Hsiao-Wang Chen

Estrogen and growth factor receptor interactions in human breast and non-small cell lung cancer cells.

Extranuclear estrogen receptors may mediate rapid effects of estradiol that communicate with nuclear receptors and contribute to proliferation of human cancers bearing these signaling proteins. To assess these growth-promoting pathways, we undertook controlled homogenization and fractionation of NIH-H23 non-small cell lung cancer cells. As many breast tumors, NIH-H23 cells express estrogen receptors (ER), with the bulk of specific estradiol binding in nuclear fractions. However, as in breast cells, a significant portion of specific, high-affinity estradiol-17beta binding-sites are also enriched in plasma membranes of lung tumor cells. These estrogen binding-sites co-purify with plasma membrane-marker enzymes and are not significantly contaminated by cytosol or nuclei. On further purification of membrane caveolae from lung tumor cells, proteins recognized by monoclonal antibodies to nuclear ER-alpha and to ER-beta were identified in close association with EGF receptor in caveolae. In parallel studies, ER-alpha and ER-beta are also detected in nuclear and extranuclear sites in archival human breast and lung tumor samples and are noted to occur in clusters at the cell membrane by using confocal microscopy to visualize fluorescent-labeled monoclonal antibodies to ER-alpha. Data on site-directed mutagenesis of cysteine-447 in ER-alpha suggest that association of ER forms with membrane sites may depend on acylation of cysteine by palmitate. Estrogen-induced growth of MCF-7 breast cancer and NIH-H23 lung cancer cells in vitro correlated closely with acute hormonal activation of mitogen-activated protein kinase signaling and was significantly reduced by treatment with Faslodex, a pure anti-estrogen. Further, combination of Faslodex with selected growth factor receptor inhibitors elicited a more pronounced inhibiton of tumor cell growth. Thus, extranuclear forms of ER play a role in promoting downstream signaling for hormone-mediated proliferation and survival of breast, as well as lung, cancers and offer a new target for anti-tumor therapy.

Estrogen receptor signaling pathways in human non-small cell lung cancer

Lung cancer is the most common cause of cancer mortality in male and female patients in the US. The etiology of non-small cell lung cancer (NSCLC) is not fully defined, but new data suggest that estrogens and growth factors promote tumor progression. In this work, we confirm that estrogen receptors (ER), both ERalpha and ERbeta, occur in significant proportions of archival NSCLC specimens from the clinic, with receptor expression in tumor cell nuclei and in extranuclear sites. Further, ERalpha in tumor nuclei was present in activated forms as assessed by detection of ER phosphorylation at serines-118 and -167, residues commonly modulated by growth factor receptor as well as steroid signaling. In experiments using small interfering RNA (siRNA) constructs, we find that suppressing expression of either ERalpha or ERbeta elicits a significant reduction in NSCLC cell proliferation in vitro. Estrogen signaling in NSCLC cells may also include steroid receptor coactivators (SRC), as SRC-3 and MNAR/PELP1 are both expressed in several lung cell lines, and both EGF and estradiol elicit serine phosphorylation of SRC-3 in vitro. EGFR and ER also cooperate in promoting early activation of p42/p44 MAP kinase in NSCLC cells. To assess new strategies to block NSCLC growth, we used Faslodex alone and with erlotinib, an EGFR kinase inhibitor. The drug tandem elicited enhanced blockade of the growth of NSCLC xenografts in vivo, and antitumor activity exceeded that of either agent given alone. The potential for use of antiestrogens alone and with growth factor receptor antagonists is now being pursued further in clinical trials.

