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CRABP1 is associated with a poor prognosis in breast cancer: adding to the complexity of breast cancer cell response to retinoic acid.

Liu RZ, Garcia E, Glubrecht DD, Poon HY, Mackey JR, Godbout R - Mol. Cancer (2015)

Bottom Line: The prognostic significance of CRABP1 is attributed to its cytoplasmic localization.We also show that CRABP1 affects the expression of genes involved in RA biosynthesis, trafficking and metabolism.We propose that these three RA-binding proteins can serve as biomarkers for predicting triple-negative breast cancer response to RA, with elevated levels of either cytoplasmic CRABP1 or FABP5 associated with RA resistance, and elevated levels of nuclear CRABP2 associated with sensitivity to RA.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Alberta, Cross Cancer Institute, 11560 University Avenue, Edmonton, T6G 1Z2, AB, Canada.

ABSTRACT

Background: Clinical trials designed to test the efficacy of retinoic acid (RA) as an adjuvant for the treatment of solid cancers have been disappointing, primarily due to RA resistance. Estrogen receptor (ER)-negative breast cancer cells are more resistant to RA than ER-positive cells. The expression and subcellular distribution of two RA-binding proteins, FABP5 and CRABP2, has already been shown to play critical roles in breast cancer cell response to RA. CRABP1, a third member of the RA-binding protein family, has not previously been investigated as a possible mediator of RA action in breast cancer.

Methods: CRABP1 and CRABP2 expression in primary breast tumor tissues was analyzed using gene expression and tissue microarrays. CRABP1 levels were manipulated using siRNAs and by transient overexpression. RA-induced subcellular translocation of CRABPs was examined by immunofluorescence microscopy and immunoblotting. RA-induced transactivation of RAR was analyzed using a RA response element (RARE)-driven luciferase reporter system. Effects of CRABP1 expression and RA treatment on downstream gene expression were investigated by semi-quantitative RT-PCR analysis.

Results: Compared to normal mammary tissues, CRABP1 expression is significantly down-regulated in ER+ breast tumors, but maintained in triple-negative breast cancers. Elevated CRABP1 levels are associated with poor patient prognosis, high Ki67 immunoreactivity and high tumor grade in breast cancer. The prognostic significance of CRABP1 is attributed to its cytoplasmic localization. We demonstrate that CRABP1 expression attenuates RA-induced cell growth arrest and inhibits RA signalling in breast cancer cells by sequestering RA in the cytoplasm. We also show that CRABP1 affects the expression of genes involved in RA biosynthesis, trafficking and metabolism.

Conclusions: CRABP1 is an adverse factor for clinical outcome in triple-negative breast cancer and a potent inhibitor of RA signalling in breast cancer cells. Our data indicate that CRABP1, in conjunction with previously identified CRABP2 and FABP5, plays a key role in breast cancer cell response to RA. We propose that these three RA-binding proteins can serve as biomarkers for predicting triple-negative breast cancer response to RA, with elevated levels of either cytoplasmic CRABP1 or FABP5 associated with RA resistance, and elevated levels of nuclear CRABP2 associated with sensitivity to RA.

No MeSH data available.


Related in: MedlinePlus

CRABP1 and CRABP2 differentially modulate RA-induced growth inhibition and RAR transcriptional activity. a, b Western blot analysis of MCF-7 cells transfected with CRABP1 or CRABP2 siRNAs using antibodies against CRABP1 or CRABP2. c Relative growth rate of MCF-7 cells treated with the indicated concentrations of RA after transfection with non-specific (control) and specific siRNAs targeting CRABP1 or CRABP2. Significance of difference was tested using two-way ANOVA. d RAR transactivation (measured by luciferase activity) of CRABP1a-depleted or CRABP2a-depleted MCF-7 cells transfected with a luciferase reporter construct under the control of a RARE. Cells were treated with DMSO (vehicle control) or RA for 6 h before harvest. Luciferase activity (a measure of RAR activation) is shown as fold change relative to cells that were cultured in the absence of RA. e The luciferase assay was repeated with a second set of siRNAs targeting CRABP1 and CRABP2 (CRABP1b, CRABP2b). f Western blots showing ectopic expression of CRABP1 in three human breast cancer cell lines. g, h, i The effects of ectopic expression of CRABP1 on RAR transactivation (measured by luciferase activity) were examined in BT-549 (g), SK-Br-3 (h) and Hs578T (i) cells co-transfected with a CRABP1 cDNA construct and a RARE-luciferase reporter construct. Cells were treated with RA for 6 h at the indicated concentrations. Luciferase activity (a measure of RAR activation) was adjusted based on protein concentrations of individual lysates and shown as fold change relative to control cells transfected with empty vector and cultured in the absence of RA. KD, denotes knockdown; *, p < 0.05; **, p < 0.01
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Fig4: CRABP1 and CRABP2 differentially modulate RA-induced growth inhibition and RAR transcriptional activity. a, b Western blot analysis of MCF-7 cells transfected with CRABP1 or CRABP2 siRNAs using antibodies against CRABP1 or CRABP2. c Relative growth rate of MCF-7 cells treated with the indicated concentrations of RA after transfection with non-specific (control) and specific siRNAs targeting CRABP1 or CRABP2. Significance of difference was tested using two-way ANOVA. d RAR transactivation (measured by luciferase activity) of CRABP1a-depleted or CRABP2a-depleted MCF-7 cells transfected with a luciferase reporter construct under the control of a RARE. Cells were treated with DMSO (vehicle control) or RA for 6 h before harvest. Luciferase activity (a measure of RAR activation) is shown as fold change relative to cells that were cultured in the absence of RA. e The luciferase assay was repeated with a second set of siRNAs targeting CRABP1 and CRABP2 (CRABP1b, CRABP2b). f Western blots showing ectopic expression of CRABP1 in three human breast cancer cell lines. g, h, i The effects of ectopic expression of CRABP1 on RAR transactivation (measured by luciferase activity) were examined in BT-549 (g), SK-Br-3 (h) and Hs578T (i) cells co-transfected with a CRABP1 cDNA construct and a RARE-luciferase reporter construct. Cells were treated with RA for 6 h at the indicated concentrations. Luciferase activity (a measure of RAR activation) was adjusted based on protein concentrations of individual lysates and shown as fold change relative to control cells transfected with empty vector and cultured in the absence of RA. KD, denotes knockdown; *, p < 0.05; **, p < 0.01

