NSCLC Progression Research Articles

Abstract 3553: Mutant KRas-mediated AKT2 activation supports lung cancer growth by activating complex II-driven mitochondrial metabolism

Abstract Non-small cell lung cancer (NSCLC), the most common type of lung cancer (comprising 80-85 %), is a leading cause of cancer-related death worldwide. KRAS mutation is s major driver oncogene in NSCLC. Constitutive activation of K-Ras via oncogenic mutations plays a key role in development and progression of NSCLC, and is an important target for drug development. Indeed, drugs that target RAF-MAPK and PI3K, the well-established downstream pathways of K-Ras, are being evaluated in the clinic. However, to date, no effective treatments specifically targeting K-Ras have reached the clinic. A new direction for anti-Ras therapies targeting Ras-mediated altered cellular metabolism is implied by recent studies. Hence, as an attempt to investigate novel therapeutic targets to suppress KRAS-driven NSCLC, we assessed metabolic changes in NSCLC cell line and mouse model systems with oncogenic K-Ras mutations. We show that activation of Ras causes metabolic alterations, leading to aerobic glycolysis. Interestingly, despite the changes in the glycolytic pathway, NSCLC cell line and mouse models carrying K-Ras mutation revealed an elevated mitochondrial membrane potential, superoxide production as well as maximal oxygen consumption rate. Moreover, blockade of mitochondrial respiration effectively reduced mutant KRas-driven neoplastic phenotypes in NSCLC cell lines vitro and lung tumor formation in vivo. In contrast, blockade of glycolysis was not effective in the mutant KRas-driven tumorigenic activities. Mechanistically, mutant KRas-mediated activation of Akt2 led to increased expression of succinate dehydrogenase subunit A (SDHA) through increased protein stability by blocking proteasome-mediated degradation, resulting in mitochondrial complex II-dependent respiration. Our study suggests that induction of mitochondrial complex II-dependent respiration is an important mechanism by which oncogenic mutant KRas induces metabolic changes and ROS stress in NSCLC cells, contributing to lung carcinogenesis. Our findings elucidate potential metabolic targets that may provide novel therapeutic strategy for treating NSCLC with oncogenic KRAS mutations. Citation Format: Kwan-Hee Park, Ho-Young Lee. Mutant KRas-mediated AKT2 activation supports lung cancer growth by activating complex II-driven mitochondrial metabolism [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 3553. doi:10.1158/1538-7445.AM2017-3553

Abstract 2871: Non-invasive monitoring of the efficacy of anticancer therapeutic agent in lung orthotopic xenograft models of NSCLC using Bio-Luminescence Imaging (BLI)

Abstract Non-Small Cell Lung Cancer (NSCLC) patients have a poor rate of survival owing to diagnosis at a later stage. This necessitates development of novel therapeutic strategies that limit rapid progression of NSCLC and imaging techniques that monitor such changes in vivo. There has been a significant growth of bioluminescence imaging applications in small laboratory animal models in recent years, owing to its high sensitivity, efficiency, and non-invasive nature. Our lab has previously demonstrated that a therapeutic agent that disrupts mitochondrial respiration effectively hampers proliferation of NSCLC cell lines in vitro. With an effort to investigate the potential of this therapeutic agent in clinical settings, we used a lung orthotopic xenograft mouse model along with bioluminescence imaging (BLI) to continuously monitor the efficacy of the drug in vivo. Briefly, we implanted NSCLC cell line transduced with lentiviral particles carrying luciferase sequence, in the left lung of 6-8 weeks old female NOD/SCID mice to generate lung orthotopic xenografts. The therapeutic agent was administered intravenously over several weeks. Tumor growth was then compared between the untreated versus the treated mice, non-invasively through bioluminescence imaging (Perkin Elmer’s IVIS Lumina III Imager). On sacrificing the mice, the lungs were harvested, formalin fixed and paraffin embedded for Immunohistochemistry. Our BLI data show that in the mice treated with the therapeutic agent, tumors are localized to the left lung (site of implantation) as opposed to the control mice where the tumors metastasized to the right lung. The BLI data indicates significant decrease in radiance (total flux in photons per second) in mice subjected to treatment when compared to control mice. We show that the therapeutic agent significantly inhibits progression and metastasis of human NSCLC cell line orthotopically injected in the lungs of NOD/SCID mice. The results suggest that it might be potent anti-cancer drug as it is effective in a model that recapitulates the natural environment of lung tumorigenesis. Immunohistochemical studies are underway to discern the mechanistic details. Citation Format: Sanchareeka Dey, Sarada Preeta Kalainayakan, Poorva Ghosh, Li Zhang. Non-invasive monitoring of the efficacy of anticancer therapeutic agent in lung orthotopic xenograft models of NSCLC using Bio-Luminescence Imaging (BLI) [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 2871. doi:10.1158/1538-7445.AM2017-2871