Targeting Aromatase and Estrogen Signaling in Human Non-Small Cell Lung Cancer

Lung cancer has become increasingly common in women, and gender differences in the physiology and pathogenesis of the disease have suggested a role for estrogens. In the lung recent data have shown local production of estrogens from androgens via the action of aromatase enzyme and higher levels of estrogen in tumor tissue as compared with surrounding normal lung tissue. High levels of aromatase expression are also maintained in metastases as compared with primary tumors. Consistent with these findings, clinical studies suggest that aromatase expression may be a useful predictive biomarker for prognosis in the management of non‐small cell lung cancer (NSCLC), the most common form of lung malignancy. Low levels of aromatase associate with a higher probability of long‐term survival in older women with early stage NSCLC. Treatment of lung NSCLC xenografts in vivo with an aromatase inhibitor (exemestane) alone or combined with standard cisplatin chemotherapy elicits a significant reduction in tumor progression as compared to paired controls. Further, lung cancer progression is also governed by complex interactions between estrogen and growth factor signaling pathways to stimulate the growth of NSCLC as well as tumor‐associated angiogenesis. We find that combination therapy with the multitargeted growth factor receptor inhibitor vandetanib and the estrogen receptor antagonist fulvestrant inhibit tumor growth more effectively than either treatment administered alone. Thus, incorporation of antiestrogen treatment strategies in standard antitumor therapies for NSCLC may contribute to improved patient outcome, an approach that deserves to be tested in clinical trials.

Estrogen receptors in membrane lipid rafts and signal transduction in breast cancer.

Regulation of breast cancer growth by estrogen is mediated by estrogen receptors (ER) in nuclear and extranuclear compartments. We assessed the structure and functions of extranuclear ER that initiate downstream signaling to the nucleus. ER, including full-length 66-kDa ER and a 46-kDa ER splice variant, are enriched in lipid rafts from MCF-7 cells with (MCF-7/HER-2) or without (MCF-7/PAR) HER-2 gene overexpression and co-localize with HER-1 and HER-2 growth factor receptors, as well as with lipid raft marker flotillin-2. In contrast, ER-negative MCF-7 cells do not express nuclear or lipid raft ER. ER knockdown with siRNA also elicits a marked loss of ER in MCF-7 cell rafts. In MCF-7/PAR cells, estrogen enhances ER association with membrane rafts and induces rapid phosphorylation of nuclear receptor coactivator AIB1, actions not detected in ER-negative cells. Thus, nuclear and lipid raft ER derive from the same transcript, and extranuclear ER co-localizes with HER receptors in membrane signaling domains that modulate downstream nuclear events leading to cell growth.

Progesterone and estrogen receptor expression and activity in human non-small cell lung cancer.

Lung cancer is the most common cause of cancer mortality in male and female patients in the US. Although it is clear that tobacco smoking is a major cause of lung cancer, about half of all women with lung cancer worldwide are never-smokers. Despite a declining smoking population, the incidence of non-small cell lung cancer (NSCLC), the predominant form of lung cancer, has reached epidemic proportions particularly in women. Emerging data suggest that factors other than tobacco, namely endogenous and exogenous female sex hormones, have a role in stimulating NSCLC progression. Aromatase, a key enzyme for estrogen biosynthesis, is expressed in NSCLC. Clinical data show that women with high levels of tumor aromatase (and high intratumoral estrogen) have worse survival than those with low aromatase. The present and previous studies also reveal significant expression and activity of estrogen receptors (ERα, ERβ) in both extranuclear and nuclear sites in most NSCLC. We now report further on the expression of progesterone receptor (PR) transcripts and protein in NSCLC. PR transcripts were significantly lower in cancerous as compared to non-malignant tissue. Using immunohistochemistry, expression of PR was observed in the nucleus and/or extranuclear compartments in the majority of human tumor specimens examined. Combinations of estrogen and progestins administered in vitro cooperate in promoting tumor secretion of vascular endothelial growth factor and, consequently, support tumor-associated angiogenesis. Further, dual treatment with estradiol and progestin increased the numbers of putative tumor stem/progenitor cells. Thus, ER- and/or PR-targeted therapies may offer new approaches to manage NSCLC.

