Correction: Lung cancer stem cells and their aggressive progeny, controlled by EGFR/MIG6 inverse expression, dictate a novel NSCLC treatment approach.

[This corrects the article DOI: 10.18632/oncotarget.26817.].

Lung cancer stem cells and their aggressive progeny, controlled by EGFR/MIG6 inverse expression, dictate a novel NSCLC treatment approach.

The lung cancer stem cell (LuCSC) model comprises an attractive framework to explore acquired drug resistance in non-small cell lung cancer (NSCLC) treatment. Here, we used NSCLC cell line model to translate cellular heterogeneity into tractable populations to understand the origin of lung cancers and drug resistance. The epithelial LuCSCs, presumably arising from alveolar bipotent stem/progenitor cells, were lineage naïve, noninvasive, and prone to creating aggressive progeny expressing AT2/AT1 markers. LuCSC-holoclones were able to initiate rimmed niches, where their specialization created pseudo-alveoli structures. Mechanistically, LuCSC transitioning from self-renewal (ß-catenin and Nanog signaling) to malignant lineage differentiation is regulated by EGFR activation and the inverse inhibition of tumor suppressor MIG6. We further identified the functional roles of endogenous EGFR signaling in mediating progeny invasiveness and their ligands in LuCSC differentiation. Importantly, drug screening demonstrated that EGFR driving progeny were strongly responsive to TKIs; however, the LuCSCs were exclusively resistant but sensitive to AMPK agonist Metformin, antibiotic Salinomycin and to a lesser degree Carboplatin. Our data reveals previously an unknown mechanism of NSCLC resistance to EGFR-TKIs, which is associated with LuCSCs bearing a silenced EGFR and inversely expressed MIG6 suppressor gene. Taken altogether, successful NSCLC treatment requires development of a novel combination of drugs, efficiently targeting both LuCSCs and heterogeneous progeny.

The Gly385(388)Arg Polymorphism of the FGFR4 Receptor Regulates Hepatic Lipogenesis Under Healthy Diet.

CONTEXT: The effect of a lifestyle intervention to reduce liver fat content in nonalcoholic fatty liver disease in humans is influenced by genetics. We hypothesized that the amino acid exchange in human Gly388Arg (mouse homolog: Gly385Arg) in fibroblast growth factor receptor 4 (FGFR4), which regulates bile acid, lipid, and glucose metabolism, could determine hepatic lipid accumulation and insulin sensitivity. Mechanisms of this substitution were studied in mice under normal chow and high-fat diets. DESIGN: In humans, the Gly388Arg polymorphism was studied for its relationship with changes in liver fat content and insulin sensitivity during 9 months of a lifestyle intervention. We also studied a knock-in mouse strain with an Arg385 allele introduced into the murine FGFR4 gene under normal chow and high-fat diets. RESULTS: In humans, the FGFR4 Arg388 allele was not associated with liver fat content or insulin sensitivity in subjects who were overweight and obese before lifestyle intervention. However, it was associated with less decrease in liver fat content and less increase in insulin sensitivity during the intervention. In mice receiving normal chow, the FGFR4 Arg385 allele was associated with elevated hepatic triglyceride content, altered hepatic lipid composition, and increased hepatic expression of genes inducing de novo lipogenesis and glycolysis. Body fat mass and distribution, glucose tolerance, and insulin sensitivity were unaltered. The FGFR4 Arg385 allele had no effect on glucose or lipid metabolism under the high-fat diet. CONCLUSION: Our data indicate that the FGFR4 Arg388(385) allele affects hepatic lipid and glucose metabolism specifically during healthy caloric intake.

Discovery of N-[4-(Quinolin-4-yloxy)phenyl]benzenesulfonamides as Novel AXL Kinase Inhibitors.

The overexpression of AXL kinase has been described in many types of cancer. Due to its role in proliferation, survival, migration, and resistance, AXL represents a promising target in the treatment of the disease. In this study we present a novel compound family that successfully targets the AXL kinase. Through optimization and detailed SAR studies we developed low nanomolar inhibitors, and after further biological characterization we identified a potent AXL kinase inhibitor with favorable pharmacokinetic profile. The antitumor activity was determined in xenograft models, and the lead compounds reduced the tumor size by 40% with no observed toxicity as well as lung metastasis formation by 66% when compared to vehicle control.

