Paul C. Pagano

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.

Loss of miR125a expression in a model of K-ras-dependent pulmonary premalignancy.

Understanding the molecular pathogenesis of lung cancer is necessary to identify biomarkers/targets specific to individual airway molecular profiles and to identify options for targeted chemoprevention. Herein, we identify mechanisms by which loss of microRNA (miRNA)125a-3p (miR125a) contributes to the malignant potential of human bronchial epithelial cells (HBEC) harboring an activating point mutation of the K-ras proto-oncogene (HBEC K-ras). Among other miRNAs, we identified significant miR125a loss in HBEC K-ras lines and determined that miR125a is regulated by the PEA3 transcription factor. PEA3 is upregulated in HBEC K-ras cells, and genetic knockdown of PEA3 restores miR125a expression. From a panel of inflammatory/angiogenic factors, we identified increased CXCL1 and vascular endothelial growth factor (VEGF) production by HBEC K-ras cells and determined that miR125a overexpression significantly reduces K-ras–mediated production of these tumorigenic factors. miR125a overexpression also abrogates increased proliferation of HBEC K-ras cells and suppresses anchorage-independent growth (AIG) of HBEC K-ras/P53 cells, the latter of which is CXCL1-dependent. Finally, pioglitazone increases levels of miR125a in HBEC K-ras cells via PEA3 downregulation. In addition, pioglitazone and miR125a overexpression elicit similar phenotypic responses, including suppression of both proliferation and VEGF production. Our findings implicate miR125a loss in lung carcinogenesis and lay the groundwork for future studies to determine whether miR125a is a possible biomarker for lung carcinogenesis and/or a chemoprevention target. Moreover, our studies illustrate that pharmacologic augmentation of miR125a in K-ras–mutated pulmonary epithelium effectively abrogates several deleterious downstream events associated with the mutation. Cancer Prev Res; 7(8); 845–55. ©2014 AACR.

Identification of a Human Airway Epithelial Cell Subpopulation with Altered Biophysical, Molecular, and Metastatic Properties

Lung cancers are documented to have remarkable intratumoral genetic heterogeneity. However, little is known about the heterogeneity of biophysical properties, such as cell motility, and its relationship to early disease pathogenesis and micrometastatic dissemination. In this study, we identified and selected a subpopulation of highly migratory premalignant airway epithelial cells that were observed to migrate through microscale constrictions at up to 100-fold the rate of the unselected immortalized epithelial cell lines. This enhanced migratory capacity was found to be Rac1-dependent and heritable, as evidenced by maintenance of the phenotype through multiple cell divisions continuing more than 8 weeks after selection. The morphology of this lung epithelial subpopulation was characterized by increased cell protrusion intensity. In a murine model of micrometastatic seeding and pulmonary colonization, the motility-selected premalignant cells exhibit both enhanced survival in short-term assays and enhanced outgrowth of premalignant lesions in longer-term assays, thus overcoming important aspects of “metastatic inefficiency.” Overall, our findings indicate that among immortalized premalignant airway epithelial cell lines, subpopulations with heritable motility-related biophysical properties exist, and these may explain micrometastatic seeding occurring early in the pathogenesis of lung cancer. Understanding, targeting, and preventing these critical biophysical traits and their underlying molecular mechanisms may provide a new approach to prevent metastatic behavior. Cancer Prev Res; 10(9); 514–24. ©2017 AACR. See related editorial by Hynds and Janes, p. 491

Abstract B22: The role of e-cigarette exposure on pulmonary epithelial cell transformation

