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Epidermal growth factor receptor (EGFR) mutations are common in lung adenocarcinomas, especially from nonsmoking women of Asian descent. We have previously shown EGFR mutations occur in >70% of lung adenocarcinoma from nonsmokers in our population with a complex mutational profile, including 13% of EGFR double mutations. In this study, we investigated the in vitro gefitinib response of four EGFR double mutants identified in untreated patients, including Q787R+L858R, E709A+G719C, T790M+L858R, and H870R+L858R. The phosphorylation profiles of EGFR and downstream effectors AKT, STAT3/5, and ERK1/2 were compared by immunoblot analyses among the single and double mutants transfected into H358 cells. Results showed that mutants responded to in vitro gefitinib treatment with different sensitivities. The G719C and L858R single mutants showed the highest gefitinib sensitivity compared with the corresponding coexisting single mutants E709A, Q787R, H870R, and T790M. The double mutants E709A+G719C, Q787R+L858R, and H870R+L858R showed attenuated responses to gefitinib in the EGFR and downstream effector phosphorylation profiles compared with G719C or L858R alone. T790M+L858R showed strong resistance to gefitinib. Clinically, the patient whose tumor contained H870R+L858R showed tumor stabilization by 250 mg oral gefitinib daily but cerebral metastasis developed 6 months later. Correlation with the in vitro phosphorylation profile of H870R+L858R suggested that treatment failure was probably due to inadequate suppression of EGFR signaling by the drug level attainable in the cerebrospinal fluid at the given oral dosage. Overall, the findings suggested that rare types of EGFR substitution mutations could confer relative gefitinib resistance when combined with the common activating mutants. [Mol Cancer Ther 2009;8(8):2142–51]

Supplementary Tables 1-2 from Double EGFR mutants containing rare EGFR mutant types show reduced in vitro response to gefitinib compared with common activating missense mutations

Tumor initiating cells (TIC) of lung cancer are mainly induced by stress-related plasticity. Calcium/Calmodulin dependent protein kinase II alpha (CAMK2A) is a key calcium signaling molecule activated by exogenous and endogenous stimuli with effects on multiple cell functions but little is known about its role on TIC. In human lung adenocarcinomas (AD), CAMK2A was aberrantly activated in a proportion of cases and was an independent risk factor predicting shorter survivals. Functionally, CAMK2A enhanced TIC phenotypes in vitro and in vivo. CAMK2A regulated SOX2 expression by reducing H3K27me3 and EZH2 occupancy at SOX2 regulatory regions, leading to its epigenetic de-repression with functional consequences. Further, CAMK2A caused kinase-dependent phosphorylation of EZH2 at T487 with suppression of EZH2 activity. Together, the data demonstrated the CAMK2A-EZH2-SOX2 axis on TIC regulation. This study provided phenotypic and mechanistic evidence for the TIC supportive role of CAMK2A, implicating a novel predictive and therapeutic target for lung cancer. Funding: This study was supported by the Research Grants Council General Research Fund (GRF) (GRF 17150916), and the Small Project Funding of the University of Hong Kong (201309176108). Declaration of Interest: The authors disclose no potential conflicts of interest. Ethical Approval: Primary lung cancer tissues from ethnic Chinese were collected in Queen Mary Hospital with informed consent after approval by HKU/HAHKWC Ethics Board. All animal experiments were approved and performed according to guidelines by the Animal Ethics Committee, the University of Hong Kong.

Tumor-initiating cells (TIC) are dynamic cancer cell subsets that display enhanced tumor functions and resilience to treatment but the mechanism of TIC induction or maintenance in lung cancer is not fully understood. In this study, we show the calcium pathway transcription factor NFATc2 is a novel regulator of lung TIC phenotypes, including tumorspheres, cell motility, tumorigenesis, as well as in vitro and in vivo responses to chemotherapy and targeted therapy. In human lung cancers, high NFATc2 expression predicted poor tumor differentiation, adverse recurrence-free and cancer-specific overall survivals. Mechanistic investigations identified NFATc2 response elements in the 3’ enhancer region of SOX2, and NFATc2/SOX2 coupling upregulates ALDH1A1 by binding to its 5’ enhancer. Through this axis, oxidative stress induced by cancer drug treatment is attenuated, leading to increased resistance in a mutation-independent manner. Targeting this axis provides a novel approach for the long-term treatment of lung cancer through TIC elimination. DOI: http://dx.doi.org/10.7554/eLife.26733.001, eLife digest Cancer develops when cells become faulty and start to grow uncontrollably. They eventually form lumps or tumors, which may spread to surrounding tissues or even to other areas in the body. One of the reasons why cancer treatment remains a challenge is that there are over 200 types of cells in the body, and there are a lot of moments in the life cycle of a cell when things could go wrong. Researchers have shown that many cancers, including lung cancer, are not only extremely different from patient to patient, but also display great differences between cancer cells within the same tumor. Increasing evidence suggest that these differences may be caused by a type of cells called tumor initiating cells, or TICs for short. These TICs behave like stem cells and can renew themselves or mature into different types of cells. They are thought to help cancers grow and spread, and even make them resistant to treatments. Previous research has shown that in many types of cancer, the protein NFATc2 helps cancer cells to grow and spread. Until now, however, it was not known if NFATc2 is also important in TICs in lung cancer. Using human lung cancer cell lines and animal models, Xiao et al. show that the protein NFATc2 stimulates the stem-cell like behavior of TICs. The results showed that TICs had higher levels of the NFATc2 protein than other lung cancer cells that were not TICs. Tumors with higher levels were also more aggressive. When NFATc2 was removed from the cells, they formed smaller tumors and were more sensitive to drug treatment compared to cancer cells with NFATc2. Further experiments revealed that NFATc2 helped to increase the levels of a protein called Sox2, which gives cells the ability to renew or develop into different cell types. Together, these two proteins stimulated the production of another protein that was already known to play a crucial role in TIC maintenance. A better understanding of the mechanisms regulating TICs in lung cancer will help scientists tackle new questions about how this cancer progresses and resists to therapy. In the longer-term, combining classic cancer treatments with new therapeutic strategies targeting NFATc2 could make treatments for lung cancer patients more effective. DOI: http://dx.doi.org/10.7554/eLife.26733.002

