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Critical role of STAT3 in melanoma metastasis through anoikis resistance.

Fofaria NM, Srivastava SK - Oncotarget (2014)

Bottom Line: 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.

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 deficient cells failed to form tumor in vivo(A) Tumor volumes and images of the mice bearing xenografts of wild type (Control), STAT3 knockout (STAT3 KO) and PL treated (5μM) SK-MEL 28 cells cultured under anchorage independent conditions for 48 hours. Values are plotted as mean ± S.E.M. *, p < 0.05 compared with control group. Cell viability of each group was evaluated using trypan blue assay and 5 × 106 live cells were injected. Once palpable tumors were observed, tumor dimensions were measured using vernier calipers. Values are plotted as mean ± S.E.M. *, p < 0.05 compared with control group. STAT3 enhances metastatic potential in melanoma cells by rendering anoikis resistance. (B) Animal weight (C) Images of lungs and liver and (D) Hematoxylin and Eosin staining of lung sections of the mice bearing metastatic nodules of wild type (Control), STAT3 knock-out (STAT3 KO) and PL treated (5μM) SK-MEL- 28 cells cultured under anchorage independent conditions for 48 hours and injected intravenously. Live cells were counted by trypan blue staining and 0.2 × 106 live cells were injected intravenously. The experiment was continued till the mice from any of the group started dying due to metastatic burden. Arrows indicate presence of metastatic nodules.
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Figure 6: STAT3 deficient cells failed to form tumor in vivo(A) Tumor volumes and images of the mice bearing xenografts of wild type (Control), STAT3 knockout (STAT3 KO) and PL treated (5μM) SK-MEL 28 cells cultured under anchorage independent conditions for 48 hours. Values are plotted as mean ± S.E.M. *, p < 0.05 compared with control group. Cell viability of each group was evaluated using trypan blue assay and 5 × 106 live cells were injected. Once palpable tumors were observed, tumor dimensions were measured using vernier calipers. Values are plotted as mean ± S.E.M. *, p < 0.05 compared with control group. STAT3 enhances metastatic potential in melanoma cells by rendering anoikis resistance. (B) Animal weight (C) Images of lungs and liver and (D) Hematoxylin and Eosin staining of lung sections of the mice bearing metastatic nodules of wild type (Control), STAT3 knock-out (STAT3 KO) and PL treated (5μM) SK-MEL- 28 cells cultured under anchorage independent conditions for 48 hours and injected intravenously. Live cells were counted by trypan blue staining and 0.2 × 106 live cells were injected intravenously. The experiment was continued till the mice from any of the group started dying due to metastatic burden. Arrows indicate presence of metastatic nodules.

Mentions: In order to validate the critical role of STAT3 in anoikis resistance in the in vivo model, melanoma tumor xenograft experiments were performed in SCID-NSG mice. Three different groups of mice (n=9/group) were taken. About 5 × 106 live anchorage-independent cells were injected subcutaneously in both the flanks of SCID-NSG mice to determine the tumor formation ability. Mice in group 1 were implanted with SK-MEL-28 wild type anoikis resistant cells (Control), group two mice received SK-MEL-28 STAT3 knockout (STAT3 KO) and group three mice received SK-MEL-28 anoikis resistant cells treated with 5 μM PL for 48 h. Once palpable tumors were formed, tumor measurements were taken twice a week using vernier calipers. Our results showed that SK-MEL-28 wild type cells formed very aggressive tumors with an average tumor volume of 440 ± 47.86 mm3 by the end of day 50 (Fig. 6A). However, STAT3 knockout or PL treated SK-MEL-28 cells completely failed to form tumors (Fig. 6A). The average tumor volume in mice from STAT3 KO group was 9 ± 2.9 mm3 and that in mice from PL treated group was 6 ± 2 mm3 (Fig. 6A). Seven mice in KO group and 5 mice in PL group were completely free of tumors (Fig. 6A). These results indicated that cells with reduced or no expression of STAT3 were highly sensitive to anoikis and, therefore, failed to grow as tumors. On the other hand, wild type cells where STAT3 was activated during anchorage-independent conditions had very high tumorigenic potential. Fig. 6A also shows the images of tumor bearing mice from the three groups. As compared to control mice, STAT3 KO mice had no palpable tumors whereas PL treated mice showed very small size tumors which failed to grow over the duration of the experiment (Fig. 6A).


