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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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The C-terminal region of hSCP3 is important for its oncogenic capability.(A) Schematic of the domains in hSCP3. Boxes represent the NLS, Gln-rich, and coiled-coil motifs, respectively. (B) Western blot analysis of levels of hSCP3 and its deletion mutants, the N-termini of which were tagged with a Flag epitope for visualization. Numbers below western blots refer to the relative values of the intensity normalized to no insert control. (C) Soft agar colony-forming capacity of NIH3T3 expressing wild type or deletion mutants of hSCP3. (D) In vitro growth curves of NIH3T3 cells expressing wild type or deletion mutants of hSCP3. Cells were counted after trypan blue staining to exclude dead cells. (E) In vivo tumorigenicity of the NIH3T3 cells. Balb/c Nude mice (n = 5) were inoculated subcutaneously with 1×105 cells/mouse of the NIH3T3 cells expressing full length or mutant hSPC3. Error bars represent the mean ± SD. (F) Representative images of skin tumors (scale bar: 60 mm). (G) Bar graph representing intensity of pAKT fluorescence image on tumor tissue sections, ImageJ densitometry software was used to quantitate the intensity of image as described in Materials and Methods. Error bars represent the mean ± SD.
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pone-0098712-g003: The C-terminal region of hSCP3 is important for its oncogenic capability.(A) Schematic of the domains in hSCP3. Boxes represent the NLS, Gln-rich, and coiled-coil motifs, respectively. (B) Western blot analysis of levels of hSCP3 and its deletion mutants, the N-termini of which were tagged with a Flag epitope for visualization. Numbers below western blots refer to the relative values of the intensity normalized to no insert control. (C) Soft agar colony-forming capacity of NIH3T3 expressing wild type or deletion mutants of hSCP3. (D) In vitro growth curves of NIH3T3 cells expressing wild type or deletion mutants of hSCP3. Cells were counted after trypan blue staining to exclude dead cells. (E) In vivo tumorigenicity of the NIH3T3 cells. Balb/c Nude mice (n = 5) were inoculated subcutaneously with 1×105 cells/mouse of the NIH3T3 cells expressing full length or mutant hSPC3. Error bars represent the mean ± SD. (F) Representative images of skin tumors (scale bar: 60 mm). (G) Bar graph representing intensity of pAKT fluorescence image on tumor tissue sections, ImageJ densitometry software was used to quantitate the intensity of image as described in Materials and Methods. Error bars represent the mean ± SD.

Mentions: As schematized in Figure 3A, hSCP3 contains a nuclear localization signal (NLS, residues 88–91), Gln-rich (residues 106–139) and coiled-coil (residues 131–236) motifs. To identify key motifs involved in hSCP3-mediated oncogenesis, we constructed four deletion mutants of hSCP3 containing different motifs (Figure 3A). The N-termini of full hSCP3 and the deletion mutants were tagged with a Flag epitope for visualization by Western blot. We then characterized the expression and the function of hSCP3 and its mutants in retrovirus-transduced NIH3T3 cells. Notably, mutants lacking the coiled-coil motif (hSCP31-80 and hSCP31-130) failed to increase phosphorylation of AKT (Figure 3B). Conversely, hSCP381-236 and hSCP3131-236 mutants containing the coiled-coil motif successfully increased levels of pAKT (Figure 3B). Consistent with these observations, NIH3T cells expressing hSCP381-236 or hSCP3131-236 exhibited an increased rate of cell proliferation and number of colony compared with those expressing hSCP31-80 and hSCP31-130 as well as no insert (Figure 3C and 3D). More importantly, as shown in Figure 3E-3G, similar phenomena were also observed in vivo with tumor xenograft experiments. The tumor-forming ability of NIH3T3 cells was drastically increased after ectopic expression of hSCP381-236 and hSCP3131-236 [no insert vs. hSCP381-236 (P<0.01) or hSCP3131-236 (P<0.003)]. Similarly, immunofluorescence staining for pAKT was increased substantially in histological sections of tumors ectopically expressing hSCP381-236 or hSCP3131-236 [no insert vs. hSCP381-236 (P<0.05) or hSCP3131-236 (P<0.003)] (Figure 3G). Taken together, our data indicate that the coiled-coil motif containing the C-terminal region is the primary contributor to the AKT-dependent oncogenic potential of hSCP3.


