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Critical role of STAT3 in melanoma metastasis through anoikis resistance.

Fofaria NM, Srivastava SK - Oncotarget (2014)

Bottom Line: When grown under anchorage-independent conditions, significant proportion of cells resisted anoikis and these resistant cells had higher rate of migration and invasion as compared to the cells grown under anchorage-dependent conditions.On the other hand, silencing STAT3 decreased the potential of cancer cells to resist anoikis and to migrate.STAT3 knock-down cells and PL treated cells did not form tumors as well as failed to metastasize in SCID-NSG mice as compared to untreated anchorage-independent cells, which formed big tumors and extensively metastasized.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences & Cancer Biology Center, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, USA.

ABSTRACT
Anoikis is an anchorage-independent cell death. Resistance to anoikis is one of the key features of metastatic cells. Here, we analyzed the role of STAT3 in anoikis resistance in melanoma cells leading to metastasis. When grown under anchorage-independent conditions, significant proportion of cells resisted anoikis and these resistant cells had higher rate of migration and invasion as compared to the cells grown under anchorage-dependent conditions. The anoikis resistant cells also had significantly higher expression and phosphorylation of STAT3 at Y705 than the cells that were attached to the basement membrane. STAT3 inhibitors, AG 490 and piplartine (PL) induced anoikis in a concentration-dependent manner in anoikis resistant cells. Over-expression of STAT3 or treatment with IL-6 not only increased anoikis resistance, but also protected the cancer cells from PL-induced anoikis. On the other hand, silencing STAT3 decreased the potential of cancer cells to resist anoikis and to migrate. STAT3 knock-down cells and PL treated cells did not form tumors as well as failed to metastasize in SCID-NSG mice as compared to untreated anchorage-independent cells, which formed big tumors and extensively metastasized. In summary, our results for the first time establish STAT3 as a critical player that renders anoikis resistance to melanoma cells and enhance their metastatic potential.

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Related in: MedlinePlus

IL-6 and STAT3 over-expression enhances anoikis resistance in cancer cells and reverses anoikis sensitization by PL(A) SK-MEL-28, MeWo, SK-MEL-2, B16-F0 and SK-MEL-5 cells were treated with IL-6 alone or in combination with 5 μM PL under anchorage independent condition. After 48 hours, cells were transferred to 24-well plate and the viability was measured by Sulforhodamine B assay. Representative bar graph shows percent anoikis resistance of cells exposed to various treatments under anchorage independent conditions. Values are plotted as mean ± S.D. *, p < 0.05 compared with control group and #, p < 0.05 when compared with PL treated cells. (B) Representative blots of pSTAT3, Bcl-2 and Cleaved PARP from of lysates of SK-MEL-28, MeWo, SK-MEL-2, B16-F0 and SK-MEL-5 cells treated with IL-6 alone or in combination with 5 μM PL for 48 hours. Actin was used as a loading control. (C-D) SK-MEL-28, SK-MEL-2, MeWo and SK-MEL-5 cells were transfected with STAT3 overexpressing plasmid. After 24 hours, cells were transferred to poly HEMA coated plates treated with or without 5 μM PL. After 48 hours, cells were replated in a 24-well plate and the viable cells were analyzed by SRB assay. Representative bar graph shows percent anoikis resistance after various treatments. Values were plotted as mean ± S.D. *, p < 0.05 compared with control cells and #, p < 0.05 when compared with PL treated cells. Fold increase in overexpression was evaluated by western blotting prior to the experiment and a representative blot is shown in the figure. Each experiment was repeated at least three times with similar results
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Figure 5: IL-6 and STAT3 over-expression enhances anoikis resistance in cancer cells and reverses anoikis sensitization by PL(A) SK-MEL-28, MeWo, SK-MEL-2, B16-F0 and SK-MEL-5 cells were treated with IL-6 alone or in combination with 5 μM PL under anchorage independent condition. After 48 hours, cells were transferred to 24-well plate and the viability was measured by Sulforhodamine B assay. Representative bar graph shows percent anoikis resistance of cells exposed to various treatments under anchorage independent conditions. Values are plotted as mean ± S.D. *, p < 0.05 compared with control group and #, p < 0.05 when compared with PL treated cells. (B) Representative blots of pSTAT3, Bcl-2 and Cleaved PARP from of lysates of SK-MEL-28, MeWo, SK-MEL-2, B16-F0 and SK-MEL-5 cells treated with IL-6 alone or in combination with 5 μM PL for 48 hours. Actin was used as a loading control. (C-D) SK-MEL-28, SK-MEL-2, MeWo and SK-MEL-5 cells were transfected with STAT3 overexpressing plasmid. After 24 hours, cells were transferred to poly HEMA coated plates treated with or without 5 μM PL. After 48 hours, cells were replated in a 24-well plate and the viable cells were analyzed by SRB assay. Representative bar graph shows percent anoikis resistance after various treatments. Values were plotted as mean ± S.D. *, p < 0.05 compared with control cells and #, p < 0.05 when compared with PL treated cells. Fold increase in overexpression was evaluated by western blotting prior to the experiment and a representative blot is shown in the figure. Each experiment was repeated at least three times with similar results

