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Metabolic reprogramming of the tumor microenvironment is recognized as a cancer hallmark. To identify new molecular processes associated with tumor metabolism, we analyzed the transcriptome of bulk and flow-sorted human primary non-small cell lung cancer (NSCLC) together with 18 FDG-PET scans, which provide a clinical measure of glucose uptake. Tumors with higher glucose uptake were functionally enriched for molecular processes associated with invasion in adenocarcinoma and cell growth in squamous cell carcinoma (SCC). Next, we identified genes correlated to glucose uptake that were predominately overexpressed in a single cell-type comprising the tumor microenvironment. For SCC, most of these genes were expressed by malignant cells, whereas in adenocarcinoma, they were predominately expressed by stromal cells, particularly cancer-associated fibroblasts (CAF). Among these adenocarcinoma genes correlated to glucose uptake, we focused on glutamine-fructose-6-phosphate transaminase 2 ( GFPT2 ), which codes for the glutamine-fructose-6-phosphate aminotransferase 2 (GFAT2), a rate-limiting enzyme of the hexosamine biosynthesis pathway (HBP), which is responsible for glycosylation. GFPT2 was predictive of glucose uptake independent of GLUT1, the primary glucose transporter, and was prognostically significant at both gene and protein level. We confirmed that normal fibroblasts transformed to CAF-like cells, following TGFβ treatment, upregulated HBP genes, including GFPT2 , with less change in genes driving glycolysis, pentose phosphate pathway, and TCA cycle. Our work provides new evidence of histology-specific tumor stromal properties associated with glucose uptake in NSCLC and identifies GFPT2 as a critical regulator of tumor metabolic reprogramming in adenocarcinoma. Significance: These findings implicate the hexosamine biosynthesis pathway as a potential new therapeutic target in lung adenocarcinoma. Cancer Res; 78(13); 3445-57. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Circulating tumor cells (CTCs) are established cancer biomarkers for the "liquid biopsy" of tumors. Molecular analysis of single CTCs, which recapitulate primary and metastatic tumor biology, remains challenging because current platforms have limited throughput, are expensive, and are not easily translatable to the clinic. Here, we report a massively parallel, multigene-profiling nanoplatform to compartmentalize and analyze hundreds of single CTCs. After high-efficiency magnetic collection of CTC from blood, a single-cell nanowell array performs CTC mutation profiling using modular gene panels. Using this approach, we demonstrated multigene expression profiling of individual CTCs from non-small-cell lung cancer (NSCLC) patients with remarkable sensitivity. Thus, we report a high-throughput, multiplexed strategy for single-cell mutation profiling of individual lung cancer CTCs toward minimally invasive cancer therapy prediction and disease monitoring., Competing Interests: S.X.W. holds two issued patents on MagSifter, which are owned by Stanford University and outlicensed for possible commercialization. S.-m.P., D.J.W., C.C.O., S.S.G., V.S.N., and S.X.W. are coinventors of a pending patent filed by Stanford University on the subject of this work.

Introduction: Circulating tumor microemboli (CTM) are potentially important cancer biomarkers, but using them for cancer detection in early-stage disease has been assay limited. We examined CTM test performance using a sensitive detection platform to identify stage I non-small-cell lung cancer (NSCLC) patients undergoing imaging evaluation., Methods: First, we prospectively enrolled patients during 18F-FDG PET-CT imaging evaluation for lung cancer that underwent routine phlebotomy where CTM and circulating tumor cells (CTCs) were identified in blood using nuclear (DAPI), cytokeratin (CK), and CD45 immune-fluorescent antibodies followed by morphologic identification. Second, CTM and CTC data were integrated with patient (age, gender, smoking, and cancer history) and imaging (tumor diameter, location in lung, and maximum standard uptake value [SUVmax]) data to develop and test multiple logistic regression models using a case-control design in a training and test cohort followed by cross-validation in the entire group., Results: We examined 104 patients with NSCLC, and the subgroup of 80 with stage I disease, and compared them to 25 patients with benign disease. Clinical and imaging data alone were moderately discriminating for all comers (Area under the Curve [AUC] = 0.77) and by stage I disease only (AUC = 0.77). However, the presence of CTM combined with clinical and imaging data was significantly discriminating for diagnostic accuracy in all NSCLC patients (AUC = 0.88, p value = 0.001) and for stage I patients alone (AUC = 0.87, p value = 0.002)., Conclusion: CTM may add utility for lung cancer diagnosis during imaging evaluation using a sensitive detection platform.