Mentions: CRABP1 and CRABP2 may play distinct roles in RA/RAR-mediated transcriptional activity [42, 45]. To investigate the role of CRABP1 in RA action, we used siRNAs to deplete the RA-responsive MCF-7 cells of either CRABP1 or CRABP2 (Fig. 4a–b). A small but statistically significant increase in growth inhibition was observed when CRABP1-depleted cells were treated with RA, with control cells showing 81 and 74 % relative growth at 0.1 μM and 0.5 μM RA, respectively, and CRABP1-depleted cells showing 65 and 66 % relative growth at 0.1 μM and 0.5 μM RA, respectively (Fig. 4c). A stronger growth inhibition effect was observed upon treatment of CRABP1-depleted cells with RA in the absence of serum (Additional file 1: Figure S2). In contrast to CRABP1, knockdown of CRABP2 had little effect on RA-mediated cell growth inhibition (91 % at 0.1 μM and 88 % at 0.5 μM) (Fig. 4c).Fig. 4


CRABP1 is associated with a poor prognosis in breast cancer: adding to the complexity of breast cancer cell response to retinoic acid.

Liu RZ, Garcia E, Glubrecht DD, Poon HY, Mackey JR, Godbout R - Mol. Cancer (2015)

CRABP1 and CRABP2 differentially modulate RA-induced growth inhibition and RAR transcriptional activity. a, b Western blot analysis of MCF-7 cells transfected with CRABP1 or CRABP2 siRNAs using antibodies against CRABP1 or CRABP2. c Relative growth rate of MCF-7 cells treated with the indicated concentrations of RA after transfection with non-specific (control) and specific siRNAs targeting CRABP1 or CRABP2. Significance of difference was tested using two-way ANOVA. d RAR transactivation (measured by luciferase activity) of CRABP1a-depleted or CRABP2a-depleted MCF-7 cells transfected with a luciferase reporter construct under the control of a RARE. Cells were treated with DMSO (vehicle control) or RA for 6 h before harvest. Luciferase activity (a measure of RAR activation) is shown as fold change relative to cells that were cultured in the absence of RA. e The luciferase assay was repeated with a second set of siRNAs targeting CRABP1 and CRABP2 (CRABP1b, CRABP2b). f Western blots showing ectopic expression of CRABP1 in three human breast cancer cell lines. g, h, i The effects of ectopic expression of CRABP1 on RAR transactivation (measured by luciferase activity) were examined in BT-549 (g), SK-Br-3 (h) and Hs578T (i) cells co-transfected with a CRABP1 cDNA construct and a RARE-luciferase reporter construct. Cells were treated with RA for 6 h at the indicated concentrations. Luciferase activity (a measure of RAR activation) was adjusted based on protein concentrations of individual lysates and shown as fold change relative to control cells transfected with empty vector and cultured in the absence of RA. KD, denotes knockdown; *, p < 0.05; **, p < 0.01
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Related In: Results  -  Collection

