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CHRONIC HYPOXIA PROMOTES ANDROGEN-INDEPENDENT GROWTH AND MALIGNANT CELL BEHAVIOR IN LNCaP HUMAN PROSTATE CANCER CELLS
Vol. 179, No. 4, Supplement, Sunday, May 18, 2008
through the stimulation of the invasive potential, and that an enhanced X3$SURGXFWLRQLQSURVWDWHFDQFHUFHOOVLQGXFHGE\7*)ȕFRXOGWKHUHIRUH be one of the most important mechanisms mediating the progression of prostate cancer after SV invasion. Source of Funding: None
286 DuCaP AND VCaP ARE ANDROGEN-DEPENDENT PROSTATE CANCER CELL LINES THAT FORM OSTEOBLASTIC BONE METASTASES George N Thalmann*, Antoinette Wetterwald, Ruth Schwaninger, Markus Germann, Urs E Studer, Marco G Cecchini. Bern, Switzerland. INTRODUCTION AND OBJECTIVE: Prostate cancer (CaP) cell lines that can easily be propagated in culture and are able to induce a truly robust osteoblastic reaction when metastatic to bone in vivo are rare. We investigated the ability of the CaP cell lines DuCaP and VCaP for their ability to form bone metastases in the intact host and their gene H[SUHVVLRQSUR¿OHVLQYLWURRIJHQHVLQYROYHGLQWKHERQHUHVSRQVHWR metastatic growth. METHODS: DuCaP and VCaP cells were injected either into the left cardiac ventricle or intraosseously in the tibia of intact SCID mice under general anesthesia. Development of bone metastases was monitored radiographically. Tridimensional bone structure of ERQH PHWDVWDVHV ZDV DQDO\]HG E\ PLFURFRPSXWHUL]HG WRPRJUDSK\ PLFUR&7 *HQH H[SUHVVLRQ SUR¿OHV IRU PHPEHUV RI WKH 7*)EHWD superfamily, the wnt family, their antagonists and osteotropic cytokines involved in bone resorption and bone turnover were determined by real time-PCR. RESULTS: Intraosseous tumor growth induced by intraosseous injection of DuCaP and VCaP resulted in a strong osteoblastic response in bone metastases as determined by radiography and Micro-CT. No metastases developed after intracardiac injection. Both cell lines expressed neither the BMP antagonist noggin nor the wnt antagonists dickkopf-1 (dkk-1) and SOST, indicative of an osteoblastic phenotype. VCaP expresses bone morphogenic protein-2 (BMP-2) and wnt2. Both cell lines do not express osteolytic cytokines such as Parathyroid hormone-related protein (PTHrP), Colony-stimulating factor 1 (CSF-1), Interleukin-8 (IL-8) or Connective Tissue Growth Factor (CTGF). Very low or no expression was found for Insulin-like Growth Factor 1 (IGF-1) and endothelin-1. CONCLUSIONS: The androgen-dependent CaP cell lines DuCaP and VCaP form osteoblastic bone metastases in vivo when injected intraosseously, but not when injected intracardiacally. Consistent with their osteoblastic phenotype they lack expression of the BMP antagonist noggin and of the wnt antagonist dkk-1 as well as of osteolytic cytokines. Source of Funding: None
287 CHRONIC HYPOXIA PROMOTES ANDROGEN-INDEPENDENT GROWTH AND MALIGNANT CELL BEHAVIOR IN LNCaP HUMAN PROSTATE CANCER CELLS Mutsushi Yamasaki*, Takeo Nomura, Fuminori Sato, Hiromitsu Mimata. Oita, Japan. INTRODUCTION AND OBJECTIVE: Prostate cancer (PC) frequently progresses from an initial androgen dependence to androgen independence, rendering the only effective androgen ablation therapy useless. Tumor hypoxia is a common feature of any cancers, including PC, and associated with tumor cell aggressiveness. The androgen ablation therapy also induces hypoxic environment