Wei-An Chang

Tumor Microenvironment: A New Treatment Target for Cancer

Recent advances in cancer therapy encounter a bottleneck. Relapsing/recurrent disease almost always developed eventually with resistance to the initially effective drugs. Tumor microenvironment has been gradually recognized as a key contributor for cancer progression, epithelial-mesenchymal transition of the cancer cells, angiogenesis, cancer metastasis, and development of drug resistance, while dysregulated immune responses and interactions between various components in the microenvironment all play important roles. Future development of anticancer treatment should take tumor microenvironment into consideration. Besides, we also discuss the limitations of current pre-clinical testing models that mainly come from the impossibility in simulating all detailed carcinogenic mechanisms in human, especially failure to create the same tumor microenvironment. With the cumulating knowledge about tumor microenvironment, the design of a novel anticancer therapy may be facilitated and may have better chance for success in cancer eradication.

Liver metastasis predicts poorer prognosis in stage IV lung adenocarcinoma patients receiving first-line gefitinib

OBJECTIVES Gefitinib is currently used as a first-line therapy in patients of advanced non-small cell lung cancer (NSCLC) with susceptible epidermal growth factor receptor (EGFR) mutations. However, treatment outcomes of these patients vary. This study was conducted to evaluate the impact of specific metastatic sites on treatment outcomes of patients with stage IV lung adenocarcinoma with susceptible EGFR mutations receiving first-line gefitinib, focusing on the impact of liver metastasis. MATERIALS AND METHODS Between October 2009 and April 2014, patients of stage IV lung adenocarcinoma harboring EGFR mutation in exon 19 or 21, who received first-line gefitinib treatment, were enrolled in two hospitals and followed until December 22, 2014. The impacts of various clinical features, including sex, age, smoking history, performance status, EGFR mutation site, metastatic sites, etc., on progression-free survival (PFS) and overall survival (OS) were analyzed. RESULTS A total of 148 patients were eligible for analysis. Patients with liver metastasis on initial diagnosis (n=19) had shorter PFS and OS than those without liver metastasis did (median of PFS, 6.7 vs. 11.2 months, p<0.0001; median of OS, 9.2 vs. 17.5 months, p<0.0001). Multivariable Cox regression analysis showed liver metastasis was an independent poor prognostic factor for PFS (HR=2.939 [95% CI: 1.729-4.997], p<0.0001) and OS (HR=3.300 [95% CI: 1.708-6.373], p=0.0004). CONCLUSION Liver metastasis predicts poorer PFS and OS in stage IV lung adenocarcinoma patients with susceptible gene mutations receiving first-line gefitinib. Further study is warranted to elucidate the underlying mechanisms and find treatment modalities to improve prognosis of these patients.

Synergistic effect of lung tumor‐associated dendritic cell‐derived HB‐EGF and CXCL5 on cancer progression

The interaction between cancer cells and their microenvironment is a paradoxical cycle that exacerbates cancer progression and results in metastasis. Our study investigated the mechanism underlying the synergistic enhancement of release of soluble factors from tumor‐associated dendritic cells and its effect on cancer development. The combination of HB‐EGF (heparin‐binding EGF‐like growth factor) and CXCL5 (CXCL5/epithelial neutrophil‐activating peptide‐78) produced a strong synergistic effect on cancer proliferation, epithelial–mesenchymal transition, migration and invasion. CXCL5 not only potentiated the classical EGFR pathway and the AKT and ERK/RSK1/2 signaling pathways but also increased the phosphorylation of heat shock protein 27 (HSP27), which was slightly increased in A549 cells treated with either HB‐EGF or CXCL5 only. Phosphorylated HSP27 stabilized sustained AKT activity by direct interaction, leading to enhanced tumor spheroid formation. Knockdown of HSP27 by shRNA decreased HB‐EGF plus CXCL5‐mediated tumor spheroid formation in a three‐dimensional culture system, suggesting that AKT/HSP27 was required for HB‐EGF/CXCL5‐mediated cancer progression. Inhibiting RSK also reduces the modulation of c‐Fos phosphorylation, Snail upregulation and cell migration by HB‐EGF plus CXCL5, suggesting a synergistic effect of ERK/RSK and HB‐EGF plus CXCL5 on cell migration. In mice, CXCL5 antibody synergistically enhances the efficiency of the tyrosine kinase inhibitor, gefitinib, without increasing its toxicity. These results provide evidence that elucidates potential cross‐points between extracellular signals affecting lung cancer progression. Targeting CXCL5 may provide therapeutic benefits for lung cancer chemotherapy or immunotherapy.

