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. 2014 Mar 20;33(12):1495-505.
doi: 10.1038/onc.2013.103. Epub 2013 Apr 22.

Targeting Homologous Recombination and Telomerase in Barrett's Adenocarcinoma: Impact on Telomere Maintenance, Genomic Instability and Tumor Growth

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Free PMC article

Targeting Homologous Recombination and Telomerase in Barrett's Adenocarcinoma: Impact on Telomere Maintenance, Genomic Instability and Tumor Growth

R Lu et al. Oncogene. .
Free PMC article

Abstract

Homologous recombination (HR), a mechanism to accurately repair DNA in normal cells, is deregulated in cancer. Elevated/deregulated HR is implicated in genomic instability and telomere maintenance, which are critical lifelines of cancer cells. We have previously shown that HR activity is elevated and significantly contributes to genomic instability in Barrett's esophageal adenocarcinoma (BAC). The purpose of this study was to evaluate therapeutic potential of HR inhibition, alone and in combination with telomerase inhibition, in BAC. We demonstrate that telomerase inhibition in BAC cells increases HR activity, RAD51 expression, and association of RAD51 to telomeres. Suppression of HR leads to shorter telomeres as well as markedly reduced genomic instability in BAC cells over time. Combination of HR suppression (whether transgenic or chemical) with telomerase inhibition, causes a significant increase in telomere attrition and apoptotic death in all BAC cell lines tested, relative to either treatment alone. A subset of treated cells also stain positive for β-galactosidase, indicating senescence. The combined treatment is also associated with decline in S-phase and a strong G2/M arrest, indicating massive telomere attrition. In a subcutaneous tumor model, the combined treatment resulted in the smallest tumors, which were even smaller (P=0.001) than those that resulted from either treatment alone. Even the tumors removed from these mice had significantly reduced telomeres and evidence of apoptosis. We therefore conclude that although telomeres are elongated by telomerase, elevated RAD51/HR assist in their maintenance/stabilization in BAC cells. Telomerase inhibitor prevents telomere elongation but induces RAD51/HR, which contributes to telomere maintenance/stabilization and prevention of apoptosis, reducing the efficacy of treatment. Combining HR inhibition with telomerase renders telomeres more vulnerable to degradation and significantly increases/expedites their attrition, leading to apoptosis. We therefore demonstrate that a therapy targeting HR and telomerase has the potential to prevent both tumor growth and genomic evolution in BAC.

Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Telomerase inhibition induces whereas nilotinib and RAD51-suppression reduce HR activity in BAC cells
(A) GRN163L, an oligonucleotide targeting RNA component of telomerase, induces HR. BAC cell lines (OE33, OE19, FLO-1) were treated with mismatch control oligonucleotide (C; 2 µM) or GRN163L (G; 2 µM) for 48 hrs, and evaluated for HR activity using the luminescence-based HR assay described in Methods. Error bars represent SEMs of triplicate assays. (B) Confirmation of increase in HR by alternate method. FLO-1 cells treated as above (in panel A) were evaluated for HR activity, using a fluorescence-based HR assay (Addgene) as described in Methods. (C) shRNA, targeting catalytic subunit of telomerase (hTERT), also induces HR. FLO-1 cells were transduced with lentivirus particles, producing control (CS) or telomerase-targeting (TS) shRNAs, and following selection evaluated for HR, using luminescence-based HR assay described in Methods. (D) Impact of TRF2 knockdown on HR. FLO-1 cells, transfected with control (CS) or TRF2 targeting (TR) siRNA, were cultured untreated or treated for 48 hrs with GRN163L (G; 2 µM), and evaluated for HR activity using the luminescence-based HR assay. (E) Inhibition of HR by nilotinib and RAD51-suppression. BAC cell lines were exposed to nilotinib (5 µM) or transduced with lentivirus-based shRNAs (CS, control; R, RAD51-targeting). Untreated cells (U), those treated with nilotinib for 48 hrs (N), and shRNA-transduced cells at day six after transduction, were evaluated for HR activity using a plasmid based assay as described in Methods. Relative HR activity in nilotinib- and shRNA-treated cells is shown as percent of activity in untreated and control shRNA-treated cells, respectively: error bar indicates SEMs of triplicate assays. (F) RAD51-suppression by lentivirus-based shRNAs. BAC cell lines, transduced with lentivirus-based shRNAs (CS, control; R, RAD51-targeting) as described in panel B, were evaluated for RAD51 expression by Western blotting.
Figure 2
Figure 2. Telomerase inhibition induces RAD51 expression and its binding to telomeres
(A) OE33 cells, untreated or treated for 48 hrs with mismatch control (C; 2 µM), GRN163L (G; 2 µM), HR inhibitor nilotinib (N; 5 µM), and combination of N and G (NG) were processed as described above. Top panel is the gel image showing RAD51-bound telomere (TEL) and internal control (IC) bands, whereas bottom is the bar graph showing relative amount of telomeric DNA bound to RAD51 following normalization with internal control DNA. (B) OE33 cells treated with control shRNA (CS), RAD51 shRNA (R), RAD51 shRNA mediating stronger suppression of RAD51 (R4), CS cells treated with GRN163L (CG), R cells treated drug (RG), or R4 cells treated with drug (R4G) were processed as described above. Top panel shows gel image of telomere (TEL) and internal control (IC) bands, whereas bottom is the bar graph showing relative amount of telomeric DNA bound to RAD51 following normalization with internal control DNA.
Figure 3
Figure 3. Impact of HR inhibition (by RAD51 knockdown) on efficacy of telomerase inhibitor in OE33 cells
OE33 cells, transduced with lentivirus particles producing control (CS) or RAD51-specific (R) shRNAs, were cultured in the presence or absence of telomerase inhibitor GRN163L (G; 2 µM) and evaluated for impact on growth, apoptosis, and telomere maintenance. (A) Impact on telomerase activity. Cells treated as above for 10 days were evaluated for telomerase activity using TRAPeze Telomerase Detection kit. CS, control shRNA treated cells; R, RAD51 suppressed cells; CG, CS cells treated with GRN163L; RG, R cells treated with GRN163L. (B) Impact on growth. The control (CS) and RAD51-suppressed (R) cells were cultured in the presence or absence of GRN163L and cell viability determined at different time points as indicated by counting the substrate attached cell number, and confirmed by trypan blue exclusion. The growth curve shows the mean of triplicate values, with S.E.M. (C) Impact on telomere length. The control and RAD51-suppressed OE33 cells were cultured in the presence or absence of GRN163L for 10 days and telomere length determined by real time PCR as described; error bars represent SEMs of triplicate assays. (D) Impact on apoptosis. Transduced OE33 cells, treated as above for 10 days, were examined for apoptosis by evaluating their ability to bind lactadherin. Extent of green fluorescence indicates early or late apoptosis. (E) Evidence of senescence. Cells treated as above were evaluated for β-galactosidase staining, a marker for senescence.
Figure 4
Figure 4. Impact of HR inhibition by nilotinib on efficacy of telomerase inhibitor in OE33 cells
(A) Nilotinib significantly enhances anti-proliferative activity of GRN163L. The BAC (OE33) cells were treated with control oligonucleotide (C), oligonucleotide GRN163L targeting telomerase (G; 2 µM), nilotinib (N; 5 µM), or combination (NG), and viable cell number determined at different time points as indicated. The growth curve shows the mean of triplicate assays, with S.E.M. (B) Impact on apoptosis. OE33 cells, treated as described above for 19 days, were examined for apoptosis by evaluating their ability to bind lactadherin. Extent of green fluorescence indicates early or late apoptosis. (C) Impact on telomere length. OE33 cells, treated as described above for 19 days, were harvested, genomic DNA purified and telomere length determined by real time PCR as described in Methods. Error bars represent SEMs of triplicate assays. (D) Impact on cell cycle. OE33 cells, treated as described above for 19 days, were examined for cell cycle by flow cytometry. (E) Impact on HR activity. OE33 cells, treated as described above for 19 days, were evaluated for HR activity which was assessed using a plasmid based assay as described in Methods. Error bars represent SEMs of triplicate assays.
Figure 5
Figure 5. Impact of HR suppression on antitumor activity of telomerase inhibitor GRN163L
Homologous recombination (HR) activity was suppressed either chemically (by nilotinib) or transgenically (by RAD51 suppression). (A) Impact of nilotinib on efficacy of GRN163L in a subcutaneous tumor model of BAC. FLO-1 cells were injected subcutaneously in the interscapular area of SCID mice. Following appearance of tumors, mice were treated with normal saline containing working dilution of DMSO (control mice), nilotinib (N; 100 mg/kg), GRN163L (G; 45 mg/kg), or combination of nilotinib and GRN163L, injecting four times per week, intraperitoneally. C, control mice; N, mice treated with nilotinib, G, mice treated with GRN163L; NG, mice treated with combination. (B) Impact of RAD51-suppression on efficacy of GRN163L in a subcutaneous tumor model of BAC. FLO-1 cells, transduced with lentivirus particles producing control (CS) or RAD51-targeting (R) shRNAs were cultured for two weeks and injected subcutaneously in the interscapular area of SCID mice. Following appearance of tumors, the mice were treated with normal saline or GRN163L (G; 45 mg/kg), injecting four times per week, intraperitoneally. CS, mice in which tumors were developed by injecting control shRNA-transduced FLO-1 cells; R, mice in which tumors were developed by injecting RAD51 shRNA-transduced FLO-1 cells; RG, R mice treated with GRN163L. (C) Summary of in vivo data. Bar graph summarizes all in vivo data and shows average tumor size in mice subjected to telomerase and/or HR inhibitors. (D) Telomere length in vivo. Tumors from group of mice treated with nilotinib and/or GRN163L were removed and telomere length evaluated by Q-PCR as described in Methods. (E) Evidence of apoptosis in vivo. To evaluate if reduced tumor size was due to apoptotic cell death, tumors from second group of mice were removed and evaluated for apoptosis using flow cytometry. CS, mice in which tumors were developed by injecting control shRNA-transduced FLO-1 cells; R, mice in which tumors were developed by injecting RAD51 shRNA-transduced FLO-1 cells; RG, R mice treated with GRN163L.

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