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Sortilin is associated with breast cancer aggressiveness and contributes to tumor cell adhesion and invasion.

Roselli S, Pundavela J, Demont Y, Faulkner S, Keene S, Attia J, Jiang CC, Zhang XD, Walker MM, Hondermarck H - Oncotarget (2015)

Bottom Line: It was found in 79% of invasive ductal carcinomas and 54% of invasive lobular carcinomas (p < 0.0001).Breast cancer cell migration and invasion were also inhibited by sortilin knockdown, with a decrease in focal adhesion kinase and SRC phosphorylation.In conclusion, sortilin participates in breast tumor aggressiveness and may constitute a new therapeutic target against tumor cell invasion.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences & Pharmacy, Faculty of Health and Medicine, University of Newcastle, Callaghan NSW 2308, Australia.

ABSTRACT
The neuronal membrane protein sortilin has been reported in a few cancer cell lines, but its expression and impact in human tumors is unclear. In this study, sortilin was analyzed by immunohistochemistry in a series of 318 clinically annotated breast cancers and 53 normal breast tissues. Sortilin was detected in epithelial cells, with increased levels in cancers, as compared to normal tissues (p = 0.0088). It was found in 79% of invasive ductal carcinomas and 54% of invasive lobular carcinomas (p < 0.0001). There was an association between sortilin expression and lymph node involvement (p = 0.0093), suggesting a relationship with metastatic potential. In cell culture, sortilin levels were higher in cancer cell lines compared to non-tumorigenic breast epithelial cells and siRNA knockdown of sortilin inhibited cancer cell adhesion, while proliferation and apoptosis were not affected. Breast cancer cell migration and invasion were also inhibited by sortilin knockdown, with a decrease in focal adhesion kinase and SRC phosphorylation. In conclusion, sortilin participates in breast tumor aggressiveness and may constitute a new therapeutic target against tumor cell invasion.

No MeSH data available.


Related in: MedlinePlus

Impact of sortilin knockdown on migration and invasion of breast cancer cellsA. Scratch assay. Breast cancer cells (MDA-MB-231, SKBR-3 and MCF-7) were transfected with siRNA against sortilin (siSORT) and universal negative control siRNA (siCONT). Scratching of the cell layer was performed 48 h after transfection and reduction in gap area was measured over 6 h. SiSORT inhibited migration only in MDA-MB-231 cells. B. Transwell invasion assay of MDA-MB-231 cells. Transwell assays were set up 48 h after siRNA transfection and cells were allowed to invade for 48 h. To take under account a potential impact of cell adhesion on the assay, cells were counted on both sides of the Transwell filter. Left panel, white columns represent the number of cells on the upper side of the filter, and the black columns the number of cells on the down side. Right panel, the percentage of invading cells in siSORT versus siCONT is represented. C. Western-blot detection of vimentin and activation of SRC, FAK, Akt and Erk1/2, 72 h after transfection with siSORT versus siCONT. Antibodies against vimentin, β-actin, SRC, phospho-SRC (Tyr416), FAK, phospho-FAK (Tyr576/577), Akt, phosphor-Akt (Ser473), Erk1/2, phosphor-Erk1/2 were used. For panel A and B, error bars represent SD. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not significant, for the difference between siCONT and siSORT.
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Figure 5: Impact of sortilin knockdown on migration and invasion of breast cancer cellsA. Scratch assay. Breast cancer cells (MDA-MB-231, SKBR-3 and MCF-7) were transfected with siRNA against sortilin (siSORT) and universal negative control siRNA (siCONT). Scratching of the cell layer was performed 48 h after transfection and reduction in gap area was measured over 6 h. SiSORT inhibited migration only in MDA-MB-231 cells. B. Transwell invasion assay of MDA-MB-231 cells. Transwell assays were set up 48 h after siRNA transfection and cells were allowed to invade for 48 h. To take under account a potential impact of cell adhesion on the assay, cells were counted on both sides of the Transwell filter. Left panel, white columns represent the number of cells on the upper side of the filter, and the black columns the number of cells on the down side. Right panel, the percentage of invading cells in siSORT versus siCONT is represented. C. Western-blot detection of vimentin and activation of SRC, FAK, Akt and Erk1/2, 72 h after transfection with siSORT versus siCONT. Antibodies against vimentin, β-actin, SRC, phospho-SRC (Tyr416), FAK, phospho-FAK (Tyr576/577), Akt, phosphor-Akt (Ser473), Erk1/2, phosphor-Erk1/2 were used. For panel A and B, error bars represent SD. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not significant, for the difference between siCONT and siSORT.

