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Supplementary Figure 4

Constitutively activated STAT3 plays a critical role in non–small cell lung carcinoma (NSCLC) progression by mediating proliferation and survival. STAT3 activation in normal cells is transient, making it an attractive target for NSCLC therapy. The therapeutic potential of blocking STAT3 in NSCLC was assessed utilizing a decoy approach by ligating a double-stranded 15-mer oligonucleotide that corresponds to the STAT3 response element of STAT3-target genes, to produce a cyclic STAT3 decoy (CS3D). The decoy was evaluated using NSCLC cells containing either wild-type EGFR (201T) or mutant EGFR with an additional EGFRi resistance mutation (H1975). These cells are resistant to EGFR inhibitors and require an alternate therapeutic approach. CS3D activity was compared with an inactive cyclic control oligonucleotide (CS3M) that differs by a single base pair, rendering it unable to bind to STAT3 protein. Transfection of 0.3 μmol/L of CS3D caused a 50% inhibition in proliferation in 201T and H1975 cells, relative to CS3M, and a 2-fold increase in apoptotic cells. Toxicity was minimal in normal cells. CS3D treatment caused a significant reduction of mRNA and protein expression of the STAT3 target gene c-Myc and inhibited colony formation by 70%. The active decoy decreased the nuclear pool of STAT3 compared with the mutant. In a xenograft model, treatments with CS3D (5 mg/kg) caused a potent 96.5% and 81.7% reduction in tumor growth in 201T (P < 0.007) and H1975 models (P < 0.0001), respectively, and reduced c-Myc and p-STAT3 proteins. Targeting STAT3 with the cyclic decoy could be an effective therapeutic strategy for NSCLC. Mol Cancer Ther; 17(9); 1917–26. ©2018 AACR.

Localization of Fluorecein-labeled CS3D by confocal Imaging

Supplementary Figure legends

Short-term CS3D treatment decreases Ki-67 expression

The STAT3 pathway is frequently overactive in non–small cell lung cancer (NSCLC), an often fatal disease with known risk factors including tobacco and chemical exposures. Whether STAT3 can be downmodulated to delay or prevent development of lung cancer resulting from an environmental exposure has not been previously tested. A circular oligonucleotide STAT3 decoy (CS3D) was used to treat mice previously exposed to the tobacco carcinogen nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. CS3D contains a double-stranded STAT3 DNA response element sequence and interrupts STAT3 signaling by binding to STAT3 dimers, rendering them unable to initiate transcription at native STAT3 DNA binding sites. An intermittent course of CS3D decreased the development of airway preneoplasias by 42% at 1 week posttreatment, reduced the progression of preneoplasia to adenomas by 54% at 8 weeks posttreatment, and reduced the size and number of resulting lung tumors by 49.7% and 29.5%, respectively, at 20 weeks posttreatment. No toxicity was detected. A mutant cyclic oligonucleotide with no STAT3 binding ability was used as a control. Chemopreventive effects were independent of the KRAS mutational status of the tumors. In lungs harvested during and after the treatment course with CS3D, airway preneoplasias had reduced STAT3 signaling. Chemopreventive effects were accompanied by decreased VEGFA expression, ablated IL6, COX-2, and p-NF-κB, and decreased pulmonary M2 macrophages and myeloid-derived suppressor cells. Thus, downmodulation of STAT3 activity using a decoy molecule both reduced oncogenic signaling in the airway epithelium and favored a lung microenvironment with reduced immunosuppression.

Long-Term CS3D treatment alters tumor cellularity

Specificity of STAT3 decoy on gene regulation

Objectives We aimed to better understand the histologic changes in vaginectomy specimens in transgender and gender-diverse (TGD) individuals after prolonged androgen administration. Methods After obtaining institutional review board approval, we reviewed clinical records for all TGD individuals who underwent vaginal tissue resection at our institution between January 2002 and July 2020. Results Ten transgender males who underwent vaginectomy for gender affirmation were identified. All patients had been assigned female gender at birth, and the median age at surgery was 41 years (range, 22-74 years). All 10 patients had received androgen for 2 to 10 years preoperatively. The corresponding pathology specimens were examined grossly and microscopically, including with immunohistochemical stains for NKX3.1, prostate-specific antigen (PSA), p501s, and androgen receptor (AR). No gross lesions were identified. Microscopically, prostate-like glands (8/10), urothelial metaplasia (4/10), and vaginal atrophy (8/10) were identified. Seven cases with prostate-like glands showed positive staining with PSA, NKX3.1, p501s, and AR in both squamous and glandular components. Conclusions Recognition of these androgen-related changes enables pathologist to avoid the overdiagnosis of dysplasia. Long-term follow-up is needed to thoroughly understand any potential future implications of these androgen-related changes.

The STAT3 pathway is frequently overactive in non–small cell lung cancer (NSCLC), an often fatal disease with known risk factors including tobacco and chemical exposures. Whether STAT3 can be downmodulated to delay or prevent development of lung cancer resulting from an environmental exposure has not been previously tested. A circular oligonucleotide STAT3 decoy (CS3D) was used to treat mice previously exposed to the tobacco carcinogen nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. CS3D contains a double-stranded STAT3 DNA response element sequence and interrupts STAT3 signaling by binding to STAT3 dimers, rendering them unable to initiate transcription at native STAT3 DNA binding sites. An intermittent course of CS3D decreased the development of airway preneoplasias by 42% at 1 week posttreatment, reduced the progression of preneoplasia to adenomas by 54% at 8 weeks posttreatment, and reduced the size and number of resulting lung tumors by 49.7% and 29.5%, respectively, at 20 weeks posttreatment. No toxicity was detected. A mutant cyclic oligonucleotide with no STAT3 binding ability was used as a control. Chemopreventive effects were independent of the KRAS mutational status of the tumors. In lungs harvested during and after the treatment course with CS3D, airway preneoplasias had reduced STAT3 signaling. Chemopreventive effects were accompanied by decreased VEGFA expression, ablated IL6, COX-2, and p-NF-κB, and decreased pulmonary M2 macrophages and myeloid-derived suppressor cells. Thus, downmodulation of STAT3 activity using a decoy molecule both reduced oncogenic signaling in the airway epithelium and favored a lung microenvironment with reduced immunosuppression.

Constitutively activated STAT3 plays a critical role in non–small cell lung carcinoma (NSCLC) progression by mediating proliferation and survival. STAT3 activation in normal cells is transient, making it an attractive target for NSCLC therapy. The therapeutic potential of blocking STAT3 in NSCLC was assessed utilizing a decoy approach by ligating a double-stranded 15-mer oligonucleotide that corresponds to the STAT3 response element of STAT3-target genes, to produce a cyclic STAT3 decoy (CS3D). The decoy was evaluated using NSCLC cells containing either wild-type EGFR (201T) or mutant EGFR with an additional EGFRi resistance mutation (H1975). These cells are resistant to EGFR inhibitors and require an alternate therapeutic approach. CS3D activity was compared with an inactive cyclic control oligonucleotide (CS3M) that differs by a single base pair, rendering it unable to bind to STAT3 protein. Transfection of 0.3 μmol/L of CS3D caused a 50% inhibition in proliferation in 201T and H1975 cells, relative to CS3M, and a 2-fold increase in apoptotic cells. Toxicity was minimal in normal cells. CS3D treatment caused a significant reduction of mRNA and protein expression of the STAT3 target gene c-Myc and inhibited colony formation by 70%. The active decoy decreased the nuclear pool of STAT3 compared with the mutant. In a xenograft model, treatments with CS3D (5 mg/kg) caused a potent 96.5% and 81.7% reduction in tumor growth in 201T (P < 0.007) and H1975 models (P < 0.0001), respectively, and reduced c-Myc and p-STAT3 proteins. Targeting STAT3 with the cyclic decoy could be an effective therapeutic strategy for NSCLC. Mol Cancer Ther; 17(9); 1917–26. ©2018 AACR.

