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. 2013 Jun;114(6):1286-93.
doi: 10.1002/jcb.24464.

Acquisition of paclitaxel resistance is associated with a more aggressive and invasive phenotype in prostate cancer

John J Kim  1 , Bo Yin, Christhunesa S Christudass, Naoki Terada, Krithika Rajagopalan, Ben Fabry, Danielle Y Lee, Takumi Shiraishi, Robert H Getzenberg, Robert W Veltri, Steven S An, Steven M Mooney
Affiliations

Acquisition of paclitaxel resistance is associated with a more aggressive and invasive phenotype in prostate cancer

John J Kim et al. J Cell Biochem. 2013 Jun.

Abstract

Drug resistance is a major limitation to the successful treatment of advanced prostate cancer (PCa). Patients who have metastatic, castration-resistant PCa (mCRPC) are treated with chemotherapeutics. However, these standard therapy modalities culminate in the development of resistance. We established paclitaxel resistance in a classic, androgen-insensitive mCRPC cell line (DU145) and, using a suite of molecular and biophysical methods, characterized the structural and functional changes in vitro and in vivo that are associated with the development of drug resistance. After acquiring paclitaxel-resistance, cells exhibited an abnormal nuclear morphology with extensive chromosomal content, an increase in stiffness, and faster cytoskeletal remodeling dynamics. Compared with the parental DU145, paclitaxel-resistant (DU145-TxR) cells became highly invasive and motile in vitro, exercised greater cell traction forces, and formed larger and rapidly growing tumors in mouse xenografts. Furthermore, DU145-TxR cells showed a discrete loss of keratins but a distinct gain of ZEB1, Vimentin and Snail, suggesting an epithelial-to-mesenchymal transition. These findings demonstrate, for the first time, that paclitaxel resistance in PCa is associated with a trans-differentiation of epithelial cell machinery that enables more aggressive and invasive phenotype and portend new strategies for developing novel biomarkers and effective treatment modalities for PCa patients.

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Figures

Fig. 1
Fig. 1
Paclitaxel resistance in PCa is associated with increases in the metastatic potential in vitro and in vivo. A: Representative photos of the cell lines using a light microscope with 10× objective. B: Would healing assays for DU145 and DU145-TxR at a 4× objective view. C: Representative 4× objective images of a soft agar and crystal violet stained Boyden chamber. D: Tumors formed by implanting DU145 or DU145-TxR cells subcutaneously with n = 5 and n = 8 per group, respectively.
Fig. 2
Fig. 2
Representative traction maps of (A) DU145 parental and (B) paclitaxel-resistant cells. Colors show the magnitude of the tractions in Pascals (Pa). Arrows show the direction and relative magnitude of the tractions. Scale bars: 50 μm. Projected cell area (C) and computed net contractile moment (D) of DU145 (parental vs. TXR) cells. Net contractile moment is a scalar measure of cell’s contractile strength and is expressed in pico-Newton meters (pNm). Data are presented as mean ± SE (n = 23 cells for parental; n = 26 cells for TXR). *, P <0.002; **, P <0.0001.
Fig. 3
Fig. 3
Rate of cytoskeletal remodeling of DU145 parental and paclitaxel-resistant cells. A: Stiffness g′ and friction g″ of DU145 (parental) and paclitaxel-resistant (TXR) cells were measured over five decades of frequency using MTC. The solid lines are the fit of experimental data to the structural damping equation with addition of a Newtonian viscous term as previously described [Fabry et al., 2001]. Fitting was carried out by nonlinear regression analysis. Closed squares and circles represent g′ of TxR and parental cells, respectively. Open squares and circles represent g″ of TxR and parental cells, respectively. Data are presented as geometric mean ± SE (n = 832 for parental; n = 575 for TXR). B: Unforced, spontaneous bead motions were quantified by their mean square displacements (MSD) as function of time.31,32 The MSDs were computed at intervals that were equally spaced in time (1.3 s).31,32 For both cells, the MSD increases with time according to a power law relationship. The coefficient D* and the exponent a of the bead motion were estimated from a least squares fit of a power law to the MSD data versus time. C: Diffusion coefficient D* of parental versus TXR cells. Data are presented as mean ± SE (n = 1,703 for parental; n = 1,547 for TXR). *, P <0.0001.
Fig. 4
Fig. 4
DU145-TxR cells have a higher ploidy than their parental counterparts. Static cytometry was used to categorize (n = 371) DU145 cells into hypoploid (G0), diploid (G1), S-Phase, tetraploid (G2), and hypertetraploid (>G2). The same size parameters were then used to assess (n = 312) DU145-TxR cells. The resulting analysis is displayed in a bar graph.
Fig. 5
Fig. 5
Keratin 8, 18, 19 are repressed in DU145-TxR. A: mRNA expression of keratins 8, 18, and 19, (B) protein expression of keratins 8, 18, and 19, (C) immunofluorescent staining of DAPI (blue) and keratins 8, 18, and 19 (green) in DU145-TxR and its parental DU145.
Fig. 6
Fig. 6
Paclitaxel resistance induces EMT. A: Classical EMT markers were immunoblotted. B: The mRNA expression of E-Cadherin showing a clear difference between DU145-TxR and DU145. C: immunofluorescence images of E-Cadherin and Vimentin taken at 10× objective magnification. E-Cadherin and Vimentin are marked with red, while DAPI is used to stain the nucleus. D: Luciferase assay shown on four β-catenin responsive promoters.
Fig. 7
Fig. 7
Schematic diagram of a proposed chemo-biomechanical pathway related with paclitaxel treatment.
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