Mithramycin Represses Basal and Cigarette Smoke-Induced Expression of ABCG2 and Inhibits Stem Cell Signaling in Lung and Esophageal Cancer Cells

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Mary Zhang1, Aarti Mathur1, Yuwei Zhang1, Sichuan Xi1, Scott Atay1, Julie A. Hong1, Nicole Datrice1, Trevor Upham1, Clinton D Kemp1, R. Taylor Ripley1, Gordon Wiegand2, Itzak Avital2, Patricia Fetsch3, Haresh Mani4, Daniel Zlott5, Robert Robey6, Susan E. Bates6, Xinmin Li7, Mahadev Rao1, David S. Schrump1

1Thoracic Oncology Section; 2 Gastrointestinal and Hepatobiliary Malignancies Section; Surgery Branch; 3Laboratory of Pathology; 5Clinical Pharmacy Department; 6Experimental Therapeutics Section, Medical Oncology Branch; Center for Cancer Research, National Cancer Institute, Bethesda, Maryland.

4Department of Pathology, Penn State Hershey Medical Center, Hershey, PA

7Clinical Micro-array Core, University of California, Los Angeles, CA

Running Title: Mithramycin and ABCG2

Key words: Lung cancer, esophageal cancer, cigarette smoke, mithramycin, ABCG2, stem cell signaling, aryl hydrocarbon receptor, Sp1, Nrf2, micro-array

Author Conflicts of Interest: None

Correspondence:

David S. Schrump, MD Head, Thoracic Oncology Section Surgery Branch, National Cancer Institute Building 10; 4-3942 10 Center Drive Bethesda, MD 20892 Tel: 301-496-2128 Fax: 301-451-6934 Email: [email protected]

Abstract Cigarette smoking at diagnosis or during therapy correlates with poor outcome in patients with lung and esophageal cancers, yet the underlying mechanisms remain unknown. In this study, we found that exposure of esophageal cancer cells to cigarette smoke condensate led to up- regulation of the xenobiotic pump ABCG2, which is expressed in cancer stem cells and confers treatment resistance in lung and esophageal carcinomas, and increased the side population of lung cancer cells containing cancer stem cells. Upregulation of ABCG2 coincided with increased occupancy of aryl hydrocarbon receptor (AhR), Sp1, and Nrf2 within its promoter, and deletion of xenobiotic response elements and/or Sp1 sites markedly attenuated ABCG2 induction. Under conditions potentially achievable in clinical settings, treatment with mithramycin diminished basal as well as cigarette smoke condensate-mediated increases in AhR, Sp1, and Nrf2 levels within the ABCG2 promoter, markedly down-regulated ABCG2, and inhibited proliferation and tumorigenicity of lung and esophageal cancer cells. Micro-array analyses revealed that mithramycin targeted multiple stem cell-related pathways in vitro and in vivo. Collectively, our findings provide a potential mechanistic link between smoking status and outcome of patients with lung and esophageal cancers and support clinical use of mithramycin for repressing ABCG2 and inhibiting stem cell signaling in thoracic malignancies.

Introduction

Lung and esophageal cancers are leading causes of cancer-related deaths worldwide (1).

In 2011, these malignancies accounted for an estimated 1.8 million deaths globally; in the United

States, nearly 160,000 deaths were attributed to lung cancer, whereas 15,000 deaths were due to

esophageal carcinoma (2). Presently, 80% of lung cancers, and 50% of esophageal carcinomas

are directly attributable to cigarette smoke (3, 4). Currently, more than 1.3 billion people smoke;

hence, the global burden of tobacco-associated thoracic malignancies will continue to increase,

with particularly devastating consequences in developing countries (5).

In addition to being a significant risk factor for major morbidity and mortality in

individuals undergoing potentially curative resections (6, 7), cigarette smoking diminishes

responses to chemo- and radiation therapy, enhances systemic metastases, and decreases survival of patients with locally advanced or disseminated lung and esophageal cancers (8-11); the mechanisms underlying these phenomena have not been fully established. Previously, we reported that under clinically relevant exposure conditions, cigarette smoke enhances tumorigenicity of lung cancer cells via polycomb-mediated repression of Dickkopf-1 (Dkk1), which encodes an antagonist of Wnt signaling (12). In unpublished studies, we observed a similar phenomenon in esophageal adenocarcinoma cells following cigarette smoke exposure.

Additionally, we have observed that cigarette smoke activates miR-31, targeting Dkk1 as well as several other Wnt antagonists in lung cancer cells; constitutive expression of this microRNA significantly enhances proliferation of lung cancer cells in-vitro and in-vivo (13). In more recent studies, we observed that cigarette smoke mediates epigenetic repression of miR-487b in lung cancer cells, resulting in over-expression of polycomb group proteins BMI1 and SUZ12, as well as Wnt5A, k-ras and C-myc, all of which have been implicated in modulating stem cell

pluripotency (14-18); consistent with these observations, knock-down of miR-487b increases

proliferation and tumorigenicity of lung cancer cells (Xi et al, submitted). The present study was

undertaken to examine if cigarette smoke activates additional stem cell-associated genes, which enhance the malignant phenotype of lung and esophageal cancers in an effort to develop novel pharmacologic strategies for treatment of these neoplasms.

Materials and Methods

Cell Lines and Treatment Conditions: Unless otherwise specified, all cancer lines were obtained from American Type Culture Collection (ATCC Manassas, VA). Cells were validated by periodic HLA typing of lab cultures relative to new cell aliquots from the repository. NCI-SB-

EsC1 and NCI-SB-EsC2 (EsC1 and EsC2, respectively) were established in our lab from two

patients with esophageal adenocarcinoma who developed disease recurrence after undergoing

induction chemo/XRT and surgery on IRB approval protocols; these cell lines exhibit HLA as

well as cytokeratin expression profiles identical to the respective primary tumors. All cancer

lines were maintained in Roswell Park Memorial Institute (RPMI) media supplemented with

10% Fetal Bovine Serum (FBS), and 1% penicillin/streptomycin (normal media; NM). Primary normal human small airway epithelial cells (SAEC) were obtained from Lonza, Inc. (Fredrick,

MD), and cultured per vendor instructions. Cigarette smoke condensates (CSC) were generated as described (19). For smoke exposure experiments, cells were cultured in appropriate normal

media with or without varying concentrations of CSC. Media and CSC were changed daily.