We have previously shown that topical opioids including morphine and its congeners promote healing of full thickness ischemic wounds in rats. We examined the contribution of mu opioid receptor (MOPr)-mediated healing of full thickness ischemic wounds using MOPr and delta or kappa opioid receptor (DOPr or KOPr) knockout (KO) mice. Wound closure in the early (day 5) as well as later phases was delayed in topical morphine or PBS treated MOPr-KO mice compared to reciprocal treatments of wounds in wild-type (WT) mice. MOPr expression was significantly upregulated at 30 min in the wound margins and colocalized with wound margins and vasculature in the epidermal and dermal layers of the skin. We next examined whether neuropeptide expression was involved in the mechanism of MOPr-mediated wound closure. Substance P (SP) and calcitonin gene related peptide (CGRP) immunoreactivity (ir) was significantly increased in the skin of MOPr-KO mice as compared to WT mice. Neuropeptide-ir was increased significantly in PBS-treated wounds of MOPr and WT mice, but morphine treatment reduced neuropeptide immunoreactivity in both as compared to PBS. Wounding of keratinocytes led to the release of opioid peptide beta-endorphin (β-END) in conditioned medium, which stimulated the proliferation of endothelial cells. MOPr-selective (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2, CTOP) and non-selective OPr antagonist naloxone inhibited endothelial proliferation induced by wounded keratinocyte conditioned medium. Additionally, accelerated wound area closure in vitro by morphine was suppressed by methylnaltrexone, a non-selective OPr antagonist with high affinity for MOPr. Morphine and its congeners stimulated the proliferation of endothelial cells from WT mice but not those from MOPr-KO mice. Furthermore, morphine-induced mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) phosphorylation in endothelial cells was significantly decreased in MOPr-KO mice as compared to WT mice. Collectively, these data suggest that MOPr plays a critical role in the proliferation phase with the formation of granulation tissue during wound healing.

BACKGROUND Activation of the mesenchymal-epidermal transition factor (MET) tyrosine kinase and its ligand, hepatocyte growth factor (HGF), is implicated in resistance to epidermal growth factor receptor (EGFR) inhibitors. In this phase 1/2 trial, rilotumumab (an anti-HGF antibody) combined with erlotinib was evaluated in patients with metastatic, previously treated non–small cell lung cancer. METHODS In phase 1, a dose de-escalation design was adopted with rilotumumab starting at 15 mg/kg intravenously every 3 weeks and oral erlotinib 150 mg daily. In phase 2, the disease control rate (DCR) (according to Response Evaluation Criteria in Solid Tumors) of the combination was evaluated using a Simon 2-stage design. The biomarkers examined included 10 plasma-circulating molecules associated with the EGFR and MET pathways. RESULTS Without indications for de-escalation, the recommended phase 2 dose was dose level 0. Overall, 45 response-evaluable patients were enrolled (13 with squamous carcinoma, 32 with adenocarcinoma; 2 had confirmed EGFR mutations, 33 had confirmed wild-type [WT] EGFR, and 7 had KRAS mutations). The DCR for all patients was 60% (90% confidence interval [CI], 47.1%-71.3%). Median progression-free survival was 2.6 months (90% CI, 1.4-2.7 months), and median overall survival was 6.6 months (90% CI, 5.6-8.9 months). Among patients with WT EGFR, the DCR was 60.6% (90% CI, 46.3%-73.3%), median progression-free survival was 2.6 months (90% CI, 1.4-2.7 months), and median overall survival was 7.0 months (90% CI, 5.6-13.4 months). Elevated baseline levels of neuregulin 1 were associated with longer progression-free survival (hazard ratio, 0.41; 95% CI, 0.19-0.87), whereas elevated amphiregulin levels were associated with more rapid progression (hazard ratio, 2.14; 95% CI, 1.48-3.08). CONCLUSIONS Combined rilotumumab and erlotinib had an acceptable safety profile, and the DCR met the prespecified criteria for success. In the EGFR WT group, the DCR exceeded published reports for erlotinib alone. High circulating levels of neuregulin 1 may indicate sensitivity to this combination. Cancer 2017;123:2936–44. © 2017 American Cancer Society.

// Jill M. Siegfried 1 , Mariya Farooqui 1 , Natalie J. Rothenberger 2 , Sanja Dacic 3 and Laura P. Stabile 2 1 Department of Pharmacology and Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA 2 Department of Pharmacology & Chemical Biology and University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA, USA 3 Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA Correspondence to: Laura P. Stabile, email: // Keywords : NSCLC, FGFR, estrogen Received : December 10, 2016 Accepted : February 18, 2017 Published : March 08, 2017 Abstract The estrogen receptor (ER) promotes non-small cell lung cancer (NSCLC) proliferation. Since fibroblast growth factors (FGFs) are known regulators of stem cell markers in ER positive breast cancer, we investigated whether a link between the ER, FGFs, and stem cell markers exists in NSCLC. In lung preneoplasias and adenomas of tobacco carcinogen exposed mice, the anti-estrogen fulvestrant and/or the aromatase inhibitor anastrozole blocked FGF2 and FGF9 secretion, and reduced expression of the stem cell markers SOX2 and nanog. Mice administered β-estradiol during carcinogen exposure showed increased FGF2, FGF9, SOX2, and Nanog expression in airway preneoplasias. In normal FGFR1 copy number NSCLC cell lines, multiple FGFR receptors were expressed and secreted several FGFs. β-estradiol caused enhanced FGF2 release, which was blocked by fulvestrant. Upon co-inhibition of ER and FGFRs using fulvestrant and the pan-FGFR inhibitor AZD4547, phosphorylation of FRS2, the FGFR docking protein, was maximally reduced, and enhanced anti-proliferative effects were observed. Combined AZD4547 and fulvestrant enhanced lung tumor xenograft growth inhibition and decreased Ki67 and stem cell marker expression. To verify a link between ERβ, the predominant ER in NSCLC, and FGFR signaling in patient tumors, mRNA analysis was performed comparing high versus low ERβ expressing tumors. The top differentially expressed genes in high ERβ tumors involved FGF signaling and human embryonic stem cell pluripotency. These results suggest interaction between the ER and FGFR pathways in NSCLC promotes a stem-like state. Combined FGFR and ER inhibition may increase the efficacy of FGFR inhibitors for NSCLC patients lacking FGFR genetic alterations.

Introduction Mounting evidence supports a role for estrogen signaling in NSCLC progression. We previously reported a seven-gene signature that predicts prognosis in estrogen receptor β positive (ERβ+) NSCLC. The signature defines a network comprised of ER and human EGFR-2/3 (HER2/HER3) signaling. Methods We tested the efficacy of combining the pan-HER inhibitor, dacomitinib, with the estrogen antagonist, fulvestrant, in ERβ+ NSCLC models with differing genotypes. We assessed the potency of this combination on xenograft growth and survival of host mice, and the ability to reverse the gene signature associated with poor outcome. Results Synergy was observed between dacomitinib and fulvestrant in three human ERβ+ NSCLC models: 201T (wild-type EGFR), A549 (KRAS mutant), and HCC827 (EGFR 19 deletion) with combination indices of 0.1-0.6. The combination, but not single agents, completely reversed the gene signature associated with poor prognosis in a mechanism that is largely mediated by activator protein 1 downregulation. In vivo, the combination also induced tumor regression and reversed the gene signature. In HCC827 xenografts treated with the combination, survival of mice was prolonged after therapy discontinuation, tumors that recurred were less aggressive, and two mechanisms of HER inhibitor resistance involving c-Met activation and PTEN loss were blocked. Conclusions The combination of an ER blocker and a pan-HER inhibitor provides synergistic efficacy in different models of ERβ+ NSCLC. Our data support the use of this combination clinically, considering its ability to induce potent antitumor effects and produce a gene signature that predicts better clinical outcomes.

Hepatocyte growth factor (HGF) is the ligand for the tyrosine kinase receptor c-Met (Mesenchymal Epithelial Transition Factor also known as Hepatocyte Growth Factor Receptor, HGFR), a receptor with expression throughout epithelial and endothelial cell types. Activation of c-Met enhances cell proliferation, invasion, survival, angiogenesis, and motility. The c-Met pathway also stimulates tissue repair in normal cells. A body of past research shows that increased levels of HGF and/or overexpression of c-Met are associated with poor prognosis in several solid tumors, including lung cancer, as well as cancers of the head and neck, gastro-intestinal tract, breast, ovary and cervix. The HGF/c-Met signaling network is complex; both ligand-dependent and ligand-independent signaling occur. This article will provide an update on signaling through the HGF/c-Met axis, the mechanism of action of HGF/c-Met inhibitors, the lung cancer patient populations most likely to benefit, and possible mechanisms of resistance to these inhibitors. Although c-Met as a target in non-small cell lung cancer (NSCLC) showed promise based on preclinical data, clinical responses in NSCLC patients have been disappointing in the absence of MET mutation or MET gene amplification. New therapeutics that selectively target c-Met or HGF, or that target c-Met and a wider spectrum of interacting tyrosine kinases, will be discussed.

