Lung Cancer Tumor Tissues Research Articles

Tumor-Infiltrating T Cells Concurrently Overexpress CD200R with Immune Checkpoints PD-1, CTLA-4, and TIM-3 in Non-Small-Cell Lung Cancer

Introduction: CD200R has been reported to be the receptor for the immune checkpoint molecule CD200 and can transduce immune-suppressive signals. In this study, we mainly focused on the expression level of CD200R in T cells in pulmonary artery (PA) blood and non-small-cell lung cancer (NSCLC) tumor tissue. Methods: Immune cells were isolated from dissected tumor samples and PA blood of NSCLC patients and analyzed with multiparameter flow cytometry. The co-expression of CD200R with other immune checkpoints, including programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), was also investigated. Results: CD200R expression was observed on the surface of approximately 75% of T cells among tumor-infiltrating leukocytes (TILs). Compared to T cells extracted from TILs, only 55% of T cells extracted from PA blood exhibited CD200R expression. Moreover, with higher expression of CD200R, the expression of other immune checkpoints, including PD-1, CTLA-4, and TIM-3, was also increased in tumor-infiltrating T cells compared to T cells in PA blood. Conclusions: Our results showed that those tumors were dominated by T cells expressing CD200R together with other checkpoints, which suggests a phenotypic change after T cell infiltration into the tumor, such as T cell exhaustion.

Circular RNA circ-PRKCI promotes lung cancer progression by binding to microRNA-1324 to regulate MECP2 expression.

We aimed to investigate the expression and specific molecular mechanism of circ-PRKCI in lung cancer (LCa). The relationship between the expression level of circ-PRKCI and the prognosis of patients was analyzed. The impacts of circ-PRKCI on the invasiveness of LCa cells were examined by Cell Counting Kit-8 (CCK-8) experiments, clone formation experiments, and transwell invasion experiments. Subcellular localization of circ-PRKCI was determined through nuclear separation experiments. Downstream target genes that can bind to circ-PRKCI was predicted through bioinformatics analysis, and was then verified by Dual-Luciferase experiments, quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) experiments, and Western blot experiments. Circ-PRKCI level was remarkably elevated in LCa tumor tissues and cell lines. At the same time, highly expressed circ-PRKCI was correlated with the poor prognosis of LCa patients. In vitro cell experiments revealed that inhibition of circ-PRKCI in LCa cell lines remarkably inhibited cell invasiveness and proliferation. In addition, circ-PRKCI can compete with MECP2 to bind microRNA-1324 and thus affect the progression of LCa. Our study shows for the first time that circ-PRKCI modulates the progression of LCa through microRNA-150-5p/MECP2 axis.

SUMO4 small interfering RNA attenuates invasion and migration via the JAK2/STAT3 pathway in non-small cell lung cancer cells.

Small ubiquitin-like modifier 4 (SUMO4) is the latest member of the sumoylation family, which enhances the stability of protein, regulates the distribution and localization of the protein, and affects the transcription activity of the protein. However, the role of SUMO4 in non-small cell lung cancer (NSCLC) has not yet been reported. The present study first demonstrated that SUMO4 was upregulated in a number of tissues from patients with NSCLC. Immunohistochemistry was performed to demonstrate the expression level of SUMO4 in lung cancer tumor tissues. Following the transfection, The EMT status and signaling pathway activation regulated by SUMO4-siRNA was assessed by western blotting. The Transwell and wound healing assays were performed to investigate the regulatory effect of SUMO4-siRNA on cell migration and invasion. Cell Counting Kit-8 assay was performed to investigate whether SUMO4-siRNA affected the chemosensitivity of the NSCLC cells to cisplatin. Statistical analysis of immunohistochemical results from the tissues showed that the overexpression of SUMO4 was significantly associated with sex, tumor type, history of smoking, T stage and poor prognosis. It was also identified that SUMO4 small interfering RNA attenuated invasion and migration in NSCLC cell lines, as well chemosensitivity to cisplatin via the inhibition of the JAK2/STAT3 pathway. In conclusion, SUMO4 may play an important role in the poor prognosis of patients with NSCLC. The present study indicates that SUMO4 may be a potential therapeutic target for NSCLC.

Xanthohumol targets the ERK1/2‑Fra1 signaling axis to reduce cyclin D1 expression and inhibit non‑small cell lung cancer.