// Jing Liu 1 , Zhijie Xiao 1 , Sunny Kit-Man Wong 1 , Vicky Pui-Chi Tin 1 , Ka-Yan Ho 1 , Junwen Wang 2 , Mai-Har Sham 2 , Maria Pik Wong 1 1 Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR China 2 Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR China Correspondence: Maria Pik Wong, email: // Keywords : lung cancer, tumor initiating cell, aldehyde dehydrogenase, CD44, drug resistance Received : August 5, 2013 Accepted : August 28, 2013 Published : August 30, 2013 Abstract Limited improvement in long term survival of lung cancer patients has been achieved by conventional chemotherapy or targeted therapy. To explore the potentials of tumor initiating cells (TIC)-directed therapy, it is essential to identify the cell targets and understand their maintenance mechanisms. We have analyzed the performance of ALDH/CD44 co-expression as TIC markers and treatment targets of lung cancer using well-validated in vitro and in vivo analyses in multiple established and patient-derived lung cancer cells. The ALDH hi CD44 hi subset showed the highest enhancement of stem cell phenotypic properties compared to ALDH hi CD44 lo , ALDH lo CD44 hi , ALDH lo CD44 lo cells and unsorted controls. They showed higher invasion capacities, pluripotency genes and epithelial-mesenchymal transition transcription factors expression, lower intercellular adhesion protein expression and higher G2/M phase cell cycle fraction. In immunosuppressed mice, the ALDH hi CD44 hi xenografts showed the highest tumor induction frequency, serial transplantability, shortest latency, largest volume and highest growth rates. Inhibition of sonic Hedgehog and Notch developmental pathways reduced ALDH + CD44 + compartment. Chemotherapy and targeted therapy resulted in higher ALDH hi CD44 hi subset viability and ALDH lo CD44 lo subset apoptosis fraction. ALDH inhibition and CD44 knockdown led to reduced stemness gene expression and sensitization to drug treatment. In accordance, clinical lung cancers containing a higher abundance of ALDH and CD44-coexpressing cells was associated with lower recurrence-free survival. Together, results suggested the ALDH hi CD44 hi compartment was the cellular mediator of tumorigenicity and drug resistance. Further investigation of the regulatory mechanisms underlying ALDH hi CD44 hi TIC maintenance would be beneficial for the development of long term lung cancer control.

BACKGROUND: The anaplastic lymphoma kinase (ALK) gene is involved frequently in chromosomal translocations, resulting in fusion genes with different partners found in various lymphoproliferative conditions. It was recently reported in nonsmall cell lung cancer (NSCLC) that the fusion protein encoded by echinoderm microtubule-associated protein-like 4-ALK (EML4-ALK) fusion gene conferred oncogenic properties. The objective of the current study was to identify other possible ALK fusion genes in NSCLC. METHODS: Immunohistochemical analysis was used to screen for aberrant ALK expression in primary NSCLC. The authors used 5′ rapid amplification of complementary DNA ends to screen for potential, novel 5′ fusion partners of ALK other than EML4-ALK. Reverse transcriptase-polymerase chain reaction and fluorescence in situ hybridization analyses were used to confirm the identity of 5′ fusion partners. The genomic breakpoint was verified using genomic sequencing. Overexpression of the novel ALK fusion gene and variants 3a and 3b of EML4-ALK was performed to assess downstream signaling and functional effects. RESULTS: The authors identified a novel gene resulting from the fusion of kinesin family member 5B (KIF5B) exon 15 to ALK exon 20 in a primary lung adenocarcinoma. Western blot analysis of clinical tumor tissues revealed the expression of a protein whose size correlated with that of the predicted KIF5B-ALK. Overexpression of KIF5B-ALK in mammalian cells led to the activation of signal transducer and activator of transcription 3 and protein kinase B and to enhanced cell proliferation, migration, and invasion. CONCLUSIONS: The discovery of the novel KIF5B-ALK variant further consolidated the role of aberrant ALK signaling in lung carcinogenesis. Cancer 2011. © 2011 American Cancer Society.

Lung cancer development is often mediated through oxidative and inflammatory stresses caused by tobacco and environmental carcinogens, suggesting dysregulation of stress mediators might play a role during carcinogenesis. The p38 MAPK pathway is activated by multiple stress stimuli and inflammatory cytokines, and mediates a wide range of cellular processes in a context-specific manner. P38α, a member of the p38 family, is known to regulate the self-renewal and differentiation of adult lung stem cells. While p38α has been reported to mediate tumor suppressive or supportive roles in different cancers, inactivation of p38α in the lung epithelium is involved in KRASG12V induced tumorigenesis. However, whether p38α could be involved in regulating tumor initiating cells (TIC) of lung cancer remains unclear. To investigate this possibility, immunohistochemistry was first used to analyse the nuclear expression of phosphorylation-activated p38α (p-p38α) in 90 human primary lung adenocarcinomas. Results showed higher expression of p-p38α was significantly correlated with longer progression free and overall survivals, as well as a more differentiated tumor histological phenotype, indicating p38α might play a tumor suppressive role and drive tumor differentiation in lung cancer. To further address its functional role on lung TIC, p38α was suppressed by siRNA or pharmacologically inhibited by SB203580. This led to increase of the TIC subset that coexpresses ALDH and CD44 (ALDH+/CD44+-TIC), as well as up-regulated expression of the pluripotency genes SOX2, OCT4, and NANOG in lung cancer cell lines. Functionally, p38α inhibition by SB203580 promoted tumor spheres formation for two consecutive generations. NFAT is a family of transcription factors reported to regulate TIC in various cancers which are activated via de-phosphorylation leading to nuclear translocation. We hypothesize the TIC supportive function of p38α inhibition might be mediated through loss of phosphorylation and subsequent activation of NFATc4. In line with this hypothesis, we showed, using Western blot, p38α inhibition increased the expression of nuclear NFATc4. Moreover, NFATc4 knockdown led to suppressed sphere formation and pluripotency genes expression in lung cancer cell lines. Together, our results indicated p38α mediates a tumor suppressor pathway through downstream NFATc4 inactivation and TIC inhibition. The p38α -NFATc4 stress pathway might serve as a potential target for long term lung cancer treatment through TIC regulation. Citation Format: Zhi-Jie Xiao, Jing Liu, Si-Qi Wang, Xu-Yuan Gao, Vicky Pui-Chi Tin, Maria Pik Wong. P38α suppresses tumor initiating cell phenotypes through inhibition of NFATc4 in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2003.

