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Increased expression of tripartite motif (TRIM) like 2 promotes tumoral growth in human oral cancer
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Increased expression of tripartite motif (TRIM) like 2 promotes tumoral growth in human oral cancer Fumihiko Hayashi a, Atsushi Kasamatsu b, *, Yosuke Endo-Sakamoto b, Keitaro Eizuka a, Kazuya Hiroshima a, Akihiro Kita b, Tomoaki Saito b, Kazuyuki Koike b, Hideki Tanzawa a, b, Katsuhiro Uzawa a, b, ** a b
Department of Oral Science, Graduate School of Medicine, Chiba University, Chiba, Japan Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, Chiba, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 November 2018 Accepted 7 December 2018 Available online xxx
Tripartite motif family-like 2 (TRIML2), a member of the TRIM proteins family, is closely related to Alzheimer's disease, however, no studies of TRIML2 have been published in the cancer research literature. In the current study, we investigated the expression level of TRIML2 and its molecular mechanisms in human oral squamous cell carcinoma (OSCC); reverse transcriptase-quantitative polymerase chain reaction, immunoblot analysis, and immunohistochemistry showed that TRIML2 is up-regulated significantly in OSCCs in vitro and in vivo. TRIML2 knockdown OSCC cells showed decreased cellular proliferation by cell-cycle arrest at G1 phase that resulted from down-regulation of CDK4, CDK6, and cyclin D1 and up-regulation of p21Cip1 and p27Kip1. Surprisingly, resveratrol, a polyphenol, led to not only down-regulation of TRIML2 but also cell-cycle arrest at G1 phase similar to TRIML2 knockdown experiments. Taken together, we concluded that TRIML2 might play a significant role in tumoral growth and that resveratrol may be a new drug for treating OSCC by interfering with TRIML2 function. © 2018 Published by Elsevier Inc.
Keywords: Cell-cycle arrest at G1 phase Cell proliferation Oral squamous cell carcinoma Resveratrol Tripartite motif family-like 2
1. Introduction The family of tripartite motif (TRIM) proteins in humans has 77 members [1], almost all of which have an N-terminal RING finger, B-boxes, and a coiled-coil domains. The TRIM family members are divided into 12 subfamilies (C-I to C-Ⅺ and an unclassified group) that were classified based on the differences in their C-terminal domains [2,3]. TRIM proteins play significant roles in a range of processes including tumoral suppression, DNA damage, stem cell differentiation, and immune reactivity against viral infections, particularly human immunodeficiency virus [4e8]. TRIM family-like 2 (TRIML2) belongs to an unclassified group because it lacks a RING-finger domain [1,3]. Polymorphisms in TRIML2 and their haplotypes are related significantly to Alzheimer's disease, suggesting that TRIML2 is a biomarker for the
* Corresponding author. Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan. ** Corresponding author. Department of Oral Science, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan.. E-mail addresses: [email protected] (A. Kasamatsu), uzawak@ faculty.chiba-u.jp (K. Uzawa).
diagnosis of Alzheimer's disease [9]. Despite increasing recognition of the significant cellular functions of TRIML2, the biochemical basis for these mechanisms is not well understood, and no reports in the cancer research literature have addressed this. In the current study, overexpressed TRIML2 was seen in human oral squamous cell carcinoma (OSCC) cells and primary OSCCs. We also reported that TRIML2 was correlated with cell-cycle arrest at G1 phase using TRIML2 knockdown models and its novel specific inhibitor, resveratrol. 2. Materials and methods 2.1. Ethics statement The ethics committee of Chiba University approved the study protocol (protocol number 680). All patients provided written informed consent, which was obtained before the experiments were conducted. 2.2. Cells and clinical OSCC tissue samples Ten OSCC cells were used for in vitro experiments [10e19]. We
https://doi.org/10.1016/j.bbrc.2018.12.060 0006-291X/© 2018 Published by Elsevier Inc.
Please cite this article as: F. Hayashi et al., Increased expression of tripartite motif (TRIM) like 2 promotes tumoral growth in human oral cancer, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.060
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Fig. 1. Evaluation of TRIML2 expression in OSCC cells and primary OSCCs. (A) RT-qPCR analysis shows significantly (*p < 0.05, Student's t-test) up-regulated TRIML2 mRNA expression in all OSCC-derived cell lines compared with the HNOKs. (B) Immunoblot analysis shows up-regulated TRIML2 protein expression in OSCC-derived cell lines compared to the HNOKs. Densitometric TRIML2 protein data are normalized to the GAPDH protein levels. The values are expressed as a percentage of the HNOKs. (C) Representative IHC results of TRIML2 in normal oral tissues and primary OSCCs. Original magnification, 400. Scale bars, 50 mm. (D) The status of TRIML2 protein expression in normal counterparts and primary OSCCs (n ¼ 100) using the IHC scoring system. The TRIML2 protein expression levels in OSCCs are significantly higher than in normal oral tissues (*p < 0.05, Student's t-test).
extracted human normal oral keratinocytes (HNOKs) from healthy young patients as control cells and cultured them as described previously [10,11]. OSCCs and normal oral tissue samples (n ¼ 100) were collected from patients with OSCC at Chiba University Hospital.