Despite a strong correlation between cigarette smoking and the onset of lung cancer, the prevalence of smoking still remains high. The electronic cigarette (ECIG) is designed to deliver nicotine without combusting tobacco. Since nicotine is widely considered the addictive component in tobacco with limited ability to initiate cancer, ECIGs have been advertised to be a safer alternative to tobacco cigarettes. However, the potential health risks and carcinogenicity of ECIGs have not previously been evaluated. In this study, we assess the impact of ECIG exposure on the carcinogenic potential of immortalized human bronchial epithelial cells on a background of silenced p53 and activated KRAS (H3mut-P53/KRAS). This model is utilized because p53 and KRAS mutations are often observed in the airway of current and former smokers at risk for lung cancer. In anchorage independent growth assays, the in vitro correlate of malignant transformation, we found enhanced colony growth in the HBEC-P53/KRAS cells following a 12-day treatment with high concentrations of ECIG-conditioned media compared to the untreated and low concentration treatment groups. We next assessed the effect of ECIG and exposure on cell invasion using three-dimensional air-liquid interface (ALI) models. HBEC-P53/KRAS cells exhibited invasion-associated morphological changes following a 12-day treatment with the high conditioned media, including increased proliferation, diminished cell-cell cohesion and the appearance of cells percolating out of and breaching the modified basement membrane. Finally, to identify the biological impact of in vitro ECIG exposure in HBECs, we profiled the gene expression of P53/KRAS cells following a 96-hour exposure to ECIG- or tobacco cigarette (TCIG)-conditioned media. We found that epithelial cells exposed to clinically relevant concentrations of ECIG vapor-conditioned media have a gene expression pattern similar to those exposed to TCIG smoke-conditioned media and whole cigarette smoke. Rank-rank hyper-geometric overlap (RRHO) analysis indicated that differential expression - based ranked genes in TCIG and ECIG exposed groups were consistently overlapped at significant levels. There were 263 differentially expressed genes in the cells treated with high ECIG media versus untreated control. Annotations of the identified genes by the Molecular Signature database revealed several enriched biological pathways involved in malignant transformation and epithelial-mesenchymal transition (EMT). We have compared the resulting list of genes to publicly available microarray datasets and identified several transformation-related gene candidates. We are in the process of evaluating their contribution to ECIG-induced dissemination and carcinogenesis in vitro and in vivo. These studies will determine the impact of ECIG exposure on lung carcinogenicity and provide needed scientific guidance to the FDA regarding the physiologic effects of ECIGs. These studies were supported by funding from the following: NIH/NCI #U01CA152751 (SMD, TCW), NCI #U01CA152751-S1 (SMD, TCW, SJP), NCI #U01CA152751-AS (SMD, KK), NCI #T32-CA009120-36 (SMD, SJP, PCP), NIH/NHLBI #T32HL072752 (SMD, EL), University of California Tobacco-Related Disease Research Program (TRDRP) #18FT-0060 (TCW), TRDRP #20KT-0055 (TCW), Prevent Cancer Foundation (SJP), Lung Cancer SPORE (P50CA70907, JDM, JEL) Citation Format: Stacy J. Park, Tonya C. Walser, Linh M. Tran, Catalina Perdomo, Teresa Wang, Long-Sheng Hong, Paul C. Pagano, Elvira L. Liclican, Jill E. Larsen, Kostyantyn Krysan, Michael C. Fishbein, John D. Minna, Marc E. Lenburg, Spira Avrum, Steven Dubinett. The role of e-cigarette exposure on pulmonary epithelial cell transformation. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B22.

Abstract B15: Cell motility and deformability in the pathogenesis of lung cancer.