Background Topically applied opioids promote angiogenesis and healing of ischemic wounds in rats. We examined if topical fentanyl stimulates wound healing in diabetic rats by stimulating growth-promoting signaling, angiogenesis, lymphangiogenesis and nerve regeneration. Methods We used Zucker diabetic fatty rats that develop obesity and diabetes on a high fat diet due to a mutation in the Leptin receptor. Fentanyl blended with hydrocream was applied topically on ischemic wounds twice daily, and wound closure was analyzed regularly. Wound histology was analyzed by hematoxylin and eosin staining. Angiogenesis, lymphangiogenesis, nerve fibers and phospho-platelet derived growth factor receptor-β (PDGFR-β) were visualized by CD31-, lymphatic vessel endothelium-1, protein gene product 9.5- and anti-phospho PDGFR-β-immunoreactivity, respectively. Nitric oxide synthase (NOS) and PDGFR-β signaling were analyzed using Western immunoblotting. Results Fentanyl significantly promoted wound closure as compared to phosphate-buffered saline (PBS). Histology scores were significantly higher in fentanyl-treated wounds, indicative of increased granulation tissue formation, reduced edema and inflammation, and increased matrix deposition. Fentanyl treatment resulted in increased wound angiogenesis, lymphatic vasculature, nerve fibers, nitric oxide, NOS and PDGFR-β signaling as compared to PBS. Phospho-PDGFR-β co-localized with CD31 co-staining for vasculature. Conclusions Topically applied fentanyl promotes closure of ischemic wounds in diabetic rats. Increased angiogenesis, lymphangiogenesis, peripheral nerve regeneration, NO and PDGFR-β signaling are associated with fentanyl-induced tissue remodeling and wound healing. 摘要 背景： 局部应用罂粟碱类可以促进大鼠血管的生成与缺血性伤口的愈合。我们的目的是调查局部芬太尼麻醉是否通过刺激促生长信号、血管生成、淋巴管生成以及神经再生来促进糖尿病大鼠伤口的愈合。 方法： 我们使用的Zucker糖尿病肥胖大鼠由于瘦素受体基因突变在高脂饮食下出现了肥胖与糖尿病。对缺血性伤口每日2次局部使用混合了芬太尼的保湿乳剂，定期分析伤口闭合情况。使用苏木精-伊红染色分析伤口组织学切片。血管生成、淋巴管生成、神经纤维以及磷酸化血小板衍生生长因子受体-β（PDGFR-β）分别使用CD31-、淋巴管内皮细胞-1、蛋白基因产物9.5-以及抗磷酸化PDGFR-β-免疫反应性抗体来显像。使用免疫印迹法分析一氧化氮合酶与PDGFR-β信号。 结果： 与磷酸盐缓冲的生理盐水相比，芬太尼可以显著地促进伤口闭合。在使用芬太尼治疗的伤口中组织学评分显著更高，提示肉芽组织形成增加，水肿与炎症减少，以及基质沉积增加。与磷酸盐缓冲的生理盐水相比，使用芬太尼治疗后可以导致伤口的血管生成、淋巴管系统、神经纤维、一氧化氮、一氧化氮合酶以及PDGFR-β信号都有所增加。脉管系统中的磷酸-PDGFR-β使用CD31来辅助染色定位。 结论： 糖尿病大鼠局部应用芬太尼可以促进缺血性伤口闭合。增加的血管生成、淋巴管生成、周围神经再生、一氧化氮以及PDGFR-β信号与芬太尼诱导的组织重构以及伤口愈合有关。

Activation of the mesenchymal-epidermal transition factor (MET) tyrosine kinase and its ligand, hepatocyte growth factor (HGF), is implicated in resistance to epidermal growth factor receptor (EGFR) inhibitors. In this phase 1/2 trial, rilotumumab (an anti-HGF antibody) combined with erlotinib was evaluated in patients with metastatic, previously treated non-small cell lung cancer.In phase 1, a dose de-escalation design was adopted with rilotumumab starting at 15 mg/kg intravenously every 3 weeks and oral erlotinib 150 mg daily. In phase 2, the disease control rate (DCR) (according to Response Evaluation Criteria in Solid Tumors) of the combination was evaluated using a Simon 2-stage design. The biomarkers examined included 10 plasma-circulating molecules associated with the EGFR and MET pathways.Without indications for de-escalation, the recommended phase 2 dose was dose level 0. Overall, 45 response-evaluable patients were enrolled (13 with squamous carcinoma, 32 with adenocarcinoma; 2 had confirmed EGFR mutations, 33 had confirmed wild-type [WT] EGFR, and 7 had KRAS mutations). The DCR for all patients was 60% (90% confidence interval [CI], 47.1%-71.3%). Median progression-free survival was 2.6 months (90% CI, 1.4-2.7 months), and median overall survival was 6.6 months (90% CI, 5.6-8.9 months). Among patients with WT EGFR, the DCR was 60.6% (90% CI, 46.3%-73.3%), median progression-free survival was 2.6 months (90% CI, 1.4-2.7 months), and median overall survival was 7.0 months (90% CI, 5.6-13.4 months). Elevated baseline levels of neuregulin 1 were associated with longer progression-free survival (hazard ratio, 0.41; 95% CI, 0.19-0.87), whereas elevated amphiregulin levels were associated with more rapid progression (hazard ratio, 2.14; 95% CI, 1.48-3.08).Combined rilotumumab and erlotinib had an acceptable safety profile, and the DCR met the prespecified criteria for success. In the EGFR WT group, the DCR exceeded published reports for erlotinib alone. High circulating levels of neuregulin 1 may indicate sensitivity to this combination. Cancer 2017;123:2936-44. © 2017 American Cancer Society.

Exposure to tobacco carcinogens such as NNK leads to mutations in genes like K-RAS in premalignant lesions, which progress to adenocarcinoma. Early events in carcinogenesis are also characterized by inflammation. Central to these processes, STAT3 activation has pleotropic pro-tumor effects on proliferation, angiogenesis, and immune evasion. We previously showed that a “decoy” cyclic STAT3 oligonucleotide (CS3D) produced robust antitumor effects in human lung adenocarcinoma models. CS3D mimics the STAT3 DNA response element and acts as a “decoy” to which STAT3 binds, blocking its transcriptional activity. Effects of CS3D were compared to an inactive mutant construct unable to bind to STAT3 (CS3M). Therapeutic effects in human lung cancer xenografts suggested that CS3D might also impair lung cancer development. To test this, mice were exposed to NNK (3mg/kg) for 4 weeks (wks) followed by a 1-wk washout phase to model ex-smokers. Mice were then treated 3 times per wk with CS3D or CS3M (5mg/kg, IV) for 8 wks. Tumor burden and size were then assessed at 8 and 20 wks after the end of treatment. To evaluate effect of CS3D on the development of tumors with K-RAS activating mutations (since STAT3 knockout has been reported to promote KRAS mutant lung cancer), we isolated DNA from tumors by laser capture microdissection and assessed the incidence of G12D activating mutations. CS3D caused a reduction in preneoplasias (P Citation Format: Christian Njatcha, Jiayi Wang, Huiyu Li, Mariya Farooqui, Jill M. Siegfried. A novel STAT3 decoy inhibitor prevents lung cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3875.

