EGFR inhibition evokes innate drug resistance in lung cancer cells by preventing Akt activity and thus inactivating Ets-1 function.

Nonsmall cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. About 14% of NSCLCs harbor mutations in epidermal growth factor receptor (EGFR). Despite remarkable progress in treatment with tyrosine kinase inhibitors (TKIs), only 5% of patients achieve tumor reduction >90%. The limited primary responses are attributed partly to drug resistance inherent in the tumor cells before therapy begins. Recent reports showed that activation of receptor tyrosine kinases (RTKs) is an important determinant of this innate drug resistance. In contrast, we demonstrate that EGFR inhibition promotes innate drug resistance despite blockade of RTK activity in NSCLC cells. EGFR TKIs decrease both the mitogen-activated protein kinase (MAPK) and Akt protein kinase pathways for a short time, after which the Ras/MAPK pathway becomes reactivated. Akt inhibition selectively blocks the transcriptional activation of Ets-1, which inhibits its target gene, dual specificity phosphatase 6 (DUSP6), a negative regulator specific for ERK1/2. As a result, ERK1/2 is activated. Furthermore, elevated c-Src stimulates Ras GTP-loading and activates Raf and MEK kinases. These observations suggest that not only ERK1/2 but also Akt activity is essential to maintain Ets-1 in an active state. Therefore, despite high levels of ERK1/2, Ets-1 target genes including DUSP6 and cyclins D1, D3, and E2 remain suppressed by Akt inhibition. Reduction of DUSP6 in combination with elevated c-Src renews activation of the Ras/MAPK pathway, which enhances cell survival by accelerating Bim protein turnover. Thus, EGFR TKIs evoke innate drug resistance by preventing Akt activity and inactivating Ets-1 function in NSCLC cells.

Drug Resistance to EGFR Inhibitors in Lung Cancer.

BACKGROUND: The discovery of mutations in epidermal growth factor receptor (EGFR) has dramatically changed the treatment of patients with non-small-cell lung cancer (NSCLC), the leading cause of cancer deaths worldwide. EGFR-targeted therapies show considerable promise, but drug resistance has become a substantial issue. METHODS: We reviewed the literature to provide an overview of the drug resistance to EGFR tyrosine kinase inhibitors (TKIs) in NSCLC. RESULTS: The mechanisms causing primary, acquired and persistent drug resistance to TKIs vary. Researchers and clinicians, who have used study findings to develop more effective therapeutic approaches, have found that the sequential use of single agents presents a formidable challenge, suggesting that multidrug combinations must be considered. CONCLUSIONS: In the era of precision medicine, oncologists should promptly obtain an accurate diagnosis of drug resistance in each patient to be able to design the most relevant combination therapy to overcome patient-specific drug resistance.

AKT inactivation causes persistent drug tolerance to EGFR inhibitors.

Drug resistance is a major obstacle to the success of EGFR-targeted therapy. We recently studied the mechanism by which a small subset of EGFR mutant lung cancer cells remains viable after EGFR inhibition. We found that this drug-tolerant subpopulation develops because EGFR inhibition prevents AKT activity and thus inactivates Ets-1 function. In this article, we discuss how changes in intrinsic cell signaling after EGFR inhibition open a new avenue to drug resistance in NSCLCs, and comment on combined TKI and MEK inhibitor treatment to reduce the probability of emergent resistance to EGFR TKIs.

Germline mutations of MEK in cardio-facio-cutaneous syndrome are sensitive to MEK and RAF inhibition: implications for therapeutic options.

Cardio-facio-cutaneous (CFC) syndrome is a sporadic developmental disorder characterized by distinctive craniofacial features, heart defects, mental retardation and ectodermal abnormalities. We recently reported missense germline mutations in the genes MEK1 and MEK2 in patients with CFC. These mutations, including F53S and Y130C MEK1, and F57C MEK2, are the first naturally occurring mutations to be identified in these genes. This study reports data concerning the biochemical functions of the novel mutants, as well as the roles of these MEK genes in the MAPK signaling cascade. Our CFC MEK variants cannot induce ERK unless they are phosphorylated by RAF at two key serine residues in the regulatory loop. When we replaced the serine residues with alanines, ERK phosphorylation was significantly reduced in the presence of RAF. We did find that F57C MEK2 activation was less dependent on RAF signaling than the other mutants. This difference results in F57C MEK2 being resistant to the selective RAF inhibitor SB-590885. All three mutants are sensitive to the MEK inhibitor U0126. The majority of CFC cases result from mutations in B-RAF. A recent report indicates the possibility that cancer cells with activated B-RAF have enhanced, selective sensitivity to MEK inhibitors. Thus, regardless of mutations identified in an individual with CFC, MEK inhibition is a potential therapeutic approach for this population.

Establishment and characterization of an oral tongue squamous cell carcinoma cell line from a never-smoking patient.

