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Cellular and molecular determinants of all-trans retinoic acid sensitivity in breast cancer: Luminal phenotype and RARα expression.

Centritto F, Paroni G, Bolis M, Garattini SK, Kurosaki M, Barzago MM, Zanetti A, Fisher JN, Scott MF, Pattini L, Lupi M, Ubezio P, Piccotti F, Zambelli A, Rizzo P, Gianni' M, Fratelli M, Terao M, Garattini E - EMBO Mol Med (2015)

Bottom Line: Luminal and ER(+) (estrogen-receptor-positive) cell lines are generally sensitive to ATRA, while refractoriness/low sensitivity is associated with a Basal phenotype and HER2 positivity.Pathway-oriented analysis of the constitutive gene-expression profiles in the cell lines identifies RARα as the member of the retinoid pathway directly associated with a Luminal phenotype, estrogen positivity and ATRA sensitivity.All this is paralleled by similar effects on ATRA-dependent inhibition of cell motility, indicating that RARα may mediate also ATRA anti-metastatic effects.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, IRCCS-Istituto di Ricerche Farmacologiche "Mario Negri", Milano, Italy.

No MeSH data available.


Related in: MedlinePlus

Over-expression of RARα1/3 in ATRA-resistant MDA-MB453 cells and silencing of RARα1/3 in ATRA-sensitive SKBR3 cellsA RARα1/3 plasmid construct and the corresponding control void vector were stably transfected into ATRA-resistant MDA-MB453 cells. Two cell clones over-expressing RARα (RARA-C5 and RARA-C7) and two appropriate control clones (Vect-C1 and Vect-C2) were isolated. A RARα3 shRNA plasmid construct and the corresponding void vector were stably transfected into ATRA-sensitive SKBR3 cells. After selection, two cell clones silenced for RARα3 (RARA-sh18 and RARA-sh19) and two appropriate control clones (Vect-C6 and Vect-C8) were isolated.A, D The indicated clones and the parental cell line (WT) were transiently transfected with the RARE-DR5-Luc retinoid reporter construct and the level of luciferase activity was measured 24 h after treatment with vehicle (DMSO) and ATRA (100 nM), as illustrated in the upper bar graph. Each value is the mean ± SD of three replicate cultures. The levels of the RARα protein measured in the indicated clones by Western blot analysis is shown under the bar graph. To demonstrate that similar levels of total proteins were loaded in each lane, the β-actin band signal obtained after re-blotting of the gel is shown. FI = Fluorescence intensity.B, E The panels illustrate the growth curves of the indicated MDA-MB453 and SKBR3 clones and the SKBR3 parental cell lines (WT) measured with the sulforhodamine assay.C The bar graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated MDA-MB453 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3, 6, and 9 days prior to the sulforhodamine assay. OD = optical density at 540 nm. Each value is the mean ± SD of five replicate culture wells.F The graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated SKBR3 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3 and 6 days prior to the sulforhodamine assay. Each value is the mean ± SD of five replicate culture wells.G The panel illustrates the effects exerted by ATRA (0.1 μM) on random cell motility of the indicated MDA-MB453 and SKBR3 clones. The results are representative of two independent experiments. Each value is the mean ± SE of the motility of at least 60 cells.Data information: * and **, significantly different from the corresponding vehicle-treated control (*P-value < 0.05, **P-value < 0.01, Student's t-test).
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fig10: Over-expression of RARα1/3 in ATRA-resistant MDA-MB453 cells and silencing of RARα1/3 in ATRA-sensitive SKBR3 cellsA RARα1/3 plasmid construct and the corresponding control void vector were stably transfected into ATRA-resistant MDA-MB453 cells. Two cell clones over-expressing RARα (RARA-C5 and RARA-C7) and two appropriate control clones (Vect-C1 and Vect-C2) were isolated. A RARα3 shRNA plasmid construct and the corresponding void vector were stably transfected into ATRA-sensitive SKBR3 cells. After selection, two cell clones silenced for RARα3 (RARA-sh18 and RARA-sh19) and two appropriate control clones (Vect-C6 and Vect-C8) were isolated.A, D The indicated clones and the parental cell line (WT) were transiently transfected with the RARE-DR5-Luc retinoid reporter construct and the level of luciferase activity was measured 24 h after treatment with vehicle (DMSO) and ATRA (100 nM), as illustrated in the upper bar graph. Each value is the mean ± SD of three replicate cultures. The levels of the RARα protein measured in the indicated clones by Western blot analysis is shown under the bar graph. To demonstrate that similar levels of total proteins were loaded in each lane, the β-actin band signal obtained after re-blotting of the gel is shown. FI = Fluorescence intensity.B, E The panels illustrate the growth curves of the indicated MDA-MB453 and SKBR3 clones and the SKBR3 parental cell lines (WT) measured with the sulforhodamine assay.C The bar graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated MDA-MB453 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3, 6, and 9 days prior to the sulforhodamine assay. OD = optical density at 540 nm. Each value is the mean ± SD of five replicate culture wells.F The graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated SKBR3 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3 and 6 days prior to the sulforhodamine assay. Each value is the mean ± SD of five replicate culture wells.G The panel illustrates the effects exerted by ATRA (0.1 μM) on random cell motility of the indicated MDA-MB453 and SKBR3 clones. The results are representative of two independent experiments. Each value is the mean ± SE of the motility of at least 60 cells.Data information: * and **, significantly different from the corresponding vehicle-treated control (*P-value < 0.05, **P-value < 0.01, Student's t-test).