Lung cancer is the leading cause of cancer mortality. Adaptive responses to microenvironmental stresses are important survival mechanisms of cancer cells that lead to long term treatment failure, with accentuated resilience in cell subsets identified by cancer stem cell markers such as CD44 and ALDH. Reactive oxidative species (ROS) are harmful by-products of both cancer cell proliferation and treatment-induced cell death, the elimination of which is critical for maintaining cellular homeostasis and survival. We previously observed the calcium/calmodulin-dependent protein kinase II alpha (CaMK2A) could enhance cancer stem cell (CSC) properties and attenuate ROS in lung cancer cells. To understand the molecular mechanisms, we focused on its relation to the master transcription factor for ROS regulation, NRF2, and the downstream pathways. To test if NRF2 could be a substrate activated by CaMK2A, total NRF2 proteins were measured in nuclear protein extracted from lung cancer cells. CaMK2A-overexpression (OE) increased, while shRNA-mediated CaMK2A-knockdown (shCaMK2A) reduced nuclear NRF2, indicating NRF2 is a mediator of CaMK2A. To identify downstream molecular pathways, screening for known NRF2 target genes was performed using qPCR. The results identified Glutathione S-transferase P1 (GSTP1), an anti-oxidant and detoxifying enzyme, was a target observed consistently in CaMK2A-OE and shCaMK2A cell lines. This finding was confirmed at the protein levels by western blot, supporting CaMK2A/NRF2/GSTP1 might be a candidate axis playing a supportive role for lung CSC. To obtain further support, we studied the expression levels of GSTP1 in 44 clinical lung cancers from our cohort and the TCGA database, respectively, which showed significant up-regulation in cancers compared to corresponding normal lung samples. Further investigations showed GSTP1 protein levels were higher in tumor-spheres than monolayers of five established lung cancer cell lines. Stable knockdown of GSTP1 led to significant reduction of tumor sphere formation, expression of CD44 and ALDH1, as well as mouse xenograft tumorigenicity. On the other hand, sensitivities to chemotherapy and mutant EGFR targeting drugs were increased. Thus, the in vitro and in vivo data supported that GSTP1 played a role in the maintenance of the stem cell-like phenotypic state in lung cancer. To assess if GSTP1 maintained the CSC phenotype through regulating oxidative stress, its effects on ROS levels was measured using cellROX deep red probes. In shGSTP1 cells, the ROS level was significantly raised while it was reciprocally reduced on GSTP1- OE. Furthermore, ROS challenge experiments using N-acetyl-L-cysteine (NAC) in shGSTP1 cell lines resulted in increased cell sphere formation. Together, the results support the CaMK2A/NRF2/GSTP1 axis increases cancer cell resilience through ROS elimination and enhancement of stem cell-like properties of lung carcinoma, indicating its potential role as a long-term treatment target. Citation Format: SIQI WANG, Zhijie Xiao, Pui Chi Tin, Xuyuan Gao, Judy Wai Ping Yam, Maria Pik Wong. Stress response through theCaMK2A/NRF2/GSTP1 axis strengthens cancer cell resilience and mediates stemness phenotype through ROS attenuation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-052.

Tumours develop from clonally expanded population of cells harbouring aberrations of oncogenes and tumour suppressor genes. In this study, metaphase and array comparative genomic hybridization showed good correlation of aberration profiles in lung adenocarcinoma cell lines from patients with different tobacco exposure. Recurrent DNA gains were found at chromosomes 1, 7, 8, 17, 20, and deletions at 1, 3, 8, 9, 10, 12, 17, 18, 19. Candidate tumour loci and encompassed genes at 7p21 (AGR2), 8q21(TPD52), 20q13 (ZNF217, WFDC2, EEF1A2) and 10p15 (KLF6) were analyzed by dual colour FISH for genomic changes and quantitative PCR for expression changes. Results indicated that EEF1A2 and KLF6 were strong candidates of oncogene and tumour suppressor genes, respectively. This study illustrates, a practical strategy for identifying candidate cancer genes from microarray data.

Aims: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been developed to treat non-small cell lung cancer (NSCLC) patients with EGFR mutation, but TKI resistance is common. Almost half of the acquired resistance patients are due to additional T790M mutation on EGFR (EGFRT790M), thus overcoming TKI resistance is important. In this study, we aim to investigate the role of reactive oxygen species (ROS) in TKI resistance as well as the molecular and biological effects of EGFRT790M after redox manipulation. Results: The basal ROS levels in EGFRT790M-containing TKI-resistant NSCLC cell lines were substantially high. Sixty-three human lung tumors showed higher NADPH oxidase isoform 2 (NOX2) expression than normal lung tissues, which may contribute to high basal ROS in cancer and poor survival. Interestingly, only NOX3 was upregulated by sanguinarine, a pharmacological agent to elevate ROS, and resulted in EGFR overoxidation, degradation, and apoptosis. By contrast, such responses were lacking in EGFRWT cells. Selective EGFRT790M degradation was manipulated by redox imbalance between NOX3 and methionine reductase A (MsrA). Furthermore, the in vivo tumor suppression effect of sanguinarine, NOX3 upregulation, and EGFR degradation were confirmed. Innovation: We have found a new treatment strategy to overcome TKI resistance by selectively inducing EGFRT790M degradation via specific stimulation of methionine 790 (M790) oxidation. It can be achieved via manipulating redox imbalance between NOX3 and MsrA. Conclusion: Targeting EGFR by elevating ROS and redox imbalance is a potential new strategy to develop a new EGFR inhibitor for TKI-resistant patients with a wide therapeutic window between EGFRT790M and EGFRWT. Antioxid. Redox Signal. 24, 263–279.

Background Bronchogenic adenocarcinoma is the predominant histologic subtype of lung cancer, which ranks top in the cancer mortality in both men and women. Female nonsmokers and adenocarcinoma have emerged as a distinct combination in patients with lung cancer in recent decades. Lung cancer cell lines established from patients with known clinical characteristics such as gender and smoking habit would be useful for future research on lung cancer. Methods Four new lung adenocarcinoma cell lines (HKULC 1–4) were established from Chinese patients with primary lung adenocarcinomas and with different clinical characteristics with respect to age, gender, smoking habits, tumor staging, and previous therapy. They were characterized by immunohistochemical and growth kinetic studies, tests for tumorigenicity in nude mice, epidermal growth factor receptor (EGFR) gene mutation analysis, and in situ hybridization, and gene expression profiling with Affymetrix GeneChip HG-U133A. Results The newly established HKULC lung adenocarcinoma cell lines were maintained for over 70 passages and demonstrated morphologic and immunohistochemical features and growth kinetics of tumor cell lines. One of the four HKULC cell lines, HKULC 3 (derived from a female nonsmoking patient with lung adenocarcinoma), was found to have a deletion at exon 19 of the EGFR gene. EGFR in situ hybridization showed no EGFR gene amplification in these cell lines. HKULC 1 and 4 formed tumor xenografts after inoculation in nude mice. A list of 71 genes that were differentially expressed or showing class predictive significance was identified. These genes included putative tumor suppressor genes ( DKK3, SERPINF1, CDH11, DSC3 , and KLF6 ), genes involved in or related to the EGFR pathways ( ERBB3, MUC1, VAV1 ), genes involved in regulation of cell cycle and proliferation ( CDKN1A and CDKN2A ), a putative oncogene ( EEF1A2 ), and a gene related to metastasis ( MTSS1 ). Discussion Four new lung adenocarcinoma cell lines were established from patients with different clinical characteristics. These characterized cell lines and their gene expression profiles will provide resources for studies of lung cancer biology and in vitro chemotherapeutic drug study.

