Nijmegen breakage syndrome protein 1 (NBS1) modulates
hypoxia inducible factor-1 (HIF-1) stability and promotes
in vitro migration and invasion under ionizing radiation
Yi-Chih Kuo1, Han-Tsang Wu2, Jung-Jyh Hung3,4, Teh-Ying Chou3,5, Shu-Chun
Teng6, and Kou-Juey Wu1,2
1Institutes of Biochemistry & Molecular Biology, and 3Clinical Medicine, National
Yang-Ming University, Taipei 112; 2Research Center for Tumor Medical Science,
Graduate Institute of Cancer Biology, China Medical University, Taichung 404;
4Division of Thoracic Surgery, Dept. of Surgery, and 5Dept. of Pathology, Taipei
Veterans General Hospital, Taipei 112; 6Graduate Institute of Microbiology, College
of Medicine, National Taiwan University, Taipei 100, Taiwan
Running title: NBS1 stabilizes IR-induced HIF-1
Correspondence should be addressed to:
Kou-Juey Wu, Research Center for Tumor Medical Science, China Medical Univ., No. 91,
Hseuh-Shih Rd., Taichung 404, Taiwan; Email: [email protected]; Tel:
Abstract
Hypoxia-inducible factor (HIF) is a heterodimer transcription factor complex that monitors the cellular response to the oxygen levels in cells. Hypoxia-inducible factor-1α (HIF-1has been shown to be stabilized by ionizing radiation (IR) and its stabilization promotes tumor progression and metastasis. Nijmegen breakage syndrome protein 1 (NBS1), a component of the MRE11-RAD50-NBS1 complex, plays an important role in the cellular response to DNA damage but its overexpression contributes to transformation and has been found to correlate with metastasis. However, whether NBS1 participates in IR-induced metastasis needs to be further determined. The aim of this study is to investigate whether radiation-induced HIF-1 stabilization is regulated by NBS1 and thereby promotes tumor cell migration/invasion. Here, we show that both NBS1 and HIF-1 expression are up-regulated after exposure to IR, and NBS1 increases HIF-1 expression at the protein level. In addition, IR treatment promotes the epithelial–mesenchymal transition (EMT) and in vitro cell migration and invasion activity, which could be abolished by suppression of NBS1. Furthermore, NBS1 directly interacts with HIF-1and reduces the ubiquitination of HIF-1Co-expression of HIF-1α and NBS1 in primary tumors of patients with lung adenocarcinoma correlates with a worse prognosis. These results provide a new function of NBS1 in stabilizing HIF-1under IR, which leads to enhanced cancer cell migration and invasion.
Keywords: ionizing radiation, HIF-1, NBS1, ubiquitination, migration/invasion Abbreviations: IR, ionizing radiation; HIF-1, hypoxia-inducible factor-1; NBS1, Nijmegen breakage syndrome protein 1, EMT, epithelial–mesenchymal transition;
ATM, ataxia telangiectasia mutated; NCS, neocarzinostatin; CHX, cycloheximide; Ub, ubiquitin
1. Introduction
Radiotherapy is a widely used treatment option for malignant tumors . Both a carcinogen and a therapeutic agent, ionizing radiation (IR), increases the risk of developing cancer at low does exposure but slow or stop tumor growth when given at a high dose . However, emerging clinical and experimental observations have indicated that IR can enhance tumor metastasis and create a microenvironment that may exert tumor-promoting effects . Different studies have focused on the mechanisms by which IR activates cellular targets to promote invasion and metastasis . IR has been shown to induce cancer cell invasion through activation of the Rho signaling pathway , induction of MMP-9 expression , and activation of the epithelial-mesenchymal transition (EMT). However, the detailed mechanism of IR-induced cancer metastasis still remains unclear.
HIF-1 (hypoxia-inducible factor-1) is a heterodimeric transcription factor complex composed of an O2-regulated HIF-1 and a constitutively expressed HIF-1 subunit .
The activity of HIF-1 is controlled primarily through post-translational modification and stabilization of the ODD (oxygen-dependent degradation) domain at its subunit . Under normoxic conditions, prolyl hydroxylases (PHDs) hydroxylate two proline residues (a.a. 402, 564) on HIF-1, which is required for binding with the von Hippel-Lindau (VHL) tumor suppressor protein and the VHL-dependent recruitment of an Elongin-C containing ubiquitin ligase complex that targets the alpha subunit for ubiquitination and degradation by the 26S proteasome . During hypoxia, the activity of PHDs is inhibited; HIF-1 is stabilized, accompanied by its nuclear translocation, heterodimerization with HIF-1, and regulation of genes expression, including essential regulators for EMT . Overexpression of HIF-1protein in cancer patients
correlates with a poor prognosis, increased risk of metastasis and decreased survival . In addition, although hypoxia is the main condition for HIF-1 accumulation, there is increasing evidence demonstrating that HIF-1 is also upregulated after radiation , but whether enrichment in HIF-1 affects EMT under IR should be investigated.
NBS1 is one component of the MRE11–RAD50–NBS1 complex (MRN) complex, which plays important roles in signal transduction related to DNA repair and cell cycle checkpoints . NBS1 is phosphorylated at serine 278 and 343 by ataxia telangiectasia mutated (ATM) kinase in response to IR both in vitro and in vivo . We previously demonstrated that c-MYC oncoprotein activates NBS1 expression and overexpression of NBS1 contributes to transformation by activating the PI3 kinase/Akt pathway . NBS1 overexpression induces EMT and promotes metastasis through the activation of Snail . Increased NBS1 expression in head and neck cancer patients is associated with significantly worse clinical outcomes . All these results indicate that NBS1 may play an important role in the process of tumorigenesis and metastasis. However, involvement of NBS1 in IR-induced cancer metastasis is largely unknown.
In this study, we demonstrated that IR increases NBS1 and HIF-1 expression and enhances in vitro migration/invasion activity of cancer cells. NBS1 regulates HIF-1 expression levels, and knockdown of NBS1 inhibits IR-induced HIF-1 expression and in vitro migration/invasion activity under normoxia or hypoxia. NBS1 also directly interacts with HIF-1 and suppresses the ubiquitination of HIF-1Co-expression of NBS1 and HIF-1in tumor samples from lung cancer patients correlates with a higher probability of metastasis and a worse prognosis. These results indicate that IR-induced cancer cell migration/invasion is mediated by a key signaling
2. Materials and Methods
2.1.
