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Tenascin-C promotes melanoma progression by maintaining the ABCB5-positive side population.

Fukunaga-Kalabis M, Martinez G, Nguyen TK, Kim D, Santiago-Walker A, Roesch A, Herlyn M - Oncogene (2010)

Bottom Line: Recent studies indicate that TNC has a role within the stem cell niche.Downmodulation of TNC by shRNA lentiviruses significantly decreased the growth of melanoma spheres.Knockdown of TNC dramatically decreased the SP fraction in melanoma spheres and lowered their resistance to doxorubicin treatment, likely because of the downregulation of multiple ATP-binding cassette (ABC) transporters, including ABCB5.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Oncogenesis Program, Division of Molecular and Cellular Biology, The Wistar Institute, Philadelphia, PA 19104, USA.

ABSTRACT
Tenascin-C (TNC) is highly expressed in melanoma; however, little is known about its functions. Recent studies indicate that TNC has a role within the stem cell niche. We hypothesized that TNC creates a specific environment for melanoma cells to show a stem cell-like phenotype, promoting tumor growth and evading conventional therapies. TNC expression was strongly upregulated in melanoma cells grown as 3D spheres (enriched for stem-like cells) when compared to adherent cells. Downmodulation of TNC by shRNA lentiviruses significantly decreased the growth of melanoma spheres. The incidence of pulmonary metastases after intravenous injection of TNC knockdown cells was significantly lower in NOD/SCID IL2Rγ() mice compared with control cells. Melanoma spheres contain an increased number of side population (SP) cells, which show stem cell characteristics, and have the potential for drug resistance due to their high efflux capacity. Knockdown of TNC dramatically decreased the SP fraction in melanoma spheres and lowered their resistance to doxorubicin treatment, likely because of the downregulation of multiple ATP-binding cassette (ABC) transporters, including ABCB5. These data suggest that TNC is critical in melanoma progression as it mediates protective signals in the therapy-resistant population of melanoma.

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TNC knockdown potentiates doxorubicin response(A) Depletion of TNC with shRNA sensitizes melanoma cells to doxorubicin treatment, demonstrated as a shift of the sh_TNC dose response curves compared to sh_Cont cells. sh_TNC and sh_Cont cells were dissociated into single cell-suspensions in hESCM4 medium, which was conditioned for 7 days with each lentiviral infected cell line, and plated in 96-well plates at a concentration of 10,000 cells/well. Twenty-four hours later, various concentrations of doxorubicin were added. The MTS assays were performed 72 hours post drug treatment. The data were normalized to the doxorubicin-untreated control. *, p<0.05 when compared to sh_Cont cells. (B) Apoptosis assessed by propidium iodide (PI) staining of untreated and 2μM doxorubicin-treated WM3734 sh_Cont cells and sh_TNC_A cells 24 hours post-treatment. Columns indicate propidium iodide-positive apoptotic cells. Twenty-four hour treatment with 2μM doxorubicin induced apoptosis in sh_TNC_A cells but did not impair survival of sh_Cont cells. (n = 3) *, p<0.05 when compared to untreated cells. (C) Exogenous TNC increases doxorubicin resistance, demonstrated as a shift in the dose response curves. WM115 sphere cells were dissociated into single cell-suspensions in hES medium and plated in 96-well plates at a concentration of 10,000 cells/well. Cont: without additional reagent. Gelatin: with 10 μg/ml gelatin matrix. TNC: with 10 μg/ml human TNC. Twenty-four hours later, various concentrations of doxorubicin were added. The MTS assays were performed 72 hours post drug treatment. The data were normalized to the doxorubicin-untreated control. *, p<0.05 when compared to Cont cells.
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Figure 6: TNC knockdown potentiates doxorubicin response(A) Depletion of TNC with shRNA sensitizes melanoma cells to doxorubicin treatment, demonstrated as a shift of the sh_TNC dose response curves compared to sh_Cont cells. sh_TNC and sh_Cont cells were dissociated into single cell-suspensions in hESCM4 medium, which was conditioned for 7 days with each lentiviral infected cell line, and plated in 96-well plates at a concentration of 10,000 cells/well. Twenty-four hours later, various concentrations of doxorubicin were added. The MTS assays were performed 72 hours post drug treatment. The data were normalized to the doxorubicin-untreated control. *, p<0.05 when compared to sh_Cont cells. (B) Apoptosis assessed by propidium iodide (PI) staining of untreated and 2μM doxorubicin-treated WM3734 sh_Cont cells and sh_TNC_A cells 24 hours post-treatment. Columns indicate propidium iodide-positive apoptotic cells. Twenty-four hour treatment with 2μM doxorubicin induced apoptosis in sh_TNC_A cells but did not impair survival of sh_Cont cells. (n = 3) *, p<0.05 when compared to untreated cells. (C) Exogenous TNC increases doxorubicin resistance, demonstrated as a shift in the dose response curves. WM115 sphere cells were dissociated into single cell-suspensions in hES medium and plated in 96-well plates at a concentration of 10,000 cells/well. Cont: without additional reagent. Gelatin: with 10 μg/ml gelatin matrix. TNC: with 10 μg/ml human TNC. Twenty-four hours later, various concentrations of doxorubicin were added. The MTS assays were performed 72 hours post drug treatment. The data were normalized to the doxorubicin-untreated control. *, p<0.05 when compared to Cont cells.

