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O-linked-N-acetylglucosamine transferase is associated with metastatic spread of human papillomavirus E6 and E7 oncoproteins to the lungs of mice
Biochemical and Biophysical Research Communications 483 (2017) 793e802
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O-linked-N-acetylglucosamine transferase is associated with metastatic spread of human papillomavirus E6 and E7 oncoproteins to the lungs of mice Sung Hwan Kim a, 1, Yoon Sook Kim b, 1, Mee Young Choi b, Minjun Kim b, Jun Ho Yang a, Hyun Oh Park a, In Seok Jang a, Sung Ho Moon a, Hyun Ok Kim c, Dae Hyun Song d, Dong Hoon Lee b, Gu Seob Roh b, Hyun Joon Kim b, Sang Soo Kang b, Gyeong Jae Cho b, Jun Young Choi a, 2, Wan Sung Choi b, *, 2 a
Department of Thoracic and Cardiovascular Surgery, Gyeongsang National University School of Medicine, Jinju, Gyeongnam, Republic of Korea Department of Anatomy and Convergence Medical Science, Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju, Gyeongnam, Republic of Korea c Department of Internal Medicine, Gyeongsang National University School of Medicine, Jinju, Gyeongnam, Republic of Korea d Department of Pathology, Gyeongsang National University School of Medicine, Jinju, Gyeongnam, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 October 2016 Accepted 26 October 2016 Available online 12 November 2016
High-risk human papilloma virus (HPV) 16/18 infections are often found in lung cancer. The cellular mechanisms involved in the metastatic spread of HPV-infected cervical cancer cells remain largely elusive. High O-linked-N-acetylglucosamine (O-GlcNAc) modification has also been observed in lung cancer. In the present study, we assessed the relationship between O-GlcNAc transferase (OGT) and HPV 16/18 E6/E7, or C-X-C chemokine receptor type 4 (CXCR4), in HeLa cells and in lungs of xenografted mice. Depleting OGT with an OGT-specific shRNA significantly decreased levels of E6 and E7 oncoproteins in HeLa cells and xenograft tumors, and reduced tumor formation in vivo. Western blotting and immunofluorescence analysis showed significantly decreased expression levels of E6, E7, and HCF-1 in the lungs of xenografted mice treated with an OGT-specific shRNA compared to those treated with nontargeting shRNA. Additionally, levels of E7 or OGT co-localized with Ki-67 were significantly decreased in the lungs of xenografted mice treated with OGT-specific shRNA compared to those treated with nontargeting shRNA. Moreover, levels of CXCR4 were significantly decreased in HeLa cells and in the lungs of xenografted mice treated with OGT-specific shRNA compared to those treated with non-targeting shRNA; this may be related to reduced adhesion or invasion of circulating HPV-positive tumor cells. These findings provide novel evidence that OGT functions in metastatic spread of HPV E6/E7-positive tumor cells to the lungs through E6/E7, HCF-1 and CXCR4, suggesting OGT might be a therapeutic target for HPV-positive lung cancer. © 2017 Elsevier Inc. All rights reserved.
Keywords: CXCR4 E6 E7 HPV-positive lung cancer Metastasis OGT
1. Introduction Viral infections affect the pathogenesis of lung cancer [1e3]. Human papillomavirus (HPV) oncogenic subtypes 16 and 18
* Corresponding author. Department of Anatomy and Convergence Medical Science, Institute of Health Sciences, Gyeongsang National University School of Medicine, 15, 816 Beongil, Jinju-daero, Jinju, Gyeongnam, 52727, Republic of Korea. E-mail address: [email protected] (W.S. Choi). 1 These authors contributed equally to this work. 2 J.Y.C. and W.S.C. contributed equally to this work. http://dx.doi.org/10.1016/j.bbrc.2016.10.156 0006-291X/© 2017 Elsevier Inc. All rights reserved.
involved in cervical cancer may be linked to the pathogenesis of lung cancer; HPV 16/18 infection is strongly associated with lung cancer development in non-smoking female lung cancer patients, but not in male lung cancer patients [3,4]. Meanwhile, the relationship between HPV 16/18 infection and lung cancer remains largely elusive. High O-linked-N-acetylglucosamine (O-GlcNAc) modification has several roles in tumor formation, maintenance, dissemination, and metastasis in many cancers, including lung cancer [5e7]. Previous studies showed that O-GlcNAc transferase (OGT), which mediates O-GlcNAc modification, promotes cancer, including
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Fig. 1. OGT depletion decreases levels of E6 and E7 in HeLa cervical cancer cells and in tumors of xenografted mice. (A) Representative Western blot and quantification of E6 and E7 in HeLa cells treated with non-targeting (shCTL) or OGT-specific shRNA (shOGT). Band intensity was normalized to b-actin. Data are presented as means ± SEM, **P < 0.005 and ***P < 0.001 by a Student's t-test. (B) Representative tumors in nude mice 10 weeks after HeLa cell injection. Scale bar, 1 cm. (C) Mean tumor volume (mm3) in nude mice injected with HeLa cells with the indicated treatment is shown at the indicated time. Data are presented as means ± SEM, *P < 0.05 and **P < 0.01. (D) Mean tumor weight in nude mice injected with HeLa cells with the indicated treatment is shown 10 weeks after HeLa cell injection. Data are presented as means ± SEM, **P < 0.005 by a Student's t-test. (E) Representative Western blot and quantification of E6 and E7 in tumors of xenografted mice with non-targeting (shCTL) or OGT-specific shRNA (shOGT). Band intensity was normalized to b-actin. Data are presented as means ± SEM, *P < 0.05 and **P < 0.005 by Student's t-test.
