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Dioxin receptor regulates aldehyde dehydrogenase to block melanoma tumorigenesis and metastasis.

Contador-Troca M, Alvarez-Barrientos A, Merino JM, Morales-Hernández A, Rodríguez MI, Rey-Barroso J, Barrasa E, Cerezo-Guisado MI, Catalina-Fernández I, Sáenz-Santamaría J, Oliver FJ, Fernandez-Salguero PM - Mol. Cancer (2015)

Bottom Line: Their tumorigenicity in vivo was analyzed using xenografts and lung metastasis assays as well as in vivo imaging.However, Aldh1a1 downmodulation in AhR-expressing B16F10 cells did not significantly affect tumor growth in vivo.Aldh1a1 knockdown reduced the high levels of CD133(+)/CD29(+)/CD44(+) cells, melanosphere size and the expression of the pluripotency marker Sox2 in sh-AhR cells.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, 06071, Badajoz, Spain. maria_cotr@hotmail.com.

ABSTRACT

Background: The dioxin (AhR) receptor can have oncogenic or tumor suppressor activities depending on the phenotype of the target cell. We have shown that AhR knockdown promotes melanoma primary tumorigenesis and lung metastasis in the mouse and that human metastatic melanomas had reduced AhR levels with respect to benign nevi.

Methods: Mouse melanoma B16F10 cells were engineered by retroviral transduction to stably downregulate AhR expression, Aldh1a1 expression or both. They were characterized for Aldh1a1 activity, stem cell markers and migration and invasion in vitro. Their tumorigenicity in vivo was analyzed using xenografts and lung metastasis assays as well as in vivo imaging.

Results: Depletion of aldehyde dehydrogenase 1a1 (Aldh1a1) impairs the pro-tumorigenic and pro-metastatic advantage of melanoma cells lacking AhR expression (sh-AhR). Thus, Aldh1a1 knockdown in sh-AhR cells (sh-AhR + sh-Aldh1a1) diminished their migration and invasion potentials and blocked tumor growth and metastasis to the lungs in immunocompetent AhR+/+ recipient mice. However, Aldh1a1 downmodulation in AhR-expressing B16F10 cells did not significantly affect tumor growth in vivo. Aldh1a1 knockdown reduced the high levels of CD133(+)/CD29(+)/CD44(+) cells, melanosphere size and the expression of the pluripotency marker Sox2 in sh-AhR cells. Interestingly, Sox2 increased Aldh1a1 expression in sh-AhR but not in sh-AhR + sh-Aldh1a1 cells, suggesting that Aldh1a1 and Sox2 may be co-regulated in melanoma cells. In vivo imaging revealed that mice inoculated with AhR + Aldh1a1 knockdown cells had reduced tumor burden and enhanced survival than those receiving Aldh1a1-expressing sh-AhR cells.

Conclusions: Aldh1a1 overactivation in an AhR-deficient background enhances melanoma progression. Since AhR may antagonize the protumoral effects of Aldh1a1, the AhR(low)-Aldh1a1(high) phenotype could be indicative of bad outcome in melanoma.

No MeSH data available.