Cell lines, oxygen deprivation, ionizing radiation and Neocarzinostatin treatmentHuman breast cancer (MCF7), lung cancer (H1299) and embryonic kidney (293T) cell lines have been described previously . Breast cancer (MDA-MB-231) and oral cancer (OECM1) cell lines were obtained from American Type Culture Collection (ATCC). OECM1-NBS1 cell line was generated by transfecting the HeBOCMVNBS plasmid (described in ) into OECM1 cells followed by G418 (200 μg/ml) selection. The H1299-NBS-si and MDA-MB-231-NBS1-si cell lines were generated by transfecting the pSUPER-NBSi plasmid (described in ) into H1299 and MDA-MB-231 cells followed by puromycin (2 g/ml) selection. The H1299-Cont. and MDA-MB-231-Cont. cell lines were generated by transfecting the pSUPER-scrambled-si plasmid into H1299 and MDA-MB-231 cells followed by puromycin (2 g/ml) selection. These cells described above were exposed to radiation using a 137Cs γ-ray source at a dose rate of 6.16 Gy/min or treated with 10 μM Neocarzinostatin (NCS). After irradiation, the cells were placed in a standard cell culture incubator (37°C, 5% CO2), or oxygen deprivation was carried out in an
incubator with 1% O2, 5% CO2 and 94% N2 for 12 to 18 hours.
2.2. Plasmid construction
The primers and restriction enzymes to digest polymerase chain reaction (PCR)-amplified fragments and to generate the different full length/truncation mutants or fusion proteins are detailed in Supplementary Table S1. The pFlag-NBS11-754,
by insertion of the indicated fragments of NBS1 into BamHI/HindIII sites of pCMV2-Flag vector. The pHA-HIF-1α1-826, pHA-HIF-1α1-400, pHA-HIF-1α401-826,
pHA-HIF-1α401-603 and pHA-HIF-1α604-826 were generated by insertion of the indicated fragments
of HIF-1α into BamHI/NotI sites of pCMV-HA vector. The pGEX-NBS1221–402 and pGEX-NBS1653–754 constructs were generated by PCR amplification of appropriate NBS1 fragments, digested, and subcloned into the pGEX-4T-1 [glutathione-S-transferase (GST)] vector (obtained from Dr. Y.C. Wu, National Taiwan University, Taiwan). The pET32a-HIF-1α1-400 and pET32a- HIF-1α401-603 constructs were generated by PCR amplification of appropriate HIF-1α fragments, digested, and subcloned into the pET32a vector (Novagen, Darmstadt, Germany). The pcDNA3-HA-HIF-1α plasmid was a gift from Dr. L.E. Huang (University of Utah, USA). The pHA-ubiquitin K48R, pHA-ubiquitin K63R and pHA-ubiquitin K48, 63R were gifts from Dr. P.H. Tseng (National Yang-Ming University, Taiwan). The pFlag-ubiquitin was a gift from Dr. Y.H. Wu-Lee (National Yang-Ming University, Taiwan).
2.3. Protein extraction, western blot analysis and co-immunoprecipitation assay These procedures were performed as described previously . For western blot analysis, 50 to 100 μg of protein extracts were loaded to 10% sodium dodecyl sulfate– polyacrylamide gel electrophoresis gels and transferred to nitrocellulose filters. The filters were probed with various antibodies. The characteristics of the antibodies used in western blot analysis were listed in Supplementary Table S2. Data shown here are representative of two or more experiments from independent cell cultures. For co-immunoprecipitation assays, 500 μg of cell lysates were incubated with antibody for 3 hours in TNTG buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1 % Triton-X 100, 10% glycerol). The immune complexes were incubated overnight with protein-A
beads, preblocked with 10% bovine serum albumin (BSA). The immunoprecipitates were washed three times with TNTG buffer, mixed with 1×Laemmli dye, boiled for 10 minutes, and loaded on SDS polyacrylamide gels (SDS-PAGE). After transfer, the filters were blocked with blocking buffer, probed with primary and secondary antibody sequentially, and developed.
2.4. Cell migration and invasiveness assay
Eight-μm pore size Boyden chamber was used for in vitro migration and invasion assays. Cells were starved for 24 hours, followed by IR treatment. After exposure to irradiation with a total dose of 5.0 Gy, cells in serum free Dulbecco’s modified Eagle’s medium (DMEM) were plated in the upper chamber and serum-free DMEM was added in the lower chamber. For invasion assay, the upper side of the filter was covered with Matrigel (Collaborative Research Inc., Boston, MA, USA) (the diluted ratio 1:3 with DMEM). After 12 hours for migration assay or 18 hours for invasion assay, cells on the upper side of the filter were removed, and cells that remained adherent to the underside of the membrane were fixed in 4% formaldehyde and stained with Hoechst 33342 dye. The number of migrated cells was counted using a fluorescence microscope.
2.5. Transient transfection and luciferase assays
The TWIST promoter region was cloned by PCR amplification of genomic DNA and inserted into HindIII/BglII sites of the pXP2 vector to generate the pXP2-TWIST reporter construct as described (Yang et al., 2008). The 293T cell was chosen to perform transient transfection. The reporter construct (pXP2-TWIST) was co-transfected into 293T cells with NBS1 plasmid and HIF-1α plasmid. At 48 hours after transfection, cells were harvested and transcriptional activity was assayed as a
function of Luciferase (Luc) activity. The values are expressed as Luc activity after normalization with β-galactosidase activity expressed from a co-transfected plasmid expressing the bacterial β-galactosidase gene (0.5 μg of pCMV-βgal). Each transfection was performed in triplicate and standard deviation bars are shown.