2.7. Cellular growth We analyzed the cellular growth in shTRIML2 and shMock cells to assess the effect of TRIML2 knockdown on cellular proliferation. The protocol was described previously [17,27,28].
2.3. Gene expression analysis We isolated total RNA from OSCC cells incubated subconfluently for generating cDNA and performed reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) as described previously [10,11,20]. The following primer sequences were used for RT-qPCR; TRIML2, 50 -CACATGGTGTGTGGGATACAA-30 and 50 eCCTCGATGTGTTCAATATTTCCT-30 ; and glyceraldehyde 3phosphate dehydrogenase (GAPDH), 50 -AGCCACATCGCTCAGACAC30 and 50 -GCCCAATACGACCAAATCC-30 .
2.8. Cell-cycle analysis We conducted cell-cycle analysis by flow cytometry using the BD Accuri™ C6 Flow Cytometer (Becton-Dickinson, San Diego, CA, USA). The protocol was described previously [14,23,29]. Table 1 Correlation between TRIML2 expression and clinical classification in OSCCs. Clinical classification
2.4. Immunoblot analysis
Results of immunostaining No. patients TRIML2 negative
We performed immunoblot analysis as described previously [17,21]. The antibodies were rabbit anti-TRIML2 polyclonal antibody (Abcam, Cambridge, UK), GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p21Cip1, p27Kip1, cyclin D1, CDK2, and CDK4 (Cell Signaling Technology Japan, Tokyo, Japan).
Age at surgery (years) < 60 26 12 S60 74 27 Gender Male 56 26 Female 44 18 T-primary tumoral size T1þT2 50 29 T3þT4 50 17 N-regional lymph node metastasis Negative 66 30 Positive 34 14 TNM Stage I þ II 32 18 III þ IV 68 24 Vascular invasion Negative 82 39 Positive 18 6 Histopathologic type Well 56 20 Moderately þ poorly 44 12
2.5. Immunohistochemistry (IHC) We performed IHC and calculated the IHC score described previously [17,21e25]. We evaluated the TRIML2 scores from 100 patients with OSCC and classified TRIML2 as positive and negative using the median score of all OSCCs as the cut-off points [26]. In this case, the IHC score over 117 was taken as TRIML2 positive. 2.6. Transfection with shRNA We established TRIML2 knockdown cells derived from the Sa3 and SAS cells using TRIML2 shRNA (shTRIML2) and control shRNA (shMock) vectors, respectively (Santa Cruz Biotechnology). The protocol was described previously [14].
Total
p value
TRIML2 positive 14 47
0.484*
30 26
0.686*
21 33
0.027*y
36 20
0.832*
14 44
0.054*
43 12
0.307*
36 32
0.396*
*
Fisher's exact test. yp < 0.05.
Please cite this article as: F. Hayashi et al., Increased expression of tripartite motif (TRIM) like 2 promotes tumoral growth in human oral cancer, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.060
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2.9. Resveratrol treatment To investigate a correlation between TRIML2 and resveratrol (Sigma-Aldrich, St. Louis, MO, USA), we treated Sa3 and SAS cells with resveratrol for 48 h. After treatment, we extracted total RNA, generated complementary DNA, and performed RT-qPCR. We also extracted protein for immunoblot analysis and evaluated whether resveratrol affected the cellular proliferation and cell-cycle by treating the Sa3 and SAS cells with resveratrol for 48 h. In the cellular proliferation assay, the cells were cultured with resveratrol, and dimethylsulfoxide served as the experimental control.
