Estrogen receptor beta (ERβ) has been detected in NSCLC cell lines and tumor specimens. The ER down-regulator, fulvestrant, blocked estradiol-stimulation of tumor growth and gene transcription in NSCLC preclinical models and showed additive effects with the EGFR inhibitor gefitinib. The safety and tolerability of combination therapy with the EGFR inhibitor, gefitinib, and fulvestrant was explored.
Post-menopausal women with advanced NSCLC received gefitinib 250 mg po daily and fulvestrant 250 mg IM monthly.
Twenty-two patients were enrolled. Eight patients had adenocarcinoma, 6 NSCLC-NOS, 4 squamous cell, and 4 BAC. Seven patients were never smokers. Eight patients received ≥ 2 lines of prior chemotherapy, 6 received one prior chemotherapy, and 8 were treatment naive. One patient experienced grade 4 dyspnea possibly related to treatment; all other grade 3/4 toxicities were unrelated to treatment. Twenty patients were evaluable for response: 3 PRs were confirmed (response rate of 15%, 95% CI: 5% – 36%). The median PFS, OS, and estimated 1 yr OS were 12 wks (3–112 wks), 38.5 weeks (7–135 wks), and 41% (95% CI 20–62%), respectively. Survival outcomes did not differ by prior lines of therapy. A subset analysis revealed that OS in the 8 patients whose tumors exhibited at least 60% ERβ nuclear IHC staining measured 65.5 weeks, while that of the 5 patients with ERβ staining of less than 60% was 21 weeks. One patient with BAC and a PR had an EGFR L858R mutation in exon 21. There was no correlation between ERβ IHC expression and histology or smoking history.
Combination therapy with gefitinib and fulvestrant in this population was well-tolerated and demonstrated disease activity.
epidermal growth factor receptor; hormonal treatment; non-small cell lung cancer; sex; age; estrogen receptor
DNA repair and cell cycle control play an important role in the repair of DNA damage caused by cigarette smoking. Given this role, functionally relevant single nucleotide polymorphisms (SNPs) in genes in these pathways may well affect the risk of smoking-related lung cancer. We examined the relationship between 240 SNPs in DNA repair and cell cycle control pathway genes and lung cancer risk in a case-control study of white current and ex-cigarette smokers (722 cases and 929 controls). Additive, dominant and recessive genetic models were evaluated for each SNP. A genetic risk summary score was also constructed. Odds ratios (OR) for lung cancer risk and 95% confidence intervals (95% CI) were estimated using logistic regression models. Thirty-eight SNPs were associated with lung cancer risk in our study population at P<0.05. The strongest associations were observed for rs2074508 in GTF2H4 (Padditive=0.003), rs10500298 in LIG1 (Precessive=2.7×10−4), rs747658 and rs3219073 in PARP1 (rs747658: Padditive=5.8×10−5; rs3219073: Padditive=4.6×10−5), and rs1799782 and rs3213255 in XRCC1 (rs1799782: Pdominant=0.006; rs3213255: Precessive=0.004). Compared to individuals with first quartile (lowest) risk summary scores, individuals with third and fourth quartile summary score results were at increased risk for lung cancer (OR: 2.21, 95% CI: 1.66–2.95 and OR: 3.44, 95% CI: 2.58–4.59, respectively; Ptrend<0.0001). Our data suggests that variation in DNA repair and cell cycle control pathway genes is associated with smoking-related lung cancer risk. Additionally, combining genotype information for SNPs in these pathways may assist in classifying current and ex-cigarette smokers according to lung cancer risk.
SNP; case-control; lung cancer
The c-Met receptor is a potential therapeutic target for non-small cell lung cancer (NSCLC). Signaling interactions between c-Met and the mutant Epidermal Growth Factor Receptor (EGFR) have been studied extensively, but signaling intermediates and biological consequences of lateral signaling to c-Met in EGFR wild-type tumors is minimally understood. Our observations indicate that delayed c-Met activation in NSCLC cell lines is initiated by wild-type EGFR, the receptor most often found in NSCLC tumors. EGFR ligands induce accumulation of activated c-Met which begins at 8 h continues for 48 h. This effect is accompanied by an increase in c-Met expression and phosphorylation of critical c-Met tyrosine residues without activation of MAPK or Akt. Gene transcription is required for delayed c-Met activation; however, phosphorylation of c-Met by EGFR occurs without production of HGF or another secreted factor, supporting a ligand-independent mechanism. Lateral signaling is blocked by two selective c-Met tyrosine kinase inhibitors (TKIs), PF2341066 and SU11274, or with gefitinib, an EGFR TKI, suggesting kinase activity of both receptors is required for this effect. Prolonged c-Src phosphorylation is observed, and c-Src pathway is essential for EGFR to c-Met communication. Pre-treatment with pan-SFK inhibitors, PP2 and dasatinib, abolishes delayed c-Met phosphorylation. A c-Src dominant-negative construct reduces EGF-induced c-Met phosphorylation compared to control, further, confirming a c-Src requirement. Inhibition of c-Met with PF2341066 and siRNA decreases EGF-induced phenotypes of invasion by ~86% and motility by ~81%, suggesting that a novel form of c-Met activation is utilized by EGFR to maximize these biological effects. Combined targeting of c-Met and EGFR leads to increased xenograft anti-tumor activity, demonstrating that inhibition of downstream and lateral signaling from the EGFR-c-Src-c-Met axis might be effective in treatment of NSCLC.
