Subcellular Localization And Function Of CCDC170/C6ORF97 In Carcinogenesis And Progression Of Breast Cancer

Background and ObjectiveCCDC170/ C6ORF97 gene is located in the region of 6q25.1 on the long arm of chromosome 6 adjacent to estrogen receptorα(ESR1) and it is the open reading frame in the upstream of ESR1. Recently, the CCDC170/C6ORF97-ESR1 locus has been documented to be a promising candidate gene involving the development for both hereditary and sporadic breast cancer. The findings from many studies suggest a clear involvement of CCDC170/C6ORF97 in breast cancer susceptibility. Recent genome-wide association studies(GWAS) suggest that a dozen variants located at the CCDC170/C6ORF97-ESR1 locus(6q25.1) are associated with risk of breast cancer and osteoporosis. In the previous research, single nucleotide polymorphisms(SNP) rs2046210 related to breast cancer risk in the CCDC170/C6ORF97-ESR1 intergenic area was identified. Numerous association studies have been carried out to investigate the relationship between this GWAS locus and breast cancer risk in various populations. Rs2046210 is found to have stronger risk-association in estrogen receptor-negative breast tumors than those in estrogen receptor-positive breast tumors, which suggests that this risk variant is likely ESR1-independent.Nonsense mutations(e.g. p E48 and p Q405) in CCDC170 have been reported in sporadic breast cancer and other cancers by researchers. Recently, a tumor-specific gene rearrangement from the second noncoding exon of ESR1 to the sixth and/or seventh exon(s) of CCDC170 was reported by several studies using high-throughput RNA-sequencing. Further study demonstrated that N-terminally truncated CCDC170 proteins(p.593-715) resulted from this ESR1-CCDC170 rearrangement. Ectopic expression of these truncated proteins increases breast cancer cell motility and enhances the transformation of normal myoepithelial cells. Numerous studies have indicated that a variety of perturbations of the CCDC170 protein played an important role in breast cancer initiation and/or progression. Therefore, it is imperative to conduct a in-depth research on functional characterization of this relatively unknown gene. But up to now, the study on subcellular localization and function of CCDC170/C6ORF97 in carcinogenesis and progression of breast cancer has not been reported in the world. This article intends to explore subcellular localization of full-length CCDC170/C6ORF97 protein and its mutants, and to investigate their roles and mechanisms in carcinogenesis and progression of breast cancer for the first time.Part 1 Subcellular localization of CCDC170/C6ORF97 protein and its mutantsObjectives To observe subcellular localization of full-length CCDC170/C6ORF97 protein and its mutants in tumor cells, and to explore the possible mechanism on subcellular localization.Materials and methods 1. By employing molecular cloning technique, The recombinant plasmids carrying GFP gene, CCDC170 gene and its truncated mutants were constructed and used for the following experimental researches. These constructed plasmids included Tight plasmids(TRE-Tight-GFP、TRE-Tight-GFP-CCDC170、TRE-Tight-GFP-Q405、TRE-Tight-GFP-E48 、 TRE-Tight-GFP-p.593-715) and non-Tight plasmid(p CMV6-GFP-p.593-715). 2. By employing side-directed mutagenesis, The recombinant plasmids carrying CCDC170 gene mutations at nucleotide 1810 site and 2047 site were constructed and used for the following experimental researches. 3. MCF7-tet-on cells were transfected with the constructed plasmids, and the expression of full-length CCDC170/C6ORF97 protein and its truncated mutants was detected by western blotting to validate that the plasmids were correctly constructed. 4. MCF7-tet-on cells and Hela cells were transfected with the constructed plasmids. Anti-GFP and anti-RCAS1 antibodies were used as probes for immuno-fluorescence, and then the reactions of them with different parts of the cells were detected and photographed by laser confocal microscope and fluorescence microscope. By this means, subcellular localization of full-length CCDC170/C6ORF97 protein and its truncated mutants in MCF7-tet-on cells and Hela cells was observed.Results 1.MCF7-tet-on cells were transfected with the constructed plasmids, and then western blotting with anti-GFP and anti-CCDC170 antibody were used to validate the expression of full-length CCDC170/C6ORF97 protein and its truncated mutants. The result showed that the fused proteins GFP-CCDC170 or GFP-truncated CCDC170 with correct molecular mass were expressed. 