Surojeet Sengupta

Defining the Conformation of the Estrogen Receptor Complex That Controls Estrogen-Induced Apoptosis in Breast Cancer

Development of acquired antihormone resistance exposes a vulnerability in breast cancer: estrogen-induced apoptosis. Triphenylethylenes (TPEs), which are structurally similar to 4-hydroxytamoxifen (4OHT), were used for mechanistic studies of estrogen-induced apoptosis. These TPEs all stimulate growth in MCF-7 cells, but unlike the planar estrogens they block estrogen-induced apoptosis in the long-term estrogen-deprived MCF7:5C cells. To define the conformation of the TPE:estrogen receptor (ER) complex, we employed a previously validated assay using the induction of transforming growth factor α (TGFα) mRNA in situ in MDA-MB 231 cells stably transfected with wild-type ER (MC2) or D351G ER mutant (JM6). The assays discriminate ligand fit in the ER based on the extremes of published crystallography of planar estrogens or TPE antiestrogens. We classified the conformation of planar estrogens or angular TPE complexes as “estrogen-like” or “antiestrogen-like” complexes, respectively. The TPE:ER complexes did not readily recruit the coactivator steroid receptor coactivator-3 (SRC3) or ER to the PS2 promoter in MCF-7 and MCF7:5C cells, and molecular modeling showed that they prefer to bind to the ER in an antagonistic fashion, i.e., helix 12 not sealing the ligand binding domain (LBD) effectively, and therefore reduce critical SRC3 binding. The fully activated ER complex with helix 12 sealing the LBD is suggested to be the appropriate trigger to initiate rapid estrogen-induced apoptosis.

The Conformation of the Estrogen Receptor Directs Estrogen-Induced Apoptosis in Breast Cancer: A Hypothesis.

Abstract Background: Estrogens are classified as type I (planar) and type II (angular) based on their structures. In this study, we used triphenylethylenes (TPEs) compounds related to 4-hydroxytamoxifen 4OHT to address the hypothesis that the conformation of the liganded estrogen receptor (ERα) can dictate the E2-induced apoptosis of the ER+ breast cancer cells. Materials and methods: ERα positive MCF7:5C cells were used to study apoptosis induced by E2, 4OHT and TPEs. Growth and apoptosis assays were used to evaluate apoptosis and the ability to reverse E2-induced apoptosis. ERα protein was measured by Western blotting to investigate the destruction of ERα by TPEs in MCF7 cells. Chromatin immunoprecipitation (ChIP) assays were performed to study the in vivo recruitment of ERα and SRC3 at classical E2-responsive promoter TFF1 (PS2) by TPEs. Molecular modeling was used to predict the binding mode of the TPE to the ERα. Results: TPEs were not only unable to induce efficient apoptosis in MCF7:5C cells but also reversed the E2-induced apoptosis similar to 4OHT. Furthermore, the TPEs and 4OHT did not reduce the ERα protein levels unlike E2. ChIP assay confirmed very weak recruitment of SRC3 despite modest recruitment of ERα in the presence of TPEs. Mole-ular modeling suggests that TPE would bind in antagonistic mode with ERα. Conclusion: Our results advances the hypothesis that the TPE liganded ERα complex structurally resembles the 4OHT bound ERα and cannot efficiently recruit co-activator SRC3. As a result, the TPE complex cannot induce apoptosis of ER+ breast cancer cells, although it can cause growth of the breast cancer cells. The conformation of the estrogen-ER complex differentially controls growth and apoptosis.

Inhibition of BET proteins impairs estrogen-mediated growth and transcription in breast cancers by pausing RNA polymerase advancement

Estrogen (E2)-induced transcription requires coordinated recruitment of estrogen receptor α (ER) and multiple factors at the promoter of activated genes. However, the precise mechanism by which this complex stimulates the RNA polymerase II activity required to execute transcription is largely unresolved. We investigated the role of bromodomain (BRD) containing bromodomain and extra-terminal (BET) proteins, in E2-induced growth and gene activation. JQ1, a specific BET protein inhibitor, was used to block BET protein function in two different ER-positive breast cancer cell lines (MCF7 and T47D). Real-time PCR and ChIP assays were used to measure RNA expression and to detect recruitment of various factors on the genes, respectively. Protein levels were measured by Western blotting. JQ1 suppressed E2-induced growth and transcription in both MCF7 and T47D cells. The combination of E2 and JQ1 down-regulated the levels of ER protein in MCF7 cells but the loss of ER was not responsible for JQ1-mediated inhibition of E2 signaling. JQ1 did not disrupt E2-induced recruitment of ER and co-activator (SRC3) at the E2-responsive DNA elements. The E2-induced increase in histone acetylation was also not altered by JQ1. However, JQ1 blocked the E2-induced transition of RNA polymerase II from initiation to elongation by stalling it at the promoter region of the responsive genes upstream of the transcription start site. This study establishes BET proteins as the key mediators of E2-induced transcriptional activation. This adds another layer of complexity to the regulation of estrogen-induced gene activation that can potentially be targeted for therapeutic intervention.

