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Hypoxia-associated p38 mitogen-activated protein kinase-mediated androgen receptor activation and increased HIF-1alpha levels contribute to emergence of an aggressive phenotype in prostate cancer.

Khandrika L, Lieberman R, Koul S, Kumar B, Maroni P, Chandhoke R, Meacham RB, Koul HK - Oncogene (2009)

Bottom Line: Our results demonstrate that hypoxia-reoxygenation resulted in increased survival, higher clonogenicity and enhanced invasiveness of these cells.Moreover, hypoxia-reoxygenation was associated with an increased AR activity independent of androgens as well as increased hypoxia inducible factor (HIF-1alpha) levels and activity.We also observed that the activation of p38 mitogen-activated protein (MAP) kinase pathway was an early response to hypoxia, and inhibition of p38 MAP kinase pathway by variety of approaches abolished hypoxia-reoxygenation induced increased AR activity as well as increased survival, clonogenicity and invasiveness.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction and Molecular Urology Laboratory-Program in Urosciences, Division of Urology, Department of Surgery, School of Medicine, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80045, USA.

ABSTRACT
Androgen receptor (AR) signaling is involved in the development and progression of prostate cancer. Tumor microvasculature contributes to continual exposure of prostate cancer cells to hypoxia-reoxygenation, however, the role of hypoxia-reoxygenation in prostate cancer progression and modulation of AR signaling is not understood. In this study, we evaluated the effects of hypoxia-reoxygenation in LNCaP cells, a line of hormone responsive human prostate cancer cells. Our results demonstrate that hypoxia-reoxygenation resulted in increased survival, higher clonogenicity and enhanced invasiveness of these cells. Moreover, hypoxia-reoxygenation was associated with an increased AR activity independent of androgens as well as increased hypoxia inducible factor (HIF-1alpha) levels and activity. We also observed that the activation of p38 mitogen-activated protein (MAP) kinase pathway was an early response to hypoxia, and inhibition of p38 MAP kinase pathway by variety of approaches abolished hypoxia-reoxygenation induced increased AR activity as well as increased survival, clonogenicity and invasiveness. These results demonstrate a critical role for hypoxia-induced p38 MAP kinase pathway in androgen-independent AR activation in prostate cancer cells, and suggest that hypoxia-reoxygenation may select for aggressive androgen-independent prostate cancer phenotype.

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Inhibition of p38 MAPK reduces survival and invasion of LNCaP cells(a) LNCaP cells were pre-treated with 10μM SB203580 for 1 hour and then incubated in hypoxia for 12h. Cells were stained with crystal violet to determine survival. Data points represent mean ± S.D of duplicate experiments done in triplicate (* indicates p<0.05 compared to normoxia control, n=6). (b) LNCaP cells were incubated in hypoxia for 8h either in absence of androgens either with or without 10μM SB203580. Equal numbers of cells were seeded in Boyden chambers and one set of cells not subjected to hypoxia, were allowed to invade in presence of 10nM DHT. 48h later invaded cells were stained with Crystal Violet (Upper Panel). Representative images from two individual experiments done in duplicate are shown. Cells that invaded through matrigel were quantified (Lower Panel) as before and each data point represents mean ± S.D (* indicates p<0.05 compared to cells incubated in hypoxia in androgen free medium and uninhibited p38 MAPK, n=4). (c) LNCaP cells were incubated in hypoxia either with or without 10μM SB203580 for 12 hours and colony formation in soft agar was assayed (Upper Panel). Representative images from duplicate experiments done in triplicate are shown. Number of colonies was quantified as before (Lower Panel). Each data point represents mean ± S.D (* indicates p<0.05 compared to control, n=6).
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Figure 5: Inhibition of p38 MAPK reduces survival and invasion of LNCaP cells(a) LNCaP cells were pre-treated with 10μM SB203580 for 1 hour and then incubated in hypoxia for 12h. Cells were stained with crystal violet to determine survival. Data points represent mean ± S.D of duplicate experiments done in triplicate (* indicates p<0.05 compared to normoxia control, n=6). (b) LNCaP cells were incubated in hypoxia for 8h either in absence of androgens either with or without 10μM SB203580. Equal numbers of cells were seeded in Boyden chambers and one set of cells not subjected to hypoxia, were allowed to invade in presence of 10nM DHT. 48h later invaded cells were stained with Crystal Violet (Upper Panel). Representative images from two individual experiments done in duplicate are shown. Cells that invaded through matrigel were quantified (Lower Panel) as before and each data point represents mean ± S.D (* indicates p<0.05 compared to cells incubated in hypoxia in androgen free medium and uninhibited p38 MAPK, n=4). (c) LNCaP cells were incubated in hypoxia either with or without 10μM SB203580 for 12 hours and colony formation in soft agar was assayed (Upper Panel). Representative images from duplicate experiments done in triplicate are shown. Number of colonies was quantified as before (Lower Panel). Each data point represents mean ± S.D (* indicates p<0.05 compared to control, n=6).