Related in: MedlinePlus

Aldh1a1 knockdown reduces the migratory and invasive phenotypes produced by AhR depletion. a sh-AhR B16F10 melanoma cells were stably transduced with a sh-RNA for Aldh1a1 to generate the sh-AhR + sh-Aldh1a1 cell line. sh-AhR and sh-AhR + sh-Aldh1a1 cells were analyzed for Aldh1a1 protein expression by immunoblotting and for Aldh1a1 activity by flow cytometry using the Aldefluor reagent kit. To normalize Aldh1a1 activity, measurements were taken in presence and absence of the Aldh1-specific inhibitor diethylamino-benzaldehyde (DEAB). Values were then referred to those obtained for sh-AhR + sh-Aldh1a1 cells. b sh-AhR and sh-AhR + sh-Aldh1a1 cells were grown to confluence and then used for wound healing migration assays. c Both cell lines were cultured in matrigel transwells and analyzed for their invasive potential by confocal microscopy. Cell invasion at a depth of 40 μm is shown. d Clone formation in 2-D was determined by growing sh-AhR and sh-AhR + sh-Aldh1a1 cells at low cell density in plain tissue culture dishes. Clones were counted as unexpanded (compact clones with a dense cellularity), expanded (cells spreading out with reduced cell-cell interactions) or intermediate clones. e Clones were photographed, counted and their spreading analyzed using the ImageJ software. Three levels of spreading were established for compact (unexpanded), intermediate and fully expanded clones. Bars correspond to 100 μm (b) and 50 μm (d). A.U. arbitrary units. Experiments were done in duplicate or triplicate in three different cultures. At least 6 fields were analyzed for each wound in panel B. Data are shown as mean ± SD. A.U. stands for arbitrary units
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Fig3: Aldh1a1 knockdown reduces the migratory and invasive phenotypes produced by AhR depletion. a sh-AhR B16F10 melanoma cells were stably transduced with a sh-RNA for Aldh1a1 to generate the sh-AhR + sh-Aldh1a1 cell line. sh-AhR and sh-AhR + sh-Aldh1a1 cells were analyzed for Aldh1a1 protein expression by immunoblotting and for Aldh1a1 activity by flow cytometry using the Aldefluor reagent kit. To normalize Aldh1a1 activity, measurements were taken in presence and absence of the Aldh1-specific inhibitor diethylamino-benzaldehyde (DEAB). Values were then referred to those obtained for sh-AhR + sh-Aldh1a1 cells. b sh-AhR and sh-AhR + sh-Aldh1a1 cells were grown to confluence and then used for wound healing migration assays. c Both cell lines were cultured in matrigel transwells and analyzed for their invasive potential by confocal microscopy. Cell invasion at a depth of 40 μm is shown. d Clone formation in 2-D was determined by growing sh-AhR and sh-AhR + sh-Aldh1a1 cells at low cell density in plain tissue culture dishes. Clones were counted as unexpanded (compact clones with a dense cellularity), expanded (cells spreading out with reduced cell-cell interactions) or intermediate clones. e Clones were photographed, counted and their spreading analyzed using the ImageJ software. Three levels of spreading were established for compact (unexpanded), intermediate and fully expanded clones. Bars correspond to 100 μm (b) and 50 μm (d). A.U. arbitrary units. Experiments were done in duplicate or triplicate in three different cultures. At least 6 fields were analyzed for each wound in panel B. Data are shown as mean ± SD. A.U. stands for arbitrary units

Mentions: As Aldh1a1 expression and activity could be manipulated in B16F10 cells, and since we sought to investigate the extent to which Aldh1a1 contributes to the increased tumorigenicity caused by AhR knockdown [23], we decided to use retroviral transduction to deplete Aldh1a1 in AhR-interfered B16F10 cells. The cell line thus generated (hereafter sh-AhR + sh-Aldh1a1) was used to analyze the effects of Aldh1a1 expression in an AhR deficient background. Control experiments revealed that sh-AhR + sh-Aldh1a1 cells had a significant reduction in both Aldh1a1 protein expression and Aldh1a1 activity as compared to sh-AhR cells (Fig. 3a). Aldh1a1 depletion moderately blocked the migration of sh-AhR + sh-Aldh1a1 cells in wound healing assays with respect to Aldh1a1 expressing sh-AhR cells (Fig. 3b). Confocal microscopy analyses of matrigel-coated culture transwells showed that sh-AhR + sh-Aldh1a1 cells had a significant impairment to invade as compared to sh-AhR melanoma cells (Fig. 3c). Both cell lines were grown at low density in 2-D cultures in order to determine their clonogenicity potential. A qualitative scale was established to account for the appearance of unexpanded (compact clones with a dense cellularity), expanded (clones in which cells spread out and had reduced cell-cell interactions) or intermediate clones. We found that sh-AhR + sh-Aldh1a1 cells formed compact clones with enhanced cell-cell interactions and with a less invasive phenotype than that exhibited by sh-AhR cells (Fig. 3d). Consistently, sh-AhR + sh-Aldh1a1 cells formed a fewer number of fully expanded clones than sh-AhR cells under the same culturing conditions (Fig. 3e). Thus, the pro-migratory and pro-invasive phenotypes observed in AhR knockdown cells may be in part dependent on Aldh1a1 activity.Fig. 3


Dioxin receptor regulates aldehyde dehydrogenase to block melanoma tumorigenesis and metastasis.