Pnck induces ligand-independent EGFR degradation by probable perturbation of the Hsp90 chaperone complex

We have recently described a novel role for pregnancy-upregulated non-ubiquitous calmodulin kinase (Pnck) in the induction of ligand-independent epidermal growth factor receptor (EGFR) degradation (Deb TB, Coticchia CM, Barndt R, Zuo H, Dickson RB, and Johnson MD. Am J Physiol Cell Physiol 295: C365-C377, 2008). In the current communication, we explore the probable mechanism by which Pnck induces ligand-independent EGFR degradation. Pnck-induced EGFR degradation is calcium/calmodulin independent and is regulated by cell density, with the highest EGFR degradation observed at low cell density. Pnck is a novel heat shock protein 90 (Hsp90) client protein that can be co-immunoprecipitated with Hsp90. Treatment of Pnck-overexpressing cells with the pharmacologic Hsp90 inhibitor geldanamycin results in enhanced EGFR degradation, and destruction of Pnck. In cells in which Pnck is inducing EGFR degradation, we observed that Hsp90 exhibits reduced electrophoretic mobility, and through mass spectrometric analysis of immunopurified Hsp90 protein we demonstrated enhanced phosphorylation at threonine 89 and 616 (in both Hsp90-α and -β) and serine 391 (in Hsp90-α). Kinase-active Pnck protein is degraded by the proteasome, concurrent with EGFR degradation. A Pnck mutant (T171A) protein with suppressed kinase activity induced EGFR degradation to essentially the same level as wild-type (WT) Pnck, suggesting that Pnck kinase activity is not required for the induction of EGFR degradation. Although EGFR is degraded, overexpression of WT Pnck paradoxically promoted cellular proliferation, whereas cells expressing mutant Pnck (T171A) were growth inhibited. WT Pnck promoted S to G(2) transition, but cells expressing the mutant exhibited higher residency time in S phase. Basal MAP kinase activity was inhibited by WT Pnck but not by mutant T171A Pnck protein. Cyclin-dependent kinase (Cdk) inhibitor p21/Cip-1/Waf-1 was transcriptionally suppressed downstream to MAP kinase inhibition by WT Pnck, but not the mutant protein. Collectively, these data suggest that 1) Pnck induces ligand-independent EGFR degradation most likely through perturbation of Hsp90 chaperone activity due to Hsp90 phosphorylation, 2) EGFR degradation is coupled to proteasomal degradation of Pnck, and 3) modulation of basal MAP kinase activity, p21/Cip-1/Waf-1 expression, and cellular growth by Pnck is independent of Pnck-induced ligand-independent EGFR degradation.

Selective estrogen-induced apoptosis in breast cancer.

Antihormone therapy remains the gold standard of care in the treatment of estrogen receptor (ER) positive breast cancer. However, development of acquired long term antihormone resistance exposes a vulnerability to estrogen that induces apoptosis. Laboratory and clinical studies indicate that successful therapy with estrogens is dependent on the duration of estrogen withdrawal and menopausal status of a woman. Interrogation of estradiol (E2) induced apoptosis using molecular studies indicate treatment of long term estrogen deprived MCF-7 breast cancer cells with estrogen causes an endoplasmic reticulum stress response that induces an unfolded protein response signal to inhibit protein translation. E2 binds to the ER and mediates apoptosis through the classical genomic pathway. Furthermore, the induction of apoptosis by estrogens is dependent on the conformation of the estrogen-ER complex. In this review, we explore the mechanism and the processes involved in the paradox of estrogen induced apoptosis and the new selectivity of estrogen action on different cell populations that is correctly been deciphered for clinical practice.

