Non-small Cell Lung Cancer Tumor Growth Research Articles

MicroRNA-34a/EGFR axis plays pivotal roles in lung tumorigenesis

MicroRNAs (miRNAs) are vital in the regulation of tumor progression and invasion. Dysregulation of miRNAs has been linked to the development of various types of human cancers, including non-small-cell lung cancer (NSCLC). However, the effect of miRNA-34a (miR-34a), a key regulator of tumor suppression, on the tumorigenesis of NSCLC has not been fully elaborated. Herein, we reveal that miR-34a is significantly downregulated in NSCLC tissues and cell lines, suggesting that miR-34a might function as a tumor suppressor in lung cancer. We also confirmed that epidermal growth factor receptor (EGFR) is a direct target of miR-34a, and our data reveal that siRNA knockdown of EGFR can inhibit cell proliferation, promote apoptosis and arrest cell-cycle progression. In addition, EGFR can reverse the suppressive function of miR-34a overexpression on proliferation and cell apoptosis. Furthermore, in vivo experiments demonstrated that miR-34a suppress tumor growth, both in the A549 xenograft model, as well as in the metastatic tumors in nude mice. Taken together, our findings suggest that miR-34a inhibits NSCLC tumor growth and metastasis through targeting EGFR.

FOXP3 promotes tumor growth and metastasis by activating Wnt/\u03b2-catenin signaling pathway and EMT in non-small cell lung cancer

BackgroundThe role of cancer cell FOXP3 in tumorigenesis is conflicting. We aimed to study FOXP3 expression and regulation, function and clinical implication in human non-small cell lung cancer (NSCLC).MethodsOne hundred and six patients with histologically-confirmed NSCLC who underwent surgery were recruited for the study. Tumor samples and NSCLC cell lines were used to examine FOXP3 and its related molecules. Various cell functions related to tumorigenesis were performed. In vivo mouse tumor xenograft was used to confirm the in vitro results.ResultsNSCLC patients with the high level of FOXP3 had a significant decrease in overall survival and recurrence-free survival. FOXP3 overexpression significantly induced cell proliferation, migration, and invasion, whereas its inhibition impaired its oncogenic function. In vivo studies confirmed that FOXP3 promoted tumor growth and metastasis. The ectopic expression of FOXP3 induced epithelial–mesenchymal transition (EMT) with downregulation of E-cadherin and upregulation of N-cadherin, vimentin, snail, slug, and MMP9. The oncogenic effects by FOXP3 could be attributed to FOX3-mediated activation of Wnt/β-catenin signaling, as FOXP3 increased luciferase activity of Topflash reporter and upregulated Wnt signaling target genes including c-Myc and Cyclin D1 in NSCLC cells. Co-immunoprecipitation results further indicated that FOXP3 could physically interacted with β-catenin and TCF4 to enhance the functions of β-catenin and TCF4, inducing transcription of Wnt target genes to promote cell proliferation, invasion and EMT induction.ConclusionsFOXP3 can act as a co-activator to facilitate the Wnt-b-catenin signaling pathway, inducing EMT and tumor growth and metastasis in NSCLC.

Abstract 1500: Evaluating the efficacy of limiting heme availability on growth and progression of lung tumor

