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Increased expression of class III beta-tubulin in castration-resistant human prostate cancer.

Terry S, Ploussard G, Allory Y, Nicolaiew N, Boissière-Michot F, Maillé P, Kheuang L, Coppolani E, Ali A, Bibeau F, Culine S, Buttyan R, de la Taille A, Vacherot F - Br. J. Cancer (2009)

Bottom Line: Overexpression of betaIII-tubulin in these tumours is associated with an unfavourable outcome and resistance to taxane-based therapies.Whereas moderate-to-strong betaIII-tubulin expression was detected in only 3 out of 74 (4%) hormone-naive tumour specimens obtained from patients who never received hormone therapy, 6 out of 24 tumour specimens (25%) from patients treated for 3 months with neoadjuvant hormone therapy and 24 out of 40 (60%) castration-resistant tumour specimens from chronic hormone-treated patients were found to express significant levels of betaIII-tubulin.These findings were supported by in vitro and in vivo settings.

View Article: PubMed Central - PubMed

Affiliation: INSERM, Unité 955, Créteil F-94000, France. stephane.terry@inserm.fr

ABSTRACT

Background: Class III beta-tubulin (betaIII-tubulin) is expressed in tissues of neuronal lineage and also in several human malignancies, including non-small-cell lung carcinoma, breast and ovarian cancer. Overexpression of betaIII-tubulin in these tumours is associated with an unfavourable outcome and resistance to taxane-based therapies. At present, betaIII-tubulin expression remains largely uncharacterised in prostate cancer.

Methods: In this report, we evaluated the expression of betaIII-tubulin in 138 different human prostate tumour specimens by immunohistochemistry from patients with hormone-treated or hormone-untreated prostate cancer. betaIII-tubulin expression was also examined in various prostatic cancer cell lines including in androgen-sensitive human prostate cancer cells, LNCaP, grown in androgen-depleted medium in 2D cultures or as tumour xenografts when the host mouse was castrated.

Results: Whereas moderate-to-strong betaIII-tubulin expression was detected in only 3 out of 74 (4%) hormone-naive tumour specimens obtained from patients who never received hormone therapy, 6 out of 24 tumour specimens (25%) from patients treated for 3 months with neoadjuvant hormone therapy and 24 out of 40 (60%) castration-resistant tumour specimens from chronic hormone-treated patients were found to express significant levels of betaIII-tubulin. These findings were supported by in vitro and in vivo settings.

Conclusion: Our data indicate that betaIII-tubulin expression is augmented in prostate cancer by androgen ablation and that the expression of this beta-tubulin isoform is associated with the progression of prostate cancer to the castration-resistant state, a stage largely responsible for mortality from prostate cancer.

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βIII-tubulin expression is regulated by androgen depletion in LNCaP cultures and is expressed in androgen-independent PCa lines. (A) Time-course expression of βIII-tubulin in LNCaPs cultivated in androgen-reduced medium. At day 0, monolayer cultured LNCaP cells were grown in the androgen-reduced medium. Each protein sample (30 μg) was resolved to SDS–PAGE, transferred onto a polyvinylidene difluoride membrane (Millipore) and incubated with a monoclonal antibody against βIII-tubulin or β-actin as an internal control. (B) The same procedure was applied to examine βIII-tubulin expression in various PCa cell lines (LNCaP, 22Rv1, DU145 and PC3) maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), AS, androgen-sensitive; AI, androgen-independent. (C) βIII-tubulin mRNA is reduced on 10 days of stimulation by 10 n DHT in LNCaP cells relative to the expression in cells maintained in the androgen-reduced medium. Columns, mean±s.e.m.
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fig2: βIII-tubulin expression is regulated by androgen depletion in LNCaP cultures and is expressed in androgen-independent PCa lines. (A) Time-course expression of βIII-tubulin in LNCaPs cultivated in androgen-reduced medium. At day 0, monolayer cultured LNCaP cells were grown in the androgen-reduced medium. Each protein sample (30 μg) was resolved to SDS–PAGE, transferred onto a polyvinylidene difluoride membrane (Millipore) and incubated with a monoclonal antibody against βIII-tubulin or β-actin as an internal control. (B) The same procedure was applied to examine βIII-tubulin expression in various PCa cell lines (LNCaP, 22Rv1, DU145 and PC3) maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), AS, androgen-sensitive; AI, androgen-independent. (C) βIII-tubulin mRNA is reduced on 10 days of stimulation by 10 n DHT in LNCaP cells relative to the expression in cells maintained in the androgen-reduced medium. Columns, mean±s.e.m.

