Phosphorylation Of Estrogen Receptor Alpha Research Articles

Silkworm (Bombyx mori L.) Has Beneficial Effects on Menopausal Symptoms by Enhancing Estrogen Receptor Signaling in Ovariectomized Mice.

Existing hormone replacement therapy for menopause has drawbacks, necessitating new treatment agents. Silkworms have demonstrated estrogenic properties, offering promising alternatives. We assessed the therapeutic effects of freeze-dried silkworm powder (SWP) on menopausal symptoms using an ovariectomized (OVX) mouse model. The experimental design comprised a sham surgery group (Sham), an OVX control group, a low-dose SWP group post-OVX (80 mg/kg, OVX-SWP-L), a high-dose SWP group post-OVX (160 mg/kg, OVX-SWP-H), and an estradiol treatment group post-OVX (OVX-E2). Treatments were administered orally thrice weekly over eight weeks; body weight was monitored weekly. The SWP-treated groups (SWP-L and SWP-H) exhibited less weight gain and increased uterine thickness than the OVX control. Molecular analyses demonstrated that SWP significantly enhanced the phosphorylation of estrogen receptor alpha (ERα), ERK, and AKT. Furthermore, biochemical assays revealed reduced serum neutral lipids across all SWP treatment groups. Notably, HDL-cholesterol levels were significantly increased in the SWP-L group compared to the OVX group. Serum estradiol concentrations were elevated in all the SWP groups, with significant increases in the high-dose group. These findings indicate that SWP may promote the activation of estrogen receptor signaling and improve symptoms associated with estrogen deficiency during menopause.

Abstract 1772: EGFR signaling as a mechanism of resistance to CDK4/6 inhibitors in Palbociclib-resistant ER+ breast cancer cells

Abstract Breast cancer driven by estrogen receptor (ER) is responsible for approximately 60-70% of cases among women. Selective inhibition of cyclin-dependent kinases 4 and 6 (CDK4/6) and ER signaling is now standard-of-care therapy for ER positive (ER+) metastatic breast cancer. CDK4/6 inhibitors, including palbociclib (PD), used in combination with endocrine therapy have shown improvement in progression-free survival compared to endocrine therapy alone in the metastatic setting. However, the inevitable development of acquired resistance significantly limits the efficacy of this targeted therapy. In ER+ breast cancer resistant cells, rewired signaling driven by different oncogenes, including the epidermal growth factor receptor (EGFR), overcomes the inhibition of CDK4/6 by PD, which, in turn, allows cell cycle progression contributing to PD-resistance. We investigated the dependency of ER+ breast cancer cell lines sensitive and resistant to PD on EGFR signaling in vitro. We evaluated the expression and activity of EGFR in two different matching pairs of ER+ palbociclib-sensitive (pS) and palbociclib-resistant (pR) breast cancer cell lines (MCF7 and T47D) and observed elevated EGFR expression in MCF7/pR cells. Increased EGFR expression in MCF7/pR cells correlated with elevated ER alpha phosphorylation at S118 and S167 sites, suggesting a direct cross-talk between EGFR and ER signaling in PD-resistant cells. Serum starvation significantly decreased EGFR activity, cdk2 and cyclin D1 expression in both MCF7 and T47D PD-resistant cells compared to the parental cell lines, confirming their dependency on the presence of growth factors. Notably, stimulation with EGF promoted ER phosphorylation and expression of cell cycle related targets: cdk2, cyclin E and wee1 in both MCF7 and T47D cell lines sensitive and resistant to PD. Furthermore, stimulation with estrogen promoted EGFR activation in MCF7/pS and MCF7/pR cells, indicating a direct cross-regulation between these molecular targets. Further, exposure to the EGFR inhibitor erlotinib dramatically inhibited the proliferation of MCF7/pR cells compared to the parental cells, suggesting that EGFR could be an important bypass signaling regulator contributing to resistance to PD. We found that dual treatment with erlotinib and PD significantly inhibited Rb phosphorylation in MCF7/pR cells improving the effect of PD on cell cycle arrest. Moreover, dual treatment of MCF7/pR cells attenuated ER expression suggesting the higher dependency of ER signaling on EGFR activity in resistant cells. Taken together, our findings suggest that EGFR signaling is a mechanism of resistance in ER+ cell lines with acquired resistance to PD in vitro and that EGFR targeting can be a promising approach to improve palbociclib efficacy in breast cancer patients. Citation Format: Nadiia Lypova, Lilibeth Lanceta, Susan M. Dougherty, Jason A. Chesney, Yoannis Imbert-Fernandez. EGFR signaling as a mechanism of resistance to CDK4/6 inhibitors in Palbociclib-resistant ER+ breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1772.