Cells were subcultured as necessary, and harvested at appropriate times for further analysis.

Mithramycin was obtained from either Sigma (St. Louis, MO) or the Developmental

Therapeutics Program (NCI). For drug exposure treatments, cells were cultured in NM with or

without CSC. Media was changed and mithramycin was added at various concentrations for 24

hours; cells were harvested at indicated time points for further analysis.

RNA Isolation, Real-time Quantitative Reverse Transcription-PCR, and Microarray Analysis:

Total RNA was isolated and real-time quantitative reverse transcription-PCR (qRT-PCR) was

performed as described (19) using primers and probes listed in Supplementary Table 1. Full

details are submitted as Supplementary Methods.

Immunoblotting and Immunofluorescence: Submitted as Supplementary Methods.

Generation of Stable Cells Expressing shRNA Constructs: A549 and EsC2 cells were

transfected with validated shRNA constructs targeting ABCG2 or sham sequences (vector

control) according to manufacturer’s protocols (Sigma). Stable cells were selected and expanded

in Puromycin (Sigma) after target gene expression was confirmed by qRT-PCR and

immunofluorescence techniques.

Proliferation, Cell Migration, and Soft Agar Clonogenicity Assays: Submitted as Supplementary

Methods.

Murine Xenograft Experiments: Athymic nude mice were injected in bilateral flanks with 1x106 parental A549 cells. Approximately ten days later when palpable tumors were present, mice were randomly assigned to receive mithramycin at 1 or 2 mg/kg body weight, or saline administered as intraperitoneal (IP) injections on Monday, Wednesday and Friday for three

5 weeks. Tumor dimensions and mouse weights were measured twice weekly. When control tumors approached maximum allowable size, all mice were euthanized, and tumors were excised, weighed, and processed for additional studies. All mouse experiments were approved by the National Cancer Institute Animal Care and Use Committee, and were in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Chromatin Immunoprecipitation (ChIP): Quantitative ChIP was performed as described (20), with minor modifications. Full methods are described in Supplementary Methods. Antibodies and primers for ChIP are listed in Supplementary Table 1.

Luciferase Promoter-Reporter Transient Transfection Experiments: Submitted as

Supplementary Methods.

Flow Cytometry: Flow cytometry for ABCG2 surface expression and side population (SP) was performed as described (21) with minor modifications. Full methods submitted as Supplementary

Methods.

Statistical Analysis: Standard error of the mean is indicated by bars on figures, and was calculated using Microsoft Office Excel 2007. All experiments were performed with at a minimum of triplicate samples, and all p-values were calculated with two-tailed t-tests.

Results

CSC Induces ABCG2 Expression in Cultured Cancer Cells: Affymetrix microarrays were used to identify gene expression profiles in cultured lung and esophageal cancer cells mediated by

CSC under clinically-relevant exposure conditions. ABCG2 [also known as breast cancer resistance protein (BCRP)], which encodes a xenobiotic pump protein highly expressed in cancer stem cells (22), was one of the most highly up-regulated genes in Calu-6, A549, EsC1 and

EsC2 cells exposed to CSC (data not shown). Subsequent qRT-PCR experiments (Figure 1A, upper panel) demonstrated that A549 and EsC2 had relatively high basal expression of ABCG2,

which was increased 2-4 fold and ~8 fold, respectively, by CSC treatment. In contrast, Calu-6

cells exhibited relatively low level basal expression of ABCG2, which was augmented approximately 25-30 fold by CSC. Normal SAEC exhibited very low basal levels, and minimal

(3-4 fold) induction of ABCG2. CSC-mediated induction of ABCG2 was observed across numerous additional non-small cell lung cancer lines, but was not observed in a panel of small cell lung cancer lines, all of which had very low levels of endogenous ABCG2 expression

(Supplementary Figure 1). The lack of validated lines precluded further induction experiments using esophageal cancer cells.

Immunoblot experiments were performed to examine ABCG2 levels in cultured cells before and after CSC exposure. Representative results are depicted in Figure 1A, lower left panel. CSC exposure increased ABCG2 levels in lung and esophageal cancer cells; this phenomenon was less dramatic in SAEC. Basal levels of ABCG2 expression in Calu-6 cells and

SAEC were considerably lower than those observed in A549 and EsC2 cells; detection of

ABCG2 in Calu-6 and SAEC required loading 3-fold more protein lysates than needed to evaluate ABCG2 in A549 and EsC2 cells (~200µg vs 60µg, respectively). Subsequent flow

cytometry experiments demonstrated that CSC exposure increased surface expression of ABCG2

in A549, Calu-6 and EsC2 cells (Figure 1A, lower right panel). This phenomenon was not

evident in SAEC, possibly due to very low basal levels of ABCG2 expression and relatively

modest up-regulation of ABCG2 following CSC exposure.

Additional qRT-PCR experiments were performed to assess timing and duration of

ABCG2 induction in cancer cells mediated by CSC. Results pertaining to Calu-6 and EsC2 cells are depicted in Figure 1B. In both cell lines, ABCG2 expression increased significantly 8-24 h following initiation of CSC exposure. In Calu-6 cells, ABCG2 expression continued to increase

throughout the 10 day smoke exposure; in EsC2 cells, ABCG2 expression peaked at 48h,

plateauing off thereafter. In both cell lines, ABCG2 expression decreased dramatically, and

approached pre-treatment levels within 1-3 days following cessation of CSC exposure.