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Fig. 1. (continued).
cervical cancer [8]. Host-cell factor (HCF)-1 is a transcriptional co-factor involved in oncogenic viral reactivation processes at the latent stage [9], and stimulates tumor formation [10]. OGT forms a complex with HCF-1, and this interaction is essential for HCF-1 cleavage and its activation [11,12]. Stromal-derived factor-1 (SDF-1a or CXCL12) and its receptor, CXCR4, have been suggested to promote cancer metastasis [13]. CXCR4 is a G-protein coupled receptor that plays critical roles in determining organ-selective metastasis, altering chemo-attraction, adhesion, and tumor cell survival. The interaction between SDF-1a and CXCR4 elicits a direct chemo-attractive action on breast cancer cells [14,15] and its pro-metastatic action broadly applies to metastatic cancer, including cervical carcinoma. In the present study, we assessed the role of OGT in HPV E6/E7 metastatic spread to the lungs and tumor growth in vivo using tumor xenograft models.
2.3. Tumor xenografts Xenograft models were made as described [16]. All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023). 2.4. Tissue collection and sample preparation
2. Materials and methods
Subcutaneous tumors and lung tissues from nude mice were obtained 10 weeks after injection of HeLa cells treated with nontargeting (shCTL) or OGT-specific shRNA. For tissue analysis (n ¼ 10 per group), mice were perfused transcardially with heparinized saline, and then with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). After 6 h post-fixation in the same fixative, the lungs were immersed sequentially in 0.1 M PBS containing 15% sucrose, and then in PBS containing 30% sucrose at 4 C until they sank. Lung tissues were cut into 40-mm coronal sections.
2.1. Cell treatment
2.5. Western blot analysis
The human cervical cancer cell line, HeLa (HPV18þ; American Type Culture Collection, Manassas, VA, USA), was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 100 mg/mL streptomycin, and 100 units/mL penicillin (Invitrogen).
For protein extraction, frozen lungs of nude mice (n ¼ 10 per group) were transferred to sterile 1.5-ml microcentrifuge tubes containing 200 ml lysis buffer (15 mM HEPES, pH 7.9, 0.25 M sucrose, 60 mM KCl, 10 mM NaCl, 1 mM EGTA, 1 mM PMSF, and 2 mM NaF). Homogenized tissues were incubated for 10 min on ice and then sonicated. Samples were centrifuged for 30 min at 2000g at 4 C, and the supernatants were transferred to clean vials. Protein concentrations were quantified using a BioRad protein assay (BioRad, Hercules, CA, USA) and samples were stored at 80 C until used. Cell lysates (30 mg) were separated by 8 or 10% sodium
2.2. Lentiviral shRNA production and infection Lentiviral shRNA production and infection were performed as described [16].
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Fig. 2. (continued).
dodecyl sulfate-polyacrylamide gel electrophoresis followed by electrophoretic transfer onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were probed with antibodies against OGT, HCF-1, E6, E7 and CXCR4 and visualized using an enhanced chemiluminescent substrate (Pierce, Rockford, IL, USA). 2.6. Antibodies Antibodies were as follows: anti-OGT (sc-74546), anti-HPV16 E6/18 E6 (sc-460), anti-HPV16 E7 (sc-365035), anti-HPV18 E7 (sc365035), anti-CXCR4 (sc-9046), from Santa Cruz Biotechnology; anti-HCF1 (A301-399A), from Bethyl Laboratories; anti-OGT (ab96718), from Abcam; and anti-Ki-67 (M7240), from DAKO. 2.7. Immunofluorescence analysis Sections of lung tissues from nude mice were incubated in blocking solution (2% normal donkey serum, 0.5% Triton X-100, 0.05% sodium azide in 0.05 M PBS, pH 7.4) for 1 h, followed by 4 C overnight incubation with primary antibodies of interest (anti-E6, -E7, -Ki-67, -OGT, and -HCF-1). After several washes in 0.1 M PBS, fluorescently labeled secondary antibodies [goat anti-mouse IgG conjugated to Alexa Fluor 488 (Thermo, A-11029), or donkey antirabbit or -mouse IgG conjugated to Alexa Fluor 594 (Thermo, A21207 or A-21203, respectively)] were applied, and sections were mounted with Mounting Medium (Invitrogen, CA). For double immunofluorescence analyses, sectioned tissues were incubated in blocking solution for 1 h, followed by 4 C overnight incubation with mixed primary antibodies. Digital images were taken by fluorescence microscopy (BX51-DSU; Olympus, Tokyo).
2.8. Statistical analysis Data are presented as the mean ± standard error of the mean (SEM). A Student's t-test was used to evaluate statistical significance. A value of P < 0.05 was considered statistically significant.