Abstract 102: IGFBP6 modulates estrogen receptor alpha phosphorylation and transcriptional activity in breast cancer cells

Abstract Insulin-like growth factors (IGFs) are signaling proteins which bind to insulin-like growth factor 1 receptor (IGF-1R) and induce a signaling cascade to promote proliferation and survival in breast cancer cells. Insulin-Like Growth Factor Binding Protein 6 (IGFBP-6) induces apoptosis by inhibiting IGF-1R upon binding to both IGF-1 and IGF-2, however, the role of IGFBP-6 in breast cancer has not been adequately studied. While IGFBP-6 can bind to both IGF-1 and IGF-2, the binding affinity for IGF2 is 20-fold higher compared to IGF1. To determine if IGFBP-6 modulates breast cancer cells, MCF-7 and T-47D cells were stably transfected with an IGFBP-6 expression plasmid. IGFBP6 over-expression resulted in decreased proliferation rates in both cell lines when compared to empty vector controls. To identify a mechanism of decreased proliferation rates, the activation of the IGF-1R-dependent AKT and MAPK mediators was measured. Reduced phosphorylation of MAPK and AKT was observed in both cell lines over-expressing IGFBP-6 compared to empty vector controls. MAPK and AKT phosphorylate ERα at serine 118 and serine 167 respectively, therefore, ERα phosphorylation at these residues was determined in the presence of IGFBP6 expression. Reduction in phosphorylation was greater in MCF-7 cells than T-47D cells when IGFBP6 was constitutively expressed. To determine if reduced ERα phosphorylation altered expression of known targets of ERα-modulated expression, such as IGF-1R and Progesterone Receptor (PR) was measured. Unexpectedly, IGF-1R and PR protein and transcript levels were greater in IGFBP6 over-expressing cells. Studies have shown that differences in ERα phosphorylation change the recruitment of transcriptional modulators associated with ERα-mediated transcription. These results suggest that despite reducing IGF-1R-dependent cell signaling, compensatory mechanisms may be induced to enhance ERα-mediated expression. Citation Format: Francisco Javier Lariz, Kevin D. Houston. IGFBP6 modulates estrogen receptor alpha phosphorylation and transcriptional activity in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 102.

OR26-2 Phosphorylation of Estrogen Receptor Alpha is Required for Mammary Gland Development and MCF-7 Breast Tumor Response to Estradiol and Tamoxifen

Serine-167 (S167) is a major phosphorylation site in estrogen receptor alpha (ERα) and a predictor for endocrine therapy response in breast cancer. We previously showed using stable transfection that mutation of S167 to alanine (S167A) impacted MCF-7 breast cancer cell growth and morphology. Using CRISPR, we evaluated S167A mutation in the endogenous ERα gene in MCF-7 breast cancer cells and xenograft tumors. In vitro, MCF-7 cells with wildtype ERα (WT) exhibited >60% increase in growth after 4 days incubation with 10-9 M 17-β estradiol (E2), and >300% decrease in growth after 4 days incubation with 10-8 M 4-hydroxy-tamoxifen (TAM) as assessed by MTS assay. In marked contrast, S167A cells showed resistance to both E2 growth stimulation and TAM growth arrest. WT cells exhibited >300% increase in migration after 24 hr E2 as assessed by Boyden Chamber assay. S167A cells exhibited >3 fold increase in basal migration compared to WT cells but were insensitive to E2 stimulation of migration. RNAseq was performed on WT and S167A cells incubated with E2 or vehicle (6 hr). Sequencing data was assessed using FastQC and sequenced libraries mapped to the human genome (UCSC hg38) using STAR RNAseq aligner. RNASeq data was normalized using TMM. Differential expression analysis by edgeR revealed significant differences in gene expression between WT and S167A cells. Vehicle-treated S167A cells exhibited 623 differentially regulated genes compared to WT cells. Notably, S167A cells exhibited marked E2 resistance with only 9 genes regulated by E2 in S167A cells compare to 93 genes in WT cells. In tumor xenograft experiments using ovariectomized nude mice, similar to WT tumors the S167A tumors did not grow in the absence of implanted 17 beta-estradiol (0.72 mg/pellet; 60 day release) demonstrating dependency of S167A tumors on E2 for tumor establishment and growth. However, unlike WT tumors that exhibited tumor regression with E2 + TAM (5 mg/pellet; 60 day release) treatment (week 4, P<0.05), S167A tumors were resistant to TAM and exhibited continued growth similar to E2 alone treatment. Serine-212 (S216 in mouse) is a phosphorylation site located between the two zinc fingers in the DNA binding domain and is highly conserved in the nuclear receptor superfamily. To understand the importance of ERα phosphorylation for mammary gland development, ERα knock-in (KI) mice (Esr1S216A) that express the ERα S216A mutation were evaluated. Mammary glands were whole-mounted with carmine-alum from 5 week-old pubertal female ERα wildtype (WT) and KI mice (n=3). KI mice exhibited a significant decrease in ductal elongation (P<0.05), number of terminal end buds (P<0.01) and branching morphogenesis (P<0.01) compared to WT mice. Collectively, these results demonstrate that S216 phosphorylation is critical for mouse mammary gland development and S167 phosphorylation is required for MCF-7 breast tumor response to estradiol and tamoxifen.

