Treatment with aromatase inhibitors stimulates the expression of Epidermal growth factor receptor-1 and neuregulin 1 in ER positive \ HER-2neu non-amplified primary breast cancers

This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.jsbmb.2016.06.011While estrogens have been shown to modulate EGFR/HER-1 and HER-2/neu expression in experimental systems, the effects of estrogen deprivation on expression levels of the HER-receptors and the neuregulin (NRG)1 ligand in breast cancers remain unknown. Here, we measured EGFR/HER-1-4 and NRG1 mRNA in ER positive tumors from 85 postmenopausal breast cancer patients before and after two weeks (n = 64) and three months (n = 85) of primary treatment with an aromatase inhibitor (AI). In tumors lacking HER-2/neu amplification, quantitative real-time PCR analyses revealed EGFR/HER-1 and NRG1 to vary significantly between the three time points (before therapy, after 2 weeks and after 3 months on treatment; P ≤ 0.001 for both). Pair-wise comparison revealed a significant increase in EGFR/HER-1 already during the first two weeks of treatment (P = 0.049) with a further increase for both EGFR/HER-1 and NRG1 after 3 months on treatment (P ≤ 0.001 and P = 0.001 for both comparing values at 3 months to values at baseline and 2 weeks respectively). No difference between tumors responding versus non-responders was recorded. Further, no significant change in any parameter was observed among HER-2/neu amplified tumors. Analyzing components of the HER-2/neu PI3K/Akt downstream pathway, the PIK3CA H1047R mutation was associated with treatment response (P = 0.035); however no association between either AKT phosphorylation status or PIK3CA gene mutations and EGFR/HER-1 or NRG1 expression levels were observed. Our results indicate primary AI treatment to modulate expression of HER-family members and the growth factor NRG1 in HER-2/neu non-amplified breast cancers in vivo. Potential implications to long term sensitivity warrants further investigations.The study was supported by the Norwegian Cancer Society (https://kreftforeningen.no), The Western Norway Regional Health Authority (http://www.helse-bergen.no/forskning/samarbeidsorganet), Odd Fellow Medisinsk Vitenskapelig Forskningsfond (oddfellow.no) and Martin Flatners legat

The Active Tamoxifen Metabolite Endoxifen (4OHNDtam) Strongly Down-Regulates Cytokeratin 6 (CK6) in MCF-7 Breast Cancer Cells

Introduction: Tamoxifen is an anti-estrogen drug used in treatment of Estrogen Receptor (ER) positive breast cancer. Effects and side effects of tamoxifen is the sumof tamoxifen and all itsmetabolites. 4-Hydroxytamoxifen (4OHtam) and 4-hydroxy-N-demethyltamoxifen (4OHNDtam, endoxifen) both have ER affinity exceeding that of the parent drug tamoxifen. 4OHNDtam is considered the main active metabolite of tamoxifen. Ndesmethyltamoxifen (NDtam) is the major tamoxifen metabolite. It has low affinity to the ER and is not believed to influence tumor growth. However, NDtam might mediate adverse effects of tamoxifen treatment. In this study we investigated the gene regulatory effects of the three metabolites of tamoxifen in MCF-7 breast cancer cells. Material and Methods: Using concentrations that mimic the clinical situation we examined effects of 4OHtam, 4OHNDtam and NDtam on global gene expression in 17β-estradiol (E2) treated MCF-7 cells. Transcriptomic responses were assessed by correspondence analysis, differential expression, gene ontology analysis and quantitative real time PCR (Q-rt-PCR). E2 deprivation and knockdown of Steroid Receptor Coactivator-3 (SRC-3)/Amplified in Breast Cancer 1 (AIB1) mRNA in MCF-7 cells were performed to further characterize specific effects on gene expression. Results: 4OHNDtam and 4OHtamcausedmajor changes in gene expression compared to treatment with E2 alone, with a stronger effect of 4OHNDtam. NDtam had nearly no effect on the global gene expression profile. Treatment ofMCF-7 cells with 4OHNDtam led to a strong down-regulation of the CytoKeratin 6 isoforms (KRT6A, KRT6B and KRT6C). The CytoKeratin 6 mRNAs were also down-regulated inMCF-7 cells after E2 deprivation and after SRC-3/ AIB1 knockdown. Conclusion: Using concentrations that mimic the clinical situation we report global gene expression changes that were most pronounced with 4OHNDtam and minimal with NDtam. Genes encoding CytoKeratin 6, were highly down-regulated by 4OHNDtam, as well as after E2 deprivation and knockdown of SRC-3/AIB1, indicating an estrogen receptor-dependent regulation.publishedVersio