2.6. GST pull-down assays
The pGST-NBS1221-402 and pGST-NBS1653-754 plasmids were transformed
respectively into Escherichia coli BL21 (DE3)-pLysS (Novagen, Gibbstown, NJ). The GST fusion proteins were induced by 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 30 °C for 3 hours, harvested and lysed by French press (a device using differential pressure to gently lyse the cells without the damage caused by mechanical or chemical methods). The cell lysates were clarified by centrifugation (20,000 r.p.m. for 30 minutes at 4°C) and the supernatant was incubated with GST-fused beads (GE Healthcare, Piscataway, NJ) at 4 °C for 3 hours. Bound proteins were washed with binding buffer and eluted with elution buffer (50 mM Tris–HCl and 10 mM reduced glutathione, pH 8.0). Purification of His-tagged HIF-1 proteins was performed by transforming pET32a- HIF-11-400 and pET32a-HIF-1401-603 plasmids, respectively,
into E. coli BL21(DE3)-pLysS followed by induction for 3 hours with IPTG and lysis by French press. The cell lysates were suspended in buffer A (PBS with 150 mM NaCl), clarified by centrifugation (20,000 r.p.m. for 30 minutes at 4°C), adjusted to 10 mM imidazole and loaded on HisTrap chelating columns (GE Healthcare). Bound proteins were washed with 50 and 100 mM imidazole in buffer A and eluted with 300 mM imidazole in buffer A. For GST pull-down assays, different His-tagged HIF-1 proteins were incubated with different GST-NBS1 proteins (or GST only as a control) and glutathione–agarose (Sigma, St Louis, MI) for 3 hours at 4°C in 600 μl of GST buffer (142.5 mM NaCl, 10 mM N-2-hydroxyethylpiperazine -N’-2-ethanesulfonic
acid, pH 7.6, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid and 2.5 mg/ml bovine serum albumin). The beads were washed five times with 1 ml GST buffer without bovine serum albumin. Finally, the bound complexes were eluted and analyzed by western blot.
2.7. RNA purification and real-time PCR analysis
The total RNA was isolated using TRIzol (Sigma) according to the manufacturer’s instructions. cDNAs were synthesized using RevertAid First Strand cDNA Synthesis Kit (Thermo scientific) according to the manufacturer’s directions. Quantitative real-time PCR analysis was performed as described . The primer sequences used in real-time PCR were:
HIF-1, TACCGAATTGATGGGATATGAGCCAG-3’ and 5’-TCCTGTACTGTCCTGTGGTGACTTG-3’; Twist1, 5’-TCTACCAGGTCCTCCAGAGC-3’ and 5’-CTCCATCCTCCAGACCGAGA -3’; 18S, 5’-GGCGGCGTTATTCCCATGA-3’ and 5’- GAGGTTTCCCGTGTTGAG-3’. 2.8. Immunofluorescence
Cells on glass coverslips were fixed with 4% paraformaldehyde for 10 minutes at room temperature, followed by treatment with 0.1% Triton X-100 for 15 minutes. The samples were blocked with PBS containing 10% bovine serum albumin at room temperature for 1 hour and incubated with primary antibodies at 4°C overnight. After this step, the cells were washed with PBS and incubated with fluorescein
isothiocyanate and rhodamine conjugated secondary antibodies at room temperature in the dark for 1 hour. Nuclei were counterstained with Hoechst 33342 (Sigma-Aldrich Crop., St Louis, MO, USA) and fluorescence images were observed by laser confocal microscope (FV10i; Olympus, Tokyo, Japan).
2.9. Study population, sample collection and tissue microarray construction
One hundred stage I lung cancer patients who underwent surgical resection at Taipei Veterans General Hospital between January 2002 and December 2006 were retrospectively analyzed. This study has been approved by the Institutional Review Board of Taipei Veterans General Hospital. The median follow-up duration was 53.7 months (range 4.7~98.2 months). The clinical characteristics of 100 lung adenocarcinoma patients are illustrated (Supplementary Table S3). Primary tumor samples and the corresponding non-cancerous matched tissue were obtained during surgery. A high-density tissue microarray (TMA) wasconstructed as described (Yang et al., 2008).
2.10. Immunohistochemistry (IHC), validation of antibodies, and scoring
The sample processing and immunohistochemistry procedure for determining the immunoreactivity of HIF-1α and NBS1 were performed as described previously . The NBS1 immunohistochemistry scoring was defined on a scale ranging from 0 to +++: 0, no appreciable staining in cells; +, only nucleus staining and no detectable cytoplasmic staining; ++, appreciable nucleus staining with cytoplasmic staining in <25% of cells; +++, significant nucleus staining and strong cytoplasmic staining. Only NBS1 level +++ staining was defined as increased NBS1 expression. HIF-1α immunoreactivity was interpreted as previously described , with greater than 50% nuclear staining scored as a positive result. All the antibodies used in IHC are listed in
the Supplementary Table S2.
2.10. Statistical analysis
An independent Student’s t-test or analysis of variance was used to compare continuous variables between two groups, and a 2 test was applied for comparison of
dichotomous variables. A Kaplan-Meier estimate was used for survival analysis, and a log-rank test was used to compare the cumulative survival durations in different patient groups. Unless otherwise specified in the figure legends, the control groups for all the statistical analyses were the first groups in the panels. Statistical significance was accepted when P <0.05 for all tests.
3. Results
3.1. Ionizing radiation (IR) increases NBS1 and HIF-1 expression and enhances in vitro migration and invasion activity
Since both NBS1 and HIF-1 are implicated in cancer metastasis and ionizing radiation (IR) also plays a role in metastasis , we firstly investigated whether NBS1 and HIF-1 are involved in IR-induced tumor metastasis. The expression levels of NBS1 and HIF-1 were examined after exposure to IR in different human cancer cell lines, including OECM1, MCF-7, MDA-MB-231, and H1299 cells. After exposure to IR, the protein levels of cellular NBS1 and HIF-1 increased under normoxic or hypoxic culture. Previous studies have shown that IR induces phosphorylation of ATM and CHK2 , and the phosphorylated form of variant histone H2AX, γH2AX, is also detected after IR exposure . In corroboration with these findings, we observed increased phosphorylation on serine 1981 of ATM and threonine 68 of CHK2, as well as increased levels of H2AX, after IR treatment (Fig. 1A & Fig. S1A). Similar results were observed in cells treated with Neocarzinostatin (NCS), a natural product which induces single- and double-strand breaks in DNA (Fig. S1B). We also determined the effect of IR on in vitro cell migration and invasion activity, using MCF7 and OECM1 cell lines, which have low baseline migration and invasion activity. IR treatment increased the migration and invasion activity of these cells (Fig. 1B). These data indicate that IR increases the expression of HIF-1α and NBS1 simultaneously and enhances cell migration and invasion activity.