c-Met; c-Src; EGFR; cross-talk
Gene promoter hypermethylation is now regarded as a promising biomarker for the risk and progression of lung cancer. The one-carbon metabolism pathway is postulated to affect deoxyribonucleic acid (DNA) methylation because it is responsible for the generation of S-adenosylmethionine (SAM), the methyl donor for cellular methylation reactions. This study investigated the association of single nucleotide polymorphisms (SNPs) in six one-carbon metabolism-related genes with promoter hypermethylation in sputum DNA from non-Hispanic white smokers in the Lovelace Smokers Cohort (LSC) (n = 907). Logistic regression was used to assess the association of SNPs with hypermethylation using a high/low methylation cutoff. SNPs in the cystathionine beta synthase (CBS) and 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR) genes were significantly associated with high methylation in males [CBS rs2850146 (-8283G > C),
OR = 4.9; 95% CI: 1.98, 12.2, P = 0.0006] and low methylation in females [MTRR rs3776467 (7068A > G), OR = 0.57, 95% CI: 0.42, 0.77, P = 0.0003]. The variant allele of rs2850146 was associated with reduced gene expression and increased plasma homocysteine (Hcy) concentrations. Three plasma metabolites, Hcy, methionine and dimethylglycine, were associated with increased risk for gene methylation. These studies suggest that SNPs in CBS and MTRR have sex-specific associations with aberrant methylation in the lung epithelium of smokers that could be mediated by the affected one-carbon metabolism and transsulfuration in the cells.
Abbreviations:CBScystathionine beta synthaseDNAdeoxyribonucleic acidHBEChuman bronchial epithelial cellHcyhomocysteineLD, linkage disequilibrium; LSClovelace Smokers CohortMAFminor allele frequencyMTHFRmethylenetetrahydrofolate reductaseMTRRmethyltransferase reductaseSNPsingle nucleotide polymorphismsSAHS-adenosylhomocysteineSAMS-adenosylmethionine
This perspective on Meireles et al. (beginning on p. XXX in this issue of the journal) discusses the increasing evidence for the role of female steroid hormones in lung-cancer development and progression. The novel work of Meireles et al. is the first evidence for the rapid upregulation by tobacco smoke of a key cytochrome P450 gene that can metabolize estrogens such as β-estradiol to potentially carcinogenic catechol and quinine forms, as well as the first evidence for the colocalization of β-estradiol and estrogen receptors in murine airway epithelium. Actions of estrogens that contribute to lung carcinogenesis, especially in the presence of tobacco smoke, may involve both reactive intermediates that damage DNA and steroid hormone-receptor signaling that promotes growth.
Lung cancer has long been thought of as a cancer that mainly affects men, but over the past several decades, because of the high increase in tobacco use by women, there has been a corresponding dramatic increase in lung cancer among women. Since 1998, lung cancer deaths in women have surpassed those caused by breast cancer in the United States. Annual lung cancer deaths among women in the US also currently surpass those caused by breast, ovarian, and cervical cancers combined. Women are more likely than men to be diagnosed with adenocarcinoma and small-cell carcinoma of the lung compared to squamous cell carcinoma, and never smokers diagnosed with lung cancer are almost three times more likely to be female than male. These observations in the population, coupled to the findings that both estrogen receptors and aromatase, the enzyme that synthesizes 17β-estradiol, are expressed by lung tumors, suggest a role for female steroid hormones in control of lung cancer growth. Pre-clinical data and clinical data are increasingly emerging to support this concept, and to suggest that a local production of estrogen and expression of ERs occurs in lung tumors that rise in men as well as women. An additional protein that recognizes 17β-estradiol with high affinity, GPR30, is also expressed in lung tumors at high levels and may be responsible for some of the proliferation signals induced by estrogen.
To assess the prognostic value of EGFR molecular characteristics of head and neck squamous cell carcinoma (HNSCC).
Patients and Methods
HNSCC tumors from patients prospectively enrolled in either an Early Detection Research Network (EDRN) study and treated with surgery without an EGFR-targeted agent (N=154) or enrolled in a chemoradiation trial involving the EGFR-targeted antibody cetuximab (N=39) were evaluated for EGFR gene amplification by fluorescence in situ hybridization (FISH) and EGFR protein by immunohistochemical (IHC) staining. Fresh-frozen tumors (EDRN) were also evaluated for EGFR protein and site-specific phosphorylation at Y992 and Y1068 using reverse-phase protein array (RPPA) (n=67). Tumor (n=50) EGFR and EGFRvIII mRNA levels were quantified using real-time PCR.