Abstract 2923: Estrogen signaling modulates cisplatin resistance in the treatment of human non-small cell lung cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Lung cancer is the most common cause of cancer mortality in male and female patients in the US. Non-small cell lung cancer (NSCLC) accounts for more than 85% of lung cancers. Standard initial chemotherapy for patients with advanced NSCLC is a platinum-based regimen, with data showing that these therapies elicit a modest improvement in patient survival. However, a major limitation to cisplatin therapy is development of drug resistance, leading to cancer progression and reduced patient survival. Cisplatin acts by interacting with DNA to form adducts that, in turn, activate signaling pathways for cell death (apoptosis). DNA damage-mediated apoptotic signals, however, can be blocked, and the resistance that ensues is a major drawback of cisplatin therapy. Resistance mechanisms, such as increased DNA adduct repair, may be modulated by interactions with other signaling pathways for cell survival, and emerging data suggest that estrogens may play a role in this process. To understand how estrogens modulate cisplatin resistance in vitro, we used two models of human NSCLC, A549 and H23 cells, that proliferate in response to estradiol-17β (E2) and express estrogen receptors and aromatase (enzyme that produces estrogens locally). Cisplatin markedly reduces tumor cell growth and enhances apoptosis in both models. However, the antitumor action of cisplatin is significantly blocked when cells are treated with estrogen in vitro. Using qRT-PCR and Western immunoblot analysis of excision repair cross-complementing-1 (ERCC1) protein (repairs cisplatin-induced DNA damage), we find that ERCC1 mRNA and protein levels are significantly increased in the presence of estrogen, thereby suggesting a potential mechanism for estrogen-induced cisplatin resistance. Analyses of the 5’-regulatory region of the human ERCC1 gene revealed putative half-estrogen responsive elements in close proximity with sp1 and AP1 sites. Chromatin immunoprecipitation assays showed binding of estrogen receptors to these promoter sites. Furthermore, treatment of human NSCLC xenografts in vivo with an aromatase inhibitor (exemestane) alone or combined with standard cisplatin chemotherapy elicits a significant reduction in tumor progression as compared to paired controls. A new strategy to block NSCLC progression may be use of cisplatin with simultaneous suppression of estrogen signaling (such as use of aromatase inhibitors). [Supported by NIH Lung Cancer SPORE, National Lung Cancer Partnership and Stiles Program funds]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2923.

MKAD-21 suppresses the oncogenic activity of the miR-21/PPP2R2A/ERK molecular network in bladder cancer

Bladder cancer represents a disease associated with significant morbidity and mortality. MiR-21 has been found to have oncogenic activity in multiple cancers, including bladder cancer, whereas inhibition of its expression suppresses tumor growth. Here, we examine the molecular network regulated by miR-21 in bladder cancer and evaluate the effects of i.v. and i.p. administration of a novel miR-21 chemical inhibitor in vivo. LNA miR-21 reduced the oncogenic potential of bladder cancer cells, whereas the MKAD-21 chemically modified antisense oligo against miR-21 dose-dependently blocked xenograft growth. I.v. administration of LNA miR-21 was more effective in suppressing tumor growth than was i.p. administration. Integration of computational and transcriptomic analyses in a panel of 28 bladder cancer lines revealed a 15-gene signature that correlates with miR-21 levels. Protein Phosphatase 2 Regulatory Subunit Balpha (PPP2R2A) was one of these 15 genes and was experimentally validated as a novel miR-21 direct target gene. Gene network and molecular analyses showed that PPP2R2A is a potent negative regulator of the ERK pathway activation and bladder cancer cell proliferation. Importantly, we show that PPP2R2A acts as a mediator of miR-21–induced oncogenic effects in bladder cancer. Integrative analysis of human bladder cancer tumors and a large panel of human bladder cancer cell lines revealed a novel 15-gene signature that correlates with miR-21 levels. Importantly, we provide evidence that PPP2R2A represents a new miR-21 direct target and regulator of the ERK pathway and bladder cancer cell growth. Furthermore, i.v. administration of the MKAD-21 inhibitor effectively suppressed tumor growth through regulation of the PPP2R2A–ERK network in mice. Mol Cancer Ther; 17(7); 1430–40. ©2018 AACR.

