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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

Associations of subcellular CRABP1 and CRABP2 levels with breast cancer patient survival and Ki67 immunoreactivity. CRABP1, CRABP2 and Ki67 protein levels were determined by immunohistochemical analysis of a TMA containing triplicate cores for each tumor from a 120 primary breast tumor cohort. a-b Association of cytoplasmic and nuclear CRABP1 levels with patient survival. c-d Association of cytoplasmic and nuclear CRABP2 levels with patient survival. e Positive correlation of cytoplasmic and nuclear CRABP1 protein levels with Ki67 immunoreactivity. f Positive correlation of CRABP1 and Ki67 mRNA levels based on gene microarray analysis. g No significant correlation was observed between subcellular CRABP2 levels and Ki67 immunoreactivity. h No correlation was evident between CRABP2 and Ki67 mRNA levels. HR, hazard ratio; p, statistical significance level; r, correlation coefficient. Scores: 0, negative; 1, weak; 2, intermediate; 3, strong. As the size of samples expressed CRABP1 is small in our TMAs, tumor samples were classified into “positive” and “negative” groups for survival analysis
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Fig3: Associations of subcellular CRABP1 and CRABP2 levels with breast cancer patient survival and Ki67 immunoreactivity. CRABP1, CRABP2 and Ki67 protein levels were determined by immunohistochemical analysis of a TMA containing triplicate cores for each tumor from a 120 primary breast tumor cohort. a-b Association of cytoplasmic and nuclear CRABP1 levels with patient survival. c-d Association of cytoplasmic and nuclear CRABP2 levels with patient survival. e Positive correlation of cytoplasmic and nuclear CRABP1 protein levels with Ki67 immunoreactivity. f Positive correlation of CRABP1 and Ki67 mRNA levels based on gene microarray analysis. g No significant correlation was observed between subcellular CRABP2 levels and Ki67 immunoreactivity. h No correlation was evident between CRABP2 and Ki67 mRNA levels. HR, hazard ratio; p, statistical significance level; r, correlation coefficient. Scores: 0, negative; 1, weak; 2, intermediate; 3, strong. As the size of samples expressed CRABP1 is small in our TMAs, tumor samples were classified into “positive” and “negative” groups for survival analysis

Mentions: We then correlated subcellular levels of CRABPs with patient survival probabilities. We found that patients with tumors that were positive for cytoplasmic, but not nuclear, CRABP1 had a significantly lower patient survival probability (p = 0.0091) (Fig. 3a–b). In contrast, nuclear, but not cytoplasmic, CRABP2 immunoreactivity was positively associated with patient survival probability (p = 0.0127) (Fig. 3c–d). Thus, our TMA analysis shows that: (i) the clinicopathological associations of CRABP1 and 2 at the protein level are in good agreement with that of the gene profiling data, (ii) CRABP1 is more frequently found in the cytoplasm whereas CRABP2 is more abundant in the nucleus of primary breast tumors, and (iii) clinical outcome and prognostic implications can be attributed to cytoplasmic CRABP1 and nuclear CRABP2, suggesting distinct subcellular functions.Fig. 3


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)

Associations of subcellular CRABP1 and CRABP2 levels with breast cancer patient survival and Ki67 immunoreactivity. CRABP1, CRABP2 and Ki67 protein levels were determined by immunohistochemical analysis of a TMA containing triplicate cores for each tumor from a 120 primary breast tumor cohort. a-b Association of cytoplasmic and nuclear CRABP1 levels with patient survival. c-d Association of cytoplasmic and nuclear CRABP2 levels with patient survival. e Positive correlation of cytoplasmic and nuclear CRABP1 protein levels with Ki67 immunoreactivity. f Positive correlation of CRABP1 and Ki67 mRNA levels based on gene microarray analysis. g No significant correlation was observed between subcellular CRABP2 levels and Ki67 immunoreactivity. h No correlation was evident between CRABP2 and Ki67 mRNA levels. HR, hazard ratio; p, statistical significance level; r, correlation coefficient. Scores: 0, negative; 1, weak; 2, intermediate; 3, strong. As the size of samples expressed CRABP1 is small in our TMAs, tumor samples were classified into “positive” and “negative” groups for survival analysis
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4491424&req=5

Fig3: Associations of subcellular CRABP1 and CRABP2 levels with breast cancer patient survival and Ki67 immunoreactivity. CRABP1, CRABP2 and Ki67 protein levels were determined by immunohistochemical analysis of a TMA containing triplicate cores for each tumor from a 120 primary breast tumor cohort. a-b Association of cytoplasmic and nuclear CRABP1 levels with patient survival. c-d Association of cytoplasmic and nuclear CRABP2 levels with patient survival. e Positive correlation of cytoplasmic and nuclear CRABP1 protein levels with Ki67 immunoreactivity. f Positive correlation of CRABP1 and Ki67 mRNA levels based on gene microarray analysis. g No significant correlation was observed between subcellular CRABP2 levels and Ki67 immunoreactivity. h No correlation was evident between CRABP2 and Ki67 mRNA levels. HR, hazard ratio; p, statistical significance level; r, correlation coefficient. Scores: 0, negative; 1, weak; 2, intermediate; 3, strong. As the size of samples expressed CRABP1 is small in our TMAs, tumor samples were classified into “positive” and “negative” groups for survival analysis
Mentions: We then correlated subcellular levels of CRABPs with patient survival probabilities. We found that patients with tumors that were positive for cytoplasmic, but not nuclear, CRABP1 had a significantly lower patient survival probability (p = 0.0091) (Fig. 3a–b). In contrast, nuclear, but not cytoplasmic, CRABP2 immunoreactivity was positively associated with patient survival probability (p = 0.0127) (Fig. 3c–d). Thus, our TMA analysis shows that: (i) the clinicopathological associations of CRABP1 and 2 at the protein level are in good agreement with that of the gene profiling data, (ii) CRABP1 is more frequently found in the cytoplasm whereas CRABP2 is more abundant in the nucleus of primary breast tumors, and (iii) clinical outcome and prognostic implications can be attributed to cytoplasmic CRABP1 and nuclear CRABP2, suggesting distinct subcellular functions.Fig. 3

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