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was up-regulated significantly (p < 0.05) in all OSCC cells compared with the HNOKs (Fig. 1A and B). Fig. 1C shows representative IHC results of TRIML2 in normal and primary OSCC tissues. The IHC scores ranged from 34 to 132 (median, 64) in normal oral tissues and 69 to 187 (median, 117) in OSCC tissues (Fig. 1D) (p < 0.05). Table 1 shows the correlation between the TRIML2 expression status and clinical classification of the patients with OSCC. Among the clinical classifications, the TRIML2 expression level was correlated significantly (p < 0.05) with the primary tumoral size (Table 1). 3.2. Establishment of TRIML2 knockdown cells
2.10. Statistical analysis Student's t-test, Fisher's exact test, and Mann-Whitney U test were performed to identify significance correlations. All data are expressed as the mean ± the standard error of the mean. 3. Results 3.1. Evaluation of TRIML2 expression OSCC cells and primary OSCCs RT-qPCR and immunoblot data showed that TRIML2 expression
We chose Sa3 and SAS cells for knockdown experiments because higher TRIML2 expression levels were observed in those OSCC cell lines. RT-qPCR and immunoblot analysis showed that TRIML2 was down-regulated in the shTRIML2 cells compared with the shMock cells (Fig. 2A and B, respectively). No morphologic changes in the TRIML2 knockdown cells were seen during microscopy. 3.3. Cellular proliferation assay of TRIML2 knockdown cells To evaluate the effect of the TRIML2 knockdown on cellular
Fig. 2. Establishment of the TRIML2 knockdown cells and cell-cycle analysis of TRIML2 knockdown cells. (A) RT-qPCR shows that the TRIML2 mRNA expression in shTRIML2 cells is significantly (*p < 0.05, Student's t-test) lower than that in shMock cells. (B) Immunoblot analysis shows that the TRIML2 protein levels in the shTRIML2 cells are decreased compared with the shMock cells. (C) The cellular growth of the shTRIML2 cells is inhibited significantly compared with the shMock cells after 4 days (96 h) (*p < 0.05, Student's ttest). (D) Flow cytometric analysis shows that the percentage of cells at G1 phase in the shTRIML2 cells is increased markedly (*p < 0.05, Mann-Whitney U test) compared with the shMock cells. (E) Immunoblot analysis shows down-regulation of CDK2, CDK4, and cyclin D1 and up-regulation of p21Cip1 and p27Kip1 in the shTRIML2 cells compared with the shMock cells.
Please cite this article as: F. Hayashi et al., Increased expression of tripartite motif (TRIM) like 2 promotes tumoral growth in human oral cancer, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.060
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growth, we performed a cellular proliferation assay that showed significantly (p < 0.05) decrease cellular growth in the shTRIML2 cells compared with the shMock cells (Fig. 2C). 3.4. Cell-cycle analysis of TRIML2 knockdown cells The cell-cycle analysis showed that the percentage of the shTRIML2 cells at G1 phase increased significantly (p < 0.05) compared with the shMock cells (Fig. 2D). Evaluation of the protein expression levels of G1 phase-related proteins in the shTRIML2 cells showed that CDK2, CDK4, and cyclin D1 were down-regulated, and p21Cip1 and p27Kip1 were up-regulated in the shTRIML2 cells (Fig. 2E). These results indicated that the shTRIML2 cells suppressed cellular proliferation by cell-cycle arrest at G1 phase. 3.5. Effect of treatment with resveratrol
Fig. 3. Effect of treatment with resveratrol. (A) RT-qPCR shows that the TRIML2 mRNA expression is significantly (*p < 0.05, Student's t-test) down-regulated after treatment with resveratrol in both the Sa3 and SAS cells compared with controls. (B) Immunoblot analysis shows down-regulated TRIML2 expression after treatment with resveratrol compared with controls.
After resveratrol treatment (50 mM, 48 h), the TRIML2 expression levels decreased in both Sa3 and SAS cell lines compared with the control (Fig. 3A and B). To assess the effect of resveratrol on cellular growth, we performed cellular proliferation assay that showed that the cellular growth of the resveratrol-treated cells was significantly (p < 0.05) lower than that of the control cells (Fig. 4A). The cell-cycle analysis, showed that the percentage of the resveratrol-treated cells in G1 phase was significantly (p < 0.05) higher than that of the control cells (Fig. 4B). Evaluation of the protein levels of CDK2, CDK4, cyclin D1, p21Cip1, and p27Kip1, showed that the cyclin D1, CDK2, and CDK4 protein levels were
Fig. 4. Effect of treatment with resveratrol. (A) The cellular growth of the resveratrol-treated cells is inhibited significantly compared with the control cells after 4 days (96 h) (*p < 0.05, Student's t-test). (B) Flow cytometric analysis shows that the percentage of cells at G1 phase in resveratrol-treated cells is increased markedly (*p < 0.05, Mann-Whitney U test) compared with controls. (C) Immunoblot analysis shows down-regulation of CDK2, CDK4, and cyclin D1, and up-regulation of p21Cip1 and p27Kip1 in resveratrol-treated cells compared with controls. (D) Schematic representation of inhibition of the p21Cip1 pathway by TRIML2. After treatment with resveratrol, the p21Cip1 expression increased with down-regulation of TRIML2, leading to cell-cycle arrest at G1 phase.