Mentions: Our observation that the reduction of TNC decreases the SP fraction expressing ABCB5 in melanoma spheres, indicated that TNC likely confers resistance to doxorubicin. We tested whether down-modulation of TNC sensitizes melanoma cells to doxorubicin. To minimize the impact of factors in the culture medium, doxorubicin resistance was tested in medium which had been conditioned for 7 days on each respective cell line. Indeed, knockdown of TNC sensitized WM3734 and WM35 cells to doxorubicin, resulting in a significant shift of the dose response curve (Figure 6A). The percentage of apoptotic cells was then assessed by propidium iodide staining. Compared to WM3734 sh_Cont cells, which were resistant when treated with 2 μM of doxorubicin for 24 hours, WM3734 sh_TNC cells showed a significantly increased rate of apoptosis (Figure 6B).


Tenascin-C promotes melanoma progression by maintaining the ABCB5-positive side population.

Fukunaga-Kalabis M, Martinez G, Nguyen TK, Kim D, Santiago-Walker A, Roesch A, Herlyn M - Oncogene (2010)

TNC knockdown potentiates doxorubicin response(A) Depletion of TNC with shRNA sensitizes melanoma cells to doxorubicin treatment, demonstrated as a shift of the sh_TNC dose response curves compared to sh_Cont cells. sh_TNC and sh_Cont cells were dissociated into single cell-suspensions in hESCM4 medium, which was conditioned for 7 days with each lentiviral infected cell line, and plated in 96-well plates at a concentration of 10,000 cells/well. Twenty-four hours later, various concentrations of doxorubicin were added. The MTS assays were performed 72 hours post drug treatment. The data were normalized to the doxorubicin-untreated control. *, p<0.05 when compared to sh_Cont cells. (B) Apoptosis assessed by propidium iodide (PI) staining of untreated and 2μM doxorubicin-treated WM3734 sh_Cont cells and sh_TNC_A cells 24 hours post-treatment. Columns indicate propidium iodide-positive apoptotic cells. Twenty-four hour treatment with 2μM doxorubicin induced apoptosis in sh_TNC_A cells but did not impair survival of sh_Cont cells. (n = 3) *, p<0.05 when compared to untreated cells. (C) Exogenous TNC increases doxorubicin resistance, demonstrated as a shift in the dose response curves. WM115 sphere cells were dissociated into single cell-suspensions in hES medium and plated in 96-well plates at a concentration of 10,000 cells/well. Cont: without additional reagent. Gelatin: with 10 μg/ml gelatin matrix. TNC: with 10 μg/ml human TNC. Twenty-four hours later, various concentrations of doxorubicin were added. The MTS assays were performed 72 hours post drug treatment. The data were normalized to the doxorubicin-untreated control. *, p<0.05 when compared to Cont cells.
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Figure 6: TNC knockdown potentiates doxorubicin response(A) Depletion of TNC with shRNA sensitizes melanoma cells to doxorubicin treatment, demonstrated as a shift of the sh_TNC dose response curves compared to sh_Cont cells. sh_TNC and sh_Cont cells were dissociated into single cell-suspensions in hESCM4 medium, which was conditioned for 7 days with each lentiviral infected cell line, and plated in 96-well plates at a concentration of 10,000 cells/well. Twenty-four hours later, various concentrations of doxorubicin were added. The MTS assays were performed 72 hours post drug treatment. The data were normalized to the doxorubicin-untreated control. *, p<0.05 when compared to sh_Cont cells. (B) Apoptosis assessed by propidium iodide (PI) staining of untreated and 2μM doxorubicin-treated WM3734 sh_Cont cells and sh_TNC_A cells 24 hours