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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 alters attachment kinetics through β1 integrin.(A) Cells fluorescently labeled green with Calcein AM were plated for specified period of time before removing non-adherent cells and quantifying the adherent fraction fluorometrically. Individual dots represent independent experiments; solid lines are fit curves with shaded region representing the 95% confidence interval. (B) Attachment rate determined from regression of each independent experiment. (C–D) Representative flow cytometry intensity histograms for cells labeled with PE-CD29 (β1 integrin) (C) as well as mean fluorescence intensity of surface β1 integrin (D), normalized to the mean of each individual experiment to account for any variations in laser intensity (N = 3). (E) Surface expression of β1 integrin shows direct linear correlation with adhesion rate (Pearson correlation coefficient ρ = 0.95, R2 = 0.902). (F–G) Neither β1 integrin (F) or adhesion rate (G) correlated with Taxol sensitivity. 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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f3: Taxol resistance alters attachment kinetics through β1 integrin.(A) Cells fluorescently labeled green with Calcein AM were plated for specified period of time before removing non-adherent cells and quantifying the adherent fraction fluorometrically. Individual dots represent independent experiments; solid lines are fit curves with shaded region representing the 95% confidence interval. (B) Attachment rate determined from regression of each independent experiment. (C–D) Representative flow cytometry intensity histograms for cells labeled with PE-CD29 (β1 integrin) (C) as well as mean fluorescence intensity of surface β1 integrin (D), normalized to the mean of each individual experiment to account for any variations in laser intensity (N = 3). (E) Surface expression of β1 integrin shows direct linear correlation with adhesion rate (Pearson correlation coefficient ρ = 0.95, R2 = 0.902). (F–G) Neither β1 integrin (F) or adhesion rate (G) correlated with Taxol sensitivity. 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: Several recent studies have shown a strong link between microtubules and focal adhesions222829. Thus, we sought to determine if changes in Taxol sensitivity produced alterations in cell adhesion. To do so, we first analyzed the attachment kinetics as cells initially attached to a collagen-coated surface (Fig. 3A). Though SKOV3 cells adhered faster overall, the Taxol-resistant populations both adhered faster than their parental counterparts (Fig. 3B). This increase in attachment rate correlated with increased spreading (Fig. S2, Fig. S3A) and was conserved on both fibronectin and Matrigel coated surfaces (Fig. S3B). Due to previous studies linking increased integrin expression and chemoresistance17. we hypothesized the increased attachment rate in Taxol-resistant cells may be due to integrin overexpression. Surface integrin expression was quantified using flow cytometry for β1 integrin responsible for binding to collagen I (Fig. 3C). For both cell lines, surface integrin expression was increased in Taxol-resistant clones, though SKOV3 cells expressed higher overall levels of β1 integrin (Fig. 3D). These results directly correlated with observed attachment kinetics, showing a positive linear relationship between β1 integrin expression and attachment rate (R2 = .90, Pearson's correlation coefficient ρ = 0.95, Fig. 3E). However, due to basal variations between SKOV3 and OVCAR3 neither attachment rate (R2 = 0.03, Fig. 3F) nor integrin expression (R2 = 0.21, Fig. 3G) correlated with IC50, suggesting though this may be a contributing factor it was not a primary mechanism of Taxol resistance.


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 alters attachment kinetics through β1 integrin.(A) Cells fluorescently labeled green with Calcein AM were plated for specified period of time before removing non-adherent cells and quantifying the adherent fraction fluorometrically. Individual dots represent independent experiments; solid lines are fit curves with shaded region representing the 95% confidence interval. (B) Attachment rate determined from regression of each independent experiment. (C–D) Representative flow cytometry intensity histograms for cells labeled with PE-CD29 (β1 integrin) (C) as well as mean fluorescence intensity of surface β1 integrin (D), normalized to the mean of each individual experiment to account for any variations in laser intensity (N = 3). (E) Surface expression of β1 integrin shows direct linear correlation with adhesion rate (Pearson correlation coefficient ρ = 0.95, R2 = 0.902). (F–G) Neither β1 integrin (F) or adhesion rate (G) correlated with Taxol sensitivity. 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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getmorefigures.php?uid=PMC4400875&req=5

f3: Taxol resistance alters attachment kinetics through β1 integrin.(A) Cells fluorescently labeled green with Calcein AM were plated for specified period of time before removing non-adherent cells and quantifying the adherent fraction fluorometrically. Individual dots represent independent experiments; solid lines are fit curves with shaded region representing the 95% confidence interval. (B) Attachment rate determined from regression of each independent experiment. (C–D) Representative flow cytometry intensity histograms for cells labeled with PE-CD29 (β1 integrin) (C) as well as mean fluorescence intensity of surface β1 integrin (D), normalized to the mean of each individual experiment to account for any variations in laser intensity (N = 3). (E) Surface expression of β1 integrin shows direct linear correlation with adhesion rate (Pearson correlation coefficient ρ = 0.95, R2 = 0.902). (F–G) Neither β1 integrin (F) or adhesion rate (G) correlated with Taxol sensitivity. 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: Several recent studies have shown a strong link between microtubules and focal adhesions222829. Thus, we sought to determine if changes in Taxol sensitivity produced alterations in cell adhesion. To do so, we first analyzed the attachment kinetics as cells initially attached to a collagen-coated surface (Fig. 3A). Though SKOV3 cells adhered faster overall, the Taxol-resistant populations both adhered faster than their parental counterparts (Fig. 3B). This increase in attachment rate correlated with increased spreading (Fig. S2, Fig. S3A) and was conserved on both fibronectin and Matrigel coated surfaces (Fig. S3B). Due to previous studies linking increased integrin expression and chemoresistance17. we hypothesized the increased attachment rate in Taxol-resistant cells may be due to integrin overexpression. Surface integrin expression was quantified using flow cytometry for β1 integrin responsible for binding to collagen I (Fig. 3C). For both cell lines, surface integrin expression was increased in Taxol-resistant clones, though SKOV3 cells expressed higher overall levels of β1 integrin (Fig. 3D). These results directly correlated with observed attachment kinetics, showing a positive linear relationship between β1 integrin expression and attachment rate (R2 = .90, Pearson's correlation coefficient ρ = 0.95, Fig. 3E). However, due to basal variations between SKOV3 and OVCAR3 neither attachment rate (R2 = 0.03, Fig. 3F) nor integrin expression (R2 = 0.21, Fig. 3G) correlated with IC50, suggesting though this may be a contributing factor it was not a primary mechanism of Taxol resistance.

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