Abstract 907: Targeting glycolysis enzyme, PFKFB3, in endocrine therapy resistant breast cancers

Reprogrammed glucose metabolism is recognized as one of the hallmarks of cancer. High energy demand in cancer cells leads to increased glycolysis to maintain anabolic processes that may be driven by altered enzyme levels. The second committed step in the glycolysis pathway is catalyzed by phosphofructokinase1 (PFK1), which converts fructose-6 phosphate (F6P) to fructose 1,6-bisphosphate (F1,6BP). Another enzyme, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), converts F6P to fructose 2,6-bisphosphate (F2,6BP) that then functions as an allosteric activator of PFK1 and drives glycolysis. PFKFB3 enzyme is upregulated in many cancers including breast cancers. PFKFB3 is regulated by estrogen in ER-positive breast cancer cells but its role in endocrine-therapy resistant breast cancer cells is largely unknown. We investigated the expression of PFKFB3 mRNA and protein in estrogen-responsive MCF7 cells and compared it with LCC9 cells that are estrogen-independent and resistant to 4-hydroxytamoxifen (4OHT) and fulvestrant (Fulv). Twofold higher PFKFB3 mRNA and protein levels were expressed in LCC9 cells compared to MCF7 cells. In addition, the estrogen-mediated stimulation of PFKFB3 that was evident in MCF7 cells was not observed in LCC9 cells. We further studied the effect of pharmacological inhibition of PFKFB3, on the growth of therapy-resistant breast cancer cells using a compound PFK158, a potent inhibitor of the PFKFB3 enzyme. Treatment with PFK158 inhibited the growth of LCC9 cells as well as another cell line, MCF7:5C that is also resistant to 4OHT and partially resistant to Fulv. PFK158 alone was effective in suppressing the growth of both the cell lines at a concentration of 5 µM or higher. Notably, combining PFK158 with either 4OHT or Fulv significantly potentiated the effect of PFK158 treatment in blocking the growth of the cells. In MCF7:5C cells the combination treatment of 2.5 µM PFK158 and Fulv (500nM) or 4OHT(500nM) inhibited cell growth by ~70% as compared to ~30% inhibition in the presence of PFK 158 alone. In LCC9 cells >70% of growth was inhibited in the combination treatment as compared to no inhibition when PFK158, 2.5 µM or 4OHT or Fulv alone was used. In publicly available datasets of ER+, node-negative breast cancer patients, high expression of PFKFB3 was associated with adverse recurrence-free survival (hazard ratio = 4.12 and p= 5.5x10-5). Our study shows an increased basal expression of PFKFB3 mRNA and protein in estrogen-independent, endocrine-therapy resistant breast cancer cells as compared to estrogen-responsive breast cancer cells. Targeting PFKFB3 in combination with anti-estrogens should be explored for potential therapeutic intervention in endocrine therapy-resistant breast cancers. Citation Format: Surojeet Sengupta, Catherine M. Sevigny, Xiaoyi Liu, Lu Jin, Paula R. Pohlmann, Robert Clarke. Targeting glycolysis enzyme, PFKFB3, in endocrine therapy resistant breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 907.

Abstract 4896: The role of SLC7A5 (LAT1) in endocrine therapy-resistant breast cancer

Breast cancer is the leading cancer diagnosis for women in the United States. Endocrine therapies, such as tamoxifen and aromatase inhibitors, are used to treat the estrogen receptor-positive (ER+) breast cancers that comprise 70% of all new cases. Unfortunately, emergence of resistance to these therapies presents a major clinical challenge. Cancer cells can adapt to dysregulate the bioenergetics of cellular metabolism and evade cell death. Solute carrier family 7 member 5 (SLC7A5 or LAT1) is expressed across the cell membrane and is a transporter of large, neutral amino acids (such as leucine or tyrosine) supporting cell proliferation. Using LCC9 breast cancer cells that are resistant to tamoxifen and fulvestrant, we studied the role of LAT1 in endocrine therapy-resistant cells as compared to sensitive MCF7 breast cancer cells. SLC7A5 expression was upregulated by estrogen in MCF7 cells that was blocked in the presence of fulvestrant treatment. A significant 2.75-fold upregulation of SLC7A5 protein and 71-fold upregulation of SLC7A5 mRNA was found in LCC9 cells (as compared with MCF7 cells) without any estrogen regulation. Fulvestrant treatment did not significantly alter SLC7A5 mRNA or protein expression. These data suggest that higher levels of SLC7A5 in resistant cells may help in transporting key amino acids from their microenvironment to support cell growth. Inhibiting the functions of SLC7A5 using a pharmacologic inhibitor, JPH203, or depleting its expression by using siRNA led to significant growth suppression of LCC9 cells. Cell cycle analysis revealed that SLC7A5 depletion increased G1 phase of cell cycle and reduced S phase cells. In five publicly available data sets, of estrogen receptor-positive and tamoxifen-treated breast cancer patients, high expression of SLC7A5 was associated with worse prognosis. Endocrine therapy resistance is partly driven by autophagy in breast cancer cells. We depleted SLC7A5 expression in LCC9s to better understand the role of this transporter in autophagy. This study uncovers a novel adaptive mechanism in endocrine therapy-resistant breast cancer cells that is facilitated by increased SLC7A5 mediated transport of large neutral amino acids enabling them to supplement their increased metabolic needs and promoting cell growth. Therefore, blocking the functions of SLC7A5, perhaps in conjunction with inhibition of autophagy, may offer an avenue of potential therapeutic intervention in endocrine therapy-resistant breast cancers. Citation Format: Catherine M. Sevigny, Surojeet Sengupta, Zhexun Luo, Lu Jin, Dominic Pearce, Robert Clarke. The role of SLC7A5 (LAT1) in endocrine therapy-resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4896.