ABCG2 Modulates Proliferation, Migration, and Clonogenicity of Cancer Cells: Additional

experiments were performed to ascertain if ABCG2 expression modulated the malignant phenotype of cultured lung and esophageal cancer cells. Briefly, lentiviral shRNA techniques were used to knock-down ABCG2 in A549 and EsC2 cells exhibiting relatively high-level

ABCG2 expression. qRT-PCR, and immunoprecipitation experiments confirmed significant reduction of ABCG2 expression in ABCG2 knockdown cells relative to respective controls

(Figure 1C). Subsequent experiments demonstrated that knock-down of ABCG2 significantly decreased proliferation and migration of A549 lung cancer cells, and to lesser extent EsC2 cells

(Figure 1D). Additionally, knock-down of ABCG2 significantly inhibited soft agar clonogenicity of A549 cells (Figure 1D). This phenomenon could not be assessed in EsC2 cells, which exhibit poor clonogenicity in soft agar.

CSC Increases Side Population of Cultured Cancer Cells: Recent studies indicate that ABCG2 is highly expressed in cancer stem cells, which are enriched within the side population (SP) identified by flow cytometry techniques (21, 23). As such, additional experiments were performed to examine if CSC increased SP of cancer cells. Representative results pertaining to

A549 and Calu-6 cells (high and low ABCG2 expressers, respectively), as well as SAEC are depicted in Figure 2A. Hoechst staining with and without verapamil revealed a SP fraction of

0.23 % in untreated A549 cells, compared to .95% (a 4-fold increase) in A549 cells exposed to

CSC for 5 days. The SP fraction in untreated Calu-6 cells (0.06%) was much lower than A549 cells, and increased to 0.98% (a 16 fold increase) following CSC exposure. In contrast, SP fraction of SAEC did not appear to be increased following CSC exposure.

Additional experiments were performed to examine if the increase in SP coincided with enhanced ABCG2 expression in cancer cells exposed to CSC. Briefly, qRT-PCR techniques were used to quantitate ABCG2 expression in sorted SP as well as non-SP fractions from A549 and Calu-6 cells cultured in NM with or without CSC. Results of this analysis are depicted in

Figure 2B, and summarized in Supplementary Table 2. In A549 cells, untreated SP fractions had

~ 2.8 fold higher ABCG2 expression compared to control non-SP. Following CSC treatment,

ABCG2 expression in A549 SP fractions increased approximately 1.6 fold (range 1.4-2.8)

relative to control SP fractions; in contrast, CSC treatment did not appear to increase ABCG2 expression in non-SP fractions from these cells. The situation was somewhat different for Calu-

6 cells. As anticipated, ABCG2 levels in SP and non-SP fractions in these cells were considerably lower than those observed for A549. Furthermore, ABCG2 expression levels in SP fractions from untreated Calu-6 cells were quite similar to levels in non-SP fractions possibly due to the very small SP fraction. Following CSC exposure, ABCG2 expression in SP fractions

increased ~26 fold, whereas ABCG2 expression in non-SP fractions increased ~3.3 fold

compared to respective untreated controls. Immunoblot analysis demonstrated that increased

ABCG2 expression coincided with increased ABCG2 levels in SP fractions of A549 cells following CSC exposure.

Role of Aryl Hydrocarbon Receptor and Sp1 in ABCG2 Up-regulation by CSC: Purified carcinogens as well as HDAC inhibitors such as romidepsin induce ABCG2 expression in cancer cells by aryl hydrocarbon receptor (AhR) signaling (24, 25). As such, additional experiments were performed to examine the mechanisms by which CSC induces ABCG2 expression in lung and esophageal cancer cells. In preliminary experiments, qRT-PCR techniques were used to evaluate ABCG2 expression in lung cancer cells exposed to CSC or various AhR agonists for 24 hours. Calu-6 as well as EsC1 cells, were chosen for these experiments, since both lines exhibit very low levels of endogenous ABCG2 expression, and comparable magnitudes of ABCG2 induction following CSC exposure (data for EsC1 available on request). The magnitude of

ABCG2 induction mediated by benzopyrene, 3-MC or TCDD relative to CSC varied somewhat between the cell lines (Figure 2C; left panel). The AhR antagonist, resveratrol, previously shown to inhibit romidepsin-mediated activation of ABCG2 (25), only partially abrogated CSC- mediated induction of ABCG2 in these cells (Figure 2C; right panel).

In additional experiments, shRNA techniques were used to knock-down HDAC6, which is required for activation and nuclear transport of AhR in response to tobacco carcinogens (26). qRT-PCR and immunofluorescence experiments confirmed decreased HDAC6 expression in knock-down cells relative to respective controls transduced with sham sequences (Figure 2D; upper panel). Knock-down of HDAC 6 only modestly diminished CSC-mediated induction of

ABCG2 in A549, EsC1, and EsC2 cells (Figure 2D; lower panel); the effects of HDAC6 inhibition could not be assessed in Calu-6 cells due to toxicity of the shRNA construct in these cells. Collectively, these findings suggested that induction of ABCG2 by CSC was not mediated

solely by AhR signaling.

In addition to xenobiotic response elements (XRE) that are binding sites for AhR, the

ABCG2 promoter contains a number of Specificity protein 1 (Sp1) sites (24) that could mediate

activation of this gene in response to cigarette smoke. As such, transient transfection

experiments using ABCG2 promoter-reporter constructs were performed to examine potential roles of AhR and Sp1 in mediating ABCG2 activation by CSC. Representative results of these experiments are depicted in Figure 3A. Following CSC exposure, luciferase activity of ABCG2 -

1662 containing 4 XRE sites was 4 fold higher than pGL3 Basic Luc control in Calu-6 cells.