Abstract 1435: Immune/inflammatory polymorphisms predict lung cancer survival

Abstract Lung cancer is the leading cause of cancer related mortality in the United States, with a median five year survival of ~16%. Recent advancements in the treatment of lung cancer focus on modulating the immune system to improve patient outcomes with markedly reduced toxicity profiles. This study aims to investigate single nucleotide polymorphisms (SNPs) within functional units of the immune system genome to identify genetic markers indicative of overall survival in a cohort of metropolitan Detroit lung cancer patients. This cohort presents a unique population of lung cancer patients for analysis, consisting of 40% African Americans, whom are known to have worse outcomes than their Caucasian counterparts. To assess SNPs within functional units of the immune genome, a hierarchical immune system gene and pathway list was constructed, incorporating genes and pathway identifiers from the Reactome database as well as previous genetic and inflammatory specific cancer studies to generate a well curated gene and pathway dataset. Genomic locations for the genes, together with proximal regulatory regions, were obtained from the UCSC genome browser for cross-referencing with the Illumina MEGA SNP array to generate SNPs for inclusion. Preliminary analysis of 848 NSCLC patients in association with 29,126 immune specific SNPs identified six SNPs as significantly associated with survival (p-value <10-4) in this lung cancer cohort, implicating the following six genes, PXN, GFRA3, HLA-DQA1, KSR2, BTBD1, and ATF2; although further gene/pathway analysis is necessary to elucidate how these associations interact in the context of genes and pathways of the immune system. PXN over expression has been previously implicated in both high risk lung epithelial dysplasia as well as in the development of lung adenocarcinoma in prior clinical studies. Additionally, GFRA3 has been identified as an activator of the RET kinase and increased GFRA3 activity in context of the Artemin pathway is a known promoter of NSCLC progression. As well, select polymorphisms in the HLA-DQA1 gene are known clinical modifiers of lung squamous cell carcinoma risk. Finally, ATF2 is a broadly active transcription factor and ATF2 upregulation has been associated with tumorigenesis and metastasis in both cell models and patient tumor samples in multiple cancer studies. The previous association of these genes in lung cancer is promising and further immune system gene/pathway analysis is warranted. Citation Format: Donovan Watza, Chrissy Lusk, Angie Wenzlaff, Christine Neslund-Dudas, Gregory Dyson, Ayman Soubani, Shirish Gadgeel, Ann G. Schwartz. Immune/inflammatory polymorphisms predict lung cancer survival [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 1435. doi:10.1158/1538-7445.AM2017-1435

Impact of atezolizumab (atezo) treatment beyond disease progression (TBP) in advanced NSCLC: Results from the randomized phase III OAK study.

9001 Background: Cancer immunotherapy (CIT) can have a positive impact on OS that exceeds response rate or PFS effects, termed post progression prolongation of survival (PPPS). This effect can also result from unconventional CIT response due to tumor immune infiltration or delayed response, reducing reliability of RECIST v1.1 (RECIST) PD as an indicator of treatment failure. In the primary analysis (N = 850) of OAK, a study of atezo vs docetaxel (doc) in 2L/3L NSCLC, OS favored atezo (HR 0.73; 95% CI: 0.62, 0.87), despite similar PFS between arms (HR 0.95; 95% CI: 0.82, 1.10). Here we evaluate clinical benefit from TBP, defined by post PD tumor regression, OS and safety. Methods: Patients (pts) received atezo 1200 mg IV q3w until PD or loss of clinical benefit per investigator or doc 75 mg/m2 IV q3w until PD per RECIST. No crossover was allowed. Primary outcome measure: OS. Atezo TBP pts were evaluated for post PD tumor change and for safety pre and post PD. OS from time of PD per RECIST was evaluated in both arms (data cutoff, July 7, 2016; minimum follow-up, 19 mo). Results: Among 332 atezo pts with PD, 51% (n = 168) continued atezo TBP; 7% (12/168) achieved subsequent response in target lesions (≥ 30% reduction from new baseline at PD); 49% (83/168) had stable target lesions (best change between +20% and −30%). mOS was 12.7 mo (95% CI: 9.3, 14.9) post PD for pts on atezo TBP (Table). Atezo TBP was not associated with increased safety risk. Conclusions: This is the first report from a Phase III study of CIT in NSCLC to evaluate post PD OS in pts continuing treatment beyond RECIST PD. Atezo TBP was associated with high frequency of stable or decreased target lesions, mOS > 1 year and a tolerable safety profile, all supporting prolonged treatment benefit consistent with PPPS. NCT02008227 Clinical trial information: NCT02008227. [Table: see text]

