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Human Cancer Biology

RRM2 Regulates Bcl-2 in Head and Neck and Lung Cancers: A Potential Target for Cancer Therapy

Mohammad Aminur Rahman, A.R.M. Ruhul Amin, Dongsheng Wang, Lydia Koenig, Sreenivas Nannapaneni, Zhengjia Chen, Zhibo Wang, Gabriel Sica, Xingming Deng, Zhuo (Georgia) Chen and Dong M. Shin
Mohammad Aminur Rahman
Authors' Affiliations: Department of Hematology and Medical Oncology, Winship Cancer Institute; Departments of Biostatistics and Bioinformatics, Pathology, and Radiation Oncology, Emory University, Atlanta, Georgia
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A.R.M. Ruhul Amin
Authors' Affiliations: Department of Hematology and Medical Oncology, Winship Cancer Institute; Departments of Biostatistics and Bioinformatics, Pathology, and Radiation Oncology, Emory University, Atlanta, Georgia
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Dongsheng Wang
Authors' Affiliations: Department of Hematology and Medical Oncology, Winship Cancer Institute; Departments of Biostatistics and Bioinformatics, Pathology, and Radiation Oncology, Emory University, Atlanta, Georgia
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Lydia Koenig
Authors' Affiliations: Department of Hematology and Medical Oncology, Winship Cancer Institute; Departments of Biostatistics and Bioinformatics, Pathology, and Radiation Oncology, Emory University, Atlanta, Georgia
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Sreenivas Nannapaneni
Authors' Affiliations: Department of Hematology and Medical Oncology, Winship Cancer Institute; Departments of Biostatistics and Bioinformatics, Pathology, and Radiation Oncology, Emory University, Atlanta, Georgia
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Zhengjia Chen
Authors' Affiliations: Department of Hematology and Medical Oncology, Winship Cancer Institute; Departments of Biostatistics and Bioinformatics, Pathology, and Radiation Oncology, Emory University, Atlanta, Georgia
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Zhibo Wang
Authors' Affiliations: Department of Hematology and Medical Oncology, Winship Cancer Institute; Departments of Biostatistics and Bioinformatics, Pathology, and Radiation Oncology, Emory University, Atlanta, Georgia
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Gabriel Sica
Authors' Affiliations: Department of Hematology and Medical Oncology, Winship Cancer Institute; Departments of Biostatistics and Bioinformatics, Pathology, and Radiation Oncology, Emory University, Atlanta, Georgia
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Xingming Deng
Authors' Affiliations: Department of Hematology and Medical Oncology, Winship Cancer Institute; Departments of Biostatistics and Bioinformatics, Pathology, and Radiation Oncology, Emory University, Atlanta, Georgia
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Zhuo (Georgia) Chen
Authors' Affiliations: Department of Hematology and Medical Oncology, Winship Cancer Institute; Departments of Biostatistics and Bioinformatics, Pathology, and Radiation Oncology, Emory University, Atlanta, Georgia
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Dong M. Shin
Authors' Affiliations: Department of Hematology and Medical Oncology, Winship Cancer Institute; Departments of Biostatistics and Bioinformatics, Pathology, and Radiation Oncology, Emory University, Atlanta, Georgia
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DOI: 10.1158/1078-0432.CCR-13-0073 Published July 2013
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    Figure 1.

    Knockdown of RRM2 induced apoptosis. Apoptosis was measured by Annexin V-PE and 7-AAD staining in (A) Tu212 and (B) A459 cell lines 72 hours after siRNA transfection (error bars, mean ± SD from three independent experiments). Cell lysates were analyzed by Western blotting with the indicated antibodies after transfection (inset). Tu212 (C) and A549 (D) cells were transfected with siC or different siRNAs against RRM2, siR2, siR2-1, and siR2-2. After 72 hours, apoptosis was measured by Annexin V-PE and 7-AAD staining. Cell lysates were analyzed by Western blotting with the indicated antibodies after transfection (inset).

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    Figure 2.

    Knockdown of RRM2 induces apoptosis via the mitochondria-mediated intrinsic pathway. A, Tu212 (left) and A549 (right) cells were transfected with siC or siR2. After 72 hours, cell lysates were analyzed by Western blotting with the indicated antibodies. A single set of blots is pictured from three independent experiments. B, Tu212 (top) and A549 (bottom) cells were transfected with siC or different siRNAs against RRM2, siR2, siR2-1, and siR2-2. After 72 hours, cell lysates were analyzed by Western blotting with the indicated antibodies. A single set of blots is pictured from three independent experiments. C, mitochondrial fraction (MF) and cytosolic fraction (CF) of Tu212 and A549 cells were isolated 72 hours after transfection with 5 nmol/L siR2, 5 nmol/L siC, or no treatment (NT). Western blotting was conducted to detect Cytochrome C and Cox-4.