Please cite this article as: F. Hayashi et al., Increased expression of tripartite motif (TRIM) like 2 promotes tumoral growth in human oral cancer, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.060
F. Hayashi et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
down-regulated and the p21Cip1 and p27Kip1 protein levels were up-regulated in the resveratrol-treated cells (Fig. 4C). The data indicated that resveratrol decreased cellular proliferation by cellcycle arrest at G1 phase similar to the effect on the TRIML2 knockdown cells. 4. Discussion The current study showed that TRIML2 expression was upregulated in OSCCs and was correlated significantly with tumoral growth. The TRIML2 knockdown cells showed inhibited cellular growth by cell-cycle arrest at G1 phase. Furthermore, resveratrol, a polyphenol, led to not only down-regulation of TRIML2 but also cell-cycle arrest at G1 phase similar to that in the TRIML2 knockdown experiments. Some TRIM proteins are associated with gastric, breast, prostate cancers [30e32]. However, the biologic functions of most TRIM proteins, including TRIML2, have not been well elucidated thus far. Although TRIML2 has been reported to be related to Alzheimer's disease, other functions and mechanisms in cancers are as yet unidentified. Since the current data are the first in the cancer research literature, the expression status of TRIML2 in other cancers need to be studied. P21Cip1, a strong CDK inhibitor, physically interacts with cyclins, CDK2, and CDK4 to control cell-cycle at G1/S phase [33,34]. The current study found that knockdown of TRIML2 resulted in overexpression of p21Cip1 and cell-cycle arrest at G1 phase, suggesting that p21Cip1 is regulated by the TRIML2 expression status. Resveratrol, an important natural compound in grapes and peanuts, have various inhibitory effects against aging and diabetes [35e44]. Resveratrol also is associated with several cancer-related genes; however, no published studies have reported a direct correlation between resveratrol and TRIML2. Furthermore, resveratrol leads to up-regulation of p21Cip1 [45,46]. Interestingly, the current data strongly indicated that TRIML2 controls p21Cip1 expression after treatment with resveratrol. In summary, overexpression of TRIML2 contributed to tumoral growth at G1 phase as seen by cell-cycle analysis, which resulted in insufficient control by down-regulation of p21Cip1 expression (Fig. 4D). Resveratrol caused up-regulation of p21Cip1 through the TRIML2 expression. Therefore, the expression status of TRIML2 might be an indicator of OSCC progression and resveratrol may be a potential new therapeutic drug for oral cancer therapy via TRIML2. Acknowledgement We thank Lynda C. Charters for editing this manuscript. We declare that all authors have no conflict of interests. References [1] S. Hatakeyama, TRIM family proteins: roles in autophagy, immunity, and carcinogenesis, Trends Biochem. Sci. 42 (2017) 297e311, https://doi.org/ 10.1016/j.tibs.2017.01.002. [2] S. Hatakeyama, TRIM proteins and cancer, Nat. Rev. Canc. 11 (2011) 792e804, https://doi.org/10.1038/nrc3139. [3] M. Watanabe, S. Hatakeyama, JB special review - recent topics in ubiquitinproteasome system and autophagy: TRIM proteins and diseases, J. Biochem. 161 (2017) 135e144, https://doi.org/10.1093/jb/mvw087. [4] G. Dellaire, D.P. Bazett-Jones, PML nuclear bodies: dynamic sensors of DNA damage and cellular stress, Bioessays 26 (2004) 963e977, https://doi.org/ 10.1002/bies.20089. [5] S. Nisole, J.P. Stoye, A. Saïb, TRIM family proteins: retroviral restriction and antiviral defence, Nat. Rev. Microbiol. 3 (2005) 799e808, https://doi.org/ 10.1038/nrmicro1248. [6] K. Ozato, D. Shin, T. Chang, H. Morse, TRIM famiy proteins and their emerging roles in innate immunity, Nat. Rev. Immunol. 8 (2012) 849e860, https:// doi.org/10.1038/nri2413.TRIM. [7] P. Salomoni, P.P. Pandolfi, The role of PML in tumor suppression, Cell 108
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Please cite this article as: F. Hayashi et al., Increased expression of tripartite motif (TRIM) like 2 promotes tumoral growth in human oral cancer, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.060
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Increased expression of tripartite motif (TRIM) like 2 promotes tumoral growth in human oral cancer Fumihiko Hayashi a, Atsushi Kasamatsu b, *, Yosuke Endo-Sakamoto b, Keitaro Eizuka a, Kazuya Hiroshima a, Akihiro Kita b, Tomoaki Saito b, Kazuyuki Koike b, Hideki Tanzawa a, b, Katsuhiro Uzawa a, b, ** a b
Department of Oral Science, Graduate School of Medicine, Chiba University, Chiba, Japan Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, Chiba, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 November 2018 Accepted 7 December 2018 Available online xxx
