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Alterations in ovarian cancer cell adhesion drive taxol resistance by increasing microtubule dynamics in a FAK-dependent manner.

McGrail DJ, Khambhati NN, Qi MX, Patel KS, Ravikumar N, Brandenburg CP, Dawson MR - Sci Rep (2015)

Bottom Line: Though Taxol-resistant cells neither effluxed more drug nor gained resistance to other chemotherapeutics, they did display increased microtubule dynamics.Adhesion strength correlated best with Taxol-sensitivity, and was found to be independent of microtubule polymerization but dependent on focal adhesion kinase (FAK), which was up-regulated in Taxol-resistant cells.FAK inhibition also decreased microtubule dynamics to equal levels in both populations, indicating alterations in adhesive signaling are up-stream of microtubule dynamics.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical &Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA.

ABSTRACT
Chemorefractory ovarian cancer patients show extremely poor prognosis. Microtubule-stabilizing Taxol (paclitaxel) is a first-line treatment against ovarian cancer. Despite the close interplay between microtubules and cell adhesion, it remains unknown if chemoresistance alters the way cells adhere to their extracellular environment, a process critical for cancer metastasis. To investigate this, we isolated Taxol-resistant populations of OVCAR3 and SKOV3 ovarian cancer cell lines. Though Taxol-resistant cells neither effluxed more drug nor gained resistance to other chemotherapeutics, they did display increased microtubule dynamics. These changes in microtubule dynamics coincided with faster attachment rates and decreased adhesion strength, which correlated with increased surface β1-integrin expression and decreased focal adhesion formation, respectively. Adhesion strength correlated best with Taxol-sensitivity, and was found to be independent of microtubule polymerization but dependent on focal adhesion kinase (FAK), which was up-regulated in Taxol-resistant cells. FAK inhibition also decreased microtubule dynamics to equal levels in both populations, indicating alterations in adhesive signaling are up-stream of microtubule dynamics. Taken together, this work demonstrates that Taxol-resistance dramatically alters how ovarian cancer cells adhere to their extracellular environment causing down-stream increases in microtubule dynamics, providing a therapeutic target that may improve prognosis by not only recovering drug sensitivity, but also decreasing metastasis.

No MeSH data available.


Related in: MedlinePlus

Taxol resistance decreases adhesion strength by altering focal adhesions.(A) Detached fraction of parent (-P) and Taxol-resistant (-T) cells allowed to adhere overnight before detachment by centrifugal force shows significantly decreased adhesion strength in Taxol-resistant clones (p < 0.01,N = 4). (B) Detached fraction of cells correlates directly with IC50 values (p < 0.01), Pearson correlation coefficient ρ = 0.99. (C) Immunofluorescent micrographs of cells labeled for paxillin (green), F-actin (red), and nuclei (blue) with zoomed versions of highlighted areas. Scale bar = 10 μm. Focal adhesion density was quantified as the integrated density of segmented focal adhesions relative to cell area (N = 3). (D–E) Total paxillin (D, N = 4) and vinculin (E, N = 3) expression, quantified by Western blot normalized to total protein, is decreased in Taxol-resistant cells but is also dependent on cell line. A cropped representative blot is shown, and full-length blots are available in the supplemental information (Fig. S6). For total protein from Coomassie, a representative region of the quantified area is shown, complete image is available in Figure S6. (F) Vinculin expression inversely correlates with IC50 values (p < 0.05), Pearson correlation coefficient ρ = −0.97.
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f4: Taxol resistance decreases adhesion strength by altering focal adhesions.(A) Detached fraction of parent (-P) and Taxol-resistant (-T) cells allowed to adhere overnight before detachment by centrifugal force shows significantly decreased adhesion strength in Taxol-resistant clones (p < 0.01,N = 4). (B) Detached fraction of cells correlates directly with IC50 values (p < 0.01), Pearson correlation coefficient ρ = 0.99. (C) Immunofluorescent micrographs of cells labeled for paxillin (green), F-actin (red), and nuclei (blue) with zoomed versions of highlighted areas. Scale bar = 10 μm. Focal adhesion density was quantified as the integrated density of segmented focal adhesions relative to cell area (N = 3). (D–E) Total paxillin (D, N = 4) and vinculin (E, N = 3) expression, quantified by Western blot normalized to total protein, is decreased in Taxol-resistant cells but is also dependent on cell line. A cropped representative blot is shown, and full-length blots are available in the supplemental information (Fig. S6). For total protein from Coomassie, a representative region of the quantified area is shown, complete image is available in Figure S6. (F) Vinculin expression inversely correlates with IC50 values (p < 0.05), Pearson correlation coefficient ρ = −0.97.

Mentions: The increased integrin expression suggested that adhesion strength would be increased in Taxol-resistant cells, but inhibition of tubulin polymerization has previously been shown to effect focal adhesion formation and steady state adhesion strength3031. Since Taxol-resistant cells displayed decreased polymerized tubulin, we next quantified their adhesion strength using a centrifugal-force based adhesion assay to test if the integrin up-regulation in Taxol-resistant clones led to increased adhesion strength. After adhering overnight, the detached fraction following centrifugation in Taxol-resistant population was nearly twice that of their parental lines (Fig. 4A), despite integrin overexpression. This result correlated well with changes in IC50 (Fig. 4B) suggesting alterations in Taxol sensitivity may be related to alterations in adhesion strength.


