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Synaptonemal complex protein 3 is a prognostic marker in cervical cancer.

Cho H, Noh KH, Chung JY, Takikita M, Chung EJ, Kim BW, Hewitt SM, Kim TW, Kim JH - PLoS ONE (2014)

Bottom Line: Functional studies using NIH3T3 cells demonstrated that the C-terminal region of human SCP3 is important for AKT activation and its oncogenic potential.High expression of SCP3 was significantly associated with tumor stage (P = 0.002) and tumor grade (P<0.001), while SCP3 expression was positively associated with pAKT protein level in cervical neoplasias.Survival times for patients with cervical cancer overexpressing both SCP3 and pAKT (median, 134.0 months, n = 68) were significantly shorter than for patients with low expression of either SCP3 or pAKT (161.5 months, n = 108) as determined by multivariate analysis (P = 0.020).

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Republic of Korea; Institute of Women's Life Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea.

ABSTRACT
Synaptonemal complex protein 3 (SCP3), a member of Cor1 family, is up-regulated in various cancer cells; however, its oncogenic potential and clinical significance has not yet been characterized. In the present study, we investigated the oncogenic role of SCP3 and its relationship with phosphorylated AKT (pAKT) in cervical neoplasias. The functional role of SCP3 expression was investigated by overexpression or knockdown of SCP3 in murine cell line NIH3T3 and human cervical cancer cell lines CUMC6, SiHa, CaSki, and HeLa both in vitro and in vivo. Furthermore, we examined SCP3 expression in tumor specimens from 181 cervical cancer and 400 cervical intraepithelial neoplasia (CIN) patients by immunohistochemistry and analyzed the correlation between SCP3 expression and clinicopathologic factors or survival. Overexpression of SCP3 promoted AKT-mediated tumorigenesis both in vitro and in vivo. Functional studies using NIH3T3 cells demonstrated that the C-terminal region of human SCP3 is important for AKT activation and its oncogenic potential. High expression of SCP3 was significantly associated with tumor stage (P = 0.002) and tumor grade (P<0.001), while SCP3 expression was positively associated with pAKT protein level in cervical neoplasias. Survival times for patients with cervical cancer overexpressing both SCP3 and pAKT (median, 134.0 months, n = 68) were significantly shorter than for patients with low expression of either SCP3 or pAKT (161.5 months, n = 108) as determined by multivariate analysis (P = 0.020). Our findings suggest that SCP3 plays an important role in the progression of cervical cancer through the AKT signaling pathway, supporting the possibility that SCP3 may be a promising novel cancer target for cervical cancer therapy.