OBJECTIVE: The rising incidence of oral tongue squamous cell carcinoma (OTSCC) in patients who have never smoked and the paucity of knowledge of its biological behavior prompted us to develop a new cell line originating from a never-smoker. MATERIALS AND METHODS: Fresh tumor tissue of keratinizing OTSCC was collected from a 44-year-old woman who had never smoked. Serum-free media with a low calcium concentration were used in cell culture, and a multifaceted approach was taken to verify and characterize the cell line, designated UCSF-OT-1109. RESULTS: UCSF-OT-1109 was authenticated by STR DNA fingerprint analysis, presence of an epithelial marker EpCAM, absence of human papilloma virus (HPV) DNA, and SCC-specific microscopic appearance. Sphere-forming assays supported its tumorigenic potential. Spectral karyotype (SKY) analysis revealed numerical and structural chromosomal abnormalities. Whole-exome sequencing (WES) identified 46 non-synonymous and 13 synonymous somatic single-nucleotide polymorphisms (SNPs) and one frameshift deletion in the coding regions. Specifically, mutations of CDKN2A, TP53, SPTBN5, NOTCH2, and FAM136A were found in the databases. Copy number aberration (CNA) analysis revealed that the cell line loses chromosome 3p and 9p, but lacks amplification of 3q and 11q (as does HPV-negative, smoking-unrelated OTSCC). It also exhibits four distinctive focal amplifications in chromosome 19p, containing 131 genes without SNPs. Particularly, 52 genes showed >3- to 4-fold amplification and could be potential oncogenic drivers. CONCLUSION: We have successfully established a novel OTSCC cell line from a never-smoking patient. UCSF-OT-1109 is potentially a robust experimental model of OTSCC in never-smokers.

ETS1 inactivation causes innate drug resistance to EGFR inhibitors.

Mutations in epidermal growth factor receptor (EGFR) are found in approximately 10% of lung cancers. Treatment with EGFR inhibitors, although promising, has surprisingly resulted in greater than 90% tumor reduction in only 5% of cases, prompting us to investigate the mechanism of innate drug resistance.

c-Kit Expression is Rate-Limiting for Stem Cell Factor-Mediated Disease Progression in Adenoid Cystic Carcinoma of the Salivary Glands.

Adenoid cystic carcinoma (ACC) is an aggressive malignant neoplasm of the salivary glands in which c-Kit is overexpressed and activated, although the mechanism for this is as yet unclear. We analyzed 27 sporadic ACC tumor specimens to examine the biologic and clinical significance of c-Kit activation. Mutational analysis revealed expression of wild-type c-Kit in all, eliminating gene mutation as a cause of activation. Because stem cell factor (SCF) is c-Kit's sole ligand, we analyzed its expression in the tumor cells and their environment. Immunohistochemistry revealed its presence in c-Kit-positive tumor cells, suggesting an activation of autocrine signaling. We observed a significant induction of ERK1/2 in the cells. SCF staining was also found in other types of non-cancerous cells adjacent to tumors within salivary glands, including stromal fibroblasts, neutrophils, peripheral nerve, skeletal muscle, vascular endothelial cells, mucous acinar cells, and intercalated ducts. Quantitative PCR showed that the top quartile of c-Kit mRNA expression distinguished ACCs from normal salivary tissues and was cross-correlated with short-term poor prognosis. Expression levels of SCF and c-Kit were highly correlated in the cases with perineural invasion. These observations suggest that c-Kit is potentially activated by receptor dimerization upon stimulation by SCF in ACC, and that the highest quartile of c-Kit mRNA expression could be a predictor of poor prognosis. Our findings may support an avenue for c-Kit-targeted therapy to improve disease control in ACC patients harboring the top quartile of c-Kit mRNA expression.

Fatty Acid binding protein 7 is a molecular marker in adenoid cystic carcinoma of the salivary glands: implications for clinical significance.

Adenoid cystic carcinoma (ACC) is an aggressive malignant neoplasm of the salivary glands. Its diagnosis is difficult due to overlapping features with other salivary tumors. Gene expression analysis may complement traditional diagnostic methods. We searched gene expression patterns in the Gene Expression Omnibus (GEO) database and in our tumor and normal samples. The biologic and prognostic potential of the identified genes was analyzed. The GEO data set of primary xenografted ACCs revealed that expression of five genes, engrailed homeobox 1 (EN1), fatty acid binding protein 7 (FABP7), hemoglobin epsilon 1, MYB, and versican (VCAN), was dramatically increased. mRNA expression of EN1, FABP7, MYB, and VCAN distinguished our sporadic ACCs from normal tissues and benign tumors. FABP7 expression appeared to be regulated differently from EN1 and MYB and was crossly correlated with poor prognosis in our ACC cohort. Immunohistochemistry showed that FABP7 protein was predominantly expressed in the nucleus of myoepithelial cells of both tubular and cribriform subtypes. In contrast, in the solid subtype, which is often associated with a lower survival rate, FABP7 protein was uniformly expressed in cancerous cells. One case with cribriform architecture and the highest level of FABP7 mRNA showed strong FABP7 staining in both duct-type epithelial and myoepithelial cells, suggesting that diffuse expression of FABP7 protein might be related to aggressive tumor behavior and poor prognosis. We propose FABP7 as a novel biomarker in ACC. The molecule may be useful in diagnosis and for identifying more effective therapies targeting this protein or upstream molecules that regulate it.

