; Lauren K McHenry; Zhenyi An; Fubaiyang Ye; Yasuhiro Takashima; James Clarke; Harold Ayetey; Florence M G Cavalli; Betty Luu; Branden S Moriarity; Shirin Ilkhanizadeh; Lukas Chavez; Chunying Yu; Kathreena M Kurian; Thierry Magnaldo; Nicolas Sevenet; Philipp Koch; Steven M Pollard; Peter Dirks; Michael P Snyder; David A Largaespada; Yoon Jae Cho; Joanna J Phillips; Fredrik J Swartling; A Sorana Morrissy; Marcel Kool; Stefan M Pfister; Michael D Taylor; Austin Smith; William A Weiss Journal: Cell Stem Cell Date: 2019-06-13 Impact factor: 24.633