Fariz Nurwidya

Epithelial Mesenchymal Transition in Drug Resistance and Metastasis of Lung Cancer

Among all types of cancer, incidence of lung cancer remains the highest with regard to cancer-related mortality. Problems contributing to recurrence of the disease include metastasis and drug resistance. Mounting evidence has demonstrated involvement of epithelial mesenchymal transition (EMT) in cancer progression. EMT is a critical mechanism ensuring tissue remodeling during morphogenesis of multicellular organisms. Therefore, understanding of the biology of this process for identification of potential EMT-targeted therapeutic strategies for the benefit cancer patients is necessary. This review describes recent evidence of EMT involvement in drug resistance and metastasis of cancers, with an emphasis on lung cancer.

Hypoxia increases gefitinib-resistant lung cancer stem cells through the activation of insulin-like growth factor 1 receptor.

Accumulating evidence indicates that a small population of cancer stem cells (CSCs) is involved in intrinsic resistance to cancer treatment. The hypoxic microenvironment is an important stem cell niche that promotes the persistence of CSCs in tumors. Our aim here was to elucidate the role of hypoxia and CSCs in the resistance to gefitinib in non-small cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutation. NSCLC cell lines, PC9 and HCC827, which express the EGFR exon 19 deletion mutations, were exposed to high concentration of gefitinib under normoxic or hypoxic conditions. Seven days after gefitinib exposure, a small fraction of viable cells were detected, and these were referred to as “gefitinib-resistant persisters” (GRPs). CD133, Oct4, Sox2, Nanog, CXCR4, and ALDH1A1–all genes involved in stemness–were highly expressed in GRPs in PC9 and HCC827 cells, and PC9 GRPs exhibited a high potential for tumorigenicity in vivo. The expression of insulin-like growth factor 1 (IGF1) was also upregulated and IGF1 receptor (IGF1R) was activated on GRPs. Importantly, hypoxic exposure significantly increased sphere formation, reflecting the self-renewal capability, and the population of CD133- and Oct4-positive GRPs. Additionally, hypoxia upregulated IGF1 expression through hypoxia-inducible factor 1α (HIF1α), and markedly promoted the activation of IGF1R on GRPs. Knockdown of IGF1 expression significantly reduced phosphorylated IGF1R-expressing GRPs under hypoxic conditions. Finally, inhibition of HIF1α or IGF1R by specific inhibitors significantly decreased the population of CD133- and Oct4-positive GRPs, which were increased by hypoxia in PC9 and HCC827 cells. Collectively, these findings suggest that hypoxia increased the population of lung CSCs resistant to gefitinib in EGFR mutation-positive NSCLC by activating IGF1R. Targeting the IGF1R pathway may be a promising strategy for overcoming gefitinib resistance in EGFR mutation-positive NSCLC induced by lung CSCs and microenvironment factors such as tumor hypoxia.

Hypoxia induces gefitinib resistance in non-small-cell lung cancer with both mutant and wild-type epidermal growth factor receptors

Somatic mutations in the epidermal growth factor receptor (EGFR) gene, such as exon 19 deletion mutations, are important factors in determining therapeutic responses to gefitinib in non‐small‐cell lung cancer (NSCLC). However, some patients have activating mutations in EGFR and show poor responses to gefitinib. In this study, we examined three NSCLC cell lines, HCC827, PC9, and HCC2935, that expressed an EGFR exon 19 deletion mutation. All cells expressed mutant EGFR, but the PC9 and HCC2935 cells also expressed wild‐type EGFR. The HCC827 cells were highly sensitive to gefitinib under both normoxia and hypoxia. However, the PC9 and HCC2935 cells were more resistant to gefitinib under hypoxic conditions compared to normoxia. Phosphorylation of EGFR and ERK was suppressed with gefitinib treatment to a lesser extent under hypoxia. The expression of transforming growth factor‐α (TGFα) was dramatically upregulated under hypoxia, and the knockdown of TGFα or hypoxia‐inducible factor‐1α (HIF1α) reversed the resistance to gefitinib in hypoxic PC9 and HCC2935 cells. Finally, introduction of the wild‐type EGFR gene into the HCC827 cells caused resistance to gefitinib under hypoxia. This phenomenon was also reversed by the knockdown of TGFα or HIF1α. Our results indicate that hypoxia causes gefitinib resistance in EGFR‐mutant NSCLC through the activation of wild‐type EGFR mediated by the upregulation of TGFα. The presence of wild‐type and mutant EGFR along with tumor hypoxia are important factors that should be considered when treating NSCLC patients with gefitinib.

