Enhanced autophagy is required for survival in EGFR-independent EGFR-mutant lung adenocarcinoma cells.

Lung cancers harboring epidermal growth factor receptor (EGFR) mutations depend on constitutive activation of the kinase for survival. Although most EGFR-mutant lung cancers are sensitive to EGFR tyrosine kinase inhibitors (TKIs) and shrink in response to treatment, acquired resistance to TKI therapy is common. We demonstrate here that two EGFR-mutated lung adenocarcinoma cell lines, HCC827 and HCC4006, contain a subpopulation of cells that have undergone epithelial-to-mesenchymal transition and survive independent of activated EGFR. These EGFR-independent cancer cells, herein termed gefitinib-resistant (GR) cells, demonstrate higher levels of basal autophagy than their parental cells and thrive under hypoxic, reduced-serum conditions in vitro; this somewhat simulates the hypoxic environment common to cancerous tissues. We show that depletion of the essential autophagy gene, ATG5, by small interfering RNA (siRNA) or chloroquine, an autophagy inhibitor, markedly reduces GR cell viability under hypoxic conditions. Moreover, we show a significant elevation in caspase activity in GR cells following knockdown of ATG5. These results suggest that GR cells can evade apoptosis and survive in hostile, hypoxic environments with constant autophagic flux. We also show the presence of autophagosomes in some cancer cells from patient samples, even in untreated EGFR-mutant lung cancer tissue samples. Together, our results indicate that autophagy inhibitors alone or in combination with EGFR TKIs may be an effective approach for the treatment of EGFR-mutant lung cancers, where basal autophagy of some cancer cells is upregulated.

Lung cancer with loss of BRG1/BRM, shows epithelial mesenchymal transition phenotype and distinct histologic and genetic features.

BRG1 and BRM, two core catalytic subunits in SWI/SNF chromatin remodeling complexes, have been suggested as tumor suppressors, yet their roles in carcinogenesis are unclear. Here, we present evidence that loss of BRG1 and BRM is involved in the progression of lung adenocarcinomas. Analysis of 15 lung cancer cell lines indicated that BRG1 mutations correlated with loss of BRG1 expression and that loss of BRG1 and BRM expression was frequent in E-cadherin-low and vimentin-high cell lines. Immunohistochemical analysis of 93 primary lung adenocarcinomas showed loss of BRG1 and BRM in 11 (12%) and 16 (17%) cases, respectively. Loss of expression of BRG1 and BRM was frequent in solid predominant adenocarcinomas and tumors with low thyroid transcription factor-1 (TTF-1, master regulator of lung) and low cytokeratin7 and E-cadherin (two markers for bronchial epithelial differentiation). Loss of BRG1 was correlated with the absence of lepidic growth patterns and was mutually exclusive of epidermal growth factor receptor (EGFR) mutations. In contrast, loss of BRM was found concomitant with lepidic growth patterns and EGFR mutations. Finally, we analyzed the publicly available dataset of 442 cases and found that loss of BRG1 and BRM was frequent in E-cadherin-low, TTF-1-low, and vimentin-high cases and correlated with poor prognosis. We conclude that loss of either or both BRG1 and BRM is involved in the progression of lung adenocarcinoma into solid predominant tumors with features of epithelial mesenchymal transition and loss of the bronchial epithelial phenotype. BRG1 loss was specifically involved in the progression of EGFR wild-type, but not EGFR-mutant tumors.

Establishment of highly metastatic KRAS mutant lung cancer cell sublines in long-term three-dimensional low attachment cultures.