3. Results 3.1. OGT depletion decreases E6 and E7 in HeLa human cervical cancer cells and reduces tumor growth in vivo Because E6 and E7 are involved in a critical step in tumorigenesis and metastasis [17,18], and OGT plays a key role in cancer proliferation and survival [8], we tested whether OGT affects levels of E6 and E7 in HeLa cells and tumor xenografts, and alters tumor growth in vivo. Western blot analysis showed that levels of E6 and E7 protein expression were significantly decreased in HeLa cells treated with an OGT-specific shRNA compared to those treated with non-targeting shRNA (Fig. 1A, P < 0.001 or P < 0.0005, respectively). Further, to test whether OGT is required for tumor growth in vivo, HeLa cells stably expressing either control or OGT shRNAs were injected subcutaneously into the back of nude (Nu/Nu) mice. After the tumors became palpable, the tumor volume was measured with a caliper. Depleting OGT with OGT-specific shRNA in HeLa cells greatly decreased tumor growth in vivo (Fig. 1B), tumor volume (Fig. 1C, P < 0.05 or P < 0.01), and tumor weight (Fig. 1D, P < 0.005). Additionally, western blot analysis showed that levels of E6 and E7 were significantly decreased in tumor of xenografted mice with OGT-specific shRNA compared to controls (Fig. 1E, P < 0.05). Therefore, these results suggest that OGT increases E6 and E7 in HeLa cells and in vivo, and promotes tumor growth in vivo.
Fig. 2. OGT depletion decreases levels of E6 and E7 in the lungs of xenografted mice. (A) Representative Western blot and quantification of E6 and E7 in the lungs of xenografted mice treated with non-targeting (shCTL) or OGT-specific shRNA (shOGT). Band intensity was normalized to b-actin. Data are presented as means ± SEM, *P < 0.05. (B) Representative images of immunofluorescence staining for E6/E7 and Ki-67 in the lungs of xenografted mice treated with non-targeting (shCTL) or OGT-specific shRNA (shOGT). (C) Representative images of double immunofluorescence staining for Ki-67 and E7 in the lungs of xenografted mice with non-targeting (shCTL) or OGT-specific shRNA (shOGT).
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3.2. OGT depletion decreases levels of E6/E7 and Ki-67 expression in the lungs of tumor xenografts To further study the impact of OGT on E6 and E7 in the lungs of
tumor xenografts, we measured levels of E6 and E7 in the lungs of xenografted mice with non-targeting or OGT-specific shRNA. Western blot analysis showed that E6 and E7 protein expression was significantly decreased in the lungs of xenografted mice with
Fig. 3. OGT depletion decreases levels of HCF-1 in the lungs of xenografted mice. (A) Representative Western blot and quantification of HCF-1 in the lungs of xenografted mice with non-targeting (shCTL) or OGT-specific shRNA (shOGT). Band intensity was normalized to b-actin. Data are presented as means ± SEM, **P < 0.005 and ***P < 0.0005 by Student's ttest. (B) Representative images of immunofluorescence staining for HCF-1 in the lungs of xenografted mice with non-targeting (shCTL) or OGT-specific shRNA (shOGT). Representative images of double immunofluorescence staining for OGT and HCF-1(C), or Ki-67 and OGT (D) in the lungs of xenografted mice with non-targeting (shCTL) or OGT-specific shRNA (shOGT).
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Fig. 3. (continued).
OGT-specific shRNA compared to those with non-targeting shRNA (Fig. 2A, P < 0.05). Consistently, immunofluorescence analysis showed decreased levels of E6 and E7 in the lungs of xenografted mice with OGT-specific shRNA compared to those treated with non-targeting shRNA, paralleling decreased levels of Ki-67, a cellular marker for proliferation [19] (Fig. 2B). Moreover, we found that E7 co-localized with Ki-67 in the lungs of tumor xenografts with non-targeting shRNA, but this co-localization mostly disappeared in the lungs of tumor xenografts with OGT-specific shRNA (Fig. 2C).
3.3. OGT depletion decreases levels of HCF-1 in the lungs of tumor xenograft Since HCF-1 is stabilized by O-GlcNAc modification [11], and a transcriptional co-factor involved in oncogenic viral reactivation processes [9], we assessed the effects of OGT depletion on HCF-1 in the lungs of xenograft mice. We found that levels of HCF-1 were significantly decreased when OGT is depleted with OGT-specific shRNA (Fig. 3A, P < 0.005), supporting a role for OGT in stabilizing the HCF-1 protein. Consistently, immunofluorescence staining for HCF-1 showed that levels of HCF-1 were markedly decreased in
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the lungs of xenografted mice with OGT-specific shRNA compared to non-targeting shRNA controls (Fig. 3B). Double immunofluorescence analysis revealed that OGT and HCF-1 co-localized in the lungs of xenografted mice with non-targeting shRNA, but this colocalization mostly disappeared in the lungs of xenografted mice with OGT-specific shRNA (Fig. 3C). These results suggest that OGT increases HCF-1, which might be associated with cell proliferation in the HPV-positive lungs of xenograft mice. Indeed, we found that OGT co-localized with Ki-67 in the lungs of xenografted mice with non-targeting shRNA, but this co-localization mostly disappeared in the lungs of xenografted mice with OGT-specific shRNA (Fig. 3D), suggesting that OGT may contribute to proliferation in the lungs of mice bearing tumor xenografts. 