post-treatment. Columns indicate propidium iodide-positive apoptotic cells. Twenty-four hour treatment with 2μM doxorubicin induced apoptosis in sh_TNC_A cells but did not impair survival of sh_Cont cells. (n = 3) *, p<0.05 when compared to untreated cells. (C) Exogenous TNC increases doxorubicin resistance, demonstrated as a shift in the dose response curves. WM115 sphere cells were dissociated into single cell-suspensions in hES medium and plated in 96-well plates at a concentration of 10,000 cells/well. Cont: without additional reagent. Gelatin: with 10 μg/ml gelatin matrix. TNC: with 10 μg/ml human TNC. Twenty-four hours later, various concentrations of doxorubicin were added. The MTS assays were performed 72 hours post drug treatment. The data were normalized to the doxorubicin-untreated control. *, p<0.05 when compared to Cont cells.
Mentions: Our observation that the reduction of TNC decreases the SP fraction expressing ABCB5 in melanoma spheres, indicated that TNC likely confers resistance to doxorubicin. We tested whether down-modulation of TNC sensitizes melanoma cells to doxorubicin. To minimize the impact of factors in the culture medium, doxorubicin resistance was tested in medium which had been conditioned for 7 days on each respective cell line. Indeed, knockdown of TNC sensitized WM3734 and WM35 cells to doxorubicin, resulting in a significant shift of the dose response curve (Figure 6A). The percentage of apoptotic cells was then assessed by propidium iodide staining. Compared to WM3734 sh_Cont cells, which were resistant when treated with 2 μM of doxorubicin for 24 hours, WM3734 sh_TNC cells showed a significantly increased rate of apoptosis (Figure 6B).

Bottom Line: Recent studies indicate that TNC has a role within the stem cell niche.Downmodulation of TNC by shRNA lentiviruses significantly decreased the growth of melanoma spheres.Knockdown of TNC dramatically decreased the SP fraction in melanoma spheres and lowered their resistance to doxorubicin treatment, likely because of the downregulation of multiple ATP-binding cassette (ABC) transporters, including ABCB5.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Oncogenesis Program, Division of Molecular and Cellular Biology, The Wistar Institute, Philadelphia, PA 19104, USA.

ABSTRACT
Tenascin-C (TNC) is highly expressed in melanoma; however, little is known about its functions. Recent studies indicate that TNC has a role within the stem cell niche. We hypothesized that TNC creates a specific environment for melanoma cells to show a stem cell-like phenotype, promoting tumor growth and evading conventional therapies. TNC expression was strongly upregulated in melanoma cells grown as 3D spheres (enriched for stem-like cells) when compared to adherent cells. Downmodulation of TNC by shRNA lentiviruses significantly decreased the growth of melanoma spheres. The incidence of pulmonary metastases after intravenous injection of TNC knockdown cells was significantly lower in NOD/SCID IL2Rγ() mice compared with control cells. Melanoma spheres contain an increased number of side population (SP) cells, which show stem cell characteristics, and have the potential for drug resistance due to their high efflux capacity. Knockdown of TNC dramatically decreased the SP fraction in melanoma spheres and lowered their resistance to doxorubicin treatment, likely because of the downregulation of multiple ATP-binding cassette (ABC) transporters, including ABCB5. These data suggest that TNC is critical in melanoma progression as it mediates protective signals in the therapy-resistant population of melanoma.

Show MeSH
Related in: MedlinePlus