Decreased cell-substratum adhesion is crucially involved in metastasis. Previous studies demonstrated that lung cancer with floating cell clusters in histology is more likely to develop metastasis. In the present study, we investigated whether cancer cells in long-term, three-dimensional low attachment cultures acquire high metastatic potential; these cells were then used to examine the mechanisms underlying metastasis. Two KRAS-mutated adenocarcinoma cell lines (A549 and H441) were cultured and selected on ultra-low attachment culture dishes, and the resulting cells were defined as FL (for floating) sublines. Cancer cells were inoculated into NOD/SCID mice via an intracardiac injection, and metastasis was evaluated using luciferase-based imaging and histopathology. In vitro cell growth (in attachment or suspension cultures), migration, and invasion were assayed. A whole genomic analysis was performed to identify key molecular alterations in FL sublines. Upon detachment on low-binding dishes, parental cells initially formed rounded spheroids with limited growth activity. However, over time in cultures, cells gradually formed smaller spheroids that grew slowly, and, after 3-4 months, we obtained FL sublines that regained prominent growth potential in suspension cultures. On ordinary dishes, FL cells reattached and exhibited a more spindle-shaped morphology than parental cells. No marked differences were observed in cell growth with attachment, migration, or invasion between FL sublines and parental cell lines; however, FL cells exhibited markedly increased growth potential under suspended conditions in vitro and stronger metastatic abilities in vivo. A genomic analysis identified epithelial-mesenchymal transition (EMT) and c-Myc amplification in A549-FL and H441-FL cells, respectively, as candidate mechanisms for metastasis. The growth potential of FL cells was markedly inhibited by lentiviral ZEB1 knockdown in A549-FL cells and by the inhibition of c-Myc through lentiviral knockdown or the pharmacological inhibitor JQ1 in H441-FL cells. Long-term three-dimensional low attachment cultures may become a useful method for investigating the mechanisms underlying metastasis mediated by decreased cell-substratum adhesion.

Stromal fibroblasts are predictors of disease-related mortality in esophageal squamous cell carcinoma.

The growth, invasiveness and metastasis of human cancers are determined not only by cancer cells, but also by their microenvironment. Activated stromal fibroblasts promote tumor progression by secreting growth factors. In the present study, we focused on interrelations between cancer and fibroblasts, the main component of tumor stroma. We retrospectively analyzed the relations of mortality to clinical, pathological, and α-smooth muscle actin (α-SMA) characteristics in 97 consecutive patients with esophageal squamous cell carcinoma (ESCC). In vitro, we used TE-11, KYSE150 and KYSE220 ESCC cell lines and isolated esophageal stromal fibroblasts, some of which were immortalized. Migration assays were conducted to assess the effects of fibroblasts on cancer-cell migration and 3-dimensional organotypic cultures. In vivo, TE-11 and KYSE220 cells plus immortalized fibroblasts were co-transplanted subcutaneously in Nod/Scid mice to assess the effects of fibroblasts on tumorigenicity. Clinicopathologically, the α-SMA expression of cancer stroma was correlated with venous invasion (p<0.01), nodal involvement (p=0.02), recurrence (p=0.01), and was a predictor of survival in patients with stage I and II ESCC (p=0.04). In vitro, the presence of fibroblasts strongly promoted the migration of TE-11, KYSE150 and KYSE220 cells. On organotypic culture, stromal invasion was observed only in the presence of immortalized fibroblasts. In vivo, tumors developed or grew in a fibroblast­dependent manner after implantation. Our findings provide evidence that stromal fibroblasts and tumor cells interact to promote tumor progression in ESCC. In patients with earlier stage ESCC, α-SMA may be a predictor of mortality. Inhibition of paracrine systems associated with tumor fibroblasts may slow or reverse tumor progression, potentially leading to the development of new targeted therapies.

Identification of CCDC6-RET fusion in the human lung adenocarcinoma cell line, LC-2/ad.

Rearranged during transfection (RET) fusions have been newly identified in approximately 1% of patients with primary lung tumors. However, patient-derived lung cancer cell lines harboring RET fusions have not yet been established or identified, and therefore, the effectiveness of an RET inhibitor on lung tumors with endogenous RET fusion has not yet been studied. In this study, we report identification of CCDC6-RET fusion in the human lung adenocarcinoma cell line LC-2/ad. LC-2/ad showed distinctive sensitivity to the RET inhibitor, vandetanib, among 39 non-small lung cancer cell lines. The xenograft tumor of LC-2/ad showed cribriform acinar structures, a morphologic feature of primary RET fusion-positive lung adenocarcinomas. LC-2/ad cells could provide useful resources to analyze molecular functions of RET-fusion protein and its response to RET inhibitors.

Molecular predictors of sensitivity to the MET inhibitor PHA665752 in lung carcinoma cells.