2. The result of subcellular localization of CCDC170 in MCF7-tet-on cell revealed that full-length CCDC170 protein was located at Golgi. CCDC170 mutation proteins resulting from CCDC170 gene point mutations at nucleotide 1810 site or 2047 site were also located at Golgi, with no significant difference from full-length CCDC170 protein. Whereas, p Q405,one of truncated CCDC170 proteins, was located in the cytoplasm. 3. The result of subcellular localization of CCDC170 in Hela cell revealed that full-length CCDC170 protein was located at Golgi. And subcellular localization of the fused proteins GFP-truncated CCDC170 was as follows. GFP-p Q 649 was located in outer nuclear periphery. GFP-p Q405 and GFP-V264 were located in the cytoplasm. GFP-p E48 and GFP- p.593-715 were located in whole cell. The findings from this study suggested that truncated CCDC170 resulted in the loss of normal subcellular localization of wild-type CCDC170. 4. The results reveal that CCDC170 attaches to the Golgi surface through its C-terminus and two sequences at C terminal of CCDC170(region a and region b) may play an important role in Golgi localization. The immediate C-terminus(region b) is necessary, but not sufficient for Golgi localization. Attachment through the immediate C-terminus(region b) may require a second sequence(region a).Part 2 The roles and mechanisms of CCDC170/C6ORF97 in carcinogenesis and progression of breast cancerObjectives To observe the influence of CCDC170 on the proliferation and motility of breast cancer cell as well as the relationship of CCDC170 with the expression of tumor molecular marks and acetylation of tubulin, and to investigate the basic expression of CCDC170 in breast cancer cell and its effect on cell proliferation, so as to explore the roles and mechanisms of CCDC170 in carcinogenesis and progression of breast cancer.Materials and methods 1.MCF7-tet-on cells were transfected with the constructed plasmids carrying wild-type CCDC170 gene and its truncated mutants, and the proportion of Ki-67 positive cells in all GFP positive MCF7-tet-on cells was detected by immunofluorescence technique. 2. MCF7-tet-on cells were transfected with the constructed plasmids carrying wild-type CCDC170 gene and its truncated mutants, and the number of cell colony as well as the proliferation and motility of transfected MCF7-tet-on cells were detected by soft agar assay. 3. MCF7-tet-on cells were transfected with the constructed plasmids carrying wild-type CCDC170 gene and its truncated mutants, and western blotting was used to investigate the relationship of CCDC170 with the expression of tumor molecular marks such as p53, Cleaved PARP-1, EGFR, Gab1, AKT, p AKT, p STAT3.4. MCF7-tet-on cells were transfected with CCDC170 Si RNA for CCDC170 knockdown, and wound healing assay with CCDC170 knockdown MCF7-tet-on cells was conducted to investigate the basic expression of CCDC170 in breast cancer cell and its effect on cell proliferation. 5. Immunofluorescence technique was employed to observe the influence of full-length CCDC170/C6ORF97 protein and its truncated mutants on acetylation of tubulin in tumor cell.Results 1.The results of immunofluorescence showed that for MCF7-tet-on cells with positive GFP, the proportion of Ki-67 positive cells in p CMV6-GFP group, p CMV6-GFP-CCDC170 group and p CMV6-GFP-Q405 group was 67%, 44% and 48%, respectively. The proportion of Ki-67 positive cells in p CMV6-GFP-CCDC170 group or p CMV6-GFP-Q405 group was significantly lower than that in p CMV6-GFP group(P<0.05). And there was no significant difference in the proportion of Ki-67 positive cells between p CMV6-GFP-CCDC170 group and p CMV6-GFP-Q405 group( P> 0.05). 2. The results of immunofluorescence showed that for MCF-7-tet-on cells with high expression of GFP, the proportion of Ki-67 positive cells in p CMV6-GFP group, p CMV6-GFP-CCDC170 group and p CMV6-GFP-p.593-715 group was 57%, 32% and 71%, respectively. The proportion of Ki-67 positive cells in p CMV6-GFP-CCDC170 group was significantly lower than that in p CMV6-GFP group(P < 0.001) and The proportion of Ki-67 positive cells in p CMV6-GFP-p.593-715 group was significantly higher than that in p CMV6-GFP group(P<0.05). However, for MCF-7-tet-on cells with low expression of GFP, The proportion of Ki-67 positive cells in p CMV6-GFP-CCDC170 group was significantly lower than that in p CMV6-GFP group(P < 0.05) and there was no significant difference in the proportion of Ki-67 positive cells between p CMV6-GFP-p.593-715 group and p CMV6-GFP group( P> 0.05). 