Treatment with aromatase inhibitors stimulates the expression of epidermal growth factor receptor-1 and neuregulin 1 in ER positive/HER-2/neu non-amplified primary breast cancers.

While estrogens have been shown to modulate EGFR/HER-1 and HER-2/neu expression in experimental systems, the effects of estrogen deprivation on expression levels of the HER-receptors and the neuregulin (NRG)1 ligand in breast cancers remain unknown. Here, we measured EGFR/HER-1-4 and NRG1 mRNA in ER positive tumors from 85 postmenopausal breast cancer patients before and after two weeks (n=64) and three months (n=85) of primary treatment with an aromatase inhibitor (AI). In tumors lacking HER-2/neu amplification, quantitative real-time PCR analyses revealed EGFR/HER-1 and NRG1 to vary significantly between the three time points (before therapy, after 2 weeks and after 3 months on treatment; P≤0.001 for both). Pair-wise comparison revealed a significant increase in EGFR/HER-1 already during the first two weeks of treatment (P=0.049) with a further increase for both EGFR/HER-1 and NRG1 after 3 months on treatment (P≤0.001 and P=0.001 for both comparing values at 3 months to values at baseline and 2 weeks respectively). No difference between tumors responding versus non-responders was recorded. Further, no significant change in any parameter was observed among HER-2/neu amplified tumors. Analyzing components of the HER-2/neu PI3K/Akt downstream pathway, the PIK3CA H1047R mutation was associated with treatment response (P=0.035); however no association between either AKT phosphorylation status or PIK3CA gene mutations and EGFR/HER-1 or NRG1 expression levels were observed. Our results indicate primary AI treatment to modulate expression of HER-family members and the growth factor NRG1 in HER-2/neu non-amplified breast cancers in vivo. Potential implications to long term sensitivity warrants further investigations.The study was supported by the Norwegian Cancer Society (https://kreftforeningen.no), The Western Norway Regional Health Authority (http://www.helse-bergen.no/forskning/samarbeidsorganet), Odd Fellow Medisinsk Vitenskapelig Forskningsfond (oddfellow.no) and Martin Flatners legat.This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.jsbmb.2016.06.01

Inverse regulation of EGFR/HER1 and HER2-4 in normal and malignant human breast tissue.

Cross-talk between the estrogen and the EGFR/HER signalling pathways has been suggested as a potential cause of resistance to endocrine therapy in breast cancer. Here, we determined HER1-4 receptor and neuregulin-1 (NRG1) ligand mRNA expression levels in breast cancers and corresponding normal breast tissue from patients previously characterized for plasma and tissue estrogen levels. In tumours from postmenopausal women harbouring normal HER2 gene copy numbers, we found HER2 and HER4, but HER3 levels in particular, to be elevated (2.48, 1.30 and 22.27 -fold respectively; P<0.01 for each) compared to normal tissue. Interestingly, HER3 as well as HER4 were higher among ER+ as compared to ER- tumours (P=0.004 and P=0.024, respectively). HER2 and HER3 expression levels correlated positively with ER mRNA (ESR1) expression levels (r=0.525, P=0.044; r=0.707, P=0.003, respectively). In contrast, EGFR/HER1 was downregulated in tumour compared to normal tissue (0.13-fold, P<0.001). In addition, EGFR/HER1 correlated negatively to intra-tumour (r=-0.633, P=0.001) as well as normal tissue (r=-0.556, P=0.006) and plasma estradiol levels (r=-0.625, P=0.002), suggesting an inverse regulation between estradiol and EGFR/HER1 levels. In ER+ tumours from postmenopausal women, NRG1 levels correlated positively with EGFR/HER1 (r=0.606, P=0.002) and negatively to ESR1 (r=-0.769, P=0.003) and E2 levels (r=-0.542, P=0.020). Our results indicate influence of estradiol on the expression of multiple components of the HER system in tumours not amplified for HER2, adding further support to the hypothesis that cross-talk between these systems may be of importance to breast cancer growth in vivo