Contador-Troca M, Alvarez-Barrientos A, Merino JM, Morales-Hernández A, Rodríguez MI, Rey-Barroso J, Barrasa E, Cerezo-Guisado MI, Catalina-Fernández I, Sáenz-Santamaría J, Oliver FJ, Fernandez-Salguero PM - Mol. Cancer (2015)

Aldh1a1 knockdown reduces the migratory and invasive phenotypes produced by AhR depletion. a sh-AhR B16F10 melanoma cells were stably transduced with a sh-RNA for Aldh1a1 to generate the sh-AhR + sh-Aldh1a1 cell line. sh-AhR and sh-AhR + sh-Aldh1a1 cells were analyzed for Aldh1a1 protein expression by immunoblotting and for Aldh1a1 activity by flow cytometry using the Aldefluor reagent kit. To normalize Aldh1a1 activity, measurements were taken in presence and absence of the Aldh1-specific inhibitor diethylamino-benzaldehyde (DEAB). Values were then referred to those obtained for sh-AhR + sh-Aldh1a1 cells. b sh-AhR and sh-AhR + sh-Aldh1a1 cells were grown to confluence and then used for wound healing migration assays. c Both cell lines were cultured in matrigel transwells and analyzed for their invasive potential by confocal microscopy. Cell invasion at a depth of 40 μm is shown. d Clone formation in 2-D was determined by growing sh-AhR and sh-AhR + sh-Aldh1a1 cells at low cell density in plain tissue culture dishes. Clones were counted as unexpanded (compact clones with a dense cellularity), expanded (cells spreading out with reduced cell-cell interactions) or intermediate clones. e Clones were photographed, counted and their spreading analyzed using the ImageJ software. Three levels of spreading were established for compact (unexpanded), intermediate and fully expanded clones. Bars correspond to 100 μm (b) and 50 μm (d). A.U. arbitrary units. Experiments were done in duplicate or triplicate in three different cultures. At least 6 fields were analyzed for each wound in panel B. Data are shown as mean ± SD. A.U. stands for arbitrary units
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Related In: Results  -  Collection

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Fig3: Aldh1a1 knockdown reduces the migratory and invasive phenotypes produced by AhR depletion. a sh-AhR B16F10 melanoma cells were stably transduced with a sh-RNA for Aldh1a1 to generate the sh-AhR + sh-Aldh1a1 cell line. sh-AhR and sh-AhR + sh-Aldh1a1 cells were analyzed for Aldh1a1 protein expression by immunoblotting and for Aldh1a1 activity by flow cytometry using the Aldefluor reagent kit. To normalize Aldh1a1 activity, measurements were taken in presence and absence of the Aldh1-specific inhibitor diethylamino-benzaldehyde (DEAB). Values were then referred to those obtained for sh-AhR + sh-Aldh1a1 cells. b sh-AhR and sh-AhR + sh-Aldh1a1 cells were grown to confluence and then used for wound healing migration assays. c Both cell lines were cultured in matrigel transwells and analyzed for their invasive potential by confocal microscopy. Cell invasion at a depth of 40 μm is shown. d Clone formation in 2-D was determined by growing sh-AhR and sh-AhR + sh-Aldh1a1 cells at low cell density in plain tissue culture dishes. Clones were counted as unexpanded (compact clones with a dense cellularity), expanded (cells spreading out with reduced cell-cell interactions) or intermediate clones. e Clones were photographed, counted and their spreading analyzed using the ImageJ software. Three levels of spreading were established for compact (unexpanded), intermediate and fully expanded clones. Bars correspond to 100 μm (b) and 50 μm (d). A.U. arbitrary units. Experiments were done in duplicate or triplicate in three different cultures. At least 6 fields were analyzed for each wound in panel B. Data are shown as mean ± SD. A.U. stands for arbitrary units
Mentions: As Aldh1a1 expression and activity could be manipulated in B16F10 cells, and since we sought to investigate the extent to which Aldh1a1 contributes to the increased tumorigenicity caused by AhR knockdown [23], we decided to use retroviral transduction to deplete Aldh1a1 in AhR-interfered B16F10 cells. The cell line thus generated (hereafter sh-AhR + sh-Aldh1a1) was used to analyze the effects of Aldh1a1 expression in an AhR deficient background. Control experiments revealed that sh-AhR + sh-Aldh1a1 cells had a significant reduction in both Aldh1a1 protein expression and Aldh1a1 activity as compared to sh-AhR cells (Fig. 3a). Aldh1a1 depletion moderately blocked the migration of sh-AhR + sh-Aldh1a1 cells in wound healing assays with respect to Aldh1a1 expressing sh-AhR cells (Fig. 3b). Confocal microscopy analyses of matrigel-coated culture transwells showed that sh-AhR + sh-Aldh1a1 cells had a significant impairment to invade as compared to sh-AhR melanoma cells (Fig. 3c). Both cell lines were grown at low density in 2-D cultures in order to determine their clonogenicity potential. A qualitative scale was established to account for the appearance of unexpanded (compact clones with a dense cellularity), expanded (clones in which cells spread out and had reduced cell-cell interactions) or intermediate clones. We found that sh-AhR + sh-Aldh1a1 cells formed compact clones with enhanced cell-cell interactions and with a less invasive phenotype than that exhibited by sh-AhR cells (Fig. 3d). Consistently, sh-AhR + sh-Aldh1a1 cells formed a fewer number of fully expanded clones than sh-AhR cells under the same culturing conditions (Fig. 3e). Thus, the pro-migratory and pro-invasive phenotypes observed in AhR knockdown cells may be in part dependent on Aldh1a1 activity.Fig. 3