Tripartite motif family-like 2 (TRIML2), a member of the TRIM proteins family, is closely related to Alzheimer's disease, however, no studies of TRIML2 have been published in the cancer research literature. In the current study, we investigated the expression level of TRIML2 and its molecular mechanisms in human oral squamous cell carcinoma (OSCC); reverse transcriptase-quantitative polymerase chain reaction, immunoblot analysis, and immunohistochemistry showed that TRIML2 is up-regulated significantly in OSCCs in vitro and in vivo. TRIML2 knockdown OSCC cells showed decreased cellular proliferation by cell-cycle arrest at G1 phase that resulted from down-regulation of CDK4, CDK6, and cyclin D1 and up-regulation of p21Cip1 and p27Kip1. Surprisingly, resveratrol, a polyphenol, led to not only down-regulation of TRIML2 but also cell-cycle arrest at G1 phase similar to TRIML2 knockdown experiments. Taken together, we concluded that TRIML2 might play a significant role in tumoral growth and that resveratrol may be a new drug for treating OSCC by interfering with TRIML2 function. © 2018 Published by Elsevier Inc.
Keywords: Cell-cycle arrest at G1 phase Cell proliferation Oral squamous cell carcinoma Resveratrol Tripartite motif family-like 2
1. Introduction The family of tripartite motif (TRIM) proteins in humans has 77 members [1], almost all of which have an N-terminal RING finger, B-boxes, and a coiled-coil domains. The TRIM family members are divided into 12 subfamilies (C-I to C-Ⅺ and an unclassified group) that were classified based on the differences in their C-terminal domains [2,3]. TRIM proteins play significant roles in a range of processes including tumoral suppression, DNA damage, stem cell differentiation, and immune reactivity against viral infections, particularly human immunodeficiency virus [4e8]. TRIM family-like 2 (TRIML2) belongs to an unclassified group because it lacks a RING-finger domain [1,3]. Polymorphisms in TRIML2 and their haplotypes are related significantly to Alzheimer's disease, suggesting that TRIML2 is a biomarker for the
* Corresponding author. Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan. ** Corresponding author. Department of Oral Science, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan.. E-mail addresses: [email protected] (A. Kasamatsu), uzawak@ faculty.chiba-u.jp (K. Uzawa).
diagnosis of Alzheimer's disease [9]. Despite increasing recognition of the significant cellular functions of TRIML2, the biochemical basis for these mechanisms is not well understood, and no reports in the cancer research literature have addressed this. In the current study, overexpressed TRIML2 was seen in human oral squamous cell carcinoma (OSCC) cells and primary OSCCs. We also reported that TRIML2 was correlated with cell-cycle arrest at G1 phase using TRIML2 knockdown models and its novel specific inhibitor, resveratrol. 2. Materials and methods 2.1. Ethics statement The ethics committee of Chiba University approved the study protocol (protocol number 680). All patients provided written informed consent, which was obtained before the experiments were conducted. 2.2. Cells and clinical OSCC tissue samples Ten OSCC cells were used for in vitro experiments [10e19]. We
https://doi.org/10.1016/j.bbrc.2018.12.060 0006-291X/© 2018 Published by Elsevier Inc.
Please cite this article as: F. Hayashi et al., Increased expression of tripartite motif (TRIM) like 2 promotes tumoral growth in human oral cancer, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.060
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Fig. 1. Evaluation of TRIML2 expression in OSCC cells and primary OSCCs. (A) RT-qPCR analysis shows significantly (*p < 0.05, Student's t-test) up-regulated TRIML2 mRNA expression in all OSCC-derived cell lines compared with the HNOKs. (B) Immunoblot analysis shows up-regulated TRIML2 protein expression in OSCC-derived cell lines compared to the HNOKs. Densitometric TRIML2 protein data are normalized to the GAPDH protein levels. The values are expressed as a percentage of the HNOKs. (C) Representative IHC results of TRIML2 in normal oral tissues and primary OSCCs. Original magnification, 400. Scale bars, 50 mm. (D) The status of TRIML2 protein expression in normal counterparts and primary OSCCs (n ¼ 100) using the IHC scoring system. The TRIML2 protein expression levels in OSCCs are significantly higher than in normal oral tissues (*p < 0.05, Student's t-test).
extracted human normal oral keratinocytes (HNOKs) from healthy young patients as control cells and cultured them as described previously [10,11]. OSCCs and normal oral tissue samples (n ¼ 100) were collected from patients with OSCC at Chiba University Hospital.