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    Figure 3.

    Apoptosis induction by knockdown of RRM2 is Bcl-2 dependent. A, cell lysates of Tu212 and A549 were collected 72 hours after transfection with 5 nmol/L siC or siR2. Western blotting was conducted to detect antiapoptotic Bcl-2 family proteins. B, cell lysates of Tu212 and A549 were collected 72 hours after transfection with 5 nmol/L siC or different siRNAs against RRM2, siR2, siR2-1, and siR2-2. Western blotting was conducted to detect Bcl-2 protein. C, apoptosis analysis (error bars, mean ± SD from three independent experiments). D, Western blotting was conducted with specific antibodies 72 hours after transfection with 5 nmol/L siC or siR2 in Tu212 and Bcl-2 overexpressing Tu212 (Tu212/Bcl-2) cell lines.

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    Figure 4.

    Apoptosis induction by knockdown of RRM2 is p53, p73, and Akt independent. A, Western blotting for p53 and p73 expression in Tu212 and A549 cells 72 hours after transfection with siC or siR2. B, Western blotting for the indicated proteins in A549 and p53-knockdown A549 (A549/sh p53) cell lines after transfection with 5 nmol/L siC or siR2. A representative blot of three independent experiments is presented. C, apoptosis analysis (error bars, mean ± SD from three independent experiments). D, Western blot analysis 72 hours after transfection with 5 nmol/L of siC or siR2 in H1299 and two clones of dominant-negative p73-expressing H1299 (H1299/dN p73 Cl-7, and Cl-10) cell lines. E, Western blotting for p-Akt and Akt in Tu212 and A549 cells 72 hours after transfection with siC or different concentrations of siR2. F, apoptosis analysis. G, Western blot of whole-cell lysates from A549 and Akt-overexpressing A549 (A549/Akt) cell lines 72 hours after siRNA transfection.

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    Figure 5.

    RRM2 regulates Bcl-2 through direct protein–protein interaction and partially influences Bcl-2 mRNA. A, RT-PCR for Bcl-2 and GAPDH mRNA levels 72 hours after transfection of Tu212 and A549 cells with 5 nmol/L siC or siR2. RRM2 was normalized to GAPDH levels within the same sample. B, Tu212 cells were stained with anti-RRM2 (green) and anti-Bcl-2 (red), and nuclei were counterstained with DAPI (blue). A z-stack of optical sections was created at 0.59-μm intervals using a confocal microscope (LSM 510; Carl Zeiss MicroImaging, Inc.). Inset, top shows magnification of white box in merged figure. Inset, bottom shows z-axis reconstructions along the bars indicated in inset top. Bar, 50 μm. (C) Tu212 and (D) A549 cells were stained with anti-RRM2 (green), anti-Bcl-2 (red), and nuclei (blue) 48 hours after transfection with 5 nmol/L siC or siR2. Bar, 50 μm. E, Co-immunoprecipitation (IP) with anti-RRM2 (left) or anti-Bcl-2 (right) and immunoblotting with anti-Bcl-2 or anti-RRM2 in Tu212 and A549 cells. IM, immunoblotting.

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    Figure 6.

    Depletion of RRM2 reduces Bcl-2 protein expression and regulates protein stability. A and B, Tu212 cells were transfected with 5 nmol/L siC or siR2, reseeded after 24 hours into 60-mm dishes, and 24 hours later treated with cyclohexamide (100 μg/mL) for 0, 1, 3, 6, 9, and 12 hours. Cell lysates were collected at indicated time points, and Western blotting was conducted with specific antibodies. C, Tu212 and A549 cells were transfected with 5 nmol/L siC or siR2 and 24 hours later treated with 10 μmol/L MG132 for 2 hours before analyzing cell lysates by Western blotting. D, Bcl-2 expression was detected in xenograft tumor tissue by IHC analysis. The animal study was conducted previously by treating with 4 doses of siCON1 (control nanoparticle) or CALAA-01 (RRM2 siRNA-nanoparticle). Representative images shown from siCON1 and CALAA-01 10 mg/kg groups (brown stain for Bcl-2 and nuclei were counterstained by hematoxylin, blue; magnification ×200). IB, immunoblot.

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    Figure 7.

