Russell E. McDaniel

C-Src modulates estrogen-induced stress and apoptosis in estrogen-deprived breast cancer cells

The emergence of anti-estrogen resistance in breast cancer is an important clinical phenomenon affecting long-term survival in this disease. Identifying factors that convey cell survival in this setting may guide improvements in treatment. Estrogen (E2) can induce apoptosis in breast cancer cells that have been selected for survival after E2 deprivation for long periods (MCF-7:5C cells), but the mechanisms underlying E2-induced stress in this setting have not been elucidated. Here, we report that the c-Src kinase functions as a key adapter protein for the estrogen receptor (ER, ESR1) in its activation of stress responses induced by E2 in MCF-7:5C cells. E2 elevated phosphorylation of c-Src, which was blocked by 4-hydroxytamoxifen (4-OHT), suggesting that E2 activated c-Src through the ER. We found that E2 activated the sensors of the unfolded protein response (UPR), IRE1α (ERN1) and PERK kinase (EIF2AK3), the latter of which phosphorylates eukaryotic translation initiation factor-2α (eIF2α). E2 also dramatically increased reactive oxygen species production and upregulated expression of heme oxygenase HO-1 (HMOX1), an indicator of oxidative stress, along with the central energy sensor kinase AMPK (PRKAA2). Pharmacologic or RNA interference-mediated inhibition of c-Src abolished the phosphorylation of eIF2α and AMPK, blocked E2-induced ROS production, and inhibited E2-induced apoptosis. Together, our results establish that c-Src kinase mediates stresses generated by E2 in long-term E2-deprived cells that trigger apoptosis. This work offers a mechanistic rationale for a new approach in the treatment of endocrine-resistant breast cancer.

Inhibition of c-Src blocks oestrogen-induced apoptosis and restores oestrogen-stimulated growth in long-term oestrogen-deprived breast cancer cells

PURPOSE Our publications demonstrate that physiological concentrations of oestrogen (E2) induce endoplasmic reticulum and oxidative stress which finally result in apoptosis in E2-deprived breast cancer cells, MCF-7:5C. c-Src is involved in the process of E2-induced stress. To mimic the clinical administration of c-Src inhibitors, we treated cells with either E2, a c-Src inhibitor PP2, or the combination for 8 weeks to further explore the apoptotic potential of the c-Src inhibitor and E2 on MCF-7:5C cells. METHODS Protein levels of receptors and signalling pathways were examined by immunoblotting. Expression of mRNA was detected through real-time polymerase chain reaction (PCR). Cell cycles were analysed by flow cytometry. RESULTS Long-term treatment with PP2 alone or E2 alone decreased cell growth. In contrast, a combination of PP2 and E2 blocked apoptosis and the resulting cell line (MCF-7:PF) was unique, as they grew vigorously in culture with physiological levels of E2, which could be blocked by the pure antioestrogen ICI182,780. One major change was that PP2 collaborated with E2 to increase the level of insulin-like growth factor-1 receptor beta (IGF-1Rβ). Blockade of IGF-1Rβ completely abolished E2-stimulated growth in MCF-7:PF cells. Furthermore, combination treatment up-regulated transcription factors, Twist1 and Snail, and repressed E-cadherin expression which made MCF-7:PF cells display a characteristic phenotype of epithelial-mesenchymal transition (EMT). CONCLUSIONS These data illustrate the role of the c-Src inhibitor to block E2-induced apoptosis and enhance E2-stimulated growth. Caution must be exercised when considering c-Src inhibitors in clinical trials following the development of acquired resistance to aromatase inhibitors, especially in the presence of the patients own oestrogen.

Modulating therapeutic effects of the c-Src inhibitor via oestrogen receptor and human epidermal growth factor receptor 2 in breast cancer cell lines

PURPOSE c-Src is an important adapter protein with oestrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2), which validates it as an attractive target for the treatment of breast cancer. A specific c-Src inhibitor, 4-amino-5-(4-chlorophenyl)-7(t-butyl)pyrazolo[3,4-d]pyrinidine (PP2), was utilised to block c-Src activity to identify targeted vulnerabilities affected by ER and HER2 in a panel of breast cancer cell lines. METHODS ER, growth factor receptors and signalling pathways were detected by Western-blot. The DNA content of the cells was determined by using a DNA fluorescence quantitation kit. Cell cycles were analysed by flow cytometry. RESULTS The antiproliferative effect of PP2 closely correlated with the inhibition of c-Src mediated extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) and/or phosphoinositide 3-kinase (PI3K)/Akt growth pathways. Inhibition of c-Src tyrosine kinase predominantly blocked ER negative breast cancer cell growth, particularly the triple (i.e. ER, progesterone receptor (PR), and HER2) negative cells. In contrast, ER negative Sk-Br-3 cells with highest HER2 phosphorylation were resistant to PP2, in which hyper-activated HER2 directly regulated growth pathways. However, blocking c-Src recovered ER expression and down-regulated HER2 which made Sk-Br-3 cells regain responsiveness to 4-hydroxytamoxifen. The majority of ER positive cells were not sensitive to PP2 regardless of wild-type or endocrine resistant cell lines. CONCLUSIONS c-Src mediates the essential role of growth pathways in ER negative breast cancer cells. The ER positive and HER2 over-activation are two important predictive biomarkers for the resistance to a c-Src inhibitor. These data provided an important therapeutic rationale for patient selection in clinical trials with c-Src inhibitors in breast cancer.