Mounting preclinical and clinical evidence suggest an involvement of estrogen signaling in promoting non-small cell lung cancer (NSCLC) progression. In estrogen receptor β-positive (ERβ) cases, we previously found seven genes within the prediction analysis of microarrays 50 gene (PAM50) panel that showed strong association in predicting prognosis of NSCLC patients, with Myc, MIA, FGFR4, CXXC5, Grb-7 and FOXC1 being upregulated while PR was downregulated in highly aggressive lung tumors. These genes described one network containing ERβ and human epidermal growth factor receptor 2/3 (HER2/HER3) signaling, which suggests that these two interacting pathways define lung tumors with more aggressive biology. Here, we sought to evaluate the therapeutic potential of combining the pan-HER tyrosine kinase inhibitor, Dacomitinib, and the ER antagonist, Fulvestrant in NSCLC. Cell viability assay in three different human NSCLC cell lines 201T (EGFR wild-type), A549 (K-ras mutant) and HCC827 (EGFR exon 19 deletion) showed strong synergy between Dacomitinib and Fulvestrant, as determined by a combination index < 1. The combination strongly inhibited p-EGFR, p-HER2, p-HER3, p-HER4, and the downstream signaling p-AKT and p-MAPK compared to single treatments. Mechanistically, the transcription activity of activator protein-1 (AP-1) family members Fos and Jun were significantly reduced, as detected by the ability of AP-1 to bind to DNA. Expression of c-Myc and CyclinD1 were also significantly inhibited by the combination. In NSCLC cell lines, the combination reversed the gene signature associated with poor prognosis with c-Myc, MIA, CXXC5, FGFR4, Grb-7, FOXC1 being downregulated while PR was upregulated. Similarly, the AP-1 inhibitor t-5224 mimicked the effects of the combination in reversing the PAM50 gene signature, suggesting that the combination’s effect is largely mediated by AP-1 downregulation. In vivo, the combination also revealed a synergistic effect, measured by the combination ratio method (> 1), and induced tumor regression in 201T and A549 xenografts, with average tumor volume significantly lower than the single treatment groups. The immunohistochemical analysis of the xenografts revealed significant downregulation in Ki67. Western blot analysis of the tumor lysates from the combination treatment groups showed an increase in cleaved caspase-3 and a decrease in both ER and HER signaling. The PAM50 gene signature was also reversed in both xenograft studies detected by real-time-qPCR. Similar effects were seen for c-Myc and PR at the protein levels detected by immunohistochemistry staining. These observations encouragingly support the use of this combination clinically in NSCLC, considering its ability to suppress tumor growth and produce a gene signature that predicts better clinical outcomes, as assessed by the PAM50 genes. Citation Format: Abdulaziz A. Almotlak, Mariya Farooqui, Jill M. Siegfried. Inhibiting pathways predicted from a steroid hormone gene signature yields synergistic antitumor effects in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1027.

Lung cancer is the number one cause of cancer-related fatalities, and to date, there are no FDA-approved chemoprevention options for lung cancer prevention. NNK, the principal carcinogen in cigarette smoke can induce mutations in oncogenes and can also act as an inflammatory mediator of the tumor microenvironment (TME) by promoting macrophage infiltration into the lungs. Macrophages play a key role in regulation of the lung TME. Pioglitazone, a synthetic peroxisome proliferator-activated receptor gamma (PPARγ) agonist belonging to the thiazolidinediones (TZDs) drug class has been used in preclinical studies to mitigate lung tumorigenesis, progression, and metastasis. A retrospective analysis found that diabetics using TZDs experienced a 33% reduction in lung cancer incidence. Cross-talk between PPARγ and estrogen receptor (ER) signaling has been previously reported, and ER has already been implicated as a driver of lung cancer severity. Both ER and PPARγ are also known to be functional in macrophages. Agents that block ER signaling such as fulvestrant, a complete ER antagonist, have shown activity against lung cancer. We therefore tested the ability of pioglitazone and fulvestrant in combination to reduce lung tumor formation and to modulate the TME. Preliminary results from a co-culture simulation of the TME in vitro using human macrophages and lung adenocarcinoma cells show pioglitazone and fulvestrant together can significantly suppress inflammatory modulators such as IL-1β, IL-10, Amphiregulin (AREG), and VEGF compared to single treatments. Similar effects were observed in a mouse model system of the TME. This drug combination also reduced number of tumor cell colonies in a soft agar assay, suggesting a direct effects on tumor cells. In vivo use of combined pioglitazone and fulvestrant in an NNK female mouse model of former smokers showed a significant reduction in tumor size by 40% compared to placebo (p= 0.0010), 13% by pioglitazone alone and 5% by fulvestrant alone. Inflammatory cytokine array analysis of the bronchio-alveolar lavage fluid (BALF) revealed maximum down-modulation of pro-tumor inflammatory mediators with combination treatment compared to single treatments. Fulvestrant alone did not suppress tumor-promoting inflammatory pathways, suggesting possible activation of compensatory signaling pathways. Further analysis of the cell cultures and the BALF confirmed EGFR compensatory activation through release of AREG with fulvestrant single treatment, that is suppressed with the addition of pioglitazone. Thus, the strong combination effect of pioglitazone and fulvestrant on lung tumor growth and macrophage function, and the ability of the combination to suppress compensatory signaling pathways, supports the hypothesis of ER/PPARγ cross-talk in lung cancer and provides a rationale for further investigation of chemopreventive effects of this combination. Citation Format: Erika Louiselle, Oshin Miranda, Huiyu Li, MiKayla Boeder, Zhijie Li, Jill M. Siegfried, Mariya Farooqui. Modulation of inflammation in the tumor microenvironment by ER and PPARγ in an NNK-induced lung carcinogenesis model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2349.

The earliest stages of malignant transformation require the acquisition of multiple phenotypes, such as proliferation, survival and migration, which contribute to tumor growth and progression. Identifying the key cellular and molecular factors that drive the formation of early breast cancer lesions will ultimately result in the development of preventive approaches for patients that are at high risk for developing invasive breast cancer. The epidermal growth factor (EGF) family of ligands has been implicated in promoting breast cancer growth and progression. The contributions of EGF family ligands and their receptors to breast cancer are complex, and the specific mechanisms through which different ligands regulate different stages of breast tumor initiation and growth are not well-defined. These studies focus on the EGF family member epiregulin (EREG) as a mediator of early stages of breast tumorigenesis. In comparison with other EGF family members, EREG was found to be highly expressed in MCF10DCIS.com cells compared with the non-transformed MCF10A cells. Increased EREG expression was also found in significantly more patient samples of human ductal carcinoma in situ (DCIS) than in samples of normal epithelium (64.5% vs. 29.4%, p Experimental studies demonstrated that treatment of MCF10A cells with exogenous EREG enhanced multiple tumorigenic phenotypes, including proliferation, migration and survival. Examination of EREG-induced signaling pathways demonstrated that EREG promotes survival of MCF10A cells primarily through activation of the signal transducer and activator of transcription 5 (STAT5) pathway. Analysis of potential STAT5 target genes revealed that the EREG-STAT5 pathway induces expression of matrix metalloproteinase-1 (MMP-1), a novel STAT5 target gene that was subsequently found to promote survival in response to EREG treatment. To assess the consequences of loss of EREG expression on tumor formation, EREG was knocked down in MCF10DCIS.com cells using shRNA strategies. Loss of EREG led to decreased tumor growth in vivo, which corresponded with decreased cell survival and decreased MMP-1 expression. To determine the relevance of these findings in human tumors, samples of DCIS were analyzed for both EREG and MMP-1 expression. MMP-1 was significantly induced in DCIS lesions in comparison with normal breast epithelium, and EREG and MMP-1 were significantly correlated in a subset of DCIS samples (p=0.011). Together, these studies suggest that EREG contributes to early stages of breast tumorigenesis through regulation of MMP-1. Based on these studies, we propose that EREG contributes to the formation of preneoplastic lesions in a subset of breast cancers and further studies are focused on characterizing the mechanisms through which the EREG-MMP-1 pathway contributes to tumor initiation and growth. Understanding these mechanisms will ultimately lead to the identification of biomarkers of aggressive disease and novel targets for therapeutic strategies for breast cancer patients. Citation Format: Kathryn L Schwertfeger, Mariya Farooqui, Laura R Bohrer, Andrew C Nelson. Epiregulin-induced matrix metalloproteinase-1 contributes to early stages of breast tumorigenesis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-06-01.

Morphine and its congener opioids are the main therapy for severe pain in cancer. However, chronic morphine treatment stimulates angiogenesis and tumour growth in mice. We examined if celecoxib (a cyclooxygenase-2 (COX-2) inhibitor) prevents morphine-induced tumour growth without compromising analgesia. The effect of chronic treatment with celecoxib (by gavage) and/or morphine (subcutaneously), or PBS on tumour prostaglandin E(2) (PGE(2)), COX-2, angiogenesis, tumour growth, metastasis, pain behaviour and survival was determined in a highly invasive SCK breast cancer model in A/J mice. Two weeks of chronic morphine treatment at clinically relevant doses stimulates COX-2 and PGE(2) (4.5-fold compared to vehicle alone) and angiogenesis in breast tumours in mice. This is accompanied by increased tumour weight ( approximately 35%) and increased metastasis and reduced survival. Co-administration of celecoxib prevents these morphine-induced effects. In addition, morphine and celecoxib together provided better analgesia than either agent alone. Celecoxib prevents morphine-induced stimulation of COX-2, PGE(2), angiogenesis, tumour growth, metastasis and mortality without compromising analgesia in a murine breast cancer model. In fact, the combination provided significantly better analgesia than with morphine or celecoxib alone. Clinical trials of this combination for analgesia in chronic and severe pain in cancer are warranted.