The estrogen receptor-alpha S118P variant does not affect breast cancer incidence or response to endocrine therapies.

Estrogen receptor-alpha (ER) is a therapeutic target of ER-positive (ER+) breast cancers. Although ER signaling is complex, many mediators of this pathway have been identified. Specifically, phosphorylation of ER at serine 118 affects responses to estrogen and therapeutic ligands and has been correlated with clinical outcomes in ER+ breast cancer patients. We hypothesized that a newly described germline variant (S118P) at this residue would drive cellular changes consistent with breast cancer development and/or hormone resistance. Isogenic human breast epithelial cell line models harboring ER S118P were developed via genome editing and characterized to determine the functional effects of this variant. We also examined the frequency of ER S118P in a case-control study (N = 536) of women with and without breast cancer with a familial risk. In heterozygous knock-in models, the S118P variant demonstrated no significant change in proliferation, migration, MAP Kinase pathway signaling, or response to the endocrine therapies tamoxifen and fulvestrant. Further, there was no difference in the prevalence of S118P between women with and without cancer relative to population registry databases. This study suggests that the ER S118P variant does not affect risk for breast cancer or hormone therapy resistance. Germline screening and modification of treatments for patients harboring this variant are likely not warranted.

Ginsenoside Rd promotes non-amyloidogenic pathway of amyloid precursor protein processing by regulating phosphorylation of estrogen receptor alpha

AimsPrevious study demonstrated that Ginsenoside Rd. (GS-Rd) could improve cognitive and memory function in animal model of Alzheimer's disease. This study was aimed to investigate whether GS-Rd could improve non-amyloidogenic pathway by activating estrogen receptor (ER). Main methods10mg/kg GS-Rd in ovariectomy (OVX)+GS-Rd group and equivalent volume of saline in sham operated group and OVX group were administrated intraperitoneally for two months, respectively. The Morris Water Maze was used to examine cognitive function of rats, with sAPPα and Aβ levels in the hippocampi measured. The culture medium of HT22 hippocampal neuronal cells were incubated with GS-Rd, ER antagonist ICI182.780, MAPK inhibitor PD98059, or PI3Kinhibitor LY294002, respectively. sAPPα levels was measured, and expression of α-secretase, sAPPα, β-secretase, Aβ, phosphorylation form of AKT (p-AKT), total AKT, p-ERK, total ERK, p-ERα, total ERα, p-ERβ and total ERβ were examined by Western blot to explore the estrogenic-like activity of GS-Rd. Key findingsGS-Rd attenuate cognitive and memory impairment, increased levels of sAPPα and reduced extracellular Aβ of OVX rats. In HT22, GS-Rd could upregulate sAPPα level, which can be inhibited by inhibitor of MAPK and PI3K pathway. In addition, inhibitor of estrogen receptor prevented GS-Rd triggered release of sAPPα and activation of MAPK and PI3K pathways. GS-Rd could increase expression of α-secretase and sAPPα, while decrease expression of β-secretase and Aβ. Besides, GS-Rd promoted phosphorylation of estrogen receptor alpha at Ser118 residue. SignificanceOur findings show that GS-Rd enhances learning and memory function of OVX rats by activating estrogen-like activity.