Estrogen-Mediated Mechanisms to Control the Growth and Apoptosis of Breast Cancer Cells. A Translational Research Success Story.

The treatment and prevention of solid tumors have proved to be a major challenge for medical science. The paradigms for success in the treatment of childhood leukemia, Hodgkins disease, Burketts lymphoma, and testicular carcinoma with cytotoxic chemotherapy did not translate to success in solid tumors--the majority of cancers that kill. In contrast, significant success has accrued for patients with breast cancer with antihormone treatments (tamoxifen or aromatase inhibitors) that are proved to enhance survivorship, and remarkably, there are now two approved prevention strategies using either tamoxifen or raloxifene. This was considered impossible 40 years ago. We describe the major clinical advances with nonsteroidal antiestrogens that evolved into selective estrogen receptor modulators (SERMs) which successfully exploited the ER target selectively inside a womans body. The standard paradigm that estrogen stimulates breast cancer growth has been successfully exploited for over 4 decades with therapeutic strategies that block (tamoxifen, raloxifene) or reduce (aromatase inhibitors) circulating estrogens in patients to stop breast tumor growth. But this did not explain why high-dose estrogen treatment that was the standard of care to treat postmenopausal breast cancer for 3 decades before tamoxifen caused tumor regression. This paradox was resolved with the discovery that breast cancer resistance to long-term estrogen deprivation causes tumor regression with physiologic estrogen through apoptosis. The new biology of estrogen action has been utilized to explain the findings in the Womens Health Initiative that conjugated equine estrogen alone given to postmenopausal women, average age 68, will produce a reduction of breast cancer incidence and mortality compared to no treatment. Estrogen is killing nascent breast cancer cells in the ducts of healthy postmenopausal women. The modulation of the ER using multifunctional medicines called SERMs has provided not only significant improvements in womens health and survivorship not anticipated 40 years ago but also has been the catalyst to enhance our knowledge of estrogens apoptotic action that can be further exploited in the future.

Integration of Downstream Signals of Insulin-like Growth Factor-1 Receptor by Endoplasmic Reticulum Stress for Estrogen-Induced Growth or Apoptosis in Breast Cancer Cells

Estrogen (E2) exerts a dual function on E2-deprived breast cancer cells, with both initial proliferation and subsequent induction of stress responses to cause apoptosis. However, the mechanism by which E2 integrally regulates cell growth or apoptosis-associated pathways remains to be elucidated. Here, E2 deprivation results in many alterations in stress-responsive pathways. For instance, E2-deprived breast cancer cells had higher basal levels of stress-activated protein kinase, c-Jun N-terminal kinase (JNK), compared with wild-type MCF-7 cells. E2 treatment further constitutively activated JNK after 24 hours. However, inhibition of JNK (SP600125) was unable to abolish E2- induced apoptosis, whereas SP600125 alone arrested cells at the G2 phase of the cell cycle and increased apoptosis. Further examination showed that inhibition of JNK increased gene expression of TNFα and did not effectively attenuate expression of apoptosis-related genes induced by E2. A notable finding was that E2 regulated both JNK and Akt as the downstream signals of insulin-like growth factor-1 receptor (IGFIR)/PI3K, but with distinctive modulation patterns: JNK was constitutively activated, whereas Akt and Akt-associated proteins, such as PTEN and mTOR, were selectively degraded. Endoplasmic reticulum–associated degradation (ERAD) was involved in the selective protein degradation. These findings highlight a novel IGFIR/PI3K/JNK axis that plays a proliferative role during the prelude to E2-induced apoptosis and that the endoplasmic reticulum is a key regulatory site to decide cell fate after E2 treatment. Implications: This study provides a new rationale for further exploration of E2-induced apoptosis to improve clinical benefit. Mol Cancer Res; 13(10); 1367–76. ©2015 AACR.