Phase II study of stereotactic radiosurgery or other local ablation followed by erlotinib for patients with EGFR mutation who have previously progressed on an EGFR tyrosine kinase inhibitor (TKI).

e20623 Background: For patients with metastatic EGFR mutated NSCLC, 1stline treatment with EGFR TKIs such as erlotinib result in a median PFS of about 10 months. In patients with a limited number of progressing lesions, local ablation therapy (LAT) of progressive lesions followed by re-initiation of TKI has shown promise in retrospective studies but to date this strategy has not been testing in prospective fashion. Methods: As part of an IRB approved open label prospective phase II trial (NCT01573702), patients with EGFR mutated NSCLC and immediate progression on a TKI in < 5 locations were treated with stereotactic radiosurgery (SRS) to progressing lesions followed by re-initiation of erlotinib. Our primary endpoint is PFS, and we hypothesize that it will be at least 3 months. Results: 25/40 planned patients have been enrolled and data is available on 23. By local reporting, 14 had exon 19, 5 had exon 21, 1 had compound exon 18 and exon 20, 1 had compound exon 19 and EML4/ALK and 2 were unknown. 65% were female, all were non-Hispanic white, median age 62.5 (range 43-85), PS0 65.2%/PS1 34.8%, median Charlson Comorbidity Index 6, and 71.4% were never smokers. Median number of lesions treated was 1 (range 1-3). Treated lesions included lung (14), bone (6), liver (3) and brain (2). There were no G3-4 AEs to SRS. Two subjects had grade 3 rash on erlotinib. Median PFS post treatment was 5.8 months (95% CI, 2.5 to 11.3) and median OS was 2.9 years (95% CI 1.1 to 2.9). Conclusions: LAT resulted in a modest extension in the duration of targeted therapy, supporting retrospective data in this population. Accrual to this study has been challenging due to the availability of 3rd generation EGFR TKIs and because LAT is often done outside of a clinical trial. Clinical trial information: NCT01573702.

Epidermal growth factor receptor (EGFR): A rising star in the era of precision medicine of lung cancer.

Lung cancer is a leading cause of cancer mortality worldwide. In tumors, the important role of noncoding RNA regulatory networks has been more and more reveal. EGFR has been identified as an oncogenic driver of NSCLC, especially activating mutations EGFR and its inhibition with specific TKIs can generate dramatic tumor responses. Studies have shown that EGFR plays significant roles in the progression of NSCLC. Subset analysis of the small proportion of patients with EGFR-mutant lung cancer showed a disease-free survival benefit, but was underpowered to detect a survival advantage. Herein, we highlight the progression of EGFR, noncoding RNA, and their roles in carcinogenesis. We also focus on anti-lung cancer drug development and EGFR-related drug resistance.

96PD_PR - Evaluation of non-classical response by immune-modified RECIST and efficacy of atezolizumab beyond disease progression in advanced NSCLC: Results from the randomized Phase II study POPLAR

96PD_PR - Evaluation of non-classical response by immune-modified RECIST and efficacy of atezolizumab beyond disease progression in advanced NSCLC: Results from the randomized Phase II study POPLAR

Patterns of initial and intracranial failure in metastatic EGFR-mutant non-small cell lung cancer treated with erlotinib

ObjectivesPatients with metastatic EGFR-mutant (mEGFRmt) NSCLC have favorable survival when treated with erlotinib. We hypothesized that treatment failure in most patients is limited to initial sites of disease, in which case incorporating local therapy such as radiation might further delay progression. We therefore analyzed patterns and predictors of failure in a large cohort of such patients. Materials and methodsWe reviewed 189 patients treated with erlotinib for mEGFRmt NSCLC. We classified first pattern of failure as involving initial sites only (ISF), new sites only (NSF), or the combination (CSF), and used competing-risks regression to identify factors associated with ISF, progression and overall survival (OS). We also separately analyzed intracranial and intrathoracic failure. ResultsOf 171 patients who progressed, 103 (60.2%) had ISF, 30 (17.5%) had NSF, and 38 (22.2%) had CSF. Younger age and lack of initial CNS involvement independently correlated with ISF, with a trend for higher T and N stage. Higher T and N stage was also a significant predictor of progression. Factors predicting shorter OS were female gender, weight loss, initial intracranial involvement, and ≥4 extracranial metastases. Intrathoracic progression was a component of first failure in 61%, and three-year cumulative incidence of brain metastasis was 30%. ConclusionThe main pattern of progression in mEGFRmt NSCLC on erlotinib is in the initial sites of disease. Younger patients and those without brain involvement are particularly likely to develop ISF. This suggests a role for incorporating local therapy into treatment of selected patients with mEGFRmt NSCLC.