Benzyl butyl phthalate increases the chemoresistance to doxorubicin/cyclophosphamide by increasing breast cancer-associated dendritic cell-derived CXCL1/GROα and S100A8/A9.

Phthalates are used as plasticizers in the manufacture of flexible vinyl, which is used in food contact applications. Phthalates have been demonstrated to have an adverse impact on human health, particularly in terms of cancer development. In the present study, we showed for the first time that benzyl butyl phthalate (BBP) potentiates the effect of tumor‑associated dendritic cells (TADCs) on the chemoresistance of breast cancer. Specific knockdown analysis revealed that S100A9 is the major factor responsible for the chemoresistance of doxorubicin/cyclophosphamide induced by BBP-stimulated TADCs in breast cancer. BBP exposure also increased tumor infiltrating myeloid-derived suppressor cell (MDSC) secretion of S100A8/A9, thereby exacerbating the resistance of breast cancer to doxorubicin with cyclophosphamide. In addition, BBP also stimulated the production of CXCL1/GROα by TADCs, which increased the angiogenesis of breast cancer in a mouse model. Inhibition of CXCL1/GROα by a neutralizing antibody, decreased the BBP-induced angiogenesis induced by BBP after chemotherapy in the mouse model. These results, for the first time, provide evidence that BBP influences the efficacy of chemotherapy by remodeling the tumor microenvironment of breast cancer.

Cysteinyl Leukotriene Receptor Antagonists Decrease Cancer Risk in Asthma Patients

Previous in vitro and in vivo studies have demonstrated the potential of using cysteinyl leukotriene receptor antagonists (LTRAs) for chemoprevention, but this has not been investigated in any clinical setting. We therefore investigated the chemopreventive effect of LTRAs in a nationwide population-based study. From the Taiwan National Health Insurance Research Database, we enrolled adults with newly-diagnosed asthma between 2001 and 2011. Among these patients, each LTRA user was matched with five randomly-selected LTRA non-users by sex, age, asthma diagnostic year and modified Charlson Comorbidity Index score. We considered the development of cancer as the outcome. Totally, 4185 LTRA users and 20925 LTRA non-users were identified. LTRA users had a significantly lower cancer incidence rate than LTRA non-users did. Multivariable Cox regression analyses adjusting for baseline characteristics and comorbidities showed LTRA use was an independent protecting factor (hazard ratio = 0.31 [95% CI: 0.24–0.39]), and cancer risk decreased progressively with higher cumulative dose of LTRAs. In conclusion, this study revealed that the LTRA use decreased cancer risk in a dose-dependent manner in asthma patients. The chemopreventive effect of LTRAs deserves further study.

Hypoxic Lung-Cancer-Derived Extracellular Vesicle MicroRNA-103a Increases the Oncogenic Effects of Macrophages by Targeting PTEN