Single Nucleotide Polymorphisms in the G-Protein Coupled Receptor Kinase 5 (GRK5) Gene are associated with Plasma LDL-Cholesterol Levels in Humans.

Genetically modified mice models suggest an important role for G-protein-coupled receptor kinase 5 (GRK5) in the pathophysiology of obesity and related disorders. We investigated whether single nucleotide polymorphisms (SNPs) in the gene encoding GRK5 affect cardiometabolic traits in humans. We genotyped 3 common SNPs in intron 1 (rs1980030, rs10466210, rs9325562) and one SNP in intron 3 (rs10886471) of GRK5 in 2332 subjects at risk for type 2 diabetes. Total- and visceral fat mass were measured by magnetic resonance (MR) tomography and liver fat content by 1H-MR spectroscopy. Insulin secretion and sensitivity were estimated during an OGTT and measured during the euglycemic, hyperinsulinemic clamp (n = 498). Carriers of the minor allele of rs10466210 and rs1980030 had higher total- and LDL-cholesterol levels (p = 0.0018 and p = 0.0031, respectively, for rs10466210; p = 0.0035 and p = 0.0081, respectively, for rs1980030), independently of gender, age, BMI and lipid-lowering drugs. The effects of rs10466210 withstood Bonferroni correction. Similar associations were observed with apolipoprotein B levels (p = 0.0034 and p = 0.0122, respectively). Carriers of the minor allele of rs10466210 additionally displayed a trend for higher intima-media thickness of the carotid artery (p = 0.075). GRK5 may represent a novel target for strategies aiming at lowering LDL-cholesterol levels and at modifying cardiovascular risk.

GAS6-expressing and self-sustaining cancer cells in 3D spheroids activate the PDK-RSK-mTOR pathway for survival and drug resistance.

AXL receptor tyrosine kinase (RTK) inhibition presents a promising therapeutic strategy for aggressive tumor subtypes, as AXL signaling is upregulated in many cancers resistant to first-line treatments. Furthermore, the AXL ligand growth arrest-specific gene 6 (GAS6) has recently been linked to cancer drug resistance. Here, we established that challenging conditions, such as serum deprivation, divide AXL-overexpressing tumor cell lines into non-self-sustaining and self-sustaining subtypes in 3D spheroid culture. Self-sustaining cells are characterized by excessive GAS6 secretion and TAM-PDK-RSK-mTOR pathway activation. In 3D spheroid culture, the activation of the TAM-PDK-RSK-mTOR pathway proves crucial following treatment with AXL/MET inhibitor BMS777607, when the self-sustaining tumor cells react with TAM-RSK hyperactivation and enhanced SRC-AKT-mTOR signaling. Thus, bidirectional activated mTOR leads to enhanced proliferation and counteracts the drug effect. mTOR activation is accompanied by an enhanced AXL expression and hyperphosphorylation following 24 h of treatment with BMS777607. Therefore, we elucidate a double role of AXL that can be assigned to RSK-mTOR as well as SRC-AKT-mTOR pathway activation, specifically through AXL Y779 phosphorylation. This phosphosite fuels the resistance mechanism in 3D spheroids, alongside further SRC-dependent EGFR Y1173 and/or MET Y1349 phosphorylation which is defined by the cell-specific addiction. In conclusion, self-sustenance in cancer cells is based on a signaling synergy, individually balanced between GAS6 TAM-dependent PDK-RSK-mTOR survival pathway and the AXLY779/EGFR/MET-driven SRC-mTOR pathway.

Metformin Triggers Autophagy to Attenuate Drug-Induced Apoptosis in NSCLC Cells, with Minor Effects on Tumors of Diabetic Patients.