Simulation with cells in vitro of tamoxifen treatment in premenopausal breast cancer patients with different CYP2D6 genotypes

Tamoxifen is a prodrug that is metabolically activated by 4‐hydroxylation to the potent primary metabolite 4‐hydroxytamoxifen (4OHT) or via another primary metabolite N‐desmethyltamoxifen (NDMTAM) to a biologically active secondary metabolite endoxifen through a cytochrome P450 2D6 variant system (CYP2D6). To elucidate the mechanism of action of tamoxifen and the importance of endoxifen for its effect, we determined the anti‐oestrogenic efficacy of tamoxifen and its metabolites, including endoxifen, at concentrations corresponding to serum levels measured in breast cancer patients with various CYP2D6 genotypes (simulating tamoxifen treatment).

Influence of the Length and Positioning of the Antiestrogenic Side Chain of Endoxifen and 4-Hydroxytamoxifen on Gene Activation and Growth of Estrogen Receptor Positive Cancer Cells

Tamoxifen has biologically active metabolites: 4-hydroxytamoxifen (4OHT) and endoxifen. The E-isomers are not stable in solution as Z-isomerization occurs. We have synthesized fixed ring (FR) analogues of 4OHT and endoxifen as well as FR E and Z isomers with methoxy and ethoxy side chains. Pharmacologic properties were documented in the MCF-7 cell line, and prolactin synthesis was assessed in GH3 rat pituitary tumor cells. The FR Z-isomers of 4OHT and endoxifen were equivalent to 4OHT and endoxifen. Other test compounds used possessed partial estrogenic activity. The E-isomers of FR 4OHT and endoxifen had no estrogenic activity at therapeutic serum concentrations. None of the newly synthesized compounds were able to down-regulate ER levels. Molecular modeling demonstrated that some compounds would each create a best fit with a novel agonist conformation of the ER. The results demonstrate modulation by the ER complex of cell replication or gene transcription in cancer.

Pharmacological Relevance of Endoxifen in a Laboratory Simulation of Breast Cancer in Postmenopausal Patients

BACKGROUND Tamoxifen is metabolically activated via a CYP2D6 enzyme system to the more potent hydroxylated derivatives 4-hydroxytamoxifen and endoxifen. This study addresses the pharmacological importance of endoxifen by simulating clinical scenarios in vitro. METHODS Clinical levels of tamoxifen metabolites in postmenopausal breast cancer patients previously genotyped for CYP2D6 were used in vitro along with clinical estrogen levels (estrone and estradiol) in postmenopausal patients determined in previous studies. The biological effects on cell growth were evaluated in a panel of estrogen receptor-positive breast cancer cell lines via cell proliferation assays and real-time polymerase chain reaction (PCR). Data were analyzed with one- and two-way analysis of variance and Students t test. All statistical tests were two-sided. RESULTS Postmenopausal levels of estrogen-induced proliferation of all test breast cancer cell lines (mean fold induction ± SD vs vehicle control: MCF-7 = 11 ± 1.74, P < .001; T47D = 7.52 ± 0.72, P < .001; BT474 = 1.75 ± 0.23, P < .001; ZR-75-1 = 5.5 ± 1.95, P = .001. Tamoxifen and primary metabolites completely inhibited cell growth regardless of the CYP2D6 genotype in all cell lines (mean fold induction ± SD vs vehicle control: MCF-7 = 1.57 ± 0.38, P = .54; T47D = 1.17 ± 0.23, P = .79; BT474 = 0.96 ± 0.2, P = .98; ZR-75-1 = 0.86 ± 0.67, P = .99). Interestingly, tamoxifen and its primary metabolites were not able to fully inhibit the estrogen-stimulated expression of estrogen-responsive genes in MCF-7 cells (P < .05 for all genes), but the addition of endoxifen was able to produce additional antiestrogenic effect on these genes. CONCLUSIONS The results indicate that tamoxifen and other metabolites, excluding endoxifen, completely inhibit estrogen-stimulated growth in all cell lines, but additional antiestrogenic action from endoxifen is necessary for complete blockade of estrogen-stimulated genes. Endoxifen is of supportive importance for the therapeutic effect of tamoxifen in a postmenopausal setting.

Abstract 5682: Modeling the pharmacological importance of endoxifen for the treatment of ER-positive breast cancer in premenopausal patients