HIF-1expression, EMT, and cell migration/ invasion
To investigate whether there is a relationship between NBS1 and HIF-1, transient expressions of HA-HIF-1α with or without Flag-NBS1 in 293T cells under NCS treatment were performed under normoxic or hypoxic conditions. We found that HIF-1 expression was increased by co-transfection with NBS1 or treatment with NCS under both normoxic and hypoxic conditions (Fig. 1C). In addition, up-regulation of HIF-1protein levels was also observed in NBS1-overexpressing OECM1 stable cells (Fig. 1D). However, HIF-1mRNA levels from control and NBS1-overexpressing OECM1 cells were not significantly different, but increased mRNA levels of Twist1, a downstream target of HIF-1were observedin cells overexpressing NBS1 (Fig. 1D). In addition, the reporter gene assays were performed to test whether NBS1 could further enhance the activation of the Twist1 promoter by increasing HIF-1expression levels. Indeed, co-transfection of HIF-1 and NBS1 expression vectors further increased Twist1 promoter activity compared to transfection of HIF-1 alone (Fig. 1E). These results demonstrated that NBS1 modulates HIF-1 protein levels.
In order to investigate whether NBS1 is required for IR-induced HIF-1expression and cell migration/invasion, knockdown of NBS1 expression was performed in the highly invasive MDA-MB-231 and H1299 cells. Knockdown of NBS1 inhibited IR-induced HIF-1expression compared to the control cells under both normoxic and hypoxic conditions (Fig. 1F). Similar results were observed after NCS treatment regardless of normoxia or hypoxia (Fig. S2A). Furthermore, knockdown of NBS1 inhibited IR-induced cell migration and invasion in MDA-MB-231 and H1299 cells (Fig. 1G). To exclude the possibility that decreased cell migration/invasion in NBS1
knockdown cells may be due to differences in cell viability, we performed alamarBlue cell viability assay. We did not detect any differences in cell viability between NBS1-knockdown and control cells with or without IR treatment (Fig. S2B). On the other hand, we also tested whether HIF-1is involved in IR-induced NBS1 expression and cell migration/invasion. Knockdown of HIF-1 in MCF-7 and H1299 cells had no effect on IR-induced NBS1 expression but inhibited cell migration/invasion activity induced by IR (Fig. S3). These results suggested that IR increases the expression of NBS1, which in turn upregulates HIF-1 protein levels and enhances cell migration/invasion.
Previous study showed that IR promotes EMT in different tumor cell lines . In addition, overexpression of HIF-1has also been found to induce EMT . Since IR upregulated NBS1 and 1expression, and NBS1 is essential for IR-induced HIF-1 expression and cell migration/invasion; we next investigated whether NBS1 is required for IR-induced EMT. Induction of EMT was first observed in OECM1 and MCF-7 cells after IR treatment (Fig. 2A, B), as shown by a shift in expression of epithelial markers (E-cadherin and plakoglobin) to mesenchymal markers (vimentin and N-cadherin). Immunofluorescence staining of E-cadherin and vimentin also confirmed the changes of EMT after IR treatment (Fig. S4A). In addition, we found that knockdown of NBS1 expression abolished IR-induced EMT compared to the control MDA-MB-231 and H1299 cells (Fig. 2C, D). Similar results were also obtained in MCF7 cells with a non-invasive phenotype (Fig. S4B). Taken together, these results indicated that NBS1 is essential for expression of HIF-1the and cell migration/invasion induced by IR.
To investigate whether NBS1 directly regulates HIF-1co-immunoprecipitation (co-IP) assays were performed to determine whether NBS1 interacts with HIF-1The results showed that endogenous NBS1 protein interacted with HIF-1in H1299 and MDA-MB-231 cells regardless of IR treatment (Fig. 3A),
suggesting that NBS1 may interact with HIF-1. The interaction between NBS1 and HIF-1was not changed by hypoxia or NCS treatment (Fig. S5A). In addition, we also performed the co-IP assays to detect whether NBS1 could interact with HIF-2. NBS1 could not interact with HIF-2 in H1299 and MDA-MB-231 cells regardless of IR treatment (Fig. S5B). To determine the possible binding regions between them, four different Flag-tagged truncated NBS1 constructs were generated, and each was co-expressed with HIF-1 in the co-IP assays. The results revealed that the NBS1 encompassing amino acid 221-402 was still able to interact with HIF-1 (Fig. 3B). Similarly, a series of HIF-1 deletion mutants were also constructed to map the interacting region of HIF-1 with NBS1. The N-terminal domain (a.a. 1-400) of HIF-1 interacted with full-length NBS1 in co-IP assays (Fig. 3C). The interaction between NBS1 and HIF-1 was verified using GST pull-down assays. GST control, GST-NBS1221-402, GST-NBS1653–754, His-HIF-11–400 and His-HIF-1were
expressed and purified from E. coli. In support of the in vivo interaction, only the GST-NBS1221–402 was able to pull down His-HIF-11–400 (Fig. 3D).In addition, we also performed the immunofluorescence staining to detect the location of NBS1 and HIF-1. The results showed that NBS1 was co-localized with HIF-1 in H1299 cells after IR treatment (Fig. S6). Together, these findings demonstrate that NBS1 shares the same compartment with HIF-1 after DNA damage and NBS1 may directly interact with HIF-1, thereby facilitating cell migration and invasion during IR
3.4. NBS1 decreases the ubiquitination levels of HIF-1α and increased the half-life of HIF-1