The role of Calcium/calmodulin-dependent protein kinase II alpha (CaMK2A) in reactive oxygen species homeostasis in lung adenocarcinoma Lung cancer is the most common cancer worldwide and patients have poor prognosis and low survival rate. The unsatisfactory outcomes of lung cancer treatment are related to multiple factors including tumor recrudescence, metastasis, cancer drug resistance, etc. Cancer stem cells are cell subsets within a cancer that excel in tumor properties, especially survival, renewal and resistance to treatment. Reactive oxygen species (ROS) are high energy molecules produced during cellular hyperproliferation and cell death. Studies have shown though a low level of ROS is required to support stem cell functions but excessive ROS damages DNA and predisposes to cell death. Therefore, the ability to attenuate oxidative damage from ROS and maintain redox homeostasis is an important property of aggressive and resilient cancer cells. The multifunctional serine/threonine kinase calcium/calmodulin- dependent protein kinase II alpha (CaMK2A) is a calcium signaling molecule involved in cell growth and stress signals integration. It has been reported to play a tumor-supportive role in osteosarcoma, prostate cancer and leukemia. However, little is known about the impact and mechanisms of CaMK2A on lung cancer and cancer redox homeostasis. This study aims to investigate the role of CaMK2A in ROS regulation in lung cancer stem cells. We measured ROS levels using H2DCFDA probes in cancer stem cells isolated from two lung cancer cell lines by the known lung cancer stem cell marker CD44 and CD166. Compared to CD44- and CD166- non-stem cells, ROS levels were lower in both CD44+ and CD166+ cancer stem cells. In cancer cells with stable CaMK2A knockdown by shRNA (CaMK2A-KD), ROS was suppressed while CaMK2A overexpression led to ROS elevation, suggesting a potential role of CaMK2A on ROS regulation in lung cancer stem cells. To understand the mechanism, activated NRF2 levels, the master transcription factor mediating ROS regulation, were measured in total nuclear protein extracts. By Western blot, nuclear NRF2 protein showed upregulation in CaMK2A overexpression cells and decreased levels in CaMK2A-KD cells, implying NRF2 might be a mediator of CaMK2A in ROS regulation. To further identify downstream regulators, mRNA expression analysis of five ROS-regulatory NRF2 target genes (GSTP1, NQO1, GCLC, HMOX1, TRXR1) was performed. Quantitative PCR results showed significant corresponding changes of some these gene expressions in cell lines, which upregulated in CaMK2A overexpression and downregulated in both CaMK2A-KD cells and CaMK2A pharmacological inhibition cells. Together, the results indicate CaMK2A might regulate ROS status through mediating downstream target genes of NRF2. Citation Format: Siqi Wang, Zhijie Xiao, Vicky Tin, Ian K. Lam, Xuyuan Gao, Maria Wong. The role of CaMK2A in reactive oxygen species homeostasis in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4787. doi:10.1158/1538-7445.AM2017-4787

Peripheral γδ T cells derived from healthy donors were found to exhibit cytotoxicity against a variety of tumor cell lines in vitro, including CNE2, which was established from nasopharyngeal carcinoma (NPC). The anti-tumor effects were further studied in a mouse model. Control nude mice inoculated s.c. with 5 × 106 CNE2 cells regularly developed hypodermal tumors, which progressively increased in size, and animals had a mean survival of 35 ± 3.4 days. Tumor growth was arrested and tumor size was reduced after animals were infused with 5 × 107 γδ T cells derived from a healthy donor. The anti-tumor effects were temporary, however, and tumor growth was resumed after about 1 week in a group of the animals that had been given a single dose of γδ T cells. In another group of animals given 2 doses of γδ cells 1 week apart, resumption of tumor growth was delayed for a further week. Mean survival of the 2 groups was increased to 61 ± 15.7 and 74 ± 12.9 days, respectively. Immunohistology revealed an accumulation of infused cells in tumors attended by focal tumor necrosis in specimens taken 2 days after infusion. Infiltrative cells virtually disappeared from tumor tissues 6 days after infusion, accompanied by increased mitotic indices of tumor cells. These temporal relationships suggested that the accumulation of infused γδ T cells in hypodermal tumors was responsible for the observed anti-tumor effects. © 2001 Wiley-Liss, Inc.

The role of p53 in the pathogenesis of gestational trophoblastic disease (GTD) was investigated. Immunohistochemical studies for p53, its regulator mdm2, and proliferation marker Ki67 were performed on paraffin-embedded tissues of 28 partial moles (PM), 57 complete moles (CM), 14 choriocarcinomas (CCA), and 31 normal placentas. Three antibodies to p53 (DO-7, Ab-2, Ab-3) were used and demonstrated immunoreactivity for wild-type p53 protein predominantly in the nuclei of cytotrophoblasts. Direct DNA sequencing of 36 hydatidiform moles using frozen tissues confirmed an absence of mutational changes in exons 5–8. CCA was found to have the highest p53 protein expression, followed by CM, PM, and normal placenta ( P < 0.001). In normal placentas ( P = 0.0001), PM, and CM ( P = 0.016), an inverse correlation between their gestational age and p53 expression was observed. p53 expression was found to correlate with proliferation index in normal placenta ( P = 0.0001) and all three groups of GTD ( P = 0.012). Significant correlation between p53 and mdm2 expression was also observed ( P < 0.01). The distinctive expression of p53 wild-type protein in the cytotrophoblasts and its positive correlation with the proliferative index suggests that its overexpression in GTD may be related to its effect on cell proliferation. The parallel expression of mdm2 and p53 also supports the presence of an autoregulatory feedback loop in the control of this process. No correlation could be found between clinical progress of the patients with hydatidiform moles, and the p53 ( P = 0.78) or mdm2 protein ( P = 0.54) expression suggesting that neither of them carries any prognostic significance.