2.7. Cellular growth We analyzed the cellular growth in shTRIML2 and shMock cells to assess the effect of TRIML2 knockdown on cellular proliferation. The protocol was described previously [17,27,28].
2.3. Gene expression analysis We isolated total RNA from OSCC cells incubated subconfluently for generating cDNA and performed reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) as described previously [10,11,20]. The following primer sequences were used for RT-qPCR; TRIML2, 50 -CACATGGTGTGTGGGATACAA-30 and 50 eCCTCGATGTGTTCAATATTTCCT-30 ; and glyceraldehyde 3phosphate dehydrogenase (GAPDH), 50 -AGCCACATCGCTCAGACAC30 and 50 -GCCCAATACGACCAAATCC-30 .
2.8. Cell-cycle analysis We conducted cell-cycle analysis by flow cytometry using the BD Accuri™ C6 Flow Cytometer (Becton-Dickinson, San Diego, CA, USA). The protocol was described previously [14,23,29]. Table 1 Correlation between TRIML2 expression and clinical classification in OSCCs. Clinical classification
2.4. Immunoblot analysis
Results of immunostaining No. patients TRIML2 negative
We performed immunoblot analysis as described previously [17,21]. The antibodies were rabbit anti-TRIML2 polyclonal antibody (Abcam, Cambridge, UK), GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p21Cip1, p27Kip1, cyclin D1, CDK2, and CDK4 (Cell Signaling Technology Japan, Tokyo, Japan).
Age at surgery (years) < 60 26 12 S60 74 27 Gender Male 56 26 Female 44 18 T-primary tumoral size T1þT2 50 29 T3þT4 50 17 N-regional lymph node metastasis Negative 66 30 Positive 34 14 TNM Stage I þ II 32 18 III þ IV 68 24 Vascular invasion Negative 82 39 Positive 18 6 Histopathologic type Well 56 20 Moderately þ poorly 44 12
2.5. Immunohistochemistry (IHC) We performed IHC and calculated the IHC score described previously [17,21e25]. We evaluated the TRIML2 scores from 100 patients with OSCC and classified TRIML2 as positive and negative using the median score of all OSCCs as the cut-off points [26]. In this case, the IHC score over 117 was taken as TRIML2 positive. 2.6. Transfection with shRNA We established TRIML2 knockdown cells derived from the Sa3 and SAS cells using TRIML2 shRNA (shTRIML2) and control shRNA (shMock) vectors, respectively (Santa Cruz Biotechnology). The protocol was described previously [14].
Total
p value
TRIML2 positive 14 47
0.484*
30 26
0.686*
21 33
0.027*y
36 20
0.832*
14 44
0.054*
43 12
0.307*
36 32
0.396*
*
Fisher's exact test. yp < 0.05.
Please cite this article as: F. Hayashi et al., Increased expression of tripartite motif (TRIM) like 2 promotes tumoral growth in human oral cancer, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.060
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2.9. Resveratrol treatment To investigate a correlation between TRIML2 and resveratrol (Sigma-Aldrich, St. Louis, MO, USA), we treated Sa3 and SAS cells with resveratrol for 48 h. After treatment, we extracted total RNA, generated complementary DNA, and performed RT-qPCR. We also extracted protein for immunoblot analysis and evaluated whether resveratrol affected the cellular proliferation and cell-cycle by treating the Sa3 and SAS cells with resveratrol for 48 h. In the cellular proliferation assay, the cells were cultured with resveratrol, and dimethylsulfoxide served as the experimental control.