High expression of cyclin D1 has a crucial role in the maintenance of unlimited cell growth in human cancer cells. The present study indicated that cyclin D1 was overexpressed in human non-small cell lung cancer (NSCLC) tumor tissues and cell lines. Knockout of cyclin D1 suppressed NSCLC cell growth, colony formation and in vivo tumor growth. Of note, the natural product xanthohumol (Xanth) inhibited NSCLC cells via the downregulation of cyclin D1. A further mechanistic study revealed that Xanth suppressed ERK1/2 signaling and reduced the protein levels of FOS-related antigen 1 (Fra1), which eventually inhibited the transcriptional activity of activator protein-1 and decreased the mRNA level of cyclin D1. Furthermore, suppression of ERK1/2 impaired Fra1 phosphorylation and enhanced Xanth-induced Fra1 ubiquitination and degradation. In addition, the S265D mutation compromised Xanth-induced Fra1 degradation. Finally, the in vivo anti-tumor effect of Xanth was validated in a xenograft mouse model. In summary, the present results indicated that targeting ERK1/2-Fra1-cyclin D1 signaling is a promising anti-tumor strategy for NSCLC treatment.

Sirtuin 7 promotes non‑small cell lung cancer progression by facilitating G1/S phase and epithelial‑mesenchymal transition and activating AKT and ERK1/2 signaling.

Increasing evidence has indicated the roles of sirtuin 7 (SIRT7) in numerous human cancers. However, the effects and the clinical significance of SIRT7 in human lung cancer is largely unknown. The present research demonstrated that SIRT7 was increased in human lung cancer tumor tissues. SIRT7 upregulation was associated with clinicopathological characteristics of lung cancer malignancy including positive lymph node metastasis, high pathologic stage and large tumor size. SIRT7 was also upregulated in human non-small cell lung cancer (NSCLC) cell lines. Furthermore SIRT7-overexpressed A549 (A549-SIRT7) and SIRT7-knocked down H292 (H292-shSIRT7) human NSCLC cell lines were established. Using these NSCLC cells and xenograft mouse models, it was revealed that SIRT7 overexpression markedly promoted growth and G1 to S cell cycle phase transition as well as migration, invasion and distant lung metastasis in A549 NSCLC cells, whereas SIRT7 knockdown suppressed these processes in H292 NSCLC cells. Mechanistically, in A549 NSCLC cells, SIRT7 overexpression significantly activated not only protein kinase B (AKT) signaling but also extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. SIRT7 overexpression also significantly downregulated cyclin-dependent kinase (CDK) inhibitors including p21 and p27 as well as upregulated cyclins including cyclin D1 and cyclin E1, and CDKs including CDK2 and CDK4. Notably, the epithelial-mesenchymal transition (EMT) process of A549 NSCLC cells was facilitated by SIRT7 overexpression, as evidenced by E-cadherin epithelial marker downregulation and mesenchymal markers (N-cadherin, vimentin, Snail and Slug) upregulation. In addition, SIRT7 knockdown in H292 NSCLC cells exhibited the opposite regulatory effects. Moreover, inhibition of AKT signaling abated the promoting effects of SIRT7 in NSCLC cell proliferation and EMT progression. The present data indicated that SIRT7 accelerated human NSCLC cell growth and metastasis possibly by promotion of G1 to S-phase transition and EMT through modulation of the expression of G1-phase checkpoint molecules and EMT markers as well as activation of AKT and ERK1/2 signaling. SIRT7 could be an innovative potential target for human NSCLC therapy.

Ultrasensitive multiplex detection of structural rearrangements in ALK, RET, ROS1 and PD-L1 using a comprehensive next-generation sequencing assay.

3572 Background: Oncogenic structural rearrangements (SR) in ALK, RET and ROS1 are well-described in lung cancer, and confer sensitivity to targeted therapy. SR disrupting the 3’UTR of PD-L1 gene have been reported in multiple cancer types and can potentially predict response to checkpoint immunotherapy. An amplicon-based next-generation sequencing (NGS) platform technology (AmpliMARK), previously optimized for detection of single nucleotide variations (SNVs), microsatellite instability and viral DNA, was extended to the multiplex detection of SR in ALK, RET, ROS1 and PD-L1 in cell-free DNA (cfDNA) and tumor tissue DNA. Methods: A hybrid primer-extension and adapter-ligation based method allowing detection of SR in a fusion-partner agnostic manner was utilized for multiplex target capture of genomic regions of ALK, RET, ROS1 and PD-L1 SR. Analytical validation was performed using admixtures of fragmented genomic DNA from an ALK SR-positive cell line, commercial standards containing RET and ROS1 SR, and synthetic PD-L1 SR gene constructs. Clinical performance was assessed in cfDNA samples from lung cancer patients and tumor tissue DNA samples from natural killer(NK)/T-cell lymphoma patients. Results: Detection of SR could be achieved to an allele frequency detection limit of 0.5% with sensitivity of 89.5% and specificity of 100% in admixture samples mimicking cfDNA. In an unselected series of 374 lung cancer cases, actionable SR for ALK, RET and ROS1 were detected in cfDNA of 9 samples, for an overall detection rate of 2.4%, and 1.8% (3 out of 168) when restricted to treatment-naive lung cancer cases only. In 29 NK/T-cell lymphoma tumor tissue samples, 9 samples were positive for PD-L1 SR, which were orthogonally confirmed by whole-genome sequencing, targeted sequencing or Sanger sequencing for a concordance rate of 100% across all samples. For 1 NK/T-cell lymphoma tumor tissue sample where matched plasma was available, the same PD-L1 SR was also detected in cfDNA. Conclusions: We have demonstrated and validated a comprehensive amplicon-based NGS assay for ultrasensitive multiplex detection of structural rearrangements in ALK, RET, ROS1 and PD-L1 across both cfDNA and tumor tissue DNA in analytical and clinical contexts. Ongoing studies will further evaluate the performance and utility of this assay across a larger number of clinical samples for the detection of these SR as well as additional cancer-associated SR involving NTRK1/2/3, FGFR2/3 and TMPRSS2.