3.4. OGT depletion decreases levels of CXCR4 in HeLa cells and the lungs of xenograft mice Because CXCR4 elicits a direct chemo-attractive action [14,15] and its pro-metastatic action applies to several metastatic cancers, including lung cancer [20], we examined whether it is affected by OGT in HeLa cells and in the lungs of xenograft mice. Notably, depleting OGT with OGT-specific shRNA significantly decreased levels of CXCR4 in HeLa cells compared to control cells with nontargeting shRNA (Fig. 4A, P < 0.005). More importantly, we observed significantly decreased levels of CXCR4 in the lungs of xenograft mice with OGT-specific shRNA compared to nontargeting shRNA (Fig. 4B, P < 0.001), suggesting that OGT could promote metastatic spread of E6/E7-infected cervical cancer cells through CXCR4 in the lungs of xenografted mice. 4. Discussion O-GlcNAcylation and OGT promote cancer, including cervical cancer [7,21,22], but whether OGT affects metastatic spread of E6/ E7 in HPV-positive lung cancer remains largely unknown. Here, we provide novel evidence that OGT increases HPV E6/E7, HCF-1, and CXCR4 in HeLa cells and in the lungs of xenografted mice, promoting metastatic spread of E6/E7-infected cervical cancer cells to the lungs, and furthering tumor growth in xenografted mice. Since E6 and E7 are involved in a critical step in tumorigenesis and metastasis [17,18], inhibition of metastatic spread of E6/E7positivie tumor cells to the lungs by reducing the metastatic cell population would be a promising strategy for treating HPV-positive lung cancer. Targeting HPV E6/E7 with siRNA induces apoptotic cell death in HPV-positive cell lines [18,23]. However, siRNAs only block HPV E6/E7 mRNAs, and they do not attack the HPV DNA in the nuclei, which is a store of escape mutants that cause resistance to siRNA. Importantly, an OGT inhibitor could reduce the potential metastatic spread of E6/E7-infected cervical cancer cells to the lungs by reducing the metastatic cell population; therefore instead of targeting RNA, designing OGT inhibitors, such as OSMI-1 [24,25] or ST045849 [26], could be a potential strategy for preventing HPVpositive lung cancer development. A recent study showed that OGT promotes gastric cancer proliferation and survival [8]; in this study, we found that OGT stimulates the metastatic spread of E6/E7-infected cervical cancer cells to the lungs, perhaps through E6/E7, HCF-1, and CXCR4. OGT depletion decreased levels of E6/E7, HCF-1, and CXCR4 in the lungs of xenografted mice, which promotes cancer [27]. Moreover, Ki-67, a nuclear protein and a proliferation marker in basal cells in cervical cancer [19,28], co-localized less often with OGT in the lungs of xenografted mice with OGT-specific shRNA than those treated with non-targeting shRNA, suggesting that OGT could contribute to tumor growth in the lungs of xenografted mice. HCF-1 is both a transcriptional co-factor and the cell-
Fig. 4. OGT depletion decreases levels of CXCR4 in HeLa cells and in the lungs of xenografted mice. (A) Representative Western blot and quantification of CXCR4 in HeLa cells treated with non-targeting (shCTL) or OGT-specific shRNA (shOGT). Band intensity was normalized to b-actin. Data are presented as means ± SEM, **P < 0.005 and ***P < 0.001 by Student's t-test. (B) Representative Western blot and quantification of CXCR4 in the lungs of xenografted mice with non-targeting (shCTL) or OGT-specific shRNA (shOGT). Band intensity was normalized to b-actin. Data are presented as means ± SEM, ***P < 0.001 by Student's t-test.
proliferation factor [10], and OGT catalyzes site-specific proteolysis of HCF-1; cleaved HCF-1 enhances the expression of viral oncogenic proteins [12]. Importantly, we observed that levels of HCF-1 were significantly decreased in the lungs of xenografted mice with OGT-specific shRNA compared to those treated with nontargeting shRNA, suggesting that OGT may both activate and increase HCF-1 in the lungs of xenografted mice. Therefore, increased HCF-1 by OGT may elevate levels of HPV 16/18 E6 and E7 expression and promote tumor growth in the lungs, similar to a previous report that demonstrated that HPV E6 and E7 promote metastatic conversion of cells [29]. Moreover, recent studies demonstrated
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that the E7 oncogene was a causative agent in early metastatic conversion [30], a direct modulator of invasiveness [31], and that E6 and E7 expression could also increase tumor cell capability to home to distant metastatic sites in conjunction with SDF-1a/CXCR4 [32]. Notably, many studies reported that CXCR4 could be a very important factor in cancer metastasis [33e36]: pulmonary adhesion and invasion leading to subsequent proliferation of HPVpositive cells into the lungs are directly mediated by the CXCR4 pathway in vivo [32], and E6/E7 gene silencing or pharmacological inhibition decreased CXCR4 expression [32]. Therefore, our results demonstrating that levels of CXCR4 were significantly decreased in the lungs of xenografted mice with OGT-specific shRNA suggest that OGT may reduce CXCR4 through lowering E6/E7. Or, rather, lung metastasis of cervical cancer may be promoted by OGT through CXCR4. However, it may be that the lungs release high levels of chemokines, such as CXCL12, that recognize CXCR4 [20] of metastatic E6-and E7-positive cells, and therefore, lung metastasis of cervical cancer may be related to chemokine expression in the lungs. In conclusion, we provide new evidence that OGT promotes progression of cervical cancer at least through E6/E7, HCF-1 and CXCR4, suggesting OGT may be a therapeutic target for HPVpositive lung cancer.
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Conflict of interest [22]
The authors declare no conflict of interest. Acknowledgments
[23]
This study was supported by the Basic Science Research Program of the National Research Foundation (NRF) of Korea Grant 20050049415 and NRF-2015R1A5A2008833 funded by the Ministry of Science, ICT and Future Planning.