A phase Ib trial of continuous once-daily oral afatinib plus sirolimus in patients with epidermal growth factor receptor mutation-positive non-small cell lung cancer and/or disease progression following prior erlotinib or gefitinib

ObjectivesDysregulation of the downstream PI3K/AKT/mTOR signaling pathway is a proposed mechanism of resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). We investigated safety and antitumor activity of afatinib plus sirolimus as a potential combination to reverse acquired resistance to EGFR-TKIs in a phase IB trial in patients with EGFR mutation-positive non-small-cell lung cancer (EGFR mut NSCLC) and/or disease progression following prior erlotinib/gefitinib. Materials and methodsPatients with EGFR mut NSCLC and/or disease progression following at least prior erlotinib/gefitinib were included in the trial. The primary endpoint was incidence of dose-limiting toxicities (DLT) to determine the maximum tolerated dose (MTD). Four initial dose cohorts were proposed to evaluate DLTs. Other endpoints included tumor response, safety, progression-free survival (PFS) and pharmacokinetics. ResultsThirty-nine patients received afatinib and sirolimus. Additional dose cohorts were added since the second cohort (afatinib 40mg/day and sirolimus 5mg/day) was considered to have excessive toxicity. All patients experienced adverse events (AE) [grade 3: 66.7%; serious AE: 56.4%]. The most frequent AEs were diarrhea (94.9%), mucosal inflammation (64.1%), asthenia (53.8%) and rash (53.8%). Discontinuations and dose reduction due to AEs occurred in 23.1% and 25.6% of patients. MTD was determined as afatinib 30mg and sirolimus 1mg. Responses were observed in 5 patients (12.8%) [2 (5.1%) with confirmed partial response (PR); 3 (7.7%) with unconfirmed PR], and stable disease in 18 patients (46.2%). Four of the 5 responses were at doses above MTD. PFS at 6 months was estimated in 33.3% (median PFS 3.4 months). Pharmacokinetic parameters of afatinib and sirolimus were similar after single administration or in combination. ConclusionThe combination of afatinib and sirolimus showed lower responses than expected. Together with increased AEs and poor tolerability, this precludes clinical use and further clinical development of this combination. No pharmacokinetic interactions were observed. ClinicalTrials.gov IdentifierNCT00993499.

PIG3 promotes NSCLC cell mitotic progression and is associated with poor prognosis of NSCLC patients

BackgroundNon-small cell lung cancer (NSCLC) is the most commonly diagnosed type of lung cancer that is associated with poor prognosis. In this study we explored the potential role of p53-induced gene 3 (PIG3) in the progression of NSCLC.MethodsImmunohistochemistry was used to determine the expression levels of PIG3 in 201 NSCLC patients. We performed in vitro studies and silenced endogenous PIG3 by using specific siRNAs that specific target PIG3. Immunofluorescent staining was performed to determine the effect of PIG3 on mitotic progression in NSCLC cells. The growth rates of microtubules were determined by microtubule nucleation analysis. Cell proliferation and chemosensitivity were analyzed by CCK8 assays. Annexin V staining and β-galactosidase activity analysis were used to evaluate PIG3 deficiency-related apoptosis and senescence, respectively.ResultsPIG3 expression levels negatively correlated with overall survival and disease-free survival of NSCLC patients. Knock down of PIG3 resulted in repressed proliferation of NSCLC cells and increased aberrant mitosis, which included misaligning and lagging chromosomes, and bi- or multi-nucleated giant cells. In addition, PIG3 contributed to mitotic spindle assembly by promoting microtubule growth. Furthermore, loss of PIG3 sensitized NSCLC cells to docetaxel by enhancing docetaxel-induced apoptosis and senescence.ConclusionsOur results indicate that PIG3 promotes NSCLC progression and therefore suggest that PIG3 may be a potential prognostic biomarker and novel therapeutic target for the treatment of NSCLC.