High expression of SRY-box transcription factor 30 associates with well differentiation, absent lymph node metastasis and predicts longer survival in nonsmall-cell lung cancer patients.

The present study aimed to investigate SRY-box transcription factor 30 (SOX30) expression in nonsmall-cell lung cancer (NSCLC) tumor tissues and adjacent noncancerous tissues, and further explore the correlation of tumor SOX30 expression with clinical characteristics and survival profiles in patients with NSCLC.Totally, 365 patients with NSCLC who underwent resection were screened, and SOX30 expression was detected in their tumor tissues and adjacent noncancerous tissues via immunohistochemistry (IHC) assay, which was assessed by a semiquantitative method considering the multiplying staining intensity score and staining density score. According to the tumor SOX30 expression, patients were categorized as tumor SOX30 low (IHC score ≤3) and high (IHC score 4–12) patients, the latter were further divided into tumor SOX30 high+ (IHC score 4–6), high++ (IHC score 7–9), and high+++ (IHC score 10–12) patients.SOX30 was downregulated in NSCLC tumor tissues compared with adjacent noncancerous tissues. Meanwhile, tumor SOX30 high expression associated with well differentiation, absent lymph node metastasis, decreased TNM stage, but did not associated with age, gender, history of smoke and drink, hypertension, hyperlipidemia, diabetes, tumor size, or carcinoembryonic antigen level. Both accumulating disease-free survival and overall survival were the longest in tumor SOX30 high+++ patients, followed by tumor SOX30 high++ patients, and tumor SOX30 high+ patients, and the shortest in tumor SOX30 low patients. Besides, tumor SOX30 high expression was an independent predictor for longer disease-free survival and overall survival.Tumor SOX30 exhibits the potential to be a novel biomarker for survival prediction of patients with NSCLC.

The B7-H4 gene induces immune escape partly via upregulating the PD-1/Stat3 pathway in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is associated with high mortality rates worldwide. The costimulatory molecule, B7-H4, a member of the B7 family, plays an important role in immune regulation, mainly by inhibiting the proliferation of T cells to achieve a negative regulatory T cell immune response. The mechanism of action of B7-H4 in non-small cell lung cancer is unknown at present. Tumor tissues from 71 patients subjected to radical pneumonectomy were examined, along with NSCLC cells and BALB/c mice. Among the 71 NSCLC cases, overall and recurrence-free survival rates were significantly lower in those displaying high B7-H4 expression. Mechanistic analyses showed that B7-H4 promoted the growth and metastasis of non-small cell lung cancer tumor tissues in mice through effects on CD8+ T cell apoptosis. Data from western blot experiments further suggested that B7-H4 induced CD8+ T cell death, both in vitro and in vivo, and affecting the PD-1/Stat3 pathway and promoting immune escape of tumor cells. Our collective findings support the potential utility of B7-H4 gene expression as a marker of NSCLC prognosis and provide a novel strategy for targeted therapy.

MicroRNA-107 may regulate lung cancer cell proliferation and apoptosis by targeting TP53 regulated inhibitor of apoptosis 1.