[24]
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O-linked-N-acetylglucosamine transferase is associated with metastatic spread of human papillomavirus E6 and E7 oncoproteins to the lungs of mice Sung Hwan Kim a, 1, Yoon Sook Kim b, 1, Mee Young Choi b, Minjun Kim b, Jun Ho Yang a, Hyun Oh Park a, In Seok Jang a, Sung Ho Moon a, Hyun Ok Kim c, Dae Hyun Song d, Dong Hoon Lee b, Gu Seob Roh b, Hyun Joon Kim b, Sang Soo Kang b, Gyeong Jae Cho b, Jun Young Choi a, 2, Wan Sung Choi b, *, 2 a
Department of Thoracic and Cardiovascular Surgery, Gyeongsang National University School of Medicine, Jinju, Gyeongnam, Republic of Korea Department of Anatomy and Convergence Medical Science, Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju, Gyeongnam, Republic of Korea c Department of Internal Medicine, Gyeongsang National University School of Medicine, Jinju, Gyeongnam, Republic of Korea d Department of Pathology, Gyeongsang National University School of Medicine, Jinju, Gyeongnam, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 October 2016 Accepted 26 October 2016 Available online 12 November 2016
High-risk human papilloma virus (HPV) 16/18 infections are often found in lung cancer. The cellular mechanisms involved in the metastatic spread of HPV-infected cervical cancer cells remain largely elusive. High O-linked-N-acetylglucosamine (O-GlcNAc) modification has also been observed in lung cancer. In the present study, we assessed the relationship between O-GlcNAc transferase (OGT) and HPV 16/18 E6/E7, or C-X-C chemokine receptor type 4 (CXCR4), in HeLa cells and in lungs of xenografted mice. Depleting OGT with an OGT-specific shRNA significantly decreased levels of E6 and E7 oncoproteins in HeLa cells and xenograft tumors, and reduced tumor formation in vivo. Western blotting and immunofluorescence analysis showed significantly decreased expression levels of E6, E7, and HCF-1 in the lungs of xenografted mice treated with an OGT-specific shRNA compared to those treated with nontargeting shRNA. Additionally, levels of E7 or OGT co-localized with Ki-67 were significantly decreased in the lungs of xenografted mice treated with OGT-specific shRNA compared to those treated with nontargeting shRNA. Moreover, levels of CXCR4 were significantly decreased in HeLa cells and in the lungs of xenografted mice treated with OGT-specific shRNA compared to those treated with non-targeting shRNA; this may be related to reduced adhesion or invasion of circulating HPV-positive tumor cells. These findings provide novel evidence that OGT functions in metastatic spread of HPV E6/E7-positive tumor cells to the lungs through E6/E7, HCF-1 and CXCR4, suggesting OGT might be a therapeutic target for HPV-positive lung cancer. © 2017 Elsevier Inc. All rights reserved.
Keywords: CXCR4 E6 E7 HPV-positive lung cancer Metastasis OGT
1. Introduction Viral infections affect the pathogenesis of lung cancer [1e3]. Human papillomavirus (HPV) oncogenic subtypes 16 and 18
* Corresponding author. Department of Anatomy and Convergence Medical Science, Institute of Health Sciences, Gyeongsang National University School of Medicine, 15, 816 Beongil, Jinju-daero, Jinju, Gyeongnam, 52727, Republic of Korea. E-mail address: [email protected] (W.S. Choi). 1 These authors contributed equally to this work. 2 J.Y.C. and W.S.C. contributed equally to this work. http://dx.doi.org/10.1016/j.bbrc.2016.10.156 0006-291X/© 2017 Elsevier Inc. All rights reserved.
involved in cervical cancer may be linked to the pathogenesis of lung cancer; HPV 16/18 infection is strongly associated with lung cancer development in non-smoking female lung cancer patients, but not in male lung cancer patients [3,4]. Meanwhile, the relationship between HPV 16/18 infection and lung cancer remains largely elusive. High O-linked-N-acetylglucosamine (O-GlcNAc) modification has several roles in tumor formation, maintenance, dissemination, and metastasis in many cancers, including lung cancer [5e7]. Previous studies showed that O-GlcNAc transferase (OGT), which mediates O-GlcNAc modification, promotes cancer, including
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Fig. 1. OGT depletion decreases levels of E6 and E7 in HeLa cervical cancer cells and in tumors of xenografted mice. (A) Representative Western blot and quantification of E6 and E7 in HeLa cells treated with non-targeting (shCTL) or OGT-specific shRNA (shOGT). Band intensity was normalized to b-actin. Data are presented as means ± SEM, **P < 0.005 and ***P < 0.001 by a Student's t-test. (B) Representative tumors in nude mice 10 weeks after HeLa cell injection. Scale bar, 1 cm. (C) Mean tumor volume (mm3) in nude mice injected with HeLa cells with the indicated treatment is shown at the indicated time. Data are presented as means ± SEM, *P < 0.05 and **P < 0.01. (D) Mean tumor weight in nude mice injected with HeLa cells with the indicated treatment is shown 10 weeks after HeLa cell injection. Data are presented as means ± SEM, **P < 0.005 by a Student's t-test. (E) Representative Western blot and quantification of E6 and E7 in tumors of xenografted mice with non-targeting (shCTL) or OGT-specific shRNA (shOGT). Band intensity was normalized to b-actin. Data are presented as means ± SEM, *P < 0.05 and **P < 0.005 by Student's t-test.