Mentions: To obtain direct proof that the RARα3 protein is mediating the action of ATRA, we over-expressed it in retinoid-resistant, HER2+/ER−, and Luminal MDA-MB453 cells. Two RARα-over-expressing (RARA-C5 and RARA-C7), two vector-transfected control (Vect-C1 and Vect-C2) clones, and the parental MDA-MB453 cells (WT) were used in comparative experiments. WT, Vect-C1, and Vect-C2 express barely detectable levels of the RARα protein, while large amounts of the product are synthesized by RARA-C5 and RARA-C7 cells (Fig10A). RARA-C5 and RARA-C7 express a transcriptionally active RARα form, as indicated by ATRA-dependent activation of the luciferase-based retinoid reporter, DR5-RARE-Luc. Over-expression of RARα does not exert major effects on the basal growth rate of the MDA-MB-453 clones (Fig10B). Upon treatment with increasing concentrations of ATRA for 3, 6, and 9 days, Vect-C1 and Vect-C2 and WT cells are equally unresponsive to retinoid-dependent growth inhibition (Fig10C). In contrast, RARA-C5/RARA-C7 proliferation is inhibited dose- and time-dependently by ATRA. Thus, stable over-expression of RARα renders MDA-MB-453 cells sensitive to the retinoid with an ∼4-fold increase in the calculated ATRA score at 9 days.


Cellular and molecular determinants of all-trans retinoic acid sensitivity in breast cancer: Luminal phenotype and RARα expression.

Centritto F, Paroni G, Bolis M, Garattini SK, Kurosaki M, Barzago MM, Zanetti A, Fisher JN, Scott MF, Pattini L, Lupi M, Ubezio P, Piccotti F, Zambelli A, Rizzo P, Gianni' M, Fratelli M, Terao M, Garattini E - EMBO Mol Med (2015)

