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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

Immunoreactivity and subcellular distribution of CRABP1 and CRABP2 in a human primary breast tumor TMA. a Western blot of CRABP1 and CRABP2 in MDA-MB-435 cells transfected with a CRABP1 or CRABP2 expression construct, respectively. b Frequency of breast tumors with different subcellular immunoreactivity scores for CRABP1 and CRABP2: 0, negative; 1, weak; 2, intermediate; 3, strong. c Selected tissue sections from a human breast cancer TMA immunostained with anti-CRABP1 and anti-CRABP2 antibodies. Nuclear (Nuc) and cytoplasmic (Cyt) scores are indicated
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Fig2: Immunoreactivity and subcellular distribution of CRABP1 and CRABP2 in a human primary breast tumor TMA. a Western blot of CRABP1 and CRABP2 in MDA-MB-435 cells transfected with a CRABP1 or CRABP2 expression construct, respectively. b Frequency of breast tumors with different subcellular immunoreactivity scores for CRABP1 and CRABP2: 0, negative; 1, weak; 2, intermediate; 3, strong. c Selected tissue sections from a human breast cancer TMA immunostained with anti-CRABP1 and anti-CRABP2 antibodies. Nuclear (Nuc) and cytoplasmic (Cyt) scores are indicated

Mentions: CRABPs serve as intracellular chaperones for RA and modulate its nuclear availability and biological activity [42, 43]. The subcellular localization of CRABPs is therefore a critical determinant of their function. To investigate whether there is any association between the subcellular distribution of CRABP1 and 2 and clinical outcomes, we conducted immunohistochemical analysis of a TMA containing primary tumor samples from 120 breast cancer patients. We first validated the specificity of our anti-CRABP1 and anti-CRABP2 antibodies by western blot analysis of whole cell lysates prepared from MDA-MB-435 cells (negative for both CRABP1 and 2) transfected with CRABP1 or CRABP2. No cross-immunoreactivity was observed for these two antibodies (Fig. 2a).Fig. 2


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)

Immunoreactivity and subcellular distribution of CRABP1 and CRABP2 in a human primary breast tumor TMA. a Western blot of CRABP1 and CRABP2 in MDA-MB-435 cells transfected with a CRABP1 or CRABP2 expression construct, respectively. b Frequency of breast tumors with different subcellular immunoreactivity scores for CRABP1 and CRABP2: 0, negative; 1, weak; 2, intermediate; 3, strong. c Selected tissue sections from a human breast cancer TMA immunostained with anti-CRABP1 and anti-CRABP2 antibodies. Nuclear (Nuc) and cytoplasmic (Cyt) scores are indicated
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4491424&req=5

Fig2: Immunoreactivity and subcellular distribution of CRABP1 and CRABP2 in a human primary breast tumor TMA. a Western blot of CRABP1 and CRABP2 in MDA-MB-435 cells transfected with a CRABP1 or CRABP2 expression construct, respectively. b Frequency of breast tumors with different subcellular immunoreactivity scores for CRABP1 and CRABP2: 0, negative; 1, weak; 2, intermediate; 3, strong. c Selected tissue sections from a human breast cancer TMA immunostained with anti-CRABP1 and anti-CRABP2 antibodies. Nuclear (Nuc) and cytoplasmic (Cyt) scores are indicated
Mentions: CRABPs serve as intracellular chaperones for RA and modulate its nuclear availability and biological activity [42, 43]. The subcellular localization of CRABPs is therefore a critical determinant of their function. To investigate whether there is any association between the subcellular distribution of CRABP1 and 2 and clinical outcomes, we conducted immunohistochemical analysis of a TMA containing primary tumor samples from 120 breast cancer patients. We first validated the specificity of our anti-CRABP1 and anti-CRABP2 antibodies by western blot analysis of whole cell lysates prepared from MDA-MB-435 cells (negative for both CRABP1 and 2) transfected with CRABP1 or CRABP2. No cross-immunoreactivity was observed for these two antibodies (Fig. 2a).Fig. 2

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