EGFR expression by IHC was significantly higher in the EDRN tumors with EGFR gene amplification (P<0.001), and a similar trend was noted in the cetuximab-treated cohort. In the EDRN and cetuximab-treated cohorts elevated EGFR by IHC was associated with reduced survival (p=0.019 and p=0.06, respectively). Elevated expression of total EGFR and EGFR PY1068 were independently significantly associated with reduced progression-free survival in the EDRN cohort (HR=2.75; 95% CI=1.26–6.00 and HR=3.29; 95% CI=1.34–8.14, respectively).
In two independent HNSCC cohorts treated with or without cetuximab, tumor EGFR levels were indicative of survival. Tumor EGFR PY1068 levels provided prognostic information independent of total EGFR.
epidermal growth factor receptor; receptor tyrosine kinase; site-specific phosphorylation; prognosis; head and neck cancer
Clinical decision-making in the setting of CT screening could benefit from accessible biomarkers that help predict the level of lung cancer risk in high-risk individuals with indeterminate pulmonary nodules.
To identify candidate serum biomarkers, we measured 70 cancer-related proteins by Luminex xMAP® multiplexed immunoassays in a training set of sera from 56 patients with biopsy-proven primary non small cell lung cancer and 56 age-, sex- and smoking-matched CT-screened controls.
We identified a panel of 10 serum biomarkers – prolactin, transthyretin, thrombospondin-1, E-selectin, C-C motif chemokine 5, macrophage migration inhibitory factor, plasminogen activator inhibitor, receptor tyrosine-protein kinase, Cyfra 21.1, and serum amyloid A – that distinguished lung cancer from controls with an estimated balanced accuracy (average of sensitivity and specificity) of 76.0%±3.8% from 20-fold internal cross-validation. We then iteratively evaluated this model in independent test and verification case/control studies confirming the initial classification performance of the panel. The classification performance of the 10-biomarker panel was also analytically validated using ELISAs in a second independent case/control population further validating the robustness of the panel.
The performance of this 10-biomarker panel based model was 77.1% sensitivity/76.2% specificity in cross-validation in the expanded training set, 73.3% sensitivity/93.3% specificity (balanced accuracy 83.3%) in the blinded verification set with the best discriminative performance in Stage I/II cases: 85% sensitivity (balanced accuracy 89.2%). Importantly, the rate of misclassification of CT-screened controls was not different in most control subgroups with or without airflow obstruction or emphysema or pulmonary nodules. These biomarkers have potential to aid in the early detection of lung cancer and more accurate interpretation of indeterminate pulmonary nodules detected by screening CT.
Lung cancer; serum protein biomarkers; CT screening; Luminex xMAP® immunoassays; pulmonary nodules
The heparan sulfate 6-O-endosulfatase (SULF2) promotes growth and metastasis of solid tumors. We recently identified that cytosine methylation of the SULF2 promoter is associated with better survival of resected lung adenocarcinoma patients and now also demonstrate a marginal improvement in survival of advanced non-small cell lung cancer (NSCLC) patients receiving standard chemotherapy (HR = 0.63, p = 0.07). Subsequent studies focused on investigating the effect of methylation on SULF2 expression and its genome-wide impact. The genes and pathways modulated by epigenetic inactivation of SULF2 and the effects on sensitivity to chemotherapy were characterized in vitro and in vivo. Silencing SULF2 through siRNA or methylation primarily increased expression of interferon-inducible genes including ISG15, a marker for increased sensitivity to topoisomerase-1 inhibitors such as camptothecin. NSCLC cell lines with methylated SULF2 (SULF2M) express 60-fold higher ISG15 compared to SULF2 unmethylated (SULF2U) NSCLC cell lines and normal human bronchial epithelial cells. In vitro, SULF2M and high ISG15 (ISG15H) expressing NSCLC cell lines were 134-fold more sensitive to camptothecin than SULF2U and low ISG15 (ISG15L) expressing cell lines. Topotecan, a soluble analogue of camptothecin and FDA approved anti-cancer drug, dramatically arrested the growth of SULF2M-ISG15H, but not SULF2U-ISG15L lung tumors in nude mice (p < 0.002). Similarly, high ISG15 expression that is comparable to the topotecan sensitive NSCLC cell lines was found in tumors from 25% of NSCLC patients compared to normal lung indicating a potential to identify and target the most sensitive NSCLC subpopulation for personalized topotecan therapy.
NSCLC; Camptothecin; SULF-2; Oncogene; Topotecan
Estrogen is known to promote proliferation and to activate the epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC). Vascular endothelial growth factor (VEGF) is a known estrogen responsive gene in breast cancer. We sought to determine if the VEGF pathway is also regulated by estrogen in lung cancer cells, and whether combining an inhibitor of the ER pathway with a dual VEGFR/EGFR inhibitor would show enhanced anti-tumor effects.