Mentions: To understand the significance of p38 MAPK activity in LNCaP cells during hypoxia, we assessed the survival, invasion and clonogenic potential of LNCaP cells incubated in hypoxia, after inhibiting p38 MAPK activity. Addition of 10μM SB203580 during 12h hypoxia incubation significantly reduced the survival of LNCaP cells (Figure 5a). A similar reduction in the proliferation of these cells was observed with MTT assay (data not shown). Invasion of LNCaP cells incubated in hypoxia for 8h in androgen free medium was comparable to the number of cells that invaded through matrigel matrix in presence of 10nM DHT in normoxia (Figure 5b). Significantly, the number of cells that invaded was reduced when 10μM SB203580 was included in the medium during 8h hypoxia incubation as well as during the invasion assay in Boyden chambers. In addition to invasiveness, clonogenic potential was also significantly reduced in cells subjected to 12h hypoxia after inhibiting p38 MAPK (Figure 5c). Moreover, the size of established colonies was also greatly reduced compared to the cells in which p38 MAPK was active during hypoxia treatment.


Hypoxia-associated p38 mitogen-activated protein kinase-mediated androgen receptor activation and increased HIF-1alpha levels contribute to emergence of an aggressive phenotype in prostate cancer.

Khandrika L, Lieberman R, Koul S, Kumar B, Maroni P, Chandhoke R, Meacham RB, Koul HK - Oncogene (2009)