Mentions: A trans-differentiation phenomenon is observed in cultured androgen-sensitive PCa cells when they are maintained in a medium depleted of androgens (Burchardt et al, 1999), and therefore we examined, using western blot analysis, whether androgen-sensitive human prostate cancer LNCaP cells upregulate βIII-tubulin when they are switched to androgen-deficient medium. Figure 2A shows that the steady-state levels of βIII-tubulin protein were strikingly elevated in the days following growth in the androgen-depleted medium with persistent expression detected up to 3 months under these conditions. The ability of growth in the androgen-depleted medium to upregulate βIII-tubulin protein expression was independent of the passage number of the LNCaP cells, as we obtained similar results using either low- (P25) or high-passage (P80) cells (data not shown). In addition, βIII-tubulin protein was expressed in various androgen-independent human PCa cell lines, including 22Rv1, PC3 and DU145 after western blot analysis of protein extracts from these cells (Figure 2B). These data agree with a previous finding that βIII-tubulin protein expression could be upregulated in LNCaP cells cultured in the androgen-deficient medium for 4 days or after transient suppression of androgen receptor expression using siRNA (Wright et al, 2003). We then tested the effect of the AR agonist, DHT on βIII-tubulin mRNA expression. To this end, LNCaP cells were incubated for 10 days in an androgen-depleted medium supplemented with 10 n DHT or with vehicle (ethanol, EtOH). The expression of βIII-tubulin was partially abrogated in chronic DHT-treated cells (Figure 2C).


Increased expression of class III beta-tubulin in castration-resistant human prostate cancer.

Terry S, Ploussard G, Allory Y, Nicolaiew N, Boissière-Michot F, Maillé P, Kheuang L, Coppolani E, Ali A, Bibeau F, Culine S, Buttyan R, de la Taille A, Vacherot F - Br. J. Cancer (2009)

βIII-tubulin expression is regulated by androgen depletion in LNCaP cultures and is expressed in androgen-independent PCa lines. (A) Time-course expression of βIII-tubulin in LNCaPs cultivated in androgen-reduced medium. At day 0, monolayer cultured LNCaP cells were grown in the androgen-reduced medium. Each protein sample (30 μg) was resolved to SDS–PAGE, transferred onto a polyvinylidene difluoride membrane (Millipore) and incubated with a monoclonal antibody against βIII-tubulin or β-actin as an internal control. (B) The same procedure was applied to examine βIII-tubulin expression in various PCa cell lines (LNCaP, 22Rv1, DU145 and PC3) maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), AS, androgen-sensitive; AI, androgen-independent. (C) βIII-tubulin mRNA is reduced on 10 days of stimulation by 10 n DHT in LNCaP cells relative to the expression in cells maintained in the androgen-reduced medium. Columns, mean±s.e.m.
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Related In: Results  -  Collection