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was up-regulated significantly (p < 0.05) in all OSCC cells compared with the HNOKs (Fig. 1A and B). Fig. 1C shows representative IHC results of TRIML2 in normal and primary OSCC tissues. The IHC scores ranged from 34 to 132 (median, 64) in normal oral tissues and 69 to 187 (median, 117) in OSCC tissues (Fig. 1D) (p < 0.05). Table 1 shows the correlation between the TRIML2 expression status and clinical classification of the patients with OSCC. Among the clinical classifications, the TRIML2 expression level was correlated significantly (p < 0.05) with the primary tumoral size (Table 1). 3.2. Establishment of TRIML2 knockdown cells
2.10. Statistical analysis Student's t-test, Fisher's exact test, and Mann-Whitney U test were performed to identify significance correlations. All data are expressed as the mean ± the standard error of the mean. 3. Results 3.1. Evaluation of TRIML2 expression OSCC cells and primary OSCCs RT-qPCR and immunoblot data showed that TRIML2 expression
We chose Sa3 and SAS cells for knockdown experiments because higher TRIML2 expression levels were observed in those OSCC cell lines. RT-qPCR and immunoblot analysis showed that TRIML2 was down-regulated in the shTRIML2 cells compared with the shMock cells (Fig. 2A and B, respectively). No morphologic changes in the TRIML2 knockdown cells were seen during microscopy. 3.3. Cellular proliferation assay of TRIML2 knockdown cells To evaluate the effect of the TRIML2 knockdown on cellular
Fig. 2. Establishment of the TRIML2 knockdown cells and cell-cycle analysis of TRIML2 knockdown cells. (A) RT-qPCR shows that the TRIML2 mRNA expression in shTRIML2 cells is significantly (*p < 0.05, Student's t-test) lower than that in shMock cells. (B) Immunoblot analysis shows that the TRIML2 protein levels in the shTRIML2 cells are decreased compared with the shMock cells. (C) The cellular growth of the shTRIML2 cells is inhibited significantly compared with the shMock cells after 4 days (96 h) (*p < 0.05, Student's ttest). (D) Flow cytometric analysis shows that the percentage of cells at G1 phase in the shTRIML2 cells is increased markedly (*p < 0.05, Mann-Whitney U test) compared with the shMock cells. (E) Immunoblot analysis shows down-regulation of CDK2, CDK4, and cyclin D1 and up-regulation of p21Cip1 and p27Kip1 in the shTRIML2 cells compared with the shMock cells.
Please cite this article as: F. Hayashi et al., Increased expression of tripartite motif (TRIM) like 2 promotes tumoral growth in human oral cancer, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.060
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growth, we performed a cellular proliferation assay that showed significantly (p < 0.05) decrease cellular growth in the shTRIML2 cells compared with the shMock cells (Fig. 2C). 3.4. Cell-cycle analysis of TRIML2 knockdown cells The cell-cycle analysis showed that the percentage of the shTRIML2 cells at G1 phase increased significantly (p < 0.05) compared with the shMock cells (Fig. 2D). Evaluation of the protein expression levels of G1 phase-related proteins in the shTRIML2 cells showed that CDK2, CDK4, and cyclin D1 were down-regulated, and p21Cip1 and p27Kip1 were up-regulated in the shTRIML2 cells (Fig. 2E). These results indicated that the shTRIML2 cells suppressed cellular proliferation by cell-cycle arrest at G1 phase. 3.5. Effect of treatment with resveratrol
Fig. 3. Effect of treatment with resveratrol. (A) RT-qPCR shows that the TRIML2 mRNA expression is significantly (*p < 0.05, Student's t-test) down-regulated after treatment with resveratrol in both the Sa3 and SAS cells compared with controls. (B) Immunoblot analysis shows down-regulated TRIML2 expression after treatment with resveratrol compared with controls.
After resveratrol treatment (50 mM, 48 h), the TRIML2 expression levels decreased in both Sa3 and SAS cell lines compared with the control (Fig. 3A and B). To assess the effect of resveratrol on cellular growth, we performed cellular proliferation assay that showed that the cellular growth of the resveratrol-treated cells was significantly (p < 0.05) lower than that of the control cells (Fig. 4A). The cell-cycle analysis, showed that the percentage of the resveratrol-treated cells in G1 phase was significantly (p < 0.05) higher than that of the control cells (Fig. 4B). Evaluation of the protein levels of CDK2, CDK4, cyclin D1, p21Cip1, and p27Kip1, showed that the cyclin D1, CDK2, and CDK4 protein levels were
Fig. 4. Effect of treatment with resveratrol. (A) The cellular growth of the resveratrol-treated cells is inhibited significantly compared with the control cells after 4 days (96 h) (*p < 0.05, Student's t-test). (B) Flow cytometric analysis shows that the percentage of cells at G1 phase in resveratrol-treated cells is increased markedly (*p < 0.05, Mann-Whitney U test) compared with controls. (C) Immunoblot analysis shows down-regulation of CDK2, CDK4, and cyclin D1, and up-regulation of p21Cip1 and p27Kip1 in resveratrol-treated cells compared with controls. (D) Schematic representation of inhibition of the p21Cip1 pathway by TRIML2. After treatment with resveratrol, the p21Cip1 expression increased with down-regulation of TRIML2, leading to cell-cycle arrest at G1 phase.