Mentions: To further strengthen the role of STAT3 in anoikis resistance, IL-6 was used to activate STAT3. IL-6 is well known to activate STAT3 by phosphorylation at Y705[28]. As we had hypothesized that phosphorylation at of STAT3 Y705 conferred anoikis resistance to melanoma cells, IL-6 treatment would enhance anoikis resistance in these cells. Moreover, since our results indicated that PL induced anoikis by inhibition of p-STAT3 (Y705), we hypothesized that IL-6 treatment would block PL-induced anoikis. To test the effect of IL-6, melanoma cells were pre-treated with IL-6 and then treated with PL under anchorage-independent conditions for 48 hours. As shown in Fig. 5A, IL-6 treatment significantly increased anoikis resistance in all the melanoma cell lines as compared to untreated controls. IL-6 treatment increased the anoikis resistance by 1.5 fold in SK-MEL-28 and SK-MEL-2, by 1.4 fold in MeWo cells and by 1.3 fold in SK-MEL-5 and B16-F0 cells (Fig. 5A). As expected, IL-6 also provided marked protection to PL treated cells (Fig. 5A). IL-6 treatment significantly blocked the reduction in anoikis resistance mediated by PL treatment in all five cell lines (Fig. 5A). For example, in SK-MEL-28 cells, PL reduced anoikis resistance by 65% (Fig. 5A). However, IL-6 treatment blocked PL mediated anoikis by almost 45% (Fig. 5A). Similarly, significant reduction in anoikis resistance by PL treatment in SK-MEL-2, SK-MEL-5, MeWo and B16-F0 cells was blocked by IL-6 treatment, confirming our hypothesis (Fig. 5A).


Critical role of STAT3 in melanoma metastasis through anoikis resistance.

Fofaria NM, Srivastava SK - Oncotarget (2014)

