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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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STAT3 inhibitors induce anoikis in cancer cells(A) Human melanoma cells SK-MEL-28, MeWo, SK-MEL-5, SK-MEL-2 and murine melanoma cells B16-F0 were cultured in plates coated with poly-HEMA as suspension culture (anchorage independent condition) and treated with DMSO or various concentrations of AG 490. After 48 hours, cells were replated in 24 well plated and the viable cells were analyzed by Sulforhodamine B assay. Representative bar graphs show the percentage anoikis resistance in different treatment conditions. Values are plotted as mean ± S.D. *, p < 0.05 compared with control group. (B) Human melanoma cells SK-MEL-28, MeWo, SK-MEL-5, SK-MEL-2, B16-F0 were cultured in plates coated with poly-HEMA under anchorage independent condition and treated with DMSO or various concentrations of piplartine (PL). After 48 hours, cells were replated in 24 well plate and the viable cells were analyzed by Sulforhodamine B assay. Representative bar graphs show the percentage anoikis resistance in different treatment conditions. Values are plotted as mean ± S.D. *, p < 0.05 compared with control group. PL reverses anoikis resistance by inhibition of STAT3. (C) Blots are representative of pSTAT3 (Y705), STAT3, Bcl-2, Mcl-1 and cleaved PARP from lysates collected from human melanoma cells SK-MEL-28, MeWo, SK-MEL-2, SK-MEL-5 and B16-F0 grown under anchorage independent conditions and treated with DMSO or various concentrations of PL for 48 hours. Actin was used as loading control. Each experiment was repeated at least three times with similar results.
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Figure 3: STAT3 inhibitors induce anoikis in cancer cells(A) Human melanoma cells SK-MEL-28, MeWo, SK-MEL-5, SK-MEL-2 and murine melanoma cells B16-F0 were cultured in plates coated with poly-HEMA as suspension culture (anchorage independent condition) and treated with DMSO or various concentrations of AG 490. After 48 hours, cells were replated in 24 well plated and the viable cells were analyzed by Sulforhodamine B assay. Representative bar graphs show the percentage anoikis resistance in different treatment conditions. Values are plotted as mean ± S.D. *, p < 0.05 compared with control group. (B) Human melanoma cells SK-MEL-28, MeWo, SK-MEL-5, SK-MEL-2, B16-F0 were cultured in plates coated with poly-HEMA under anchorage independent condition and treated with DMSO or various concentrations of piplartine (PL). After 48 hours, cells were replated in 24 well plate and the viable cells were analyzed by Sulforhodamine B assay. Representative bar graphs show the percentage anoikis resistance in different treatment conditions. Values are plotted as mean ± S.D. *, p < 0.05 compared with control group. PL reverses anoikis resistance by inhibition of STAT3. (C) Blots are representative of pSTAT3 (Y705), STAT3, Bcl-2, Mcl-1 and cleaved PARP from lysates collected from human melanoma cells SK-MEL-28, MeWo, SK-MEL-2, SK-MEL-5 and B16-F0 grown under anchorage independent conditions and treated with DMSO or various concentrations of PL for 48 hours. Actin was used as loading control. Each experiment was repeated at least three times with similar results.

Mentions: To test our hypothesis that STAT3 plays a role in anoikis resistance, we wanted to determine whether STAT3 inhibitors can overcome aniokis resistance in cancer cells. For this purpose, we used a known synthetic STAT3 inhibitor AG 490. Five melanoma cell lines (SK-MEL-28, SK-MEL-2, SK-MEL-5, MeWo and B16-F0) were treated with 50μM or 100μM AG 490 under anchorage independent conditions for 48 hours, after which the cells were re-cultured on an adherent plate where only the anoikis resistant cells attached and survived. The survival of the treated cells was evaluated using the SRB assay and compared with the untreated anoikis resistant cells. Our results demonstrated that AG 490 was able to substantially reduce anoikis resistance in all the melanoma cell lines (Fig. 3A). Anoikis resistance by treatment with 50-100μM AG 490 was reduced by 20-60% in SK-MEL-28 cells (Fig. 3A). Similar concentrations of AG 490 reduced anoikis resistance by 45-65% in SK-MEL-2, 25-50% in SK-MEL-5, 30-65% in MeWo and 70-75% in B16-F0 cells (Fig. 3A).


Critical role of STAT3 in melanoma metastasis through anoikis resistance.