Mutations in the c-Kit gene disrupt mitogen-activated protein kinase signaling during tumor development in adenoid cystic carcinoma of the salivary glands.

The Ras/mitogen-activated protein kinase (MAPK) pathway is considered to be a positive regulator of tumor initiation, progression, and maintenance. This study reports an opposite finding: we have found strong evidence that the MAPK pathway is inhibited in a subset of adenoid cystic carcinomas (ACCs) of the salivary glands. ACC tumors consistently overexpress the receptor tyrosine kinase (RTK) c-Kit, which has been considered a therapeutic target. We performed mutational analysis of the c-Kit gene (KIT in 17 cases of ACC and found that 2 cases of ACC had distinct missense mutations in KIT at both the genomic DNA and messenger RNA levels. These mutations caused G664R and R796G amino acid substitutions in the kinase domains. Surprisingly, the mutations were functionally inactive in cultured cells. We observed a significant reduction of MAPK (ERK1/2) activity in tumor cells, as assessed by immunohistochemistry. We performed further mutational analysis of the downstream effectors in the c-Kit pathway in the genes HRAS, KRAS, NRAS, BRAF, PIK3CA, and PTEN. This analysis revealed that two ACC tumors without KIT mutations had missense mutations in either KRAS or BRAF, causing S17N K-Ras and V590I B-Raf mutants, respectively. Our functional analysis showed that proteins with these mutations were also inactive in cultured cells. This is the first time that MAPK activity from the RTK signaling has been shown to be inhibited by gene mutations during tumor development. Because ACC seems to proliferate despite inactivation of the c-Kit signaling pathway, we suggest that selective inhibition of c-Kit is probably not a suitable treatment strategy for ACC.

Genetic profiling reveals cross-contamination and misidentification of 6 adenoid cystic carcinoma cell lines: ACC2, ACC3, ACCM, ACCNS, ACCS and CAC2.

Adenoid cystic carcinoma (ACC) is the second most common malignant neoplasm of the salivary glands. Most patients survive more than 5 years after surgery and postoperative radiation therapy. The 10 year survival rate, however, drops to 40%, due to locoregional recurrences and distant metastases. Improving long-term survival in ACC requires the development of more effective systemic therapies based on a better understanding of the biologic behavior of ACC. Much preclinical research in this field involves the use of cultured cells and, to date, several ACC cell lines have been established. Authentication of these cell lines, however, has not been reported. We performed DNA fingerprint analysis on six ACC cell lines using short tandem repeat (STR) examinations and found that all six cell lines had been contaminated with other cells. ACC2, ACC3, and ACCM were determined to be cervical cancer cells (HeLa cells), whereas the ACCS cell line was composed of T24 urinary bladder cancer cells. ACCNS and CAC2 cells were contaminated with cells derived from non-human mammalian species: the cells labeled ACCNS were mouse cells and the CAC2 cells were rat cells. These observations suggest that future studies using ACC cell lines should include cell line authentication to avoid the use of contaminated or non-human cells.

A critical role for FBXW8 and MAPK in cyclin D1 degradation and cancer cell proliferation.

Cyclin D1 regulates G1 progression. Its transcriptional regulation is well understood. However, the mechanism underlying cyclin D1 ubiquitination and its subsequent degradation is not yet clear. We report that cyclin D1 undergoes increased degradation in the cytoplasm during S phase in a variety of cancer cells. This is mediated by phosphorylation at Thr286 through the activity of the Ras/Raf/MEK/ERK cascade and the F-box protein FBXW8, which is an E3 ligase. The majority of FBXW8 is expressed in the cytoplasm during G1 and S phase. In contrast, cyclin D1 accumulates in the nucleus during G1 phase and exits into the cytoplasm in S phase. Increased cyclin D1 degradation is linked to association with FBXW8 in the cytoplasm, and enhanced phosphorylation of cyclin D1 through sustained ERK1/2 signaling. Depletion of FBXW8 caused a significant accumulation of cyclin D1, as well as sequestration of CDK1 in the cytoplasm. This resulted in a severe reduction of cell proliferation. These effects could be rescued by constitutive nuclear expression of cyclin D1-T286A. Thus, FBXW8 plays an essential role in cancer cell proliferation through proteolysis of cyclin D1. It may present new opportunities to develop therapies targeting destruction of cyclin D1 or its regulator E3 ligase selectively.