The biologic plausibility of an association between type 2 diabetes mellitus (T2D) and lung cancer has received increasing attention, but the results of investigations remain largely inconclusive. In the present study we investigated the influence of the anti-diabetic drug metformin on the cytotoxic effects of EGFR targeted therapy and chemotherapy in 7 non-small cell lung cancer (NSCLC) cell lines and a cohort of lung cancer patients with/without T2D. In vitro cell viability assays indicated that metformin didn't potentiate the growth inhibitory effects of erlotinib at different doses in cell lines that are of distinct genetic background. EGFR downstream signaling evaluation further demonstrated that metformin, at its IC50 value, modified apoptosis caused in erlotinib or chemotherapeutic agent-treated cells via AKT activation and the inhibition of caspase 3 and PARP cleavages. These regulations were driven independently from EGFR, LKB1, KRAS, PTEN and p53 status. Metformin triggered autophagy (LC3B expression) was identified to interplay with apoptosis to attenuate the drug effect and postpone cancer cell death. In the retrospective study of 8 NSCLC patients, the administration of metformin did not induce statistically significant changes as assessed by immunohistochemical staining of pERK, pAKT and cleaved PARP. Consequently, the application of metformin for T2D NSCLC patients receiving chemo or EGFR targeted therapy should be considered with caution.

Sunitinib specifically augments glucose-induced insulin secretion.

The tyrosine kinase inhibitor sunitinib is used for the treatment of numerous cancers in humans. In diabetic patients, sunitinib lowers blood glucose levels and improves glycaemic control. This study aims to analyse whether sunitinib has specific and direct effects on insulin secreting ß-cells. Regulation of insulin secretion, of cellular cAMP levels and activation of signalling pathways were examined upon exposure of rat insulinoma INS-1E cells to sunitinib under specific stimulatory and inhibitory conditions. Secreted insulin and cellular cAMP levels were measured using RIA and ELISA, respectively. Protein phosphorylations were examined on western blots. Sunitinib enhanced glucose-induced insulin secretion (GIIS) concentration-dependently, reaching a maximal stimulation at 2µM. Sunitinib further augmented insulin secretion in the presence of elevated cAMP levels and the FFAR1 agonists. Adrenaline and the PKA inhibitor H89 counteracted the stimulatory effect of sunitinib on secretion. However, sunitinib altered neither the cellular levels of cAMP nor the phosphorylation of PKA. Sunitinib did not reduce IGF-1-induced phosphorylation of AKT/PKB and ERK1/2. In conclusion, these results suggest that sunitinib stimulates GIIS by a direct effect on ß-cells, which may contribute to the glucose-lowering action of the tyrosine kinase inhibitor in humans.

Salinomycin co-treatment enhances tamoxifen cytotoxicity in luminal A breast tumor cells by facilitating lysosomal degradation of receptor tyrosine kinases.

Luminal A breast cancer is the most common breast cancer subtype which is usually treated with selective estrogen receptor modulators (SERMS) like tamoxifen. Nevertheless, one third of estrogen receptor positive breast cancer patients initially do not respond to endocrine therapy and about 40% of luminal A breast tumors recur in five years. In this study, we investigated an alternative treatment approach by combining tamoxifen and salinomycin in luminal A breast cancer cell lines. We have found that salinomycin induces an additional cytotoxic effect by inhibiting the ligand independent activation of ERα. Thereby salinomycin increases the intracellular calcium level. This leads to a premature fusion of endosomes with lysosomes and thus to the degradation of Egfr family members. Since this process is essential for luminal A breast cancer cells to circumvent tamoxifen treatment, the combination of both drugs induces cytotoxicity in tamoxifen sensitive as well as resistant luminal A breast cancer cell lines.

Membrane-proximal binding of STAT3 revealed by cancer-associated receptor variants.

In cancer biology, somatic mutations in the extracellular (ligand binding) and cytosolic (functional/catalytic) domains are pursued with great interest. However, in our recent publication we report that germline mutations in the membrane-proximal region of type I receptors are able to modulate the amplitude of signal transducer and activator of transcription 3 (STAT3) signaling in cells. This unexpected finding has implications for the prognosis of heritable cancer.

Inhibiting receptor tyrosine kinase AXL with small molecule inhibitor BMS-777607 reduces glioblastoma growth, migration, and invasion in vitro and in vivo.