Orphan nuclear receptor TR2 is a preadipocyte proliferator. Knockdown of TR2 in 3T3-L1 preadipocytes reduced their proliferation efficiency, whereas specific elevation of TR2 in these cells facilitated their proliferation. All-trans retinoic acid (RA) stimulates cellular proliferation in 3T3-L1 preadipocytes by activating TR2 through an IR0-type RA response element, which further activates c-Myc expression. In post-differentiated adipocytes, RA becomes a repressive signal for TR2 and rapidly down-regulates its expression. The biphasic effect of RA on TR2 expression in 3T3-L1 is mediated by differential RA-dependent coregulator recruitment to the receptor/Glucocorticoid Receptor-Interacting Protein 1 (GRIP1) complex that binds IR0 on the TR2 promoter. RA induces the recruitment of histone acetyl transferase-containing/GRIP1/p300/CBP-associated factor (PCAF) complex to the TR2 promoter in undifferentiated cells, whereas it triggers recruitment of histone deacetylase-containing/GRIP1/receptor-interacting protein 140 (RIP140) complex in differentiated cells. GRIP1 directly interacts with RIP140 through its carboxyl terminal AD2 domain. GRIP1 interacts with PCAF and RIP140 directly and differentially, functioning as a platform molecule to mediate differential RA-induced coregulator recruitment to TR2 promoter target. This results in a biphasic effect of RA on the expression of TR2 in undifferentiated and differentiated cells, which is required for RA-stimulated preadipocyte proliferation.

Background The epidermal growth factor (EGF) family of ligands has been implicated in promoting breast cancer initiation, growth and progression. The contributions of EGF family ligands and their receptors to breast cancer are complex, and the specific mechanisms through which different ligands regulate breast tumor initiation and growth are not well-defined. These studies focus on the EGF family member epiregulin (EREG) as a mediator of early stage breast tumorigenesis. Methods EREG expression levels were assessed in both cell lines and human samples of ductal carcinoma in situ (DCIS) using quantitative RT-PCR, ELISA and immunohistochemistry. Gene knock-down approaches using shRNA-based strategies were used to determine the requirement of EREG for growth of MCF10DCIS cells in vivo, and for identifying mechanisms through which EREG promotes tumor cell survival. Experiments were performed using a combination of two-dimensional culture, three-dimensional culture and tumor growth in vivo. Results In comparison with other EGF family members, EREG was induced in MCF10DCIS cells compared with MCF10A and MCF10AT cells and its expression was partially regulated by fibroblast growth factor receptor (FGFR) activity. Reduced EREG expression in MCF10DCIS cells led to decreased tumor growth in vivo, which was associated with reduced cell survival. Furthermore, treatment of MCF10A cells with exogenous EREG enhanced cell survival both in three-dimensional culture and in response to chemotherapeutic agents. Examination of EREG-induced signaling pathways demonstrated that EREG promoted survival of MCF10A cells through regulating expression of matrix metalloproteinase-1 (MMP-1). To determine the relevance of these findings in human tumors, samples of DCIS were analyzed for EREG and MMP-1 expression. EREG was induced in DCIS lesions compared to normal breast epithelium, and EREG and MMP-1 were correlated in a subset of DCIS samples. Conclusions Together, these studies lead to identification of a novel pathway involving EREG and MMP-1 that contributes to the formation of early stage breast cancer. Understanding these complex pathways could ultimately lead to the development of novel biomarkers of neoplastic progression and/or new therapeutic strategies for patients with early stage cancer. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0408-z) contains supplementary material, which is available to authorized users.

Estrogen promotes the growth of breast cancer via estrogen receptors (ER). Earlier studies showed that the opioid receptor antagonist naloxone inhibits MCF-7 breast cancer growth in mice. We examined the cellular and molecular mechanism of naloxone antagonism of ERα activity in human MCF-7 cells. Naloxone (100 nmol/L) inhibited 17β-estradiol (E2)–induced (10 nmol/L) MCF-7 cell proliferation by 65% and mitogen-activated protein kinase/extracellular signal-regulated kinase phosphorylation. Naloxone blocked the E2-induced activation of ERα, with 85% inhibition after 5 minutes and 100% recovery after 60 minutes. This assay is based on quantitation of E2-activated nuclear ERα binding to the immobilized coactivator peptide. A significant decrease in E2-induced ERα transactivation was observed in the presence of naloxone in the estrogen response element-luciferase reporter assay (P < 0.05, E2 versus E2 + naloxone). Naloxone also blocked E2-induced down-regulation of ERα mRNA at 30 minutes and 6 hours. Although naloxone inhibits ERα activity directly, it also induces a cross-talk between μ-opioid receptor (MOR) and ERα. Immunoprecipitates with anti-MOR antibody showed the presence of ERα in cells incubated with E2 in the presence of naloxone but not in cells incubated with E2 or naloxone alone. Higher amounts of ERα associated with MOR after 60 minutes compared with 10 minutes of incubation. Naloxone inhibited E2-bovine serum albumin-FITC binding to plasma membrane–associated ERα and also inhibited the direct binding of [3H]E2 to ERα. Thus, naloxone modulates ERα activity directly as well as indirectly via MOR. This study suggests that naloxone-like compounds can be developed as novel therapeutic molecules for breast cancer therapy. [Mol Cancer Ther 2006;5(3):611–20]

Gene induction by retinoic acid (RA) is suppressed by overexpression of receptor-interacting protein 140 (RIP140). RIP140-mediated suppression was reversed most effectively by overexpressing the coactivator p300/CREB-binding protein-associated factor (P/CAF). Immunoprecipitation demonstrated coexistence of holoreceptors complexed with RIP140 or P/CAF. Chromatin immunoprecipitation revealed rapid RA-enhanced recruitment of RIP140, but delayed P/CAF recruitment, to an RA-targeted promoter in COS-1 cells supplemented with RIP140. In RA-induced P19 cells, endogenous RIP140 was rapidly (within 4 h) and significantly recruited to both the RARβ2 and TR2 genes, whereas the peak of endogenous P/CAF recruitment occurred much later (48 h) and to a lesser degree. Consistent with these observations, significant histone acetylation of endogenous RA receptor (RAR) targets was only observed 48 h following RA treatment. In vitro experiments confirmed RA-induced transcription from a chromatin template, which was reduced by adding RIP140. This study presents evidence for coexistence of multiple RAR-coregulator complexes and a preferential RA-induced recruitment of RIP140 to endogenous RAR-targeted promoters after short term RA treatment, which correlates with suppressed induction of RA-regulated gene expression in the presence of RIP140.

EGFR (Epidermal Growth Factor Receptor) is a major target for the treatment of Non-Small Cell Lung Cancer (NSCLC), but patients lacking EGFR mutation are insensitive to EGFR inhibition. Patients with EGFR mutations initially respond to EGFR tyrosine kinase inhibitors (TKIs), but eventually develop acquired resistance as a result of secondary mutations in the EGFR tyrosine kinase domain. Upregulation of Signal Transducer and Activator of Transcription (STAT3), a key oncogenic molecule in NSCLC that is downstream of EGFR signaling, contributes to intrinsic and acquired resistance to EGFR TKIs. In the clinic, agents targeting STAT3 have not been satisfactory to date. To address the limitations of available STAT3 inhibitors, we are currently testing a cyclic oligonucleotide molecule with a novel mechanism that acts as a STAT3 “decoy” (Cyclic STAT3 decoy, CS3D). CS3D mimics the DNA consensus sequence in the promoter region of STAT3-responsive genes, causing the binding of STAT3 dimers and preventing transcriptional regulation of STAT3-target genes. The effects of CS3D are compared to mutant cyclic STAT3 decoy (CS3M), which differs from CS3D by one base-pair. We have tested the ability of CS3D to produce anti-tumor effects in NSCLC cells that are EGFR WT (201T) and in EGFR mutant cells that also carry the T790M resistant mutation (H1975). Initial in vitro studies showed that CS3D caused a 50% inhibition in cell proliferation in 201T and H1975 relative to CS3M using MTS assays. Flow cytometry studies also demonstrated that CS3D caused a 2-fold increase in the percent of apoptotic cells as compared to CS3M. CS3D also caused a 2-fold reduction in expression of STAT3-target genes c-Myc, Bcl-xL and IL-6. Compared to CS3M, CS3D inhibited colony formation by 70%. Using an in vivo mouse xenograft model of 201T and H1975, CS3D caused a 96.5% reduction in tumor growth in 201T (P Citation Format: Christian Njatcha, Mariya Farooqui, Jennifer R. Grandis, Jill M. Siegfried. Targeting the EGFR/STAT3 axis in NSCLC with resistance to EGFR tyrosine kinase inhibitors using an oligonucleotide-based decoy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4101. doi:10.1158/1538-7445.AM2017-4101