Lung cancer causes over 1.6 million mortalities worldwide annually. MicroRNAs (miRs) are involved in various types of cancer-associated processes. The present study investigated the possible mechanism of miR-107 in the development of lung cancer in order to identify novel targets for clinical treatment. The expression levels of miR-107 and its putative target gene TP53 regulated inhibitor of apoptosis 1 (TRIAP1) were measured in lung cancer tumor tissues and non-tumor adjacent tissues. Subsequently, the association between TRIAP1 and miR-107 was investigated using a dual-luciferase reporter assay. Following transfection, the effects of miR-107 and TRIAP1 on the proliferation and apoptosis of lung cancer cell lines in vitro were investigated using Cell Counting Kit-8 and flow cytometry assays, respectively. Furthermore, the regulatory effect of miR-107 on the expression levels of TRIAP1 and associated proteins was analyzed using a western blot assay. The results revealed lower expression levels of miR-107 and higher expression levels of TRIAP1 in lung cancer tumor tissues compared with non-tumor adjacent tissues. The dual-luciferase reporter assay demonstrated that TRIAP1 is a target gene of miR-107. Additionally, the results revealed that overexpression of miR-107 resulted in a lower proliferation rate and higher apoptosis rate of A549 cells, compared with the negative control (NC) and control groups (P<0.01). The variation of cell proliferation and apoptosis induced by miR-107 mimics was reversed by co-transfection with pcDNA3.1-TRIAP1. Furthermore, the expression levels of cyclin D1 and proliferating cell nuclear antigen were markedly decreased in the miR-107 mimics group compared with the NC group (P<0.01). The expression levels of BCL2 associated X apoptosis regulator, tumor protein p53 and caspase 3 were upregulated and the expression levels of TRIAP1 and BCL2 apoptosis regulator were significantly reduced in the miR-107 mimics group compared with the NC group (P<0.01). The results of the present study suggested that miR-107 regulates lung cancer cell proliferation and apoptosis by targeting TRIAP1.

Downregulation of CPA4 inhibits non small-cell lung cancer growth by suppressing the AKT/c-MYC pathway.

Carboxypeptidase A4 (CPA4) is a member of the metallocarboxypeptidase family. A previous study indicated that CPA4 may participate in the modulation of peptide hormone activity and hormone‐regulated tissue growth and differentiation. However, the role of CPA4 in lung tumorigenesis remains unclear. Our study revealed that CPA4 expression was higher in both lung cancer cells and tumor tissues. We performed 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assays, colony‐formation assays, and Cellomics ArrayScan Infinity analysis to demonstrate that CPA4 knockdown inhibited non small–cell lung cancer (NSCLC) cell proliferation. Conversely, ectopic expression of CPA4 enhanced lung cancer cell proliferation. Consistent with these observations, we generated xenograft tumor models to confirm that CPA4 downregulation suppressed NSCLC cell growth. Mechanistically, we revealed that CPA4 downregulation may induce apoptosis and G1‐S arrest by suppressing the protein kinase B/c‐MYC pathway. These results suggest that CPA4 has an oncogenic effect on lung cancer growth. Taken together, we identified a novel gene in lung cancer that might provide a basis for new therapeutic targets.

Anti-CTLA-4 Activates Intratumoral NK Cells and Combined with IL15/IL15Rα Complexes Enhances Tumor Control.

Antibodies targeting CTLA-4 induce durable responses in some patients with melanoma and are being tested in a variety of human cancers. However, these therapies are ineffective for a majority of patients across tumor types. Further understanding the immune alterations induced by these therapies may enable the development of novel strategies to enhance tumor control and biomarkers to identify patients most likely to respond. In several murine models, including colon26, MC38, CT26, and B16 tumors cotreated with GVAX, anti-CTLA-4 efficacy depends on interactions between the Fc region of CTLA-4 antibodies and Fc receptors (FcR). Anti-CTLA-4 binding to FcRs has been linked to depletion of intratumoral T regulatory cells (Treg). In agreement with previous studies, we found that Tregs infiltrating CT26, B16-F1, and autochthonous Braf V600E Pten -/- melanoma tumors had higher expression of surface CTLA-4 (sCTLA-4) than other T-cell subsets, and anti-CTLA-4 treatment led to FcR-dependent depletion of Tregs infiltrating CT26 tumors. This Treg depletion coincided with activation and degranulation of intratumoral natural killer cells. Similarly, in non-small cell lung cancer (NSCLC) and melanoma patient-derived tumor tissue, Tregs had higher sCTLA-4 expression than other intratumoral T-cell subsets, and Tregs infiltrating NSCLC expressed more sCTLA-4 than circulating Tregs. Patients with cutaneous melanoma who benefited from ipilimumab, a mAb targeting CTLA-4, had higher intratumoral CD56 expression, compared with patients who received little to no benefit from this therapy. Furthermore, using the murine CT26 model we found that combination therapy with anti-CTLA-4 plus IL15/IL15Rα complexes enhanced tumor control compared with either monotherapy.

Downregulation of TCEAL7 expression induces CCND1 expression in non-small cell lung cancer.