We examined activation of EGFR and expression of VEGF in response to β-estradiol, and the anti-tumor activity of the multi-targeted VEGFR/EGFR/RET inhibitor, vandetanib, when combined with the anti-estrogen fulvestrant both in vitro and in vivo.
NSCLC cells expressed VEGFR-3 and EGFR. Vandetanib treatment of NSCLC cells resulted in inhibition of EGFR and VEGFR-3 and inhibition of β-estradiol-induced P-MAPK activation, demonstrating that vandetanib blocks β-estradiol-induced EGFR signaling. Treatment with β-estradiol stimulated VEGFA mRNA and protein (P<0.0001 over baseline), suggesting estrogenic signaling causes heightened VEGFA pathway activation. This estrogenic induction of VEGFA mRNA appears largely dependent on cross-talk with EGFR. Long-term vandetanib treatment also significantly increased ERβ protein expression. The combination of vandetanib with fulvestrant maximally inhibited cell growth compared to single agents (P<0.0001) and decreased tumor xenograft volume by 64%, compared to 51% for vandetanib (P<0.05) and 23% for fulvestrant (P<0.005). Antitumor effects of combination therapy were accompanied by a significant increase in apoptotic cells compared to single agents.
Fulvestrant may enhance effects of vandetanib in NSCLC by blocking estrogen-driven activation of the EGFR pathway.
EGFR; Estrogen; NSCLC; VEGF; VEGFR; Tyrosine kinase inhibitor
Bradykinin (BK) has been shown to promote growth and migration of head and neck squamous cell carcinoma (HNSCC) cells via epidermal growth factor receptor (EGFR) transactivation. It has also been reported that BK can cause the induction of cyclooxygenase-2 (COX-2), a pro-tumorigenic enzyme, via the MAPK (Mitogen-Activated Protein Kinase) pathway in human airway cells. To determine whether COX-2 is up-regulated by BK in HNSCC, the current study investigated BK- induced EGFR transactivation, MAPK activation, and cyclooxygenase-2 (COX-2) expression in human HNSCC cells. BK induced a concentration- and time-dependent induction of COX-2 protein in HNSCC, which was preceded by phosphorylation of EGFR and MAPK. These effects were abolished by the B2 receptor (B2R) antagonist Hoe 140 but not the B1 receptor (B1R) antagonist, Lys-[Leu8]des-Arg9-BK. COX-2 induction was accompanied by increased release of PGE2. No effect of a B1R agonist (des-Arg9-BK) on p-MAPK or COX-2 expression was observed. B2R protein was found to be expressed in all four head and neck cell lines tested. Immunohistochemical analysis and immunoblot analysis revealed that B2R, but not B1R, was significantly over-expressed in HNSCC tumors compared to levels in normal mucosa from the same patient. In HNSCC cells, the BK-induced expression of COX-2 was inhibited by the EGFR kinase inhibitor gefitinib or mitogen activated protein kinase kinases (MEK) inhibitors (PD98059 or U0126). These results suggest that EGFR and MAPK are required for COX-2 induction by BK. Up-regulation of the B2R in head and neck cancers suggests this pathway is involved in HNSCC tumorigenesis.
Bradykinin; B2 receptor; EGFR; MAPK; cyclooxygenase-2; head and neck cancer
The detection of tumor suppressor gene promoter methylation in sputum-derived exfoliated cells predicts early lung cancer. Here we identified genetic determinants for this epigenetic process and examined their biological effects on gene regulation. A two-stage approach involving discovery and replication was employed to assess the association between promoter hypermethylation of a 12-gene panel and common variation in 40 genes involved in carcinogen metabolism, regulation of methylation, and DNA damage response in members of the Lovelace Smokers Cohort (n=1434). Molecular validation of three identified variants was conducted using primary bronchial epithelial cells. Association of study-wide significance (P<8.2×10−5) was identified for rs1641511, rs3730859, and rs1883264 in TP53, LIG1, and BIK, respectively. These SNPs were significantly associated with altered expression of the corresponding genes in primary bronchial epithelial cells. In addition, rs3730859 in LIG1 was also moderately associated with increased risk for lung cancer among Caucasian smokers. Together, our findings suggest that genetic variation in DNA replication and apoptosis pathways impacts the propensity for gene promoter hypermethylation in the aerodigestive tract of smokers. The incorporation of genetic biomarkers for gene promoter hypermethylation with clinical and somatic markers may improve risk assessment models for lung cancer.
DNA damage response; promoter hypermethylation; single nucleotide polymorphism; sputum; smoker
Rationale: As computed tomography (CT) screening for lung cancer becomes more widespread, volumetric analyses, including doubling times, of CT-screen detected lung nodules and lung cancers may provide useful information in the follow-up and management of CT-detected lung nodules and cancers.
