Osteopontin selectively regulates p70S6K/mTOR phosphorylation leading to NF-kappaB dependent AP-1-mediated ICAM-1 expression in breast cancer cells

Abstract

Background: Breast cancer is one of the most frequently diagnosed cancer and accounts for over 400,000 deaths each year worldwide. It causes premature death in women, despite progress in early detection, treatment, and advances in understanding the molecular basis of the disease. Therefore, it is important to understand the in depth mechanism of tumor progression and develop new strategies for the treatment of breast cancer. Thus, this study is aimed at gaining an insight into the molecular mechanism by which osteopontin (OPN), a member of SIBLING (Small Integrin Binding LIgand N-linked Glycoprotein) family of protein regulates tumor progression through activation of various transcription factors and expression of their downstream effector gene(s) in breast cancer.

Results: In this study, we report that purified native OPN induces ICAM-1 expression in breast cancer cells. The data revealed that OPN induces NF-kappaB activation and NF-kappaB dependent ICAM-1 expression. We also observed that OPN-induced NF-kappaB further controls AP-1 transactivation, suggesting that there is cross talk between NF-kappaB and AP-1 which is unidirectional towards AP-1 that in turn regulates ICAM-1 expression in these cells. We also delineated the role of mTOR and p70S6 kinase in OPN-induced ICAM-1 expression. The study suggests that inhibition of mTOR by rapamycin augments whereas overexpression of mTOR/p70S6 kinase inhibits OPN-induced ICAM-1 expression. Moreover, overexpression of mTOR inhibits OPN-induced NF-kappaB and AP-1-DNA binding and transcriptional activity. However, rapamycin further enhanced these OPN-induced effects. We also report that OPN induces p70S6 kinase phosphorylation at Thr-421/Ser-424, but not at Thr-389 or Ser-371 and mTOR phosphorylation at Ser-2448. Overexpression of mTOR has no effect in regulation of OPN-induced phosphorylation of p70S6 kinase at Thr-421/Ser-424. Inhibition of mTOR by rapamycin attenuates Ser-371 phosphorylation but does not have any effect on Thr-389 and Thr-421/Ser-424 phosphorylation of p70S6 kinase. However, OPN-induced phosphorylation of p70S6 kinase at Thr-421/Ser-424 is being controlled by MEK/ERK pathway.

Conclusion: These results suggest that blocking of OPN-induced ICAM-1 expression through mTOR/p70S6 kinase signaling pathway may be an important therapeutic strategy for the treatment of breast cancer.

Figure 1

OPN-induces ICAM-1 expression in breast cancer cells. A and C. MCF-7 or MDA-MB-468 cells were treated with 0.5 μM OPN in serum free medium for 0-24 h. B and D. In a separate experiments, MCF-7 or MDA-MB-468 cells were treated with various concentrations of OPN (0-5.0 μM) for 24 h. Cell lysates containing equal amount of total proteins were analyzed by western blot using anti-ICAM-1 antibody. Actin was used as loading control.

Figure 2

Both mTOR and p70S6 kinase suppress OPN-induced NF-κB and AP-1 mediated ICAM-1 expression. A. MCF-7 cells were transfected with wild type or rapamycin resistant mTOR or pretreated with 20 nM rapamycin for 1 h and then treated with 0.5 μM OPN for 24 h. B. In another experiments, MCF-7 cells were transfected with wild type or rapamycin resistant p70S6 kinase or pretreated with 20 nM rapamycin and then treated with OPN under the same condition as mentioned above. Cell lysates were analyzed by western blot using anti-ICAM-1 antibody. Actin was used as loading control. C.MCF-7 cells were transfected with ICAM-1 luciferase reporter construct along with Renilla luciferase and then treated with 0.5 μM OPN for 24 h or pretreated with rapamycin (20 nM) for 1 h and then treated with OPN. The transfection efficiency was normalized and fold changes in luciferase activity with respect to control were calculated, and mean ± S.D. of triplicate determinations are plotted. The values were also analyzed by One Way ANOVA (*, p > 0.05; **, p > 0.05). D. MCF-7 cells were individually transfected with IκBα sup. rep., wild type and dominant negative c-Jun or A-Fos and then treated with 0.5 μM OPN for 24 h. Cell lysates were analyzed by western blot using anti-ICAM-1 antibody. Actin was used as loading control.