Transcription Elongation Factor A-like 7 (TCEAL7) was first reported as a candidate tumor suppressor gene because of its inactivation in ovarian cancer as a result of promoter methylation. Down-regulation of the TCEAL7 gene expression was also associated with other cancers such as endometrial, breast, brain, prostate, gastric cancers, glioblastoma and linked to tumor phenotypes and clinical outcomes. However, there is no report in the literature investigating the role of TCEAL7 in non-small cell lung cancer. Cyclin D1 is an important molecule in the transition from G1 to S phase of the cell cycle, and is frequently deregulated in cancers. Cylin D1 (CCND1) gene is amplified or overexpressed in a variety of tumors. In our previous study we reported that CCND1 over-expression was not associated with amplification in non-small cell lung cancer. Recently, it has been reported that TCEAL7 regulates CCND1 expression through myc-binding E-box sequences. The aim of this study was to investigate the expression of TCEAL7 gene in non-small cell lung cancer and to determine its effect on the CCND1 expression level. For this purpose, expression levels of TCEAL7 and CCND1 genes were investigated in 50 patients with non-small cell lung cancer by quantitative real time polymerase chain reaction (qRT-PCR). TCEAL7 was under-expressed (68%) in non-small cell lung cancer tumor tissues while CCND1 was over-expressed (42%). The TCEAL7 levels negatively correlated with increased CCND1 expression (p = 0.002).

Abstract 2750: Physical and functional landscape of deubiquitinating enzymes (DUBs) in KRAS mutant lung cancer

Abstract DUBs are involved in tumorigenesis and are of high interest as potential targets for cancer therapy. However, target discovery is problematic due to the absence of clear DUB activity within subsets of human cancers. Our goal was to profile DUBs and their activity in KRAS-mutated lung cancer cell lines using chemical biology strategies. Approximately 25% of patients with Lung adenocarcinoma have tumor associated KRAS mutations in non-small lung cancers (NSCLC), yet specific RAS inhibitors against KRAS-mutated lung cancer have not yet been successfully developed. We used activity-based protein profiling (ABPP) to profile DUBs in 25 distinct human lung cancer cell lines harboring KRAS mutations. ABPP uses chemical probes that are active-site directed and covalently bind to a class of enzymes in complex proteome. Whole cell lysates were incubated with HA-UB-VME and HA-UB-PA probes and ABPP pull-down was performed. Enriched proteins were trypsin digested and peptides were analyzed using liquid chromatography and tandem mass spectrometry (LC-MS/MS). MAXQUANT software was used to quantify DUBs. We related activity of observed DUBs to effects on cell viability by examining publicly available RNAi (Project DRIVE) and CRISPR (PICKLES) databases. A total of 50 DUBs were identified in our ABPP screen. Each cell line expressed at least 32 different DUBs and 22 DUBs were represented in all cell lines. The identified DUBs include 48% USP, 36% OTU, 8% JAMM and 8% UCH &amp; MJD family. USP5, USP7, USP14, USP15, UCHL5, UCHL3, OTUB1, PRP8, PSMD7 and PSMD14 are the highly expressed DUBs. Functional enrichment approach and protein-protein interaction network reveal that 70% of the active DUBs are involved in cell cycle regulation. DDR, RNA splicing, TGF-beta receptor, NF-kB and Wnt signaling are some other enriched pathways. Of the 35 cell cycle DUBs, CRISPR screens identify 14 DUBs and DRIVE data identified 11 DUBS as having effects on cell viability. Combining the ABPP data, shRNA and CRISPR results, OTUD5, BRCC3, UCHL5, UFD1L, USP5, YY1, USP3, USP39, USP37, OTUB1, USP9X and COPS5 have activity in KRAS mutant lung cancer cell lines and demonstrate reduced cell viability with loss of function through CRISPR or RNAi. We have identified and prioritized DUB targets in KRAS mutant lung cancer. Future studies will further validate DUB as targets and identify mechanisms of activity in KRAS mutant lung cancers, as well as examine DUB activity using ABPP in human lung cancer tumor tissues, including adenocarcinoma (KRAS mutant and wild-type) and squamous cell lung cancer. Citation Format: Emma Adhikari, Shikha Mahajan, Harshani Lawrence, Yan Yang, Bin Fang, Eric Haura. Physical and functional landscape of deubiquitinating enzymes (DUBs) in KRAS mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2750.

Nociceptin Receptor Is Overexpressed in Non-small Cell Lung Cancer and Predicts Poor Prognosis.