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)

The C-terminal region of hSCP3 is important for its oncogenic capability.(A) Schematic of the domains in hSCP3. Boxes represent the NLS, Gln-rich, and coiled-coil motifs, respectively. (B) Western blot analysis of levels of hSCP3 and its deletion mutants, the N-termini of which were tagged with a Flag epitope for visualization. Numbers below western blots refer to the relative values of the intensity normalized to no insert control. (C) Soft agar colony-forming capacity of NIH3T3 expressing wild type or deletion mutants of hSCP3. (D) In vitro growth curves of NIH3T3 cells expressing wild type or deletion mutants of hSCP3. Cells were counted after trypan blue staining to exclude dead cells. (E) In vivo tumorigenicity of the NIH3T3 cells. Balb/c Nude mice (n = 5) were inoculated subcutaneously with 1×105 cells/mouse of the NIH3T3 cells expressing full length or mutant hSPC3. Error bars represent the mean ± SD. (F) Representative images of skin tumors (scale bar: 60 mm). (G) Bar graph representing intensity of pAKT fluorescence image on tumor tissue sections, ImageJ densitometry software was used to quantitate the intensity of image as described in Materials and Methods. Error bars represent the mean ± SD.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4048308&req=5

pone-0098712-g003: The C-terminal region of hSCP3 is important for its oncogenic capability.(A) Schematic of the domains in hSCP3. Boxes represent the NLS, Gln-rich, and coiled-coil motifs, respectively. (B) Western blot analysis of levels of hSCP3 and its deletion mutants, the N-termini of which were tagged with a Flag epitope for visualization. Numbers below western blots refer to the relative values of the intensity normalized to no insert control. (C) Soft agar colony-forming capacity of NIH3T3 expressing wild type or deletion mutants of hSCP3. (D) In vitro growth curves of NIH3T3 cells expressing wild type or deletion mutants of hSCP3. Cells were counted after trypan blue staining to exclude dead cells. (E) In vivo tumorigenicity of the NIH3T3 cells. Balb/c Nude mice (n = 5) were inoculated subcutaneously with 1×105 cells/mouse of the NIH3T3 cells expressing full length or mutant hSPC3. Error bars represent the mean ± SD. (F) Representative images of skin tumors (scale bar: 60 mm). (G) Bar graph representing intensity of pAKT fluorescence image on tumor tissue sections, ImageJ densitometry software was used to quantitate the intensity of image as described in Materials and Methods. Error bars represent the mean ± SD.
Mentions: As schematized in Figure 3A, hSCP3 contains a nuclear localization signal (NLS, residues 88–91), Gln-rich (residues 106–139) and coiled-coil (residues 131–236) motifs. To identify key motifs involved in hSCP3-mediated oncogenesis, we constructed four deletion mutants of hSCP3 containing different motifs (Figure 3A). The N-termini of full hSCP3 and the deletion mutants were tagged with a Flag epitope for visualization by Western blot. We then characterized the expression and the function of hSCP3 and its mutants in retrovirus-transduced NIH3T3 cells. Notably, mutants lacking the coiled-coil motif (hSCP31-80 and hSCP31-130) failed to increase phosphorylation of AKT (Figure 3B). Conversely, hSCP381-236 and hSCP3131-236 mutants containing the coiled-coil motif successfully increased levels of pAKT (Figure 3B). Consistent with these observations, NIH3T cells expressing hSCP381-236 or hSCP3131-236 exhibited an increased rate of cell proliferation and number of colony compared with those expressing hSCP31-80 and hSCP31-130 as well as no insert (Figure 3C and 3D). More importantly, as shown in Figure 3E-3G, similar phenomena were also observed in vivo with tumor xenograft experiments. The tumor-forming ability of NIH3T3 cells was drastically increased after ectopic expression of hSCP381-236 and hSCP3131-236 [no insert vs. hSCP381-236 (P<0.01) or hSCP3131-236 (P<0.003)]. Similarly, immunofluorescence staining for pAKT was increased substantially in histological sections of tumors ectopically expressing hSCP381-236 or hSCP3131-236 [no insert vs. hSCP381-236 (P<0.05) or hSCP3131-236 (P<0.003)] (Figure 3G). Taken together, our data indicate that the coiled-coil motif containing the C-terminal region is the primary contributor to the AKT-dependent oncogenic potential of hSCP3.

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