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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

Microtubule dynamics are increased in Taxol-resistant cells.(A) Microtubules were depolymerized with nocodazole for four hours and then drug was washed out for indicated time at which point cells were immunostained for microtubules (green), actin (red), and nuclei (blue). Scale bar = 10 μm. (B–C) Live-cell microtubule dynamics as determined from plus-end tracking of fluorescent EB3 (see Videos 1 and 2). Following transfection with mCherry-EB3, growing microtubule plus-ends were imaged at 1 Hz for 2 minutes to determine growth rate (B) as well as growth density (C), defined as the total number of growing ends normalized to cell area. Each dot represents the average of over 100 tracked plus-ends from one cell collected from a total of 4 independent experiments. (D) Taxol-resistant cells have less polymerized tubulin. Cells were lysed following 4 hour pretreatment with Taxol and separated into polymerized (P) and soluble (S) tubulin fractions for Western blot analysis. Percent polymerized tubulin was quantified as polymerized tubulin divided by the sum of polymerized and soluble tubulin. A cropped representative blot from 4 independent experiments is shown, and the full-length blot is available in the supplemental information (Fig. S6). Values given as mean ± SEM; significance is indicated relative to control parent population unless otherwise noted, *P < 0.05, **P < 0.01,***P < 0.001.
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f2: Microtubule dynamics are increased in Taxol-resistant cells.(A) Microtubules were depolymerized with nocodazole for four hours and then drug was washed out for indicated time at which point cells were immunostained for microtubules (green), actin (red), and nuclei (blue). Scale bar = 10 μm. (B–C) Live-cell microtubule dynamics as determined from plus-end tracking of fluorescent EB3 (see Videos 1 and 2). Following transfection with mCherry-EB3, growing microtubule plus-ends were imaged at 1 Hz for 2 minutes to determine growth rate (B) as well as growth density (C), defined as the total number of growing ends normalized to cell area. Each dot represents the average of over 100 tracked plus-ends from one cell collected from a total of 4 independent experiments. (D) Taxol-resistant cells have less polymerized tubulin. Cells were lysed following 4 hour pretreatment with Taxol and separated into polymerized (P) and soluble (S) tubulin fractions for Western blot analysis. Percent polymerized tubulin was quantified as polymerized tubulin divided by the sum of polymerized and soluble tubulin. A cropped representative blot from 4 independent experiments is shown, and the full-length blot is available in the supplemental information (Fig. S6). Values given as mean ± SEM; significance is indicated relative to control parent population unless otherwise noted, *P < 0.05, **P < 0.01,***P < 0.001.

Mentions: Based on the increase in viability seen with low-dose Taxol in Taxol-resistant cells, we next evaluated if Taxol-resistant cells displayed enhanced microtubule dynamics. First, we performed a microtubule regrowth assay where microtubules were depolymerized with nocodazole and then allowed to regrow following washout (Fig. 2A). By 10 minutes SKOV3-P cells had begun to nucleate whereas microtubule networks had already begun to form in SKOV3-T cells, which was not seen until 30 minutes in the SKOV3-P cells. To verify this difference in live cells in absence of chemical perturbation we transfected cells with fluorescent end-binding protein (mApple-EB3) which binds to the growing plus ends of microtubules allowing for quantification of microtubule growth rates (Supp. Vid. 1–2)27. Not only were microtubule growth rates significantly faster in the SKOV3-T cells (p < 0.0001, Fig. 2B), but they also showed an increased number of growing plus ends (p < 0.0001, Fig. 2C), indicating significantly increased dynamics. Finally, we performed a microtubule pelleting assay to determine if the cells natively had different levels of polymerized microtubules revealing SKOV3-P cells had significantly more polymerized tubulin than SKOV3-T cells (Fig. 2D). Treating SKOV3-T with 10 nM Taxol returned levels of polymerized tubulin to those of parental cells, while 100 nM Taxol was required to increase tubulin polymerization above parent levels. With similar findings in OVCAR3-T cells (Fig. S1), these results indicate that Taxol-resistant cells have decreased polymerized microtubules and increased microtubule growth rates consistent with previous reports91011.