Classic opioid receptors, mu (μ), delta (δ), and kappa (κ), have been reported to be expressed in non-small cell lung cancer (NSCLC) cell lines and tumor tissues and to play a role in tumor prognosis. However, the expression and role of the non-classic opioid receptor, nociceptin receptor (NOP) in cancer are unclear. Our hypothesis was that NOP was also highly expressed in NSCLC tumor tissues and this could be correlated with patients' prognostic characters. Expression of NOP was examined in archived cancer tissues from 129 enrolled NSCLC patients by immunohistochemistry and was further analyzed with the patients' outcomes. NOP expression in NSCLC cell lines was also detected. The dataset from Kaplan-Meier Plotter was used to explore the correlation between the levels of NOP mRNA in cancerous tissue and the prognosis of NSCLC patients. Cell functional assays were performed to detect the effect of NOP activation on tumor aggressive furthers. Results showed NOP expression was highly expressed in cancer tissues and human cancer cell lines. NOP expression was not associated with patients' opioid requirement but closely with some clinicopathological indicators which reflected the malignancy. Moreover, NOP staining level was the independent poor prognostic factor for NSCLC patients receiving lobectomy, which was further verified by determining the mRNA expression levels through the online dataset. In vitro experiments revealed that NOP activation promotes the proliferation and invasion of A549 cells via PI3K/Akt signaling pathway. We conclude that NOP is overexpressed in NSCLC and is inversely correlated with patient's postoperative survival.

Inhibiting Glycine Decarboxylase Suppresses Pyruvate-to-Lactate Metabolism in Lung Cancer Cells.

Glycine decarboxylase (GLDC) gene is frequently upregulated in various types of cancer including lung, prostate and brain. It catabolizes glycine to yield 5,10-methylenetetrahydrofolate, an important substrate in one-carbon metabolism for nucleotide synthesis. In this study, we used exon splicing modulating steric hindrance antisense oligonucleotide (shAON) to suppress GLDC expression and investigated its effect on pyruvate metabolism via hyperpolarized carbon-13 magnetic resonance spectroscopy (MRS). The MRS technique allows us to study in vivo metabolic flux in tumor tissues with/without GLDC-shAON intervention. Here, we show that GLDC-shAON treatment is able to suppress lung cancer cell growth and tumorigenesis, both in vitro and in vivo. The carbon-13 MRS results indicated that the conversion of pyruvate into lactate in GLDC-shAON-treated tumor tissues was significantly reduced, when compared with the control groups. This observation corroborated with the reduced activity of lactate dehydrogenase and pyruvate dehydrogenase in GLDC-shAON-treated lung cancer cells and tumor tissues. Glycolysis stress test showed that extracellular acidification rate was significantly suppressed after GLDC-shAON treatment. Besides lung cancer, the antitumor effect of GLDC-shAON was also observed in brain, liver, cervical, and prostate cancer cell lines. Furthermore, it enhanced the treatment efficacy of cisplatin in lung cancer cells. Taken together, our findings illustrate that pyruvate metabolism decreases upon GLDC inhibition, thereby starving cancer cells from critical metabolic fuels.

Preclinical Evaluation of SCC244 (Glumetinib), a Novel, Potent, and Highly Selective Inhibitor of c-Met in MET-dependent Cancer Models.

Because the receptor tyrosine kinase c-Met plays a critical role in tumor growth, metastasis, tumor angiogenesis, and drug resistance, the c-Met axis represents an attractive therapeutic target. Herein, we report the first preclinical characterization of SCC244, a novel, potent, and highly selective inhibitor of c-Met kinase. SCC244 showed subnanomolar potency against c-Met kinase activity and high selectivity versus 312 other tested protein kinases, making it one of the most selective c-Met inhibitors described to date. Moreover, this inhibitor profoundly and specifically inhibits c-Met signal transduction and thereby suppresses the c-Met-dependent neoplastic phenotype of tumor and endothelial cells. In xenografts of human tumor cell lines or non-small cell lung cancer and hepatocellular carcinoma patient-derived tumor tissue driven by MET aberration, SCC244 administration exhibits robust antitumor activity at the well-tolerated doses. In addition, the in vivo antitumor activity of SCC244 involves the inhibition of c-Met downstream signaling via a mechanism of combined antiproliferation and antiangiogenic effects. The results of the current study provide a strong foundation for the clinical investigation of SCC244 in patients with tumors harboring c-Met pathway alterations. Mol Cancer Ther; 17(4); 751-62. ©2017 AACR.