Over-expression of RARα1/3 in ATRA-resistant MDA-MB453 cells and silencing of RARα1/3 in ATRA-sensitive SKBR3 cellsA RARα1/3 plasmid construct and the corresponding control void vector were stably transfected into ATRA-resistant MDA-MB453 cells. Two cell clones over-expressing RARα (RARA-C5 and RARA-C7) and two appropriate control clones (Vect-C1 and Vect-C2) were isolated. A RARα3 shRNA plasmid construct and the corresponding void vector were stably transfected into ATRA-sensitive SKBR3 cells. After selection, two cell clones silenced for RARα3 (RARA-sh18 and RARA-sh19) and two appropriate control clones (Vect-C6 and Vect-C8) were isolated.A, D The indicated clones and the parental cell line (WT) were transiently transfected with the RARE-DR5-Luc retinoid reporter construct and the level of luciferase activity was measured 24 h after treatment with vehicle (DMSO) and ATRA (100 nM), as illustrated in the upper bar graph. Each value is the mean ± SD of three replicate cultures. The levels of the RARα protein measured in the indicated clones by Western blot analysis is shown under the bar graph. To demonstrate that similar levels of total proteins were loaded in each lane, the β-actin band signal obtained after re-blotting of the gel is shown. FI = Fluorescence intensity.B, E The panels illustrate the growth curves of the indicated MDA-MB453 and SKBR3 clones and the SKBR3 parental cell lines (WT) measured with the sulforhodamine assay.C The bar graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated MDA-MB453 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3, 6, and 9 days prior to the sulforhodamine assay. OD = optical density at 540 nm. Each value is the mean ± SD of five replicate culture wells.F The graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated SKBR3 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3 and 6 days prior to the sulforhodamine assay. Each value is the mean ± SD of five replicate culture wells.G The panel illustrates the effects exerted by ATRA (0.1 μM) on random cell motility of the indicated MDA-MB453 and SKBR3 clones. The results are representative of two independent experiments. Each value is the mean ± SE of the motility of at least 60 cells.Data information: * and **, significantly different from the corresponding vehicle-treated control (*P-value < 0.05, **P-value < 0.01, Student's t-test).
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fig10: Over-expression of RARα1/3 in ATRA-resistant MDA-MB453 cells and silencing of RARα1/3 in ATRA-sensitive SKBR3 cellsA RARα1/3 plasmid construct and the corresponding control void vector were stably transfected into ATRA-resistant MDA-MB453 cells. Two cell clones over-expressing RARα (RARA-C5 and RARA-C7) and two appropriate control clones (Vect-C1 and Vect-C2) were isolated. A RARα3 shRNA plasmid construct and the corresponding void vector were stably transfected into ATRA-sensitive SKBR3 cells. After selection, two cell clones silenced for RARα3 (RARA-sh18 and RARA-sh19) and two appropriate control clones (Vect-C6 and Vect-C8) were isolated.A, D The indicated clones and the parental cell line (WT) were transiently transfected with the RARE-DR5-Luc retinoid reporter construct and the level of luciferase activity was measured 24 h after treatment with vehicle (DMSO) and ATRA (100 nM), as illustrated in the upper bar graph. Each value is the mean ± SD of three replicate cultures. The levels of the RARα protein measured in the indicated clones by Western blot analysis is shown under the bar graph. To demonstrate that similar levels of total proteins were loaded in each lane, the β-actin band signal obtained after re-blotting of the gel is shown. FI = Fluorescence intensity.B, E The panels illustrate the growth curves of the indicated MDA-MB453 and SKBR3 clones and the SKBR3 parental cell lines (WT) measured with the sulforhodamine assay.C The bar graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated MDA-MB453 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3, 6, and 9 days prior to the sulforhodamine assay. OD = optical density at 540 nm. Each value is the mean ± SD of five replicate culture wells.F The graphs illustrate the effect of increasing concentrations of ATRA on the growth of the indicated SKBR3 clones and the parental cell line. The cell lines were challenged with vehicle (DMSO) or ATRA for 3 and 6 days prior to the sulforhodamine assay. Each value is the mean ± SD of five replicate culture wells.G The panel illustrates the effects exerted by ATRA (0.1 μM) on random cell motility of the indicated MDA-MB453 and SKBR3 clones. The results are representative of two independent experiments. Each value is the mean ± SE of the motility of at least 60 cells.Data information: * and **, significantly different from the corresponding vehicle-treated control (*P-value < 0.05, **P-value < 0.01, Student's t-test).
Mentions: To obtain direct proof that the RARα3 protein is mediating the action of ATRA, we over-expressed it in retinoid-resistant, HER2+/ER−, and Luminal MDA-MB453 cells. Two RARα-over-expressing (RARA-C5 and RARA-C7), two vector-transfected control (Vect-C1 and Vect-C2) clones, and the parental MDA-MB453 cells (WT) were used in comparative experiments. WT, Vect-C1, and Vect-C2 express barely detectable levels of the RARα protein, while large amounts of the product are synthesized by RARA-C5 and RARA-C7 cells (Fig10A). RARA-C5 and RARA-C7 express a transcriptionally active RARα form, as indicated by ATRA-dependent activation of the luciferase-based retinoid reporter, DR5-RARE-Luc. Over-expression of RARα does not exert major effects on the basal growth rate of the MDA-MB-453 clones (Fig10B). Upon treatment with increasing concentrations of ATRA for 3, 6, and 9 days, Vect-C1 and Vect-C2 and WT cells are equally unresponsive to retinoid-dependent growth inhibition (Fig10C). In contrast, RARA-C5/RARA-C7 proliferation is inhibited dose- and time-dependently by ATRA. Thus, stable over-expression of RARα renders MDA-MB-453 cells sensitive to the retinoid with an ∼4-fold increase in the calculated ATRA score at 9 days.

Bottom Line: Luminal and ER(+) (estrogen-receptor-positive) cell lines are generally sensitive to ATRA, while refractoriness/low sensitivity is associated with a Basal phenotype and HER2 positivity.Pathway-oriented analysis of the constitutive gene-expression profiles in the cell lines identifies RARα as the member of the retinoid pathway directly associated with a Luminal phenotype, estrogen positivity and ATRA sensitivity.All this is paralleled by similar effects on ATRA-dependent inhibition of cell motility, indicating that RARα may mediate also ATRA anti-metastatic effects.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, IRCCS-Istituto di Ricerche Farmacologiche "Mario Negri", Milano, Italy.

No MeSH data available.


Related in: MedlinePlus