BACKGROUND: No comprehensive data are available on the molecular predictors of sensitivity to MET inhibitor in lung carcinomas. METHODS: We examined the efficacy of the MET inhibitor PHA665752 in 41 cell lines of non-small lung carcinoma to determine whether sensitivity to the MET inhibitor is correlated with the (1) genetic statuses of MET, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2, and KRAS, (2) MET phosphorylation and its downstream signaling pathways, or (3) epithelial-mesenchymal transition. RESULTS: Of the 41 cells, 8 were highly or intermediately sensitive to PHA665752, and the remainder were PHA665752 resistant. The sensitive cells (n = 8) included not only 4 of 4 MET-amplified cell lines but also 2 of 11 KRAS-mutated cell lines and 1 of 6 EGFR-mutated cell lines. Unlike the MET-amplified cell lines, both the MET-mutated cell lines were PHA665752 resistant. High phospho-MET was not restricted to the four MET-amplified cell lines. To the contrary, it was also found in 9 of 37 MET-nonamplified cell lines, including 3 of 6 EGFR-mutated cell lines and 4 of 11 KRAS-mutated cell lines. High phospho-MET was correlated with PHA665752 sensitivity in the entire panel of cell lines, especially in the KRAS-mutated cells. The AKT and ERK pathways in the high phospho-MET cell lines were dependent on MET activation in MET-amplified and KRAS-mutated cells but not in EGFR-mutated and human epidermal growth factor receptor 2-amplified cells. CONCLUSIONS: MET amplification is an excellent predictor of PHA665752 sensitivity but not the sole predictor. High phospho-MET and dependence of the AKT and ERK pathways on MET activation may predict sensitivity to PHA665752, especially in KRAS-mutated cell lines.

c-kit gene mutations in intracranial germinomas.

Gain-of-function mutations of the c-kit gene and the expression of phosphorylated KIT are found in most gastrointestinal stromal tumors and mastocytosis. Further, almost all gonadal seminomas/dysgerminomas exhibit KIT membranous staining, and several reports have clarified that some (10-25%) have a c-kit gene mutation. But, whether intracranial germinomas also have a c-kit gene mutation remains unsolved. To elucidate the presence, frequency, and location of c-kit gene mutations in intracranial germinomas, we analyzed five mutational hot spots (exons 9, 10, 11, 13, and 17) in the c-kit genomic DNA of 16 germinomas using polymerase chain reaction and direct sequencing. We found c-kit gene mutations at exon 11 (W557C) or 17 (D816V, D820V, and N822Y) in four germinomas (25.0%), although no statistically significant difference in any clinicopathological factor was found between patients with or without mutations. These results are similar to those seen in gonadal seminoma/dysgerminoma patients, and confirm that intracranial germinomas are exact counterparts of gonadal seminomas/dysgerminomas, as would be expected on histological and immunohistochemical grounds. Moreover, molecular targeting drugs such as imatinib mesylate (STI571), which is a selective inhibitor of KIT, might be promising agents for the treatment of intracranial germinomas with c-kit gene mutations.

Alterations of the c-kit gene in testicular germ cell tumors.

Expression and gain-of-function mutation of the c-kit gene, that encodes a receptor tyrosine kinase (KIT), have been reported in mast cell tumors and gastrointestinal stromal tumors (GISTs). Among human testicular germ cell tumors (GCTs), seminomas and seminoma components of mixed GCTs have also been shown to express KIT, but only one study has found the c-kit gene mutation at exon 17 in seminoma. To elucidate the frequency and location of the c-kit gene mutation of testicular GCTs, we analyzed the whole coding region of the c-kit complementary DNA along with 4 mutational hot spots (exons 9, 11, 13 and 17) of the c-kit genomic DNA by polymerase chain reaction and direct sequencing. Somatic mutations were found in 4 pure seminomas of 34 testicular GCTs (11.8%). One mutation was found in exon 11 (W557R) and the others were observed in exon 17 (D816H and D816V). These types of mutations were reported in GISTs (W557R), seminoma (D816H) and mastocytosis (D816V) and were considered to be gain-of-function mutations, although there were no differences of any clinicopathological factors or outcome between patients with and without mutations. Additionally, we also demonstrated coexpression of Gly-Asn-Asn-Lys510-513 (GNNK) + and GNNK - isoforms of the c-kit gene with dominance of the GNNK - transcript in all testicular GCTs. The mutations and/or preferential expression of GNNK - isoform of the c-kit gene might play an important role in the development of testicular GCTs, and these tumors may also be targets for STI571, which is a promising drug for advanced and metastatic GISTs.