Abstract Numerous studies support the importance of mitochondrial respiration in tumorigenesis. Studies from our lab have demonstrated that mitochondrial respiration is intensified, and the levels of intracellular heme and hemoproteins are elevated in Non-small cell lung cancer (NSCLC) cells. We also demonstrated that lowering intracellular heme levels selectively decreases oxygen consumption in NSCLC cells and inhibits cell migration and colony formation. Heme is a central molecule for mitochondrial function and for all processes involved in oxygen utilization. It serves as a prosthetic group in several oxidative phosphorylation enzymes and other oxygen-utilizing hemoproteins. Most human cells can synthesize heme de novo and uptake heme from the circulation. Further, a number of epidemiological studies have shown that high heme intake is associated with increased risk of several cancers, including lung cancer. The purpose of the study is to determine the extent to which altering heme availability impacts NSCLC tumor growth and development. To this end, we devised a two-pronged approach to target both heme intake and heme synthesis to limit the heme available to cells. Our lab synthesized a peptide to sequester heme from circulation and thereby limit heme available for uptake. A well-known inhibitor of heme synthesis is used to limit heme synthesis. NSCLC cells that express luciferase were implanted in NOD/SCID mice to generate subcutaneous and lung orthotopic xenografts. The mice were treated with the peptide alone and in combination with an inhibitor. The tumor growth was monitored by non-invasive bioluminescence imaging (BLI) using Perkin Elmer’s IVIS Lumina III Imager. In subcutaneously implanted xenografts, our BLI data show that there is a considerable reduction in radiance (total flux in photons per second) in the treatment groups that received the peptide and a combination of peptide and inhibitor. This trend was also observed in tumor volumes measured by calipers. This observation is further corroborated in lung orthotopic xenografts, where there is a significant reduction in radiance in the treatment groups that received the peptide and peptide-inhibitor combination. In all the cases the combination of the peptide and the inhibitor had a greater effect than the peptide alone. Our results indicate that combination of heme lowering agents works effectively in limiting lung tumor growth and progression by limiting both heme uptake and synthesis. Further studies are underway to discern the role of heme in lung cancer growth and progression. Citation Format: Poorva Ghosh, Sarada Preeta Kalainayakan, Sanchareeka Dey, Sagar Sohoni, Li Zhang. Evaluating the efficacy of limiting heme availability on growth and progression of lung tumor [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 1500. doi:10.1158/1538-7445.AM2017-1500

Selective targeting p53WT lung cancer cells harboring homozygous p53 Arg72 by an inhibitor of CypA

TP53 plays essential roles in tumor initiation and progression, and is frequently mutated in cancer. However, pharmacological stabilization and reactivation of p53 have not been actively explored for targeted cancer therapies. Herein, we identify a novel Cyclophilin A (CypA) small molecule inhibitor (HL001) that induces non-small cell lung cancer (NSCLC) cell cycle arrest and apoptosis via restoring p53 expression. We find that HL001 stabilizes p53 through inhibiting the MDM2-mediated p53 ubiquitination. Further mechanistic studies reveal that the downregulation of G3BP1 and the induction of reactive oxygen species and DNA damage by HL001 contribute to p53 stabilization. Surprisingly, HL001 selectively suppresses tumor growth in p53 wild-type NSCLC harboring Arg72 homozygous alleles (p53-72R) through disrupting interaction between MDM2 and p53-72R in a CypA-dependent manner. Moreover, combining HL001 with cisplatin synergistically enhance tumor regression in orthotopic NSCLC mouse model. Collectively, this study demonstrates that pharmacologic inhibition of CypA offers a potential therapeutic strategy via specific activation of p53-72R in NSCLC.

PSCA and MUC1 in non-small-cell lung cancer as targets of chimeric antigen receptor T cells

ABSTRACTIn recent years, immunotherapies, such as those involving chimeric antigen receptor (CAR) T cells, have become increasingly promising approaches to non-small-cell lung cancer (NSCLC) treatment. In this study, we explored the antitumor potential of prostate stem cell antigen (PSCA)-redirected CAR T and mucin 1 (MUC1)-redirected CAR T cells in tumor models of NSCLC. First, we generated patient-derived xenograft (PDX) mouse models of human NSCLC that maintained the antigenic profiles of primary tumors. Next, we demonstrated the expression of PSCA and MUC1 in NSCLC, followed by the generation and confirmation of the specificity and efficacy of PSCA- and MUC1-targeting CAR T cells against NSCLC cell lines in vitro. Finally, we demonstrated that PSCA-targeting CAR T cells could efficiently suppress NSCLC tumor growth in PDX mice and synergistically eliminate PSCA+MUC1+ tumors when combined with MUC1-targeting CAR T cells. Taken together, our studies demonstrate that PSCA and MUC1 are both promising CAR T cell targets in NSCLC and that the combinatorial targeting of these antigens could further enhance the antitumor efficacy of CAR T cells.

