Mi-Heon Lee

IL-27 inhibits epithelial-mesenchymal transition and angiogenic factor production in a STAT1-dominant pathway in human non-small cell lung cancer

BackgroundInterleukin-27 signaling is mediated by the JAK-STAT pathway via activation of STAT1 and STAT3, which have tumor suppressive and oncogenic activities, respectively. Epithelial–mesenchymal transition (EMT) and angiogenesis are key processes in carcinogenesis. Although IL-27 has been shown to have potent anti-tumor activity in various cancer models, the role of IL-27 in EMT and angiogenesis is poorly understood. In this study, we investigated the role of IL-27 in regulating EMT and angiogenesis through modulation of the STAT pathways in human non-small cell lung carcinoma (NSCLC) cells.MethodsSTAT activation following IL-27 exposure was measured in human NSCLC cell lines. Expression of epithelial (E-cadherin, γ-catenin) and mesenchymal (N-cadherin, vimentin) markers were assessed by Western blot analysis. Production of pro-angiogenic factors (VEGF, IL-8/CXCL8, CXCL5) were examined by ELISA. Cell motility was examined by an in vitro scratch and transwell migration assays. Selective inhibitors of STAT1 (STAT1 siRNAs) and STAT3 (Stattic) were used to determine whether both STAT1 and STAT3 are required for IL-27 mediated inhibition of EMT and secretion of angiogenic factors.ResultsOur results demonstrate that IL-27 stimulation in NSCLC resulted in 1) STAT1 and STAT3 activation in a JAK-dependent manner, 2) development of epithelial phenotypes, including a decrease in the expression of a transcriptional repressor for E-cadherin (SNAIL), and mesenchymal marker (vimentin) with a reciprocal increase in the expression of epithelial markers, 3) inhibition of cell migration, and 4) reduced production of pro-angiogenic factors. STAT1 inhibition in IL-27–treated cells reversed the IL-27 effect with resultant increased expression of Snail, vimentin and the pro-angiogenic factors. The inhibition of STAT3 activation had no effect on the development of the epithelial phenotype.ConclusionIL-27 induces mesenchymal to epithelial transition and inhibits the production of pro-angiogenic factors in a STAT1–dominant pathway. These findings highlight the importance of STAT1 in repressing lung carcinogenesis and describe a new anti-tumor mechanism of IL-27.

Combination Treatment with Apricoxib and IL-27 Enhances Inhibition of Epithelial-Mesenchymal Transition in Human Lung Cancer Cells through a STAT1 Dominant Pathway

BACKGROUND The cyclooxygenase 2 (COX-2) pathway has been implicated in the molecular pathogenesis of many malignancies, including lung cancer. Apricoxib, a selective COX-2 inhibitor, has been described to inhibit epithelial-mesenchymal transition (EMT) in human malignancies. The mechanism by which apricoxib may alter the tumor microenvironment by affecting EMT through other important signaling pathways is poorly defined. IL-27 has been shown to have anti-tumor activity and our recent study showed that IL-27 inhibited EMT through a STAT1 dominant pathway. OBJECTIVE The purpose of this study is to investigate the role of apricoxib combined with IL-27 in inhibiting lung carcinogenesis by modulation of EMT through STAT signaling. METHODS AND RESULTS Western blot analysis revealed that IL-27 stimulation of human non-small cell lung cancer (NSCLC) cell lines results in STAT1 and STAT3 activation, decreased Snail protein and mesenchymal markers (N-cadherin and vimentin) and a concomitant increase in expression of epithelial markers (E-cadherin, β-and γ-catenins), and inhibition of cell migration. The combination of apricoxib and IL-27 resulted in augmentation of STAT1 activation. However, IL-27 mediated STAT3 activation was decreased by the addition of apricoxib. STAT1 siRNA was used to determine the involvement of STAT1 pathway in the enhanced inhibition of EMT and cell migration by the combined IL-27 and apricoxib treatment. Pretreatment of cells with STAT1 siRNA inhibited the effect of combined IL-27 and apricoxib in the activation of STAT1 and STAT3. In addition, the augmented expression of epithelial markers, decreased expression mesenchymal markers, and inhibited cell migration by the combination treatment were also inhibited by STAT1 siRNA, suggesting that the STAT1 pathway is important in the enhanced effect from the combination treatment. CONCLUSION Combined apricoxib and IL-27 has an enhanced effect in inhibition of epithelial-mesenchymal transition and cell migration in human lung cancer cells through a STAT1 dominant pathway.