via reduction of EORRGÀRZWR3&WLVVXH$OWKRXJKVRPHUHSRUWVDUHDYDLODEOHRQDFXWH hypoxia-response in PC cells aggressiveness, little is known about chronic hypoxia-response. METHODS: The human androgen-dependent PC cell line (LNCaP) was cultured under normoxia (21% O2, LNCaP-N), acute hypoxia (1% O2, LNCaP-AH) or chronic hypoxia (1% O2 over 6 months, LNCaP-CH). The cell growth and cycle of these cells, cultured in QRUPDORUDQGURJHQDEODWHGFRQGLWLRQZHUHDQDO\]HGE\FHOOFRXQWDQG ÀRZF\WRPHWU\DQDO\VLV7KHFHOOEHKDYLRUZDVWHVWHGXVLQJPLJUDWLRQ
THE JOURNAL OF UROLOGY®
101
and invasion assay. The hypoxia-induced intracellular signalling was DQDO\]HGE\ZHVWHUQEORWWLQJ RESULTS: LNCaP-CH cells grew androgen-independent and hypoxia-resistant manner (Fig. A). The sensitivity of androgen ablation was kept in LNCaP-CH cells. The androgen ablation induced G1-arrest in LNCaP-N and -AH cells, but not in LNCaP-CH cells (Fig. B). The expression of p27 Kip1 was decreased in LNCaP-CH cells. Cyclin D1 and phospho-Rb was decreased in LNCaP-AH cells compared with in LNCaP-N cells, but re-increased in LNCaP-CH cells. The cell migration and invasion were accelerated about twice in LNCaP-CH cells compared with LNCaP-N cells, whereas the cell migration was decreased in LNCaP-AH cells (Fig. C). The HIF-1, PI3K/Akt and Jak/Stat pathways were activated and Bcl-2 was overexpressed in LNCaP-CH cells. CONCLUSIONS: In this study, chronic hypoxia promoted androgen-independent growth and malignant cell behavior in human PC cells. These results suggest that chronic hypoxia plays an important role in enhancement of malignant potential during androgen independent PC progression via these signalling pathways.
Source of Funding: None
288 INTERLEUKIN-4 ACTIVATES ANDROGEN RECEPTOR THROUGH CBP/p300 MEDIATED ACETYLATION AND STIMULATES ANDROGEN INDEPENDENT GROWTH Soo Lee, Jae Chun, Meng Sun, Allen Gao*. Buffalo, NY. INTRODUCTION AND OBJECTIVE: Previous studies demonstrated that IL-4 enhances AR activation in the absence of androgen. In the present study, the mechanism of IL-4 mediated AR activation and the role of IL-4 in promoting the growth of androgen independent prostate cancer cells were investigated. METHODS: The levels of CBP/p300 protein and the effect of IL-4 on AR acetylation were examined by Western blot analysis. The effect of IL-4 on the interactions between androgen receptor and CBP/p300 proteins was examined by co-immunoprecipitation and by chromatin immunoprecipitation assays. LNCaP cells were transfected with a full length IL-4 cDNA and stable clones expressing IL-4 were VHOHFWHG7KHJURZWKRI/1&D3FHOOVH[SUHVVLQJ,/ZDVDQDO\]HGin vitro and in vivo both in the presence and absence of androgen. RESULTS: We showed that IL-4 increases the coactivator CBP/p300 protein expression and enhances the interactions between AR and CBP/p300 proteins. IL-4 increases the recruitment of CBP/ p300 protein to the androgen responsive elements in the promoter of androgen responsive genes. Downregulation of CBP/p300 expression XVLQJ&%3SVSHFL¿FVL51$DEROLVKHG,/PHGLDWHG$5DFWLYDWLRQ suggesting that CBP/p300 is responsible for AR activation induced by IL-4. Furthermore, AR activation can be enhanced by AR acetylation induced by IL-4 in prostate cancer cells. The IL-4 mediated AR acetylation can be blocked by knocking down CBP/p300 using interference RNA. Overexpression of IL-4 enhances the growth of androgen sensitive LNCaP cells in culture media containing charcoal stripped FBS condition,