PURPOSE: Receptor tyrosine kinase AXL (RTK-AXL) is regarded as suitable target in glioma therapy. Here we evaluate the anti-tumoral effect of small molecule inhibitor BMS-777607 targeting RTK-AXL in a preclinical glioma model and provide evidence that RTK-AXL is expressed and phosphorylated in primary and recurrent glioblastoma multiforme (GBM). EXPERIMENTAL DESIGN: We studied the impact of BMS-777607 targeting RTK-AXL in GBM models in vitro and in vivo utilizing glioma cells SF126 and U118MG. Impact on proliferation, apoptosis and angiogenesis was investigated by immunohistochemistry (IHC) and functional assays in vitro and in vivo. Tumor growth was assessed with MRI. Human GBM tissue was analyzed in terms of RTK-AXL phosphorylation by immunoprecipitation and immunohistochemistry. RESULTS: BMS-777607 displayed various anti-cancer effects dependent on increased apoptosis, decreased proliferation and migration in vitro and ex vivo in SF126 and U118 GBM cells. In vivo we observed a 56% tumor volume reduction in SF126 xenografts and remission in U118MG xenografts of more than 91%. The tube formation assay confirmed the anti-angiogenic effect of BMS-777607, which became also apparent in tumor xenografts. IHC of human GBM tissue localized phosphorylated RTK-AXL in hypercellular tumor regions, the migratory front of tumor cells in pseudo-palisades, and in vascular proliferates within the tumor. We further proved RTK-AXL phosphorylation in primary and recurrent disease state. CONCLUSION: Collectively, these data strongly suggest that targeting RTK-AXL with BMS-777607 could represent a novel and potent regimen for the treatment of primary and recurrent GBM.

Novel compounds reducing IRS-1 serine phosphorylation for treatment of diabetes.

Activation of various interacting stress kinases, particularly the c-Jun N-terminal kinases (JNK), and a concomitant phosphorylation of insulin receptor substrate 1 (IRS-1) at serine 307 play a central role both in insulin resistance and in ß-cell dysfunction. IRS-1 phosphorylation is stimulated by elevated free fatty acid levels through different pathways in obesity. A series of novel pyrido[2,3-d]pyrimidin-7-one derivatives were synthesized as potential antidiabetic agents, preventing IRS-1 phosphorylation at serine 307 in a cellular model of lipotoxicity and type 2 diabetes.

Germline variant FGFR4 p.G388R exposes a membrane-proximal STAT3 binding site.

Variant rs351855-G/A is a commonly occurring single-nucleotide polymorphism of coding regions in exon 9 of the fibroblast growth factor receptor FGFR4 (CD334) gene (c.1162G>A). It results in an amino-acid change at codon 388 from glycine to arginine (p.Gly388Arg) in the transmembrane domain of the receptor. Despite compelling genetic evidence for the association of this common variant with cancers of the bone, breast, colon, prostate, skin, lung, head and neck, as well as soft-tissue sarcomas and non-Hodgkin lymphoma, the underlying biological mechanism has remained elusive. Here we show that substitution of the conserved glycine 388 residue to a charged arginine residue alters the transmembrane spanning segment and exposes a membrane-proximal cytoplasmic signal transducer and activator of transcription 3 (STAT3) binding site Y(390)-(P)XXQ(393). We demonstrate that such membrane-proximal STAT3 binding motifs in the germline of type I membrane receptors enhance STAT3 tyrosine phosphorylation by recruiting STAT3 proteins to the inner cell membrane. Remarkably, such germline variants frequently co-localize with somatic mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Using Fgfr4 single nucleotide polymorphism knock-in mice and transgenic mouse models for breast and lung cancers, we validate the enhanced STAT3 signalling induced by the FGFR4 Arg388-variant in vivo. Thus, our findings elucidate the molecular mechanism behind the genetic association of rs351855 with accelerated cancer progression and suggest that germline variants of cell-surface molecules that recruit STAT3 to the inner cell membrane are a significant risk for cancer prognosis and disease progression.

Activation of Cardiac Fibroblast Growth Factor Receptor 4 Causes Left Ventricular Hypertrophy.