Genomic abnormalities are the hallmark of cancers and may harbor potential candidate genes important for cancer development and progression. We performed array comparative genomic hybridization (array CGH) on 36 cases of primary lung adenocarcinoma (AD) using an array containing 2621 BAC or PAC clones spanning the genome at an average interval of 1 Mb. Array CGH identified the commonest aberrations consisting of DNA gains within 1p, 1q, 5p, 5q, 7p, 7q, 8q, 11q, 12p, 13q, 16p, 17q, 20q, and losses with 6q, 9p, 10q and 18q. High-level copy gains involved mainly 7p21-p15 and 20q13.3. Dual color fluorescence in situ hybridization (FISH) was performed on a selective locus for validation of array CGH results. Genomic aberrations were compared with different clinicopathological features and a trend of higher number of aberrations in tumors with aggressive phenotypes and current tobacco exposure was identified. According to array CGH data, 23 candidate genes were selected for quantitative PCR (qPCR) analysis. The concordance observed between the genomic and expression changes in most of the genes suggested that they could be candidate cancer-related genes that contributed to the development of lung AD.

Background: This study investigated whether there were differential survival outcomes to first-line tyrosine kinase inhibitors (TKI) in patients with metastatic non–small-cell lung cancer harboring different subtypes of exon 19 and exon 21 mutations on epidermal growth factor receptor ( EGFR ). Methods: Of 452 patients with stage IIIB and IV non–small-cell lung cancer, 192 patients (42.5%) harbored EGFR mutation and 170 (37.5%) received TKI as first-line treatment. EGFR mutation analysis was performed by direct sequencing. Survival and response outcome were compared among different subtypes of exon 19 and exon 21 EGFR mutations in these 170 patients. Results: Patients harboring exon 19 18-nucleotide deletion (delL747_P753insS) had the shortest median progression-free survival (PFS) (6.5 months), followed by those with 15-nucleotide deletion (delE746_A750) (12.4 months) and mixed insertion/substitution mutations (22.3 months; p = 0.012). However, patients who had exon 19 deletions starting on codon E746 had better median PFS (14.2 months) than those starting on L747 (6.5 months; hazard ratio, 0.445; 95% confidence interval [0.219–0.903]; p = 0.021). Besides, exon 21 L858R derived a longer median PFS than L861R/L861Q (11.4 months versus 2.1 months, respectively; hazard ratio, 0.298; 95% confidence interval [0.090–0.980]; p = 0.034). Conclusions: Different subtypes of EGFR exon 19 and 21 mutations exhibited differential survival to first-line TKI therapy. Detailed sequence evaluation of exon 19 deletions may provide important prognostic information on survival outcome after TKI.

Personalized therapy is becoming a dominant cancer therapeutic strategy, Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been developed to treat non-small-cell lung cancer (NSCLC) patients with EGFR mutation but they only inhibit EGFR phosphorylation, acquired-resistance still remains unavoidable without EGFRT790M removal. Here, we report a new treatment strategy to overcome such drug resistance by selectively inducing EGFRT790M degradation via specific stimulation of methionine 790 (M790) oxidation. The basal reactive oxygen species (ROS) levels in EGFRT790M containing gefitinib-resistant NSCLC cell lines were substantially high and 63 clinical lung tumors showed higher NADPH oxidase 2 (NOX2) expression than 61 normal lung tissues which may contribute to high basal ROS in cancer. Interestingly, only NOX3 was upregulated by sanguinarine, a pharmacological agent to elevate ROS, resulted in EGFR over-oxidation and degradation as well as apoptosis. By contrast, such responses were lacking in EGFRWild-type (WT) cells, suggesting a new promising drug development strategy with wide therapeutic window between EGFRT790M and EGFRWT. Selective EGFRT790M degradation was manipulated by REDOX imbalance between NOX3 and methionine reductase (MsrA). When NOX3 was activated, NADPH, the key substrate of MsrA, was depleted to generate excessive ROS, resulting in inhibition of MsrA physiological reduction function on oxidized-methionine, thus localized methionine 790 oxidation of EGFRT790M occurred. Furthermore, the in vivo tumor suppression effect of sanguinarine, NOX3 upregulation and EGFR degradation were confirmed. These findings indicate that specific inducing EGFRT790M degradation via manipulating REDOX imbalance between NOX3 and MsrA would be promising strategy for treating gefitinib-resistant NSCLC. Citation Format: Elaine Lai-Han Leung, Xing-Xing Fan, Maria Pik Wong, Zhi-Hong Jiang, Zhong-Qiu Liu, Xiao-Jun Yao, Lin-Lin Lu, Yan-Ling Zhou, Li-Fong Yau, Vicky Pui-Chi Tin, Liang Liu. Overcoming gefitinib resistance in non-small-cell lung cancer by inducing epidermal growth factor receptor (EGFR) degradation via methionine 790 oxidation. [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 3034.

Background: The cancer stem cell theory hypothesizes that cancers are perpetuated by cancer stem cells (CSC) or tumor initiating cells (TIC) possessing self-renewal and other stem cell-like properties while differentiated non-stem/initiating cells have a finite life span. To investigate whether the hypothesis is applicable to lung cancer, identification of lung CSC and demonstration of these capacities is essential. Methodology/Principal Finding: The expression profiles of five stem cell markers (CD34, CD44, CD133, BMI1 and OCT4) were screened by flow cytometry in 10 lung cancer cell lines. CD44 was further investigated by testing for in vitro and in vivo tumorigenecity. Formation of spheroid bodies and in vivo tumor initiation ability were demonstrated in CD44+ cells of 4 cell lines. Serial in vivo tumor transplantability in nude mice was demonstrated using H1299 cell line. The primary xenografts initiated from CD44+ cells consisted of mixed CD44+ and CD44- cells in similar ratio as the parental H1299 cell line, supporting in vivo differentiation. Semi-quantitative Real-Time PCR (RT-PCR) showed that both freshly sorted CD44+ and CD44+ cells derived from CD44+-initiated tumors expressed the pluripotency genes OCT4/POU5F1, NANOG, SOX2. These stemness markers were not expressed by CD44- cells. Furthermore, freshly sorted CD44+ cells were more resistant to cisplatin treatment with lower apoptosis levels than CD44- cells. Immunohistochemical analysis of 141 resected non-small cell lung cancers showed tumor cell expression of CD44 in 50.4% of tumors while no CD34, and CD133 expression was observed in tumor cells. CD44 expression was associated with squamous cell carcinoma but unexpectedly, a longer survival was observed in CD44-expressing adenocarcinomas. Conclusion/Significance: Overall, our results demonstrated that stem cell-like properties are enriched in CD44-expressing subpopulations of some lung cancer cell lines. Further investigation is required to clarify the role of CD44 in tumor cell renewal and cancer propagation in the in vivo environment.© 2010 Leung et al., published_or_final_version

