Mol. Cells 2019; 42(7): 557-567
Published online July 20, 2019
https://doi.org/10.14348/molcells.2019.0015
© The Korean Society for Molecular and Cellular Biology
Correspondence to : cuixiaobo@hrbmu.edu.cn
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
TSPAN12, a member of the tetraspanin family, has been highly connected with the pathogenesis of cancer. Its biological function, however, especially in ovarian cancer (OC), has not been well elucidated. In this study, The Cancer Genome Atlas (TCGA) dataset analysis revealed that upregulation of
Keywords cell cycle, ovarian cancer, proliferation, TSPAN12
As a member of the tetraspanin family, Tetraspanin 12 (TSPAN12) physically binds to a large number of partners, including immunoglobulin proteins, integrins, growth factors, and other tetraspanin family members (Bailey et al., 2011; Hemler, 2003). The most broadly-known clinical significance of TSPAN12 is its association with familial exudative vitreoretinopathy (Junge et al., 2009). The correlation of tetraspanin proteins (e.g., CD151, NET-6, CD82, and CD9) and cancer progression has been frequently reported (Lafleur et al., 2009; Wang et al., 2011). These data suggest that TSPAN12 may also be correlated with carcinogenesis.
Ovarian cancer (OC) is among the top five of the most lethal gynecological malignancies (Thibault et al., 2014). Mounting evidence has revealed the clinicopathological and molecular mechanisms of OC. Multiple potential causative genes, such as
Cancer cells obtain a growth advantage through uncontrolled cell proliferation (Hanahan and Weinberg, 2011), which may be caused by mutations that help them adapt to the microenvironment through selective pressure. Uncontrolled cell proliferation underlies tumor progression, including tumor initiation and metastasis (Feitelson et al., 2015). Targeted therapy against uncontrolled cancer cell growth could become an effective treatment for cancer patients (Feitelson et al., 2015).
In this study, we investigated how TSPAN12 regulates OC cell proliferation both
Human OC cell lines SKOV3, A2780, and OVCAR3 were obtained from the American Type Culture Collection (ATCC, USA) and maintained as previously described (Li et al., 2019; Yang et al., 2019). CDK inhibitor (#S1524, AT7519) was purchased from Selleck (China).
Lenti-viral vectors (#EX-A6476-Lv203-GS) from GeneCopoeia (China) were used to overexpress TSPAN12 in OVCAR3 and SKOV3 cells according to standard protocol.
Three expression profile datasets (TCGA_OV_exp_HiSeqV2_PANCAN-2015-02-24; TCGA_OV_exp_G4502A_07_ 3-2015-02-24; TCGA_OV_exp_u133a-2015-02-24) were downloaded from The Cancer Genome Atlas (TCGA;
Total RNA was extracted using Trizol following the manufacturer’s protocol (Invitrogen, China). cDNA was reverse transcribed with random primers using the High Capacity cDNA Reverse Transcription kit (Thermo Fisher, China). Real-time PCR was used to quantify mRNA expression using the LightCycler® 480 SYBR Green I Master (Roche, China). β-Actin was used as the control gene. Gene specific primers are:
5′-GTCCCTCATCCAAGCAGAAAC-3′ (reverse);
5′-GAGGAACGGTGACATGCTCAT-3′ (reverse);
5′-CCTCCTTCTGCACACATTTGAA-3′ (reverse);
5′-TGACATCCTGGGTAGTTTTCCTC-3′ (reverse);
5′-TTCATCCAGGGGAGGTACAAC-3′ (reverse);
5′-CATTGGGGACTCTCACACTCT-3′ (reverse).