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Fig. 1. (continued).
cervical cancer [8]. Host-cell factor (HCF)-1 is a transcriptional co-factor involved in oncogenic viral reactivation processes at the latent stage [9], and stimulates tumor formation [10]. OGT forms a complex with HCF-1, and this interaction is essential for HCF-1 cleavage and its activation [11,12]. Stromal-derived factor-1 (SDF-1a or CXCL12) and its receptor, CXCR4, have been suggested to promote cancer metastasis [13]. CXCR4 is a G-protein coupled receptor that plays critical roles in determining organ-selective metastasis, altering chemo-attraction, adhesion, and tumor cell survival. The interaction between SDF-1a and CXCR4 elicits a direct chemo-attractive action on breast cancer cells [14,15] and its pro-metastatic action broadly applies to metastatic cancer, including cervical carcinoma. In the present study, we assessed the role of OGT in HPV E6/E7 metastatic spread to the lungs and tumor growth in vivo using tumor xenograft models.
2.3. Tumor xenografts Xenograft models were made as described [16]. All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023). 2.4. Tissue collection and sample preparation
2. Materials and methods
Subcutaneous tumors and lung tissues from nude mice were obtained 10 weeks after injection of HeLa cells treated with nontargeting (shCTL) or OGT-specific shRNA. For tissue analysis (n ¼ 10 per group), mice were perfused transcardially with heparinized saline, and then with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). After 6 h post-fixation in the same fixative, the lungs were immersed sequentially in 0.1 M PBS containing 15% sucrose, and then in PBS containing 30% sucrose at 4 C until they sank. Lung tissues were cut into 40-mm coronal sections.
2.1. Cell treatment
2.5. Western blot analysis
The human cervical cancer cell line, HeLa (HPV18þ; American Type Culture Collection, Manassas, VA, USA), was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 100 mg/mL streptomycin, and 100 units/mL penicillin (Invitrogen).
For protein extraction, frozen lungs of nude mice (n ¼ 10 per group) were transferred to sterile 1.5-ml microcentrifuge tubes containing 200 ml lysis buffer (15 mM HEPES, pH 7.9, 0.25 M sucrose, 60 mM KCl, 10 mM NaCl, 1 mM EGTA, 1 mM PMSF, and 2 mM NaF). Homogenized tissues were incubated for 10 min on ice and then sonicated. Samples were centrifuged for 30 min at 2000g at 4 C, and the supernatants were transferred to clean vials. Protein concentrations were quantified using a BioRad protein assay (BioRad, Hercules, CA, USA) and samples were stored at 80 C until used. Cell lysates (30 mg) were separated by 8 or 10% sodium
2.2. Lentiviral shRNA production and infection Lentiviral shRNA production and infection were performed as described [16].
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Fig. 2. (continued).
dodecyl sulfate-polyacrylamide gel electrophoresis followed by electrophoretic transfer onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were probed with antibodies against OGT, HCF-1, E6, E7 and CXCR4 and visualized using an enhanced chemiluminescent substrate (Pierce, Rockford, IL, USA). 2.6. Antibodies Antibodies were as follows: anti-OGT (sc-74546), anti-HPV16 E6/18 E6 (sc-460), anti-HPV16 E7 (sc-365035), anti-HPV18 E7 (sc365035), anti-CXCR4 (sc-9046), from Santa Cruz Biotechnology; anti-HCF1 (A301-399A), from Bethyl Laboratories; anti-OGT (ab96718), from Abcam; and anti-Ki-67 (M7240), from DAKO. 2.7. Immunofluorescence analysis Sections of lung tissues from nude mice were incubated in blocking solution (2% normal donkey serum, 0.5% Triton X-100, 0.05% sodium azide in 0.05 M PBS, pH 7.4) for 1 h, followed by 4 C overnight incubation with primary antibodies of interest (anti-E6, -E7, -Ki-67, -OGT, and -HCF-1). After several washes in 0.1 M PBS, fluorescently labeled secondary antibodies [goat anti-mouse IgG conjugated to Alexa Fluor 488 (Thermo, A-11029), or donkey antirabbit or -mouse IgG conjugated to Alexa Fluor 594 (Thermo, A21207 or A-21203, respectively)] were applied, and sections were mounted with Mounting Medium (Invitrogen, CA). For double immunofluorescence analyses, sectioned tissues were incubated in blocking solution for 1 h, followed by 4 C overnight incubation with mixed primary antibodies. Digital images were taken by fluorescence microscopy (BX51-DSU; Olympus, Tokyo).
2.8. Statistical analysis Data are presented as the mean ± standard error of the mean (SEM). A Student's t-test was used to evaluate statistical significance. A value of P < 0.05 was considered statistically significant.