    RRM2 and Bcl-2 proteins colocalize, and their expression is positively correlated in tumor tissues from patients with HNSCC and NSCLC. A and D, staining of RRM2 and Bcl-2 in paraffin-embedded formalin-fixed HNSCC (A) and NSCLC (D) tissue sections using primary antibodies with QD-secondary antibody conjugates. A representative QD-image is shown (×400 magnification). B and E, quantification of QD signals. Average signals of RRM2 and Bcl-2 expression in each HNSCC (B) and NSCLC (E) patient sample were plotted. C and F, Spearman correlation coefficient was estimated between Bcl-2 and RRM2 levels in HNSCC (C) and NSCLC (F) tumors. A linear regression was used to plot their relationship with 95% confidence interval bound.

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    • Supplementary Figures 1 - 6 - PDF file - 427K, Figure S1. (A) Western blotting was performed for the indicated proteins in Tu212 and Bcl-2 overexpressing Tu212 (Tu212/Bcl-2) cell lines (upper panel) and in Tu686 and Bcl-2 overexpressing Tu686 (Tu686/Bcl-2) cell lines (lower panel) (B) Apoptosis analysis (error bars are mean � SD from 3 independent experiments) of indicated cell lines upon treatment with siC or siR2. Figure S2. (A) Western blotting was performed for the indicated proteins in H1299 and two clones of dominant negative p73 overexpressing H1299 (H1299/dN p73 cl-7 and cl-10) cell lines (upper panel) and in H358 dominant negative p73 overexpressing H358 (H358/dN) cell lines (lower panel). (B) Apoptosis analysis (error bars are mean � SD from 3 independent experiments) of indicated cell lines upon treatment with siC or siR2. Figure S3. (A) Western blotting was performed for the indicated proteins in A549 and Akt overexpressing A549 (A549/Akt) cell lines (upper panel) and in Tu686 and Akt overexpressing Tu686 (Tu686/Akt) cell lines (lower panel) (B) Apoptosis analysis (error bars are mean � SD from 3 independent experiments) of indicated cell lines upon treatment with siC or siR2. Figure S4. Gallery images of a z-stack. Tu212 cells were stained with anti-RRM2 (green), anti-Bcl-2 (red) and DAPI (blue). A z-stack of optical sections was created at 0.59 μm intervals using a confocal microscope (LSM 510; Carl Zeiss MicroImaging, Inc ) collected Inc.). A total of 32 images were collected, shown in a gallery of merged images collected in the z-stack in a single display. Figure S5. The relationship between RRM2 (IHC) and RRM2 (QD-IHF) in HNSCC. Spearman's correlation coefficient was estimated between RRM2 (IHC) and RRM2 (QD-IHF) expression level in tumor tissues from HNSCC patients. Figure S6. The relationship between RRM2 (IHC) and RRM2 (QD-IHF) in NSCLC. Spearman's correlation coefficient was estimated between RRM2 (IHC) and RRM2 (QD-IHF) expression levels in tumor tissues from NSCLC patients.
    • Supplementary Tables 1 - 3 - PDF file - 94K, Supplementary Table 1: Clinico-pathological features of the HNSCC and NSCLC patient groups. Supplementary Table 2: Average signal of RRM2 and Bcl-2 in tumor tissues from HNSCC patients. Supplementary Table 3: Average signal of RRM2 and Bcl-2 in tumor tissues from NSCLC patients.
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Clinical Cancer Research: 19 (13)
July 2013
Volume 19, Issue 13
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RRM2 Regulates Bcl-2 in Head and Neck and Lung Cancers: A Potential Target for Cancer Therapy
Mohammad Aminur Rahman, A.R.M. Ruhul Amin, Dongsheng Wang, Lydia Koenig, Sreenivas Nannapaneni, Zhengjia Chen, Zhibo Wang, Gabriel Sica, Xingming Deng, Zhuo (Georgia) Chen and Dong M. Shin
Clin Cancer Res July 1 2013 (19) (13) 3416-3428; DOI: 10.1158/1078-0432.CCR-13-0073

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RRM2 Regulates Bcl-2 in Head and Neck and Lung Cancers: A Potential Target for Cancer Therapy
Mohammad Aminur Rahman, A.R.M. Ruhul Amin, Dongsheng Wang, Lydia Koenig, Sreenivas Nannapaneni, Zhengjia Chen, Zhibo Wang, Gabriel Sica, Xingming Deng, Zhuo (Georgia) Chen and Dong M. Shin
Clin Cancer Res July 1 2013 (19) (13) 3416-3428; DOI: 10.1158/1078-0432.CCR-13-0073
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