CD103+CD8+ T lymphocytes in non-small cell lung cancer are phenotypically and functionally primed to respond to PD-1 blockade

CD103+CD8+ tumor infiltrating lymphocytes (TILs) have been linked to prolonged survival in various types of cancer including non-small cell lung cancer (NSCLC). However, the factors associated with the retention of CD103+CD8+ TILs in lung cancer tissues remain largely unknown. Additionally, the contribution of CD103+CD8+ TILs to effective PD-1 based immunotherapy has not been fully elucidated. In this study, we identified that the expression levels of E-cadherin and TGF-β were significantly correlated with the distribution and the density of CD103+ TILs in lung cancer tumor tissues. Unexpectedly, we observed that CD103+CD8+ TILs that expressed higher levels of PD-1 co-express Ki-67. Moreover, CD103+CD8+ TILs expressed an increased level of T-bet compared to their counterparts, indicating these cells may be better armed for immunotherapy. Lastly, PD-1 pathway blockade led to a significantly increased production of IFN-γ by CD103+CD8+ TILs, suggesting CD103+CD8+ TILs could serve as a predictive biomarker for PD-1 based immunotherapy.

Epigenetic silencing of miR-493 increases the resistance to cisplatin in lung cancer by targeting tongue cancer resistance-related protein 1(TCRP1)

BackgroundThe potential mechanisms regarding how methylation of microRNA(miRNA) CpG Island could regulate cancer cell chemo-resistance remains unclear. This study aims to explore the epigenetic dysregulation mechanism of miRNA-493 and the ability to modulate lung cancer cell chemotherapy resistance.MethodsReal-time quantitative PCR (qRT-PCR) and In situ hybridization (ISH) were used to analyze the expression of miR-493 in lung cancer cell lines and tumor tissue, respectively. Bisulfite sequencing PCR (BSP) was used to exam the promoter CpG Island of miR-493. The effect of miR-493 on chemosensitivity was evaluated by cell viability assays, apoptosis assays and in vivo experiment. The DNA damage was measured by γ-H2AX immunofluorescence. Luciferase reporter assay was used to assess the target genes of miR-493. Expression of target proteins and downstream molecules were analyzed by Western blot.ResultsmiR-493 is silenced in resistant lung cancer cell due to the aberrant DNA methylation. Enforced expression of miR-493 in lung cancer cells promotes chemotherapy sensitivity to cisplatin through impairing the DNA damage repair and increasing the cells apoptosis in vitro and in vivo. Furthermore, we identify that TCRP1 is a direct functional target of miR-493. Ectopic expression of TCRP1 attenuated increased apoptosis in miR-493-overexpressing lung cancer cells upon cisplatin treatment. Meanwhile, miR-493 level is negatively correlated with TCRP1 expression in lung cancer patients and TCRP1 expression were correlated with poor survival.ConclusionsOur results highlight that hyper-methylation of miR-493CpG island might play important roles in the development of lung cancer chemo-resistance by targeting TCRP1, which might be used as a potential therapeutic target in preventing the chemo-resistance of lung cancer.

Abstract 5474: Sphingosine kinase 2/sphingosine 1-phosphate signaling regulates p16 mediated accelerated aging in normal somatic tissues and tcf21 mediated tumor suppression in lung cancer

Abstract Sphingosine 1-phosphate (S1P), a pro-proliferative sphingolipid generated by Sphingosine Kinases is upregulated in many cancers. Telomerase (hTERT), is a ribonucleoprotein that extends the ends of chromosomes (telomeres), to promote lung cancer growth. We discovered that SK2 generated S1P binds and stabilizes telomerase in the nuclear periphery by allosterically mimicking protein phosphorylation. Mechanistically, S1P binding protected telomerase from MKRN1 mediated degradation thereby preventing telomere dysfunction and senescence. The objective of this study is to delineate the molecular mechanism of SK2-S1P mediated telomere dysfunction in aging and lung cancer. SK2 knockout mice tissues showed increased telomeric DNA damage and senescence associated-beta galactosidase activity. Moreover, SK2 knockout mice displayed aging phenotypes by reduced subcutaneous fat, atrophy in spleens and hypoplasia in mice testes. Also, SK2 knockout fibroblasts showed increased senescence and robust p16 expression and interestingly, p16 ablation rescued SK2 knockout fibroblasts from senescence. Importantly, we found SK2 and telomerase protein levels to be upregulated in lung cancer tumor tissues (n=48). Mechanistically, pharmacological inhibition of SK2 by ABC294640 (in Phase II clinical trials) leads to telomere dysfunction in A549 cells and interestingly wild type and a phosphomimicking S921D-hTERT mutant of human telomerase overexpression prevented telomere dysfunction whereas S1P defective mutant D684A-hTERT did not. In vivo, inhibition of SK2 by ABC294640 or shRNA knockdown displayed reduced tumor volume, decreased telomerase protein levels and increased TUNEL positive cells. Interestingly, qPCR based gene array revealed robust increase in TCF21 tumor suppressor in stable shSK2 tumors. Furthermore, shRNA against TCF21 following SCID mice xenograft studies showed protection against ABC294640 induced tumor suppression. Interestingly, SK2 inhibition showed ATM/ATR kinase mediated p-CHK1 increase and subsequent activation of caspase-3 in both in vivo and in vitro experiments and stable TCF21 knockdown prevents apoptotic cell death. Importantly, p16 overexpression in p16 deficient A549 lung cancer cells prevented caspase-3 activation and leading to senescence induction following inhibition of SK2-S1P by ABC294640 or shRNA against SK2. Overall, our data suggest that SK2-S1P regulates telomere dysfunction through p16 mediated aging in normal somatic tissues and TCF21 mediated tumor suppression in lung cancer and interestingly SK2-S1P and p16 acts as a molecular switch between senescence and apoptosis in normal and cancer cells respectively. Citation Format: Shanmugam Panneer Selvam, Marion Cooley, Kristi Helke, Elizabeth Garrett-Mayer, Charles D. Smith, Besim Ogretmen. Sphingosine kinase 2/sphingosine 1-phosphate signaling regulates p16 mediated accelerated aging in normal somatic tissues and tcf21 mediated tumor suppression in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5474. doi:10.1158/1538-7445.AM2017-5474