Inhibition of p38 MAPK reduces survival and invasion of LNCaP cells(a) LNCaP cells were pre-treated with 10μM SB203580 for 1 hour and then incubated in hypoxia for 12h. Cells were stained with crystal violet to determine survival. Data points represent mean ± S.D of duplicate experiments done in triplicate (* indicates p<0.05 compared to normoxia control, n=6). (b) LNCaP cells were incubated in hypoxia for 8h either in absence of androgens either with or without 10μM SB203580. Equal numbers of cells were seeded in Boyden chambers and one set of cells not subjected to hypoxia, were allowed to invade in presence of 10nM DHT. 48h later invaded cells were stained with Crystal Violet (Upper Panel). Representative images from two individual experiments done in duplicate are shown. Cells that invaded through matrigel were quantified (Lower Panel) as before and each data point represents mean ± S.D (* indicates p<0.05 compared to cells incubated in hypoxia in androgen free medium and uninhibited p38 MAPK, n=4). (c) LNCaP cells were incubated in hypoxia either with or without 10μM SB203580 for 12 hours and colony formation in soft agar was assayed (Upper Panel). Representative images from duplicate experiments done in triplicate are shown. Number of colonies was quantified as before (Lower Panel). Each data point represents mean ± S.D (* indicates p<0.05 compared to control, n=6).
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Figure 5: Inhibition of p38 MAPK reduces survival and invasion of LNCaP cells(a) LNCaP cells were pre-treated with 10μM SB203580 for 1 hour and then incubated in hypoxia for 12h. Cells were stained with crystal violet to determine survival. Data points represent mean ± S.D of duplicate experiments done in triplicate (* indicates p<0.05 compared to normoxia control, n=6). (b) LNCaP cells were incubated in hypoxia for 8h either in absence of androgens either with or without 10μM SB203580. Equal numbers of cells were seeded in Boyden chambers and one set of cells not subjected to hypoxia, were allowed to invade in presence of 10nM DHT. 48h later invaded cells were stained with Crystal Violet (Upper Panel). Representative images from two individual experiments done in duplicate are shown. Cells that invaded through matrigel were quantified (Lower Panel) as before and each data point represents mean ± S.D (* indicates p<0.05 compared to cells incubated in hypoxia in androgen free medium and uninhibited p38 MAPK, n=4). (c) LNCaP cells were incubated in hypoxia either with or without 10μM SB203580 for 12 hours and colony formation in soft agar was assayed (Upper Panel). Representative images from duplicate experiments done in triplicate are shown. Number of colonies was quantified as before (Lower Panel). Each data point represents mean ± S.D (* indicates p<0.05 compared to control, n=6).
Mentions: To understand the significance of p38 MAPK activity in LNCaP cells during hypoxia, we assessed the survival, invasion and clonogenic potential of LNCaP cells incubated in hypoxia, after inhibiting p38 MAPK activity. Addition of 10μM SB203580 during 12h hypoxia incubation significantly reduced the survival of LNCaP cells (Figure 5a). A similar reduction in the proliferation of these cells was observed with MTT assay (data not shown). Invasion of LNCaP cells incubated in hypoxia for 8h in androgen free medium was comparable to the number of cells that invaded through matrigel matrix in presence of 10nM DHT in normoxia (Figure 5b). Significantly, the number of cells that invaded was reduced when 10μM SB203580 was included in the medium during 8h hypoxia incubation as well as during the invasion assay in Boyden chambers. In addition to invasiveness, clonogenic potential was also significantly reduced in cells subjected to 12h hypoxia after inhibiting p38 MAPK (Figure 5c). Moreover, the size of established colonies was also greatly reduced compared to the cells in which p38 MAPK was active during hypoxia treatment.

Bottom Line: Our results demonstrate that hypoxia-reoxygenation resulted in increased survival, higher clonogenicity and enhanced invasiveness of these cells.Moreover, hypoxia-reoxygenation was associated with an increased AR activity independent of androgens as well as increased hypoxia inducible factor (HIF-1alpha) levels and activity.We also observed that the activation of p38 mitogen-activated protein (MAP) kinase pathway was an early response to hypoxia, and inhibition of p38 MAP kinase pathway by variety of approaches abolished hypoxia-reoxygenation induced increased AR activity as well as increased survival, clonogenicity and invasiveness.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction and Molecular Urology Laboratory-Program in Urosciences, Division of Urology, Department of Surgery, School of Medicine, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80045, USA.

ABSTRACT
Androgen receptor (AR) signaling is involved in the development and progression of prostate cancer. Tumor microvasculature contributes to continual exposure of prostate cancer cells to hypoxia-reoxygenation, however, the role of hypoxia-reoxygenation in prostate cancer progression and modulation of AR signaling is not understood. In this study, we evaluated the effects of hypoxia-reoxygenation in LNCaP cells, a line of hormone responsive human prostate cancer cells. Our results demonstrate that hypoxia-reoxygenation resulted in increased survival, higher clonogenicity and enhanced invasiveness of these cells. Moreover, hypoxia-reoxygenation was associated with an increased AR activity independent of androgens as well as increased hypoxia inducible factor (HIF-1alpha) levels and activity. We also observed that the activation of p38 mitogen-activated protein (MAP) kinase pathway was an early response to hypoxia, and inhibition of p38 MAP kinase pathway by variety of approaches abolished hypoxia-reoxygenation induced increased AR activity as well as increased survival, clonogenicity and invasiveness. These results demonstrate a critical role for hypoxia-induced p38 MAP kinase pathway in androgen-independent AR activation in prostate cancer cells, and suggest that hypoxia-reoxygenation may select for aggressive androgen-independent prostate cancer phenotype.

Show MeSH
Related in: MedlinePlus