Objectives: To analyze doubling times in CT screen detected lung cancers and compare prevalent and nonprevalent cancers and different cell types on non small cell lung cancer.
Methods: We performed volumetric and doubling time analysis on 63 non–small cell lung cancers detected as part of the Pittsburgh Lung Screening Study using a commercially available VITREA 2 workstation and VITREA VITAL nodule segmentation software.
Measurements and Main Results: Doubling times (DT) were divided into three groups: rapid (DT < 183 d), typical (DT 183–365 d), and slow (DT > 365 d). Adenocarcinoma/bronchioloalveolar carcinoma comprised 86.7% of the slow DT group compared with 20% of the rapid DT group. Conversely, squamous cell cancer comprised 60% of the rapid DT group compared with 3.3% of the slow DT group. Twenty-eight of 42 (67%) prevalent and 2 of 21 (10%) nonprevalent cancers were in the slow DT group (P < 0.0001; Fisher's exact test). Twenty-four of 32 (75%) prevalent and 1 of 11 (9%) nonprevalent adenocarcinomas were in the slow DT group (P < 0.0002; Fisher's exact test).
Conclusions: Volumetric analysis of CT-detected lung cancers is particularly useful in AC/BAC. Prevalent cancers have a significantly slower DT than nonprevalent cancers and a higher percentage of adenocarcinoma/bronchioloalveolar carcinoma. These results should affect the management of indeterminant lung nodules detected on screening CT scans.
lung cancer; doubling times; lung cancer screening
Gastrin-releasing peptide receptor (GRPR) and the epidermal growth factor receptor (EGFR) are expressed in several cancers including non-small cell lung cancer (NSCLC). Here we demonstrate the activation of EGFR by the GRPR ligand, gastrin-releasing peptide (GRP), in NSCLC cells. GRP induced rapid activation of p44/42 MAPK in lung cancer cells through EGFR. GRP-mediated activation of MAPK in NSCLC cells was abrogated by pretreatment with the anti-EGFR-neutralizing antibody, C225. Pretreatment of NSCLC cells with neutralizing antibodies to the EGFR ligands, TGF-α or HB-EGF, also decreased GRP-mediated MAPK activation. On matrix metalloproteinase (MMP) inhibition, GRP failed to activate MAPK in NSCLC cells. EGF and GRP both stimulated NSCLC proliferation, and inhibition of either EGFR or GRPR resulted in cell death. Combining a GRPR antagonist with the EGFR tyrosine kinase inhibitor, gefitinib, resulted in additive cytotoxic effects. Additive effects were seen at gefitinib concentrations from 1 to 18 µM, encompassing the ID50 values of both gefitinib-sensitive and gefitinib-resistant NSCLC cell lines. Because a major effect of GRPR appears to be promoting the release of EGFR ligand, this study suggests that a greater inhibition of cell proliferation may occur by abrogating EGFR ligand release in consort with inhibition of EGFR.
EGFR; GRPR; MAPK; signal transduction; non-small cell lung cancer
Regional quantitative analysis of airway morphological abnormalities is of great interest in lung disease investigation. Considering that pulmonary lobes are relatively independent functional unit, we develop and test a novel and efficient computerized scheme in this study to automatically and robustly classify the airways into different categories in terms of pulmonary lobe. Given an airway tree, which could be obtained using any available airway segmentation scheme, the developed approach consists of four basic steps: (1) airway skeletonization or centerline extraction, (2) individual airway branch identification, (3) initial rule-based airway classification/labeling, and (4) self-correction of labeling errors. In order to assess the performance of this approach, we applied it to a dataset consisting of 300 chest CT examinations in a batch manner and asked an image analyst to subjectively examine the labeled results. Our preliminary experiment showed that the labeling accuracy for the right upper lobe, the right middle lobe, the right lower lobe, the left upper lobe, and the left lower lobe is 100%, 99.3%, 99.3%, 100%, and 100%, respectively. Among these, only two cases are incorrectly labeled due to the failures in airway detection. It takes around 2 minutes to label an airway tree using this algorithm.
Increased expression and/or activation of epidermal growth factor receptor (EGFR) is associated with tumor progression and poor prognosis in many cancers including head and neck squamous cell carcinoma (HNSCC). Src family kinases, including c-Src, mediate a variety of intra- or extracellular signals that contribute to tumor formation and progression. This study was undertaken to elucidate the role of c-Src in the growth and invasion of HNSCC and to determine the effects of combined targeting of EGFR and Src kinases in HNSCC cell lines.
HNSCC cells were engineered to stably express a dominant-active (DA) form of c-Src and investigated in cell growth and invasion assays. The biochemical effects of combined treatment with the Src inhibitor, AZD0530, a potent, orally active Src inhibitor with Bcr/Abl activity and the EGFR kinase inhibitor, gefitinib, were examined as well as the consequences of dual Src/EGFR targeting on the growth and invasion of a panel of HNSCC cell lines.