3. The results of soft agar assay showed that the number of cell colony in p CMV6-GFP-CCDC170 group or p CMV6-GFP-Q405 group was less than that in p CMV6-GFP group. And the number of cell colony in p CMV6-GFP-p.593-715 group was more than that in p CMV6-GFP group. The number of cell colony in p CMV6-GFP- CCDC170 group was similar to that in p CMV6-GFP-Q405 group. 4. The results of western blotting showed that the expression intensity of cleaved PARP-1 in p CMV6-GFP-CCDC170 group was higher than that in p CMV6-GFPp.593-715 group or p CMV6-GFP group. However, the expression intensity of p53, p STAT3, Gab1, AKT, p AKT and EGFR in p CMV6-GFP-CCDC170 group was similar to that in p CMV6-GFP- p.593-715 group or p CMV6-GFP group. 5. The results of CCDC170 knockdown and wound healing assay showed that with the healing time going on, wound area in si RNA control group decreased obviously and wound areas in si RNA A, si RNA C group also decreased with the lower decrement. In si RNA control group, the wound healing rate 24, 48, 72, 96 hours after creating a wound was 17.80%, 34.68%, 48.71%, 61.35%, respectively. 24, 48, 72, 96 hours after creating a wound, the wound healing rate in si RNA A group was 7.23%、16.98%、26.37%、38.90%, respectively and the wound healing rate in si RNA C group was 14.25%、24.25%、31.61%、43.21%, respectively. The speed of wound healing in si RNA A group or si RNA C group was significantly slower than that in si RNA control group. 6. The results of immunofluorescence showed that acetylated tubulin was expressed in different expression intensity in hela cells transfected with p CMV6-GFP, p CMV6-GFP-CCDC170, p CMV6-GFP-Q405, p CMV6-GFP-p.593-715. And the corrected total cell fluorescence(CTCF) of the red fluorescence corresponding to acetylated tubulin was calculated. The results indicated that the CTCF of p CMV6-GFP-CCDC170 group was the highest, that of p CMV6-GFP-Q405 group was second, and that of p CMV6-GFP group and p CMV6-GFP-p.593-715 group was the lowest. The CTCF of p CMV6-GFP-CCDC170 group or p CMV6-GFP-Q405 group was significantly higher than that of p CMV6-GFP group(P<0.05) and there was no significant difference in the CTCF between p CMV6-GFP-p.593-715 group and p CMV6-GFP group( P> 0.05).Conclusions 1.This study revealed that wild-type CCDC170 showed clear localization to the Golgi and CCDC170 was a member of a group of resident Golgi coiled-coil proteins. The results suggest that truncated CCDC170 can result in the loss of normal cellular localization of wild-type CCDC170. 2. The results suggest that CCDC170 attaches to the Golgi surface through its C-terminus and two sequences at C terminal of CCDC170 may play an important role in Golgi localization. 3. Overexpression of wild-type CCDC170 could apparently inhibit cell proliferation, motility, invasion and p Q405 could also inhibit cell proliferation. However, p.593-715 could promote cell proliferation. The results suggest that wild-type CCDC170 or truncated CCDC170 may play different role in the regulation of proliferation, motility, invasion of breast cancer cell. 4. There were some difference in both subcellular localization and function between wild-type CCDC170 and truncated CCDC170, which suggested that there was a correlation between subcellular localization and tumorigenic potential of CCDC170. The results reveal that disruption of the normal localization at the Golgi of wild-type CCDC170 resulting from gene mutation will lead to the changes in proliferation, invasion of breast cancer cell and consequently, will influence the progression of breast cancer. 5. The basic expression of wild-type CCDC170 could promote proliferation of breast cancer cell and the overexpression of wild-type CCDC170 could apparently inhibit proliferation of breast cancer cell. The results suggest that it is possible that the overexpression of wild-type CCDC170 can inhibit the tumor cell proliferation by regulating the decomposition of PARP-1 and therefore accelerating breast cancer cell apoptosis or by promoting acetylation of tubulin. Different effect of wild-type CCDC170 or truncated CCDC170 on breast cancer cell proliferation may relate with acetylation of tubulin. 6. The results of this study first elaborate subcellular localization and function of CCDC170, a new protein related with breast cancer and reveal the roles and mechanisms of CCDC170 in carcinogenesis and progression of breast cancer, which fill the gaps in this research field. It is expected as a novel possible strategy for breast cancer treatment, targeting abnormalities in CCDC170 associated pathways will uncover a new way for the targeted therapy in breast cancer and consequently improve the therapeutic effect of breast cancer.