The estrogen receptor (ER) promotes cell proliferation in non-small cell lung cancer (NSCLC). Since fibroblast growth factors (FGFs) are known regulators of stem cell markers in ER positive breast cancer, we investigated whether a link between the estrogen pathway, FGFs, and stem cell markers could be demonstrated in NSCLC animal models, human cells lines and tumor tissue. In the lungs of female FVB/N mice exposed to the tobacco carcinogen 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone, NNK, treatment with the anti-estrogen fulvestrant and/or the aromatase inhibitor anastrozole blocked secretion of both FGF2 and FGF9, and reduced expression of the stem cell markers SOX2 and nanog. These effects were observed in the normal airway epilthelium as well as in both lung preneoplasias and lung adenomas. To investigate the effects of β-estradiol (E2), male mice were administered E2 in the drinking water during exposure to NNK for 4 weeks. In this mouse model, the incidence of carcinogen-induced lung preneoplasias increased 1.8-fold (p Citation Format: Laura P. Stabile, Natalie J. Rothenberger, Mariya Farooqui, Sanja Dacic, Jill M. Siegfried. Interaction between the estrogen receptor and fibroblast growth factor receptor pathways in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1005. doi:10.1158/1538-7445.AM2017-1005

Receptor-interacting protein 140 (RIP140) encodes a histone deacetylase (HDAC) inhibitor-sensitive repressive activity. Direct interaction of RIP140 with HDAC1 and HDAC3 occurs in vitro and in vivo as demonstrated in co-immunoprecipitation and glutathione S-transferase pull-down experiments. The HDAC-interacting domain of RIP140 is mapped to its N-terminal domain, between amino acids 78 and 303 based upon glutathione S-transferase pull-down experiments. In chromatin immunoprecipitation assays, it is demonstrated that histone deacetylation occurs at the chromatin region of the Gal4 binding sites as a result of Gal4 DNA binding domain-tethered RIP expression. The immunocomplexes of RIP140 from cells transfected with RIP140 and HDAC are able to deacetylate histone proteins in vitro. This study presents the first evidence for RIP140 as a negative coregulator for nuclear receptor actions by directly recruiting histone deacetylases and categorizes RIP140 as a novel negative coregulator that is able to directly interact with HDACs.

Background Pain and renal dysfunction occur in sickle cell disease. Morphine used to treat pain also co-activates platelet-derived growth factor receptor-β (PDGFR-β), which can adversely affect renal disease. We examined the influence of morphine in mesangial cells in vitro and in mouse kidneys in vivo. Methods Mouse mesangial cells treated with 1 μM morphine in vitro or kidneys of transgenic homozygous or hemizygous sickle or control mice (n=3 for each), treated with morphine (0.75, 1.4, 2.14, 2.8, 3.6, and 4.3 mg kg−1 day−1 in two divided doses during the first, second, third, fourth, fifth, and sixth weeks, respectively), were used. Western blotting, bromylated deoxy uridine incorporation-based cell proliferation assay, reverse transcriptase-polymerase chain reaction, immunofluorescent microscopy, and blood/urine chemistry were used to analyse signalling, cell proliferation, opioid receptor (OP) expression, and renal function. Results Morphine stimulated phosphorylation of PDGFR-β and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) to the same extent as induced by platelet-derived growth factor-BB (PDGF-BB) and promoted a two-fold increase in mesangial cell proliferation. The PDGFR-β inhibitor, AG1296, OP antagonists, and silencing of μ- and κ-OP abrogated morphine-induced MAPK/ERK phosphorylation and proliferation by ∼100%. Morphine treatment of transgenic mice resulted in phosphorylation of PDGFR-β, MAPK/ERK, and signal transducer and activator of transcription 3 (Stat3) in the kidneys. Morphine inhibited micturition and blood urea nitrogen (BUN) clearance and increased BUN and urinary protein in sickle mice. Conclusion Morphine stimulates mitogenic signalling leading to mesangial cell proliferation and promotes renal dysfunction in sickle mice.

e20647Background: We previously reported a 59% disease control rate among EGFR wild-type (WT) patients in a phase II trial of the combination of rilotumumab (an HGF neutralizing antibody) and erlot...

BACKGROUND: The carbohydrate sialyl Lewis X (sLeX) is expressed on leukocytes and carcinoma cells and binds to selectins during inflammatory processes and early metastasis. Synthesis of sLeX depends on activity of enzymes, including α(1,3/1,4) fucosyltransferase (FucT-III). Tumor necrosis factor-α (TNF-α) up-regulates FucT-III, resulting in increased sLeX in the airways of patients with respiratory disease; however, the mechanisms that regulate sLeX in the inflammatory tumor microenvironment are not well understood. METHODS: The authors stably transfected human lung carcinoma cell lines with the FucT-III gene and exposed them to TNF-α to investigate its role in regulation of sLeX expression and selectin-binding ability using semiquantitative real-time polymerase chain reaction and flow cytometry. Cytokine expression was examined in transfected cells using chemiluminescent arrays and enzyme-linked immunosorbent assays, and invasion was studied using Matrigel assays and alterations in morphology. Human lung tissue arrays were analyzed for immunohistochemical detection of sLeX and neutrophils. RESULTS: Stimulation of FucT-III–transfected cells with recombinant human (rh) TNF-α up-regulated sLeX expression and increased E-selectin binding. Transfected cells secreted high levels of interleukin 8, growth-regulated oncogene-α, and mast cell proteinase-1. Cells exposed to rhTNF-α, neutrophil-conditioned media, and cultures with a 5:1 ratio of neutrophils to cancer cells had significantly increased sLeX expression and invasiveness and underwent nonadherent morphologic changes. In lung carcinomas, but not in normal lung tissues, 71% of tumors were highly positive for sLeX expression in areas of increased neutrophil infiltration. CONCLUSIONS: The current results indicated that neutrophils may be recruited to areas of FucT-III activity and sLeX expression in lung carcinomas to enhance the invasive and metastatic potential of lung cancer cells. Cancer 2011. © 2011 American Cancer Society.

Morphine sulfate (MS) stimulates mesangial cell (MC) proliferation, a process central to development of glomerular disease. The purpose of this study was to examine whether specific opioid receptors (OR) and signal transducer and activators of transcription 3 (STAT3) signaling are associated with MS-induced MC proliferation. C57Bl/6J and OR-specific knockout (KO) mice were treated for up to 6 wk with PBS, MS (0.7–2.14 mg/kg), naloxone (equimolar to MS), or MS+naloxone ( n = 6 per group). Glomerular volume and expression of PCNA, Thy1, and ED1/CD68 were analyzed in kidney sections. Cell proliferation and STAT3 phosphorylation were analyzed by bromodeoxyuridine (BrdU) ELISA and Western blot, respectively, in MCs in vitro. MS treatment led to enlarged kidneys and glomerulopathy and naloxone reversed these effects. MS treatment increased glomerular volume in both μ-OR (MOR) KO and δ-OR (DOR) KO mice, but not in κ-OR (KOR) KO mice. To ascertain that MS-induced glomerulopathy in vivo was due to MC proliferation, we further examined the OR-specific effects of MS in MCs in vitro. MS-induced MC proliferation in vitro was inhibited by KOR-specific nor-BNI, but not by DOR or MOR-specific antagonists naltrindol or CTOP, respectively. KOR-specific agonist U50488H stimulated proliferation of MCs, but DOR-specific agonist DPDPE and MOR-specific agonist DAMGO did not. MS failed to stimulate proliferation of MCs from KOR KO mice. MS and KOR agonists induced STAT3 phosphorylation, and STAT3 inhibitor blocked KOR agonist-induced MC proliferation. We show that MS stimulates glomerulopathy and MC proliferation via KOR and STAT3 signaling.