HNSCC cells expressing DA c-Src demonstrated increased growth and invasion compared with vector-transfected controls. Combined treatment with AZD0530 and gefitinib resulted in greater inhibition of HNSCC cell growth and invasion compared with either agent alone.
These results suggest that increased expression and activation of c-Src promotes HNSCC progression where combined targeting of EGFR and c-Src may be an efficacious treatment approach.
head and neck cancer; epidermal growth factor receptor; Src family kinases; protein kinase inhibitors
To determine the effect of tyrosine-phosphorylated signal transducer and activator of transcription 3 (pSTAT3) immunoexpression on survival in two independent cohorts of patients with squamous cell carcinoma of the head and neck (SCCHN) and to evaluate pSTAT3, trans-forming growth factor-α (TGF-α), epidermal growth factor receptor (EGFR), and gastrin-releasing peptide receptor (GRPR) expression in matched tumor and lymph node metastases in one of these cohorts.
Immunostaining for pSTAT3, TGF-α, EGFR, and GRPR was done in two SCCHN cohorts (cohort 1, 61 tumors; cohort 2, 69 paired primary tumors and lymph node metastases). Semiquantitative scores derived from the product of staining intensity (scale 0–3) score and percentage of positive tumor cells were correlated with clinical outcome.
Immunoexpression of pSTAT3 did not correlate with clinical outcome in either cohort (cohort 1, P = 0.914; cohort 2, P = 0.312). Incohort 2, TGF-α and EGFR expression in the primary tumors showed some association with decreased disease-free survival (P = 0.0306 and P = 0.0985, respectively). Both pSTAT3 and EGFR showed a correlation of expression between tumor and matched lymph node metastasis (P < 0.0001 and P = 0.0046, respectively). In addition, the expression of EGFR and GRPR in the primary tumors correlated with TGF-α expression in paired nodal metastases (P = 0.0043 and P = 0.0268, respectively). In the nodal metastases, TGF-α expression correlated with EGFR expression (P = 0.0069). In primary tumors, GRPR expression correlated with TGF-α and EGFR expression(P =0.0378 and P =0.0026, respectively).
These findings support an autocrine signaling pathway involving TGF-α, EGFR, and pSTAT3 in metastatic SCCHN as well as transactivation of EGFR by GRPR via TGF-α, but fails to identify an independent prognostic role for pSTAT3 immunoexpression.
Head and neck squamous cell carcinoma (HNSCC) is usually fatal, and innovative approaches targeting growth pathways are necessary to effectively treat this disease. Both the epidermal growth factor receptor (EGFR) and the hepatocyte growth factor (HGF)/c-Met pathways are overexpressed in HNSCC and initiate similar downstream signaling pathways. c-Met may act in consort with EGFR and/or be activated as a compensatory pathway in the presence of EGFR blockade.
Expression levels of EGFR and c-Met were determined by Western analysis in HNSCC cell lines and correlated with anti-tumor responses to inhibitors of these pathways.
Combining the c-Met inhibitor PF2341066 with the EGFR inhibitor gefitinib abrogated HNSCC cell proliferation, invasion and wound healing significantly more than inhibition of each pathway alone in HNSCC cell lines. When both HGF and the EGFR ligand, transforming growth factor-alpha (TGF-α), were present in vitro, P-AKT and P-MAPK expression were maximally inhibited by targeting both EGFR and c-Met pathways, suggesting that c-Met or EGFR can compensate when phosphorylation of the other receptor is inhibited. We also demonstrated that TGF-α can induce phosphorylation of c-Met over 6-fold by 8 hours in the absence of HGF, supporting a ligand-independent mechanism. Combined targeting of c-Met and EGFR resulted in an enhanced inhibition of tumor volumes accompanied by a decreased number of proliferating cells and increased apoptosis compared to single agent treatment in vivo.
Together these results suggest that dual blockade of c-Met and EGFR may be a promising clinical therapeutic strategy for treating HNSCC.
Steroid hormones and growth factors affect lung cancer, and it is possible they act in concert to influence patient outcome.
Primary lung tumors and normal lung tissue were analyzed for expression and localization of estrogen receptor α and β–1 ERα and ERβ), aromatase, progesterone receptor (PR), and epidermal growth factor receptor (EGFR).
Tumors expressed higher levels of ERβ compared to matched normal lung, while the reverse was true of PR. High cytoplasmic ERβ expression was identified as an independent negative prognostic predictor of overall survival (OS) (HR=1.67), and low total PR was identified as an independent negative predictor of time to progression (TTP) (HR=1.59). After adjusting for stage, age, sex and smoking, combined high cytoplasmic ERβ and low total PR showed enhanced effects on OS (HR=2.64) and on TTP (HR=6.02). Further effects on OS were observed when EGFR expression was included (HR=5.32). Patients with low cytoplasmic ERβ, low aromatase, low EGFR and high total PR had shorter OS than patients with the opposite pattern (HR= 6.60). Contribution of these markers to survival showed no significant sex differences in a multivariable model. ERα was elevated in tumors but was not predictive of survival, and appears to represent a variant ERα protein that is only recognized by a C-terminal antibody.