3. Results 3.1. OGT depletion decreases E6 and E7 in HeLa human cervical cancer cells and reduces tumor growth in vivo Because E6 and E7 are involved in a critical step in tumorigenesis and metastasis [17,18], and OGT plays a key role in cancer proliferation and survival [8], we tested whether OGT affects levels of E6 and E7 in HeLa cells and tumor xenografts, and alters tumor growth in vivo. Western blot analysis showed that levels of E6 and E7 protein expression were significantly decreased in HeLa cells treated with an OGT-specific shRNA compared to those treated with non-targeting shRNA (Fig. 1A, P < 0.001 or P < 0.0005, respectively). Further, to test whether OGT is required for tumor growth in vivo, HeLa cells stably expressing either control or OGT shRNAs were injected subcutaneously into the back of nude (Nu/Nu) mice. After the tumors became palpable, the tumor volume was measured with a caliper. Depleting OGT with OGT-specific shRNA in HeLa cells greatly decreased tumor growth in vivo (Fig. 1B), tumor volume (Fig. 1C, P < 0.05 or P < 0.01), and tumor weight (Fig. 1D, P < 0.005). Additionally, western blot analysis showed that levels of E6 and E7 were significantly decreased in tumor of xenografted mice with OGT-specific shRNA compared to controls (Fig. 1E, P < 0.05). Therefore, these results suggest that OGT increases E6 and E7 in HeLa cells and in vivo, and promotes tumor growth in vivo.
Fig. 2. OGT depletion decreases levels of E6 and E7 in the lungs of xenografted mice. (A) Representative Western blot and quantification of E6 and E7 in the lungs of xenografted mice treated with non-targeting (shCTL) or OGT-specific shRNA (shOGT). Band intensity was normalized to b-actin. Data are presented as means ± SEM, *P < 0.05. (B) Representative images of immunofluorescence staining for E6/E7 and Ki-67 in the lungs of xenografted mice treated with non-targeting (shCTL) or OGT-specific shRNA (shOGT). (C) Representative images of double immunofluorescence staining for Ki-67 and E7 in the lungs of xenografted mice with non-targeting (shCTL) or OGT-specific shRNA (shOGT).
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3.2. OGT depletion decreases levels of E6/E7 and Ki-67 expression in the lungs of tumor xenografts To further study the impact of OGT on E6 and E7 in the lungs of
tumor xenografts, we measured levels of E6 and E7 in the lungs of xenografted mice with non-targeting or OGT-specific shRNA. Western blot analysis showed that E6 and E7 protein expression was significantly decreased in the lungs of xenografted mice with
Fig. 3. OGT depletion decreases levels of HCF-1 in the lungs of xenografted mice. (A) Representative Western blot and quantification of HCF-1 in the lungs of xenografted mice with non-targeting (shCTL) or OGT-specific shRNA (shOGT). Band intensity was normalized to b-actin. Data are presented as means ± SEM, **P < 0.005 and ***P < 0.0005 by Student's ttest. (B) Representative images of immunofluorescence staining for HCF-1 in the lungs of xenografted mice with non-targeting (shCTL) or OGT-specific shRNA (shOGT). Representative images of double immunofluorescence staining for OGT and HCF-1(C), or Ki-67 and OGT (D) in the lungs of xenografted mice with non-targeting (shCTL) or OGT-specific shRNA (shOGT).
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Fig. 3. (continued).
OGT-specific shRNA compared to those with non-targeting shRNA (Fig. 2A, P < 0.05). Consistently, immunofluorescence analysis showed decreased levels of E6 and E7 in the lungs of xenografted mice with OGT-specific shRNA compared to those treated with non-targeting shRNA, paralleling decreased levels of Ki-67, a cellular marker for proliferation [19] (Fig. 2B). Moreover, we found that E7 co-localized with Ki-67 in the lungs of tumor xenografts with non-targeting shRNA, but this co-localization mostly disappeared in the lungs of tumor xenografts with OGT-specific shRNA (Fig. 2C).
3.3. OGT depletion decreases levels of HCF-1 in the lungs of tumor xenograft Since HCF-1 is stabilized by O-GlcNAc modification [11], and a transcriptional co-factor involved in oncogenic viral reactivation processes [9], we assessed the effects of OGT depletion on HCF-1 in the lungs of xenograft mice. We found that levels of HCF-1 were significantly decreased when OGT is depleted with OGT-specific shRNA (Fig. 3A, P < 0.005), supporting a role for OGT in stabilizing the HCF-1 protein. Consistently, immunofluorescence staining for HCF-1 showed that levels of HCF-1 were markedly decreased in
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the lungs of xenografted mice with OGT-specific shRNA compared to non-targeting shRNA controls (Fig. 3B). Double immunofluorescence analysis revealed that OGT and HCF-1 co-localized in the lungs of xenografted mice with non-targeting shRNA, but this colocalization mostly disappeared in the lungs of xenografted mice with OGT-specific shRNA (Fig. 3C). These results suggest that OGT increases HCF-1, which might be associated with cell proliferation in the HPV-positive lungs of xenograft mice. Indeed, we found that OGT co-localized with Ki-67 in the lungs of xenografted mice with non-targeting shRNA, but this co-localization mostly disappeared in the lungs of xenografted mice with OGT-specific shRNA (Fig. 3D), suggesting that OGT may contribute to proliferation in the lungs of mice bearing tumor xenografts. 