Abstract 5045: Characterization of estrogen receptor alpha phosphorylation sites as a marker for tamoxifen resistant breast cancer

Abstract A major focus of breast cancer research is to understand the mechanisms responsible for disease progression and drug resistance. Most treatment options target endocrine therapy, however, the processes that modulate these signaling pathways are poorly understood. Here, we seek to gain additional understanding about the phosphorylation status of the estrogen receptor as it relates to tamoxifin drug treatment. Tamoxifen, a selective ER inhibitor, was the first targeted breast cancer therapy developed. Unfortunately, tamoxifen therapy was shown to be ineffective for certain kinds of cancers and even in affected tumors an acquired resistance was eventually seen. Previous research has shown that the estrogen receptor protein is phosphorylated at more than 20 different sites and some of these sites are linked with poor prognosis in tamoxifen treated patients. For example, phosphorylation at the S305 site may lead to an activation of ERα and to transcription of ERα-responsive gene after tamoxifen treatment, leading to tamoxifen resistance. In addition, other phosphorylation sites have also been found to correlate with poor prognosis of tamoxifen treated patients. Thus, differential phosphorylation of ERα may serve as a biomarker to identify patients unlikely to respond to tamoxifen treatment. Here, utilizing a newly developed medium throughput IHC test system we have interrogated multiple sites of ERα for phosphorylation status by screening an extensive panel of different breast cancer patient samples and other non-breast cancer tissue microarray (TMA) samples. These patient samples were classified according to their clinical grade (ER positive or negative, Her2, progesterone positive or negative, and Ki67 index) and tamoxifen treatment and then screened for their ERα phosphorylation status. The results show unique staining patterns depending upon the tumor classification and conclusions about the utilization of this information as a potential for diagnostic applications will be discussed. Citation Format: Wayne A. Speckmann, Robert Brockett, Chun Yang, Chandra Mohan, Kevin Long, Michele Hatler, Xiaoyi Xu. Characterization of estrogen receptor alpha phosphorylation sites as a marker for tamoxifen resistant breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5045. doi:10.1158/1538-7445.AM2015-5045

Function of RasGRP3 in the formation and progression of human breast cancer.

IntroductionRas guanine nucleotide exchange factors (RasGEFs) mediate the activation of the Ras signaling pathway that is over activated in many human cancers. The RasGRP3, an activator of H-Ras and R-Ras protein exerts oncogenic effects and the overexpression of the protein is observed in numerous malignant cancer types. Here, we investigated the putative alteration of expression and potential function of RasGRP3 in the formation and progression of human breast cancer.MethodsThe RasGRP3 and phosphoRasGRP3 expressions were examined in human invasive ductal adenocarcinoma derived samples and cell lines (BT-474, JIMT-1, MCF7, SK-BR-3, MDA-MB-453, T-47D) both in mRNA (Q-PCR) and protein (Western blot; immunohistochemistry) levels. To explore the biological function of the protein, RasGRP3 knockdown cultures were established. To assess the role of RasGRP3 in the viability of cells, annexin-V/PI staining and MitoProbe™ DilC1 (5) assay were performed. To clarify the function of the protein in cell proliferation and in the development of chemotherapeutic resistance, CyQuant assay was performed. To observe the RasGRP3 function in tumor formation, the Severe combined immunodeficiency (SCID) mouse model was used. To investigate the role of the protein in Ras-related signaling Q-PCR and Western blot experiments were performed.ResultsRasGRP3 expression was elevated in human breast tumor tissue samples as well as in multiple human breast cancer cell lines. Down-regulation of RasGRP3 expression in breast cancer cells decreased cell proliferation, induced apoptosis in MCF7 cells, and sensitized T-47D cells to the action of drugs Tamoxifen and trastuzumab (Herceptin). Gene silencing of RasGRP3 reduced tumor formation in mouse xenografts as well. Inhibition of RasGRP3 expression also reduced Akt, ERK1/2 and estrogen receptor alpha phosphorylation downstream from IGF-I insulin like growth factor-I (IGF-I) or epidermal growth factor (EGF) stimulation confirming the functional role of RasGRP3 in the altered behavior of these cells.ConclusionsTaken together, our results suggest that the Ras activator RasGRP3 may have a role in the pathological behavior of breast cancer cells and may constitute a therapeutic target for human breast cancer.

