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Osteopontin promotes a cancer stem cell-like phenotype in hepatocellular carcinoma cells via an integrin-NF-κB-HIF-1α pathway.

Cao L, Fan X, Jing W, Liang Y, Chen R, Liu Y, Zhu M, Jia R, Wang H, Zhang X, Zhang Y, Zhou X, Zhao J, Guo Y - Oncotarget (2015)

Bottom Line: Depletion of OPN in HCC cell lines resulted in a reduction in the proportion of side population fractions, formation of hepato-spheroids, expression of stem-cell-associated genes and decreased tumorigenecity in immunodeficient mice.Suppression of the αvβ3-NF-κB-HIF-1α pathway decreased OPN-mediated self-renewal capabilities.Levels of OPN protein expression were significantly correlated with HIF-1α protein levels in HCC tumor tissue samples.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Shanghai Jiao Tong University, Shanghai, 200025, People's Republic of China.

ABSTRACT
There is increasing evidence to suggest that hepatocellular carcinomas (HCCs) are sustained by a distinct subpopulation of self-renewing cells known as cancer stem cells. However, the precise signals required for maintenance of stemness-like properties of these cells are yet to be elucidated. Here, we demonstrated that the level of oncoprotein osteopontin (OPN) in tumor cells of the edge of bulk tumors was significantly correlated with the clinical prognosis of patients with HCC. OPN was highly expressed in side population fractions of HCC cell lines, as well as in dormant cells, spheroids and chemo-resistant cancer cells, all of which are considered as having stemness-like cellular features. Depletion of OPN in HCC cell lines resulted in a reduction in the proportion of side population fractions, formation of hepato-spheroids, expression of stem-cell-associated genes and decreased tumorigenecity in immunodeficient mice. Mechanistically, OPN was demonstrated to bind to integrin αvβ3 and activate the transcription factor NF-κB, which resulted in upregulation of HIF-1α transcription and its downstream gene, BMI1, to mediate maintenance of the stemness-like phenotype. Suppression of the αvβ3-NF-κB-HIF-1α pathway decreased OPN-mediated self-renewal capabilities. Levels of OPN protein expression were significantly correlated with HIF-1α protein levels in HCC tumor tissue samples. OPN might promote a cancer stem cell-like phenotype via the αvβ3-NF-κB-HIF-1α pathway. Our findings offer strong support for OPN requirement in maintaining stem-like properties in HCC cells.

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OPN expression is high in a side population of HCC cells(A) The side population fraction of HCC cells is higher in metastatic HCC cell lines (HCCLM3, HCCC97H, HCC97L) than in non-metastatic HCC cell lines (Hep3B, PLC/PRF/5, HuH7). Side population cells are indicated by the gate. (B) Secreted OPN protein is increased in metastatic HCC cell lines compared with non-metastatic HCC cell lines. (C) Cells in the side population fractions have higher levels of OPN expression than cells in the main population fraction either by western blot assay or (D) by immunofluoresence. Magnification ×40.
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Figure 1: OPN expression is high in a side population of HCC cells(A) The side population fraction of HCC cells is higher in metastatic HCC cell lines (HCCLM3, HCCC97H, HCC97L) than in non-metastatic HCC cell lines (Hep3B, PLC/PRF/5, HuH7). Side population cells are indicated by the gate. (B) Secreted OPN protein is increased in metastatic HCC cell lines compared with non-metastatic HCC cell lines. (C) Cells in the side population fractions have higher levels of OPN expression than cells in the main population fraction either by western blot assay or (D) by immunofluoresence. Magnification ×40.

Mentions: Side-population fractions of six HCC cell lines, including HCCLM3, HCC97H, HCC97L, Hep3B, PLC/PRF/5 and HuH7 were analyzed (Figure 1A). In metastatic HCC lines (HCCLM3, HCC97H and HCC97L), the side-population fractions ranged from 23.7% to 7.86%. By comparison, proportion of side-population cells in non-metastatic HCC lines (Hep3B, PLC/PRF/5 and HuH7) were markedly lower and ranged from 1.53% to 0.5%. We then assessed the relationship between side-population fractions and levels of secreted OPN in HCC cell lines. Consistent with a role for OPN in driving metastasis, levels of secreted OPN were much higher in metastatic cell lines with high side-population fractions than those in the non-metastatic cell lines with low side-population fractions (Figure 1B). The self-renewal capacity of cells in the side and main populations of HCCLM3 cells was also examined. In both sphere-forming and colony-forming assays, cells in the side population fraction formed great numbers of both spheres and colonies than cells in the main population. This finding indicates that cells in the side population have greater self-renewal capacity than those in the main population of the HCCLM3 cell line (Supplementary Figures S1A and S1B). To further delineate the association of OPN production and cells in the side-population fraction, we sorted the side-population and main-population from HCCLM3 cells and assessed levels of OPN protein expression. Western blot revealed a great increase of OPN protein levels in side-population cells compared with main-population cells (Figure 1C). This finding was supported by semi-quantitative immunofluorescence analysis, which suggested that more OPN protein was present in side-population cells versus main-population cells (Figure 1D).