The most effective therapy for lung cancer is surgical resection, but it recurs in approximately 50% of patients with early stage disease, with recurrence most commonly presenting as metastatic disease. This suggests that micrometastatic disease is often already present at the time of surgery, but below the level of detection of our current imaging studies. This is consistent with the reports of circulating tumor cells in patients with stage-I non-small cell lung cancer (NSCLC). Although metastatic behavior is often considered a late stage event, these clinical findings suggest that the metastatic process is also operative early in the pathogenesis of the disease. These clinical observations are also consistent with recent laboratory-based investigations indicating that dissemination may occur during early tumor development, particularly in the context of the genetic program associated with epithelial-mesenchymal transition (EMT). Our preliminary studies indicate that a key regulator of the EMT program, Snail, is responsible for both transformation and enhanced motility of human bronchial epithelial cells (HBECs). Cells that over-express Snail (HBEC-Snail) show structural features indicative of enhanced motility, including filopodia, lamellipodia, membrane ruffling, and front-rear polarity. HBEC-Snail cells move with up to 50% greater velocity than vector control cells. We find that HBECs expressing genes associated with EMT or bearing common NSCLC driver mutations (p53/KRAS), show marked heterogeneity in their capacity for cell motility. Greater than an order of magnitude difference between the fastest and slowest moving cells was consistently observed in these cells. We hypothesize that enhanced epithelial cell motility is operative during premalignancy and is a driver of early metastatic dissemination. The overarching objective of this research is to elucidate the fundamental mechanisms involved in epithelial cell motility and their potential impact on disease onset and progression by coupling novel in vitro and in vivo models of human lung carcinogenesis and an innovative motility-based cell isolation technique. As a result of isolating these fast moving cells, we have shown that the selected cells move through transwell membranes at a 45- to 120-fold increased frequency compared to their unselected counterparts. We have also found that cellular deformability relates to a motile phenotype. By using atomic force microscopy (AFM) and deformability cytometry (DC), we have determined that highly migratory HBEC-Snail cells are more deformable than unselected cells. In the future, we intend to implant selected cells into mice to determine if there is a physiologically-relevant transformation- or cancer-associated phenotype, such as decreased time to metastasis or increased final metastatic burden. These are the first investigations to tie the physical traits of cell motility and deformability to lung carcinogenesis and early metastatic behavior. We anticipate that further investigation, including transcriptome analysis of selected cells by RNAseq, will yield a more complete understanding of the molecular determinants of lung cancer pathogenesis, which will stimulate development of more effective chemoprevention and early detection, benefiting those at risk for lung cancer and those with early stage disease. These studies were supported by funding from the following: NIH/NCI #T32-CA009120-36 (SMD, PCP, SJP), NCI #U01CA152751 (SMD, TCW), NCI #U01CA152751-S1 (SMD, TCW, SJP), NCI #U01CA152751-AS (SMD, KK), NIH/NHLBI #T32HL072752 (SMD, EL), Department of Veteran Affairs #5I01BX000359 (SMD), University of California Tobacco-Related Disease Research Program (TRDRP) #22DT-0005 (PCP), TRDRP #18FT-0060 (TCW), and TRDRP #20KT-0055 (TCW), Lung Cancer SPORE #P50CA70907 (JDM, JEL), Packard Foundation Fellowship (DDC). Citation Format: Paul C. Pagano, Shivani Sharma, Sean O9Byrne, Henry T. Tse, Stacy J. Park, Elvira L. Liclican, Kostyantyn Krysan, Tonya C. Walser, Jill E. Larsen, John D. Minna, James K. Gimzewski, Dino Di Carlo, Steven M. Dubinett. Cell motility and deformability in the pathogenesis of lung cancer. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B15.

Abstract 932: The impact of e-cigarette exposure on pulmonary epithelium gene expression and transformation

Lung cancer is the leading cause of cancer-related mortality in the United States. Despite a strong correlation between cigarette smoking and the onset of lung cancer, the prevalence of smoking still remains high. The electronic cigarette (ECIG) is designed to deliver nicotine without combusting tobacco. Since nicotine is widely considered the addictive component in tobacco with limited ability to initiate cancer, ECIGs have been advertised to be a safer alternative to tobacco cigarettes (TCIGs). However, the potential health risks and carcinogenicity of ECIGs have not previously been evaluated. In this study, we assess the impact of in vitro ECIG exposure on the carcinogenic potential of immortalized human bronchial epithelial cells on a background of silenced p53 and activated KRAS (H-P53/KRAS). This model is utilized because p53 and KRAS mutations are often observed in the airway of current and former smokers at risk for lung cancer. Our preliminary results suggest that ECIG-induced alterations may be dependent upon the mutational landscape of the airway cells being exposed. In anchorage independent growth assays, the in vitro correlate of malignant transformation, we found enhanced colony growth in the H-P53/KRAS cells following a 10-day treatment with the high nicotine ECIG-conditioned media compared to the untreated and low nicotine treatment groups. We next assessed the effect of ECIG exposure on cell invasion using three-dimensional air-liquid interface (ALI) models. H-P53KRAS cells exhibited invasion-associated morphological changes following a 10-day treatment with the high nicotine conditioned media, including increased proliferation, diminished cell-cell cohesion and the appearance of cells percolating out of and breaching the modified basement membrane. Finally, gene expression profiles show 263 differentially expressed genes following exposure to high nicotine ECIG-conditioned media for 96hrs. The ECIG-conditioned media induced a gene expression pattern similar to TCIG-conditioned media and whole cigarette smoke exposure in the H-P53/KRAS cells. We will next compare the ECIG-induced gene expression signature to carcinogenicity-related gene signatures established in previous and ongoing clinical investigations and test ECIG-altered candidate genes for their ability to drive the malignant transformation of airway epithelial cells. These studies will determine the impact of ECIG exposure on lung carcinogenicity and provide needed scientific guidance to the FDA regarding the physiologic effects of ECIGs. These studies were supported by funding from the following: NIH/NCI #U01CA152751 (SMD, TCW), NCI #U01CA152751-S1 (SMD, TCW, SJP), NCI #U01CA152751-AS (SMD, KK), NCI #T32-CA009120-36 (SMD, SJP, PCP), NIH/NHLBI #T32HL072752 (SMD, EL), University of California Tobacco-Related Disease Research Program (TRDRP) #18FT-0060 (TCW), TRDRP #20KT-0055 (TCW), Lung Cancer SPORE (P50CA70907, JDM, JEL) Citation Format: Stacy J. Park, Tonya C. Walser, Catalina Perdomo, Teresa Wang, Long-Sheng Hong, Paul C. Pagano, Elvira L. Liclican, Kostyantyn Krysan, Jill E. Larsen, Michael C. Fishbein, John D. Minna, Marc E. Lenburg, Avrum Spira, Steven M. Dubinett. The impact of e-cigarette exposure on pulmonary epithelium gene expression and transformation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 932. doi:10.1158/1538-7445.AM2014-932

