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The aldehyde dehydrogenase enzyme 7A1 is functionally involved in prostate cancer bone metastasis.

van den Hoogen C, van der Horst G, Cheung H, Buijs JT, Pelger RC, van der Pluijm G - Clin. Exp. Metastasis (2011)

Bottom Line: Knockdown of ALDH7A1 resulted in decreased intra-bone growth and inhibited experimentally induced (bone) metastasis, while intra-prostatic growth was not affected.In line with these observations, evidence is presented that TGF-β, a key player in cancer invasiveness and bone metastasis, strongly induced ALDH activity while BMP7 (an antagonist of TGF-β signaling) down-regulated ALDH activity.Our findings show, for the first time, that the ALDH7A1 enzyme is functionally involved in the formation of bone metastases and that the effect appeared dependent on the microenvironment, i.e., bone versus prostate.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Leiden University Medical Centre, The Netherlands.

ABSTRACT
High aldehyde dehydrogenase (ALDH) activity can be used to identify tumor-initiating and metastasis-initiating cells in various human carcinomas, including prostate cancer. To date, the functional importance of ALDH enzymes in prostate carcinogenesis, progression and metastasis has remained elusive. Previously we identified strong expression of ALDH7A1 in human prostate cancer cell lines, primary tumors and matched bone metastases. In this study, we evaluated whether ALDH7A1 is required for the acquisition of a metastatic stem/progenitor cell phenotype in human prostate cancer. Knockdown of ALDH7A1 expression resulted in a decrease of the α2(hi)/αv(hi)/CD44(+) stem/progenitor cell subpopulation in the human prostate cancer cell line PC-3M-Pro4. In addition, ALDH7A1 knockdown significantly inhibited the clonogenic and migratory ability of human prostate cancer cells in vitro. Furthermore, a number of genes/factors involved in migration, invasion and metastasis were affected including transcription factors (snail, snail2, and twist) and osteopontin, an ECM molecule involved in metastasis. Knockdown of ALDH7A1 resulted in decreased intra-bone growth and inhibited experimentally induced (bone) metastasis, while intra-prostatic growth was not affected. In line with these observations, evidence is presented that TGF-β, a key player in cancer invasiveness and bone metastasis, strongly induced ALDH activity while BMP7 (an antagonist of TGF-β signaling) down-regulated ALDH activity. Our findings show, for the first time, that the ALDH7A1 enzyme is functionally involved in the formation of bone metastases and that the effect appeared dependent on the microenvironment, i.e., bone versus prostate.

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Characterization of ALDH7A1-kd-Pro4luc prostate cancer cells. a Western blot analysis of ALDH7A1 expression in NT-Pro4luc (first lane) and ALDH7A1-kd-Pro4luc cells (cell clone #1 and #2 derived from different shRNAi constructs). b The number of colonies per 96-well plate in the single cell diluted cultures after 2 weeks in the ALDH7A1-kd-Pro4luc and NT-Pro4luc cells. c Mean number of migrated ALDH7A1-kd-Pro4luc and NT-Pro4luc cells per field. d Absorbance measured at 490 nm after 24, 48, and 72 h of incubation in ALDH7A1-kd-Pro4luc (filled circle) and control NT-Pro4luc cells (open circle). Data are representative for three independent experiments
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Fig1: Characterization of ALDH7A1-kd-Pro4luc prostate cancer cells. a Western blot analysis of ALDH7A1 expression in NT-Pro4luc (first lane) and ALDH7A1-kd-Pro4luc cells (cell clone #1 and #2 derived from different shRNAi constructs). b The number of colonies per 96-well plate in the single cell diluted cultures after 2 weeks in the ALDH7A1-kd-Pro4luc and NT-Pro4luc cells. c Mean number of migrated ALDH7A1-kd-Pro4luc and NT-Pro4luc cells per field. d Absorbance measured at 490 nm after 24, 48, and 72 h of incubation in ALDH7A1-kd-Pro4luc (filled circle) and control NT-Pro4luc cells (open circle). Data are representative for three independent experiments

Mentions: Different shRNAi constructs from the Sigma MISSION library were used to knockdown ALDH7A1 activity and two constructs showed a strong down-regulation of ALDH7A1 compared to the non-targeted control prostate cancer cells (Fig. 1a cell clone #1 and #2). Based on the high level of knockdown, we selected cell clone #1 (ALDH7A1-kd-Pro4luc) for further characterization in vitro and functional analysis in vivo and compared all results to the non-targeted control prostate cancer cells (NT-Pro4luc). Cell viability was confirmed by trypan blue exclusion prior to each experiment, and no differences among experimental groups were observed (data not shown). Potential impact on other ALDH isoforms was investigated by q-PCR analysis (Supplemental Figure S1). The expression levels of the ALDH isoforms 3A2, 5A1, 9A1 and 18A1 were not significantly affected by the ALDH7 knock down, whereas the expression levels of ALDH4A1 and 6A1 displayed a minor increase.Fig. 1