Chronic kidney disease (CKD) is a worldwide public health threat that increases risk of death due to cardiovascular complications, including left ventricular hypertrophy (LVH). Novel therapeutic targets are needed to design treatments to alleviate the cardiovascular burden of CKD. Previously, we demonstrated that circulating concentrations of fibroblast growth factor (FGF) 23 rise progressively in CKD and induce LVH through an unknown FGF receptor (FGFR)-dependent mechanism. Here, we report that FGF23 exclusively activates FGFR4 on cardiac myocytes to stimulate phospholipase Cγ/calcineurin/nuclear factor of activated T cell signaling. A specific FGFR4-blocking antibody inhibits FGF23-induced hypertrophy of isolated cardiac myocytes and attenuates LVH in rats with CKD. Mice lacking FGFR4 do not develop LVH in response to elevated FGF23, whereas knockin mice carrying an FGFR4 gain-of-function mutation spontaneously develop LVH. Thus, FGF23 promotes LVH by activating FGFR4, thereby establishing FGFR4 as a pharmacological target for reducing cardiovascular risk in CKD.

Erk2 but not Erk1 regulates crosstalk between Met and EGFR in squamous cell carcinoma cell lines.

BACKGROUND: Squamous cell carcinoma (SCC) is the most common type of tongue and larynx cancer and a common type of lung cancer. In this study, we attempted to specifically evaluate the signaling pathway underlying HGF/Met induced EGFR ligand release in SSCs. The Met proto-oncogene encodes for a tyrosine kinase receptor which is often hyperactivated in human cancers. Met activation correlates with poor patient outcome. Several studies revealed a role of Met in receptor-crosstalk inducing either activation of other receptors, or inducing their resistance to targeted cancer treatments. In an epithelial tumor cell line screen we recently showed that the Met ligand HGF blocks the EGFR tyrosine kinase and at the same time activates transcriptional upregulation and accumulation in the supernatant of the EGFR ligand amphiregulin (Oncogene 32:3846-56, 2013). In the present work we describe the pathway responsible for the amphiregulin induction. FINDINGS: Amphiregulin is transcriptionally upregulated and is released into the supernatant. We show that Erk2 but not Erk1 mediates amphiregulin upregulation upon treatment with monocyte derived HGF. A siRNA knockdown of Erk2 completely abolishes amphiregulin release in squamous cell carcinomas. CONCLUSIONS: These results identify Erk2 as the key downstream signal transducer between Met activation and EGFR ligand upregulation in squamous cell carcinoma cell lines derived from tongue, larynx and lung.

Combined inhibition of AXL, Lyn and p130Cas kinases block migration of triple negative breast cancer cells.

Blocking the migration of metastatic cancer cells is a major goal in the therapy of cancer. The receptor tyrosine kinase AXL is one of the main triggers for cancer cell migration in neoplasia of breast, colon, skin, thyroid and prostate. In our study we analyzed the effect of AXL inhibition on cell motility and viability in triple negative breast cancer cell lines overexpressing AXL. Thereby we reveal that the compound BMS777607, exhibiting the lowest IC50 values for inhibition of AXL kinase activity in the studied cell lines, attenuates cell motility to a lower extent than the kinase inhibitors MPCD84111 and SKI606. By analyzing the target kinases of MPCD84111 and SKI606 with kinase profiling assays we identified Lyn, a Src family kinase, as a target of both compounds. Knockdown of Lyn and the migration-related CRK-associated substrate (p130Cas), had a significant inhibitory effect on cell migration. Taken together, our findings highlight the importance of combinatorial or multikinase inhibition of non-receptor tyrosine kinases and AXL receptor tyrosine kinase in the therapy of triple negative breast cancer.

Metformin and salinomycin as the best combination for the eradication of NSCLC monolayer cells and their alveospheres (cancer stem cells) irrespective of EGFR, KRAS, EML4/ALK and LKB1 status.