through the stimulation of the invasive potential, and that an enhanced X3$SURGXFWLRQLQSURVWDWHFDQFHUFHOOVLQGXFHGE\7*)ȕFRXOGWKHUHIRUH be one of the most important mechanisms mediating the progression of prostate cancer after SV invasion. Source of Funding: None
286 DuCaP AND VCaP ARE ANDROGEN-DEPENDENT PROSTATE CANCER CELL LINES THAT FORM OSTEOBLASTIC BONE METASTASES George N Thalmann*, Antoinette Wetterwald, Ruth Schwaninger, Markus Germann, Urs E Studer, Marco G Cecchini. Bern, Switzerland. INTRODUCTION AND OBJECTIVE: Prostate cancer (CaP) cell lines that can easily be propagated in culture and are able to induce a truly robust osteoblastic reaction when metastatic to bone in vivo are rare. We investigated the ability of the CaP cell lines DuCaP and VCaP for their ability to form bone metastases in the intact host and their gene H[SUHVVLRQSUR¿OHVLQYLWURRIJHQHVLQYROYHGLQWKHERQHUHVSRQVHWR metastatic growth. METHODS: DuCaP and VCaP cells were injected either into the left cardiac ventricle or intraosseously in the tibia of intact SCID mice under general anesthesia. Development of bone metastases was monitored radiographically. Tridimensional bone structure of ERQH PHWDVWDVHV ZDV DQDO\]HG E\ PLFURFRPSXWHUL]HG WRPRJUDSK\ PLFUR&7 *HQH H[SUHVVLRQ SUR¿OHV IRU PHPEHUV RI WKH 7*)EHWD superfamily, the wnt family, their antagonists and osteotropic cytokines involved in bone resorption and bone turnover were determined by real time-PCR. RESULTS: Intraosseous tumor growth induced by intraosseous injection of DuCaP and VCaP resulted in a strong osteoblastic response in bone metastases as determined by radiography and Micro-CT. No metastases developed after intracardiac injection. Both cell lines expressed neither the BMP antagonist noggin nor the wnt antagonists dickkopf-1 (dkk-1) and SOST, indicative of an osteoblastic phenotype. VCaP expresses bone morphogenic protein-2 (BMP-2) and wnt2. Both cell lines do not express osteolytic cytokines such as Parathyroid hormone-related protein (PTHrP), Colony-stimulating factor 1 (CSF-1), Interleukin-8 (IL-8) or Connective Tissue Growth Factor (CTGF). Very low or no expression was found for Insulin-like Growth Factor 1 (IGF-1) and endothelin-1. CONCLUSIONS: The androgen-dependent CaP cell lines DuCaP and VCaP form osteoblastic bone metastases in vivo when injected intraosseously, but not when injected intracardiacally. Consistent with their osteoblastic phenotype they lack expression of the BMP antagonist noggin and of the wnt antagonist dkk-1 as well as of osteolytic cytokines. Source of Funding: None
287 CHRONIC HYPOXIA PROMOTES ANDROGEN-INDEPENDENT GROWTH AND MALIGNANT CELL BEHAVIOR IN LNCaP HUMAN PROSTATE CANCER CELLS Mutsushi Yamasaki*, Takeo Nomura, Fuminori Sato, Hiromitsu Mimata. Oita, Japan. INTRODUCTION AND OBJECTIVE: Prostate cancer (PC) frequently progresses from an initial androgen dependence to androgen independence, rendering the only effective androgen ablation therapy useless. Tumor hypoxia is a common feature of any cancers, including PC, and associated with tumor cell aggressiveness. The androgen ablation therapy also induces hypoxic environment via reduction of EORRGÀRZWR3&WLVVXH$OWKRXJKVRPHUHSRUWVDUHDYDLODEOHRQDFXWH hypoxia-response in PC cells aggressiveness, little is known about chronic hypoxia-response. METHODS: The human androgen-dependent PC cell line (LNCaP) was cultured under normoxia (21% O2, LNCaP-N), acute hypoxia (1% O2, LNCaP-AH) or chronic hypoxia (1% O2 over 6 months, LNCaP-CH). The cell growth and cycle of these cells, cultured in QRUPDORUDQGURJHQDEODWHGFRQGLWLRQZHUHDQDO\]HGE\FHOOFRXQWDQG ÀRZF\WRPHWU\DQDO\VLV7KHFHOOEHKDYLRUZDVWHVWHGXVLQJPLJUDWLRQ