Minced frozen tissue and cells were lysed with 100 [Symbol]ml lysis buffer (Cell Signaling Technology, USA) supplemented with proteinase inhibitors (5 mg/ml aprotinin, 1 mg/ml leupeptin, and 10 mM PMSF; Sigma, USA) and phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA). Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membrane. Blots were incubated with antibodies against TSPAN12 (1:400, anti-rabbit, Av46887; Sigma), Cyclin D1, Cyclin E2, CDK2, CDK4 (1:400, anti-rabbit, Cell Cycle Antibody Sampler kit #9932, #9870; Cell Signaling Technology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1,000, anti-mouse, 60004-1; Proteintech, USA). Staining was detected using the SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific). GAPDH was used as control for equal protein loading.
All animal experiments were conducted with five-week-old female BALB/c nude mice (NCI-Charles River, China) in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animal. The animal protocol was approved by the Institutional Review Board of Harbin Medical University (approval No. HMUIRB20170033). Eight mice were employed to establish subcutaneous xenograft experiments with cells injected on either side flank of mice. Subcutaneous tumor size was measured every three days, and mice were euthanized three weeks after injection. Tumor volume was measured by caliper and calculated as (L × W2) / 2 (mm3) (L, length; W, width). Five orthotopic mice were generated according to previous reports (Yi et al., 2014). Orthotopic mice were euthanized four week after inoculation.
Cells were first seeded in six-well plates. Then relative cell proliferation were visualized by crystal violet staining (0.5% w/v), and reflected by absorbance of crystal violet dissolved in dimethyl sulfoxide (DMSO), which was recorded at 490 nm with Sunrise microplate reader (Tecan, Switzerland).
Cells were seeded in six-well plates. Cell clones were fixed with ice-cold methanol, stained by crystal violet (0.5% w/v) and counted. The colony is defined by containing a minimum of 50 cells.
Cells were synchronized with serum-free medium for 24 h, and then harvested at time point 0 h, 4 h, 8 h, and 16 h after released in regular cell culture medium containing 10% fetal bovine serum. Then cells were fixed with ethanol and analyzed by flow cytometry with 488 nm excitation laser line following the manufacturer’s protocol of FxCycle™ PI/RNase Staining kit (Thermo Fisher, USA).
Overall survival (OS) was determined using Kaplan–Meier analysis and compared via the log-rank test. Differences between control and treatment groups were determined using the Student’s
To understand the potential function of TSPAN12 in OC, we extracted the profiling data of three cohorts of human tumor specimens from TCGA including 208 OC patients (Grade 2) in total. Statistical analysis based on open-source data from TCGA database unraveled that high expression of
The most common characteristic of cancer cells is their unlimited growth. To understand whether TSPAN12 may involve in the proliferation of OC, we manipulated
To get further understanding on how
To further confirm that TSPAN12 regulated cell proliferation by controlling the cell cycle, we suppressed CDK activity with AT7519, a specific CDK2 and CDK4 inhibitor (Figs. 6E–6F). We found that cell proliferation (Figs. 2E and 2F) and colony formation (Figs. 3E and 3F) was significantly reversed in the presence of this inhibitor. Meanwhile, cell cycle progression was also compromised following AT7519 treatment in the context of TSPAN12 overexpression (Figs. 5I–5L). These data suggested that TSPAN12 may contribute to cell proliferation by controlling the cell cycle through cyclins and CDKs regulation, which was further supported at the mRNA level by data from the TCGA database analysis (
In our investigation of the clinical relevance of TSPAN12 in OC, we found that high expression of TSPAN12 was significantly correlated with poor prognosis in this disease. Thus, we hypothesized that TSPAN12 might be an oncogene in OC development.