Abstract 1918: Gene characterization of lung-tumorspheres for their usage as an in vitro screening platform for testing new therapeutic strategies

Abstract Background: Lung cancer features like treatment resistance or tumor relapse have been linked to cancer stem cells (CSCs), a population of cells with self-renewal properties, and the ability to grow forming tumorspheres in non-adherent conditions. The aim of this study was to isolate and characterize tumorspheres from lung cancer cell lines and tumor tissue from resectable non-small cell lung cancer (NSCLC) patients and to use them as an in vitro platform for drug screening. Methods: This study was performed on cells from seven NSCLC tumor samples and five cell lines (H1650, H1993, H1395, A549 and PC9) grown in monolayer and as spheroids. The expression of 60 genes, including CSC-markers, pluripotency inducers, cell cycle regulators, invasion promoters and components of Notch, Wnt and Hedgehog pathways was analyzed by RTqPCR. Drugs commonly used in clinical guidelines (Cisplatin, Paclitaxel, Gefitinib, Erlotinib, Afatinib, Pemetrexed and Vinorelbine) and specific inhibitors of Wnt and Hedgehog pathways (Iwp2, Xav939, LDE225 and Vismodegib and Salynomicin) were tested in triplicates at 4 different concentrations. Cell viability was measured after 48h and 72h using MTS Assay, normalized to the respective mock-treated control cells and presented as percentage of control. Statistical analyses were considered significant at p&amp;lt;0.05. Results: Lung tumorspheres had significant increased expression of CSC-related genes EPCAM1, CD44, CCND1, KLF4 and CDKN1A, compared to their paired-adherent cells. Likewise, epithelial to mesenquimal transition (EMT) inducer SNAI1 and integrin ITGA6 were overexpressed in spheroids too. Regarding stemness pathway, Notch pathway ligands JAG1 and DLL4 and receptors NOTCH1, NOTCH2 and NOTCH3 showed higher expression in lungspheres. In Wnt pathway, higher expression levels of WNT3, CTNNB1 and GSK3B were found in tumorspheres. No significant differences were found for the rest of genes. Drug screening showed classical anticancer drugs, such as Cisplatin, Vinorelbine or Pemetrexed, had mild cytotoxic effects on lungspheres, obtaining significant differences with the adherent-cultured cells. In contrast, the stemness pathways inhibitors IWP2, XAV939, Salinomycin and Vismodegib showed higher cytotoxic effects on spheroids than in cells grown in monolayers. Conclusions: Lung-tumorspheres derived from cancer cell lines and primary tumor tissues show increased levels of CSC markers and components of Notch and Wnt signaling pathways compared to the cells grown in adherence. Spheroids showed resistance to classical anticancer drugs, and a greater response to inhibitors from Notch, Wnt and Hedgehog pathways, strengthening its possible use as a short-term culture platform for a simple, and cost-effective screening to investigate novel therapeutic approaches. Supported by grants RD12/0036/0025 from RTICC-FEDER, PI12-02838/PI15-00753 from ISCIII and SEOM/2012. Citation Format: Alejandro Herreros-Pomares, Ester Munera-Maravilla, Silvia Calabuig-Fariñas, Rut Lucas, Rosa Farràs, Ana Blasco, Susana Torres, Jose Ferri, Ricardo Guijarro, Miguel Martorell, Eloísa Jantus-Lewintre, Camps Carlos. Gene characterization of lung-tumorspheres for their usage as an in vitro screening platform for testing new therapeutic strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1918. doi:10.1158/1538-7445.AM2017-1918