Phase I Trial of Intratumoral Injection of CCL21 Gene–Modified Dendritic Cells in Lung Cancer Elicits Tumor-Specific Immune Responses and CD8+ T-cell Infiltration

Purpose: A phase I study was conducted to determine safety, clinical efficacy, and antitumor immune responses in patients with advanced non–small cell lung carcinoma (NSCLC) following intratumoral administration of autologous dendritic cells (DC) transduced with an adenoviral (Ad) vector expressing the CCL21 gene (Ad-CCL21-DC). We evaluated safety and tumor antigen–specific immune responses following in situ vaccination (ClinicalTrials.gov: NCT01574222). Experimental Design: Sixteen stage IIIB/IV NSCLC subjects received two vaccinations (1 × 106, 5 × 106, 1 × 107, or 3 × 107 DCs/injection) by CT- or bronchoscopic-guided intratumoral injections (days 0 and 7). Immune responses were assessed by tumor antigen–specific peripheral blood lymphocyte induction of IFNγ in ELISPOT assays. Tumor biopsies were evaluated for CD8+ T cells by IHC and for PD-L1 expression by IHC and real-time PCR (RT-PCR). Results: Twenty-five percent (4/16) of patients had stable disease at day 56. Median survival was 3.9 months. ELISPOT assays revealed 6 of 16 patients had systemic responses against tumor-associated antigens (TAA). Tumor CD8+ T-cell infiltration was induced in 54% of subjects (7/13; 3.4-fold average increase in the number of CD8+ T cells per mm2). Patients with increased CD8+ T cells following vaccination showed significantly increased PD-L1 mRNA expression. Conclusions: Intratumoral vaccination with Ad-CCL21-DC resulted in (i) induction of systemic tumor antigen–specific immune responses; (ii) enhanced tumor CD8+ T-cell infiltration; and (iii) increased tumor PD-L1 expression. Future studies will evaluate the role of combination therapies with PD-1/PD-L1 checkpoint inhibition combined with DC-CCL21 in situ vaccination. Clin Cancer Res; 23(16); 4556–68. ©2017 AACR.

Abstract 339: Apricoxib, a selective COX-2 inhibitor, suppresses IL-27-mediated STAT3 activation and potentiates its inhibition of epithelial to mesenchymal transition in human non-small cell lung cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction The cyclooxygenase 2 (COX-2) pathway has been implicated in the molecular pathogenesis of many cancers. Apricoxib, a selective COX-2 inhibitor, has recently been demonstrated to inhibit epithelial to mesenchymal transition (EMT) in human malignancies. EMT is a critical process in cancer progression and metastasis whereby epithelial cells undergo changes to a migratory, mesenchymal phenotype. The mechanism by which apricoxib may alter the tumor microenvironment by impacting other important pathways in EMT is poorly defined. To investigate this concept, we utilized Interleukin-27 (IL-27), a member of the IL-12 family, which signals through the JAK-STAT pathway and activates transcriptional factors with opposing roles in carcinogenesis, STAT1 (tumor suppressor) and STAT3 (oncogene). IL-27 has been shown to have anti-tumor activity, but understanding of its mechanism is limited. We previously demonstrated that IL-27 activates both STAT1 and STAT3 pathways in human non-small cell lung cancer (NSCLC) and that the balance in STAT1 and STAT3 activation is important in inhibiting EMT. Here, we studied the effect of apricoxib on IL-27-mediated STAT activation and EMT inhibition. We hypothesize that apricoxib modulates STAT1 and STAT3 activation and regulates EMT. Methods A human NSCLC cell line, A549, was pre-treated with apricoxib (80nM-10µM) for up to 24 hours prior to IL-27 stimulation (50ng/mL). Activation of STAT1 and STAT3 proteins, demonstrated by tyrosine phosphorylation, was measured by Western Blot analysis. Additionally, epithelial markers (E-cadherin, γ-catenin, β-catenin) and mesenchymal markers (N-cadherin, vimentin, snail) were evaluated also by Western blotting. Results IL-27 alone induced phosphorylation of STAT1 and STAT3 proteins compared to untreated control. Pre-treatment with apricoxib resulted in decreased levels of IL-27-mediated STAT1 and STAT3 phosphorylation compared to the IL-27 alone group. Apricoxib alone did not cause STAT1 or STAT3 activation. IL-27 treatment alone increased the levels of E-cadherin, γ-catenin, and β-catenin and decreased the levels of N-cadherin, vimentin, and snail compared to untreated control. Pre-treatment with apricoxib for 24 hours prior to IL-27 exposure resulted in potentiation of IL-27-mediated reduction of N-cadherin, vimentin, and snail and further increased the levels of E-cadherin when compared to the IL-27 alone group. There appeared to be no impact on the expression of γ-catenin or β-catenin with the addition of apricoxib to IL-27. Conclusions Apricoxib inhibits IL-27-mediated activation of STAT1 and STAT3, and potentiates the IL-27-mediated inhibition of epithelial to mesenchymal transition. These findings suggest that apricoxib may regulate EMT through modulation of the STAT pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 339. doi:1538-7445.AM2012-339