Long term adjuvant tamoxifen therapy for five years is the antiestrogenic standard of care for ER-positive breast cancer in premenopausal patients. The metabolic activation of tamoxifen by CYP2D6 to endoxifen remains controversial to plan the treatment of patients with breast cancer. However, all retrospective studies focus entirely on postmenopausal patients and no studies have been undertaken in the relevant premenopausal treatment population. We have addressed the issue of the pharmacological importance of endoxifen to control the estrogen-stimulated growth of four ER-positive breast cancer cell lines (MCF-7, T47-D, BT-474 and ZR-75). We have modeled the actual estrogen environment in the laboratory (estradiol plus estrone) based on previous data from premenopausal patients taking tamoxifen (Jordan et al, JNCI 2001; 83:1488-91). Our strategy was to evaluate the anti-proliferative actions of actual concentrations of tamoxifen, N-desmethyltamoxifen and 4-hydroxytamoxifen combined, based on the actual measurements reported for these metabolites in patients who were extensive metabolizers (EM), intermediate metabolizers (IM) or poor metabolizers (PM) (Murdter et al, Clin Pharmacol Ther 2011; 89:708-17). These data were then compared with similar anti-proliferative experiments in the four cell lines, using endoxifen obtained from four sources (Mayo Clinic, University of Indiana, Institut fur Klinische Pharmakologie (IKP) and the Fox Chase Cancer Center). Each cell line was evaluated with the clinically relevant circulating levels of estrogen, with and without the inclusion of the different endoxifens at the published concentrations from EM, IM and PM. It is important to note that all synthetic endoxifen currently available is an impure mixture of geometric isomers. Therefore, results obtained are only an estimate of the efficacy of endoxifen in the patient. To address this issue, we have specifically synthesized the appropriate isomer of endoxifen as a fixed ring compound that can no longer isomerize. We have previously used this strategy to discover the actual pharmacology of the two geometric isomers of 4-hydroxytamoxifen (Murphy et al, Mol Pharm 1990; 38:737-43). The results demonstrate the essential requirement with the appropriate concentration of endoxifen necessary to block estrogen-mediated cell replication. Acknowledgements: This work (VCJ) was supported the subcontract under the SU2C (AACR) Grant number SU2C-AACR-DT0409; the Susan G Komen For The Cure Foundation under Award number SAC100009. 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 5682. doi:1538-7445.AM2012-5682

Abstract 830: Transcriptional modulation of estrogen-induced apoptosis through activation of c-Fos/c-Jun in long-term estrogen deprived breast cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Recent clinical trials have demonstrated that estrogen (E2) alone reduces breast cancer incidence in postmenopausal women and has therapeutic effects on aromatase inhibitor resistant patients which both are related with the effect of E2-induced apoptosis. We have shown that E2 induces apoptosis in long-term E2 deprived breast cancer cells (MCF-7:5C) through stress responses, but the molecular mechanism underlying E2-induced stress remains to be elucidated. Here, we report that E2 activated the sensors of unfolded protein response (UPR) inositol-requiring protein 1 alpha (IRE1α) and PRK-like endoplasmic reticulum kinase (PERK) within 24 hours. Knockdown of PERK and IRE-1α through small interferon RNAs (siRNA) partially prevented E2-induced apoptosis, which suggested that endoplasmic reticulum stress was involved in the E2-induced apoptosis. Further examination showed E2 activated both classical estrogen responsive element (ERE) pathway and nonclassical activating protein-1 (AP-1) pathway in MCF-7:5C cells. Classical ERE regulated genes were not directly involved in E2-induced apoptosis. However, the transcription factor c-Fos acted as a critical trigger involved in stress responses induced by E2 in MCF-7:5C cells. E2 immediately elevated c-Fos expression in MCF-7:5C cells but not in wild-type breast cancer cells and 4-hydroxytamoxifen blocked this stimulation which suggested that E2 activated c-Fos through estrogen receptor (ER). E2 increased phosphorylation of c-Jun after 24 hours treatment but did not significantly enhance the abundance of c-Jun as c-Fos. c-Fos protein forms stable heterodimers with c-Jun which preferentially bind to phorbol 12-O-tetradecanoate-13-acetate (TPA)-responsive element (TRE). Interestingly, low doses of TPA which activated TRE activity could induce apoptosis and activated apoptosis-related genes similar to E2 in MCF-7:5C cells. Knockdown of c-Fos and c-Jun with specific siRNAs blocked E2-induced apoptosis, which confirmed the activation of AP-1 played a critical role in the process of apoptosis induced by E2. Although c-Fos closely associates with c-Jun to form a stable heterodimer, they had differential functions in modulation of UPR and other nuclear transcription factors. Knockdown of c-Fos but not c-Jun could inhibit the activation of UPR by E2 in MCF-7:5C cells. Furthermore, downregulation of c-Fos abolished the expression of multiple nuclear transcription factors such as SRC-3, NF-κB, and p300 etc in MCF-7:5C cells. Overall, these data illustrate that c-Jun might provide a dimerizing site for c-Fos, whereas c-Fos was identified as a pivotal regulatory target of E2 in MCF-7:5C cells to trigger the apoptotic cascades. This study provides an important rationale for further exploration of E2-induced apoptosis in endocrine resistant breast cancer to improve clinical benefit. Citation Format: Ping Fan, Fadeke Agboke, Obi L. Griffith, Russell E. McDaniel, Xiaojun Zou, Karen Creswell, Joe W. Gray, Virgil Craig Jordan. Transcriptional modulation of estrogen-induced apoptosis through activation of c-Fos/c-Jun in long-term estrogen deprived breast cancer cells. [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 830. doi:10.1158/1538-7445.AM2013-830