Sarah Statt

Characterization of a Novel Long Noncoding RNA, SCAL1, Induced by Cigarette Smoke and Elevated in Lung Cancer Cell Lines

The incidence of lung diseases and cancer caused by cigarette smoke is increasing. The molecular mechanisms of gene regulation induced by cigarette smoke that ultimately lead to cancer remain unclear. This report describes a novel long noncoding RNA (lncRNA) that is induced by cigarette smoke extract (CSE) both in vitro and in vivo and is elevated in numerous lung cancer cell lines. We have termed this lncRNA the smoke and cancer-associated lncRNA-1 (SCAL1). This lncRNA is located in chromosome 5, and initial sequencing analysis reveals a transcript with four exons and three introns. The expression of SCAL1 is regulated transcriptionally by nuclear factor erythroid 2-related factor (NRF2), as determined by the small, interfering RNA (siRNA) knockdown of NRF2 and kelch-like ECH-associated protein 1 (KEAP1). A nuclear factor erythroid-derived 2 (NF-E2) motif was identified in the promoter region that shows binding to NRF2 after its activation. Functionally, the siRNA knockdown of SCAL1 in human bronchial epithelial cells shows a significant potentiation of cytotoxicity induced by CSE in vitro. Altogether, these results identify a novel and intriguing new noncoding RNA that may act downstream of NRF2 to regulate gene expression and mediate oxidative stress protection in airway epithelial cells.

High mobility group box 1-induced epithelial mesenchymal transition in human airway epithelial cells

Epithelial–mesenchymal transition (EMT) is implicated in bronchial remodeling and loss of lung function in chronic inflammatory airway diseases. Previous studies showed the involvement of the high mobility group box 1 (HMGB1) protein in the pathology of chronic pulmonary inflammatory diseases. However, the role of HMGB1 in EMT of human airway epithelial cells is still unclear. In this study, we used RNA sequencing to show that HMGB1 treatment regulated EMT-related gene expression in human primary-airway epithelial cells. The top five upregulated genes were SNAI2, FGFBP1, VIM, SPARC (osteonectin), and SERPINE1, while the downregulated genes included OCLN, TJP1 (ZO-1), FZD7, CDH1 (E-cadherin), and LAMA5. We found that HMGB1 induced downregulation of E-cadherin and ZO-1, and upregulation of vimentin mRNA transcription and protein translation in a dose-dependent manner. Additionally, we observed that HMGB1 induced AKT phosphorylation, resulting in GSK3β inactivation, cytoplasmic accumulation, and nuclear translocation of β-catenin to induce EMT in human airway epithelial cells. Treatment with PI3K inhibitor (LY294006) and β-catenin shRNA reversed HMGB1-induced EMT. Moreover, HMGB1 induced expression of receptor for advanced glycation products (RAGE), but not that of Toll-like receptor (TLR) 2 or TLR4, and RAGE shRNA inhibited HMGB1-induced EMT in human airway epithelial cells. In conclusion, we found that HMGB1 induced EMT through RAGE and the PI3K/AKT/GSK3β/β-catenin signaling pathway.

Statin-conferred enhanced cellular resistance against bacterial pore-forming toxins in airway epithelial cells.

Statins are widely used to prevent cardiovascular disease. In addition to their inhibitory effects on cholesterol synthesis, statins have beneficial effects in patients with sepsis and pneumonia, although molecular mechanisms have mostly remained unclear. Using human airway epithelial cells as a proper in vitro model, we show that prior exposure to physiological nanomolar serum concentrations of simvastatin (ranging from 10-1,000 nM) confers significant cellular resistance to the cytotoxicity of pneumolysin, a pore-forming toxin and the main virulence factor of Streptococcus pneumoniae. This protection could be demonstrated with a different statin, pravastatin, or on a different toxin, α-hemolysin. Furthermore, through the use of gene silencing, pharmacological inhibitors, immunofluorescence microscopy, and biochemical and metabolic rescue approaches, we demonstrate that the mechanism of protection conferred by simvastatin at physiological nanomolar concentrations could be different from the canonical mevalonate pathways seen in most other mechanistic studies conducted with statins at micromolar levels. All of these data are integrated into a protein synthesis-dependent, calcium-dependent model showing the interconnected pathways used by statins in airway epithelial cells to elicit an increased resistance to pore-forming toxins. This research fills large gaps in our understanding of how statins may confer host cellular protection against bacterial infections in the context of airway epithelial cells without the confounding effect from the presence of immune cells. In addition, our discovery could be potentially developed into a host-centric strategy for the adjuvant treatment of pore-forming toxin associated bacterial infections.

