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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

Alterations in adhesion and microtubule dynamics in Taxol-resistant cells are reversible with FAK inhibition.(A) SKOV3 parent (-P) and Taxol-resistant (-T) were pre-incubated with 1:1 serial dilutions ranging of Taxol ranging from 3.13–100 nM for 4 hours to stabilize microtubules before repeating the adhesion strength assay and displayed no change from the untreated controls (shaded region) (N = 3). (B) The adhesion strength assay was repeated using 4 hour nocodazole pre-treatment with 1:1 serial dilutions ranging from 0.16–10 μM to depolymerize microtubules (N = 3). In order to prevent changes from increased Rho activity upon nocodazole upon washout the experiment was carried out in the drug-containing media instead, producing a slightly higher baseline for SKOV3-T. (C) To inhibit focal adhesion signaling cells were pre-incubated with 1:1 serial dilutions of focal adhesion kinase inhibitor PF228 ranging from 2.5–40 μM for 4 hours before running the adhesion strength assay demonstrating selective recovery of adhesion force in Taxol-resistant cells relative to their untreated controls (red shaded region) to become equivalent with parent control cells (gray shaded region) (N = 3). (D) To determine the effects of FAK inhibition on attachment kinetics, cells were pre-incubated with 10 μM PF228 for 30 minutes in solution and allowed to adhere for 30 minutes revealing FAK inhibition decreased attachment kinetics in Taxol-resistant cells (N = 3). (E) Cells transfected with mCherry-EB3 were imaged and then treated with 10 μM PF228 for four hours before re-imaging revealing a significant (p < 0.0001) decrease in both parent and Taxol-resistant cells to a statistically equal value. Values listed in parenthesis are given as mean ± std. All other values given as mean ± SEM; significance is indicated relative to matched parent population with *′s and relative to solvent treated control #′s. *P < 0.05, **P < 0.01,***P < 0.001.
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f5: Alterations in adhesion and microtubule dynamics in Taxol-resistant cells are reversible with FAK inhibition.(A) SKOV3 parent (-P) and Taxol-resistant (-T) were pre-incubated with 1:1 serial dilutions ranging of Taxol ranging from 3.13–100 nM for 4 hours to stabilize microtubules before repeating the adhesion strength assay and displayed no change from the untreated controls (shaded region) (N = 3). (B) The adhesion strength assay was repeated using 4 hour nocodazole pre-treatment with 1:1 serial dilutions ranging from 0.16–10 μM to depolymerize microtubules (N = 3). In order to prevent changes from increased Rho activity upon nocodazole upon washout the experiment was carried out in the drug-containing media instead, producing a slightly higher baseline for SKOV3-T. (C) To inhibit focal adhesion signaling cells were pre-incubated with 1:1 serial dilutions of focal adhesion kinase inhibitor PF228 ranging from 2.5–40 μM for 4 hours before running the adhesion strength assay demonstrating selective recovery of adhesion force in Taxol-resistant cells relative to their untreated controls (red shaded region) to become equivalent with parent control cells (gray shaded region) (N = 3). (D) To determine the effects of FAK inhibition on attachment kinetics, cells were pre-incubated with 10 μM PF228 for 30 minutes in solution and allowed to adhere for 30 minutes revealing FAK inhibition decreased attachment kinetics in Taxol-resistant cells (N = 3). (E) Cells transfected with mCherry-EB3 were imaged and then treated with 10 μM PF228 for four hours before re-imaging revealing a significant (p < 0.0001) decrease in both parent and Taxol-resistant cells to a statistically equal value. Values listed in parenthesis are given as mean ± std. All other values given as mean ± SEM; significance is indicated relative to matched parent population with *′s and relative to solvent treated control #′s. *P < 0.05, **P < 0.01,***P < 0.001.

