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HIF-1α stimulates aromatase expression driven by prostaglandin E2 in breast adipose stroma.

Samarajeewa NU, Yang F, Docanto MM, Sakurai M, McNamara KM, Sasano H, Fox SB, Simpson ER, Brown KA - Breast Cancer Res. (2013)

Bottom Line: We have identified the presence of a consensus HIF-1α binding motif overlapping with the proximal CRE of aromatase PII.Treatments included PGE2 or DMOG ((dimethyloxalglycine), HIF-1α stabilizer).Results also demonstrate that HIF-1α significantly increases PII activity, and aromatase transcript expression and activity, in the presence of DMOG and/or PGE2, and that HIF-1α and CREB1 act co-operatively on PII.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: The majority of postmenopausal breast cancers are estrogen-dependent. Tumor-derived factors, such as prostaglandin E2 (PGE2), stimulate CREB1 binding to cAMP response elements (CREs) on aromatase promoter II (PII), leading to the increased expression of aromatase and biosynthesis of estrogens within human breast adipose stromal cells (ASCs). Hypoxia inducible factor-1α (HIF-1α), a key mediator of cellular adaptation to low oxygen levels, is emerging as a novel prognostic marker in breast cancer. We have identified the presence of a consensus HIF-1α binding motif overlapping with the proximal CRE of aromatase PII. However, the regulation of aromatase expression by HIF-1α in breast cancer has not been characterized. This study aimed to characterize the role of HIF-1α in the activation of aromatase PII.

Methods: HIF-1α expression and localization were examined in human breast ASCs using quantitative PCR (QPCR), Western blotting, immunofluorescence and high content screening. QPCR and tritiated water-release assays were performed to assess the effect of HIF-1α on aromatase expression and activity. Reporter assays and chromatin immunoprecipitation (ChIP) were performed to assess the effect of HIF-1α on PII activity and binding. Treatments included PGE2 or DMOG ((dimethyloxalglycine), HIF-1α stabilizer). Double immunohistochemistry for HIF-1α and aromatase was performed on tissues obtained from breast cancer and cancer-free patients.

Results: Results indicate that PGE2 increases HIF-1α transcript and protein expression, nuclear localization and binding to aromatase PII in human breast ASCs. Results also demonstrate that HIF-1α significantly increases PII activity, and aromatase transcript expression and activity, in the presence of DMOG and/or PGE2, and that HIF-1α and CREB1 act co-operatively on PII. There is a significant increase in HIF-1α positive ASCs in breast cancer patients compared to cancer-free women, and a positive association between HIF-1α and aromatase expression.

Conclusions: This study is the first to identify HIF-1α as a modulator of PII-driven aromatase expression in human breast tumor-associated stroma and provides a novel mechanism for estrogen regulation in obesity-related, post-menopausal breast cancer. Together with our on-going studies on the role of AMP-activated protein kinase (AMPK) in the regulation of breast aromatase, this work provides another link between disregulated metabolism and breast cancer.

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Effect of PGE2 on HIF-1α expression and nuclear localization in primary human breast ASCs. PGE2 caused a significant increase in HIF-1α transcript (A) and nuclear protein expression (B). Confocal microscopy demonstrated that HIF-1α (green) is mainly perinuclear in breast ASCs under basal conditions (C, top left) and that PGE2 stimulates the translocation of HIF-1α to the nucleus (C, top right). Treatment with DMOG (C, bottom left) and DMOG with PGE2 (C, bottom right) caused a much higher HIF-1α staining in the nucleus. The merged lamin B1+B2 nuclear stain (red) and HIF-1α are found as insets at the bottom right of each image. The percentage of cells positive for nuclear HIF-1α was also shown to be significantly increased with PGE2 treatment (D). vc = vehicle control, n = 3, repeated twice. Confocal images are representative of the majority of cells examined.
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Figure 1: Effect of PGE2 on HIF-1α expression and nuclear localization in primary human breast ASCs. PGE2 caused a significant increase in HIF-1α transcript (A) and nuclear protein expression (B). Confocal microscopy demonstrated that HIF-1α (green) is mainly perinuclear in breast ASCs under basal conditions (C, top left) and that PGE2 stimulates the translocation of HIF-1α to the nucleus (C, top right). Treatment with DMOG (C, bottom left) and DMOG with PGE2 (C, bottom right) caused a much higher HIF-1α staining in the nucleus. The merged lamin B1+B2 nuclear stain (red) and HIF-1α are found as insets at the bottom right of each image. The percentage of cells positive for nuclear HIF-1α was also shown to be significantly increased with PGE2 treatment (D). vc = vehicle control, n = 3, repeated twice. Confocal images are representative of the majority of cells examined.

Mentions: The effect of PGE2 on HIF-1α transcript and protein expression was examined in primary breast ASCs. HIF-1α mRNA was significantly increased with PGE2 treatment (Figure 1A). Moreover, Western blotting demonstrated that PGE2 also caused a significant increase in HIF-1α protein abundance in the nucleus (Figure 1B). The subcellular localization of endogenously expressed HIF-1α was examined using immunofluorescence and confocal microscopy after treating with PGE2 and/or DMOG. Results demonstrated that punctate staining for HIF-1α appears in the nucleus after PGE2 treatment (Figure 1C, top right) compared to cytoplasmic localization under basal conditions (Figure 1C, top left). Furthermore, DMOG (Figure 1C, bottom left) and DMOG with PGE2 (Figure 1C, bottom right) resulted in more intense staining for HIF-1α in the nucleus. Results from high content screening also demonstrated that the percentage cells positive for HIF-1α in the nucleus (Figure 1D) was significantly increased in response to PGE2 treatment.