Hormonal and EGFR pathways together may contribute to lung cancer prognosis. Lung tumors with high ERβ–1 /low PR may define patients with aggressive biology. A validation study is necessary to fully assess the predictive value of these markers.
estrogen receptor; progesterone receptor; aromatase; epidermal growth factor receptor; lung cancer
Targeted glycoproteomics represents an attractive approach for conducting peripheral blood based cancer biomarker discovery due to the well-known altered pattern of protein glycosylation in cancer and the reduced complexity of the resultant glycoproteome. Here we report its application to a set of pooled non-small cell lung cancer (NSCLC) case sera (9 adenocarcinoma and 6 squamous cell carcinoma pools from 54 patients) and matched controls pools, including 8 clinical control pools with computed tomography detected nodules but being non-malignant as determined by biopsy from 54 patients, and 8 matched healthy control pools from 106 cancer-free subjects. The goal of the study is to discover biomarkers which may enable improved early detection and diagnosis of lung cancer. Immunoaffinity subtraction was used to first deplete the top most abundant serum proteins; the remaining serum proteins were then subjected to hydrazide chemistry based glycoprotein capture and enrichment. Hydrazide resin in situ trypsin digestion was used to release non-glycosylated peptides. Formerly N-linked glycosylated peptides were released by peptide-N-glycosidase F (PNGase F) treatment and were subsequently analyzed by liquid chromatography (LC)-tandem mass spectrometry (MS/MS). A MATLAB® based in-house tool was developed to facilitate retention time alignment across different LC-MS/MS runs, determination of precursor ion m/z values and elution profiles, and the integration of mass chromatograms based on determined parameters for identified peptides. A total of 38 glycopeptides from 22 different proteins were significantly differentially abundant across the case/control pools (P<0.01, Student’s t test) and their abundances led to a near complete separation of case and control pools based on hierarchical clustering. The differential abundances of three of these candidate proteins were verified by commercially available ELISAs applied in the pools. Strong positive correlations between glycopeptide mass chromatograms and ELISA-measured protein abundance was observed for all of the selected glycoproteins.
Lung cancer; serum biomarkers; glycoproteomics; LC-MS/MS; mass chromatogram
We determined hepatocyte growth factor (HGF) and c-Met expression and signaling in human head and neck squamous cell carcinoma (HNSCC) cells and primary tissues and tested the ability of c-Met tyrosine kinase inhibitors (TKI) to block HGF-induced biological signaling.
Expression and signaling were determined using immunoblotting, ELISA, and immunohistochemistry. Biological end points included wound healing, cell proliferation, and invasion. c-Met TKIs were tested for their ability to block HGF-induced signaling and biological effects in vitro and in xenografts established in nude mice.
c-Met was expressed and functional in HNSCC cells. HGF was secreted by HNSCC tumor-derived fibroblasts, but not by HNSCC cells. Activation of c-Met promoted phosphorylation of AKT and mitogen-activated protein kinase as well as release of the inflammatory cytokine interleukin-8. Cell growth and wound healing were also stimulated by HGF. c-Met TKIs blocked HGF-induced signaling, interleukin-8 release, and wound healing. Enhanced invasion of HNSCC cells induced by the presence of tumor-derived fibroblasts was completely blocked with a HGF-neutralizing antibody. PF-2341066, a c-Met TKI, caused a 50% inhibition of HNSCC tumor growth in vivo with decreased proliferation and increased apoptosis within the tumors. In HNSCC tumor tissues, both HGF and c-Met protein were increased compared with expression in normal mucosa.
These results show that HGF acts mainly as a paracrine factor in HNSCC cells, the HGF/c-Met pathway is frequently up-regulated and functional in HNSCC, and a clinically relevant c-Met TKI shows antitumor activity in vivo. Blocking the HGF/c-Met pathway may be clinically useful for the treatment of HNSCC.
Aberrant promoter hypermethylation of tumor suppressor genes is a promising marker for lung cancer detection. We investigated the likelihood of detecting aberrant DNA methylation of tumor suppressor genes, in plasma samples of patients with abnormalities of the lung detected upon CT-scan.
In a small evaluation cohort, 4 gene promoters (DCC, Kif1a, NISCH, Rarb) were found to be methylated with increased frequency in samples from cancer patients specifically. We then examined DNA from 93 plasma samples from patients with abnormal findings in the lung detected upon CT scan for aberrant methylation of these 4 gene promoters by quantitative fluorogenic real-time PCR (QMSP). The patients were divided into 2 groups, ground glass opacity (GGO n=23) and cancerous tumors (n=70). Plasma DNA from age-matched nodule-free individuals were used as controls (n=80).