Alterations in ovarian cancer cell adhesion drive taxol resistance by increasing microtubule dynamics in a FAK-dependent manner.

McGrail DJ, Khambhati NN, Qi MX, Patel KS, Ravikumar N, Brandenburg CP, Dawson MR - Sci Rep (2015)

Taxol resistance decreases adhesion strength by altering focal adhesions.(A) Detached fraction of parent (-P) and Taxol-resistant (-T) cells allowed to adhere overnight before detachment by centrifugal force shows significantly decreased adhesion strength in Taxol-resistant clones (p < 0.01,N = 4). (B) Detached fraction of cells correlates directly with IC50 values (p < 0.01), Pearson correlation coefficient ρ = 0.99. (C) Immunofluorescent micrographs of cells labeled for paxillin (green), F-actin (red), and nuclei (blue) with zoomed versions of highlighted areas. Scale bar = 10 μm. Focal adhesion density was quantified as the integrated density of segmented focal adhesions relative to cell area (N = 3). (D–E) Total paxillin (D, N = 4) and vinculin (E, N = 3) expression, quantified by Western blot normalized to total protein, is decreased in Taxol-resistant cells but is also dependent on cell line. A cropped representative blot is shown, and full-length blots are available in the supplemental information (Fig. S6). For total protein from Coomassie, a representative region of the quantified area is shown, complete image is available in Figure S6. (F) Vinculin expression inversely correlates with IC50 values (p < 0.05), Pearson correlation coefficient ρ = −0.97.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4: Taxol resistance decreases adhesion strength by altering focal adhesions.(A) Detached fraction of parent (-P) and Taxol-resistant (-T) cells allowed to adhere overnight before detachment by centrifugal force shows significantly decreased adhesion strength in Taxol-resistant clones (p < 0.01,N = 4). (B) Detached fraction of cells correlates directly with IC50 values (p < 0.01), Pearson correlation coefficient ρ = 0.99. (C) Immunofluorescent micrographs of cells labeled for paxillin (green), F-actin (red), and nuclei (blue) with zoomed versions of highlighted areas. Scale bar = 10 μm. Focal adhesion density was quantified as the integrated density of segmented focal adhesions relative to cell area (N = 3). (D–E) Total paxillin (D, N = 4) and vinculin (E, N = 3) expression, quantified by Western blot normalized to total protein, is decreased in Taxol-resistant cells but is also dependent on cell line. A cropped representative blot is shown, and full-length blots are available in the supplemental information (Fig. S6). For total protein from Coomassie, a representative region of the quantified area is shown, complete image is available in Figure S6. (F) Vinculin expression inversely correlates with IC50 values (p < 0.05), Pearson correlation coefficient ρ = −0.97.
Mentions: The increased integrin expression suggested that adhesion strength would be increased in Taxol-resistant cells, but inhibition of tubulin polymerization has previously been shown to effect focal adhesion formation and steady state adhesion strength3031. Since Taxol-resistant cells displayed decreased polymerized tubulin, we next quantified their adhesion strength using a centrifugal-force based adhesion assay to test if the integrin up-regulation in Taxol-resistant clones led to increased adhesion strength. After adhering overnight, the detached fraction following centrifugation in Taxol-resistant population was nearly twice that of their parental lines (Fig. 4A), despite integrin overexpression. This result correlated well with changes in IC50 (Fig. 4B) suggesting alterations in Taxol sensitivity may be related to alterations in adhesion strength.

Bottom Line: Though Taxol-resistant cells neither effluxed more drug nor gained resistance to other chemotherapeutics, they did display increased microtubule dynamics.Adhesion strength correlated best with Taxol-sensitivity, and was found to be independent of microtubule polymerization but dependent on focal adhesion kinase (FAK), which was up-regulated in Taxol-resistant cells.FAK inhibition also decreased microtubule dynamics to equal levels in both populations, indicating alterations in adhesive signaling are up-stream of microtubule dynamics.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical &Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA.

ABSTRACT
Chemorefractory ovarian cancer patients show extremely poor prognosis. Microtubule-stabilizing Taxol (paclitaxel) is a first-line treatment against ovarian cancer. Despite the close interplay between microtubules and cell adhesion, it remains unknown if chemoresistance alters the way cells adhere to their extracellular environment, a process critical for cancer metastasis. To investigate this, we isolated Taxol-resistant populations of OVCAR3 and SKOV3 ovarian cancer cell lines. Though Taxol-resistant cells neither effluxed more drug nor gained resistance to other chemotherapeutics, they did display increased microtubule dynamics. These changes in microtubule dynamics coincided with faster attachment rates and decreased adhesion strength, which correlated with increased surface β1-integrin expression and decreased focal adhesion formation, respectively. Adhesion strength correlated best with Taxol-sensitivity, and was found to be independent of microtubule polymerization but dependent on focal adhesion kinase (FAK), which was up-regulated in Taxol-resistant cells. FAK inhibition also decreased microtubule dynamics to equal levels in both populations, indicating alterations in adhesive signaling are up-stream of microtubule dynamics. Taken together, this work demonstrates that Taxol-resistance dramatically alters how ovarian cancer cells adhere to their extracellular environment causing down-stream increases in microtubule dynamics, providing a therapeutic target that may improve prognosis by not only recovering drug sensitivity, but also decreasing metastasis.

No MeSH data available.


Related in: MedlinePlus