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fig2: βIII-tubulin expression is regulated by androgen depletion in LNCaP cultures and is expressed in androgen-independent PCa lines. (A) Time-course expression of βIII-tubulin in LNCaPs cultivated in androgen-reduced medium. At day 0, monolayer cultured LNCaP cells were grown in the androgen-reduced medium. Each protein sample (30 μg) was resolved to SDS–PAGE, transferred onto a polyvinylidene difluoride membrane (Millipore) and incubated with a monoclonal antibody against βIII-tubulin or β-actin as an internal control. (B) The same procedure was applied to examine βIII-tubulin expression in various PCa cell lines (LNCaP, 22Rv1, DU145 and PC3) maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), AS, androgen-sensitive; AI, androgen-independent. (C) βIII-tubulin mRNA is reduced on 10 days of stimulation by 10 n DHT in LNCaP cells relative to the expression in cells maintained in the androgen-reduced medium. Columns, mean±s.e.m.
Mentions: A trans-differentiation phenomenon is observed in cultured androgen-sensitive PCa cells when they are maintained in a medium depleted of androgens (Burchardt et al, 1999), and therefore we examined, using western blot analysis, whether androgen-sensitive human prostate cancer LNCaP cells upregulate βIII-tubulin when they are switched to androgen-deficient medium. Figure 2A shows that the steady-state levels of βIII-tubulin protein were strikingly elevated in the days following growth in the androgen-depleted medium with persistent expression detected up to 3 months under these conditions. The ability of growth in the androgen-depleted medium to upregulate βIII-tubulin protein expression was independent of the passage number of the LNCaP cells, as we obtained similar results using either low- (P25) or high-passage (P80) cells (data not shown). In addition, βIII-tubulin protein was expressed in various androgen-independent human PCa cell lines, including 22Rv1, PC3 and DU145 after western blot analysis of protein extracts from these cells (Figure 2B). These data agree with a previous finding that βIII-tubulin protein expression could be upregulated in LNCaP cells cultured in the androgen-deficient medium for 4 days or after transient suppression of androgen receptor expression using siRNA (Wright et al, 2003). We then tested the effect of the AR agonist, DHT on βIII-tubulin mRNA expression. To this end, LNCaP cells were incubated for 10 days in an androgen-depleted medium supplemented with 10 n DHT or with vehicle (ethanol, EtOH). The expression of βIII-tubulin was partially abrogated in chronic DHT-treated cells (Figure 2C).

Bottom Line: Overexpression of betaIII-tubulin in these tumours is associated with an unfavourable outcome and resistance to taxane-based therapies.Whereas moderate-to-strong betaIII-tubulin expression was detected in only 3 out of 74 (4%) hormone-naive tumour specimens obtained from patients who never received hormone therapy, 6 out of 24 tumour specimens (25%) from patients treated for 3 months with neoadjuvant hormone therapy and 24 out of 40 (60%) castration-resistant tumour specimens from chronic hormone-treated patients were found to express significant levels of betaIII-tubulin.These findings were supported by in vitro and in vivo settings.

View Article: PubMed Central - PubMed

Affiliation: INSERM, Unité 955, Créteil F-94000, France. stephane.terry@inserm.fr

ABSTRACT

Background: Class III beta-tubulin (betaIII-tubulin) is expressed in tissues of neuronal lineage and also in several human malignancies, including non-small-cell lung carcinoma, breast and ovarian cancer. Overexpression of betaIII-tubulin in these tumours is associated with an unfavourable outcome and resistance to taxane-based therapies. At present, betaIII-tubulin expression remains largely uncharacterised in prostate cancer.

Methods: In this report, we evaluated the expression of betaIII-tubulin in 138 different human prostate tumour specimens by immunohistochemistry from patients with hormone-treated or hormone-untreated prostate cancer. betaIII-tubulin expression was also examined in various prostatic cancer cell lines including in androgen-sensitive human prostate cancer cells, LNCaP, grown in androgen-depleted medium in 2D cultures or as tumour xenografts when the host mouse was castrated.

Results: Whereas moderate-to-strong betaIII-tubulin expression was detected in only 3 out of 74 (4%) hormone-naive tumour specimens obtained from patients who never received hormone therapy, 6 out of 24 tumour specimens (25%) from patients treated for 3 months with neoadjuvant hormone therapy and 24 out of 40 (60%) castration-resistant tumour specimens from chronic hormone-treated patients were found to express significant levels of betaIII-tubulin. These findings were supported by in vitro and in vivo settings.

Conclusion: Our data indicate that betaIII-tubulin expression is augmented in prostate cancer by androgen ablation and that the expression of this beta-tubulin isoform is associated with the progression of prostate cancer to the castration-resistant state, a stage largely responsible for mortality from prostate cancer.

Show MeSH
Related in: MedlinePlus