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hSCP3 increases tumorigenesis through AKT pathway.(A) Western blot analysis of levels of pAKT, pERK, and pp38 in NIH3T3 cells ectopically expressing hSCP3 or no insert. Numbers below western blots refer to the relative values of the intensity normalized to no insert control. (B) In vitro growth curves of NIH3T3/hSCP3 cells treated with DMSO or API2 (Akt inhibitor), PD98059 (Erk inhibitor), or SB203580 (p38 inhibitor). (C) Soft agar colony-forming capacity of NIH3T3/hSPC3 cells in the presence of API2, PD98059, or SB203580; (C, Right) Representative images of average colony size in each group; (C, Left) bar graph showing the number of colonies with diameters greater than 2 mm in soft agar (scale bar: 1 mm). (D) Western blot analysis of levels of pAKT in NIH3T3/hSCP3 cells transfected with a siRNA targeting GFP or AKT (siGFP or siAKT) to confirm the reduction of AKT protein level. Numbers below western blots refer to the relative values of the intensity normalized to no insert control. (E) In vitro growth curves of NIH3T3/hSCP3 cells transfected with siGFP or siAKT. (F) Soft agar colony-forming capacity of NIH3T3/hSPC3 cells treated with siGFP or siAKT; (Left) Representative images of average colony size in each group; (Right) Bar graph representing the number of colonies with diameters greater than 2 mm in soft agar (scale bar: 1 mm). Error bars represent the mean ± SD.
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pone-0098712-g002: hSCP3 increases tumorigenesis through AKT pathway.(A) Western blot analysis of levels of pAKT, pERK, and pp38 in NIH3T3 cells ectopically expressing hSCP3 or no insert. Numbers below western blots refer to the relative values of the intensity normalized to no insert control. (B) In vitro growth curves of NIH3T3/hSCP3 cells treated with DMSO or API2 (Akt inhibitor), PD98059 (Erk inhibitor), or SB203580 (p38 inhibitor). (C) Soft agar colony-forming capacity of NIH3T3/hSPC3 cells in the presence of API2, PD98059, or SB203580; (C, Right) Representative images of average colony size in each group; (C, Left) bar graph showing the number of colonies with diameters greater than 2 mm in soft agar (scale bar: 1 mm). (D) Western blot analysis of levels of pAKT in NIH3T3/hSCP3 cells transfected with a siRNA targeting GFP or AKT (siGFP or siAKT) to confirm the reduction of AKT protein level. Numbers below western blots refer to the relative values of the intensity normalized to no insert control. (E) In vitro growth curves of NIH3T3/hSCP3 cells transfected with siGFP or siAKT. (F) Soft agar colony-forming capacity of NIH3T3/hSPC3 cells treated with siGFP or siAKT; (Left) Representative images of average colony size in each group; (Right) Bar graph representing the number of colonies with diameters greater than 2 mm in soft agar (scale bar: 1 mm). Error bars represent the mean ± SD.

Mentions: We previously reported that pAKT level is increased in hSCP3-expressing cervical cell lines [10]. To evaluate changes in various signaling molecules that may play a role in the global control of cell proliferation, we performed Western blot analysis to measure the levels of Ser 473 phosphorylated AKT, Thr 202/Tyr 204 phosphorylated ERK, and Thr 180/Tyr 182 phosphorylated p38 MAP kinase in NIH3T3 cells expressing hSCP3 or no insert (empty vector). We observed that the expression of pAKT was significantly increased in cells transduced with hSCP3 compared to control cells (Figure 2A). Next, to explore the relationship between hSCP3-mediated tumorigenic potential and the AKT signaling, we compared both cell proliferation and soft agar colony forming capability of NIH3T3/hSCP3 cells in the presence of control (DMSO), 10 µM API2 (an inhibitor of AKT), 50 µM PD98059 (an inhibitor of MEK/ERK) or 10 µM SB203580 (an inhibitor of p38-MAPK) (Figure 2B and 2C). Treatment with PD 98059 and SB203580 did not significantly affect the proliferation and the colony formation capacity of NIH3T3/hSCP3 cells compared with that of DMSO. On the contrary, API2 treated NIH3T3/hSCP3 cells exhibited a significantly decreased proliferation rate compared with DMSO-treated control (P<0.001). Consistently, API2-treated NIH3T3/hSCP3 cells also formed smaller colonies, the number of which was almost 5-times less than colonies formed by DMSO-treated NIH3T3/hSCP3 cells. Thus, it is obvious that both proliferation and colony formation in these cells are dependent on the AKT signaling. To exclude potential off-target effects of API2, we performed in vitro cell proliferation and soft agar colony formation experiments using siRNA-targeting AKT (siAKT) to block the AKT pathway. siRNA targeting to irrelevant GFP (GFP siRNA) was used as a negative control. Consistent with API2 data, siAKT-treated NIH3T3/hSCP3 cells exhibited a decreased rate of cell proliferation (Figure 2D and 2E) and colony number compared with control siGFP-treated NIH3T3/hSCP3 cells (Figure 2F). Similar AKT dependency was also observed in NIH3T3 cells expressing mXLR, which displayed the most potent tumorigenic potential among members of the murine Cor1 family (Figure S3). Thus, our data demonstrate that SCP3 and the other Cor1 members could confer tumorigenic potentials upon NIH3T3 cell through AKT pathway.