CSC-mediated luciferase activity of ABCG2-1662 del 198, in which one of the XRE sites has been deleted was ~50% lower than that observed for ABCG2-1662. CSC-mediated luciferase activity was also diminished following transfection with ABCG2-245 containing two XRE sites and five Sp1 elements present in ABCG2-1662. Mutation of the five Sp1 sites significantly diminished luciferase activity of ABCG2-245 to a level less than that seen for ABCG2 del 198 luc. CSC-mediated luciferase activities of ABCG2-245 following deletion of one of the remaining XRE (ABCG2-XRE del 245 luc) or combined deletion of this XRE with mutation of the 5 Sp1 sites (ABCG2-Sp1 mut-XRE del 198 luc) were further decreased, approximating those seen for PGL3 basic luc. These data suggested that in addition to AhR, Sp1 contributes significantly to CSC-mediated activation of ABCG2 in cancer cells.

Effects of Mithramycin on CSC-Mediated induction of ABCG2: Additional studies were

performed to examine if mithramycin, a pharmacologic agent that inhibits Sp1-mediated

activation of a variety of genes including DHFR and AhR (27, 28), could repress ABCG2 expression in cancer cells. Briefly, cancer cells were cultured in NM with or without CSC in the presence or absence of escalating doses of mithramycin. qRT-PCR analysis revealed that 24h treatment with mithramycin decreased basal levels of ABCG2, and markedly attenuated CSC- mediated induction of ABCG2 in lung and esophageal cancer cells (Figure 3B). Down- regulation of these genes persisted for at least 16 hours following removal of mithramycin from culture media (data not shown). Additional analysis revealed that mithramycin inhibited basal levels as well as CSC-mediated up-regulation of Sp1 and AhR in these cells. Furthermore, mithramycin decreased basal as well as CSC-mediated expression of Nuclear Factor Erythroid

Related Factor 2 (Nrf2), which has been shown recently to modulate ABCG2 expression (29).

Immunoblot experiments demonstrated that mithramycin mediated dose-dependent decreases in

ABCG2, Sp1, AhR, and Nrf2 expression, and partially abrogated CSC-mediated increases in levels of these transcription factors in A549 and Calu-6 cells (Figure 3C).

Chromatin immunoprecipitation (ChIP) experiments were performed to examine if mithramycin modulates basal as well as CSC-mediated levels of Sp1, AhR, and Nrf2 within the

ABCG2 promoter. ChIP primers encompassed a region containing AhR and Sp1 recognition elements adjacent to the transcription start site; additional ChIP primers amplified a region containing the Nrf2 antioxidant response element (ARE) within the ABCG2 promoter (29).

Results pertaining to Calu-6 cells (simultaneously processed for qRT-PCR experiments in Figure

3B) are depicted in Figure 3D. CSC-mediated activation of ABCG2 coincided with increased levels of RNA polymerase II (pol II) and H3K9Ac, with decreased levels of H3K9Me3

(activation and repression marks, respectively) within the ABCG2 promoter. CSC induced recruitment of Sp1, AhR, and Nrf2 to the ABCG2 promoter; these results were most dramatic for

Sp1, and were consistent with aforementioned luciferase experiments. Mithramycin diminished

CSC-mediated occupancy of these transcription factors within the ABCG2 promoter; these effects coincided with appropriate alterations in pol II, H3K9Ac and H3K9Me3.

Effects of Mithramycin on Proliferation and Tumorigenicity of Cancer Cells: Additional experiments were undertaken to examine the effects of mithramycin on proliferation and tumorigenicity of lung and esophageal cancer cells. Results of this analysis are shown in Figure

4. MTS experiments demonstrated that 24h mithramycin exposure mediated profound growth inhibitory effects in cultured lung and esophageal cancer cells (Figure 4A). Apo-BrdU experiments revealed no appreciable increase in apoptosis in mithramycin-treated cells,

suggesting that the growth inhibitory effects of this agent were mediated by cell cycle arrest

rather than apoptosis (data not shown). Additional flow cytometry experiments using A549 cells

suggested that mithramycin decreased SP fraction (Figure 4B).

Further experiments were performed to examine if mithramycin diminished growth of

established tumor xenografts. As shown in Figure 4C, mithramycin mediated significant dose-

dependent growth inhibition of A549 xenografts without appreciable systemic toxicities such as

decreased activity, skin changes, or significant weight loss. Histopathologic analysis revealed

that tumors from mithramycin treated mice were less glandular in appearance with somewhat

less stroma. Furthermore, tumors from mice treated with 2mg/kg mithramycin had 50% fewer

mitoses relative to control tumors (data not shown). Immunofluorescence experiments confirmed that mithramycin decreased ABCG2 expression in tumor xenografts (Figure 4D).

Because Sp1 has diverse potential targets (30), Affymetrix micro-array experiments were

performed to examine global gene expression profiles in Calu-6 and A549 cultured in NM with or without mithramycin for 24 hours. Under conditions potentially achievable in clinical settings

(50-200 nM x 24h), mithramycin mediated dramatic dose-dependent alterations in gene expression in cultured lung cancer cells. Highly reproducible results were noted among cell lines and within treatment groups (Figure 5A, upper left panel). Using stringent criteria of fold change >3 and adjusted p<0.01 for drug treatment vs. control, 1582 and 3771 genes were simultaneously modulated in A549 and Calu-6 cells following 50 nM and 200 nM mithramycin exposures, respectively (Figure 5A; lower left panel). 1258 genes were commonly regulated by mithramycin across two cell lines and two drug concentrations; the majority of differentially regulated genes were down-regulated in both cell lines (Figure 5A right panel; Supplementary

Figure 2). Sixteen of the top cancer-associated pathways, which were down-regulated in cultured lung cancer cells by mithramycin are listed in Table 1A; eight of these canonical pathways are related to stem cell signaling.