Anthracyclines and taxanes have remarkable anticancer efficacy, but have poor selectivity and high toxicity. Targeted delivery of chemotherapeutics has emerged as a strategy to achieve higher drug levels at the tumor site, to spare noncancerous tissue and potentially to use lower systemic drug doses, thus preventing side effects. In this study, we targeted the HER2 receptor using the monoclonal antibody (mAb) Herceptin (Trastuzumab) chemically conjugated to Doxorubicin or Taxol. In vitro, drug-Herceptin conjugates exhibited cytotoxicity comparable to equimolar concentrations of free drugs, with the benefit that the cytotoxicity of the conjugates was selective for cells expressing the HER2 target. In vivo, treatment of tumor-bearing mice with Taxol-Herceptin conjugates had a reduction of primary tumors comparable to equivalent doses of free drugs. However, Taxol-Herceptin conjugates significantly reduced metastasis compared with equivalent doses of free drugs. Thus, the data support the concept that conjugates might target metastasis better than primary tumors. This would offer a potential therapeutic approach for management of metastatic breast cancer.

A large number of epidemiologic studies have been undertaken to identify potential risk factors for cancer, amongst which the association with cadmium has received considerable attention. There is compelling evidence in support of positive associations between cadmium and risk of lung cancer. In present study we measured the concentration of Cd in whole blood and scalp hair samples of 120 male lung cancer patients (smokers) and 150 controls or referents (smokers and nonsmokers) from different cities of Pakistan. Both referents and patients were of same age group (ranged 40-70 years), socio-economic status, localities and dietary habits. The scalp hair and whole blood samples were oxidized by 65% nitric acid: 30% hydrogen peroxide (2:1) ratio in microwave oven. To check the validity of the proposed method, a conventional wet acid digestion method was used to obtain total Cd concentration in certified samples of human hair BCR 397 and Clincheck control-lyophilized human whole blood. All digests were analyzed for Cd concentration by electrothermal atomic absorption spectrometer (ETAAS). The results of this study showed that the average Cd concentration was higher in the blood and scalp hair of lung cancer patients at different stages as compared to controls (p<001). The smoker referents have high level of Cd in both biological samples as compared to nonsmoker subjects. These results illustrate that the patients who continued smoking after confirmed diagnosis of lung cancer have 34.2-67.26 and 22.4-57.3% more Cd in blood samples and scalp hair than lung cancer patients who cease smoking. This study is compelling evidence in support of positive associations between cadmium, cigarette smoking and lung cancer risk.

Aim: To assess H pylori seroprevalence in lung cancer and determine whether there is a potential association between lung cancer and H pylori infection., Methods: The study was conducted on forty consecutive patients with lung cancer, confirmed by pathology (32 men, 8 women; mean age 55.50+/-11.91 years, range 16-77 years). Forty healthy subjects (25 men, 15 women; mean age 43.08+/-12.60 years, range 20-79 years) from the patients' family members were matched to each case subject on the basis of age and socioeconomic status. H pylori infection was detected with a commercially available immunoglobulin G (IgG) enzyme-linked immunosorbent assay (Trinity kit, Biotech co., USA), previously validated in adults (86% sensitivity, 96% specificity) against a gold standard of culture and histology., Results: H pylori seropositivity was present in 52.5% of patients with lung cancer in comparison to 45.0% of healthy control subjects. Although H pylori seropositivity was more frequent in lung cancer patients than in controls, the difference did not reach statistical significance (OR=1.35, 95% CI=0.56-3.25; P=0.65). In addition, there was no significant difference between cases and controls in terms of gastrointestinal symptoms., Conclusion: The earlier described association between H pylori infection and lung cancer was not supported in this study. Further studies with larger sample sizes should be undertaken to assess the frequency of H pylori infection in patients with lung cancer and their potential association.

The enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) expressed by the parasite Trypanosoma brucei (Tb) can convert allopurinol, a purine analogue, to corresponding nucleotides with greater efficiency than its human homologue. We have developed a retroviral system that expresses the parasitic enzyme and tested its capacity to activate the prodrug allopurinol to a cytotoxic metabolite. Cytotoxicity assays demonstrated that five non-small cell lung carcinoma cell lines transduced with the construct were sensitized to the prodrug by 2.1- to 7.6-fold compared with control values. This selectivity was not observed in seven other cell lines also expressing the construct, such as breast carcinoma. Assays indicated that enhanced cytotoxicity to allopurinol correlated with induction of apoptosis in lung cancer cells. The selectivity of this suicide gene was not explained either by the TbHGPRT expression or by the allopurinol accumulation. Our study shows that this novel system may represent a therapeutic tool for gene prodrug targeting of lung cancer, considering the fact that allopurinol is well tolerated in humans.