The estrogen signaling pathway induces proliferation in non-small cell lung cancer (NSCLC) and represents a novel therapeutic target for lung cancer. Estrogen receptor β (ERβ) is the predominant ER isoform in lung cancer and we have previously shown that high cytoplasmic ERβ combined with low progesterone receptor (PR) expression is a poor prognostic marker for NSCLC patients. To further elucidate the biological differences between ERβ high/PR low versus ERβ low/PR high expressing lung tumors, a mRNA microarray analysis using the Illumina HT-12 V4 Bead Chip platform was performed using RNA extracted from 64 cases. 165 genes were differentially expressed between these two groups at P Citation Format: Laura P. Stabile, Natalie J. Rothenberger, Marjorie Romkes, Lisa Koodie, Mariya Farooqui, Sanja Dacic, Jill M. Siegfried. Estrogen modulation of fibroblast growth factor signaling in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1861. doi:10.1158/1538-7445.AM2015-1861

Go/Gi coupled G-protein receptor mediated transactivation is critical in the activation of receptor tyrosine kinases (RTK). Here we show that mu opioid receptor (MOR) transactivates Flk1 and platelet-derived growth factor-beta (PDGF-beta) receptors and its agonist morphine stimulates pro-angiogenic and survival-promoting signaling in mouse retinal endothelial cells (mREC). Morphine stimulates mREC proliferation in a dose dependent fashion and promotes survival to the same extent as vascular endothelial growth factor164 (VEGF164). Morphine stimulates mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and Akt phosphorylation in a time dependent manner like VEGF in mREC. Moreover, analogous to VEGF, morphine stimulates oncogenic signal transducer and activator of transcription 3 (STAT3) signaling. Morphine as well as VEGF-induced phospho-STAT3 and phospho-Flk1 immunoprecipitated with MOR-associated proteins. In addition morphine also stimulated MOR associated PDGF-beta receptor phosphorylation. Consistent with the relationship between VEGF and MOR we found that VEGF upregulates MOR protein and RNA expression in mREC. These data suggest that MOR associates and transactivates RTKs for Flk1 and PDGF-beta, which may have a compounding effect on angiogenic signaling in endothelium. Therefore, G-Protein coupled receptors including MOR provide novel targets to develop anti-angiogenic agents.

Receptor interacting protein 140 (RIP140) interacts with retinoic acid receptor (RAR) and retinoid X receptor (RXR) constitutively, but hormone binding enhances this interaction. The ligand-independent interaction is mediated by the amino and central regions of RIP140 which contain a total of nine copies of the LXXLL motif, whereas the agonist-induced interaction is mediated by its carboxyl terminus which contains a novel motif (1063-1076, LTKTNPILYYMLQK). The ligand-independent interaction could be enhanced slightly by agonists, whereas the ligand-dependent interaction was strictly agonist dependent for both RAR and RXR. In the context of heterodimers, ligand occupancy of RXR played a more dominant role for both molecular interaction and biological activity of RIP140. Competition and mutation studies demonstrated an essential role for 1 0 6 7 Asn and 1 0 7 3 Met for a ligand-dependent interaction. A model was proposed to address the constitutive and agonist-dependent interaction of RIP140 with RAR/RXR.

A combination of in vivo and in vitro assays was employed to describe the ligand-independent interaction of the orphan nuclear receptor TR2 and histone deacetylase proteins. The repressive effect of TR2 on transcription of a luciferase reporter driven by a promoter containing a direct repeat-5 (DR5) derived from the human RARbeta gene was suppressed by the addition of the histone deacetylase inhibitor trichostatin A. Immunoprecipitation with FLAG-epitope (MDYKDDDDK)-tagged histone deacetylase proteins was used to demonstrate that TR2 and histone deacetylases 3 or 4 are present in the same immunoprecipitated complex. Deacetylase activity was demonstrated for these coimmunoprecipitates, further confirming the in vivo interaction of TR2 and histone deacetylases. Immunoprecipitation with anti-TR2 antibody was used to demonstrate interaction of TR2 with endogenously expressed histone deacetylases 3 and 4 in COS-1 cells. Dissection of TR2 domains showed that the DNA binding domain of the receptor was responsible for interaction with both histone deacetylases 3 and 4 in glutathione-S-transferase pull-down assays, while the ligand binding domain did not interact. The pull-down data were confirmed with far Western blots that also showed a direct interaction between labeled histone deacetylase proteins and TR2. It is suggested that repression mediated by unliganded TR2 is mediated, in part, by a direct interaction of this receptor with histone deacetylase proteins.

Genomic profiling studies have demonstrated that a number of potentially targetable pathways are capable of contributing to breast cancer, including the fibroblast growth factor receptor (FGFR) pathway. Our studies focus on understanding the mechanisms through which FGFR1 activation in tumor cells leads to pro-tumorigenic changes in the microenvironment. Specifically, using an in an in vivo model of fibroblast growth factor receptor 1 (FGFR1)-induced mammary tumorigenesis, we have found that following FGFR1 activation in mammary epithelial cells, macrophages are recruited to the hyperplastic regions where they are required for angiogenesis and epithelial cell proliferation. To identify the factors that contribute to the pro-tumorigenic microenvironment, we have developed two- and three-dimensional co-culture in vitro models to mimic what is observed in vivo. Using these in vitro models, we have identified that soluble factors derived from FGFR1-activated epithelial cells lead to the upregulation of target genes in macrophages that promote tumor formation, invasion, and migration. Findings from the in vitro studies are further assessed using a mouse mammary tumor model utilizing orthotopic transplants into immunocompetent mice. These model systems will help us identify novel regulators of mammary tumorigenesis and provide new insight into the mechanisms by which macrophages promote tumor formation. Citation Format: Nicholas J. Brady, Mariya Farooqui, Laura Bohrer, Kaylee Schwertfeger. Contribution of macrophages to FGFR-dependent breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A098.

Sialyl Lewis X (sLeX) is a carbohydrate epitope expressed on leukocytes and carcinoma cells that promotes binding of both cell types to vascular endothelium by selectin mediated processes. These binding events are crucial for leukocyte trafficking to lymphoid tissues and sites of inflammation and metastasis of cancer cells. Increased sLeX expression is correlated with advanced disease and a poor prognosis for several types of carcinomas. Synthesis of sLeX is dependent on activity of the enzymes α-(1,3)-fucosyltransferase (FucT-III) and α2,3-sialyltransferase. Exposure to tumor necrosis factor alpha (TNF-α) has been described to upregulate FucT-III resulting in increased sLeX in the airways of patients with respiratory disease, however, little is known about the molecular mechanisms involved in regulation of sLeX expression in the inflammatory tumor microenvironment. We stably transfected lung carcinoma cells with FucT-III to investigate the role of TNF-α in regulation of sLeX expression, selectin binding ability, and metastatic potential of lung carcinoma cells. Stimulation of FucT-III transfected but not mock-transfected H1299 and A549 lung carcinoma cells with recombinant TNF-α resulted in a concentration dependent upregulation of sLeX expression, increased binding of carcinoma cells to E-selectin, and increased migration of transfectants through Matrigel. Lung carcinoma cells transfected with FucT-III demonstrated high secretion of the neutrophil chemoattractant cytokines IL-8, GRO-α and MCP-1. Co-culture of lung carcinoma cells with neutrophil conditioned media and neutrophils isolated from blood at an optimal ratio of 1:5 resulted in significantly increased sLeX expression (p = 0.02 and p = 0.002 respectively). Exposure of transfected carcinoma cells to neutrophil conditioned media and neutrophils resulted in morphological changes consistent with increased invasive potential and a TNF-α dependent increased cellular migration through Matrigel. In patient lung carcinoma specimens but not in normal lung tissues, we observed areas of high sLeX expression that correlated with increased neutrophil infiltration in 80% of tumors. We conclude that FucT-III activity and sLeX expression in lung carcinomas may recruit neutrophils in the tumor microenvironment that enhance the metastatic potential of lung tumor cells. This work was supported in part by funds from the National Institutes of Health/National Cancer Institute grant K08 CA111829-04. 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 2339.