3.4. OGT depletion decreases levels of CXCR4 in HeLa cells and the lungs of xenograft mice Because CXCR4 elicits a direct chemo-attractive action [14,15] and its pro-metastatic action applies to several metastatic cancers, including lung cancer [20], we examined whether it is affected by OGT in HeLa cells and in the lungs of xenograft mice. Notably, depleting OGT with OGT-specific shRNA significantly decreased levels of CXCR4 in HeLa cells compared to control cells with nontargeting shRNA (Fig. 4A, P < 0.005). More importantly, we observed significantly decreased levels of CXCR4 in the lungs of xenograft mice with OGT-specific shRNA compared to nontargeting shRNA (Fig. 4B, P < 0.001), suggesting that OGT could promote metastatic spread of E6/E7-infected cervical cancer cells through CXCR4 in the lungs of xenografted mice. 4. Discussion O-GlcNAcylation and OGT promote cancer, including cervical cancer [7,21,22], but whether OGT affects metastatic spread of E6/ E7 in HPV-positive lung cancer remains largely unknown. Here, we provide novel evidence that OGT increases HPV E6/E7, HCF-1, and CXCR4 in HeLa cells and in the lungs of xenografted mice, promoting metastatic spread of E6/E7-infected cervical cancer cells to the lungs, and furthering tumor growth in xenografted mice. Since E6 and E7 are involved in a critical step in tumorigenesis and metastasis [17,18], inhibition of metastatic spread of E6/E7positivie tumor cells to the lungs by reducing the metastatic cell population would be a promising strategy for treating HPV-positive lung cancer. Targeting HPV E6/E7 with siRNA induces apoptotic cell death in HPV-positive cell lines [18,23]. However, siRNAs only block HPV E6/E7 mRNAs, and they do not attack the HPV DNA in the nuclei, which is a store of escape mutants that cause resistance to siRNA. Importantly, an OGT inhibitor could reduce the potential metastatic spread of E6/E7-infected cervical cancer cells to the lungs by reducing the metastatic cell population; therefore instead of targeting RNA, designing OGT inhibitors, such as OSMI-1 [24,25] or ST045849 [26], could be a potential strategy for preventing HPVpositive lung cancer development. A recent study showed that OGT promotes gastric cancer proliferation and survival [8]; in this study, we found that OGT stimulates the metastatic spread of E6/E7-infected cervical cancer cells to the lungs, perhaps through E6/E7, HCF-1, and CXCR4. OGT depletion decreased levels of E6/E7, HCF-1, and CXCR4 in the lungs of xenografted mice, which promotes cancer [27]. Moreover, Ki-67, a nuclear protein and a proliferation marker in basal cells in cervical cancer [19,28], co-localized less often with OGT in the lungs of xenografted mice with OGT-specific shRNA than those treated with non-targeting shRNA, suggesting that OGT could contribute to tumor growth in the lungs of xenografted mice. HCF-1 is both a transcriptional co-factor and the cell-
Fig. 4. OGT depletion decreases levels of CXCR4 in HeLa cells and in the lungs of xenografted mice. (A) Representative Western blot and quantification of CXCR4 in HeLa cells treated with non-targeting (shCTL) or OGT-specific shRNA (shOGT). Band intensity was normalized to b-actin. Data are presented as means ± SEM, **P < 0.005 and ***P < 0.001 by Student's t-test. (B) Representative Western blot and quantification of CXCR4 in the lungs of xenografted mice with non-targeting (shCTL) or OGT-specific shRNA (shOGT). Band intensity was normalized to b-actin. Data are presented as means ± SEM, ***P < 0.001 by Student's t-test.
proliferation factor [10], and OGT catalyzes site-specific proteolysis of HCF-1; cleaved HCF-1 enhances the expression of viral oncogenic proteins [12]. Importantly, we observed that levels of HCF-1 were significantly decreased in the lungs of xenografted mice with OGT-specific shRNA compared to those treated with nontargeting shRNA, suggesting that OGT may both activate and increase HCF-1 in the lungs of xenografted mice. Therefore, increased HCF-1 by OGT may elevate levels of HPV 16/18 E6 and E7 expression and promote tumor growth in the lungs, similar to a previous report that demonstrated that HPV E6 and E7 promote metastatic conversion of cells [29]. Moreover, recent studies demonstrated
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that the E7 oncogene was a causative agent in early metastatic conversion [30], a direct modulator of invasiveness [31], and that E6 and E7 expression could also increase tumor cell capability to home to distant metastatic sites in conjunction with SDF-1a/CXCR4 [32]. Notably, many studies reported that CXCR4 could be a very important factor in cancer metastasis [33e36]: pulmonary adhesion and invasion leading to subsequent proliferation of HPVpositive cells into the lungs are directly mediated by the CXCR4 pathway in vivo [32], and E6/E7 gene silencing or pharmacological inhibition decreased CXCR4 expression [32]. Therefore, our results demonstrating that levels of CXCR4 were significantly decreased in the lungs of xenografted mice with OGT-specific shRNA suggest that OGT may reduce CXCR4 through lowering E6/E7. Or, rather, lung metastasis of cervical cancer may be promoted by OGT through CXCR4. However, it may be that the lungs release high levels of chemokines, such as CXCL12, that recognize CXCR4 [20] of metastatic E6-and E7-positive cells, and therefore, lung metastasis of cervical cancer may be related to chemokine expression in the lungs. In conclusion, we provide new evidence that OGT promotes progression of cervical cancer at least through E6/E7, HCF-1 and CXCR4, suggesting OGT may be a therapeutic target for HPVpositive lung cancer.
[12]
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[16]
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[21]
Conflict of interest [22]
The authors declare no conflict of interest. Acknowledgments
[23]
This study was supported by the Basic Science Research Program of the National Research Foundation (NRF) of Korea Grant 20050049415 and NRF-2015R1A5A2008833 funded by the Ministry of Science, ICT and Future Planning.
[24]
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