Synaptonemal complex protein 3 is a prognostic marker in cervical cancer.

Cho H, Noh KH, Chung JY, Takikita M, Chung EJ, Kim BW, Hewitt SM, Kim TW, Kim JH - PLoS ONE (2014)

hSCP3 increases tumorigenesis through AKT pathway.(A) Western blot analysis of levels of pAKT, pERK, and pp38 in NIH3T3 cells ectopically expressing hSCP3 or no insert. Numbers below western blots refer to the relative values of the intensity normalized to no insert control. (B) In vitro growth curves of NIH3T3/hSCP3 cells treated with DMSO or API2 (Akt inhibitor), PD98059 (Erk inhibitor), or SB203580 (p38 inhibitor). (C) Soft agar colony-forming capacity of NIH3T3/hSPC3 cells in the presence of API2, PD98059, or SB203580; (C, Right) Representative images of average colony size in each group; (C, Left) bar graph showing the number of colonies with diameters greater than 2 mm in soft agar (scale bar: 1 mm). (D) Western blot analysis of levels of pAKT in NIH3T3/hSCP3 cells transfected with a siRNA targeting GFP or AKT (siGFP or siAKT) to confirm the reduction of AKT protein level. Numbers below western blots refer to the relative values of the intensity normalized to no insert control. (E) In vitro growth curves of NIH3T3/hSCP3 cells transfected with siGFP or siAKT. (F) Soft agar colony-forming capacity of NIH3T3/hSPC3 cells treated with siGFP or siAKT; (Left) Representative images of average colony size in each group; (Right) Bar graph representing the number of colonies with diameters greater than 2 mm in soft agar (scale bar: 1 mm). Error bars represent the mean ± SD.
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Related In: Results  -  Collection

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pone-0098712-g002: hSCP3 increases tumorigenesis through AKT pathway.(A) Western blot analysis of levels of pAKT, pERK, and pp38 in NIH3T3 cells ectopically expressing hSCP3 or no insert. Numbers below western blots refer to the relative values of the intensity normalized to no insert control. (B) In vitro growth curves of NIH3T3/hSCP3 cells treated with DMSO or API2 (Akt inhibitor), PD98059 (Erk inhibitor), or SB203580 (p38 inhibitor). (C) Soft agar colony-forming capacity of NIH3T3/hSPC3 cells in the presence of API2, PD98059, or SB203580; (C, Right) Representative images of average colony size in each group; (C, Left) bar graph showing the number of colonies with diameters greater than 2 mm in soft agar (scale bar: 1 mm). (D) Western blot analysis of levels of pAKT in NIH3T3/hSCP3 cells transfected with a siRNA targeting GFP or AKT (siGFP or siAKT) to confirm the reduction of AKT protein level. Numbers below western blots refer to the relative values of the intensity normalized to no insert control. (E) In vitro growth curves of NIH3T3/hSCP3 cells transfected with siGFP or siAKT. (F) Soft agar colony-forming capacity of NIH3T3/hSPC3 cells treated with siGFP or siAKT; (Left) Representative images of average colony size in each group; (Right) Bar graph representing the number of colonies with diameters greater than 2 mm in soft agar (scale bar: 1 mm). Error bars represent the mean ± SD.
Mentions: We previously reported that pAKT level is increased in hSCP3-expressing cervical cell lines [10]. To evaluate changes in various signaling molecules that may play a role in the global control of cell proliferation, we performed Western blot analysis to measure the levels of Ser 473 phosphorylated AKT, Thr 202/Tyr 204 phosphorylated ERK, and Thr 180/Tyr 182 phosphorylated p38 MAP kinase in NIH3T3 cells expressing hSCP3 or no insert (empty vector). We observed that the expression of pAKT was significantly increased in cells transduced with hSCP3 compared to control cells (Figure 2A). Next, to explore the relationship between hSCP3-mediated tumorigenic potential and the AKT signaling, we compared both cell proliferation and soft agar colony forming capability of NIH3T3/hSCP3 cells in the presence of control (DMSO), 10 µM API2 (an inhibitor of AKT), 50 µM PD98059 (an inhibitor of MEK/ERK) or 10 µM SB203580 (an inhibitor of p38-MAPK) (Figure 2B and 2C). Treatment with PD 98059 and SB203580 did not significantly affect the proliferation and the colony formation capacity of NIH3T3/hSCP3 cells compared with that of DMSO. On the contrary, API2 treated NIH3T3/hSCP3 cells exhibited a significantly decreased proliferation rate compared with DMSO-treated control (P<0.001). Consistently, API2-treated NIH3T3/hSCP3 cells also formed smaller colonies, the number of which was almost 5-times less than colonies formed by DMSO-treated NIH3T3/hSCP3 cells. Thus, it is obvious that both proliferation and colony formation in these cells are dependent on the AKT signaling. To exclude potential off-target effects of API2, we performed in vitro cell proliferation and soft agar colony formation experiments using siRNA-targeting AKT (siAKT) to block the AKT pathway. siRNA targeting to irrelevant GFP (GFP siRNA) was used as a negative control. Consistent with API2 data, siAKT-treated NIH3T3/hSCP3 cells exhibited a decreased rate of cell proliferation (Figure 2D and 2E) and colony number compared with control siGFP-treated NIH3T3/hSCP3 cells (Figure 2F). Similar AKT dependency was also observed in NIH3T3 cells expressing mXLR, which displayed the most potent tumorigenic potential among members of the murine Cor1 family (Figure S3). Thus, our data demonstrate that SCP3 and the other Cor1 members could confer tumorigenic potentials upon NIH3T3 cell through AKT pathway.