Hypoxia, the most commonly observed characteristic in cancers, is implicated in the establishment of an immunosuppressive niche. Recent studies have indicated that extracellular vesicle (EV)-mediated cancer-stroma interactions are considered to play a critical role in the regulation of various cellular biological functions, with phenotypic consequences in recipient cells. However, the mechanisms underlying the relationship between EVs and hypoxia during cancer progression remain largely unknown. In this study, we found that EVs derived from hypoxic lung cancers increased M2-type polarization by miR-103a transfer. Decreased PTEN levels caused by hypoxic cancer-cell-derived EV miR-103a increased activation of AKT and STAT3 as well as expression of several immunosuppressive and pro-angiogeneic factors. In contrast, inhibition of miR-103a by an miRNA inhibitor effectively decreased hypoxic cancer-mediated M2-type polarization, improving the cytokine prolife of tumor infiltration macrophages. Macrophages received cancer-cell-derived EV miR-103a feedback to further enhance cancer progression and tumor angiogenesis. Finally, circulating EV miR-103a levels were higher in patients with lung cancer and closely associated with the M2 polarization. In conclusion, our results delineate a novel mechanism by which lung cancer cells induce immunosuppressive and pro-tumoral macrophages through EVs and inspire further research into the clinical application of EV inhibition or PTEN restoration for immunotherapy.

Secreted protein acidic and rich in cysteine (SPARC) induces cell migration and epithelial mesenchymal transition through WNK1/snail in non-small cell lung cancer

The extracellular matrix is a component of physiological microenvironment and a regulator of cellular processes such as migration and proliferation. Secreted Protein Acidic and Rich in Cysteine (SPARC/osteonectin) is an extracellular matrix-associated glycoprotein involved in the regulation of cell proliferation and cell migration in several types of cancers. However, the role of SPARC in lung cancer is paradoxical and details of the regulatory mechanism are not well-known. In this study, we investigated novel SPARC-mediated signaling pathways. Treatment of SPARC increased cell proliferation, migration, and mesenchymal phenotype in two non-small cell lung cancer cell lines, CL1-5 and H1299. We found that these phenotypes were not regulated by focal adhesion kinase and Src kinase, but were mediated by with no lysine (K) kinase 1 (WNK1). Suppression of WNK1 expression decreased the expression of SPARC-induced N-cadherin and smooth muscle actin. Moreover, Snail, an important transcription factor for regulating epithelial–mesenchymal transition, is also involved in SPARC/WNK1 pathway. In a murine tumor model, SPARC treatment significantly induced phosphorylation of Akt and WNK1 in lung tumor nodules when compared to control mice. In conclusion, these data suggest that WNK1 is a novel molecule in SPARC-mediated mesenchymal signaling pathway in non-small cell lung cancer.The extracellular matrix is a component of physiological microenvironment and a regulator of cellular processes such as migration and proliferation. Secreted Protein Acidic and Rich in Cysteine (SPARC/osteonectin) is an extracellular matrix-associated glycoprotein involved in the regulation of cell proliferation and cell migration in several types of cancers. However, the role of SPARC in lung cancer is paradoxical and details of the regulatory mechanism are not well-known. In this study, we investigated novel SPARC-mediated signaling pathways. Treatment of SPARC increased cell proliferation, migration, and mesenchymal phenotype in two non-small cell lung cancer cell lines, CL1-5 and H1299. We found that these phenotypes were not regulated by focal adhesion kinase and Src kinase, but were mediated by with no lysine (K) kinase 1 (WNK1). Suppression of WNK1 expression decreased the expression of SPARC-induced N-cadherin and smooth muscle actin. Moreover, Snail, an important transcription factor for regulating epithelial-mesenchymal transition, is also involved in SPARC/WNK1 pathway. In a murine tumor model, SPARC treatment significantly induced phosphorylation of Akt and WNK1 in lung tumor nodules when compared to control mice. In conclusion, these data suggest that WNK1 is a novel molecule in SPARC-mediated mesenchymal signaling pathway in non-small cell lung cancer.