Abstract B064: Anti-phospho-estrogen receptor alpha (Ser305), clone 124.9.4, is a potential clinical antibody marker for tamoxifen-resistant breast cancer

Abstract Resistance to tamoxifen is one of the major challenges in breast cancer treatment. Different mechanisms related to tamoxifen sensitivity have been widely investigated, including the phosphorylation of ERα. It has been shown experimentally that phosphorylation of estrogen receptor alpha at serine 305 (ERαS305-P) by protein kinase A (PKA) leads to an activation of ERα and to transcription of ERα-responsive gene after tamoxifen treatment [EMBO J 26:3534-3544][EMBO J 21:5437-5447], leading to tamoxifen resistance. More recently, clinical studies also show that ERαS305-P may be a good biomarker to identify patients unlikely to respond to tamoxifen [J Pathol 217:372-379][Breast Cancer Res Treat 125(1):1-12]. Here we developed and validated a rabbit monoclonal anti-phospho-estrogen receptor alpha (Ser305) antibody, EMD Millipore 05-922R., with immunohistochemistry (IHC) validation on different breast cancer patient samples and other non-breast cancer tissue micro array (TMA) samples. Citation Format: Wayne Speckmann, Robert Brockett, Chun Yang, Alejandra Solache. Anti-phospho-estrogen receptor alpha (Ser305), clone 124.9.4, is a potential clinical antibody marker for tamoxifen-resistant breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B064.

Abstract 3571: Inhibition of Cdk9 blocks ligand induced phosphorylation of Estrogen receptor alpha at Ser294.

Abstract In response to ligand binding, the estrogen receptor (ER, alpha isoform) becomes phosphorylated on several serines including Ser294 and Ser118 to initiate an estrogenic transcriptional program that has been well studied. On the other hand, growth factor stimulation of ER also promotes serine phosphorylation at several sites including Ser305 and Ser118, but not Ser294, to drive a transcriptional program different from that induced by estrogenic ligands. Our previous work demonstrated that following estrogen or tamoxifen stimulation, cyclin dependent kinase (Cdk) mediated phosphorylation of the ER hinge region at Ser294 was essential for full transcriptional activation of ER. Current studies, using a recently developed rabbit monoclonal antibody to phospho-Ser294 ER, show that the Cdk9 inhibitor SCH727956/Dinaciclib, now in phase II clinical trials, completely prevents Ser294 phosphorlyation induced by estrogen stimulation of MCF-7 cells without influencing phospho-Ser118 formation which remains robust. Immunoprecipitation of ER from MCF-7 cells following estrogen treatment results in the co-immunoprecipitaton of cdk9. Further SCH727965/Dinaciclib has no influence upon epidermal growth factor (EGF) induced ER phosphorylation at Ser305, consistent with previous studies linking phospho-Ser305 formation to the activated kinases PKA and PAK. Ongoing studies evaluating siRNA induced knockdown of Cdk9 in MCF-7 cells followed by estrogen and/or EGF stimulation are designed to confirm the specific role of Cdk9 in mediating ligand-induced ER phosphorylation at Ser294. As Cdk9 is essential to promote transcriptional elongation by RNA polymerase II, the Ser294 specific recruitment of Cdk9 to ER following ligand stimulation offers rationale for the clinical use of Cdk9 inhibitors such as SCH727965/Dinaciclib in the treatment of ER-positive breast cancers. In particular, for those ER-positive breast cancers responsive to endocrine therapy the combined use of a Cdk9 inhibitor with antiestrogenic therapy is likely to prove clinically beneficial. Citation Format: Gary K. Scott, Ravneet Kaur, Daniel Rothschild, C. C. Benz. Inhibition of Cdk9 blocks ligand induced phosphorylation of Estrogen receptor alpha at Ser294. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3571. doi:10.1158/1538-7445.AM2013-3571

Abstract 2494: Liquid chromatography-multiple reaction monitoring mass spectrometry biomarker quantification for breast cancer patient assessment.