Fofaria NM, Srivastava SK - Oncotarget (2014)

STAT3 inhibitors induce anoikis in cancer cells(A) Human melanoma cells SK-MEL-28, MeWo, SK-MEL-5, SK-MEL-2 and murine melanoma cells B16-F0 were cultured in plates coated with poly-HEMA as suspension culture (anchorage independent condition) and treated with DMSO or various concentrations of AG 490. After 48 hours, cells were replated in 24 well plated and the viable cells were analyzed by Sulforhodamine B assay. Representative bar graphs show the percentage anoikis resistance in different treatment conditions. Values are plotted as mean ± S.D. *, p < 0.05 compared with control group. (B) Human melanoma cells SK-MEL-28, MeWo, SK-MEL-5, SK-MEL-2, B16-F0 were cultured in plates coated with poly-HEMA under anchorage independent condition and treated with DMSO or various concentrations of piplartine (PL). After 48 hours, cells were replated in 24 well plate and the viable cells were analyzed by Sulforhodamine B assay. Representative bar graphs show the percentage anoikis resistance in different treatment conditions. Values are plotted as mean ± S.D. *, p < 0.05 compared with control group. PL reverses anoikis resistance by inhibition of STAT3. (C) Blots are representative of pSTAT3 (Y705), STAT3, Bcl-2, Mcl-1 and cleaved PARP from lysates collected from human melanoma cells SK-MEL-28, MeWo, SK-MEL-2, SK-MEL-5 and B16-F0 grown under anchorage independent conditions and treated with DMSO or various concentrations of PL for 48 hours. Actin was used as loading control. Each experiment was repeated at least three times with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: STAT3 inhibitors induce anoikis in cancer cells(A) Human melanoma cells SK-MEL-28, MeWo, SK-MEL-5, SK-MEL-2 and murine melanoma cells B16-F0 were cultured in plates coated with poly-HEMA as suspension culture (anchorage independent condition) and treated with DMSO or various concentrations of AG 490. After 48 hours, cells were replated in 24 well plated and the viable cells were analyzed by Sulforhodamine B assay. Representative bar graphs show the percentage anoikis resistance in different treatment conditions. Values are plotted as mean ± S.D. *, p < 0.05 compared with control group. (B) Human melanoma cells SK-MEL-28, MeWo, SK-MEL-5, SK-MEL-2, B16-F0 were cultured in plates coated with poly-HEMA under anchorage independent condition and treated with DMSO or various concentrations of piplartine (PL). After 48 hours, cells were replated in 24 well plate and the viable cells were analyzed by Sulforhodamine B assay. Representative bar graphs show the percentage anoikis resistance in different treatment conditions. Values are plotted as mean ± S.D. *, p < 0.05 compared with control group. PL reverses anoikis resistance by inhibition of STAT3. (C) Blots are representative of pSTAT3 (Y705), STAT3, Bcl-2, Mcl-1 and cleaved PARP from lysates collected from human melanoma cells SK-MEL-28, MeWo, SK-MEL-2, SK-MEL-5 and B16-F0 grown under anchorage independent conditions and treated with DMSO or various concentrations of PL for 48 hours. Actin was used as loading control. Each experiment was repeated at least three times with similar results.
Mentions: To test our hypothesis that STAT3 plays a role in anoikis resistance, we wanted to determine whether STAT3 inhibitors can overcome aniokis resistance in cancer cells. For this purpose, we used a known synthetic STAT3 inhibitor AG 490. Five melanoma cell lines (SK-MEL-28, SK-MEL-2, SK-MEL-5, MeWo and B16-F0) were treated with 50μM or 100μM AG 490 under anchorage independent conditions for 48 hours, after which the cells were re-cultured on an adherent plate where only the anoikis resistant cells attached and survived. The survival of the treated cells was evaluated using the SRB assay and compared with the untreated anoikis resistant cells. Our results demonstrated that AG 490 was able to substantially reduce anoikis resistance in all the melanoma cell lines (Fig. 3A). Anoikis resistance by treatment with 50-100μM AG 490 was reduced by 20-60% in SK-MEL-28 cells (Fig. 3A). Similar concentrations of AG 490 reduced anoikis resistance by 45-65% in SK-MEL-2, 25-50% in SK-MEL-5, 30-65% in MeWo and 70-75% in B16-F0 cells (Fig. 3A).

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