The anaplastic lymphoma kinase (ALK) gene is involved frequently in chromosomal translocations, resulting in fusion genes with different partners found in various lymphoproliferative conditions. It was recently reported in nonsmall cell lung cancer (NSCLC) that the fusion protein encoded by echinoderm microtubule-associated protein-like 4-ALK (EML4-ALK) fusion gene conferred oncogenic properties. The objective of the current study was to identify other possible ALK fusion genes in NSCLC.Immunohistochemical analysis was used to screen for aberrant ALK expression in primary NSCLC. The authors used 5' rapid amplification of complementary DNA ends to screen for potential, novel 5' fusion partners of ALK other than EML4-ALK. Reverse transcriptase-polymerase chain reaction and fluorescence in situ hybridization analyses were used to confirm the identity of 5' fusion partners. The genomic breakpoint was verified using genomic sequencing. Overexpression of the novel ALK fusion gene and variants 3a and 3b of EML4-ALK was performed to assess downstream signaling and functional effects.The authors identified a novel gene resulting from the fusion of kinesin family member 5B (KIF5B) exon 15 to ALK exon 20 in a primary lung adenocarcinoma. Western blot analysis of clinical tumor tissues revealed the expression of a protein whose size correlated with that of the predicted KIF5B-ALK. Overexpression of KIF5B-ALK in mammalian cells led to the activation of signal transducer and activator of transcription 3 and protein kinase B and to enhanced cell proliferation, migration, and invasion.The discovery of the novel KIF5B-ALK variant further consolidated the role of aberrant ALK signaling in lung carcinogenesis.

Epidermal growth factor receptor (EGFR) mutations are common in lung adenocarcinomas, especially from nonsmoking women of Asian descent. We have previously shown EGFR mutations occur in >70% of lung adenocarcinoma from nonsmokers in our population with a complex mutational profile, including 13% of EGFR double mutations. In this study, we investigated the in vitro gefitinib response of four EGFR double mutants identified in untreated patients, including Q787R+L858R, E709A+G719C, T790M+L858R, and H870R+L858R. The phosphorylation profiles of EGFR and downstream effectors AKT, STAT3/5, and ERK1/2 were compared by immunoblot analyses among the single and double mutants transfected into H358 cells. Results showed that mutants responded to in vitro gefitinib treatment with different sensitivities. The G719C and L858R single mutants showed the highest gefitinib sensitivity compared with the corresponding coexisting single mutants E709A, Q787R, H870R, and T790M. The double mutants E709A+G719C, Q787R+L858R, and H870R+L858R showed attenuated responses to gefitinib in the EGFR and downstream effector phosphorylation profiles compared with G719C or L858R alone. T790M+L858R showed strong resistance to gefitinib. Clinically, the patient whose tumor contained H870R+L858R showed tumor stabilization by 250 mg oral gefitinib daily but cerebral metastasis developed 6 months later. Correlation with the in vitro phosphorylation profile of H870R+L858R suggested that treatment failure was probably due to inadequate suppression of EGFR signaling by the drug level attainable in the cerebrospinal fluid at the given oral dosage. Overall, the findings suggested that rare types of EGFR substitution mutations could confer relative gefitinib resistance when combined with the common activating mutants. [Mol Cancer Ther 2009;8(8):2142–51]

Molecular-targeted therapy using tyrosine kinase inhibitors against epidermal growth factor receptor (EGFR) is an effective therapy for non–small cell lung cancer that harbor EGFR mutations. This study aimed to investigate the role of Src, a close EGFR associator, as a drug target in NSCLC cells with different EGFR genomic statuses. Src inhibition was achieved using 4-(4′-Phenoxyanilino)-6,7-dimethoxyquinazolinee (SKI-1) and the specificity of action was verified by RNA interference. The results showed that SKI-1 induced significant apoptosis in a dose-dependent manner in cancer cells with high basal Src activation. Activation of FAK and p130Cas was involved in Src-mediated invasion in SKI-1–sensitive cells. SKI-1 inhibited phosphorylation of EGFR as well as EGFR downstream effectors, such as signal transducers and activators of transcription 3/5, extracellular signal-regulated kinase 1/2 and AKT in the mutant cells but not the wild-type cells. This inhibition profile of EGFR implicates that induction of apoptosis and sensitivity of mutant cells to SKI treatment is mediated by EGFR and EGFR downstream pathways. Cotreatment with SKI-1 and gefitinib enhanced apoptosis in cancer cells that contained EGFR mutation and/or amplification. SKI-1 treatment alone induced significant apoptosis in H1975 cells known to be resistant to gefitinib. Src phosphorylation was shown by immunohistochemistry in around 30% of primary lung carcinomas. In 152 adenocarcinomas studied, p-Src was associated with EGFR mutations (P = 0.029). Overall, the findings indicated that Src could be a useful target for treatment of non–small cell lung cancer. Besides EGFR genomic mutations, other forms of EGFR and related family member abnormalities such as EGFR amplification might enhance SKI sensitivity. (Mol Cancer Res 2009;7(6):923–32)

Nicotine and its derivatives, by binding to nicotinic acetylcholine receptors (nAChR) on bronchial epithelial cells, can regulate cellular proliferation and apoptosis via activating the Akt pathway. Delineation of nAChR subtypes in non–small-cell lung cancers (NSCLC) may provide information for prevention or therapeutic targeting. Expression of nAChR subunit genes in 66 resected primary NSCLCs, 7 histologically non-involved lung tissues, 13 NSCLC cell lines, and 6 human bronchial epithelial cell lines (HBEC) was analyzed with quantitative PCR and microarray analysis. Five nonmalignant HBECs were exposed to nicotine in vitro to study the variation of nAChR subunit gene expression with nicotine exposure and removal. NSCLCs from nonsmokers showed higher expression of nAChR α6 (P < 0.001) and β3 (P = 0.007) subunit genes than those from smokers, adjusted for gender. In addition, nAChR α4 (P < 0.001) and β4 (P = 0.029) subunit gene expression showed significant difference between NSCLCs and normal lung. Using Affymetrix GeneChip U133 Sets, 65 differentially expressed genes associated with NSCLC nonsmoking nAChR α6β3 phenotype were identified, which gave high sensitivity and specificity of prediction. nAChR α1, α5, and α7 showed significant reversible changes in expression levels in HBECs upon nicotine exposure. We conclude that between NSCLCs from smokers and nonsmokers, different nAChR subunit gene expression patterns were found, and a 65-gene expression signature was associated with nonsmoking nAChR α6β3 expression. Finally, nicotine exposure in HBECs resulted in reversible differences in nAChR subunit gene expression. These results further implicate nicotine in bronchial carcinogenesis and suggest targeting nAChRs for prevention and therapy in lung cancer. [Cancer Res 2007;67(10):4638–47]