Bottom Line: Functional studies using NIH3T3 cells demonstrated that the C-terminal region of human SCP3 is important for AKT activation and its oncogenic potential.High expression of SCP3 was significantly associated with tumor stage (P = 0.002) and tumor grade (P<0.001), while SCP3 expression was positively associated with pAKT protein level in cervical neoplasias.Survival times for patients with cervical cancer overexpressing both SCP3 and pAKT (median, 134.0 months, n = 68) were significantly shorter than for patients with low expression of either SCP3 or pAKT (161.5 months, n = 108) as determined by multivariate analysis (P = 0.020).

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Republic of Korea; Institute of Women's Life Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea.

ABSTRACT
Synaptonemal complex protein 3 (SCP3), a member of Cor1 family, is up-regulated in various cancer cells; however, its oncogenic potential and clinical significance has not yet been characterized. In the present study, we investigated the oncogenic role of SCP3 and its relationship with phosphorylated AKT (pAKT) in cervical neoplasias. The functional role of SCP3 expression was investigated by overexpression or knockdown of SCP3 in murine cell line NIH3T3 and human cervical cancer cell lines CUMC6, SiHa, CaSki, and HeLa both in vitro and in vivo. Furthermore, we examined SCP3 expression in tumor specimens from 181 cervical cancer and 400 cervical intraepithelial neoplasia (CIN) patients by immunohistochemistry and analyzed the correlation between SCP3 expression and clinicopathologic factors or survival. Overexpression of SCP3 promoted AKT-mediated tumorigenesis both in vitro and in vivo. Functional studies using NIH3T3 cells demonstrated that the C-terminal region of human SCP3 is important for AKT activation and its oncogenic potential. High expression of SCP3 was significantly associated with tumor stage (P = 0.002) and tumor grade (P<0.001), while SCP3 expression was positively associated with pAKT protein level in cervical neoplasias. Survival times for patients with cervical cancer overexpressing both SCP3 and pAKT (median, 134.0 months, n = 68) were significantly shorter than for patients with low expression of either SCP3 or pAKT (161.5 months, n = 108) as determined by multivariate analysis (P = 0.020). Our findings suggest that SCP3 plays an important role in the progression of cervical cancer through the AKT signaling pathway, supporting the possibility that SCP3 may be a promising novel cancer target for cervical cancer therapy.

Show MeSH
Related in: MedlinePlus