Steroid receptor coactivators, HER-2 and HER-3 expression is stimulated by tamoxifen treatment in DMBA-induced breast cancer

Background: Steroid receptor coactivators (SRCs) may modulate estrogen receptor (ER) activity and the response to endocrine treatment in breast cancer, in part through interaction with growth factor receptor signaling pathways. In the present study the effects of tamoxifen treatment on the expression of SRCs and human epidermal growth factor receptors (HERs) were examined in an animal model of ER positive breast cancer. Methods: Sprague-Dawley rats with DMBA-induced breast cancer were randomized to 14 days of oral tamoxifen 40 mg/kg bodyweight/day or vehicle only (controls). Tumors were measured throughout the study period. Blood samples and tumor tissue were collected at sacrifice and tamoxifen and its main metabolites were quantified using LC-MS/MS. The gene expression in tumor of SRC-1, SRC-2/transcription intermediary factor-2 (TIF-2), SRC-3/amplified in breast cancer 1 (AIB1), ER, HER-1, -2, -3 and HER-4, as well as the transcription factor Ets-2, was measured by real-time RT-PCR. Protein levels were further assessed by Western blotting. Results: Tamoxifen and its main metabolites were detected at high concentrations in serum and accumulated in tumor tissue in up to tenfolds the concentration in serum. Mean tumor volume/rat decreased in the tamoxifen treated group, but continued to increase in controls. The mRNA expression levels of SRC-1 (P=0.035), SRC-2/TIF-2 (P=0.002), HER-2 (P = 0.035) and HER-3 (P = 0.006) were significantly higher in tamoxifen treated tumors compared to controls, and the results were confirmed at the protein level using Western blotting. SRC-3/AIB1 protein was also higher in tamoxifen treated tumors. SRC-1 and SRC-2/TIF-2 mRNA levels were positively correlated with each other and with HER-2 (P≤0.001), and the HER-2 mRNA expression correlated with the levels of the other three HER family members (P<0.05). Furthermore, SRC-3/AIB1 and HER-4 were positively correlated with each other and Ets-2 (P<0.001). Conclusions: The expression of SRCs and HER-2 and -3 is stimulated by tamoxifen treatment in DMBA-induced breast cancer. Stimulation and positive correlation of coactivators and HERs may represent an early response to endocrine treatment. The role of SRCs and HER-2 and -3 should be further studied in order to evaluate their effects on response to long-term tamoxifen treatment