Please cite this article as: F. Hayashi et al., Increased expression of tripartite motif (TRIM) like 2 promotes tumoral growth in human oral cancer, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.060
F. Hayashi et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
down-regulated and the p21Cip1 and p27Kip1 protein levels were up-regulated in the resveratrol-treated cells (Fig. 4C). The data indicated that resveratrol decreased cellular proliferation by cellcycle arrest at G1 phase similar to the effect on the TRIML2 knockdown cells. 4. Discussion The current study showed that TRIML2 expression was upregulated in OSCCs and was correlated significantly with tumoral growth. The TRIML2 knockdown cells showed inhibited cellular growth by cell-cycle arrest at G1 phase. Furthermore, resveratrol, a polyphenol, led to not only down-regulation of TRIML2 but also cell-cycle arrest at G1 phase similar to that in the TRIML2 knockdown experiments. Some TRIM proteins are associated with gastric, breast, prostate cancers [30e32]. However, the biologic functions of most TRIM proteins, including TRIML2, have not been well elucidated thus far. Although TRIML2 has been reported to be related to Alzheimer's disease, other functions and mechanisms in cancers are as yet unidentified. Since the current data are the first in the cancer research literature, the expression status of TRIML2 in other cancers need to be studied. P21Cip1, a strong CDK inhibitor, physically interacts with cyclins, CDK2, and CDK4 to control cell-cycle at G1/S phase [33,34]. The current study found that knockdown of TRIML2 resulted in overexpression of p21Cip1 and cell-cycle arrest at G1 phase, suggesting that p21Cip1 is regulated by the TRIML2 expression status. Resveratrol, an important natural compound in grapes and peanuts, have various inhibitory effects against aging and diabetes [35e44]. Resveratrol also is associated with several cancer-related genes; however, no published studies have reported a direct correlation between resveratrol and TRIML2. Furthermore, resveratrol leads to up-regulation of p21Cip1 [45,46]. Interestingly, the current data strongly indicated that TRIML2 controls p21Cip1 expression after treatment with resveratrol. In summary, overexpression of TRIML2 contributed to tumoral growth at G1 phase as seen by cell-cycle analysis, which resulted in insufficient control by down-regulation of p21Cip1 expression (Fig. 4D). Resveratrol caused up-regulation of p21Cip1 through the TRIML2 expression. Therefore, the expression status of TRIML2 might be an indicator of OSCC progression and resveratrol may be a potential new therapeutic drug for oral cancer therapy via TRIML2. Acknowledgement We thank Lynda C. Charters for editing this manuscript. We declare that all authors have no conflict of interests. References [1] S. Hatakeyama, TRIM family proteins: roles in autophagy, immunity, and carcinogenesis, Trends Biochem. Sci. 42 (2017) 297e311, https://doi.org/ 10.1016/j.tibs.2017.01.002. [2] S. Hatakeyama, TRIM proteins and cancer, Nat. Rev. Canc. 11 (2011) 792e804, https://doi.org/10.1038/nrc3139. [3] M. Watanabe, S. Hatakeyama, JB special review - recent topics in ubiquitinproteasome system and autophagy: TRIM proteins and diseases, J. Biochem. 161 (2017) 135e144, https://doi.org/10.1093/jb/mvw087. [4] G. Dellaire, D.P. Bazett-Jones, PML nuclear bodies: dynamic sensors of DNA damage and cellular stress, Bioessays 26 (2004) 963e977, https://doi.org/ 10.1002/bies.20089. [5] S. Nisole, J.P. Stoye, A. Saïb, TRIM family proteins: retroviral restriction and antiviral defence, Nat. Rev. Microbiol. 3 (2005) 799e808, https://doi.org/ 10.1038/nrmicro1248. [6] K. Ozato, D. Shin, T. Chang, H. Morse, TRIM famiy proteins and their emerging roles in innate immunity, Nat. Rev. Immunol. 8 (2012) 849e860, https:// doi.org/10.1038/nri2413.TRIM. [7] P. Salomoni, P.P. Pandolfi, The role of PML in tumor suppression, Cell 108
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Please cite this article as: F. Hayashi et al., Increased expression of tripartite motif (TRIM) like 2 promotes tumoral growth in human oral cancer, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.060