We next investigated the mechanism of how NBS1 regulates HIF-1 protein. According to the previous data, we found that NBS1 regulates HIF-1 expression at the protein level but not the mRNA level. This prompted us to explore the post-translational modification of HIF-1, i.e. ubiquitination, because the ubiquitin-proteasome pathway has so far been the most well-known mechanism that regulates HIF-1 protein stability . HEK293T cells were transfected with expression vectors for HIF-1 and HA-tagged ubiquitin, and incubated with MG-132 (a proteasome inhibitor) so HA-tagged ubiquitin can be conjugated to HIF-1. After incubation with MG132, a smear detected by anti-HIF-1 antibodies (lane 3, Fig. 4A), which indicates ubiquitinated HIF-1, appeared in the blots of lysates of cells transfected with HIF-1 and HA-tagged ubiquitin compared to the control or DMSO treated group. But the ubiquitination levels of HIF-1 were markedly decreased by co-expression of NBS1 (lane 4, Fig. 4A). An alternative way to demonstrate the ubiquitinated HIF-1 was to detect it by anti-HA antibodies. When HIF-1α was immunoprecipitated, the amount of ubiquitins linked to HIF-1α, as detected by anti-HA antibodies, robustly decreased in cells co-transfected with NBS1 (lane 1 vs. lane 2, Fig. 4B). These data revealed that HIF-1α ubiquitination significantly decreases when NBS1 is co-expressed, indicating that NBS1 stabilizes HIF-1α by interfering with the ubiquitin–proteasome pathway. The resulting confirmation of the polyubiquitin chains depends on which lysines within ubiquitin are used for the
isopeptide bond formation, and the linkage also provides an additional layer of specificity . K48-linked chains are usually associated with proteasome degradation, but K63 linkage is involved in a variety of other processes, including endocytosis and DNA repair . In order to further confirm the linkage form of HIF-1 ubiquitination, the K48R (a.a. position 48 in ubiquitin, lysine changed to arginine) or K63R (a.a. position 63 in ubiquitin, lysine changed to arginine) ubiquitin mutant and HIF-1 expression plasmid were co-transfected with or without NBS1. We found that ubiquitination of HIF-1 occurred through K48 but not through K63 since only the wild type and K63R mutant ubiquitin was conjugated to HIF-1 under MG132 treatment, and this ubiquitination was also inhibited by co-transfection with NBS1 (lane 1 vs. lane 2 for wild type ubiquitin, lane 5 vs. lane 6 for K63R ubiquitin, Fig. 4C). Furthermore, we investigated whether NBS1 can prolong the half-life of endogenous HIF-1. We overexpressed Flag-NBS1 in H1299 cells and treated cells with a protein synthesis inhibitor, cycloheximide, for different time periods. As shown in Fig. 4D, HIF-1protein decayed with a half-life of 2.5 minutes in H1299 cells compared with >10 minutes in cells transfected with Flag-NBS1. These results suggested that NBS1 decreases K48-linked polyubiquitination of HIF-1 and prolongs the half-life of HIF-1. Finally, we tested whether NBS1 can compete with RACK1 for binding to HIF-1 since RACK1 has been shown to compete with HSP90 to mediate HIF-1 degradation (Liu et al., 2007). Co-transfection of HIF-1 together with increasing amount of Flag-NBS1 into cells followed by co-immunoprecipitation by anti-HA antibodies under MG132 treatment was performed. The result showed that NBS1 competed with RACK1 for binding to HIF-1 and increasing amount of NBS1 decreased RACK1 binding to HIF-1 (Fig. 4E). This result indicates that NBS1 modulates HIF-1 stability through competition with RACK1.
3.5. Co-expression of HIF-1α and NBS1 in lung adenocarcinoma samples is associated with worse clinical outcomes
Stabilization of HIF-1α through intratumoral hypoxia has been found in various cancers and correlates with metastasis and poor survival in tumor patients . Overexpression of NBS1 also correlates with poor prognosis for cancer patients . We wanted to investigate whether NBS1 indeed stabilizes HIF-1α in human cancers and to evaluate the prognostic significance of HIF-1α/NBS1 co-expression. Tissue-microarray immunohistochemical (IHC) analysis of HIF-1α and NBS1 expression was performed in 100 sets of resected stage I lung adenocarcinoma patient samples (Supplementary Table S3, IHC staining from a representative case was shown in Fig. 5A). Tumors with increased HIF-1 expression significantly correlated with NBS1 overexpression (P<0.001, Fig. 5B). Prognostic prediction analysis showed that co-expression of HIF-1 and NBS1 had a significantly worse overall survival than non-co-expression cases (P=0.005; Fig. 5C). The prognostic effect of HIF-1α and NBS1 co-expression was independent of other prognostic markers (gender, tumor size) (P=0.002; Supplementary Table S4). Collectively, the correlation analysis indicates that NBS1 expression correlates with HIF-1α levels in lung adenocarcinoma samples, and survival analysis supports the prognostic value of co-expression of HIF-1 and NBS1 in lung cancer cases.
4. Discussion
Increasing evidence indicates that IR might promote a metastatic behavior of cancer cells , but data addressing the mechanism of IR-induced cancer metastasis are relatively scarce. Our results demonstrate a novel signaling pathway in IR-induced metastasis and highlight the critical role of NBS1 and HIF-1 in metastatic tumors.
Increased HIF-1 expression has been identified in different types of human cancer , and is regarded as one of the most important factors to promote tumor metastasis . Although several studies have shown that HIF-1 expression was increased after IR , the detailed mechanism still needs to be unequivocally demonstrated. In this report, we showed that increased HIF-1 expression was accompanied by an elevated level of NBS1 in different cancer cells after IR treatment, and NBS1 regulates HIF-1α expression at the protein level. This result was consistent with the findings of previous study in which NBS1 was shown to be increased after exposure to IR . NBS1, as with many other DNA checkpoint proteins, was initially known to guard the integrity of the genome. However, recent studies showed that overexpression of NBS1 induces EMT via Snail induction, leading to the development of metastasis and poor prognosis of tumor patients . In addition, IR has also been found to promote EMT in different tumor cell lines . Here, we demonstrate that NBS1 is required for IR-induced HIF-1α expression, EMT, and cell migration/invasion, but knockdown of HIF-1α has no effect on IR-induced NBS1 expression. Hence, we suggest that IR-induced EMT and cell migration/invasion should be through regulation of HIF-1α by NBS1. Our results uncovered the new function of NBS1 in IR-induced HIF-1α stabilization and cancer cell migration/invasion.