Abstract Introduction: A quantitative mass spectrometry platform has been developed for measuring biomarkers of protein expression and phosphorylation in breast cancer. The quantification of numerous biomarkers from a single tissue specimen has the potential to impact patient care. Specifically, the focus of initial development was on the expression and phosphorylation of estrogen receptor alpha (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2). However, immunohistochemical assessments of Ki-67 and vimentin are also widely used for monitoring proliferation in breast cancer. The integration of tissue quality control measurements, quantification of proliferation biomarkers, and ER/PR/HER2 evaluation could improve patient assessment and open novel avenues for patient classification, prognosis, and selection of therapy in breast cancer. Experimental Procedures: Breast cancer cell lines MCF7, BT474, and T47D were used to develop peptide-based quantitative assays, including stable isotope-labeled standard peptides, (n = 31) for protein expression and phosphorylation biomarkers using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) of digested whole cell lysates or SDS-PAGE fractionated proteins followed by immunoprecipitation to examine expression and phosphorylation of ER/PR/HER2 (IP-LC-MRM). Using 2 mg of total protein, the sample is divided into three parts. Tissue QC biomarkers and expression levels of Ki67, vimentin, and HER2 are measured in digests of whole cell lysate or tissue homogenate (1% of sample). Then, parallel IP strategies are used for HER2 (10% of sample) and ER/PR (90%) before LC-MRM analysis of expression and phosphorylation. Multiplex IP of the three proteins was not successful, because the amplification of HER2 could not be accommodated. The sensitivity of each assay was tested using serial dilutions of cell lysate to evaluate requirements for analysis of clinical specimens. Data Summary: LC-MRM assays for both unmodified and phosphorylated peptides have been developed and characterized for each protein using cell line models. Assays have been implemented in cell lines to study Estradiol/EGF stimulation as well as lapatinib inhibition of HER2. Data has been generated for pre-treatment frozen tissue specimens from breast cancer patients that are either HER2+ or ER+ (n = 12 per group). The analysis of a larger group of patients and in clinical scenarios (e.g. comparison of naïve and drug resistant tissues from the same patient) will establish the clinical utility of these assays. Conclusions: The ability to examine protein expression as well as phosphorylation status in these biomarkers with quantitative mass spectrometry has the potential to improve the selection of targeted therapeutics. Citation Format: Yi Chen, David J. Britton, Elizabeth R. Wood, Anthony M. Magliocco, Ian Pike, John M. Koomen. Liquid chromatography-multiple reaction monitoring mass spectrometry biomarker quantification for breast cancer patient assessment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2494. doi:10.1158/1538-7445.AM2013-2494

Rhodacyanine Derivative Selectively Targets Cancer Cells and Overcomes Tamoxifen Resistance

MKT-077, a rhodacyanine dye, was shown to produce cancer specific cell death. However, complications prevented the use of this compound beyond clinical trials. Here we describe YM-1, a derivative of MKT-077. We found that YM-1 was more cytotoxic and localized differently than MKT-077. YM-1 demonstrated this cytotoxicity across multiple cancer cell lines. This toxicity was limited to cancer cell lines; immortalized cell models were unaffected. Brief applications of YM-1 were found to be non-toxic. Brief treatment with YM-1 restored tamoxifen sensitivity to a refractory tamoxifen-resistant MCF7 cell model. This effect is potentially due to altered estrogen receptor alpha phosphorylation, an outcome precipitated by selective reductions in Akt levels (Akt/PKB). Thus, modifications to the rhodocyanine scaffold could potentially be made to improve efficacy and pharmacokinetic properties. Moreover, the impact on tamoxifen sensitivity could be a new utility for this compound family.

Abstract 3933: ERα phosphorylation patterns at hinge domain Ser294 and Ser305 differentiate ligand-dependent from ligand-independent receptor activation