Alectinib reduces CNS progression in NSCLC

Alectinib reduces CNS progression in NSCLC

Epigenetic activation of SIN1 promotes NSCLC cell proliferation and metastasis by affecting the epithelial–mesenchymal transition

Stress-activated protein kinase (SAPK) interacting protein 1 (SIN1) is an essential component of mTORC2. Previous studies have shown that SIN1 is a key regulator of Akt pathway which plays an important role in various pathological conditions including cancer. While its effects and mechanisms on the progression of NSCLC remain unknown. In this study, we report that SIN1 is able to promote the growth and migration of NSCLC cells both in vitro and in vivo. Overexpression of SIN1 promoted A549 and H1299 cells proliferation by both MTT and colony formation assays. Consistently, knockdown of SIN1 inhibited the proliferation of these cells. In transwell assay, overexpression of SIN1 increased the migration of A549 and H1299 cells, while SIN1 knockdown reduced their migration. In a tumor xenograft model, overexpression of SIN1 promoted tumor growth of A549 cells in vivo, while SIN1 knockdown suppresses the tumor growth. We also found a mechanistic link between SIN1 and H3K4me3, H3K4me3 is involved in SIN1 upregulation. Moreover, SIN1 can significantly promote the in vitro migration and invasion of NSCLC cells via induction epithelial mesenchymal transition (EMT) process, which subsequently leads to transcriptional downregulation of epithelial marker E-cadherin and upregulation of mesenchymal markers N-cadherin and Vimentin expression. Together, our results reveal that SIN1 plays an important role in NSCLC and SIN1 is a potential biomarker and a promising target in the treatment of NSCLC.

Pathway Targeted Immunotherapy: Rationale and Evidence of Durable Clinical Responses with a Novel, EGF-directed Agent for Advanced NSCLC

Abnormalities in the epidermal growth factor (EGF) and EGFR pathway promote progression of NSCLC. Immunization with EGF vaccine induces specific, neutralizing anti-EGF antibodies that prevent binding of the ligand to its receptor. This concept of pathway targeted immunotherapy (PTI) was validated in vitro by dose-related suppression of EGFR, Akt, and Erk1/2 phosphorylation in cell lines with different mutations. A randomized phase II trial showed improved overall survival (OS) in subgroups with advanced NSCLC showing a clear immunologic response. By per-protocol analysis of the ensuing phase IIb trial, patients receiving EGF PTI survived 3 months longer than controls (12.43 versus 9.43 months; hazard ratio = 0.77 [95% confidence interval, 0.61–0.98]). These data were confirmed in a larger trial showing an OS benefit over control of >3 months. The variable most strongly correlated with efficacy was circulating EGF at enrolment. Patients with serum EGF levels >250 pg/mL benefited most from treatment with EGF PTI. Of 188 patients tested, 94 were above this biomarker threshold. The OS benefit from active versus control treatment was 6.7 months. More than 15% of patients had responses for >5 years. Long-term survivors are seen in all EGF PTI trials. Treatment is well-tolerated, induces high anti-EGF antibody titers, reduces levels of circulating serum EGF, achieves durable responses, and significantly prolongs OS. A threshold of 250 pg/mL has been set to enrich the study population in the ongoing pivotal trial. This biomarker-guided study in an enriched population of patients with both squamous and nonsquamous stage IV NSCLC aims to replicate the favorable efficacy/tolerability balance of earlier studies.

MiR-541-3p reverses cancer progression by directly targeting TGIF2 in non-small cell lung cancer.

Lung cancer remains a leading cause of cancer-associated mortality worldwide, and non-small lung cancer (NSCLC) is responsible for over 80% of lung cancer-related deaths. Identifying novel molecular biomarker that can inhibit the progression of lung cancer will facilitate the development of new treatment strategies. Herein, we demonstrated that miR-541-3p is a tumor-suppressor microRNA (miRNA) in NSCLC progression. We found that expression of miR-541-3p was decreased obviously in NSCLC tissues and plasma. Down-regulation of miR-541-3p was associated with TNM stage and postoperative survival. Overexpression of miR-541-3p inhibited the growth and metastasis of NSCLC cells. The TGIF2 was a direct target of miR-541-3p and promoted the growth and metastasis of NSCLC cells. Further study showed that TGIF2 could reverse the inhibitory effect of miR-541-3p on growth and metastasis of NSCLC cells. Taken together, our data highlight the pivotal role of miR-541-3p in the progression of NSCLC. Thus, miR-541-3p may be a potential prognostic marker and of treatment relevance for NSCLC progression intervention.