Abstract 1206: Preclinical evaluation of targeting Notch-3 in breast cancer

Introduction: Notch-3 overexpression has been implicated in the development of breast cancer (BC) and is associated with poor outcomes. A critical challenge to eliminating treatment resistance in breast cancer likely relates to the presence of cancer stem cells (CSCs) that maintain the ability to differentiate and divide indefinitely. We postulate that targeted eradication of CSCs is possible using a Notch3 antibody drug conjugate (ADC) without irreversibly reducing stem cell viability in vital normal tissues. PF-06650808, is an ADC comprised of a humanized anti-Notch-3 antibody linked to an auristatin-based cytotoxic agent. To better understand the therapeutic index of targeting Notch-3, we evaluated PF-06650808 across a large panel of BC lines and normal cells and correlated response with Notch-3 levels. PF-06650808 was also evaluated in a murine BC xenograft model. Methods: Response to PF-06650808 and control ADC was evaluated across a panel of BC and normal cell lines by a 2D proliferation assay. Notch-3 mRNA expression was measured by flow cytometry (FC) and RPPA. MDA-MB-468 (triple negative BC, TNBC) tumor bearing mice were randomized into 4 arms of 8 mice and treated with 3 mg/kg PF-06650808 or control-ADC (days 0, 4, 8 & 12), 10 mg/kg docetaxel (q week) or vehicle control. Results: High expression of Notch-3 was detected in multiple BC cell lines by RPPA and FC. BC cell lines with elevated levels of Notch-3 were sensitive to PF-06650808 (HCC1187, MDA-MB-468, HCC1143, HCC70, EFM-19, HCC202). Responders were also enriched for TNBC. All normal cell lines were resistant to PF-06650808 ADC. When treated with a control ADC against a non-relevant target, all cell lines exhibited IC50s between 5-50ug/ml, indicating that the sub-0.5ug/ml responses seen with the Notch-3 ADC were target-dependent. Durable complete tumor regressions were observed in PF-06650808-treated mice bearing MDA-MB-468 TNBC cell line xenografts. Conclusions: Sensitivity to a novel anti-Notch-3-ADC is associated with high expression of Notch-3 in BC cell lines. Normal cells are resistant to PF-06650808, possibly predicting a better therapeutic index than seen with other Notch inhibitors. Xenograft studies evaluating the in vivo efficacy of PF-06650808 in a panel of xenografts with varying levels of Notch-3 expression will be presented. Ongoing experiments are exploring the potency of PF-06650808 on CSCs. Our data will help identify breast cancer subtypes most likely to respond to a Notch3-ADC based on high tumor/normal target concentration as well as its effects on CSCs. Citation Format: Sara A. Hurvitz, Erika von Euw, Neil O’Brien, Dylan Conklin, Chuhong Hu, Jiaying Zhuo, Alice Zhao, Frank Calzone, Hsiao-Wang Chen, Judy Dering, Ken Geles, Puja Sapra, Dennis J. Slamon. Preclinical evaluation of targeting Notch-3 in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1206.

Abstract 3491: Development of a new approach to kill non-small cell lung cancer cells with resistance to standard chemotherapy using parthenolide analogues

Lung cancer is the leading cause of cancer death in men and women worldwide. The poor prognosis of advanced non-small cell lung cancer (NSCLC) is due, in part, to emergence of tumor resistance to chemotherapy. Recent data indicate that human tumors including NSCLC contain a small subset of cancer stem/ progenitor cells (CSC) responsible for drug resistance and tumor maintenance. If such minute subsets of CSC drive tumor formation and drug resistance, therapies targeting the bulk tumor mass but not CSC will fail. We now confirm identification of subpopulations of chemotherapy-resistant human NSCLC cells with enrichment for CSC biomarkers and exhibiting significant CSC activity. We identified CD133+/ALDH+ tumor stem/progenitor cells from human lung cancer cells in vitro using established Aldefluor assays in combination with labeled anti-CD133 antibodies. Estrogen, a known risk factor for lung cancer progression, stimulated a modest increase in the numbers of CSC. In contrast to control CD133-/ALDH- tumor cell subsets, CSC subpopulations grew as tumor spheres and maintained self-renewal capacity in vitro and exhibited a greater tumorigenic capability than non-CSC subsets in vivo, properties indicative of CSC. Furthermore, resistance of CSC-like cells to cisplatin (a standard chemotherapy for NSCLC treatment) was fully reversed by treatment with parthenolide (PTL), a naturally-occurring sesquiterpene lactone compound with strong antitumor activity in leukemia and prostate cancer while sparing normal cells. The antitumor effect of PTL appears due to its action as a potent inhibitor of nuclear factor-βB (NF-κB) which is markedly activated by chemotherapy. To target CSC and suppress tumor progression, we synthesized and tested novel analogs of PTL with improved antitumor properties and aqueous solubility. PTL analogs inhibit proliferation of H157 NSCLC cells using both bulk cell preparations and CSC-subpopulations, with effects significantly different from control at P 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 3491. doi:1538-7445.AM2012-3491