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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: Compared to normal mammary tissues, CRABP1 expression is significantly down-regulated in ER+ breast tumors, but maintained in triple-negative breast cancers.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

a Subcellular localization of CRABP1 and CRABP2 in MCF-7 cells treated with RA. Cells were cultured in medium with serum for 24 h and then treated with 0.5 μM RA in serum-free medium for 6 h. An equivalent amount of DMSO was added to control cells. Cells were immunostained with anti-CRABP1 (red, upper panel) or anti-CRABP2 (red, lower panel) antibodies as described in Materials and Methods. DAP1 (blue) staining was used to visualize the nucleus. b Expression of RA-responsive genes in MCF-7. MCF-7 cells underwent two rounds of transfection with scrambled or CRABP1 siRNAs. Cells were treated with increasing concentrations of RA [lanes 1 to 6 (0, 5 × 10−5, 5 × 10−4, 5 × 10−3, 5 × 10−2, 5 × 10−1 μM RA)]. RNA was purified from each culture and semi-quantitative RT-PCR was carried out using gene-specific primers (Additional file 1: Table S1). c Summary of the effect of CRABP1 and RA on downstream genes and pathways. d Western blots showing the subcellular distribution of CRABP2 in SK-Br-3 cells upon CRABP1 overexpression and RA treatment. Densitometric analysis was used to quantitate CRABP2 signal intensity in the cytoplasm and nucleus relative to the cytoplasmic marker (β-tubulin) and nuclear marker (lamin A/C), respectively. Changes in band intensities are shown as fold change in relation to lane 1 (for cytoplasmic CRABP2) and lane 5 (for nuclear CRABP2)
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Fig5: a Subcellular localization of CRABP1 and CRABP2 in MCF-7 cells treated with RA. Cells were cultured in medium with serum for 24 h and then treated with 0.5 μM RA in serum-free medium for 6 h. An equivalent amount of DMSO was added to control cells. Cells were immunostained with anti-CRABP1 (red, upper panel) or anti-CRABP2 (red, lower panel) antibodies as described in Materials and Methods. DAP1 (blue) staining was used to visualize the nucleus. b Expression of RA-responsive genes in MCF-7. MCF-7 cells underwent two rounds of transfection with scrambled or CRABP1 siRNAs. Cells were treated with increasing concentrations of RA [lanes 1 to 6 (0, 5 × 10−5, 5 × 10−4, 5 × 10−3, 5 × 10−2, 5 × 10−1 μM RA)]. RNA was purified from each culture and semi-quantitative RT-PCR was carried out using gene-specific primers (Additional file 1: Table S1). c Summary of the effect of CRABP1 and RA on downstream genes and pathways. d Western blots showing the subcellular distribution of CRABP2 in SK-Br-3 cells upon CRABP1 overexpression and RA treatment. Densitometric analysis was used to quantitate CRABP2 signal intensity in the cytoplasm and nucleus relative to the cytoplasmic marker (β-tubulin) and nuclear marker (lamin A/C), respectively. Changes in band intensities are shown as fold change in relation to lane 1 (for cytoplasmic CRABP2) and lane 5 (for nuclear CRABP2)

Mentions: Next, we analysed CRABP1 and CRABP2 subcellular localization in response to RA in MCF-7 cells. Both proteins were primarily found in the cytoplasm in the absence of RA (Fig. 5a). In contrast to CRABP2 which translocated to the nucleus upon RA treatment, there was no change in CRABP1 subcellular localization upon RA treatment (Fig. 5a). These results are consistent with a general role for CRABP1 in sequestering RA in the cytoplasm, rendering it unavailable for RAR activation.Fig. 5


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)

a Subcellular localization of CRABP1 and CRABP2 in MCF-7 cells treated with RA. Cells were cultured in medium with serum for 24 h and then treated with 0.5 μM RA in serum-free medium for 6 h. An equivalent amount of DMSO was added to control cells. Cells were immunostained with anti-CRABP1 (red, upper panel) or anti-CRABP2 (red, lower panel) antibodies as described in Materials and Methods. DAP1 (blue) staining was used to visualize the nucleus. b Expression of RA-responsive genes in MCF-7. MCF-7 cells underwent two rounds of transfection with scrambled or CRABP1 siRNAs. Cells were treated with increasing concentrations of RA [lanes 1 to 6 (0, 5 × 10−5, 5 × 10−4, 5 × 10−3, 5 × 10−2, 5 × 10−1 μM RA)]. RNA was purified from each culture and semi-quantitative RT-PCR was carried out using gene-specific primers (Additional file 1: Table S1). c Summary of the effect of CRABP1 and RA on downstream genes and pathways. d Western blots showing the subcellular distribution of CRABP2 in SK-Br-3 cells upon CRABP1 overexpression and RA treatment. Densitometric analysis was used to quantitate CRABP2 signal intensity in the cytoplasm and nucleus relative to the cytoplasmic marker (β-tubulin) and nuclear marker (lamin A/C), respectively. Changes in band intensities are shown as fold change in relation to lane 1 (for cytoplasmic CRABP2) and lane 5 (for nuclear CRABP2)
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Fig5: a Subcellular localization of CRABP1 and CRABP2 in MCF-7 cells treated with RA. Cells were cultured in medium with serum for 24 h and then treated with 0.5 μM RA in serum-free medium for 6 h. An equivalent amount of DMSO was added to control cells. Cells were immunostained with anti-CRABP1 (red, upper panel) or anti-CRABP2 (red, lower panel) antibodies as described in Materials and Methods. DAP1 (blue) staining was used to visualize the nucleus. b Expression of RA-responsive genes in MCF-7. MCF-7 cells underwent two rounds of transfection with scrambled or CRABP1 siRNAs. Cells were treated with increasing concentrations of RA [lanes 1 to 6 (0, 5 × 10−5, 5 × 10−4, 5 × 10−3, 5 × 10−2, 5 × 10−1 μM RA)]. RNA was purified from each culture and semi-quantitative RT-PCR was carried out using gene-specific primers (Additional file 1: Table S1). c Summary of the effect of CRABP1 and RA on downstream genes and pathways. d Western blots showing the subcellular distribution of CRABP2 in SK-Br-3 cells upon CRABP1 overexpression and RA treatment. Densitometric analysis was used to quantitate CRABP2 signal intensity in the cytoplasm and nucleus relative to the cytoplasmic marker (β-tubulin) and nuclear marker (lamin A/C), respectively. Changes in band intensities are shown as fold change in relation to lane 1 (for cytoplasmic CRABP2) and lane 5 (for nuclear CRABP2)
Mentions: Next, we analysed CRABP1 and CRABP2 subcellular localization in response to RA in MCF-7 cells. Both proteins were primarily found in the cytoplasm in the absence of RA (Fig. 5a). In contrast to CRABP2 which translocated to the nucleus upon RA treatment, there was no change in CRABP1 subcellular localization upon RA treatment (Fig. 5a). These results are consistent with a general role for CRABP1 in sequestering RA in the cytoplasm, rendering it unavailable for RAR activation.Fig. 5

Bottom Line: Compared to normal mammary tissues, CRABP1 expression is significantly down-regulated in ER+ breast tumors, but maintained in triple-negative breast cancers.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