Many human epithelial carcinomas are characterized by the overexpression and constitutive activation of the epidermal growth factor receptor (EGF-R) via an autocrine signaling loop. We have investigated the effects of a ligand-blocking monoclonal antibody (mAb) against the EGF-R LA1 on selected parameters of human lung cancer cell lines (H322 and H661) and normal human bronchial epithelial (NHBE) cells. Using Western blot analysis, we show that H322 and NHBE cell lines express comparable levels of EGF-R/p170erbB-1. The LA1 mAb against EGF-R inhibits growth, induces differentiation to a more epithelial phenotype, reduces the constitutive activation of EGF-R, and upregulates epithelial cadherin glycoprotein expression in H322 and NHBE cells. In contrast, LA1 had no effect on either growth, differentiation, receptor tyrosine phosphorylation, or the expression of adhesion molecules in H661 cells, which is consistent with our finding that this cell line does not express detectable levels of EGF-R. These studies demonstrate that a blocking anti-EGF-R mAb can regulate proliferation, differentiation, and the expression of cell adhesion molecules in human bronchial epithelial cells. Our findings suggest possible therapeutic avenues for the treatment of invasive carcinomas via the blockade of EGF-R with antibodies.

The receptors erbB-3 and erbB-4 are members of the type 1 tyrosine kinase receptor family which also comprises epidermal growth factor receptor (EGF-R) and erbB-2. ErbB-3 and erbB-4 receptors are known to bind a family of related proteins termed heregulins. In this study, we report differential expression of P185erbB-2, P160erbB-3 and P180erbB-4, and their ligand heregulin alpha, in normal bronchial epithelial, and non-small cell lung carcinoma (NSCLC) cell lines. Expression of P185erbB-2 and P160erbB-3 vary from very low to a high level in NSCLC cell lines and a low level in normal bronchial cells. In contrast, P180erbB-4 was detected only in NSCLC cell lines but not in normal bronchial cells. Heregulin alpha is expressed at intermediate levels in the normal and cancer cell lines studied. Immunoprecipitation, using antibodies to erbB-2, erbB-3 or erbB-4 receptors, coupled to phosphotyrosine Western blot analysis indicates that these three receptors are constitutively tyrosine phosphorylated in lung cancer cell lines, but only erbB-2 and erbB-3 are autophosphorylated in normal cells. These data suggest that constitutive activation of erbB-2, erbB-3 and erbB-4 receptors could be induced by heregulin alpha via an autocrine loop mechanism, and that the active forms of erbB-4 may cooperate with the other members of the EGF-receptor family in human lung carcinogenesis.

We compared the biological activity of a new group of keto-C-glycosides to that of a narrow spectrum of unsaturated ketonucleosides in a panel of non-small-cell lung cancer (NSCLC) cells with various levels of intrinsic resistance to standard chemotherapy drugs. Unlike cisplatin, etoposide, adriamycin, or taxol, for which a significant difference in the cytotoxic effect was observed between sensitive cell lines (H460, H125, and MGH4) and drug-resistant cell lines (H661, MGH7, and FADU), nucleoside analogs were equally cytotoxic in NSCLC cell lines, with compound 92 being 10-fold more active than compound 43, 44, 81, or 161, while compound 3 was the least active. Apoptotic measurements with flow cytometric analysis of terminal uridine deoxynucleotide nick end-labeled cells revealed that the cytotoxic activity of these nucleosides correlated with their potency to induce apoptosis. Compound 92 triggered death in cells with wild-type p53, mutated p53, or p53 gene deletion. Our findings suggest that keto-C-glycosides may be promising alternative anticancer agents which merit further studies in in vivo cancer models refractory to standard chemotherapy drugs.

Non-small cell lung cancer (N-SCLC) is generally unresponsive to chemotherapy even without previous drug treatment, as opposed to small cell lung cancer (SCLC), which is initially responsive to chemotherapy. The mechanisms of this intrinsic resistance are unknown. This study was designed to investigate the role of DNA repair in intrinsic resistance of N-SCLC to cisplatin. A panel of primary N-SCLC cell cultures and established cell lines were examined and compared to SCLC cell lines established previously from untreated patients. The overall DNA repair capacity was estimated by the ability of cells to reactivate the pRSV-CAT plasmid damaged by cisplatin ("host cell reactivation" assay). Cytotoxicity was determined for cisplatin in vitro. N-SCLC cells were found to be significantly more resistant to cisplatin than SCLC cell lines isolated from untreated patients (P < 0.01). The capacity of N-SCLC cells to reactivate pRSV-CAT plasmid damaged with cisplatin and transfected into cells was higher in N-SCLC cells than in SCLC cells originating from patients who were untreated previously (P < 0.05). Correlation was also observed between chloramphenicol acetyltransferase activity and intrinsic resistance to cisplatin. However, no significant difference was observed between primary N-SCLC cultures and established cell lines. This study indicates that elevated DNA repair capacity is associated with drug resistance in lung cancer and suggests that modulation of DNA repair mechanism(s), such as the incorporation of specific DNA repair inhibitor(s) in therapeutic regimens, may help to improve therapeutic strategies of N-SCLC.