Mentions: The functional analysis was performed on the highly invasive and triple negative MDA-MB-231 breast cancer cell line, the HER2 overexpressing SKBR-3, and the luminal A type MCF-7 cells. Breast cancer cell lines were transfected with siRNA against sortilin versus control siRNA and the impact on cell growth, survival, adhesion, migration and invasion was measured. The efficacy of siRNA was assessed by Western-blotting at 24, 48 and 72 h after transfection (Fig. 4A). In MDA-MB-231 cells, a strong decrease in sortilin protein was observed from 24 h and was maintained after 72 h. In SKBR-3 and MCF-7 cells, the inhibition was complete only at 48 h, but was also maintained at 72 h. Microscopic observation 72 h after transfection (Fig. 4B) suggested that there were fewer cells in siRNA sortilin than in control siRNA, with a lower attachment (higher proportion of round cells). The decrease in cell number was confirmed by cell counting (Fig. 4C). This has prompted us to analyze cell cycle and apoptosis. Flow cytometry after propidium iodide incorporation (Fig. 4D) indicated no change in the proportion of cells in each phase of the cell cycle (G1/G0, S, G2M) between the siRNA sortilin and the siRNA control conditions. This demonstrated that the sortilin siRNA had no impact on cell proliferation. In addition there was also no change in subG0/G1, suggesting that siRNA against sortilin did not induce cell death. This was confirmed by Hoechst staining (Fig. 4D), as no particular nuclei fragmentation or condensation could be observed in the anti-sortilin siRNA condition. About 5% of apoptosis could be observed for all cell lines with or without anti-sortilin siRNA. Therefore, the decrease in cell number observed after sortilin siRNA transfection was not due to a decrease in cell proliferation or an increase in cell death. This has prompted us to test the impact of the sortilin siRNA on cell adhesion. Interestingly, breast cancer cell adhesion was affected by sortilin siRNA knockdown (Fig. 4E). SiRNA against sortilin resulted in 30% inhibition of MCF-7 cell adhesion, as measured 20 h after cell seeding. In SKBR3, the inhibition of cell adhesion was ~50% and it reached ~80% in MDA-MB-231 cells. These results indicated that sortilin is involved in breast cancer cell adhesion. We then investigated the impact of sortilin knockdown on breast cancer cell migration and invasion (Fig. 5). In wound healing assay, anti-sortilin siRNA inhibited the migration of MDA-MB-231 cells (Fig. 5A). In contrast the migration of MCF-7 and SKBR-3 cells was not affected (Fig. 5A). We then looked for the invasive property of MDA-MB-231 in Transwell assays. The invasion of MDA-MB-231 cells was significantly inhibited by anti-sortilin siRNA (Fig. 5B). To take under account the potential impact of the inhibition of cell adhesion on the invasion of MDA-MB-231 cells, we counted the number of cells attached in both the upper part and the down-side of the Transwell filters (Fig. 5B left panel). We then expressed the percentage (%) of invading cells, as compared to attached cells (Fig. 5B right panel). The results show that the invasion of cancer cells that had attached was inhibited and therefore, siRNA against sortilin had a direct inhibitory effect on MDA-MB-231 cell invasion. We have then explored the level of activation of various cell invasion-related signaling pathways (Fig. 5C) (the entire blots are shown in Supplemental Data). Western-blotting experiments revealed that the level of vimentin was not affected by siRNA against sortilin, indicating that the EMT (epithelial-mesenchymal transition) phenotype of MDA-MB-231 was not altered. Akt and Erk1/2 phosphorylation was also not modified, but in contrast, the activation of SRC and focal adhesion kinase (FAK) was inhibited by anti-sortilin siRNA. Therefore, the sortilin knockdown-induced inhibition of MDA-MB-231 breast cancer cell invasion involves a decrease in SRC/FAK signaling pathways.