Osteopontin promotes a cancer stem cell-like phenotype in hepatocellular carcinoma cells via an integrin-NF-κB-HIF-1α pathway.

Cao L, Fan X, Jing W, Liang Y, Chen R, Liu Y, Zhu M, Jia R, Wang H, Zhang X, Zhang Y, Zhou X, Zhao J, Guo Y - Oncotarget (2015)

OPN expression is high in a side population of HCC cells(A) The side population fraction of HCC cells is higher in metastatic HCC cell lines (HCCLM3, HCCC97H, HCC97L) than in non-metastatic HCC cell lines (Hep3B, PLC/PRF/5, HuH7). Side population cells are indicated by the gate. (B) Secreted OPN protein is increased in metastatic HCC cell lines compared with non-metastatic HCC cell lines. (C) Cells in the side population fractions have higher levels of OPN expression than cells in the main population fraction either by western blot assay or (D) by immunofluoresence. Magnification ×40.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4466639&req=5

Figure 1: OPN expression is high in a side population of HCC cells(A) The side population fraction of HCC cells is higher in metastatic HCC cell lines (HCCLM3, HCCC97H, HCC97L) than in non-metastatic HCC cell lines (Hep3B, PLC/PRF/5, HuH7). Side population cells are indicated by the gate. (B) Secreted OPN protein is increased in metastatic HCC cell lines compared with non-metastatic HCC cell lines. (C) Cells in the side population fractions have higher levels of OPN expression than cells in the main population fraction either by western blot assay or (D) by immunofluoresence. Magnification ×40.
Mentions: Side-population fractions of six HCC cell lines, including HCCLM3, HCC97H, HCC97L, Hep3B, PLC/PRF/5 and HuH7 were analyzed (Figure 1A). In metastatic HCC lines (HCCLM3, HCC97H and HCC97L), the side-population fractions ranged from 23.7% to 7.86%. By comparison, proportion of side-population cells in non-metastatic HCC lines (Hep3B, PLC/PRF/5 and HuH7) were markedly lower and ranged from 1.53% to 0.5%. We then assessed the relationship between side-population fractions and levels of secreted OPN in HCC cell lines. Consistent with a role for OPN in driving metastasis, levels of secreted OPN were much higher in metastatic cell lines with high side-population fractions than those in the non-metastatic cell lines with low side-population fractions (Figure 1B). The self-renewal capacity of cells in the side and main populations of HCCLM3 cells was also examined. In both sphere-forming and colony-forming assays, cells in the side population fraction formed great numbers of both spheres and colonies than cells in the main population. This finding indicates that cells in the side population have greater self-renewal capacity than those in the main population of the HCCLM3 cell line (Supplementary Figures S1A and S1B). To further delineate the association of OPN production and cells in the side-population fraction, we sorted the side-population and main-population from HCCLM3 cells and assessed levels of OPN protein expression. Western blot revealed a great increase of OPN protein levels in side-population cells compared with main-population cells (Figure 1C). This finding was supported by semi-quantitative immunofluorescence analysis, which suggested that more OPN protein was present in side-population cells versus main-population cells (Figure 1D).

Bottom Line: Depletion of OPN in HCC cell lines resulted in a reduction in the proportion of side population fractions, formation of hepato-spheroids, expression of stem-cell-associated genes and decreased tumorigenecity in immunodeficient mice.Suppression of the αvβ3-NF-κB-HIF-1α pathway decreased OPN-mediated self-renewal capabilities.Levels of OPN protein expression were significantly correlated with HIF-1α protein levels in HCC tumor tissue samples.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Shanghai Jiao Tong University, Shanghai, 200025, People's Republic of China.

ABSTRACT
There is increasing evidence to suggest that hepatocellular carcinomas (HCCs) are sustained by a distinct subpopulation of self-renewing cells known as cancer stem cells. However, the precise signals required for maintenance of stemness-like properties of these cells are yet to be elucidated. Here, we demonstrated that the level of oncoprotein osteopontin (OPN) in tumor cells of the edge of bulk tumors was significantly correlated with the clinical prognosis of patients with HCC. OPN was highly expressed in side population fractions of HCC cell lines, as well as in dormant cells, spheroids and chemo-resistant cancer cells, all of which are considered as having stemness-like cellular features. Depletion of OPN in HCC cell lines resulted in a reduction in the proportion of side population fractions, formation of hepato-spheroids, expression of stem-cell-associated genes and decreased tumorigenecity in immunodeficient mice. Mechanistically, OPN was demonstrated to bind to integrin αvβ3 and activate the transcription factor NF-κB, which resulted in upregulation of HIF-1α transcription and its downstream gene, BMI1, to mediate maintenance of the stemness-like phenotype. Suppression of the αvβ3-NF-κB-HIF-1α pathway decreased OPN-mediated self-renewal capabilities. Levels of OPN protein expression were significantly correlated with HIF-1α protein levels in HCC tumor tissue samples. OPN might promote a cancer stem cell-like phenotype via the αvβ3-NF-κB-HIF-1α pathway. Our findings offer strong support for OPN requirement in maintaining stem-like properties in HCC cells.

Show MeSH
Related in: MedlinePlus