The presence of cancer stem cells (CSCs) is linked to preexisting or acquired drug resistance and tumor relapse. Therefore, targeting both differentiated tumor cells and CSCs was suggested as an effective approach for non-small cell lung cancer (NSCLC) treatment. After screening of chemotherapeutic agents, tyrosine kinase inhibitors (TKIs) or monoclonal antibody in combination with the putative stem cell killer Salinomycin (SAL), we found Metformin (METF), which modestly exerted a growth inhibitory effect on monolayer cells and alveospheres/CSCs of 5 NSCLC cell lines regardless of their EGFR, KRAS, EML4/ALK and LKB1 status, interacted synergistically with SAL to effectively promote cell death. Inhibition of EGFR (AKT, ERK1/2) and mTOR (p70 s6k) signaling with the combination of METF and SAL can be augmented beyond that achieved using each agent individually. Phospho-kinase assay further suggested the multiple roles of this combination in reducing oncogenic effects of modules, such as ß-catenin, Src family kinases (Src, Lyn, Yes), Chk-2 and FAK. Remarkably, significant reduction of sphere formation was seen under combinatorial treatment in all investigated NSCLC cell lines. In conclusion, METF in combination with SAL could be a promising treatment option for patients with advanced NSCLC irrespective of their EGFR, KRAS, EML4/ALK and LKB1 status.

PTK7 as a potential prognostic and predictive marker of response to adjuvant chemotherapy in breast cancer patients, and resistance to anthracycline drugs.

Biomarkers predicting resistance to particular chemotherapy regimens could play a key role in optimally individualized treatment concepts. PTK7 (protein tyrosine kinase 7) belongs to the receptor tyrosine kinase family involved in several physiological, but also malignant, cell behaviors. Recent studies in acute myeloid leukemia have associated PTK7 expression with resistance to anthracycline therapy. PTK7 mRNA expression in primary tumor tissue (PTT) and corresponding lymph node tissue (LNT) were retrospectively measured in 117 patients with early breast cancer; PTK7 expression was available in 103 PTT and 108 LNT samples. Median age was 60 years (range, 27-87 years). At a median follow-up of 28.5 months, 6 deaths and 16 recurrences had occurred. PTK7 expression correlations with clinicopathological features were computed and PTK7 expression effects on patient outcome were analyzed in three cohorts defined by adjuvant treatment: anthracycline-based treatment, other chemotherapy regimens (including taxane or other substances), or no chemotherapy. Association of PTK7 expression with clinicopathological features was seen only for age in PTT and nodal stage in LNT. High LN PTK7 was associated with poorer disease-free survival (DFS) in the total population (3-year DFS: low [81.7%] versus high [70.4%]; P=0.016) and in patients without adjuvant chemotherapy (3-year DFS: low [91.7%] versus high [22.3%]; P<0.001), but not in patients receiving adjuvant chemotherapy (P=0.552). DFS stratified by PTK7 expression was compared in treatment cohorts: In patients with low LN PTK7 expression, neither chemotherapy cohort showed significantly better survival than the no-chemotherapy cohort. In patients with high LN PTK7 expression, those receiving chemotherapy, including substances other than anthracyclines, but not those receiving only anthracycline-based chemotherapy, showed significantly better DFS than those receiving no chemotherapy (P=0.001). Our results support earlier findings that PTK7 may be a prognostic and predictive marker associated with resistance to anthracycline-based chemotherapy. Further investigations are needed to validate these findings in breast cancer.

Elimination of B-RAF in oncogenic C-RAF-expressing alveolar epithelial type II cells reduces MAPK signal intensity and lung tumor growth.

Tumors are often greatly dependent on signaling cascades promoting cell growth or survival and may become hypersensitive to inactivation of key components within these signaling pathways. Ras and RAF mutations found in human cancer confer constitutive activity to these signaling molecules thereby converting them into an oncogenic state. RAF dimerization is required for normal Ras-dependent RAF activation and is required for the oncogenic potential of mutant RAFs. Here we describe a new mouse model for lung tumor development to investigate the role of B-RAF in oncogenic C-RAF-mediated adenoma initiation and growth. Conditional elimination of B-RAF in C-RAF BxB-expressing embryonic alveolar epithelial type II cells did not block adenoma formation. However, loss of B-RAF led to significantly reduced tumor growth. The diminished tumor growth upon B-RAF inactivation was due to reduced cell proliferation in absence of senescence and increased apoptosis. Furthermore, B-RAF elimination inhibited C-RAF BxB-mediated activation of the mitogenic cascade. In line with these data, mutation of Ser-621 in C-RAF BxB abrogated in vitro the dimerization with B-RAF and blocked the ability to activate the MAPK cascade. Taken together these data indicate that B-RAF is an important factor in oncogenic C-RAF-mediated tumorigenesis.