Identification of novel gene expression signature in lung adenocarcinoma by using next-generation sequencing data and bioinformatics analysis

Lung adenocarcinoma is one of the leading causes of cancer-related death worldwide. We showed transcriptomic profiles in three pairs of tumors and adjacent non-tumor lung tissues using next-generation sequencing (NGS) to screen protein-coding RNAs and microRNAs. Combined with meta-analysis from the Oncomine and Gene Expression Omnibus (GEO) databases, we identified a representative genetic expression signature in lung adenocarcinoma. There were 9 upregulated genes, and 8 downregulated genes in lung adenocarcinoma. The analysis of the effects from each gene expression on survival outcome indicated that 6 genes (AGR2, SPDEF, CDKN2A, CLDN3, SFN, and PHLDA2) play oncogenic roles, and 7 genes (PDK4, FMO2, CPED1, GNG11, IL33, BTNL9, and FABP4) act as tumor suppressors in lung adenocarcinoma. In addition, we also identified putative genetic interactions, in which there were 5 upregulated microRNAs with specific targets - hsa-miR-183-5p-BTNL9, hsa-miR-33b-5p-CPED1, hsa-miR-429-CPED1, hsa-miR-182-5p-FMO2, and hsa-miR-130b-5p-IL33. These 5 microRNAs have been shown to be associated with tumorigenesis in lung cancer. Our findings suggest that these genetic interactions play important roles in the progression of lung adenocarcinoma. We propose that this molecular change of genetic expression may represent a novel signature in lung adenocarcinoma, which may be developed for diagnostic and therapeutic strategies in the future.

Montelukast Induces Apoptosis-Inducing Factor-Mediated Cell Death of Lung Cancer Cells

Developing novel chemo-prevention techniques and advancing treatment are key elements to beating lung cancer, the most common cause of cancer mortality worldwide. Our previous cohort study showed that cysteinyl leukotriene receptor antagonists, mainly montelukast, decreased the lung cancer risk in asthma patients. In the current study, we conducted in vivo and in vitro experiments to demonstrate the inhibiting effect of montelukast on lung cancer and to investigate the underlying mechanisms. Using Lewis lung carcinoma-bearing mice, we showed that feeding montelukast significantly delayed the tumor growth in mice (p < 0.0001). Montelukast inhibited cell proliferation and colony formation and induced the cell death of lung cancer cells. Further investigation showed the down-regulation of B-cell lymphoma 2 (Bcl-2), up-regulation of Bcl-2 homologous antagonist/killer (Bak), and nuclear translocation of apoptosis-inducing factor (AIF) in montelukast-treated lung cancer cells. Montelukast also markedly decreased the phosphorylation of several proteins, such as with no lysine 1 (WNK1), protein kinase B (Akt), extracellular signal-regulated kinase 1/2 (Erk1/2), MAPK/Erk kinase (MEK), and proline-rich Akt substrate of 40-kDa (PRAS40), which might contribute to cell death. In conclusion, montelukast induced lung cancer cell death via the nuclear translocation of AIF. This study confirmed the chemo-preventive effect of montelukast shown in our previous cohort study. The utility of montelukast in cancer prevention and treatment thus deserves further studies.

Laricitrin ameliorates lung cancer-mediated dendritic cell suppression by inhibiting signal transducer and activator of transcription 3

Natural polyphenolic compounds of grapes and their seeds are thought to be therapeutic adjuvants in a variety of diseases, including cancer prevention. This study was carried out to investigate the effect of grape phenolic compounds on the regulation of cancer-mediated immune suppression. Laricitrin exhibits the greatest potential to ameliorate the suppressive effects of lung cancer on dendritic cells’ (DCs’) differentiation, maturation and function. Human lung cancer A549 and CL1-5 cells change the phenotype of DCs that express to high levels of IL-10 and prime T cells towards an immune suppression type-2 response (Th2). Laricitrin treatment stimulated DC differentiation and maturation in the condition media of cancer cells, a finding supported by monocyte marker CD14s disappearance and DC marker CD1as upregulation. Laricitrin decreases expression of IL-10 in cancer-conditioned DCs, and subsequently switches CD4+ T cell response from Th2 to Th1 in vitro and in vivo. Reversal of laricitrin on lung cancer-induced DCs’ paralysis was via inhibiting the phosphorylation of signal transducer and activator of transcription 3 (STAT3). Laricitrin also potentiated the anticancer activity of cisplatin in mouse models. Thus, laricitrin could be an efficacious immunoadjuvant and have a synergistic effect when combined with chemotherapy.