Bottom Line: Their tumorigenicity in vivo was analyzed using xenografts and lung metastasis assays as well as in vivo imaging.However, Aldh1a1 downmodulation in AhR-expressing B16F10 cells did not significantly affect tumor growth in vivo.Aldh1a1 knockdown reduced the high levels of CD133(+)/CD29(+)/CD44(+) cells, melanosphere size and the expression of the pluripotency marker Sox2 in sh-AhR cells.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, 06071, Badajoz, Spain. maria_cotr@hotmail.com.

ABSTRACT

Background: The dioxin (AhR) receptor can have oncogenic or tumor suppressor activities depending on the phenotype of the target cell. We have shown that AhR knockdown promotes melanoma primary tumorigenesis and lung metastasis in the mouse and that human metastatic melanomas had reduced AhR levels with respect to benign nevi.

Methods: Mouse melanoma B16F10 cells were engineered by retroviral transduction to stably downregulate AhR expression, Aldh1a1 expression or both. They were characterized for Aldh1a1 activity, stem cell markers and migration and invasion in vitro. Their tumorigenicity in vivo was analyzed using xenografts and lung metastasis assays as well as in vivo imaging.

Results: Depletion of aldehyde dehydrogenase 1a1 (Aldh1a1) impairs the pro-tumorigenic and pro-metastatic advantage of melanoma cells lacking AhR expression (sh-AhR). Thus, Aldh1a1 knockdown in sh-AhR cells (sh-AhR + sh-Aldh1a1) diminished their migration and invasion potentials and blocked tumor growth and metastasis to the lungs in immunocompetent AhR+/+ recipient mice. However, Aldh1a1 downmodulation in AhR-expressing B16F10 cells did not significantly affect tumor growth in vivo. Aldh1a1 knockdown reduced the high levels of CD133(+)/CD29(+)/CD44(+) cells, melanosphere size and the expression of the pluripotency marker Sox2 in sh-AhR cells. Interestingly, Sox2 increased Aldh1a1 expression in sh-AhR but not in sh-AhR + sh-Aldh1a1 cells, suggesting that Aldh1a1 and Sox2 may be co-regulated in melanoma cells. In vivo imaging revealed that mice inoculated with AhR + Aldh1a1 knockdown cells had reduced tumor burden and enhanced survival than those receiving Aldh1a1-expressing sh-AhR cells.

Conclusions: Aldh1a1 overactivation in an AhR-deficient background enhances melanoma progression. Since AhR may antagonize the protumoral effects of Aldh1a1, the AhR(low)-Aldh1a1(high) phenotype could be indicative of bad outcome in melanoma.

No MeSH data available.


Related in: MedlinePlus