Estrogens Correlate with <i>PELP1</i> Expression in ER Positive Breast Cancer

<div><p>The Proline-, glutamic acid- and leucine-rich protein 1 (<i>PELP1</i>) is an estrogen receptor (ER) coactivator and a proto-oncogene known to be deregulated in endocrine cancers. In breast cancer, <i>PELP1</i> overexpression has been associated with endocrine therapy resistance. Although <i>PELP1</i> is known to be regulated by estrogens <i>in vitro</i>, its association with estrogen levels within the tissue of breast cancer patients has not previously been assessed. Here, we determined <i>PELP1</i> mRNA expression levels in paired samples of normal and malignant breast tissue obtained from 32 postmenopausal and 11 premenopausal women. In the total sample set, <i>PELP1</i> levels were higher in tumors compared to normal breast tissue (<i>P</i> = 0.041). Among postmenopausal women, <i>PELP1</i> tumor levels correlated positively with estrone (E₁) and estradiol (E₂) levels in both normal tissue (r = 0.543, <i>P</i> = 0.003 and r = 0.601, <i>P</i> = 0.001, respectively) and plasma (r = 0.392, <i>P</i> = 0.053 and r = 0.403, <i>P</i> = 0.046, respectively). Analyzing all ER+ tumors (n = 26), <i>PELP1</i> correlated positively with E₁ and E₂ in tumor tissue (r = 0.562, <i>P</i> = 0.003 and r = 0.411, <i>P</i> = 0.037, respectively) and normal tissue (r = 0.461, <i>P</i> = 0.018 and r = 0.427, <i>P</i> = 0.030, respectively) in addition to plasma E₁, E₂ and estrone sulphate (E₁S) concentrations (r = 0.576, <i>P</i> = 0.003, r = 0.456, <i>P</i> = 0.025 and r = 0.406, <i>P</i> = 0.049, respectively). Finally, <i>PELP1</i> correlated positively with ER mRNA (<i>ESR1)</i> (r = 0.553, <i>P</i> = 0.026) in ER+ tumors, whereas a negative association between <i>PELP1</i> and <i>ESR1</i> (r = -0.733, <i>P</i> = 0.010) was observed in ER- breast tumors. Taken together, tumor <i>PELP1</i> mRNA expression is associated with estrogen levels in breast cancer, suggesting a potentially important role of <i>PELP1</i> in ER+ breast cancer growth <i>in vivo</i>.</p></div

Intratumoral correlations of <i>PELP1</i> with <i>HER1</i> and <i>NRG1</i>.

<p>Tumor <i>PELP1</i> mRNA levels were correlated with <i>HER1</i> (A) and <i>NRG1</i> (B) tumor levels. Relative <i>PELP1</i> expression levels were analyzed using RT- real-time PCR whereas <i>HER1</i> and <i>NRG1</i> tumor levels are published before [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134351#pone.0134351.ref020" target="_blank">20</a>]. Correlations were evaluated using Spearman Rank (two-tailed) test.</p

Intratumoural correlations of growth factor receptors with estrogen receptor mRNA levels (<i>ESR1</i>).

<p>Scatterplots illustrate correlations of <i>HER2</i> (A), <i>HER3</i> (B) and <i>EGFR/HER1</i> (C) with <i>ESR1</i> among postmenopausal women. <i>HER2</i> amplified tumours are excluded. Significant correlations were evaluated using the Spearman rank (two-tailed) test.</p

Correlations of <i>PELP1</i> with ER mRNA (<i>ESR1</i>) levels.

<p>Scatterplot illustrating correlations of <i>PELP1</i> with <i>ESR1</i> in ER- (open circle) and ER+ (filled circle) tumors. Relative <i>PELP1</i> expression levels were analyzed using RT- real-time PCR whereas <i>ESR1</i> tumor levels are published before [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134351#pone.0134351.ref021" target="_blank">21</a>]. Correlations were evaluated using Spearman Rank (two-tailed) test. Association between <i>PELP1</i> and <i>ESR1</i> levels when ER status was taken into account was verified using a multivariate logistic regression model (<i>P</i> ≤ 0.001).</p

Patient, tumor and treatment characteristics.

<p>Abbreviations: ER, estrogen receptor; HRT, hormone replacement therapy</p><p>^a Both normal breast and cancer tissue were available for gene expression analyses from 11 premenopausal and 32 postmenopausal patients.</p><p>^b These four patients were excluded from the correlation analysis with the estrogen levels.</p><p>Patient, tumor and treatment characteristics.</p