Additional micro-array experiments were performed to examine effects of mithramycin in A549 xenografts (9 each from drug-treated or control mice). Similar to what was observed following in-vitro drug treatment, mithramycin mediated highly reproducible, dose-dependent alterations in gene expression in A549 tumor xenografts (Figure 5B; left panel, and upper right panel). Using criteria of fold change >2 and p< 0.05 for drug treatment vs. control, 351 and

1896 genes were differentially expressed in xenografts from mice receiving mithramycin at 1 mg/kg and 2 mg/kg, respectively, relative to control tumors (Figure 5B; lower right panel). 299 genes were modulated by mithramycin under both doses, 100 of which are listed in Table 2.

ABCG2 was down-regulated > 2 fold in xenografts from mice receiving 2mg/kg but not 1mg/kg

mithramycin. All eight of the stem-cell related pathways modulated in-vitro by mithramycin,

were also targeted in tumor xenografts by systemic drug treatment, albeit to a somewhat lesser

degree (Table 1B). A similar phenomenon was observed regarding the remaining 8 canonical

pathways listed in Table 1A. A variety of networks regulating intracelleular signaling, DNA

damage response, chromatin remodeling, and chromosomal replication were inhibited in A549

tumor xenografts following mithramycin treatment (Supplementary Figure 3).

Further analysis was undertaken to correlate in-vivo effects of mithramycin with in-vitro

drug exposures. A progressive dose-dependent increase in genes commonly regulated in-vitro

and in-vivo by mithramycin was observed (Figure 5C). Two to ten percent (average 5%) of

genes modulated in-vitro overlapped with 13-24% (average 18%) of genes altered by in-vivo

mithramycin across various treatment comparisons. 337 genes were simultaneously modulated

in cultured A549 and Calu-6 cells following 200 nM mithramycin and A549 xenografts from

mice receiving 2 mg/kg mithramycin IP. The top 100 repressed, and all 43 up-regulated genes from these 337 commonly regulated genes are listed in Supplementary Table 3. Top molecular and cellular functions mediated by these 337 genes included cell cycle progression, gene expression, stem cell pluripotency, cellular morphology, and death. Representative networks mediating chromatin remodeling and TGF-β/BMP-3 signaling are depicted in Supplementary

Figure 4.

Discussion

The vast majority of lung and esophageal carcinomas are directly attributable to tobacco

abuse (3, 4). Cigarette smoking not only facilitates initiation and preclinical progression of lung

and esophageal cancers, but also enhances treatment resistance and dissemination of established

malignancies, thereby decreasing overall survival of patients with these neoplasms (8, 10, 11,

31). Delineation of the mechanisms by which cigarette smoke promotes proliferation of lung

and esophageal carcinomas may facilitate development of more efficacious treatment regimens

for these malignancies.

Our previous studies have shown that cigarette smoke increases the malignant phenotype

of lung and esophageal cancer cells, in part, by up-regulating genes, which mediate stem cell

phenotype (12, 13). Consistent with these observations, our current experiments revealed that

cigarette smoke mediates time- and dose-dependent up-regulation of ABCG2 in lung and

esophageal cancer cells; this phenomenon appeared to be considerably less pronounced in

cultured normal aerodigestive tract epithelia. As expected, CSC-mediated up-regulation of

ABCG2 in cancer cells coincided with increased SP fraction; these findings suggest, although

certainly do not prove that cigarette smoke promotes expansion of pluripotent tumor cells (21).

Further analysis revealed that CSC-mediated activation of ABCG2 coincided with recruitment of

AhR and Nrf2, as well as Sp1 to the ABCG2 promoter. Mithramycin repressed basal as well as

CSC-mediated induction of ABCG2; these effects may be attributable to direct inhibition of binding of Sp1 to DNA (27), and decreased expression of Sp1, as well as AhR and Nrf2 (two potential targets of Sp1) (28), which are known to activate ABCG2. Additionally, mithramycin dramatically decreased proliferation and tumorigenicity of cancer cells. Growth inhibition coincided with down-regulation of ABCG2, as well as numerous other genes mediating

“stemness”, proliferation, and metastatic potential of cancer cells. To the best of our knowledge, these experiments represent the most comprehensive analysis of mechanisms regulating ABCG2 expression in thoracic malignancies following exposure to cigarette smoke, and are the first to

demonstrate the potential of mithramycin for targeting ABCG2 as well as other stem cell-related genes in thoracic malignancies.

ABCG2 (BCRP) is a member of the ATP binding cassette (ABC) transporters, which functions as a xenobiotic pump in many normal tissues (32). Its substrates include numerous environmental toxins as well as chemotherapeutic agents. Our experiments demonstrated that knock-down of ABCG2 inhibits proliferation, migration, and clonogenicity of lung and esophageal cancer cells; these findings suggest that ABCG2 modulates intracellular processes other than extrusion of xenobiotics. Several recent studies suggest that ABCG2 is a critical mediator of stem cell homeostasis. For example, ABCG2 is an essential upstream mediator of sonic-hedgehog signaling, which has been implicated in maintenance of stemness (33).

Furthermore, ABCG2 binds to heme, thereby diminishing intracellular porphyrin levels, rendering stem cells resistant to hypoxia (34). Constitutive expression of ABCG2 protects cardiac stem cells from oxidative stress (35), and enhances expansion, while impairing maturation of hematopoietic progenitor cells (36). Of particular relevance regarding our current study are recent reports demonstrating that increased expression of ABCG2 correlates with

chemo-resistance and stem-like phenotype of lung and esophageal carcinomas (21, 23, 37-39),

and decreased survival of patients with these neoplasms (40-42).

Despite the fact that knock-down of ABCG2 decreased proliferation, migration and

clonogenicity of lung and esophageal cancer cells, our current data do not establish, nor imply

that down-regulation of ABCG2 is the primary mechanism by which mithramycin inhibits

proliferation and tumorigenicity of these cancer cells. Indeed, our micro-array analysis revealed that mithramycin significantly down-regulated hundreds of genes mediating stem cell signaling, cell cycle progression, chromatin remodeling, and DNA damage response. Although

experiments are underway to examine this issue, it seems unlikely that constitutive over-

expression of ABCG2 would significantly diminish mithramycin-mediated cytotoxicity in cultured cancer cells, unless this agent is a substrate for ABCG2. In all likelihood, the antitumor effects of mithramycin are mediated by direct inhibition of Sp1 binding to promoters of master

genes regulating diverse cellular functions, with subsequent repression of down-stream targets by

direct as well as indirect mechanisms (30, 43).