Targeting Myristoylated Alanine-Rich C Kinase Substrate Phosphorylation Site Domain in Lung Cancer. Mechanisms and Therapeutic Implications

RATIONALE Phosphorylation of myristoylated alanine-rich C kinase substrate (phospho-MARCKS) at the phosphorylation site domain (PSD) is crucial for mucus granule secretion and cell motility, but little is known concerning its function in lung cancer. OBJECTIVES We aimed to determine if MARCKS PSD activity can serve as a therapeutic target and to elucidate the molecular basis of this potential. METHODS The clinical relevance of phospho-MARCKS was first confirmed. Next, we used genetic approaches to verify the functionality and molecular mechanism of phospho-MARCKS. Finally, cancer cells were pharmacologically inhibited for MARCKS activity and subjected to functional bioassays. MEASUREMENTS AND MAIN RESULTS We demonstrated that higher phospho-MARCKS levels were correlated with shorter overall survival of lung cancer patients. Using shRNA silencing and ectopic expression of wild-type and PSD-mutated (S159/163A) MARCKS, we showed that elevated phospho-MARCKS promoted cancer growth and erlotinib resistance. Further studies demonstrated an interaction of phosphoinositide 3-kinase with MARCKS, but not with phospho-MARCKS. Interestingly, phospho-MARCKS acted in parallel with increased phosphatidylinositol (3,4,5)-triphosphate pools and AKT activation in cells. Through treatment with a 25-mer peptide targeting the MARCKS PSD motif (MPS peptide), we were able to suppress tumor growth and metastasis in vivo, and reduced levels of phospho-MARCKS, phosphatidylinositol (3,4,5)-triphosphate, and AKT activity. This peptide also enhanced the sensitivity of lung cancer cells to erlotinib treatment, especially those with sustained activation of phosphoinositide 3-kinase/AKT signaling. CONCLUSIONS These results suggest a key role for MARCKS PSD in cancer disease and provide a unique strategy for inhibiting the activity of MARCKS PSD as a treatment for lung cancer.

Dual-specificity phosphatase 6 deficiency regulates gut microbiome and transcriptome response against diet-induced obesity in mice.

The gut microbiota plays profound roles in host metabolism and the inflammatory response associated with the development of obesity. Dusp6-deficient mice have been shown to be resistant to diet-induced obesity, but the mechanism behind this remains unclear. 16S ribosomal RNA gene analysis demonstrated that dusp6-deficient mice harbour unique gut microbiota with resistance to diet-induced-obesity-mediated alteration of the gut microbiome. Using a germ-free mouse model, we found that faecal/gut microbiota derived from dusp6-deficient mice significantly increased energy expenditure and reduced weight gain in recipient wild-type mice fed on a high-fat diet. On analysis of the intestinal transcriptome of dusp6-deficient mice, we found that dusp6 deficiency mainly induced biological processes involved in metabolism and the extracellular matrix, particularly the peroxisome proliferator-activated receptor gamma (Pparγ) pathway and tight-junction genes. Furthermore, dusp6-deficient mice have a high-fat-diet-specific transcriptomic response to reverse the expression of genes associated with intestinal barrier functions and mucosal immunity involved in microbiome homeostasis. This study demonstrates that dusp6 deficiency is a strong genetic factor shaping gut microbiota, and that it confers obesity protection by ameliorating the gut microbiota response to diet-mediated stress.

Lipidome and Transcriptome Profiling of Pneumolysin Intoxication Identifies Networks Involved in Statin-Conferred Protection of Airway Epithelial Cells

Pneumonia remains one of the leading causes of death in both adults and children worldwide. Despite the adoption of a wide variety of therapeutics, the mortality from community-acquired pneumonia has remained relatively constant. Although viral and fungal acute airway infections can result in pneumonia, bacteria are the most common cause of community-acquired pneumonia, with Streptococcus pneumoniae isolated in nearly 50% of cases. Pneumolysin is a cholesterol-dependent cytolysin or pore-forming toxin produced by Streptococcus pneumonia and has been shown to play a critical role in bacterial pathogenesis. Airway epithelium is the initial site of many bacterial contacts and its barrier and mucosal immunity functions are central to infectious lung diseases. In our studies, we have shown that the prior exposure to statins confers significant resistance of airway epithelial cells to the cytotoxicity of pneumolysin. We decided to take this study one step further, assessing changes in both the transcriptome and lipidome of human airway epithelial cells exposed to toxin, statin or both. Our current work provides the first global view in human airway epithelial cells of both the transcriptome and the lipid interactions that result in cellular protection from pneumolysin.