3p21 is an important locus harbouring critical tumour suppressor genes (TSG), which are implicated in the pathogenesis of multiple tumours, including oesophageal carcinoma. RASSF1A is a 3p21.3 candidate TSG frequently inactivated by promoter methylation in multiple tumours. We investigated RASSF1A promoter methylation and gene expression in Chinese oesophageal squamous cell carcinoma (ESCC) to compare it to data from Japanese patients. Methylation-specific PCR (MSP) showed that RASSF1A was partially methylated in 3/7 (43%) cell lines; 22/64 (34%) primary tumours and 3/64 (5%) corresponding non-tumour samples; and was not methylated in 2 immortalized normal oesophageal epithelial cell lines and 6 normal oesophageal epithelium samples. Bisulfite genome sequencing confirmed the MSP results. Promoter hypermethylation correlated well with RASSF1A mRNA down-regulation. Treatment of cell lines with 5-aza-2'-deoxycytidine activated RASSF1A mRNA expression along with promoter demethylation. RASSF1A hypermethylation in the Chinese cohort was much lower than in a published report of Japanese ESCC patients (52%) and cell lines (74%). Our own analysis of Japanese ESCC cell lines for direct comparison also detected a high frequency of RASSF1A hypermethylation (8/10; 80%) and high levels of hypermethylation at each CpG site. No significant association between RASSF1A hypermethylation and histological differentiation (p=0.953), tumour staging (p=0.117), or survival (p=0.7571) was found in Chinese ESCC, unlike the results of Japanese patients. The incidence of oesophageal cancer shows marked variation by geographic area and ethnic group; it is almost three times higher in China than in Japan, indicating possible different pathogenetic mechanisms. Our results show that RASSF1A hypermethylation in ESCC has epidemiological/ethnic differences, and suggest that Chinese ESCC may result from different pathogenetic mechanisms.

The BCL10 gene, originally identified through its recurrent involvement in t(1;14)(p22;q32) of the mucosa-associated lymphoid tissue (MALT) lymphoma, encodes a cytosolic protein composed of 233 amino acid residues with proapoptotic and proinflammatory activities.[1][1] B-cell chronic lymphocytic

BACKGROUND Fabry's disease is an X-linked recessive inborn error of glycosphingolipid catabolism resulting from deficient activity of lysosomal enzyme alpha-galactosidase A causing occlusive microvascular diseases affecting the kidney, heart, peripheral nerves and brain. It is an uncommon disease in the Oriental population. METHODS AND RESULTS We report a Chinese kindred of Fabry's disease and the relevant clinical features are discussed. The diagnosis of Fabry's disease was based on serum alpha-galactosidase A activity and typical histological features from renal biopsy in the index patient. Genetic analysis of two hemizygous male patients revealed a missense mutation predicting a leucine to proline substitution (L14P) in the alpha-galactosidase gene causing classical Fabry's disease in this family. This is a novel point mutation not described previously in the literature and the second report describing novel genetic mutations for Fabry's disease in Chinese patients. CONCLUSIONS Fabry's disease is rare in Chinese patients but this diagnosis should be considered in patients with positive family history of kidney disease and relevant clinical features.

The p38 MAPK cascade regulates body responses to a wide range of stress stimuli in a context-dependent and tissue-specific manner. The isoform p38alpha (p38) mediates both tumor suppressive and tumor-supportive functions in different cancer models. However, its role in lung cancer is not clear. Since EGFR is frequently mutated in lung adenocarcinomas in non-smoking women and p38 is a downstream target of EGFR signaling, it is possible that p38 plays a tumor-supportive role. Using immunohistochemistry to analyze nuclear activated p38 expression in 90 lung adenocarcinomas, we observed p38 expression was significantly associated with well differentiated compared to poorly-differentiated carcinomas (p Citation Format: Jing Liu, Lai-han Leung, Zhi-jie Xiao, Vicky PC Tin, Maria Pik Wong. P38MAPK plays a tumor suppressive role through differentiation of ALDHhigh/CD44high tumor iniating cells in primary human lung adenocarcinoma. [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 1949. doi:10.1158/1538-7445.AM2014-1949

Background: Targeting specific pathways responsible for Tumor Initiating Cells (TIC) maintenance is postulated to be an important therapeutic strategy for more effective control of tumors. Nuclear Factors of Activated T-cells (NFATs), consisting of 4 homologues (NFATc1-c4), are calcium-dependent transcription factors that are activated upon dephosphorylation by the protein phosphatase calcineurin. NFAT is known to be involved in T cell activation and differentiation. Recently, its roles in regulating embryonic stem cell proliferation and cancer survival have been reported. However, the involvement of Calcineurin/NFAT signaling in TIC maintenance remains unknown. Objective: To study the role of Calcineurin/NFAT in regulating TIC properties in non-small cell lung cancer (NSCLC). Methods and results: Analysis of NFAT mRNA expression in a panel of established and early passage patient derived NSCLC cell lines showed NFATc2 and NFATc4 were up-regulated in 92.6% (25 of 27) and 72% (20 of 27) of cancer cells compared to normal bronchial cell line (BEAS-2B), respectively. To study its role in TIC, tumor spheres or flow cytometry-isolated ALDHhigh/CD44high cells were obtained and NFAT expressions and activities were compared between TIC and non-TIC. The expression of NFAT and downstream targets were significantly higher in tumor spheres and ALDHhigh/CD44high cells compared to the corresponding monolayers and ALDHlow/CD44low cells. Correspondingly, luciferase reporter assay also showed higher NFAT activities in tumor spheres compared to monolayers. To study its functional importance, calcineurin and NFAT were inhibited and effects on TIC properties were analyzed. Inhibition of calcineurin by pharmacological inhibitors cyclosporin A (CSA) and FK506 resulted in reduced sphere formation ability, down-regulation of pluripotent genes (SOX2, OCT4, NANOG) and TIC markers (ALDH1A1, CD133, CD166) expression, as well as decreased TIC proportions (ALDHhigh/CD44high and CD133+) by flow cytometry. These effects were verified using siRNA knockdown of PPP3R1 which encodes one of the calcineurin regulatory subunits. Apart from expression changes, stable knockdown of NFATc2 using lentiviral shRNA inhibited in vitro and in vivo tumorigenecity as shown in soft agar colony formation and mice xenografts compared to shRNA control. To explore the clinical relevance of NFAT, NFAT expression levels were analyzed in human resected lung cancers by quantitative PCR and immunohistochemsitry. Results showed that NFATs were significantly up-regulated in clinical NSCLC compared to corresponding normal lung, and higher NFAT expression predicted shorter recurrence free survival. Conclusions: Calcineurin/NFAT signaling is active in both NSCLC and lung TIC. It plays an important role in TIC maintenance. Inhibition of this pathway is a promising approach for long term lung cancer control. Citation Format: Zhi-Jie Xiao, Jing Liu, Vicky Pui-Chi Tin, Maria Pik Wong. Calcineurin/NFAT signaling regulates tumor initiating cell properties in non-small cell lung cancer. [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 1939. doi:10.1158/1538-7445.AM2014-1939