Chronic intermittent hypoxia/reoxygenation facilitate amyloid-β generation in mice.

Previous studies have shown a high prevalence of obstructive sleep apnea (OSA) among patients with Alzheimers disease (AD). However, it is poorly assessed whether chronic intermittent hypoxia (CIH), which is a characteristic of OSA, affects the pathophysiology of AD. We aimed to investigate the direct effect of intermittent hypoxia (IH) in pathophysiology of AD in vivo and in vitro. In vivo, 15 male triple transgenic AD mice were exposed to either CIH or normoxia (5% O2 and 21% O2 every 10 min, 8 h/day for 4 weeks). Amyloid-β (Aβ) profile, cognitive brain function, and brain pathology were evaluated. In vitro, human neuroblastoma SH-SY5Y cells stably expressing wild-type amyloid-β protein precursor were exposed to either IH (8 cycles of 1% O2 for 10 min followed by 21% O2 for 20 min) or normoxia. The Aβ profile in the conditioned medium was analyzed. CIH significantly increased levels of Aβ42 but not Aβ40 in the brains of mice without the increase in hypoxia-inducible factor 1, alpha subunit (HIF-1α) expression. Furthermore, CIH significantly increased intracellular Aβ in the brain cortex. There were no significant changes in cognitive function. IH significantly increased levels of Aβ42 in the medium of SH-SY5Y cells without the increase in the HIF-1α expression. CIH directly and selectively increased levels of Aβ42 in the AD model. Our results suggest that OSA would aggravate AD. Early detection and intervention of OSA in AD may help to alleviate the progression of the disease.

Acquired resistance of non-small cell lung cancer to epidermal growth factor receptor tyrosine kinase inhibitors

Activation of epidermal growth factor receptor (EGFR) triggers anti-apoptotic signaling, proliferation, angiogenesis, invasion, metastasis, and drug resistance, which leads to development and progression of human epithelial cancers, including non-small cell lung cancer (NSCLC). Inhibition of EGFR by tyrosine kinase inhibitors such as gefitinib and erlotinib has provided a new hope for the cure of NSCLC patients. However, acquired resistance to gefitinib and erlotinib via EGFR-mutant NSCLC has occurred through various molecular mechanisms such as T790M secondary mutation, MET amplification, hepatocyte growth factor (HGF) overexpression, PTEN downregulation, epithelial-mesenchymal transition (EMT), and other mechanisms. This review will discuss the biology of receptor tyrosine kinase inhibition and focus on the molecular mechanisms of acquired resistance to tyrosine kinase inhibitors of EGFR-mutant NSCLC.

Treatment with insulin-like growth factor 1 receptor inhibitor reverses hypoxia-induced epithelial-mesenchymal transition in non-small cell lung cancer

Insulin-like growth factor 1 receptor (IGF1R) is expressed in many types of solid tumors including non-small cell lung cancer (NSCLC), and enhanced activation of IGF1R is thought to reflect cancer progression. Epithelial-mesenchymal transition (EMT) has been established as one of the mechanisms responsible for cancer progression and metastasis, and microenvironment conditions, such as hypoxia, have been shown to induce EMT. The purposes of this study were to address the role of IGF1R activation in hypoxia-induced EMT in NSCLC and to determine whether inhibition of IGF1R might reverse hypoxia-induced EMT. Human NSCLC cell lines A549 and HCC2935 were exposed to hypoxia to investigate the expression of EMT-related genes and phenotypes. Gene expression analysis was performed by quantitative real-time PCR and cell phenotypes were studied by morphology assessment, scratch wound assay, and immunofluorescence. Hypoxia-exposed cells exhibited a spindle-shaped morphology with increased cell motility reminiscent of EMT, and demonstrated the loss of E-cadherin and increased expression of fibronectin and vimentin. Hypoxia also led to increased expression of IGF1, IGF binding protein-3 (IGFBP3), and IGF1R, but not transforming growth factor β1 (TGFβ1). Inhibition of hypoxia-inducible factor 1α (HIF1α) with YC-1 abrogated activation of IGF1R, and reduced IGF1 and IGFBP3 expression in hypoxic cells. Furthermore, inhibition of IGF1R using AEW541 in hypoxic condition restored E-cadherin expression, and reduced expression of fibronectin and vimentin. Finally, IGF1 stimulation of normoxic cells induced EMT. Our findings indicated that hypoxia induced EMT in NSCLC cells through activation of IGF1R, and that IGF1R inhibition reversed these phenomena. These results suggest a potential role for targeting IGF1R in the prevention of hypoxia-induced cancer progression and metastasis mediated by EMT.