A perspective on the impact of radiation therapy on the immune rheostat

The advent and success of immune checkpoint inhibitors (ICIs) in cancer treatment has broadened the spectrum of tumours that might be considered “immunogenic” and susceptible to immunotherapeutic (IT) intervention. Not all cancer types are sensitive, and not all patients with any given type respond. Combination treatment of ICIs with an established cytotoxic modality such as radiation therapy (RT) is a logical step towards improvement. For one, RT alone has been shown to be genuinely immunomodulatory and secondly pre-clinical data generally support combined ICI-RT approaches. This new integrated therapy for cancer treatment holds much promise, although there is still a lot to be learned about how best to schedule the treatments, manage the toxicities and determine what biomarkers might predict response, as well as many other issues. This review examines how RT alters the immune rheostat and how it might best be positioned to fully exploit IT.

Increased PD-L1 expression in KRAS mutated premalignant human bronchial epithelial cells is enhanced by LKB1 loss and mediated by ERK activation

Meeting abstracts PD-1/PD-L1 immune checkpoint pathway mediates tumor evasion from the immune system, and may be associated with poor prognosis in lung cancer. Activating KRAS mutations and LKB1 loss are common mutations in non-small cell lung carcinoma (NSCLC). Patients with mutated KRAS

Abstract B20: Premalignant lung lesions demonstrate enhanced PD-L1 upregulation in response to interferon-gamma exposure

Background: Interferon-gamma (IFN-g) is known to play a pivotal role in PD-L1 expression and immune evasion in cancer cells, but there is little known regarding its interactions with premalignant lung lesions. Methods: Immortalized human bronchial epithelial cells (HBEC-vector control), KRAS-mutated (KRASv12) HBEC cells (HBEC-KRAS), p53 knockdown HBEC cells (HBEC-p53), and p53 knockdown/KRAS mutated cells (HBEC-p53/KRAS) were used to assess mRNA expression as well as surface and total protein expression levels of PD-L1 by RT-PCR, flow cytometry, and Western blot before and after treatment with IFN-g. For STAT-1 knockdown, cells were transiently transfected using Lipofectamine RNAiMAX (Thermo Scientific). After 48 hours of transfection, cells were incubated with IFN-g (50 ng/mL) or PBS with 0.1% BSA for 48 hours, then harvested and analyzed by RT-PCR, flow cytometry, and Western blot. An FFPE tissue block from a patient with known premalignant lesions and lung adenocarcinoma was obtained from the UCLA Lung Cancer Tissue Repository and sectioned to create slides for HE Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B20.