Abstract Domain-specific phosphorylation of estrogen receptor alpha (ERα) is critical for its transcription factor activity and may determine its predictive and therapeutic biomarker value. Recent attention has turned to the poorly understood ERα hinge domain in light of preliminary evidence that phosphorylation at serine 305 (pSer305) associates with poor clinical outcome and endocrine resistance. Using multiple reaction monitoring (MRM) mass spectrometry (MS), we have studied a number of ERα overexpressing human breast cancer cells lines (MCF-7, BT474, T47D) and found that robust ERα phosphorylation occurs on a neighboring hinge domain site at Ser294, but only upon ligand (estradiol, tamoxifen) stimulation of the receptor since growth factor cell stimulation (EGF, insulin, heregulin-α, forskolin) fails to induce pSer294. As well, pSer294 formation appears to be essential for full ligand induction of ERα gene transcription in experimental models. Selective inhibition of cyclin-dependent kinases (CDKs) by roscovitine or SNS-032 in the breast cancer cell lines blocks ligand induced pSer294 formation, indicating that this hinge domain phosphorylation is mediated by CDKs; likewise, cell-free studies with recombinant ERα and specific cyclin-CDK complexes reveal that Ser294 phosphorylation is most effectively induced by the transcription-regulating and cell cycle-independent kinases, CDK7 and CDK9. In contrast to this ligand specific induction of pSer294, western analysis of immunopurified ERα from MCF-7 probed with a newly available pSer305 monoclonal demonstrates that pSer305 formation occurs in response to growth factor stimulation (EGF, insulin, forskolin) but not in response to cell stimulation by ligand alone. Interestingly, co-treatment with ligand and growth factor reduces pSer294 formation relative to ligand stimulation alone while co-treatment reduces pSer305 formation relative to growth factor stimulation alone, suggesting that these two hinge phosphorylation sites not only respond reciprocally to specify different aspects of ERα function but are also antagonistically responsive to crosstalk. Unlike the better studied N-terminal domain phosphorylation sites at Ser118 and Ser167, which are phosphorylated jointly and additively upon combined ligand-dependent and ligand-independent ERα activation, the two hinge domain sites at Ser294 and Ser305 appear to respond in a reciprocal fashion differentiating ligand-dependent from ligand-independent ERα activation. Pending current development of a pSer294 monoclonal, future immunohistochemical interrogation of the ERα hinge domain phosphorylation status at Ser294 and Ser305 is expected to improve the biomarker value of this receptor by better predicting ERα overexpressing breast cancers most likely to respond to endocrine agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3933. doi:1538-7445.AM2012-3933

Evidence of a positive correlation between accelerated oestrogen receptor phosphorylation and breast cancer progression

Estrogen receptor beta (ERβ) is predicted to play an important role in prevention of breast cancer development and metastasis. Phosphorylation of Estrogen receptor alpha (ERα) has been proven to be involved in the progression of breast cancer and it is believed oestrogen receptor beta too phosphorylated at multiple sites within the protein upon ligand binding although the exact function of this site-specific phosphorylation is unknown. Nevertheless it is assumed that the site-specific phosphorylation of ERβ may be involved in the progression of human breast cancer. To test this hypothesis we developed novel monoclonal antibodiesusing synthetic peptide specific for putative serine phosphorylation site in human ERs (S87). These antibodies tested on human cancerous breast tissue samples provided clear evidence of phosphorylation of ERβ at S87 progressively as cancer advanced. These antibodies could be used in targeting the phosphorylation site which could help in treatment strategies and control of cancer progression. Key words: Oestrogen receptor beta, breast cancer, monoclonal antibodies, phospho specific anti-peptide antibodies.

Evidence of a positive correlation between accelerated oestrogen receptor phosphorylation and breast cancer progression

Estrogen receptor beta (ERβ) is predicted to play an important role in prevention of breast cancer development and metastasis. Phosphorylation of Estrogen receptor alpha (ERα) has been proven to be involved in the progression of breast cancer and it is believed oestrogen receptor beta too phosphorylated at multiple sites within the protein upon ligand binding although the exact function of this site-specific phosphorylation is unknown. Nevertheless it is assumed that the site-specific phosphorylation of ERβ may be involved in the progression of human breast cancer. To test this hypothesis we developed novel monoclonal antibodiesusing synthetic peptide specific for putative serine phosphorylation site in human ERs (S87). These antibodies tested on human cancerous breast tissue samples provided clear evidence of phosphorylation of ERβ at S87 progressively as cancer advanced. These antibodies could be used in targeting the phosphorylation site which could help in treatment strategies and control of cancer progression. Key words: Oestrogen receptor beta, breast cancer, monoclonal antibodies, phospho specific anti-peptide antibodies.

Estrogen Receptor-α Phosphorylation at Serine-118 and Tamoxifen Response in Breast Cancer