Retrospective analysis of previous studies demonstrated that TSPAN12 was associated with the progression of breast, lung, and colon cancer (Knoblich et al., 2013; Lafleur et al., 2009; Liu et al., 2017; Otomo et al., 2014; Wang et al., 2011). Downregulation of TSPAN12 expression in human MDA-MB-231 breast cancer cells significantly decreased primary tumor growth, increased tumor apoptosis, and inhibited metastasis (Knoblich et al., 2013). In addition, silencing of TSPAN12 inhibited the growth of non-small cell lung carcinoma cells both
In the current study, we performed mechanistic studies to determine how TSPAN12 may regulate the proliferation of OC cells. We identified cell cycle control as the mechanism by which TSPAN12 could contribute to OC progression. Cell cycle control is regulated by a panel of enzymes (i.e., cyclins and CDKs) to maintain accurate DNA replication and chromosome segregation (Bendris et al., 2015). It is widely accepted that cyclin D1 leads to progression through the G1 phase of the cell cycle by activating CDK4 in multiple cancer types (Ewen and Lamb, 2004); cyclin E2-CDK2 is implicated in the transition of human cells from G1 to S (Gudas et al., 1999; Payton and Coats, 2002). Furthermore, cyclin A2 regulates cell cycle progression by interacting with CDK2 during S phase and the G2/M transition (Blanchard, 2000; Pagano et al., 1992). The results of our study indicated that TSPAN12 could trigger the upregulation of a broad spectrum of cyclin and CDK proteins, which promoted cell cycle progression. These results were supported by correlation studies at the mRNA level from tissue samples in the TCGA database and cell lines established in this study. However, we did not find a positive correlation between the cyclins or CDKs and TSPAN12 in xenograft tumors, which may be due to the limited sample size of the xenograft tumors. Therefore, in this study, cyclin and CDK proteins were identified as potential targets for TSPAN12, which have not been evaluated in previous studies focusing on the role of TSPAN12 in carcinogenesis.
Our data demonstrated that TSPAN12 could induce cell proliferation in OC cells, making it a potential therapeutic target in OC. How the interplay between TSPAN12 and cell cycle proteins regulate OC development requires further exploration.
Mol. Cells 2019; 42(7): 557-567
Published online July 31, 2019 https://doi.org/10.14348/molcells.2019.0015
Copyright © The Korean Society for Molecular and Cellular Biology.
Guohua Ji1,2, Hongbin Liang1, Falin Wang1,3, Nan Wang1,4, Songbin Fu1,2,5, and Xiaobo Cui1,2,*
1Laboratory of Medical Genetics, Harbin Medical University, Harbin, China, 2Key Laboratory of Medical Genetics, Harbin Medical University, Heilongjiang Higher Education Institutions, Harbin, China, 3The Gynecology and Obstetrics Department, The Fourth Affiliated Hospital of Harbin Medical University, Harbin, China, 4The Organization Department, The Fourth Affiliated Hospital of Harbin Medical University, Harbin, China, 5Key Laboratory of Preservation of Human Genetic Resources and Disease Control, Chinese Ministry of Education, Harbin, China
Correspondence to:cuixiaobo@hrbmu.edu.cn
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
TSPAN12, a member of the tetraspanin family, has been highly connected with the pathogenesis of cancer. Its biological function, however, especially in ovarian cancer (OC), has not been well elucidated. In this study, The Cancer Genome Atlas (TCGA) dataset analysis revealed that upregulation of
Keywords: cell cycle, ovarian cancer, proliferation, TSPAN12
As a member of the tetraspanin family, Tetraspanin 12 (TSPAN12) physically binds to a large number of partners, including immunoglobulin proteins, integrins, growth factors, and other tetraspanin family members (Bailey et al., 2011; Hemler, 2003). The most broadly-known clinical significance of TSPAN12 is its association with familial exudative vitreoretinopathy (Junge et al., 2009). The correlation of tetraspanin proteins (e.g., CD151, NET-6, CD82, and CD9) and cancer progression has been frequently reported (Lafleur et al., 2009; Wang et al., 2011). These data suggest that TSPAN12 may also be correlated with carcinogenesis.
Ovarian cancer (OC) is among the top five of the most lethal gynecological malignancies (Thibault et al., 2014). Mounting evidence has revealed the clinicopathological and molecular mechanisms of OC. Multiple potential causative genes, such as
Cancer cells obtain a growth advantage through uncontrolled cell proliferation (Hanahan and Weinberg, 2011), which may be caused by mutations that help them adapt to the microenvironment through selective pressure. Uncontrolled cell proliferation underlies tumor progression, including tumor initiation and metastasis (Feitelson et al., 2015). Targeted therapy against uncontrolled cancer cell growth could become an effective treatment for cancer patients (Feitelson et al., 2015).