Mithramycin, a polyauroleic acid isolated from streptomyces, was initially evaluated as a

chemotherapeutic agent in cancer patients during the 1960’s and 70’s (44), but was discontinued

due to excessive systemic toxicities (45). Recently there has been renewed interest in clinical

development of mithramycin and its derivatives because of their ability to specifically inhibit

binding of Sp1 to GC-rich DNA (27), and down-regulate numerous genes mediating

proliferation, invasion and metastasis of cancer cells (46-48). Of particular interest in this regard

are recent studies indicating that currently approved agents such as cyclo-oxygenase inhibitors

markedly enhance mithramycin mediated-inhibition of Sp1 expression/ activity in cancer cells

(49). Such combinational strategies could enable reduction of mithramycin doses, and possibly

decrease systemic toxicities in clinical settings.

Our current findings have direct translational implications regarding evaluation of

mithramycin in patients with thoracic malignancies. Extrapolation of data from previous animal

studies (50) suggests that mithramycin levels achieved in our xenograft experiments were in the

50-200 nM range over 24h; these exposure conditions, which closely approximated those used

for our in-vitro experiments, are potentially achievable using previous mithramycin dosing

schedules in humans (44). Collectively our findings support the clinical evaluation of

18 mithramycin as a strategy to repress ABCG2 and inhibit signaling pathways mediating

“stemness” in thoracic malignancies.

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Figure Legends

Figure 1: Effects of CSC on ABCG2 expression in cultured cells.

A. Upper panel: qRT-PCR analysis demonstrating dose-dependent induction of ABCG2 expression by CSC in A549, Calu-6 and EsC2 cancer cells, as well as normal SAEC.

Lower panel: Left; immunoblot demonstrating increased ABCG2 expression in A549, Calu-6,

EsC2 and SAEC cells following CSC exposure. Right; flow cytometry analysis demonstrating increased cell surface ABCG2 expression in A549, Calu-6, and EsC2 cancer cells, not SAEC following CSC exposure (red: negative control antibody; blue: ABCG2 antibody).

B. qRT-PCR analysis of ABCG2 expression in lung and esophageal cancer cells following exposure to, and removal of CSC.

C. Left panel: qRT-PCR analysis of ABCG2 expression in cells transduced with shRNA targeting

ABCG2 or control shRNA. Right panel: Representative immunoprecipitation experiment depicting decreased ABCG2 expression in A549 cells transduced with shRNA targeting ABCG2.

D. Left panel: MTS assay showing that ABCG2 knock-down inhibits cancer cell proliferation.

Middle panel: scratch assay demonstrating that ABCG2 knock-down diminishes cancer cell migration. Right panel: ABCG2 knock-down reduces clonogenicity of A549 cells.

Figure 2: CSC modulates ABCG2 expression and SP fraction in lung and esophageal cancer cells.

A. Representative flow cytometry analysis demonstrating that CSC increases SP fraction in

A549 and Calu-6 lung cancer cells, but not SAEC.

B. Left panel: qRT-PCR analysis of ABCG2 expression in SP and non-SP fractions in A549 and

Calu-6 cells cultured in NM+/- CSC. Right panel: representative immunoblot demonstrating that

CSC exposure increases ABCG2 protein levels in SP.

C. Left panel: qRT-PCR analysis of ABCG2 expression in Calu-6 and EsC1 cells following

exposure to CSC or purified carcinogens, which activate AhR signaling. Right panel: qRT-PCR

analysis demonstrating relatively modest inhibition of CSC-mediated induction of ABCG2 by

resveratrol.

D. Upper panel: qRT-PCR analysis depicting shRNA-mediated knock-down of HDAC6 in lung and esophageal cancer cells. Lower panel: qRT-PCR analysis demonstrating effects of HDAC6

knock-down on CSC-mediated induction of ABCG2 in lung and esophageal cancer cells.

Figure 3: Role of AhR, Sp1 and Nrf2 in ABCG2 activation by CSC.

A. Luciferase activity of ABCG2 promoter reporter constructs following transient transfection

into Calu-6 cells. Relative to full length promoter (ABCG2-1662-LUC), luciferase activities of

ABCG2 promoter constructs decreased following serial deletions or mutations of XRE and Sp1

elements.

B. qRT-PCR analysis of ABCG2, AhR, Sp1, and Nrf2 expression in A549 and Calu-6 cells

cultured in NM with or without mithramycin in the presence or absence of CSC.

C. Immunoblot analysis of ABCG2, SP1, AHR Nrf2 levels in A549 and Calu-6 cells cultured in

the presence or absence of mithramycin with or without CSC.

D. Quantitative ChIP analysis of the ABCG2 promoter region in Calu-6 cells cultured in NM with or without CSC in the presence or absence of mithramycin. The dose of mithramycin for

ChIP was optimized in preliminary experiments.

Figure 4: Effects of mithramycin in lung and esophageal cancer cells.

A. MTS assays depicting effects of 24h mithramycin exposure on proliferation of lung and esophageal cancer cells.

B. Flow cytometry analysis demonstrating that mithramycin decreases SP in A549 cells.

C. Effects of IP mithramycin (1 mg/kg or 2 mg/kg Monday-Wednesday-Friday x3) on growth of established subcutaneous A549 xenografts. Left panel: tumor volumes; middle panel: tumor masses; right panel: effects of mithramycin on body mass.

D. Representative tissue sections from A549 xenografts from control and mithramycin-treated mice. Upper panel: H&E stains. Lower panel: representative immunofluorescence results depicting ABCG2 expression in control tumors, and xenografts from mice treated with mithramycin.