Abstract 4235: Choline metabolism is an early predictor of EGFR-mediated survival in NSCLC

Abstract Oncogenic signalling and metabolic reprograming are hallmarks of tumour progression, yet little is known about the regulatory elements that coordinate their interface. Aberrant choline and phospholipid metabolism are strongly correlated to malignant progression in NSCLC and provide the essential components required by both hallmarks and yet mechanistic links to either remain scarce. Choline kinase alpha (ChoKα) regulates the conversion of choline to phosphocholine and although its regulatory cascade has not been described, it is thought to act in conjuction with EGFR. We used an integrated systems approach and queried whether pharmacoproteomic pathway mapping could identify regulators of the cholinic phenotype. Proteomic and phosphoproteomic Stable isotope labelling by amino acids in cell culture (SILAC) analysis was used to describe the interactome following ChoKα or EGFR inhibition. Bioinformatic analysis was used to identify the significant (Significance-B test) subset of targets for each condition. These subsets were clustered according to GeneOntology, Reactome and KEGG databases and the resulting maps identifed the potential regulators of choline metabolism. Choline uptake, phosphorylation and efflux were further evaluated in vitro in response to erlotinib, cisplatin, pemetrexed and paclitaxel using radio-labelled Choline analogues. Derived metabolites were characterised using radio-HPLC. Uptake was further characterised under hypoxic and nutrient deficient conditions. In vivo, [18F]-D4-Choline PET dynamic imaging was performed following treatment. Pharmacoproteomic analysis revealed a 40% overlap between ChoKα and EGFR inhibition providing direct evidence of the pathways and targets involved in, mostly, biosynthesis. Rapid modulation of the cholinic phenotype was directly dependent on ChoKα activity. Intracellular uptake was induced by nutrient deprivation, hypoxia and reversed through second messenger signalling or growth factor stimulation. Choline uptake within 3 hours of treatment correlated to survival at 72 hours. In vivo, [18F]-D4-Choline tracer kinetics were diagnostic of choline kinase expression and sensitive to treatment. Significant correspondence between ChoKα and EGFR inhibition provided mechanistic evidence that ChoKα and lipid metabolism are effectors of the EGFR signalling cascade in NSCLC. Choline can act as a sensor by synchronizing the survival response via metabolic and signalling reprograming and is thus an early predictor of therapeutic efficiency in vitro and in vivo. Citation Format: Rosy Favicchio, Nicos Angelopoulos, Diana Brickute, Robin Fortt, Frazer Twyman, Georgios Giamas, Juan Carlos Lacal, Eric O. Aboagye. Choline metabolism is an early predictor of EGFR-mediated survival in NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4235.

Abstract 2496: Novel role of YAP1 in tumor angiogenesis and vascular mimicry

Abstract Non-small cell lung cancer (NSCLC) is highly correlated with smoking and has very low survival rates. Multiple studies have shown that cancer stem-like cells (CSCs) contribute to the genesis and progression of NSCLC. These cells have been shown to form differentiated tumor cells upon receiving appropriate micro-environmental cues and can give rise to multiple components of the tumor, including the vasculature. We isolated the CSCs from NSCLC cell lines and PDX tumors based on the side population (SP) phenotype. SP cells displayed high self-renewal capacity, were highly drug resistant, and could form metastatic tumors in immunocompromised mice. The present study shows that the transcriptional co-activator YAP1, which is the oncogenic component of the Hippo signaling pathway, is elevated in the stem-like cells from NSCLC and contributes to their self-renewal and the ability to form angiogenic tubule-like structures in matrigel, a unique feature of cancer stem-like cells that is also called as vascular mimicry. Inhibition of YAP1 by a small molecule inhibitor Visudyne or depletion of YAP1 by siRNAs suppressed self-renewal and vascular mimicry of SP cells. The stem -like SP cells from NSCLC were found to have higher mRNA expression of VEGF receptor II (KDR) and Angiopoietin-2 (AngPT-2) which are crucial genes during angiogenic tubule regression and growth in addition to YAP1. Further, depletion of YAP1 with siRNAs reduced the expression of VEGF, KDR and AngPT-2 mRNA levels. Overexpression of YAP1 resulted in increase in the promoter activity of both KDR and AngPT-2 in transient transfection experiments. This suggests that YAP1 might play a role in regulating VEGF, KDR and AngPT-2 mediated angiogenic functions in cancer cells. In addition to vascular mimicry, YAP1 appears to play a unique role in regulating the expression of genes involved in angiogenesis. Hypoxia is known to contribute towards angiogenesis as well as cancer progression and metastasis. Preliminary experiments showed that cells grown in hypoxic conditions or treated with hypoxia mimetic compounds like DMOG resulted in increase in YAP1 mRNA and protein expression. However, such an increase was not observed in YAP1 orthologue, TAZ or other canonical Hippo pathway proteins. Hypoxia-inducible factor-1alpha (HIF-1α) is the key transcription factor that regulates the expression of various hypoxia response genes like VEGF. We find that YAP1 directly interacts with HIF-1α as detected by co-immunoprecipitation experiments and proximity ligation assays (PLA). YAP1 also associated with VEGF promoter as seen in ChIP RT-PCRs, and this interaction was elevated under hypoxic environment. Our data suggest a distinct role for YAP1 in regulating hypoxia response, promoting tumor angiogenesis and vascular mimicry. Citation Format: Namrata Bora Singhal, Srikumar Chellappan. Novel role of YAP1 in tumor angiogenesis and vascular mimicry. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2496.