Abstract P3-05-11: The evolution of the estrogen receptor (ER) complex conformation governs estrogen-induced apoptosis in antihormone-resistant breast cancer cells

Over the past decade new insights have been gained into the acquired resistance to tamoxifen and the Aromatase Inhibitors (AIs) with the discovery of the new biology of estrogen-induced apoptosis. However, it has also been learned that estrogens can be classified into planar class I and angular class II estrogens. Using model systems of long-term estrogen-deprived breast cancer cells in vitro (MCF-7:5C), it was previously shown that class I estrogens cause immediate apoptosis over a 3-4 day period. Paradoxically, class II estrogens actually block apoptosis caused by planar class I estrogens. To gain insight into this paradox we have successfully crystallized an angular class II triphenylethylene (TPE) estrogen bound to the ligand binding domain (LBD) of the ER and derived a new conformation for the TPE:ER complex (code 3Q97 in the PDB). Surprisingly, Helix 12 seals the LBD with the class II estrogen, but not the same conformation is observed with the planar class I estrogen 17β-estradiol (E2). There would seem to be no reason why the 3Q97 complex would not cause immediate apoptosis. To address this issue we have used Western blot analysis for protein and qRT-PCR for mRNA levels for the ER. ER parameters were monitored for up to 72 hours and results compared and contrasted between E2, the Class II estrogens, 4-hydroxytamoxifen (4OHT) and endoxifen (Endox). ER protein and mRNA levels with 4OHT or Endox accumulated and remained high throughout the study period. In contrast, the planar estrogen E2 produced a rapid decline in the protein and mRNA levels for the ER complex. The angular class II estrogens initially produced an accumulation of the ER protein complex, which decreased with time. Using a chromatin immunoprecipitation (ChIP) technique we demonstrated that the class II estrogens recruit only half of the ER to the estrogen-responsive genes promoters (TFF1 And BREB1) and less than half co-activator binding compared to E2. The TPEs were only partial agonists compared to planar estrogen. These results explain why the Class II estrogens induce delayed apoptosis. We conclude that, for the first time, we have observed the binding of a ligand to a receptor that changes conformation against time and evolves from an antagonist to an agonist conformation to trigger apoptosis. These observations have current relevance to the decryption of the protective effects of estrogen alone therapy against breast cancer incidence in the Women9s Health Initiative (WHI) trial. This work was supported by the Susan G. Komen for the Cure Foundation award SAC100009. Citation Format: Maximov PY, Sengupta S, Fernandes DJ, Fan P, Curpan RF, Rajan SS, Greene GL, Jordan VC. The evolution of the estrogen receptor (ER) complex conformation governs estrogen-induced apoptosis in antihormone-resistant breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-11.