The aldehyde dehydrogenase enzyme 7A1 is functionally involved in prostate cancer bone metastasis.

van den Hoogen C, van der Horst G, Cheung H, Buijs JT, Pelger RC, van der Pluijm G - Clin. Exp. Metastasis (2011)

Characterization of ALDH7A1-kd-Pro4luc prostate cancer cells. a Western blot analysis of ALDH7A1 expression in NT-Pro4luc (first lane) and ALDH7A1-kd-Pro4luc cells (cell clone #1 and #2 derived from different shRNAi constructs). b The number of colonies per 96-well plate in the single cell diluted cultures after 2 weeks in the ALDH7A1-kd-Pro4luc and NT-Pro4luc cells. c Mean number of migrated ALDH7A1-kd-Pro4luc and NT-Pro4luc cells per field. d Absorbance measured at 490 nm after 24, 48, and 72 h of incubation in ALDH7A1-kd-Pro4luc (filled circle) and control NT-Pro4luc cells (open circle). Data are representative for three independent experiments
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Related In: Results  -  Collection

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Fig1: Characterization of ALDH7A1-kd-Pro4luc prostate cancer cells. a Western blot analysis of ALDH7A1 expression in NT-Pro4luc (first lane) and ALDH7A1-kd-Pro4luc cells (cell clone #1 and #2 derived from different shRNAi constructs). b The number of colonies per 96-well plate in the single cell diluted cultures after 2 weeks in the ALDH7A1-kd-Pro4luc and NT-Pro4luc cells. c Mean number of migrated ALDH7A1-kd-Pro4luc and NT-Pro4luc cells per field. d Absorbance measured at 490 nm after 24, 48, and 72 h of incubation in ALDH7A1-kd-Pro4luc (filled circle) and control NT-Pro4luc cells (open circle). Data are representative for three independent experiments
Mentions: Different shRNAi constructs from the Sigma MISSION library were used to knockdown ALDH7A1 activity and two constructs showed a strong down-regulation of ALDH7A1 compared to the non-targeted control prostate cancer cells (Fig. 1a cell clone #1 and #2). Based on the high level of knockdown, we selected cell clone #1 (ALDH7A1-kd-Pro4luc) for further characterization in vitro and functional analysis in vivo and compared all results to the non-targeted control prostate cancer cells (NT-Pro4luc). Cell viability was confirmed by trypan blue exclusion prior to each experiment, and no differences among experimental groups were observed (data not shown). Potential impact on other ALDH isoforms was investigated by q-PCR analysis (Supplemental Figure S1). The expression levels of the ALDH isoforms 3A2, 5A1, 9A1 and 18A1 were not significantly affected by the ALDH7 knock down, whereas the expression levels of ALDH4A1 and 6A1 displayed a minor increase.Fig. 1

Bottom Line: Knockdown of ALDH7A1 resulted in decreased intra-bone growth and inhibited experimentally induced (bone) metastasis, while intra-prostatic growth was not affected.In line with these observations, evidence is presented that TGF-β, a key player in cancer invasiveness and bone metastasis, strongly induced ALDH activity while BMP7 (an antagonist of TGF-β signaling) down-regulated ALDH activity.Our findings show, for the first time, that the ALDH7A1 enzyme is functionally involved in the formation of bone metastases and that the effect appeared dependent on the microenvironment, i.e., bone versus prostate.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Leiden University Medical Centre, The Netherlands.

ABSTRACT
High aldehyde dehydrogenase (ALDH) activity can be used to identify tumor-initiating and metastasis-initiating cells in various human carcinomas, including prostate cancer. To date, the functional importance of ALDH enzymes in prostate carcinogenesis, progression and metastasis has remained elusive. Previously we identified strong expression of ALDH7A1 in human prostate cancer cell lines, primary tumors and matched bone metastases. In this study, we evaluated whether ALDH7A1 is required for the acquisition of a metastatic stem/progenitor cell phenotype in human prostate cancer. Knockdown of ALDH7A1 expression resulted in a decrease of the α2(hi)/αv(hi)/CD44(+) stem/progenitor cell subpopulation in the human prostate cancer cell line PC-3M-Pro4. In addition, ALDH7A1 knockdown significantly inhibited the clonogenic and migratory ability of human prostate cancer cells in vitro. Furthermore, a number of genes/factors involved in migration, invasion and metastasis were affected including transcription factors (snail, snail2, and twist) and osteopontin, an ECM molecule involved in metastasis. Knockdown of ALDH7A1 resulted in decreased intra-bone growth and inhibited experimentally induced (bone) metastasis, while intra-prostatic growth was not affected. In line with these observations, evidence is presented that TGF-β, a key player in cancer invasiveness and bone metastasis, strongly induced ALDH activity while BMP7 (an antagonist of TGF-β signaling) down-regulated ALDH activity. Our findings show, for the first time, that the ALDH7A1 enzyme is functionally involved in the formation of bone metastases and that the effect appeared dependent on the microenvironment, i.e., bone versus prostate.

Show MeSH
Related in: MedlinePlus