MicroRNA-663a is downregulated in non-small cell lung cancer and inhibits proliferation and invasion by targeting JunD.

BackgroundMicroRNA-663a expression is downregulated in several tumors. However, its functions and mechanisms in human non-small cell lung (NSCLC) cancer remain obscure. The present study aimed to identify the expression pattern, biological roles and potential mechanisms by which miR-663a dysregulation is associated with NSCLC.MethodsWe examined expression level of miR-663a in 62 cases of NSCLC tissues and 5 NSCLC cell lines by reverse transcription PCR. In vitro, gain-of-function and loss-of-function experiments were performed to examine the impact of miR-663a on proliferation, cell cycle progression and invasion of NSCLC cells. Using fluorescence reporter assays, we also explored the potential targets and possible mechanisms of miR-663a in NSCLC cells.ResultsDownregulation of miR-663a was observed in 42 of 62 of lung cancer tissues compared with paired normal tissues (mean cancer/normal value = 0.745) and its downregulation correlated with nodal metastasis. Transfection of miR-663a mimic suppressed cell proliferation, cell cycle progression and invasion, with downregulation of cyclin D1, cyclin E and MMP9 in both H460 and H1299 cell lines. Transfection of miR-663a inhibitor in both H460 and H1299 cell lines exhibited the opposite effects. In addition, we confirmed that miR-663a could inhibit AP-1 activity and AP-1 component JunD was a direct target of miR-663a in lung cancer cells. Transfection of miR-663a mimic downregulated JunD expression. In addition, JunD siRNA treatment abrogated miR-663a inhibitor-induced expression of cyclin D1, cyclin E and MMP9. Above all, both miRNA mimic and inhibitor in two different NSCLC cell lines demonstrated that miR-663a inhibits proliferation and invasion by targeting AP-1 transcription factor JunD.ConclusionsThis study indicates that miR-663a downregulation might be associated with NSCLC progression. MiR-663a suppresses proliferation and invasion by targeting AP-1 component JunD in NSCLC cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2350-x) contains supplementary material, which is available to authorized users.

MicroRNA-1976 functions as a tumor suppressor and serves as a prognostic indicator in non-small cell lung cancer by directly targeting PLCE1

Lung cancer remains a leading cause of cancer-associated mortality worldwide and non-small lung cancer (NSCLC) is responsible for over 80% of lung cancer-related deaths. Identifying novel molecular biomarker that can inhibit the progression of lung cancer will facilitate the development of new treatment strategies. Phospholipase C epsilon 1 (PLCE1) is a susceptibility gene in NSCLC. Nevertheless, the role of PLCE1 in NSCLC tumorigenesis has not been elucidated. Herein, we demonstrated that miR-1976 is a tumor-suppressor miRNA in NSCLC progression. We found that expression of miR-1976 was decreased obviously in NSCLC tissues. Down-regulation of miR-1976 was associated with TNM stage and postoperative survival. Overexpression of miR-1976 inhibited the growth and metastasis of NSCLC cells. The PLCE1 was a direct target of miR-1976 and promoted the growth and metastasis of NSCLC cells. Further study showed that PLCE1 could reverse the inhibitory effect of miR-1976 on growth and metastasis of NSCLC cells. Taken together, our data highlight the pivotal role of miR-1976 in the progression of NSCLC. Thus, miR-1976 may be a potential prognostic marker and of treatment relevance for NSCLC progression intervention.

Long noncoding RNAs: new insights into non-small cell lung cancer biology, diagnosis and therapy.