Abstract 5642: Transcriptional deregulation of cMYC as a critical determinant of estrogen independence and aromatase inhibitor resistance in breast cancers.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Aromatase inhibitors (AI), which block the synthesis of estrogen and render an “estrogen-deprived” environment, are used extensively to treat postmenopausal ER positive breast cancers (BC). Unfortunately, 15-60% of breast cancer patients will relapse within 3-5 years of AI treatment. Therefore, it is crucial to understand the mechanisms involved in AI-resistance to identify new therapeutic targets. Our laboratory has previously reported the development of an estrogen-deprivation (ED) resistant cell line known as MCF7:5C cells which mimic the AI-resistant BC and can grow spontaneously in vitro as well as form xenograft tumors in the athymic mice in absence of estrogen (ref: Lewis et al., JNCI, 2005; 97(23):1746-59). Recent clinical studies have indicated over-expression of a transcription factor, cMYC oncogene and the genes regulated by cMYC as one of the major predictor in the aromatase inhibitor resistant BC. However, the mechanism by which the cMYC oncogene is over-expressed remains largely elusive. Using MCF7:5C cells as a model for ER positive, AI-resistant BC cells; we investigated the levels of cMYC oncogene and compared it with the parental MCF7 cells which do not proliferate in the absence of estrogen. We found that cMYC protein as well as mRNA levels were approximately 3 fold higher in MCF7:5C cells as compared to MCF7 cells. Treating the cells with a cMYC inhibitor, 10058-F4, or with cMYC si RNA decreased the proliferation and the number of ‘S’ phase cells in MCF7:5C cells. To elucidate the underlying mechanism responsible for the elevated transcription of cMYC we examined the promoter of the cMYC gene and found that the recruitment of phospho-serine-2-RNA pol II (a marker of RNA elongation) was significantly higher in MCF7:5C cells as compared to the parental MCF7 cells. Further investigation revealed that activated CDK9 levels were responsible for phosphorylating serine 2 RNA pol II in MCF7:5C cells. Inhibition of CDK9 decreased the levels of total phospho-serine-2 RNA pol II and the cMYC mRNA and also inhibited the estrogen-independent proliferation of MCF7:5C cells. This study strongly suggests CDK9 as a potential therapeutic target which mediates elevated transcription of cMYC, which in turn is responsible for estrogen-independent growth of AI-resistant BC cells. Grant Support: This work (VCJ) was supported by the Department of Defense Breast Program under Award no: W81XWH-06-1-0590 Center of Excellence; the Susan G Komen For The Cure Foundation under Award no: SAC100009; National Cancer Institute, Award no: P30CA051008. Citation Format: Surojeet Sengupta, Michael C. Biarnes, V. Craig Jordan. Transcriptional deregulation of cMYC as a critical determinant of estrogen independence and aromatase inhibitor resistance in breast cancers. [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 5642. doi:10.1158/1538-7445.AM2013-5642

Abstract 955: Role of cMYC as a critical determinant of estrogen-independent growth of estrogen deprivation-resistant breast cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Around two-third of all breast cancers express estrogen receptor alpha (ER), which depend upon estrogen for proliferation of the ER positive breast cancer cells. Aromatase inhibitors (AI) are used extensively to treat postmenopausal patients, which block the synthesis of estrogen and render an “estrogen-deprived” environment. Unfortunately, long-term treatment with AIs is associated with “AI-resistant” breast cancers, which by-passes the dependency on estrogen for the growth of the resistant breast cancer cells. To mimic these resistant breast cancers we have developed an estrogen-deprivation (ED) resistant cell line known as MCF7:5C cells from the parental ER positive MCF7 cells by propagating them in estrogen-free conditions for more than a year. MCF7:5C cells are ER positive and grow spontaneously in vitro as well as form xenograft tumors in the athymic mice in absence of estrogen (ref: Lewis et al., JNCI, 2005; 97(23):1746-59). To understand the underlying mechanism and identify the molecular determinant responsible for the estrogen-independent growth of these breast cancer cells we found that a critical estrogen-regulated gene, cMYC, is upregulated in these ED-resistant MCF7:5C cells. Basal levels of cMYC mRNA as well as the protein level were elevated three to four fold as compared to parental MCF7 cells which are dependent on estrogen for growth. Cell cycle analysis revealed twice as many “S” phase MCF7:5C cells as compared to MCF7 cells under basal condition. Treating the cells with a cMYC inhibitor, 10058-F4, decreased the number of ‘S’ phase cells and increased the cells in ‘G1′ phase in MCF7:5C cells but not in parental MCF7 cells, in a dose dependent manner. Interestingly, treatment with fulvestrant, a complete anti-estrogen, which inhibits the growth of the MCF7:5C cells by 50% also decreased the cMYC mRNA levels by similar extent. To understand the precise mechanism by which high levels of cMYC transcripts are synthesized in MCF7:5C cells we investigated the promoter of the cMYC gene and found that under basal conditions the recruitment of phosphor-serine-2-RNA polymeraseII, which is a marker of RNA elongation, was significantly higher in MCF7:5C cells as compared to the parental MCF7 cells. This study strongly indicates cMYC as a critical determinant responsible for estrogen-independent growth of ED-resistant breast cancer cells. Grant Support: This work (VCJ) was supported by the Department of Defense Breast Program under Award number W81XWH-06-1-0590 Center of Excellence; 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 955. doi:1538-7445.AM2012-955