Although estrogen receptor-alpha (ER) [corrected] is a marker used to identify breast cancer patients most likely to benefit from endocrine therapy, approximately 50% of ER-positive [corrected] breast carcinomas are resistant to tamoxifen. Preclinical studies have shown that phosphorylation of ER [corrected] at serine-118 (ER alpha S118-P) is required for tamoxifen-mediated inhibition of ER-induced [corrected] gene expression. We evaluated the association between recurrence-free survival after tamoxifen treatment and ER alpha S118-P expression by use of Cox proportional hazards regression. Data were from 239 premenopausal patients with breast cancer who participated in a randomized trial of 2 years of adjuvant tamoxifen treatment vs no systemic treatment. ER alpha S118-P expression was assessed by immunohistochemistry and categorized by use of the Allred score (low expression = score of 0-6; high expression = score of 7-8). All statistical tests were two-sided. Compared with systemically untreated patients, we found evidence of a benefit from adjuvant tamoxifen among patients whose tumors had high ER alpha S118-P expression (23.7 recurrences per 1000 person-years versus 72.2 recurrences per 1000 person-years, hazard ratio [HR] of recurrence = 0.36, 95% confidence interval [CI] = 0.20 to 0.65) but not among patients whose tumors had low expression (51.0 recurrences per 1000 person-years versus 57.0 recurrences per 1000 person-years, HR of recurrence = 0.87, 95% CI = 0.51 to 1.48), a statistically significant difference (P for interaction = .037). ER alpha 118-P was not associated with recurrence-free survival among untreated patients. Thus, ER alpha S118-P expression appears to be associated with response to tamoxifen. [corrected]

Estrogenic and Neuroprotective Properties of Scutellarin fromErigeron breviscapus: A Drug against Postmenopausal Symptoms and Alzheimer's Disease

Besides the classical hormonal effect, estrogen possesses neuroprotective effects in the brain, which leads to the searching of novel treatments for neurodegenerative diseases such as Alzheimer's disease. Scutellarin is a major flavone derived from Herba Erigerontis, a Chinese medicine derived from Erigeron breviscapus, which has been shown here to possess both estrogenic and neuroprotective properties. Scutellarin showed the estrogenic effects by activating the estrogen responsive elements and phosphorylation of estrogen receptor alpha in cultured MCF-7 cells: the activation was in a dose-dependent manner. On the other hand, scutellarin inhibited the aggregation of beta-amyloid in vitro, and prevented the cell death mediated by beta-amyloid when applied to cultured neuronal PC12 cells. These results therefore suggested that Herba Erigerontis and its component scutellarin might have therapeutic effects against postmenopausal symptoms and Alzheimer's disease.

Retrospective analysis of phosphorylation status of the estrogen receptor in patients with early stage disease

21034 Background: The AKT kinase, when phosphorylated to its activated form (pAKT), mediates many proliferative and survival effects of growth factor receptors such as members of the epidermal growth factor receptor (EGFR) family. Overexpression of AKT in breast neoplasms suggests that AKT activation is involved in breast cancer development and/or progression, and contributes to poor outcome. pAKT is frequently upregulated in Her-2/neu positive breast cancers. Activated AKT is able to phosphorylate in vitro serine 167 (pS167) of estrogen receptor alpha (ERa), resulting in ER-mediated transcriptional activity in the absence of estrogen and possibly contributing to tamoxifen resistance in ERa positive breast cancers. In addition, ERa is phosphorylated at S118 (pS118) by MAPK. Thus, it is possible that pAKT- mediated ERa phosphorylation is implicated in tumorigenesis and resistance to treatment with hormonal therapy in patients with breast cancer. Methods: Utilizing commercially available antibodies, IHC analysis of 125 paraffin-embedded, ERa positive, early breast cancer cases, stratified for stage, were tested for pAKT, PTEN, pS167, and pS118. Staining intensity and number of positive cells were assessed by 4 independent graders. Fisher's exact test was used to determine the significance of associations with a two-sided type I error of 5%. Results: Phosphorylation of AKT occurred in approximately one-half of cases. pS167 was phosphorylated in 66% of pAKT positive cases compared to 35% of pAKT negative cases (p&lt;0.0001). When we analyzed nuclear pAKT staining, this association was more pronounced: 76% of pAKT(+) vs. 37% of pAKT(-) (p&lt;0.0001). 69% of pS118(+) cases were pS167(+) compared to only 15% pS167(+) when pS118 was negative (p&lt;0.0001). In approx. 32% of cases pAKT was present in both the cytoplasm and nucleus. 20% of samples had only cytoplasmic pAKT, while in 4% of cases pAKT was nuclear only, supporting the hypothesis that cytoplasmic phosphorylation of AKT may precede nuclear accumulation. Conclusions: Phosphorylation of pS167ERa is significantly associated with AKT activation (especially in the nucleus) in early breast cancer, supporting the hypothesis that these two events may be causally linked and contribute to breast cancer pathogenesis. No significant financial relationships to disclose.