In plasma, 73% of patients with cancerous tumors showed methylation of at least one gene with a specificity of 71% (p=0.0001). Only 22% patients with GGO exhibited methylation of at least one gene. When smoking history was taken into account, 72% of cancer patients with no smoking history or those who smoked <20 pack years, showed methylation of at least one gene with 100% specificity (p=0.05) when compared to matched controls. Among heavy smokers with 20+ pack years of smoking history, 30% of the control group and 73% of the patients with cancerous tumors showed methylation (p=0.0001).
These biomarkers can distinguish between cancerous and non-cancerous abnormal CT findings.
EGFR and c-Met are both overexpressed in lung cancer and initiate similar downstream signaling, which may be redundant. To determine how frequently ligands that initiate signaling of both pathways are found in lung cancer, we analyzed serum for hepatocyte growth factor (HGF), transforming growth factor-alpha, and amphiregulin (AREG) in lung cancer cases and tobacco-exposed controls. HGF and AREG were both significantly elevated in cases compared to controls, suggesting that both HGF/c-Met and AREG/EGFR pathways are frequently active. When both HGF and AREG are present in vitro, downstream signaling to MAPK and Akt in non-small cell lung cancer (NSCLC) cells can only be completely inhibited by targeting both pathways. To test if dual blockade of the pathways could better suppress lung tumorigenesis in an animal model than single blockade, mice transgenic for airway expression of human HGF were treated with inhibitors of both pathways alone and in combination after exposure to a tobacco carcinogen. Mean tumor number in the group using both the HGF neutralizing antibody L2G7 and the EGFR inhibitor gefitinib was significantly lower than with single agents. A higher tumor K-ras mutation rate was observed with L2G7 alone compared to controls, suggesting that agents targeting HGF may be less effective against mutated K-ras lung tumors. This was not observed with combination treatment. A small molecule c-Met inhibitor decreased formation of both K-ras wild-type and mutant tumors and showed additive anti-tumor effects when combined with gefitinib. Dual targeting of c-Met/EGFR may have clinical benefit for lung cancer.
lung cancer; EGFR; HGF; c-Met
To characterize estrogen receptor (ER) expression and signaling in head and neck squamous cell carcinoma (HNSCC) cell lines and patient tissues and evaluate ER and epidermal growth factor (EGF) receptor (EGFR) cross-activation in HNSCC.
ER expression and signaling in HNSCC cell lines were assessed by immunoblotting. In vitro proliferation and invasion were evaluated in HNSCC cell lines in response to ER and EGFR ligands or inhibitors. ER and EGFR protein expression in patient tissues was assessed by immunohistochemical (IHC) staining.
Phospho-MAP kinase (P-MAPK) levels were significantly increased following combined estrogen (E2) and EGF treatment. Treatment of HNSCC cells with E2 and EGF significantly increased cell invasion compared to either treatment alone while inhibiting these two pathways resulted in reduced invasion compared to inhibiting either pathway alone. EGFR (P=0.008) and nuclear ERα (ERαnuc) (P<0.001) levels were significantly increased in HNSCC tumors (n=56) compared to adjacent mucosa (n=30) while ERβnuc levels did not differ (P=0.67). Patients with high ERαnuc and EGFR tumor levels had significantly reduced PFS compared to patients low tumor ERαnuc and EGFR levels (H.R. = 4.09, P = 0.01; Cox proportional hazards). In contrast, high ERβnuc tumor levels were not associated with reduced PFS alone or when combined with EGFR.
ERα and ERβ were expressed in HNSCC and stimulation with ER ligands resulted in both cytoplasmic signal transduction and transcriptional activation. ER and EGFR cross-talk was observed. Collectively, these studies indicate ER and EGFR together may contribute to HNSCC development and disease progression.
estrogen receptor; epidermal growth factor receptor; head and neck squamous cell carcinoma
The high mortality rate of lung cancer is largely due to the spread of disease to other organs. However, the molecular changes driving lung cancer invasion and metastasis remain unclear. In this study, we identified fibulin-5, a vascular ligand for integrin receptors, as a suppressor of lung cancer invasion and metastasis. Fibulin-5 was silenced by promoter hypermethylation in a majority of lung cancer cell lines and primary tumors. It inhibited lung cancer cell invasion and downregulated matrix metalloproteinase-7 (MMP-7), which promoted lung cancer cell invasion. Knockdown of fibulin-5 was sufficient to stimulate cell invasion and MMP-7 expression. The expression levels of fibulin-5 and MMP-7 were inversely correlated in lung tumors. Suppression of MMP-7 expression by fibulin-5 was mediated by an integrin-binding RGD motif via the extracellular signal-regulated kinase (ERK) pathway. Furthermore, overexpression of fibulin-5 in H460 lung cancer cells inhibited metastasis in mice. Collectively, these results suggest that epigenetic silencing of fibulin-5 promotes lung cancer invasion and metastasis by activating MMP-7 expression through the ERK pathway.
Fibulin-5; MMP-7; integrin; invasion; lung cancer