Abstract 1469: Functional roles of SCAL1 expression in lung cancer and progression

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Our group has identified a novel long non-coding RNA (lncRNA), SCAL1 (smoke and cancer-associated lncRNA 1), whose expression is associated with smoking and related to cancer malignancy (Thai et al., 2013, AJRCMB). Currently, we have found that SCAL1 expression is necessary in lung epithelial cells during injury in order to initiate repair after exposure to smoke. In lung cancer cells, SCAL1 expression is needed for cancer cells to combat DNA double stranded breaks associated with chemotherapy treatment, especially cisplatin, doxyrubcin, and possibly other DNA damaging reagents. Most interestingly, the induction of SCAL1 expression is regulated by NRF-2. While NRF2 may protect normal cells from injury, it becomes persistently activated in some cancers and may enhance their malignant characteristics. Therefore, SCAL1 expression is an early marker for lung cellular injury and may be involved in lung cancer progression. We believe that smoke-induced SCAL1 expression leads to epithelial repair and enhances cancer cell resistance to chemotherapy. Our hypothesis is that SCAL1 expression in lung cancer is associated with increased malignant potential and is a poor prognostic marker. Citation Format: Sarah Statt, Philip Thai, Ching-Hsien Chen, Ya-Wen Cheng, Steven Belinsky, Reen Wu. Functional roles of SCAL1 expression in lung cancer and progression. [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 1469. doi:10.1158/1538-7445.AM2014-1469

Abstract 5574: A comprehensive, targeted next-generation sequencing method that rapidly and accurately detects circulating tumor DNA variants at 0.1% frequency in plasma samples

Mutation analysis of circulating tumor DNA (ctDNA) in blood-based liquid biopsies provides a minimally invasive approach to detect and monitor disease. Existing next-generation sequencing (NGS) liquid biopsy techniques have laborious and/or inefficient workflows, heuristic error-correction algorithms, and variable performance with clinical tumor-plasma samples. We present a method that combines an efficient wet-bench workflow with accurate drybench analytics to reduce costs and turnaround time and is relevant to clinical research and patient testing.

Abstract 2596: Suppression of lung cancer malignancy by peptides targeting MARCKS phosphorylation site domain: A comparative study

We have recently reported elevated MARCKS phosphorylation (p-MARCKS) in lung cancer tissues but not in their adjacent normal non-cancer tissue sections. We have extended this study to a tumor tissue array from a cohort of 110 human patients with lung cancer. Immunohistochemistry results have shown a significant association of elevated p-MARCKS with advanced-stage, lymph node metastasis and malignant phenotypes of lung cancer, confirming the importance of p-MARCKS in lung cancer progression. To see if this newly discover biomarker can be a target for therapeutic treatment for the suppression of lung cancer malignancy, we have employed two peptides for this potential; one is the MANS peptide developed by our collaborator many years ago to target myristoylated N-terminal amino acid region for the control of airway mucus granule secretion, the other peptide is MPS that directly targets the MARCKS phosphorylation site domain. For MANS peptide, our recent publication has shown it is effective in blocking lung cancer cell migration and invasiveness in vitro as well as in vivo metastasis. A similar observation was seen for MPS peptide treatment. However, in contrast to MANS peptide, MPS treatment can directly block PIP3-dependent AKT signaling and repress lung tumor growth in vivo in addition to the suppression of metastasis, while MANS can only reduce cancer metastasis. These results support the initial notion on the involvement of p-MARCKS as a biomarker in lung cancer and the potential to target the phosphorylation site domain for controlling cancer progression. Our data have also suggested a direct targeting MARCKS phosphorylation site domain by MPS peptide is more effective in the suppression of lung cancer malignancy. Citation Format: Ching-Hsien Chen, Sarah Statt, Phillip Thai, Jasmine G. Lee, Kenneth B. Adler, Pan-Chyr Yang, Reen Wu. Suppression of lung cancer malignancy by peptides targeting MARCKS phosphorylation site domain: A comparative study. [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 2596. doi:10.1158/1538-7445.AM2014-2596