THE JOURNAL OF UROLOGY®
101
and invasion assay. The hypoxia-induced intracellular signalling was DQDO\]HGE\ZHVWHUQEORWWLQJ RESULTS: LNCaP-CH cells grew androgen-independent and hypoxia-resistant manner (Fig. A). The sensitivity of androgen ablation was kept in LNCaP-CH cells. The androgen ablation induced G1-arrest in LNCaP-N and -AH cells, but not in LNCaP-CH cells (Fig. B). The expression of p27 Kip1 was decreased in LNCaP-CH cells. Cyclin D1 and phospho-Rb was decreased in LNCaP-AH cells compared with in LNCaP-N cells, but re-increased in LNCaP-CH cells. The cell migration and invasion were accelerated about twice in LNCaP-CH cells compared with LNCaP-N cells, whereas the cell migration was decreased in LNCaP-AH cells (Fig. C). The HIF-1, PI3K/Akt and Jak/Stat pathways were activated and Bcl-2 was overexpressed in LNCaP-CH cells. CONCLUSIONS: In this study, chronic hypoxia promoted androgen-independent growth and malignant cell behavior in human PC cells. These results suggest that chronic hypoxia plays an important role in enhancement of malignant potential during androgen independent PC progression via these signalling pathways.
Source of Funding: None
288 INTERLEUKIN-4 ACTIVATES ANDROGEN RECEPTOR THROUGH CBP/p300 MEDIATED ACETYLATION AND STIMULATES ANDROGEN INDEPENDENT GROWTH Soo Lee, Jae Chun, Meng Sun, Allen Gao*. Buffalo, NY. INTRODUCTION AND OBJECTIVE: Previous studies demonstrated that IL-4 enhances AR activation in the absence of androgen. In the present study, the mechanism of IL-4 mediated AR activation and the role of IL-4 in promoting the growth of androgen independent prostate cancer cells were investigated. METHODS: The levels of CBP/p300 protein and the effect of IL-4 on AR acetylation were examined by Western blot analysis. The effect of IL-4 on the interactions between androgen receptor and CBP/p300 proteins was examined by co-immunoprecipitation and by chromatin immunoprecipitation assays. LNCaP cells were transfected with a full length IL-4 cDNA and stable clones expressing IL-4 were VHOHFWHG7KHJURZWKRI/1&D3FHOOVH[SUHVVLQJ,/ZDVDQDO\]HGin vitro and in vivo both in the presence and absence of androgen. RESULTS: We showed that IL-4 increases the coactivator CBP/p300 protein expression and enhances the interactions between AR and CBP/p300 proteins. IL-4 increases the recruitment of CBP/ p300 protein to the androgen responsive elements in the promoter of androgen responsive genes. Downregulation of CBP/p300 expression XVLQJ&%3SVSHFL¿FVL51$DEROLVKHG,/PHGLDWHG$5DFWLYDWLRQ suggesting that CBP/p300 is responsible for AR activation induced by IL-4. Furthermore, AR activation can be enhanced by AR acetylation induced by IL-4 in prostate cancer cells. The IL-4 mediated AR acetylation can be blocked by knocking down CBP/p300 using interference RNA. Overexpression of IL-4 enhances the growth of androgen sensitive LNCaP cells in culture media containing charcoal stripped FBS condition,