Critical role of STAT3 in melanoma metastasis through anoikis resistance.

Fofaria NM, Srivastava SK - Oncotarget (2014)

STAT3 deficient cells failed to form tumor in vivo(A) Tumor volumes and images of the mice bearing xenografts of wild type (Control), STAT3 knockout (STAT3 KO) and PL treated (5μM) SK-MEL 28 cells cultured under anchorage independent conditions for 48 hours. Values are plotted as mean ± S.E.M. *, p < 0.05 compared with control group. Cell viability of each group was evaluated using trypan blue assay and 5 × 106 live cells were injected. Once palpable tumors were observed, tumor dimensions were measured using vernier calipers. Values are plotted as mean ± S.E.M. *, p < 0.05 compared with control group. STAT3 enhances metastatic potential in melanoma cells by rendering anoikis resistance. (B) Animal weight (C) Images of lungs and liver and (D) Hematoxylin and Eosin staining of lung sections of the mice bearing metastatic nodules of wild type (Control), STAT3 knock-out (STAT3 KO) and PL treated (5μM) SK-MEL- 28 cells cultured under anchorage independent conditions for 48 hours and injected intravenously. Live cells were counted by trypan blue staining and 0.2 × 106 live cells were injected intravenously. The experiment was continued till the mice from any of the group started dying due to metastatic burden. Arrows indicate presence of metastatic nodules.
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Figure 6: STAT3 deficient cells failed to form tumor in vivo(A) Tumor volumes and images of the mice bearing xenografts of wild type (Control), STAT3 knockout (STAT3 KO) and PL treated (5μM) SK-MEL 28 cells cultured under anchorage independent conditions for 48 hours. Values are plotted as mean ± S.E.M. *, p < 0.05 compared with control group. Cell viability of each group was evaluated using trypan blue assay and 5 × 106 live cells were injected. Once palpable tumors were observed, tumor dimensions were measured using vernier calipers. Values are plotted as mean ± S.E.M. *, p < 0.05 compared with control group. STAT3 enhances metastatic potential in melanoma cells by rendering anoikis resistance. (B) Animal weight (C) Images of lungs and liver and (D) Hematoxylin and Eosin staining of lung sections of the mice bearing metastatic nodules of wild type (Control), STAT3 knock-out (STAT3 KO) and PL treated (5μM) SK-MEL- 28 cells cultured under anchorage independent conditions for 48 hours and injected intravenously. Live cells were counted by trypan blue staining and 0.2 × 106 live cells were injected intravenously. The experiment was continued till the mice from any of the group started dying due to metastatic burden. Arrows indicate presence of metastatic nodules.
Mentions: In order to validate the critical role of STAT3 in anoikis resistance in the in vivo model, melanoma tumor xenograft experiments were performed in SCID-NSG mice. Three different groups of mice (n=9/group) were taken. About 5 × 106 live anchorage-independent cells were injected subcutaneously in both the flanks of SCID-NSG mice to determine the tumor formation ability. Mice in group 1 were implanted with SK-MEL-28 wild type anoikis resistant cells (Control), group two mice received SK-MEL-28 STAT3 knockout (STAT3 KO) and group three mice received SK-MEL-28 anoikis resistant cells treated with 5 μM PL for 48 h. Once palpable tumors were formed, tumor measurements were taken twice a week using vernier calipers. Our results showed that SK-MEL-28 wild type cells formed very aggressive tumors with an average tumor volume of 440 ± 47.86 mm3 by the end of day 50 (Fig. 6A). However, STAT3 knockout or PL treated SK-MEL-28 cells completely failed to form tumors (Fig. 6A). The average tumor volume in mice from STAT3 KO group was 9 ± 2.9 mm3 and that in mice from PL treated group was 6 ± 2 mm3 (Fig. 6A). Seven mice in KO group and 5 mice in PL group were completely free of tumors (Fig. 6A). These results indicated that cells with reduced or no expression of STAT3 were highly sensitive to anoikis and, therefore, failed to grow as tumors. On the other hand, wild type cells where STAT3 was activated during anchorage-independent conditions had very high tumorigenic potential. Fig. 6A also shows the images of tumor bearing mice from the three groups. As compared to control mice, STAT3 KO mice had no palpable tumors whereas PL treated mice showed very small size tumors which failed to grow over the duration of the experiment (Fig. 6A).

Bottom Line: 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.

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