In this study, we investigated how TSPAN12 regulates OC cell proliferation both
Human OC cell lines SKOV3, A2780, and OVCAR3 were obtained from the American Type Culture Collection (ATCC, USA) and maintained as previously described (Li et al., 2019; Yang et al., 2019). CDK inhibitor (#S1524, AT7519) was purchased from Selleck (China).
Lenti-viral vectors (#EX-A6476-Lv203-GS) from GeneCopoeia (China) were used to overexpress TSPAN12 in OVCAR3 and SKOV3 cells according to standard protocol.
Three expression profile datasets (TCGA_OV_exp_HiSeqV2_PANCAN-2015-02-24; TCGA_OV_exp_G4502A_07_ 3-2015-02-24; TCGA_OV_exp_u133a-2015-02-24) were downloaded from The Cancer Genome Atlas (TCGA;
Total RNA was extracted using Trizol following the manufacturer’s protocol (Invitrogen, China). cDNA was reverse transcribed with random primers using the High Capacity cDNA Reverse Transcription kit (Thermo Fisher, China). Real-time PCR was used to quantify mRNA expression using the LightCycler® 480 SYBR Green I Master (Roche, China). β-Actin was used as the control gene. Gene specific primers are:
5′-GTCCCTCATCCAAGCAGAAAC-3′ (reverse);
5′-GAGGAACGGTGACATGCTCAT-3′ (reverse);
5′-CCTCCTTCTGCACACATTTGAA-3′ (reverse);
5′-TGACATCCTGGGTAGTTTTCCTC-3′ (reverse);
5′-TTCATCCAGGGGAGGTACAAC-3′ (reverse);
5′-CATTGGGGACTCTCACACTCT-3′ (reverse).
Minced frozen tissue and cells were lysed with 100 [Symbol]ml lysis buffer (Cell Signaling Technology, USA) supplemented with proteinase inhibitors (5 mg/ml aprotinin, 1 mg/ml leupeptin, and 10 mM PMSF; Sigma, USA) and phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA). Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membrane. Blots were incubated with antibodies against TSPAN12 (1:400, anti-rabbit, Av46887; Sigma), Cyclin D1, Cyclin E2, CDK2, CDK4 (1:400, anti-rabbit, Cell Cycle Antibody Sampler kit #9932, #9870; Cell Signaling Technology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1,000, anti-mouse, 60004-1; Proteintech, USA). Staining was detected using the SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific). GAPDH was used as control for equal protein loading.
All animal experiments were conducted with five-week-old female BALB/c nude mice (NCI-Charles River, China) in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animal. The animal protocol was approved by the Institutional Review Board of Harbin Medical University (approval No. HMUIRB20170033). Eight mice were employed to establish subcutaneous xenograft experiments with cells injected on either side flank of mice. Subcutaneous tumor size was measured every three days, and mice were euthanized three weeks after injection. Tumor volume was measured by caliper and calculated as (L × W2) / 2 (mm3) (L, length; W, width). Five orthotopic mice were generated according to previous reports (Yi et al., 2014). Orthotopic mice were euthanized four week after inoculation.
Cells were first seeded in six-well plates. Then relative cell proliferation were visualized by crystal violet staining (0.5% w/v), and reflected by absorbance of crystal violet dissolved in dimethyl sulfoxide (DMSO), which was recorded at 490 nm with Sunrise microplate reader (Tecan, Switzerland).
Cells were seeded in six-well plates. Cell clones were fixed with ice-cold methanol, stained by crystal violet (0.5% w/v) and counted. The colony is defined by containing a minimum of 50 cells.