diverse discoveries on HIF-1α and NBS1 expression have been reported. For instance, it has been reported that HIF-1α is critical for NBS1 downregulation by hypoxia , whereas other evidence showed that NBS1 expression does not change under hypoxia and it has also been found that hypoxia induces phosphorylation of NBS1 through ATR kinase . By western blot analysis using two different cells, H1299 and MDA-MB-231, we found that the expression of NBS1 is not changed by hypoxia. These results were consistent with the outcome of knockdown experiments, in which suppression of HIF-1α does not increase NBS1 expression in MCF-7 and H1299 cells. In addition, although it has shown that the phosphorylation status of the PAS-B domain distinguishes HIF-1α from HIF-2α to repress NBS1 expression , whether other kinases modify HIF-1α after exposure to IR cannot be excluded thus far. For example, it has been reported that IR can activate DNA-dependent protein kinase (DNA-PK) which is involved in phosphorylation of HIF-1α . Furthermore, this phenomenon was confirmed in different cancer cells and also consistent with previous studies demonstrating that IR can upregulate HIF-1α and increase NBS1 expression . In addition, the expression levels of NBS1 and HIF-1α were also validated in clinical human tumors since we confirmed that increased HIF-1 expression significantly correlated with NBS1 overexpression in lung adenocarcinoma samples and co-expression of HIF-1 and NBS1 was associated with a poor prognosis of patients. According to the above findings, we suggest that IR can increase the expression of HIF-1α and NBS1 simultaneously.
This report demonstrated that that IR-induced EMT and cell migration/invasion may be mediated through stabilization of HIF-1α by NBS1. Although most studies for intracellular regulation of HIF-1α indicate that post-translational modifications of HIF-1α are located in the ODD domain via an oxygen-dependent pathway, RACK1
has been shown to bind to Elongin C and recruit other components of E3 ubiquitin ligases to facilitate HIF-1α ubiquitination and degradation in an oxygen-independent manner . This report shows that NBS1 increases HIF-1α protein levels under both normoxic and hypoxic conditions and decreases the ubiquitination of HIF-1α. Furthermore, it is interesting that HIF-1α uses the same N-terminal region (a.a. 1-400) to interact with NBS1 and RACK1. Previous results showed that RHBDF1 promotes HIF-1α stability by preventing RACK1 binding to HIF-1α, thus attenuating HIF-1α ubiquitination and proteasome degradation . The role of NBS1 is similar to RHBDF1 since NBS1 also competes with RACK1 for binding to HIF-1 to modulate HIF-1 stability.
Radiotherapy is the traetment of choice for primary tumors combined with surgery and chemotherapy . Malignant foci or residual cancer cells are observed in a number of patients with breast cancer after surgery and radiotherapy . These residual cancer cells could be responsible for distant metastases associated with deaths from cancer . Due to the ability of IR to induce NBS1 and stabilize HIF-1α to promote EMT and cell migration/invasion, radiotherapy needs to be seriously planned to avoid detrimental effects in cancer therapy
Acknowledgments
We thank Drs. L.E. Huang, P.H. Tseng, Y.H. Wu-Lee, and Y.C. Wu for the
generous gifts of various plasmids. This work was supported in part to K.J.W. by
Ministry of Science and Technology Summit grant (MOST
103-2745-B-039-001-ASP), National Science Council Frontier grant (NSC102-231-B-010-001), center of
excellence for cancer research at Taipei Veterans General Hospital
(MOHW104-TDU-B-211-124-001), and National Health Research Institutes
(NHRI-EX104-10230SI).
Author contributions
K.J.W. conceived the project and designed the experiments. Y.C.K. and H.T.W.
carried out experiments. J.J.H., and T.Y.C. provided the tumor samples and
performed patient sample staining and analysis. K.J.W., Y.C.K., H.T.W. and S.C.T.
analyzed the data, K.J.W. wrote the manuscript with the help of S.C.T.
Conflict of interests
References
Adams JM, Difazio LT, Rolandelli RH, Lujan JJ, Hasko G, Csoka B, et al. HIF-1: a key mediator in hypoxia. Acta physiologica Hungarica. 2009;96:19-28.
Andarawewa KL, Erickson AC, Chou WS, Costes SV, Gascard P, Mott JD, et al. Ionizing radiation predisposes nonmalignant human mammary epithelial cells to undergo transforming growth factor beta induced epithelial to mesenchymal transition. Cancer research. 2007;67:8662-70.
Bakkenist CJ, Kastan MB. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature. 2003;421:499-506.
BarcellosHoff MH, Park C, Wright EG. Radiation and the microenvironment -tumorigenesis and therapy. Nature reviews Cancer. 2005;5:867-75.
Bruick RK, McKnight SL. A conserved family of prolyl-4-hydroxylases that modify HIF. Science. 2001;294:1337-40.
Carney JP, Maser RS, Olivares H, Davis EM, Le Beau M, Yates JR, 3rd, et al. The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell. 1998;93:477-86.
Chen YC, Chiang HY, Yang MH, Chen PM, Chang SY, Teng SC, et al. Activation of phosphoinositide 3-kinase by the NBS1 DNA repair protein through a novel activation motif. Journal of molecular medicine. 2008;86:401-12.
Chen YC, Su YN, Chou PC, Chiang WC, Chang MC, Wang LS, et al. Overexpression of NBS1 contributes to transformation through the activation of phosphatidylinositol 3-kinase/Akt. The Journal of biological chemistry. 2005;280:32505-11.
Cheng JC, Chou CH, Kuo ML, Hsieh CY. Radiation-enhanced hepatocellular carcinoma cell invasion with MMP-9 expression through PI3K/Akt/NF-kappaB signal transduction pathway. Oncogene. 2006;25:7009-18.
Cheng L, Wu Q, Huang Z, Guryanova OA, Huang Q, Shou W, et al. L1CAM regulates DNA damage checkpoint response of glioblastoma stem cells through NBS1. The EMBO journal. 2011;30:800-13.
Chiang YC, Teng SC, Su YN, Hsieh FJ, Wu KJ. c-Myc directly regulates the transcription of the NBS1 gene involved in DNA double-strand break repair. The Journal of biological chemistry. 2003;278:19286-91.
Cockman ME, Masson N, Mole DR, Jaakkola P, Chang GW, Clifford SC, et al. Hypoxia inducible factor-alpha binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein. The Journal of biological chemistry. 2000;275:25733-41. Costanzo V, Robertson K, Bibikova M, Kim E, Grieco D, Gottesman M, et al. Mre11 protein complex prevents double-strand break accumulation during chromosomal DNA replication. Molecular cell. 2001;8:137-47.