Sortilin is associated with breast cancer aggressiveness and contributes to tumor cell adhesion and invasion.

Roselli S, Pundavela J, Demont Y, Faulkner S, Keene S, Attia J, Jiang CC, Zhang XD, Walker MM, Hondermarck H - Oncotarget (2015)

Impact of sortilin knockdown on migration and invasion of breast cancer cellsA. Scratch assay. Breast cancer cells (MDA-MB-231, SKBR-3 and MCF-7) were transfected with siRNA against sortilin (siSORT) and universal negative control siRNA (siCONT). Scratching of the cell layer was performed 48 h after transfection and reduction in gap area was measured over 6 h. SiSORT inhibited migration only in MDA-MB-231 cells. B. Transwell invasion assay of MDA-MB-231 cells. Transwell assays were set up 48 h after siRNA transfection and cells were allowed to invade for 48 h. To take under account a potential impact of cell adhesion on the assay, cells were counted on both sides of the Transwell filter. Left panel, white columns represent the number of cells on the upper side of the filter, and the black columns the number of cells on the down side. Right panel, the percentage of invading cells in siSORT versus siCONT is represented. C. Western-blot detection of vimentin and activation of SRC, FAK, Akt and Erk1/2, 72 h after transfection with siSORT versus siCONT. Antibodies against vimentin, β-actin, SRC, phospho-SRC (Tyr416), FAK, phospho-FAK (Tyr576/577), Akt, phosphor-Akt (Ser473), Erk1/2, phosphor-Erk1/2 were used. For panel A and B, error bars represent SD. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not significant, for the difference between siCONT and siSORT.
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Figure 5: Impact of sortilin knockdown on migration and invasion of breast cancer cellsA. Scratch assay. Breast cancer cells (MDA-MB-231, SKBR-3 and MCF-7) were transfected with siRNA against sortilin (siSORT) and universal negative control siRNA (siCONT). Scratching of the cell layer was performed 48 h after transfection and reduction in gap area was measured over 6 h. SiSORT inhibited migration only in MDA-MB-231 cells. B. Transwell invasion assay of MDA-MB-231 cells. Transwell assays were set up 48 h after siRNA transfection and cells were allowed to invade for 48 h. To take under account a potential impact of cell adhesion on the assay, cells were counted on both sides of the Transwell filter. Left panel, white columns represent the number of cells on the upper side of the filter, and the black columns the number of cells on the down side. Right panel, the percentage of invading cells in siSORT versus siCONT is represented. C. Western-blot detection of vimentin and activation of SRC, FAK, Akt and Erk1/2, 72 h after transfection with siSORT versus siCONT. Antibodies against vimentin, β-actin, SRC, phospho-SRC (Tyr416), FAK, phospho-FAK (Tyr576/577), Akt, phosphor-Akt (Ser473), Erk1/2, phosphor-Erk1/2 were used. For panel A and B, error bars represent SD. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not significant, for the difference between siCONT and siSORT.
Mentions: The functional analysis was performed on the highly invasive and triple negative MDA-MB-231 breast cancer cell line, the HER2 overexpressing SKBR-3, and the luminal A type MCF-7 cells. Breast cancer cell lines were transfected with siRNA against sortilin versus control siRNA and the impact on cell growth, survival, adhesion, migration and invasion was measured. The efficacy of siRNA was assessed by Western-blotting at 24, 48 and 72 h after transfection (Fig. 4A). In MDA-MB-231 cells, a strong decrease in sortilin protein was observed from 24 h and was maintained after 72 h. In SKBR-3 and MCF-7 cells, the inhibition was complete only at 48 h, but was also maintained at 72 h. Microscopic observation 72 h after transfection (Fig. 4B) suggested that there were fewer cells in siRNA sortilin than in control siRNA, with a lower attachment (higher proportion of round cells). The decrease in cell number was confirmed by cell counting (Fig. 4C). This has prompted us to analyze cell cycle and apoptosis. Flow cytometry after propidium iodide incorporation (Fig. 4D) indicated no change in the proportion of cells in each phase of the cell cycle (G1/G0, S, G2M) between the siRNA sortilin and the siRNA control conditions. This demonstrated that the sortilin siRNA had no impact on cell proliferation. In addition there was also no change in subG0/G1, suggesting that siRNA against sortilin did not induce cell death. This was confirmed by Hoechst staining (Fig. 4D), as no particular nuclei fragmentation or condensation could be observed in the anti-sortilin siRNA condition. About 5% of apoptosis could be observed for all cell lines with or without anti-sortilin siRNA. Therefore, the decrease in cell number observed after sortilin siRNA transfection was not due to a decrease in cell proliferation or an increase in cell death. This has prompted us to test the impact of the sortilin siRNA on cell adhesion. Interestingly, breast cancer cell adhesion was affected by sortilin siRNA knockdown (Fig. 4E). SiRNA against sortilin resulted in 30% inhibition of MCF-7 cell adhesion, as measured 20 h after cell seeding. In SKBR3, the inhibition of cell adhesion was ~50% and it reached ~80% in MDA-MB-231 cells. These results indicated that sortilin is involved in breast cancer cell adhesion. We then investigated the impact of sortilin knockdown on breast cancer cell migration and invasion (Fig. 5). In wound healing assay, anti-sortilin siRNA inhibited the migration of MDA-MB-231 cells (Fig. 5A). In contrast the migration of MCF-7 and SKBR-3 cells was not affected (Fig. 5A). We then looked for the invasive property of MDA-MB-231 in Transwell assays. The invasion of MDA-MB-231 cells was significantly inhibited by anti-sortilin siRNA (Fig. 5B). To take under account the potential impact of the inhibition of cell adhesion on the invasion of MDA-MB-231 cells, we counted the number of cells attached in both the upper part and the down-side of the Transwell filters (Fig. 5B left panel). We then expressed the percentage (%) of invading cells, as compared to attached cells (Fig. 5B right panel). The results show that the invasion of cancer cells that had attached was inhibited and therefore, siRNA against sortilin had a direct inhibitory effect on MDA-MB-231 cell invasion. We have then explored the level of activation of various cell invasion-related signaling pathways (Fig. 5C) (the entire blots are shown in Supplemental Data). Western-blotting experiments revealed that the level of vimentin was not affected by siRNA against sortilin, indicating that the EMT (epithelial-mesenchymal transition) phenotype of MDA-MB-231 was not altered. Akt and Erk1/2 phosphorylation was also not modified, but in contrast, the activation of SRC and focal adhesion kinase (FAK) was inhibited by anti-sortilin siRNA. Therefore, the sortilin knockdown-induced inhibition of MDA-MB-231 breast cancer cell invasion involves a decrease in SRC/FAK signaling pathways.