Figure 5: Microarray analysis of mithramycin effects on gene expression cultured A549 and

Calu-6 cells, and A549 xenografts related to respective controls.

A. Left panel (top): Principal component analysis (PCA) demonstrating highly reproducible results of triplicate samples for each cell line and each treatment condition.

Left panel (bottom): Venn diagram demonstrating overlap of genes simultaneously modulated in

A549 and Calu-6 cells under two in-vitro exposure conditions.

Right panel: Heat map depicting 1258 differentially expressed genes modulated by mithramycin.

A marked dose-dependent alteration of gene expression profiles was observed in these cells

(triplicate samples).

B. Left panel: Heat map revealing dose-dependent modulation of gene expression by mithramycin in tumor xenografts.

Right panel: top, PCA demonstrating highly reproducible results of triplicate samples (derived from 9 tumors for such conditions); bottom, Venn diagram depicting overlap of genes modulated in-vivo under both mithramycin doses.

C. Venn diagrams showing overlap of differentially expressed genes in cultured A549 and Calu-

6 cells exposed to either 50 or 200 nM mithramycin for 24h, or A549 tumor xenografts from mice treated with 1 mg/kg or 2 mg/kg mithramycin.

Table 1: Genes Differentially Expressed in vitro and in vivo by Mithramycin

A. Ingenuity Canonical Pathways - in vitro Differentially expressed genes MAPK1, SMAD3, ARHGEF7, LRP6, BMPR2, MAP3K5, RBL1, EP300, PTK2, BMPR1A, SOS1, TGFB2, PRKCE, AKT3, GSK3B, HIPK2, PRKD1, PRKCA, Molecular Mechanisms of Cancer PMAIP1, GNA12, CREBBP, SMAD6, GNAQ, CDK6, TCF3, FZD8, BMPR1B, PLCB4, NF1, IRS1, PIK3CB, ARHGEF18, CFLAR MAPK1, SMAD3, SKI, CREBBP, SMAD6, BMPR2, SMURF1, EP300, BMPR1B, TGF-β Signaling * BMPR1A, SOS1, TGFB2, SMURF2 SLC2A1, NCOA3, EP300, LDLR, SREBF1, AKT3, STRBP, NCOR1, ACACA, TR/RXR Activation PIK3CB, NCOR2, TBL1XR1, RXRA SOS1, CDK6, PRKCE, AKT3, PIK3CB, MAP3K5, GSK3B, PARD3, PRKD1, HER-2 Signaling in Breast Cancer PRKCA, EGFR Non-Small Cell Lung Cancer Signaling STK4, MAPK1, SOS1, CDK6, AKT3, PDPK1, PIK3CB, RXRA, PRKCA, EGFR MAPKAP1, MAPK1, PPP2R5C, PPP2CA, PDPK1, RICTOR, EIF4E, IRS1, mTOR Signaling * PRKAA1, EIF3A, PRKCE, AKT3, PIK3CB, PPP2R5E, PRKD1, PRKCA BMPR1B, MAPK1, BMPR1A, CREB1, SOS1, CREBBP, SMAD6, BMPR2, BMP signaling pathway * SMURF1, ATF2 Cyclins and Cell Cycle Regulation HDAC4, PPP2R5C, PPP2CA, CDK6, TGFB2, GSK3B, PPP2R5E PPP2R5C, PPP2CA, CSNK1G2, TGFBR3, CREBBP, LRP6, GNAQ, CSNK1A1, Wnt/β-catenin Signaling * TCF3, EP300, FZD8, TGFB2, AKT3, GSK3B, PPP2R5E, TCF7L2 SMAD3, SMAD6, BMPR2, PDPK1, TCF3, FZD8, BMPR1B, BMPR1A, TGFB2, Human Embryonic Stem Cell Pluripotency * AKT3, PIK3CB, GSK3B, TCF7L2 DNA Methylation and Transcriptional Repression Signaling MECP2, ARID4B, SAP18 Role of Oct4 in Mammalian Embryonic Stem Cell Pluripotency * C3orf63, CCNF, JARID2, IGF2BP1, WWP2 Role of NANOG in Mammalian Embryonic Stem Cell Pluripotency * FZD8, BMPR1B, MAPK1, BMPR1A, SOS1, BMPR2, AKT3, PIK3CB, GSK3B Notch Signaling * MAML2, NUMB, MAML3, JAG1 TRAF3, TNFRSF1A, TGFBR3, CREBBP, BMPR2, MAP4K4, MALT1, EP300, NF-κB Signaling BMPR1B, BMPR1A, IGF1R, AKT3, PIK3CB, GSK3B, EGFR p53 Signaling PMAIP1, STAG1, AKT3, PIK3CB, GSK3B, HIPK2, EP300

B. Ingenuity Canonical Pathways - in vivo Differentially expressed genes MAP2K6, PMAIP1, TCF4, TGFBR1, BMPR2, CRK, RBL1, TGFBR2, FZD8, Molecular Mechanisms of Cancer BMPR1B, HIPK2, BRCA1, PRKCA TGF-β Signaling * MAP2K6, TGFBR2, BMPR1B, TGFBR1, BMPR2 TR/RXR Activation LDLR, TBL1XR1, RXRA, NCOA3 HER-2 Signaling in Breast Cancer PARD3, PRKCA Non-Small Cell Lung Cancer Signaling STK4, RXRA, PRKCA mTOR Signaling * PRKAB2, PPP2R5C, PPP2R5E, PDGFC, PRKCA BMP signaling pathway * BMPR1B, BMPR2 Cyclins and Cell Cycle Regulation HDAC4, PPP2R5C, CCNB2, PPP2R5E Wnt/β-catenin Signaling * TGFBR2, FZD8, TCF4, TGFBR1, PPP2R5C, PPP2R5E Human Embryonic Stem Cell Pluripotency * TGFBR2, FZD8, BMPR1B, TCF4, TGFBR1, BMPR2, PDGFC DNA Methylation and Transcriptional Repression Signaling SAP30 Role of Oct4 in Mammalian Embryonic Stem Cell Pluripotency * JARID2, BRCA1 Role of NANOG in Mammalian Embryonic Stem Cell Pluripotency * FZD8, BMPR1B, BMPR2 Notch Signaling * MAML2 NF-κB Signaling MAP2K6, TGFBR2, BMPR1B, TGFBR1, BMPR2 p53 Signaling PMAIP1, TP53INP1, HIPK2, BRCA1