Gibson SL, Bindra RS, Glazer PM. Hypoxia-induced phosphorylation of Chk2 in an ataxia telangiectasia mutated-dependent manner. Cancer research. 2005;65:10734-41. Gupta GP, Massague J. Cancer metastasis: building a framework. Cell. 2006;127:679-95.
Hammond EM, Green SL, Giaccia AJ. Comparison of hypoxia-induced replication arrest with hydroxyurea and aphidicolin-induced arrest. Mutation research. 2003;532:205-13.
Harris AL. Hypoxia--a key regulatory factor in tumour growth. Nature reviews Cancer. 2002;2:38-47.
Huang LE, Arany Z, Livingston DM, Bunn HF. Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit. The Journal of biological chemistry. 1996;271:32253-9.
Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ, et al. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science. 2001;292:468-72.
Janssen HL, Haustermans KM, Balm AJ, Begg AC. Hypoxia in head and neck cancer: how much, how important? Head & neck. 2005;27:622-38.
Kim RK, Cui YH, Yoo KC, Kim IG, Lee M, Choi YH, et al. Radiation promotes malignant phenotypes through SRC in breast cancer cells. Cancer science. 2015;106:78-85.
Kim YH, Yoo KC, Cui YH, Uddin N, Lim EJ, Kim MJ, et al. Radiation promotes malignant progression of glioma cells through HIF-1alpha stabilization. Cancer letters. 2014;354:132-41.
Li F, Sonveaux P, Rabbani ZN, Liu S, Yan B, Huang Q, et al. Regulation of HIF-1alpha stability through S-nitrosylation. Molecular cell. 2007;26:63-74.
Lim DS, Kim ST, Xu B, Maser RS, Lin J, Petrini JH, et al. ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway. Nature. 2000;404:613-7.
Liu W, Huang YJ, Liu C, Yang YY, Liu H, Cui JG, et al. Inhibition of TBK1 attenuates radiation-induced epithelial-mesenchymal transition of A549 human lung cancer cells via activation of GSK-3beta and repression of ZEB1. Laboratory investigation; a journal of technical methods and pathology. 2014;94:362-70.
Liu YV, Baek JH, Zhang H, Diez R, Cole RN, Semenza GL. RACK1 competes with HSP90 for binding to HIF-1alpha and is required for O(2)-independent and HSP90 inhibitor-induced degradation of HIF-1alpha. Molecular cell. 2007;25:207-17.
Liu YV, Semenza GL. RACK1 vs. HSP90: competition for HIF-1 alpha degradation vs. stabilization. Cell cycle. 2007;6:656-9.
Lu H, Liang K, Lu Y, Fan Z. The anti-EGFR antibody cetuximab sensitizes human head and neck squamous cell carcinoma cells to radiation in part through inhibiting
radiation-induced upregulation of HIF-1alpha. Cancer letters. 2012;322:78-85.
Lutz ST, Jones J, Chow E. Role of Radiation Therapy in Palliative Care of the Patient With Cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2014;32:2913-9.
Madani I, De Neve W, Mareel M. Does ionizing radiation stimulate cancer invasion and metastasis? Bulletin du cancer. 2008;95:292-300.
Mak P, Leav I, Pursell B, Bae D, Yang X, Taglienti CA, et al. ERbeta impedes prostate cancer EMT by destabilizing HIF-1alpha and inhibiting VEGF-mediated snail nuclear localization: implications for Gleason grading. Cancer cell. 2010;17:319-32.
Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC, Cockman ME, et al. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature. 1999;399:271-5.
Moeller BJ, Cao Y, Li CY, Dewhirst MW. Radiation activates HIF-1 to regulate vascular radiosensitivity in tumors: role of reoxygenation, free radicals, and stress granules. Cancer cell. 2004;5:429-41.
Nagarajan D, Melo T, Deng Z, Almeida C, Zhao W. ERK/GSK3beta/Snail signaling mediates radiation-induced alveolar epithelial-to-mesenchymal transition. Free radical biology & medicine. 2012;52:983-92.
Pickart CM. Ubiquitin in chains. Trends in biochemical sciences. 2000;25:544-8. Pickart CM. Mechanisms underlying ubiquitination. Annual review of biochemistry. 2001;70:503-33.
Pugh CW, Gleadle J, Maxwell PH. Hypoxia and oxidative stress in breast cancer. Hypoxia signalling pathways. Breast cancer research : BCR. 2001;3:313-7.
Redon CE, Dickey JS, Bonner WM, Sedelnikova OA. gamma-H2AX as a biomarker of DNA damage induced by ionizing radiation in human peripheral blood lymphocytes and artificial skin. Advances in space research : the official journal of the Committee on Space Research. 2009;43:1171-8.
Semenza GL. Targeting HIF-1 for cancer therapy. Nature reviews Cancer. 2003;3:721-32.
To KK, Sedelnikova OA, Samons M, Bonner WM, Huang LE. The phosphorylation status of PAS-B distinguishes HIF-1alpha from HIF-2alpha in NBS1 repression. The EMBO journal. 2006;25:4784-94.
Um JH, Kang CD, Bae JH, Shin GG, Kim DW, Kim DW, et al. Association of DNA-dependent protein kinase with hypoxia inducible factor-1 and its implication in resistance to anticancer drugs in hypoxic tumor cells. Experimental & molecular medicine. 2004;36:233-42.
basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proceedings of the National Academy of Sciences of the United States of America. 1995;92:5510-4. Ward IM, Wu X, Chen J. Threonine 68 of Chk2 is phosphorylated at sites of DNA strand breaks. The Journal of biological chemistry. 2001;276:47755-8.
Wu X, Ranganathan V, Weisman DS, Heine WF, Ciccone DN, O'Neill TB, et al. ATM phosphorylation of Nijmegen breakage syndrome protein is required in a DNA damage response. Nature. 2000;405:477-82.
Yajima H, Lee KJ, Chen BP. ATR-dependent phosphorylation of DNA-dependent protein kinase catalytic subunit in response to UV-induced replication stress. Molecular and cellular biology. 2006;26:7520-8.