Bottom Line: It was found in 79% of invasive ductal carcinomas and 54% of invasive lobular carcinomas (p < 0.0001).Breast cancer cell migration and invasion were also inhibited by sortilin knockdown, with a decrease in focal adhesion kinase and SRC phosphorylation.In conclusion, sortilin participates in breast tumor aggressiveness and may constitute a new therapeutic target against tumor cell invasion.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences & Pharmacy, Faculty of Health and Medicine, University of Newcastle, Callaghan NSW 2308, Australia.

ABSTRACT
The neuronal membrane protein sortilin has been reported in a few cancer cell lines, but its expression and impact in human tumors is unclear. In this study, sortilin was analyzed by immunohistochemistry in a series of 318 clinically annotated breast cancers and 53 normal breast tissues. Sortilin was detected in epithelial cells, with increased levels in cancers, as compared to normal tissues (p = 0.0088). It was found in 79% of invasive ductal carcinomas and 54% of invasive lobular carcinomas (p < 0.0001). There was an association between sortilin expression and lymph node involvement (p = 0.0093), suggesting a relationship with metastatic potential. In cell culture, sortilin levels were higher in cancer cell lines compared to non-tumorigenic breast epithelial cells and siRNA knockdown of sortilin inhibited cancer cell adhesion, while proliferation and apoptosis were not affected. Breast cancer cell migration and invasion were also inhibited by sortilin knockdown, with a decrease in focal adhesion kinase and SRC phosphorylation. In conclusion, sortilin participates in breast tumor aggressiveness and may constitute a new therapeutic target against tumor cell invasion.

No MeSH data available.


Related in: MedlinePlus