Abstract A08: The role of miR-125a in the pathogenesis of lung cancer

Lung cancer is the leading cause of cancer-related mortality in the U.S., and despite focused research pertaining to conventional therapies, the five-year survival rate remains only 17%. The lack of a systematic approach for chemoprevention agent selection and the disregard for individual differences when randomizing to intervention groups have led to clinical prevention trials that have failed or, in some cases, increased the risk for lung cancer. In this regard, understanding the molecular pathogenesis of lung cancer development in a given individual can facilitate identification of targets and/or biomarkers specific to their unique airway molecular profile and the application of targeted chemoprevention. This is expected to yield more effective lung cancer prevention and control. microRNAs (miRNA) have the potential to be robust biomarkers for lung cancer. Our work focuses on preclinical studies to identify mechanisms by which loss of miRNA-125a-3p (miR-125a) expression contributes to the malignant potential of pulmonary epithelial cells harboring an activating point mutation of the K-ras proto-oncogene, one of the most clinically challenging genetic changes commonly found in current and former smokers. Normal human bronchial epithelial cells (HBECs) isolated from large airways of patients were immortalized with hTERT and Cdk4 and subsequently manipulated to express oncogenic K-ras (HBEC K-ras). Compared to their respective vector controls (HBEC Vector), the basal expression of miR-125a is significantly reduced by 3- to 4-fold in premalignant HBEC K-ras lines. Subsequent studies demonstrated that the loss of miR-125a expression in K-ras-mutated HBECs is regulated by the PEA3 transcription factor. Compared with Vector, PEA3 expression is upregulated in HBEC K-ras cells at both the mRNA and protein level, and genetic knockdown of PEA3 with siRNA in HBEC K-ras cells restores miR-125a expression. To assess the impact of miR-125a loss on tumor-promoting factors in K-ras-mutated HBECs, we evaluated a panel of inflammatory and angiogenic proteins and found that the basal protein expression of CXCL1 and VEGF was significantly elevated in HBEC3 K-ras cells compared with Vector. Importantly, overexpression of miR-125a significantly reduced the levels of these tumorigenic factors in HBEC K-ras cells, while no changes were observed in Vector. miR-125a overexpression also reduced the increased proliferation rate of HBEC K-ras cells to the level of Vector and suppressed anchorage-independent growth of K-ras/P53-mutated HBECs by 50%, an inhibition found to be dependent on CXCL1. Importantly, our data also show that pioglitazone, a model chemopreventive agent, increases levels of miR-125a in HBEC K-ras cells through downregulation of PEA3. Moreover, pioglitazone exhibits similar anti-tumor activity as miR-125a overexpression. Treatment of HBEC K-ras cells with pioglitazone downregulates expression of VEGF, suppresses proliferation and induces morphological changes indicative of mesenchymal-to-epithelial transition. Taken together, our novel findings identify the loss of miR-125a expression as a putative biomarker for lung carcinogenesis and possible chemoprevention target. Moreover, our studies illustrate how augmentation of miR-125a by a model chemoprevention agent, in the setting of the K-ras-mutated pulmonary epithelium, can abrogate some of the deleterious downstream events associated with this mutation. These studies were supported by NHLBI #T32HL072752 (EL) and Lung Cancer SPORE #P50CA70907 (JDM, JEL). Citation Format: Elvira L. Liclican, Saswati Hazra, Jill E. Larsen, Stacy J. Park, Paul C. Pagano, Tonya C. Walser, Kostyantyn Krysan, John D. Minna, Steven M. Dubinett. The role of miR-125a in the pathogenesis of lung cancer. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr A08.