* Stem cell related signaling pathway

Table 2: Top 100 Genes Modulated in Lung Cancer Xenografts by both Doses of IP Mithramycin

Downregulated genes Upregulated genes Fold change Fold change Fold change Fold change Probeset ID Gene Symbol Probeset ID Gene Symbol (1mg/kg vs. C) (2mg/kg vs. C) (1mg/kg vs. C) (2mg/kg vs. C) 225846_at ESRP1 -3.7 -8.4 237737_at LOC727770 7.6 17.2 204283_at FARS2 -2.5 -7.7 223218_s_at NFKBIZ 3.8 13.2 214106_s_at GMDS -6.6 -7.1 213348_at CDKN1C 4.8 11.4 203474_at IQGAP2 -2.0 -6.5 229331_at SPATA18 4.2 11.1 213432_at MUC5B -3.4 -6.5 1557765_at LOC643401 2.8 9.9 223278_at GJB2 -2.0 -6.2 232164_s_at EPPK1 3.7 9.8 219077_s_at WWOX -9.8 -5.9 1554195_a_at C5orf46 3.3 8.6 204623_at TFF3 -2.6 -5.9 204803_s_at RRAD 2 8.2 232603_at DCDC5 -2.7 -5.9 212667_at SPARC 3.7 7.2 212942_s_at KIAA1199 -5.4 -5.7 209283_at CRYAB 2.4 7 219410_at TMEM45A -2.7 -5.6 213764_s_at MFAP5 2.6 6.3 227059_at GPC6 -4.4 -5.0 211340_s_at MCAM 3.2 6.2 211596_s_at LRIG1 -2.2 -4.8 221276_s_at SYNC 2.9 5.8 225864_at FAM84B -3.0 -4.7 229275_at IGFN1 2.2 5.5 218788_s_at SMYD3 -2.9 -4.7 227870_at IGDCC4 2.3 5.2 204857_at MAD1L1 -4.6 -4.7 217562_at FAM5C 2.3 4.8 204066_s_at AGAP1 -5.6 -4.6 203889_at SCG5 3.8 4.8 1552673_at RFX6 -3.0 -4.5 203477_at COL15A1 2.7 4.6 218872_at TESC -2.5 -4.4 207173_x_at CDH11 2.3 4.5 204646_at DPYD -4.3 -4.4 1563498_s_at SLC25A45 2.5 4.5 224926_at EXOC4 -2.7 -4.3 201860_s_at PLAT 3.4 4.5 213243_at VPS13B -3.2 -4.3 229711_s_at MDM2 2.3 4.5 203908_at SLC4A4 -3.1 -4.3 223540_at PVRL4 2.7 4.4 229030_at CAPN8 -2.1 -4.2 200974_at ACTA2 3.1 4.4 203699_s_at DIO2 -3.2 -4.2 236656_s_at LOC100130506 2.1 4.3 206010_at HABP2 -2.4 -4.1 218611_at IER5 2.6 4.3 227202_at CNTN1 -2.1 -4.0 215033_at TM4SF1 2.3 4.1 204690_at STX8 -2.8 -4.0 209462_at APLP1 2.2 4 212692_s_at LRBA -2.1 -4.0 203002_at AMOTL2 2.2 4 219732_at RP11-35N6.1 -3.1 -3.9 1552309_a_at NEXN 2.1 3.7 202068_s_at LDLR -2.4 -3.9 201939_at PLK2 2.4 3.6 225081_s_at CDCA7L -2.1 -3.8 225604_s_at GLIPR2 2.9 3.6 212338_at MYO1D -2.4 -3.8 205483_s_at ISG15 2.1 3.5 238440_at CLYBL -2.4 -3.7 205691_at SYNGR3 2.2 3.5 201622_at SND1 -2.9 -3.7 236561_at TGFBR1 2 3.5 201911_s_at FARP1 -2.2 -3.7 223484_at C15orf48 2.2 3.4 211429_s_at SERPINA1 -3.0 -3.7 206857_s_at FKBP1B 2.4 3.4 214303_x_at MUC5AC -2.8 -3.6 225973_at TAP2 2.5 3.4 228570_at BTBD11 -2.2 -3.6 206907_at TNFSF9 2.3 3.4 219307_at PDSS2 -2.0 -3.6 225912_at TP53INP1 2.2 3.4 212093_s_at MTUS1 -2.1 -3.6 202284_s_at CDKN1A 2.3 3.2 228038_at SOX2 -3.5 -3.5 36711_at MAFF 2.1 3.2 243681_at SHANK2 -4.0 -3.5 203153_at IFIT1 2 3.2 212098_at LOC151162 /// MGAT5 -2.1 -3.5 228531_at SAMD9 2.2 3.1 220488_s_at BCAS3 -2.8 -3.5 204430_s_at SLC2A5 2 3.1 203006_at INPP5A -2.8 -3.4 228293_at DEPDC7 2.1 3 223681_s_at INADL -3.2 -3.4 207813_s_at FDXR 2.5 3 205603_s_at DIAPH2 -3.0 -3.3 209277_at TFPI2 2 2.9 228486_at SLC44A1 -2.6 -3.3 209183_s_at C10orf10 2.4 2.8 207414_s_at PCSK6 -2.1 -3.2 204483_at ENO3 2.9 2.8