Mentions: We next sought to determine what intracellular signaling cascades could be responsible for the alterations in adhesion strength and microtubule dynamics. Based on our observations, we hypothesized that either (1) alterations in microtubule dynamics were decreasing focal adhesion formation or (2) decreased focal adhesion formation was altering microtubule dynamics. To determine which mechanism was ultimately altering the adhesive strength of Taxol-resistant cells we chemically modified both pathways and repeated the adhesion strength assay. First, to test if the decreased adhesion strength was due to increased microtubule dynamics we pre-incubated cells with either Taxol to stabilize (Fig. 5A) microtubules or nocodazole to depolymerize microtubules (Fig. 5B) and repeated the adhesion strength assay. In both cases, there was no change in the adhesive strength of either the parent or Taxol-resistant cells across a wide array of concentrations indicating the changes in adhesion strength are not due to microtubule dynamics. To investigate if these changes were due to altered focal adhesion signaling we inhibited the increased FAK phosphorylation observed in Taxol-resistant cells with PF228 (Fig. S4B–C), which has been shown to block focal adhesion turnover34. In parental cells FAK inhibition did not alter adhesion strength at any tested concentration (Fig. 5C, black line). In contrast to this, the detached fraction was significantly lower than untreated control for all concentrations in Taxol-resistant cells and no longer significantly different than the parental cell line for all concentrations greater than 5 μM (Fig. 5C, red line). To probe if FAK inhibition could also reverse changes in attachment kinetics, we allowed cells to adhere for 30 minutes after pretreatment with PF228 and found FAK inhibition significantly reduced Taxol-resistant cell attachment with equal fractions of both parental and Taxol-resistant cells adhering in this short time scale (Fig. 5D). Finally, to verify that changes in adhesive signaling were the up-stream cause we repeated the microtubule plus-tip tracking with EB3 after FAK inhibition and found a significant (p < 0.001) decrease in microtubule growth rate for both SKOV3-P and SKOV3-T cells (Fig. 5E). Notably, this produced equivalent growth rates in both cell populations through a larger growth rate decrease in the Taxol-resistant cells (45% vs. 28%). Taken together, these results suggest a model where changes in focal adhesion signaling cause alterations in microtubule dynamics leading to increased resistance to microtubule-stabilizing drugs such as Taxol.


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)