Recent advances in tiling array and high throughput analyses revealed that at least 87.3% of the human genome is actively transcribed, though <3% of the human genome encodes proteins. This unexpected truth suggests that most of the transcriptome is constituted by noncoding RNA. Among them, high-resolution microarray and massively parallel sequencing analyses identified long noncoding RNAs (lncRNAs) as nonprotein-coding transcripts. lncRNAs are largely polyadenylated and >200 nucleotides in length transcripts, involved in gene expression through epigenetic and transcriptional regulation, splicing, imprinting and subcellular transport. Although lncRNAs functions are largely uncharacterized, accumulating data indicate that they are involved in fundamental biological functions. Conversely, their dysregulation has increasingly been recognized to contribute to the development and progression of several human malignancies, especially lung cancer, which represents the leading cause of cancer-related deaths worldwide. We conducted a comprehensive review of the published literature focusing on lncRNAs function and disruption in nonsmall cell lung cancer biology, also highlighting their value as biomarkers and potential therapeutic targets. lncRNAs are involved in NSCLC pathogenesis, modulating fundamental cellular processes such as proliferation, cell growth, apoptosis, migration, stem cell maintenance and epithelial to mesenchymal transition, also serving as signaling transducers, molecular decoys and scaffolds. Also, lncRNAs represent very promising biomarkers in early-stage NSCLC patients and may become particularly useful in noninvasive screening protocols. lncRNAs may be used as predictive biomarkers for chemotherapy and targeted therapies sensitivity. Furthermore, selectively targeting oncogenic lncRNAs could provide a new therapeutic tool in treating NSCLC patients. lncRNAs disruption plays a pivotal role in NSCLC development and progression. These molecules also serve as diagnostic, prognostic and predictive biomarkers. Characterization of lncRNA genes and their mechanisms of action will enable us to develop a more comprehensive clinical approach, with the final goal to benefit our patients.

Abstract C93: RYBP predicts survival of patients with non-small cell lung cancer and sensitizes chemotherapy

Abstract Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers and associates with a poor prognosis. Despite several targeted therapies have been developed with promising results, the therapeutic options for NSCLC are limited and the 5-year survival rate remains poor (∼15%). Therefore, there is an urgent need for a better understanding of its pathogenesis and exploring novel therapeutic targets. Ring1 and YY1 binding protein (RYBP) is a member of polycomb group (PcG) proteins that typically act as transcriptional repressors via epigenetic modification of chromatin and participate in the establishment and maintenance of cell fates. Recent studies have shown that RYBP also possesses PcG-independent functions that promote apoptosis and cell cycle arrest in cancer cells. Our previous study has shown that RYBP stabilizes p53 through inhibiting MDM2 activity. Recent study indicates that RYBP is downregulated in human cancers, including NSCLC, but the underlying mechanisms are largely unknown. The present study was designed to demonstrate the molecular role of RYBP in NSCLC development and progression, chemotherapy, and patient survival. We systemically investigated the expression of RYBP in human NSCLC tissues and matched non-cancerous samples and evaluated the associations between the RYBP expression in NSCLCs and patient survial. We also generated a replication-deficient recombinant adenovirus driving the expression of RYBP (Adv-RYBP) and carried out in vitro and in vivo studies to explore the molecular basis for the tumor suppressing role of RYBP in NSCLC. We first demonstrated that the RYBP mRNA and protein expressions levels were significantly down-regulated in NSCLC tissues compared with matched noncancerous lung tissues. The low expression of RYBP was significantly associated with the poor prognosis in NSCLC patients. We then identified that enforced RYBP expression decreased cell viability, inhibited colony formation and induced apoptosis in NSCLC cells, while RYBP knockdown led to the opposite effects. RYBP affected the expression of several apoptosis-related proteins; and promoting apoptosis was one of the main mechanisms of RYBP-mediated cell growth inhibition in NSCLC. Moreover, AdRYBP led to a decreased NSCLC tumor xenograft growth. Additionally, clinically used chemotherapeutic agents induced the expression of RYBP, and RYBP sensitized NSCLC cells to chemotherapy in vitro and in vivo. In conclusion, our results reveal that RYBP is a potential biomarker for the diagnosis and prognosis of NSCLC and that reactivating RYBP in cancer cells may provide an effective and safe therapeutic approach to NSCLC therapy. (Supported by NIH/NCI grants R01 CA186662). Citation Format: wei wang, Sukesh Voruganti, Jiang-Jiang Qin, Biyun Qian, Ruiwen Zhang. RYBP predicts survival of patients with non-small cell lung cancer and sensitizes chemotherapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C93.