Cells were synchronized with serum-free medium for 24 h, and then harvested at time point 0 h, 4 h, 8 h, and 16 h after released in regular cell culture medium containing 10% fetal bovine serum. Then cells were fixed with ethanol and analyzed by flow cytometry with 488 nm excitation laser line following the manufacturer’s protocol of FxCycle™ PI/RNase Staining kit (Thermo Fisher, USA).
Overall survival (OS) was determined using Kaplan–Meier analysis and compared via the log-rank test. Differences between control and treatment groups were determined using the Student’s
To understand the potential function of TSPAN12 in OC, we extracted the profiling data of three cohorts of human tumor specimens from TCGA including 208 OC patients (Grade 2) in total. Statistical analysis based on open-source data from TCGA database unraveled that high expression of
The most common characteristic of cancer cells is their unlimited growth. To understand whether TSPAN12 may involve in the proliferation of OC, we manipulated
To get further understanding on how
To further confirm that TSPAN12 regulated cell proliferation by controlling the cell cycle, we suppressed CDK activity with AT7519, a specific CDK2 and CDK4 inhibitor (Figs. 6E–6F). We found that cell proliferation (Figs. 2E and 2F) and colony formation (Figs. 3E and 3F) was significantly reversed in the presence of this inhibitor. Meanwhile, cell cycle progression was also compromised following AT7519 treatment in the context of TSPAN12 overexpression (Figs. 5I–5L). These data suggested that TSPAN12 may contribute to cell proliferation by controlling the cell cycle through cyclins and CDKs regulation, which was further supported at the mRNA level by data from the TCGA database analysis (
In our investigation of the clinical relevance of TSPAN12 in OC, we found that high expression of TSPAN12 was significantly correlated with poor prognosis in this disease. Thus, we hypothesized that TSPAN12 might be an oncogene in OC development.
Retrospective analysis of previous studies demonstrated that TSPAN12 was associated with the progression of breast, lung, and colon cancer (Knoblich et al., 2013; Lafleur et al., 2009; Liu et al., 2017; Otomo et al., 2014; Wang et al., 2011). Downregulation of TSPAN12 expression in human MDA-MB-231 breast cancer cells significantly decreased primary tumor growth, increased tumor apoptosis, and inhibited metastasis (Knoblich et al., 2013). In addition, silencing of TSPAN12 inhibited the growth of non-small cell lung carcinoma cells both
In the current study, we performed mechanistic studies to determine how TSPAN12 may regulate the proliferation of OC cells. We identified cell cycle control as the mechanism by which TSPAN12 could contribute to OC progression. Cell cycle control is regulated by a panel of enzymes (i.e., cyclins and CDKs) to maintain accurate DNA replication and chromosome segregation (Bendris et al., 2015). It is widely accepted that cyclin D1 leads to progression through the G1 phase of the cell cycle by activating CDK4 in multiple cancer types (Ewen and Lamb, 2004); cyclin E2-CDK2 is implicated in the transition of human cells from G1 to S (Gudas et al., 1999; Payton and Coats, 2002). Furthermore, cyclin A2 regulates cell cycle progression by interacting with CDK2 during S phase and the G2/M transition (Blanchard, 2000; Pagano et al., 1992). The results of our study indicated that TSPAN12 could trigger the upregulation of a broad spectrum of cyclin and CDK proteins, which promoted cell cycle progression. These results were supported by correlation studies at the mRNA level from tissue samples in the TCGA database and cell lines established in this study. However, we did not find a positive correlation between the cyclins or CDKs and TSPAN12 in xenograft tumors, which may be due to the limited sample size of the xenograft tumors. Therefore, in this study, cyclin and CDK proteins were identified as potential targets for TSPAN12, which have not been evaluated in previous studies focusing on the role of TSPAN12 in carcinogenesis.
Our data demonstrated that TSPAN12 could induce cell proliferation in OC cells, making it a potential therapeutic target in OC. How the interplay between TSPAN12 and cell cycle proteins regulate OC development requires further exploration.
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