BACKGROUND: The anaplastic lymphoma kinase (ALK) gene is frequently involved in chromosomal translocations resulting in fusion genes with different partners found in various lympho-proliferative conditions. In non-small cell lung cancers (NSCLC), the fusion protein encoded by echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion gene was recently reported to confer oncogenic properties. This study aimed to identify other possible ALK fusion genes and analyze their oncogenic potentials. METHODS: Immunohistochemical analysis was used to screen for aberrant ALK expression in primary NSCLC. 5’ rapid amplification of cDNA ends was used to screen for potential novel 5’ fusion partners of ALK other than EML4-ALK. Reverse transcription polymerase chain reaction and fluorescence in situ hybridization were used to confirm the identity of 5’fusion partner. The genomic breakpoint was verified using genomic sequencing. Overexpression of the novel ALK fusion gene and variants 3a and 3b of EML4-ALK was performed to assess downstream signaling and functional effects. RESULTS: We identified a novel gene resulting from fusion of kinesin family member 5B (KIF5B) exon 15 to ALK exon 20 in a primary lung adenocarcinoma. Western blot of the clinical tumor tissues showed expression of a protein whose size correlated with that of the predicted KIF5B-ALK. Overexpression of KIF5B-ALK in mammalian cells led to STAT3 and AKT activation, enhanced cell proliferation, migration and invasion. These oncogenic properties were comparable to those of EML4-ALK variants. CONCLUSIONS: The discovery of the novel KIF5B-ALK variant with transforming functions further consolidates the role of aberrant ALK signaling in lung carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1121. doi:10.1158/1538-7445.AM2011-1121

The cancer stem cell (CSC) theory suggests that cancer is maintained by a subpopulation of cancer cells which possesses stem cell characteristics, including self renewal, tumor initiation and pluripotent differentiation abilities. A large series of CSC markers has been reported in various cancer types. CD133 is the most frequently used marker and has been applied to isolate cancer stem cells from cancers of the liver, brain, colon and lung, etc. CD44+/CD24-/low were used as CSC markers in breast cancers. CD34 and Sca-1 were used for identification of murine lung stem cells but Sca-1 is not expressed in human tissues. Expression of embryonic genes such as BMI1 and POU5F1 were found in CSC from different tissues and both proteins are key components in maintaining ‘stemness’ of embryonic stem (ES) cells and induced-pluripotent stem (iPS) cells. It still remains unclear whether additional markers are needed to detect lung CSC. To our knowledge, limited studies using CSC markers on a panel of non-small cell lung cancer (NSCLC) cell lines including those raised from Chinese lung cancer patients have been documented. This study aimed to examine the expression profiles of 6 CSC markers (CD24, CD34, CD44, CD133, BMI1 and POU5F1) in a panel of 10 NSCLC cell lines, 163 NSCLC tumor specimens, fetal lung, reactive and regenerating lung tissues. NSCLC cell lines showed expression of CSC markers at various frequencies that ranged from 0-99.7%, suggesting absence of a single universal marker. Six cell lines expressed CD44 while only HCC1833 expressed CD133. Expression of CD44 and CD133 were mutually exclusive. The CD133+ and CD44+ populations showed distinct properties from CD133- and CD44- cells, respectively. Both CD133+ and CD44+ expanded into tumor-spheroids in vitro in non-adherent medium but not CD133- and CD44- cells. CD44+ H1299 cells expressed ‘stemness’ markers including Oct4, Nanog and Sox2 by RT-PCR and immunofluorescence but were lost after induced-differentiation. The clonogenicity of CD44+ cells is significantly higher than CD44- cells and self-renewal ability was maintained after serial passages. CD44+ cells were cisplatin-resistant and initiated tumor formation in nude mice. The xenograft tumors showed poor differentiation that resembled the parental cell line. Freshly sorted tumor cells showed both CD44+ and CD44- populations, suggesting in vivo differentiation. Immunohistochemistry (IHC) results showed CD44 expression in epithelia of developing airways and regenerating lung. In lung cancers, CD44 expression was associated with squamous cell carcinoma (SCC) (P=0.046). When only 96 adenocarcinomas (AD) were included, CD44 expression was associated with non-smokers (P=0.024). Overall, CD44+cells in NSCLC are capable of tumor-initiation and possess stem cell characteristics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-251.

Introduction: Anaplastic lymphoma kinase ( ALK ) rearrangements are present in an important subset of non–small-cell lung cancer (NSCLC) and predict for response to the tyrosine kinase inhibitor crizotinib. In this study, we evaluated the yet unknown frequency and functional role of ALK splicing isoforms in NSCLC. Methods: We analyzed 270 cases of NSCLC for ALK kinase domain splicing aberrations and in addition generated constructs with full-length echinoderm microtubule-associated protein-like 4 ( EML4 )- ALK (E13;A20) and a splicing isoform. Results: Splicing isoforms of the kinase domain of ALK —including complete skipping of exon 23 ( ALK del23, ALK p.I1171fs*42) and exon 27 ( ALK del27, ALK p.T1312fs*0)—were identified in 11.1% (30 of 270 cases) of NSCLC, and these changes coexisted with ALK rearrangements, KRAS mutations, and EGFR mutations. ALK splicing isoforms were observed with full-length EML4-ALK in crizotinib-naive and treated NSCLCs. ALK T1312fs*0 was unable to render cells solely dependent on ALK signaling. Unlike EML4-ALK and EML4-ALK p.L1196M, EML4-ALK T1312fs*0 did not autophosphorylate ALK or other phosphotyrosine sites. Coexpression of equal amounts of EML4-ALK T1312fs*0 and EML4-ALK did not result in resistance to crizotinib, whereas coexpression of EML4-ALK L1196M with EML4-ALK resulted in resistance to inhibition of ALK by crizotinib. Conclusions: ALK kinase splicing isoforms were present in NSCLC and even if translated seemed to be nonfunctional variants of ALK.