IL-6 and STAT3 over-expression enhances anoikis resistance in cancer cells and reverses anoikis sensitization by PL(A) SK-MEL-28, MeWo, SK-MEL-2, B16-F0 and SK-MEL-5 cells were treated with IL-6 alone or in combination with 5 μM PL under anchorage independent condition. After 48 hours, cells were transferred to 24-well plate and the viability was measured by Sulforhodamine B assay. Representative bar graph shows percent anoikis resistance of cells exposed to various treatments under anchorage independent conditions. Values are plotted as mean ± S.D. *, p < 0.05 compared with control group and #, p < 0.05 when compared with PL treated cells. (B) Representative blots of pSTAT3, Bcl-2 and Cleaved PARP from of lysates of SK-MEL-28, MeWo, SK-MEL-2, B16-F0 and SK-MEL-5 cells treated with IL-6 alone or in combination with 5 μM PL for 48 hours. Actin was used as a loading control. (C-D) SK-MEL-28, SK-MEL-2, MeWo and SK-MEL-5 cells were transfected with STAT3 overexpressing plasmid. After 24 hours, cells were transferred to poly HEMA coated plates treated with or without 5 μM PL. After 48 hours, cells were replated in a 24-well plate and the viable cells were analyzed by SRB assay. Representative bar graph shows percent anoikis resistance after various treatments. Values were plotted as mean ± S.D. *, p < 0.05 compared with control cells and #, p < 0.05 when compared with PL treated cells. Fold increase in overexpression was evaluated by western blotting prior to the experiment and a representative blot is shown in the figure. Each experiment was repeated at least three times with similar results
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Figure 5: IL-6 and STAT3 over-expression enhances anoikis resistance in cancer cells and reverses anoikis sensitization by PL(A) SK-MEL-28, MeWo, SK-MEL-2, B16-F0 and SK-MEL-5 cells were treated with IL-6 alone or in combination with 5 μM PL under anchorage independent condition. After 48 hours, cells were transferred to 24-well plate and the viability was measured by Sulforhodamine B assay. Representative bar graph shows percent anoikis resistance of cells exposed to various treatments under anchorage independent conditions. Values are plotted as mean ± S.D. *, p < 0.05 compared with control group and #, p < 0.05 when compared with PL treated cells. (B) Representative blots of pSTAT3, Bcl-2 and Cleaved PARP from of lysates of SK-MEL-28, MeWo, SK-MEL-2, B16-F0 and SK-MEL-5 cells treated with IL-6 alone or in combination with 5 μM PL for 48 hours. Actin was used as a loading control. (C-D) SK-MEL-28, SK-MEL-2, MeWo and SK-MEL-5 cells were transfected with STAT3 overexpressing plasmid. After 24 hours, cells were transferred to poly HEMA coated plates treated with or without 5 μM PL. After 48 hours, cells were replated in a 24-well plate and the viable cells were analyzed by SRB assay. Representative bar graph shows percent anoikis resistance after various treatments. Values were plotted as mean ± S.D. *, p < 0.05 compared with control cells and #, p < 0.05 when compared with PL treated cells. Fold increase in overexpression was evaluated by western blotting prior to the experiment and a representative blot is shown in the figure. Each experiment was repeated at least three times with similar results
Mentions: To further strengthen the role of STAT3 in anoikis resistance, IL-6 was used to activate STAT3. IL-6 is well known to activate STAT3 by phosphorylation at Y705[28]. As we had hypothesized that phosphorylation at of STAT3 Y705 conferred anoikis resistance to melanoma cells, IL-6 treatment would enhance anoikis resistance in these cells. Moreover, since our results indicated that PL induced anoikis by inhibition of p-STAT3 (Y705), we hypothesized that IL-6 treatment would block PL-induced anoikis. To test the effect of IL-6, melanoma cells were pre-treated with IL-6 and then treated with PL under anchorage-independent conditions for 48 hours. As shown in Fig. 5A, IL-6 treatment significantly increased anoikis resistance in all the melanoma cell lines as compared to untreated controls. IL-6 treatment increased the anoikis resistance by 1.5 fold in SK-MEL-28 and SK-MEL-2, by 1.4 fold in MeWo cells and by 1.3 fold in SK-MEL-5 and B16-F0 cells (Fig. 5A). As expected, IL-6 also provided marked protection to PL treated cells (Fig. 5A). IL-6 treatment significantly blocked the reduction in anoikis resistance mediated by PL treatment in all five cell lines (Fig. 5A). For example, in SK-MEL-28 cells, PL reduced anoikis resistance by 65% (Fig. 5A). However, IL-6 treatment blocked PL mediated anoikis by almost 45% (Fig. 5A). Similarly, significant reduction in anoikis resistance by PL treatment in SK-MEL-2, SK-MEL-5, MeWo and B16-F0 cells was blocked by IL-6 treatment, confirming our hypothesis (Fig. 5A).

Bottom Line: When grown under anchorage-independent conditions, significant proportion of cells resisted anoikis and these resistant cells had higher rate of migration and invasion as compared to the cells grown under anchorage-dependent conditions.On the other hand, silencing STAT3 decreased the potential of cancer cells to resist anoikis and to migrate.STAT3 knock-down cells and PL treated cells did not form tumors as well as failed to metastasize in SCID-NSG mice as compared to untreated anchorage-independent cells, which formed big tumors and extensively metastasized.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences & Cancer Biology Center, Texas Tech University Health Sciences Center, Amarillo, Texas 79106, USA.

ABSTRACT
Anoikis is an anchorage-independent cell death. Resistance to anoikis is one of the key features of metastatic cells. Here, we analyzed the role of STAT3 in anoikis resistance in melanoma cells leading to metastasis. When grown under anchorage-independent conditions, significant proportion of cells resisted anoikis and these resistant cells had higher rate of migration and invasion as compared to the cells grown under anchorage-dependent conditions. The anoikis resistant cells also had significantly higher expression and phosphorylation of STAT3 at Y705 than the cells that were attached to the basement membrane. STAT3 inhibitors, AG 490 and piplartine (PL) induced anoikis in a concentration-dependent manner in anoikis resistant cells. Over-expression of STAT3 or treatment with IL-6 not only increased anoikis resistance, but also protected the cancer cells from PL-induced anoikis. On the other hand, silencing STAT3 decreased the potential of cancer cells to resist anoikis and to migrate. STAT3 knock-down cells and PL treated cells did not form tumors as well as failed to metastasize in SCID-NSG mice as compared to untreated anchorage-independent cells, which formed big tumors and extensively metastasized. In summary, our results for the first time establish STAT3 as a critical player that renders anoikis resistance to melanoma cells and enhance their metastatic potential.

Show MeSH
Related in: MedlinePlus