Alterations in adhesion and microtubule dynamics in Taxol-resistant cells are reversible with FAK inhibition.(A) SKOV3 parent (-P) and Taxol-resistant (-T) were pre-incubated with 1:1 serial dilutions ranging of Taxol ranging from 3.13–100 nM for 4 hours to stabilize microtubules before repeating the adhesion strength assay and displayed no change from the untreated controls (shaded region) (N = 3). (B) The adhesion strength assay was repeated using 4 hour nocodazole pre-treatment with 1:1 serial dilutions ranging from 0.16–10 μM to depolymerize microtubules (N = 3). In order to prevent changes from increased Rho activity upon nocodazole upon washout the experiment was carried out in the drug-containing media instead, producing a slightly higher baseline for SKOV3-T. (C) To inhibit focal adhesion signaling cells were pre-incubated with 1:1 serial dilutions of focal adhesion kinase inhibitor PF228 ranging from 2.5–40 μM for 4 hours before running the adhesion strength assay demonstrating selective recovery of adhesion force in Taxol-resistant cells relative to their untreated controls (red shaded region) to become equivalent with parent control cells (gray shaded region) (N = 3). (D) To determine the effects of FAK inhibition on attachment kinetics, cells were pre-incubated with 10 μM PF228 for 30 minutes in solution and allowed to adhere for 30 minutes revealing FAK inhibition decreased attachment kinetics in Taxol-resistant cells (N = 3). (E) Cells transfected with mCherry-EB3 were imaged and then treated with 10 μM PF228 for four hours before re-imaging revealing a significant (p < 0.0001) decrease in both parent and Taxol-resistant cells to a statistically equal value. Values listed in parenthesis are given as mean ± std. All other values given as mean ± SEM; significance is indicated relative to matched parent population with *′s and relative to solvent treated control #′s. *P < 0.05, **P < 0.01,***P < 0.001.
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f5: Alterations in adhesion and microtubule dynamics in Taxol-resistant cells are reversible with FAK inhibition.(A) SKOV3 parent (-P) and Taxol-resistant (-T) were pre-incubated with 1:1 serial dilutions ranging of Taxol ranging from 3.13–100 nM for 4 hours to stabilize microtubules before repeating the adhesion strength assay and displayed no change from the untreated controls (shaded region) (N = 3). (B) The adhesion strength assay was repeated using 4 hour nocodazole pre-treatment with 1:1 serial dilutions ranging from 0.16–10 μM to depolymerize microtubules (N = 3). In order to prevent changes from increased Rho activity upon nocodazole upon washout the experiment was carried out in the drug-containing media instead, producing a slightly higher baseline for SKOV3-T. (C) To inhibit focal adhesion signaling cells were pre-incubated with 1:1 serial dilutions of focal adhesion kinase inhibitor PF228 ranging from 2.5–40 μM for 4 hours before running the adhesion strength assay demonstrating selective recovery of adhesion force in Taxol-resistant cells relative to their untreated controls (red shaded region) to become equivalent with parent control cells (gray shaded region) (N = 3). (D) To determine the effects of FAK inhibition on attachment kinetics, cells were pre-incubated with 10 μM PF228 for 30 minutes in solution and allowed to adhere for 30 minutes revealing FAK inhibition decreased attachment kinetics in Taxol-resistant cells (N = 3). (E) Cells transfected with mCherry-EB3 were imaged and then treated with 10 μM PF228 for four hours before re-imaging revealing a significant (p < 0.0001) decrease in both parent and Taxol-resistant cells to a statistically equal value. Values listed in parenthesis are given as mean ± std. All other values given as mean ± SEM; significance is indicated relative to matched parent population with *′s and relative to solvent treated control #′s. *P < 0.05, **P < 0.01,***P < 0.001.
Mentions: We next sought to determine what intracellular signaling cascades could be responsible for the alterations in adhesion strength and microtubule dynamics. Based on our observations, we hypothesized that either (1) alterations in microtubule dynamics were decreasing focal adhesion formation or (2) decreased focal adhesion formation was altering microtubule dynamics. To determine which mechanism was ultimately altering the adhesive strength of Taxol-resistant cells we chemically modified both pathways and repeated the adhesion strength assay. First, to test if the decreased adhesion strength was due to increased microtubule dynamics we pre-incubated cells with either Taxol to stabilize (Fig. 5A) microtubules or nocodazole to depolymerize microtubules (Fig. 5B) and repeated the adhesion strength assay. In both cases, there was no change in the adhesive strength of either the parent or Taxol-resistant cells across a wide array of concentrations indicating the changes in adhesion strength are not due to microtubule dynamics. To investigate if these changes were due to altered focal adhesion signaling we inhibited the increased FAK phosphorylation observed in Taxol-resistant cells with PF228 (Fig. S4B–C), which has been shown to block focal adhesion turnover34. In parental cells FAK inhibition did not alter adhesion strength at any tested concentration (Fig. 5C, black line). In contrast to this, the detached fraction was significantly lower than untreated control for all concentrations in Taxol-resistant cells and no longer significantly different than the parental cell line for all concentrations greater than 5 μM (Fig. 5C, red line). To probe if FAK inhibition could also reverse changes in attachment kinetics, we allowed cells to adhere for 30 minutes after pretreatment with PF228 and found FAK inhibition significantly reduced Taxol-resistant cell attachment with equal fractions of both parental and Taxol-resistant cells adhering in this short time scale (Fig. 5D). Finally, to verify that changes in adhesive signaling were the up-stream cause we repeated the microtubule plus-tip tracking with EB3 after FAK inhibition and found a significant (p < 0.001) decrease in microtubule growth rate for both SKOV3-P and SKOV3-T cells (Fig. 5E). Notably, this produced equivalent growth rates in both cell populations through a larger growth rate decrease in the Taxol-resistant cells (45% vs. 28%). Taken together, these results suggest a model where changes in focal adhesion signaling cause alterations in microtubule dynamics leading to increased resistance to microtubule-stabilizing drugs such as Taxol.

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