The family of selectin adhesion molecules, CD62-E and P-selectin expressed on vascular endothelium, and L-selectin expressed on leukocytes, bind sialyl-Lewis X (sLeX)-related glycans and are critical for leukocyte trafficking to lymphoid tissues and sites of inflammation. The sLeX-modified core 2 O-glycans (C2-O-sLeX) on leukocytes promote significantly higher affinity selectin binding than sLeX. Our objectives were to determine whether C2-O-sLeX glycans conferred carcinoma cells with high binding activity to the selectins and participated in metastasis. We investigated C2-O-sLeX expression in human colorectal adenocarcinomas and metastatic liver tumors by immunohistochemistry and RT-PCR. C2-O-sLeX was abundantly expressed in adenocarcinomas and mRNA expression of C2GnT1, the enzyme responsible for C2-O-sLeX synthesis, was greater than 15-fold upregulated. C2-O-sLeX was also present in metastatic liver tumors but not normal colorectal or liver tissues. We evaluated human lung (H1299), pancreatic (AsPC-1), colon (LS174T), and hepatic (HepG2) carcinoma cell lines for their expression of C2-O-sLeX or sLeX glycans and ability to bind to selectins. We also examined mRNA expression of genes coding for the enzymes alpha-2,3 fucosyltransferases (FucT-III) and core 2 beta-1,6 N-acetylglucosaminyltransferase (C2GnT1). FucT-III is responsible for sLeX synthesis and the formation of the core 2 branched O-glycan structures is dependent on C2GnT1. The activity of both enzymes is necessary for C2-O-sLeX synthesis. All four carcinoma cell lines expressed similar levels of C2GnT1 mRNA but FucT-III mRNA levels were lower in H1299 and AsPC-1 cells by RT-PCR. LS174T and HepG2 cell lines were 70 to 90% positive for cell surface expression of C2-O-sLeX and between 70 to 97% positive for sLeX by flow cytometry. C2-O-sLeX and sLeX were also detected in these cells by Western blotting. H1299 and AsPC-1 cells did not express these glycans. H1299 and AsPC-1 cells stably transfected with FucT-III were 43% and 7% positive for C2-O-sLeX respectively. Transfected (H1299/FucT-III and AsPC-1/FucTIII) and LS174T and HepG2 cell lines bound avidly to E-selection but not P- and L-selectin. We used shRNA vectors to silence gene expression of C2GnT1 in H1299/FucT-III and LS174T cells. We observed a 40–90% decrease in C2-O-sLeX but not sLeX expression and a 30–50% decrease in binding to E-selectin on cells containing C2GnT1 shRNA compared to unmanipulated cells. Migration of H1299/FucT-III cells across a reconstituted basement membrane was significantly increased compared to untransfected cells using a matrigel invasion assay. In contrast, the migration of H1299 cells containing C2GnT1 shRNA was reduced. Similarly, LS174T cells transfected with C2GnT1 shRNA exhibited significantly reduced migration and proliferation. Our findings demonstrate that C2-O-sLeX glycans are tumor-associated antigens expressed on carcinoma cells. Importantly, C2-O-sLeX glycans are high affinity ligands for E-selectin and play a role in the adhesion of carcinoma cells to endothelium and tumor invasiveness. These findings are relevant to the development of novel biomarkers and allow further insight into the role of sLeX-containing glycans in carcinoma metastasis. C2-O-sLeX may prove useful to predict patient prognosis and survival. This work was supported in part by funds from the National Institutes of Health/National Cancer Institute grant K08 CA111829-04. Citation Information: Cancer Res 2009;69(23 Suppl):C19.

Like VEGF, morphine stimulates MAPK/ERK and Akt, leading to the promotion of angiogenesis via NO dependent signaling (Cancer Res62: 4491, 2002). Morphine acts via pertussis toxin (PT)-dependent G-protein coupled receptors (GPCRS), while VEGF acts via receptor tyrosine kinases (RTKs). We showed that PT-dependent GPCRs transactivate VEGF receptor-2/Flk1 via small GTPase RhoA (JBC277: 4679, 2002; JBC278:20738, 2003). Therefore, we hypothesized that morphine via the mu opioid receptor (MOR) transactivates Flk1 and promotes a pro-angiogenic microenvironment. Morphine-induced proliferation of human umbilical vein endothelial cells (HUVEC) was completely abrogated by Y-27632 (100 μM), a highly selective and potent inhibitor of Rho-associated protein kinases, suggesting the activation of Rho signaling by morphine. Addition of 1 μM morphine potentiated VEGF-induced (10 ng/ml) proliferation of HUVEC by 25%. We observed a 30% increase in intracellular calcium release after VEGF stimulation of HUVEC pre-incubated with morphine as compared to HUVEC pre-incubated with PBS, detected by a change in the fluorescence ratio of the Fura-2 AM dye. These findings show that morphine, via MOR and Rho signaling, transactivates Flk1 leading to the stimulation of calcium signaling and endothelial cell proliferation. To functionally corroborate our hypothesis, we used MOR knockout (MOR-KO) mice and injected them with MOR-replete T241 fibrosarcoma cells. T241 fibrosarcoma tumor growth in vivo showed appearance of palpable and measurable tumors 2 days earlier in wild type (wt) as compared to MOR-KO mice. Tumor growth and angiogenesis were decreased by 20–35% in MOR-KO mice as compared to wt littermates during 3 weeks of tumor growth. None of the MOR-KO showed signs of lung metastasis versus 40% wt mice with metastasis. Morphine (1.42 for the first 2 wks and 2.14 mg/Kg/day later, respectively) stimulated 20–35% tumor growth in wt, but not in MOR-KO mice. Western immunoblotting showed a 10-fold increase in the expression of phospho-Flk1 in morphine treated wt tumors as compared to PBS-treated wt mice. Morphine did not stimulate phospho-Flk1 expression in MOR-KO mice. Western analysis of immunoprecipitates obtained with α-MOR antibody showed the expression of Flk1 and phospho-Flk1 in wt, but were not expressed in MOR-KO tumors. Thus, MOR stimulates the transactivation of Flk1 in wt mice but not in MOR-KO. These in vitro and in vivo data using MOR-KO mice and the MOR agonist, morphine, show that MOR stimulates endothelial proliferation, angiogenesis and promotes tumor growth and metastasis directly as well as by transactivating Flk1 phosphorylation. We speculate that MOR is a critical component of the ‘angiogenic switch’, which regulates the pro-angiogenic and growth promoting tumor microenvironment. Thus, MOR provides a novel target for developing anti-angiogenic therapies.

Background The metastasis of cancer cells and leukocyte extravasation into inflamed tissues share common features. Specialized carbohydrates modified with sialyl Lewis x (sLex) antigens on leukocyte membranes are ligands for selectin adhesion molecules on activated vascular endothelial cells at inflammatory sites. The activity of the enzyme core 2 β1,6 N-acetylglucosaminyltransferase (C2GnT1) in leukocytes greatly increases their ability to bind to endothelial selectins. C2GnT1 is essential for the synthesis of core 2-branched O-linked carbohydrates terminated with sLex (C2-O-sLex). Our goal was to determine the expression profiles of C2-O-sLex in the malignant progression and metastasis of colorectal adenocarcinomas. The well characterized CHO-131 monoclonal antibody (mAb) specifically recognizes C2-O-sLex present in human leukocytes and carcinoma cells. Using CHO-131 mAb, we investigated whether C2-O-sLex was present in 113 human primary colorectal adenocarcinomas, 10 colorectal adenomas, 46 metastatic liver tumors, 28 normal colorectal tissues, and 5 normal liver tissues by immunohistochemistry. We also examined mRNA levels of the enzyme core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT1) in 20 well, 15 moderately, and 2 poorly differentiated colorectal adenocarcinomas, and in 5 normal colorectal tissues by using quantitative real-time polymerase chain reactions (RT-PCR). Results We observed high reactivity with CHO-131 mAb in approximately 70% of colorectal carcinomas and 87% of metastatic liver tumors but a lack of reactivity in colorectal adenomas and normal colonic and liver tissues. Positive reactivity with CHO-131 mAb was very prominent in neoplastic colorectal glands of well to moderately differentiated adenocarcinomas. The most intense staining with CHO-131 mAb was observed at the advancing edge of tumors with the deepest invasive components. Finally, we analyzed C2GnT1 mRNA levels in 37 colorectal adenocarcinomas and 5 normal colorectal tissues by RT-PCR. Significantly, we observed a greater than 15-fold increase in C2GnT1 mRNA levels in colorectal adenocarcinomas compared to normal colorectal tissues. Conclusion C2-O-sLex, detected by the CHO-131 mAb, is a tumor associated antigen whose expression is highly upregulated in colorectal adenocarcinomas and metastatic liver tumors compared to normal tissues. C2-O-sLex is a potentially useful early predictor of metastasis.