HIF-1α stimulates aromatase expression driven by prostaglandin E2 in breast adipose stroma.

Samarajeewa NU, Yang F, Docanto MM, Sakurai M, McNamara KM, Sasano H, Fox SB, Simpson ER, Brown KA - Breast Cancer Res. (2013)

Effect of PGE2 on HIF-1α expression and nuclear localization in primary human breast ASCs. PGE2 caused a significant increase in HIF-1α transcript (A) and nuclear protein expression (B). Confocal microscopy demonstrated that HIF-1α (green) is mainly perinuclear in breast ASCs under basal conditions (C, top left) and that PGE2 stimulates the translocation of HIF-1α to the nucleus (C, top right). Treatment with DMOG (C, bottom left) and DMOG with PGE2 (C, bottom right) caused a much higher HIF-1α staining in the nucleus. The merged lamin B1+B2 nuclear stain (red) and HIF-1α are found as insets at the bottom right of each image. The percentage of cells positive for nuclear HIF-1α was also shown to be significantly increased with PGE2 treatment (D). vc = vehicle control, n = 3, repeated twice. Confocal images are representative of the majority of cells examined.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672802&req=5

Figure 1: Effect of PGE2 on HIF-1α expression and nuclear localization in primary human breast ASCs. PGE2 caused a significant increase in HIF-1α transcript (A) and nuclear protein expression (B). Confocal microscopy demonstrated that HIF-1α (green) is mainly perinuclear in breast ASCs under basal conditions (C, top left) and that PGE2 stimulates the translocation of HIF-1α to the nucleus (C, top right). Treatment with DMOG (C, bottom left) and DMOG with PGE2 (C, bottom right) caused a much higher HIF-1α staining in the nucleus. The merged lamin B1+B2 nuclear stain (red) and HIF-1α are found as insets at the bottom right of each image. The percentage of cells positive for nuclear HIF-1α was also shown to be significantly increased with PGE2 treatment (D). vc = vehicle control, n = 3, repeated twice. Confocal images are representative of the majority of cells examined.
Mentions: The effect of PGE2 on HIF-1α transcript and protein expression was examined in primary breast ASCs. HIF-1α mRNA was significantly increased with PGE2 treatment (Figure 1A). Moreover, Western blotting demonstrated that PGE2 also caused a significant increase in HIF-1α protein abundance in the nucleus (Figure 1B). The subcellular localization of endogenously expressed HIF-1α was examined using immunofluorescence and confocal microscopy after treating with PGE2 and/or DMOG. Results demonstrated that punctate staining for HIF-1α appears in the nucleus after PGE2 treatment (Figure 1C, top right) compared to cytoplasmic localization under basal conditions (Figure 1C, top left). Furthermore, DMOG (Figure 1C, bottom left) and DMOG with PGE2 (Figure 1C, bottom right) resulted in more intense staining for HIF-1α in the nucleus. Results from high content screening also demonstrated that the percentage cells positive for HIF-1α in the nucleus (Figure 1D) was significantly increased in response to PGE2 treatment.

Bottom Line: We have identified the presence of a consensus HIF-1α binding motif overlapping with the proximal CRE of aromatase PII.Treatments included PGE2 or DMOG ((dimethyloxalglycine), HIF-1α stabilizer).Results also demonstrate that HIF-1α significantly increases PII activity, and aromatase transcript expression and activity, in the presence of DMOG and/or PGE2, and that HIF-1α and CREB1 act co-operatively on PII.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: The majority of postmenopausal breast cancers are estrogen-dependent. Tumor-derived factors, such as prostaglandin E2 (PGE2), stimulate CREB1 binding to cAMP response elements (CREs) on aromatase promoter II (PII), leading to the increased expression of aromatase and biosynthesis of estrogens within human breast adipose stromal cells (ASCs). Hypoxia inducible factor-1α (HIF-1α), a key mediator of cellular adaptation to low oxygen levels, is emerging as a novel prognostic marker in breast cancer. We have identified the presence of a consensus HIF-1α binding motif overlapping with the proximal CRE of aromatase PII. However, the regulation of aromatase expression by HIF-1α in breast cancer has not been characterized. This study aimed to characterize the role of HIF-1α in the activation of aromatase PII.

Methods: HIF-1α expression and localization were examined in human breast ASCs using quantitative PCR (QPCR), Western blotting, immunofluorescence and high content screening. QPCR and tritiated water-release assays were performed to assess the effect of HIF-1α on aromatase expression and activity. Reporter assays and chromatin immunoprecipitation (ChIP) were performed to assess the effect of HIF-1α on PII activity and binding. Treatments included PGE2 or DMOG ((dimethyloxalglycine), HIF-1α stabilizer). Double immunohistochemistry for HIF-1α and aromatase was performed on tissues obtained from breast cancer and cancer-free patients.

Results: Results indicate that PGE2 increases HIF-1α transcript and protein expression, nuclear localization and binding to aromatase PII in human breast ASCs. Results also demonstrate that HIF-1α significantly increases PII activity, and aromatase transcript expression and activity, in the presence of DMOG and/or PGE2, and that HIF-1α and CREB1 act co-operatively on PII. There is a significant increase in HIF-1α positive ASCs in breast cancer patients compared to cancer-free women, and a positive association between HIF-1α and aromatase expression.

Conclusions: This study is the first to identify HIF-1α as a modulator of PII-driven aromatase expression in human breast tumor-associated stroma and provides a novel mechanism for estrogen regulation in obesity-related, post-menopausal breast cancer. Together with our on-going studies on the role of AMP-activated protein kinase (AMPK) in the regulation of breast aromatase, this work provides another link between disregulated metabolism and breast cancer.

Show MeSH
Related in: MedlinePlus