Yang MH, Chang SY, Chiou SH, Liu CJ, Chi CW, Chen PM, et al. Overexpression of NBS1 induces epithelial-mesenchymal transition and co-expression of NBS1 and Snail predicts metastasis of head and neck cancer. Oncogene. 2007;26:1459-67. Yang MH, Chiang WC, Chou TY, Chang SY, Chen PM, Teng SC, et al. Increased NBS1 expression is a marker of aggressive head and neck cancer and overexpression of NBS1 contributes to transformation. Clinical cancer research : an official journal of the American Association for Cancer Research. 2006;12:507-15.
Yang MH, Wu KJ. TWIST activation by hypoxia inducible factor-1 (HIF-1): implications in metastasis and development. Cell cycle. 2008;7:2090-6.
Yang MH, Wu MZ, Chiou SH, Chen PM, Chang SY, Liu CJ, et al. Direct regulation of TWIST by HIF-1alpha promotes metastasis. Nature cell biology. 2008;10:295-305. Zhai GG, Malhotra R, Delaney M, Latham D, Nestler U, Zhang M, et al. Radiation enhances the invasive potential of primary glioblastoma cells via activation of the Rho signaling pathway. Journal of neuro-oncology. 2006;76:227-37.
Zhang P, Wei Y, Wang L, Debeb BG, Yuan Y, Zhang J, et al. ATM-mediated stabilization of ZEB1 promotes DNA damage response and radioresistance through CHK1. Nature cell biology. 2014;16:864-75.
Zhong H, De Marzo AM, Laughner E, Lim M, Hilton DA, Zagzag D, et al. Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases. Cancer research. 1999;59:5830-5.
Zhou Z, Liu F, Zhang ZS, Shu F, Zheng Y, Fu L, et al. Human rhomboid family-1 suppresses oxygen-independent degradation of hypoxia-inducible factor-1alpha in breast cancer. Cancer research. 2014;74:2719-30.
Figure Legends
Fig. 1. IR increases NBS1 and HIF-1α expression and enhances the in vitro migration/invasion activity and NBS1 regulates HIF-1α expression and is critical for IR-induced HIF-1α expression.
(A) IR of two different cell lines (OECM1 and MCF7) increased the levels of HIF-1, NBS1 and DNA double-strand break markers (ATM pS1981, CHK2 pT68 and γH2AX) under normoxic or hypoxic culture. -actin was used as a loading control. (B) The percentage change of migration and invasion activity of OECM1 and MCF-7 cells after exposure to IR. Data from three independent experiments are expressed as mean ± s.d. (C) HIF-1 levels were increased when co-expression of NBS1 or treatment with NCS under normoxic or hypoxic condition. (D) HIF-1 protein levels were increased in NBS1 overexpression cells (left panel). Overexpression of NBS1 did not change the endogenous HIF-1 mRNA levels, but increased the endogenous Twist1 mRNA levels (n=3, right panel). (E) HIF-1protein levels were increased by NBS1 to further activate Twist1 promoter driven reporter construct in transient transfection assays. β-galactosidase normalized Twist1 promoter-driven luciferase activity was analyzed. Data from three independent experiments are expressed as mean ± s.d. (F) Knockdown of NBS1 inhibited IR-induced HIF-1α expression in H1299 and MDA-MB-231 cells under normoxic or hypoxic condition. (G) The percentage change of migration and invasion activity of NBS1 knockdown clones (MDA-MB-231-NBS1i and H1299-NBS1i) and control clones (MDA-MB-231 control and H1299 control) after exposure to IR. Data from three independent experiments are expressed as mean ± s.d. The asterisk (*) indicated statistical significance (P < 0 .05) between experimental and control clones.
Fig. 2. Knockdown of NBS1 abolishes IR-induced epithelial-mesenchymal transition.
(A, B) Western blot analysis of epithelial markers (E-cadherin and plakoglobin) and
mesenchymal markers (vimentin and N-cadherin) in OECM1 and MCF7 cells after exposure to IR. (C, D) Western blot analysis of epithelial and mesenchymal markers in MDA-MB-231-NBS1-si (upper panel) or H1299-NBS1-si (lower panel) cells and control clones (MDA-MB-231-control, as well as H1299-control) following exposure to IR.
Fig. 3. Interaction between NBS1 and HIF-1α.
(A) Co-immunoprecipitation assays using anti-HIF-1 antibodies pulled down NBS1 in H1299 and MDA-MB-231 cells under IR treatment or not. IgG was used as a control antibody. (B) Upper panel: schematic diagram of NBS1 and NBS1 deletion mutants. Co-immunoprecipitation assays showed the interaction of the internal domain (a.a. 221-402) of NBS1 with HIF-1. (C) Upper panel: schematic diagram of HIF-1α and HIF-1α deletion mutants. Co-immunoprecipitation assays showed the interaction of the N-terminal domain (a.a. 1-400) of HIF-1 with NBS1. (D) GST pull down assays showed that the direct interaction between the N-terminal domain of HIF-1 (a.a. 1-400) and the internal domain (a.a. 221-402) of NBS1.
Fig. 4. NBS1 decreases K48-linked ubiquitination of HIF-1α, increases the half-life of HIF-1,and competes with RACK1 for binding to HIF-1
(A) The ubiquitination levels of HIF-1 were decreased by co-expression of NBS1. MG132 was a proteasome inhibitor to inhibit HIF-1 degradation. (B) Co-immunoprecipitation assays showed that NBS1 decreased the ubiquitination levels of
1C) Co-expression of NBS1 decreased the K48-linked ubiquitination of HIF-1D) Co-expression of NBS1 increased the half-life of HIF-1Cycloheximide was used to inhibit new protein synthesis. (E) Co-transfection of Flag-NBS1 with HA-HIF-1 expression vectors into 293T cells followed by anti-HA antibodies showed that increasing amount of NBS1 decreased the binding of HIF-1 to endogenous RACK1.
.
Fig. 5. Co-expression of HIF-1α and NBS1 correlates with a worse outcome of lung adenocarcinoma cases.
(A) IHC analysis of co-expression of HIF-1α and NBS1 in tumor samples. The
samples prepared for co-expression analysis were cut and examined at the same region. Scale bars, 200 m. (B) Correlation of the IHC Expression of HIF-1α and NBS1 in 100 patients of resected stage I lung adenocarcinoma. (C) Survival difference in lung adenocarcinoma cases with or without HIF-1α/NBS1 co-expression. P value of the comparison between these two groups was shown in the inset.