Figure 3

mTOR suppresses OPN-induced NF-κB Activation. A and B. MCF-7 cells were treated with 0.5 μM OPN for 0-240 min or transiently transfected with wild type or rapamycin resistant mTOR and then treated with OPN. The non-transfected or transfected cells were treated with rapamycin alone or along with OPN under the same condition as described in Materials and Methods. Nuclear extracts were prepared and analyzed by EMSA. C. MCF-7 cells were transfected with wt or rapamycin resistant mTOR along with NF-κB luciferase reporter construct and then treated with 0.5 μM OPN. The non-transfected or transfected cells were treated with rapamycin alone or along with OPN. The transfection efficiency was normalized by Renilla and fold changes in luciferase activity with respect to control were calculated, and mean ± S.D. of triplicate determinations are plotted. The values were also analyzed by One Way ANOVA (*, p < 0.05; **, p < 0.05).

Figure 4

mTOR down reguates OPN-induced AP-1 activation. A and B. MCF-7 cells were treated with 0.5 μM OPN for 0-240 min or transiently transfected with wild type or rapamycin resistant mTOR and then treated with OPN. The non-transfected or transfected cells were treated with rapamycin alone or along with OPN. Nuclear extracts were prepared and analyzed by EMSA. C. MCF-7 cells were transiently transfected with wild type or rapamycin resistant mTOR along with pAP-1-Luc. The non-transfected or transfected cells were treated with rapamycin alone or along with OPN. The transfection efficiency was normalized by Renilla and fold changes in luciferase activity with respect to control were calculated, and mean ± S.D. of triplicate determinations are plotted. The values were also analyzed by One Way ANOVA (*, p < 0.05; **, p < 0.05).

Figure 5

NF-κB regulates OPN-induced AP-1 activation. A. MCF-7 cells were transfected with pAP-1-Luc along with Renilla luciferase and then either treated with OPN alone or pretreated with anti-α_vβ₃ integrin blocking antibody along with OPN. In separate experiments, cells were transfected with IκBα super-repressor along with pAP-1-Luc and then treated with OPN. The transfection efficiency was normalized by Renilla and fold changes in luciferase activity with respect to control were calculated, and mean ± S.D. of triplicate determinations are plotted. The values were also analyzed by One Way ANOVA (*, p < 0.05; **, p < 0.05). B. MCF-7 cells were individually transfected with wild type and dominant negative c-Jun, wild type c-Fos, A-Fos and then treated with OPN for 30 min. Nuclear extracts were prepared and analyzed by EMSA. C. MCF-7 cells were individually transfected with wild type and dominant negative c-Jun, wild type c-Fos, A-Fos along with pNF-κB-Luc and then treated with OPN for 24 h. The transfection efficiency was normalized by Renilla and fold changes in luciferase activity with respect to control were calculated, and mean ± S.D. of triplicate determinations are plotted. The values were also analyzed by One Way ANOVA (*, p < 0.05; **, p < 0.05).

Figure 6

OPN induces phosphorylation of p70S6 kinase at Thr-421/Ser-424, but not at Thr-389 and Ser-371 and has no effect on mTOR phosphorylation. A-D. MCF-7 cells were either treated with 0.5 μM OPN for 0-120 min or pretreated with 20 nM rapamycin for 1 h and then treated with 0.5 μM OPN for 10 min. Cell lysates containing equal amount of total proteins were resolved by SDS-PAGE and immunoblotted with anti-p-mTOR or anti-p-p70S6 kinase (Thr-421/Ser-424, Ser-371 or Thr-389) antibody. These blots were reprobed with anti-mTOR or anti-p70S6 kinase antibody.

Figure 7

OPN induces mTOR independent p70S6 kinase phosphorylation at Thr-421/Ser-424 through MEK/ERK pathway. A. MCF-7 cells were transiently transfected with wild type or rapamycin resistant mTOR. After 48 h, cells were either treated with OPN or pretreated with rapamycin and then treated with 0.5 μM OPN for 10 min. Cell lysates containing equal amount of total proteins were analyzed by western blot with anti-p-p70S6 kinase (Thr-421/Ser-424) or anti-mTOR antibody. The blots were reprobed with anti-p70S6 kinase antibody. B. MCF-7 cells were pretreated with 0-500 μM U0126 (MEK inhibitor) for 1 h and then treated with 0.5 μM OPN for 10 min. Cell lysates were analyzed by western blot with anti-p-p70S6 kinase (Thr-421/Ser-424) or anti-p-ERK1/2 antibody. The same blots were reprobed with anti-p70S6 kinase or anti-ERK2 antibody.

Figure 8

Molecular mechanism of OPN-induced p70S6 kinase/mTOR regulated NF-κB/AP-1 mediated ICAM-1 expression in breast cancer cells. OPN induces α_vβ₃ integrin-mediated NF-κB/AP-1 dependent ICAM-1 expression. Overexpression of mTOR/p70S6 kinase suppresses OPN-induced ICAM-1 expression in breast cancer cells.