From tumor hypoxia to cancer progression: the implications of hypoxia-inducible factor-1 expression in cancers

Hypoxia, defined as a decrease of tissue oxygen levels, represents a fundamental pathophysiological condition in the microenvironment of solid tumors. Tumor hypoxia is known to be associated with radio/chemo-resistance and metastasis that eventually lead to cancer progression contributing to poor prognosis in cancer patients. Among transcription factors that accumulated under hypoxic conditions, hypoxia-inducible factor-1 (HIF-1) is a master transcription factor that has received the most intense attention in this field of research due to its capacity to modulate several hundred genes. With a clearer understanding of the HIF-1 pathway, efforts are directed at manipulation of this complex genetic process in order to ultimately decrease cellular HIF-1 levels. Some novel agents have been shown to have HIF-1 inhibition activity through a variety of molecular mechanisms and have provided promising results in the preclinical setting.

Oct4 plays a crucial role in the maintenance of gefitinib-resistant lung cancer stem cells.

Several recent studies have suggested that cancer stem cells (CSCs) are involved in resistance to gefitinib in non-small cell lung cancer (NSCLC). Oct4, a member of the POU-domain transcription factor family, has been shown to be involved in CSC properties of various cancers. We previously reported that Oct4 and the putative lung CSC marker CD133 were highly expressed in gefitinib-resistant persisters (GRPs) in NSCLC cells, and GRPs exhibited characteristic features of the CSCs phenotype. The aim of this study was to elucidate the role of Oct4 in the resistance to gefitinib in NSCLC cells with an activating epidermal growth factor receptor (EGFR) mutation. NSCLC cell lines, PC9, which express the EGFR exon 19 deletion mutation, were transplanted into NOG mice, and were treated with gefitinib in vivo. After 14-17 days of gefitinib treatment, the tumors still remained; these tumors were referred to as gefitinib-resistant tumors (GRTs). PC9-GRTs showed higher expression of Oct4 and CD133. To investigate the role of Oct4 in the maintenance of gefitinib-resistant lung CSCs, we introduced the Oct4 gene into PC9 and HCC827 cells carrying an activating EGFR mutation by lentiviral infection. Transfection of Oct4 significantly increased CD133-positive GRPs and the number of sphere formation, reflecting the self-renewal activity, of PC9 and HCC827 cells under the high concentration of gefitinib in vitro. Furthermore, Oct4-overexpressing PC9 cells (PC9-Oct4) significantly formed tumors at 1 × 10 cells/injection in NOG mice as compared to control cells. In addition, PC9-Oct4 tumors were more resistant to gefitinib treatment as compared to control cells in vivo. Finally, immunohistochemical analysis revealed that Oct4 was highly expressed in tumor specimens of EGFR-mutant NSCLC patients with acquired resistance to gefitinib. Collectively, these findings suggest that Oct4 plays a pivotal role in the maintenance of lung CSCs resistant to gefitinib in EGFR mutation-positive NSCLC.

The Role of Innate and Adaptive Immune Cells in the Immunopathogenesis of Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a chronic and progressive inflammatory disease of the airways and lungs that results in limitations of continuous airflow and is caused by exposure to noxious gasses and particles. A major cause of morbidity and mortality in adults, COPD is a complex disease pathologically mediated by many inflammatory pathways. Macrophages, neutrophils, dendritic cells, and CD8+ T-lymphocytes are the key inflammatory cells involved in COPD. Recently, the non-coding small RNA, micro-RNA, have also been intensively investigated and evidence suggest that it plays a role in the pathogenesis of COPD. Here, we discuss the accumulated evidence that has since revealed the role of each inflammatory cell and their involvement in the immunopathogenesis of COPD. Mechanisms of steroid resistance in COPD will also be briefly discussed.

Meeting report: Current cancer perspectives from the 9th Annual Meeting of the Japanese Society of Medical Oncology

The purposes of Annual Meeting of the Japanese Society of Medical Oncology (JSMO) are to provide a scientific forum among scientists and oncologist, and to bridge research findings from the molecular to clinical application. Cancer treatments can immediately benefit from all areas of oncology: the discovery and clinical application of biomarkers; development of more personalized anticancer therapy including molecular targeted agents; recent findings from clinical trials; and strategies for overcoming drug resistance. International sessions at the 9th Annual Meeting of the JSMO, held in Yokohama, Japan from July 21 to 23, 2011 addressed these issues. The meeting also held a joint symposium with the American Society of Clinical Oncology. Due to space constraints this report will highlight only topics related to thoracic cancer, including controversies in the treatment of advanced cancer, thoracic‐related cancer, such as lung cancer and esophageal carcinoma, and biomarkers.