Abstract 2324: FRA1 contributes to ERK-mediated increased PD-L1 expression in KRAS mutated premalignant human bronchial epithelial cells

BACKGROUND: Activating KRAS mutations are common driver mutations in non-small cell lung carcinoma. Patients with mutated KRAS demonstrate less benefit from adjuvant chemotherapy and resistance to tyrosine kinase inhibitors. KRAS mutations are known to activate the RAF-MEK-ERK pathway. Fos-related antigen-1 (FRA1) is a MEK/ERK-dependent transcription factor and member of the AP-1 transcription factor superfamily. Immune checkpoint pathways including the PD-1/PD-L1 pathway are involved in immune-mediated tumor evasion. We hypothesize that KRAS mutation directly regulates the PD-1/PD-L1 pathway through ERK activation and FRA1 may be an important transcriptional mediator. METHODS: In order to assess the role of KRAS mutation independent of other somatic mutations and to begin to understand potential immune suppressive pathways operative in pulmonary premalignancy, premalignant human bronchial epithelial cell lines (HBEC) were used instead of human lung cancer cell lines. Four immortalized HBEC (2, 3, 7, and 11 cell lines) with KRAS v12-mutation (HBEC-KRAS) compared to control (HBEC-vector) were used to assess mRNA and protein expression levels of PD-L1 by RT-qPCR, flow cytometry, and western blot. Cell-lines were treated with MEK (ERK kinase) inhibitor (PD0325901) for 24 hours (h) up to 1μM. FRA1 was silenced by transfection with siRNA at 100nM. RESULTS: In comparing HBEC-KRAS to HBEC-vector (wild-type) cells, PD-L1 mRNA (1.3∼3.4 fold) and surface protein expressions (1.2∼2.3 fold by flow cytometry) were increased in all 4 cell lines and confirmed by western blot analyses. PD-L1 mRNA and protein levels were highest in HBEC3-KRAS. MEK inhibition resulted in decreased PD-L1 expression by 10 fold (HBEC3-vector) and 11 fold (HBEC3-KRAS) in mRNA levels and 3 fold (HBEC3-vector and HBEC3-KRAS) in surface protein levels. In comparing HBEC3-KRAS to HBEC3-vector, FRA1 increased mRNA (2.7 fold) and protein level expression (2.8 fold). In comparing HBEC3-KRAS to HBEC3-vector cells, FRA1 silencing (FRA1 siRNA) resulted in reduced PD-L1 (53% at 48 h; 73% at 72 h) and FRA1 (94% at 48 h; 99% at 72 h) by western blot. In comparing HBEC3-KRAS to HBEC3-vector cells, ERK phosphorylation increased 7.9 fold and FRA1 silencing led to inhibition of ERK phosphorylation by 2.8 fold at 72 h by western blot. CONCLUSIONS: Here, we demonstrate that PD-L1 expression is elevated in premalignant KRAS mutated human bronchial epithelial cells, and ERK activation mediates KRAS mutation driven up regulation of PD-L1 and at least in part through FRA1 dependence. Our data suggest that KRAS mutation may directly regulate the PD-1/PD-L1 immune checkpoint pathway through FRA1 and MEK/ERK-dependent transcriptional regulation. Further understanding of KRAS driven molecular pathways that modulate immune checkpoints may elucidate therapeutic targets for potential new combination immune therapies. Citation Format: Mi-Heon Lee, Jane Yanagawa, Tonya C Walser, Jonathan W. Goldman, Edward B. Garon, Gang Zeng, Sherven Sharma, Boning Gao, John Minna, Steven M. Dubinett, Jay M Lee. FRA1 contributes to ERK-mediated increased PD-L1 expression in KRAS mutated premalignant human bronchial epithelial cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2324.

PD-L1 expression correlates with immune response in a Phase I trial of CCL21 gene modified dendritic cell therapy in lung cancer

Amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) has become widely popular for mapping protein-ligand interfaces, for understanding protein-protein interactions, and for discovering dynamic allostery. Several platforms are now available which provide large data sets of amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) data. Although many of these platforms provide some down-stream processing, a comprehensive software that provides the most commonly used down-stream processing tools such as automatic back-exchange correction options, analysis of overlapping peptides, calculations of relative deuterium uptake into regions of the protein after such corrections, rigorous statistical analysis of the significance of uptake differences, and generation of high quality figures for data presentation is not yet available. Here we describe the Deuterium Exchange Correction and Analysis (DECA) software package, which provides all these downstream processing options for data from the most popular mass spectrometry platforms. The major functions of the software are demonstrated on sample data.