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Fig4: CRABP1 and CRABP2 differentially modulate RA-induced growth inhibition and RAR transcriptional activity. a, b Western blot analysis of MCF-7 cells transfected with CRABP1 or CRABP2 siRNAs using antibodies against CRABP1 or CRABP2. c Relative growth rate of MCF-7 cells treated with the indicated concentrations of RA after transfection with non-specific (control) and specific siRNAs targeting CRABP1 or CRABP2. Significance of difference was tested using two-way ANOVA. d RAR transactivation (measured by luciferase activity) of CRABP1a-depleted or CRABP2a-depleted MCF-7 cells transfected with a luciferase reporter construct under the control of a RARE. Cells were treated with DMSO (vehicle control) or RA for 6 h before harvest. Luciferase activity (a measure of RAR activation) is shown as fold change relative to cells that were cultured in the absence of RA. e The luciferase assay was repeated with a second set of siRNAs targeting CRABP1 and CRABP2 (CRABP1b, CRABP2b). f Western blots showing ectopic expression of CRABP1 in three human breast cancer cell lines. g, h, i The effects of ectopic expression of CRABP1 on RAR transactivation (measured by luciferase activity) were examined in BT-549 (g), SK-Br-3 (h) and Hs578T (i) cells co-transfected with a CRABP1 cDNA construct and a RARE-luciferase reporter construct. Cells were treated with RA for 6 h at the indicated concentrations. Luciferase activity (a measure of RAR activation) was adjusted based on protein concentrations of individual lysates and shown as fold change relative to control cells transfected with empty vector and cultured in the absence of RA. KD, denotes knockdown; *, p < 0.05; **, p < 0.01
Mentions: CRABP1 and CRABP2 may play distinct roles in RA/RAR-mediated transcriptional activity [42, 45]. To investigate the role of CRABP1 in RA action, we used siRNAs to deplete the RA-responsive MCF-7 cells of either CRABP1 or CRABP2 (Fig. 4a–b). A small but statistically significant increase in growth inhibition was observed when CRABP1-depleted cells were treated with RA, with control cells showing 81 and 74 % relative growth at 0.1 μM and 0.5 μM RA, respectively, and CRABP1-depleted cells showing 65 and 66 % relative growth at 0.1 μM and 0.5 μM RA, respectively (Fig. 4c). A stronger growth inhibition effect was observed upon treatment of CRABP1-depleted cells with RA in the absence of serum (Additional file 1: Figure S2). In contrast to CRABP1, knockdown of CRABP2 had little effect on RA-mediated cell growth inhibition (91 % at 0.1 μM and 88 % at 0.5 μM) (Fig. 4c).Fig. 4

Bottom Line: The prognostic significance of CRABP1 is attributed to its cytoplasmic localization.We also show that CRABP1 affects the expression of genes involved in RA biosynthesis, trafficking and metabolism.We propose that these three RA-binding proteins can serve as biomarkers for predicting triple-negative breast cancer response to RA, with elevated levels of either cytoplasmic CRABP1 or FABP5 associated with RA resistance, and elevated levels of nuclear CRABP2 associated with sensitivity to RA.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Alberta, Cross Cancer Institute, 11560 University Avenue, Edmonton, T6G 1Z2, AB, Canada.

ABSTRACT

Background: Clinical trials designed to test the efficacy of retinoic acid (RA) as an adjuvant for the treatment of solid cancers have been disappointing, primarily due to RA resistance. Estrogen receptor (ER)-negative breast cancer cells are more resistant to RA than ER-positive cells. The expression and subcellular distribution of two RA-binding proteins, FABP5 and CRABP2, has already been shown to play critical roles in breast cancer cell response to RA. CRABP1, a third member of the RA-binding protein family, has not previously been investigated as a possible mediator of RA action in breast cancer.

Methods: CRABP1 and CRABP2 expression in primary breast tumor tissues was analyzed using gene expression and tissue microarrays. CRABP1 levels were manipulated using siRNAs and by transient overexpression. RA-induced subcellular translocation of CRABPs was examined by immunofluorescence microscopy and immunoblotting. RA-induced transactivation of RAR was analyzed using a RA response element (RARE)-driven luciferase reporter system. Effects of CRABP1 expression and RA treatment on downstream gene expression were investigated by semi-quantitative RT-PCR analysis.

Results: Compared to normal mammary tissues, CRABP1 expression is significantly down-regulated in ER+ breast tumors, but maintained in triple-negative breast cancers. Elevated CRABP1 levels are associated with poor patient prognosis, high Ki67 immunoreactivity and high tumor grade in breast cancer. The prognostic significance of CRABP1 is attributed to its cytoplasmic localization. We demonstrate that CRABP1 expression attenuates RA-induced cell growth arrest and inhibits RA signalling in breast cancer cells by sequestering RA in the cytoplasm. We also show that CRABP1 affects the expression of genes involved in RA biosynthesis, trafficking and metabolism.

Conclusions: CRABP1 is an adverse factor for clinical outcome in triple-negative breast cancer and a potent inhibitor of RA signalling in breast cancer cells. Our data indicate that CRABP1, in conjunction with previously identified CRABP2 and FABP5, plays a key role in breast cancer cell response to RA. We propose that these three RA-binding proteins can serve as biomarkers for predicting triple-negative breast cancer response to RA, with elevated levels of either cytoplasmic CRABP1 or FABP5 associated with RA resistance, and elevated levels of nuclear CRABP2 associated with sensitivity to RA.

No MeSH data available.


Related in: MedlinePlus