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)

Microtubule dynamics are increased in Taxol-resistant cells.(A) Microtubules were depolymerized with nocodazole for four hours and then drug was washed out for indicated time at which point cells were immunostained for microtubules (green), actin (red), and nuclei (blue). Scale bar = 10 μm. (B–C) Live-cell microtubule dynamics as determined from plus-end tracking of fluorescent EB3 (see Videos 1 and 2). Following transfection with mCherry-EB3, growing microtubule plus-ends were imaged at 1 Hz for 2 minutes to determine growth rate (B) as well as growth density (C), defined as the total number of growing ends normalized to cell area. Each dot represents the average of over 100 tracked plus-ends from one cell collected from a total of 4 independent experiments. (D) Taxol-resistant cells have less polymerized tubulin. Cells were lysed following 4 hour pretreatment with Taxol and separated into polymerized (P) and soluble (S) tubulin fractions for Western blot analysis. Percent polymerized tubulin was quantified as polymerized tubulin divided by the sum of polymerized and soluble tubulin. A cropped representative blot from 4 independent experiments is shown, and the full-length blot is available in the supplemental information (Fig. S6). Values given as mean ± SEM; significance is indicated relative to control parent population unless otherwise noted, *P < 0.05, **P < 0.01,***P < 0.001.
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Related In: Results  -  Collection

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f2: Microtubule dynamics are increased in Taxol-resistant cells.(A) Microtubules were depolymerized with nocodazole for four hours and then drug was washed out for indicated time at which point cells were immunostained for microtubules (green), actin (red), and nuclei (blue). Scale bar = 10 μm. (B–C) Live-cell microtubule dynamics as determined from plus-end tracking of fluorescent EB3 (see Videos 1 and 2). Following transfection with mCherry-EB3, growing microtubule plus-ends were imaged at 1 Hz for 2 minutes to determine growth rate (B) as well as growth density (C), defined as the total number of growing ends normalized to cell area. Each dot represents the average of over 100 tracked plus-ends from one cell collected from a total of 4 independent experiments. (D) Taxol-resistant cells have less polymerized tubulin. Cells were lysed following 4 hour pretreatment with Taxol and separated into polymerized (P) and soluble (S) tubulin fractions for Western blot analysis. Percent polymerized tubulin was quantified as polymerized tubulin divided by the sum of polymerized and soluble tubulin. A cropped representative blot from 4 independent experiments is shown, and the full-length blot is available in the supplemental information (Fig. S6). Values given as mean ± SEM; significance is indicated relative to control parent population unless otherwise noted, *P < 0.05, **P < 0.01,***P < 0.001.
Mentions: Based on the increase in viability seen with low-dose Taxol in Taxol-resistant cells, we next evaluated if Taxol-resistant cells displayed enhanced microtubule dynamics. First, we performed a microtubule regrowth assay where microtubules were depolymerized with nocodazole and then allowed to regrow following washout (Fig. 2A). By 10 minutes SKOV3-P cells had begun to nucleate whereas microtubule networks had already begun to form in SKOV3-T cells, which was not seen until 30 minutes in the SKOV3-P cells. To verify this difference in live cells in absence of chemical perturbation we transfected cells with fluorescent end-binding protein (mApple-EB3) which binds to the growing plus ends of microtubules allowing for quantification of microtubule growth rates (Supp. Vid. 1–2)27. Not only were microtubule growth rates significantly faster in the SKOV3-T cells (p < 0.0001, Fig. 2B), but they also showed an increased number of growing plus ends (p < 0.0001, Fig. 2C), indicating significantly increased dynamics. Finally, we performed a microtubule pelleting assay to determine if the cells natively had different levels of polymerized microtubules revealing SKOV3-P cells had significantly more polymerized tubulin than SKOV3-T cells (Fig. 2D). Treating SKOV3-T with 10 nM Taxol returned levels of polymerized tubulin to those of parental cells, while 100 nM Taxol was required to increase tubulin polymerization above parent levels. With similar findings in OVCAR3-T cells (Fig. S1), these results indicate that Taxol-resistant cells have decreased polymerized microtubules and increased microtubule growth rates consistent with previous reports91011.

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