Magnolol Inhibits the Growth of Non-Small Cell Lung Cancer via Inhibiting Microtubule Polymerization

Background: The tubulin/microtubule system, which is an integral component of the cytoskeleton, plays an essential role in mitosis. Targeting mitotic progression by disturbing microtubule dynamics is a rational strategy for cancer treatment. Methods: Microtubule polymerization assay was performed to examine the effect of Magnolol (a novel natural phenolic compound isolated from Magnolia obovata) on cellular microtubule polymerization in human non-small cell lung cancer (NSCLC) cells. Cell cycle analysis, mitotic index assay, cell proliferation assay, colony formation assay, western blotting analysis of cell cycle regulators, Annexin V-FITC/PI staining, and live/dead viability staining were carried out to investigate the Magnolol’s inhibitory effect on proliferation and viability of NSCLS cells in vitro. Xenograft model of human A549 NSCLC tumor was used to determine the Magnolol’s efficacy in vivo. Results: Magnolol treatment effectively inhibited cell proliferation and colony formation of NSCLC cells. Further study proved that Magnolol induced the mitotic phase arrest and inhibited G2/M progression in a dose-dependent manner, which were mechanistically associated with expression alteration of a series of cell cycle regulators. Furthermore, Magnolol treatment disrupted the cellular microtubule organization via inhibiting the polymerization of microtubule. We also found treatment with NSCLC cells with Magnolol resulted in apoptosis activation through a p53-independent pathway, and autophgy induction via down-regulation of the Akt/mTOR pathway. Finally, Magnolol treatment significantly suppressed the NSCLC tumor growth in mouse xenograft model in vivo. Conclusion: These findings identify Magnolol as a promising candidate with anti-microtubule polymerization activity for NSCLC treatment.

P2.01-068 Analysis of Epithelial-Stromal Interactions and their Relevance to Lung Cancer

The communication between epithelial cells and their underlying stroma is an important but poorly understood aspect of organismal biology. If aberrantly regulated, these interactions can prove to be tumorigenic. Although it has been known for years that cancer-associated fibroblasts (CAFs) promote and sustain the growth of tumors, the underlying mechanisms remain incompletely understood. Previous work in our lab has identified a novel mechanism of communication in which CAFs secrete cardiotrophin-like cytokine factor 1 (CLCF1), a cytokine that binds ciliary neurotrophic factor receptor (CNTFR) on tumor cells and promotes neoplastic growth. CNTFR is a component of the tripartite receptor complex formed by CNTFR-gp130-LIFR and is capable of activating several oncogenic signaling cascades, including JAK-STAT. Patient tumor and normal samples were collected and plated as CAFs and normal lung fibroblasts (NLFs), respectively. The effect of CNTFR knockdown was assessed by shRNA in A549 xenografts, and the CNTFR decoy receptor was evaluated using patient-derived xenograft (PDX) models. Independent TCGA analyses of CNTFR and CLCF1 expression levels in non-small cell lung cancer (NSCLC) patients revealed that increased levels of both genes correlate with poor patient outcomes. In isolated pairs of human CAFs and NLFs from the same patient, gene expression analyses consistently demonstrated a higher level of CLCF1 in CAFs. In vitro studies using three NSCLC cell lines–A549, H23, and H358–showed that CNTFR knockdown decreases proliferation whereas exogenous recombinant CLCF1 increases growth. We repeatedly observed decreased protein levels of phosphorylated STAT3 and phosphorylated ERK upon CNTFR knockdown, thus implicating two canonical oncogenic pathways: Jak-STAT and Ras-Raf-MEK-ERK. Using xenograft models, we found that CLCF1 overexpression by CAFs increases tumor growth while knockdown of CNTFR in lung tumor cells decreases overall growth. With the use of advanced protein engineering technology, we generated a high-affinity CNTFR decoy that inhibits CLCF1-CNTFR signaling and are currently testing this novel reagent to elucidate the mechanism by which CNTFR activation alters intercellular signaling to increase tumor cell growth. Through in vivo studies with cell lines and PDXs, we are also exploring the efficacy of this CNTFR decoy as a form of lung cancer therapy. In sum, these data indicate that CLCF1-CNTFR signaling is important for NSCLC tumor growth and is a viable therapeutic target.

LKB1 promotes metabolic flexibility in response to energy stress

The Liver Kinase B1 (LKB1) tumor suppressor acts as a metabolic energy sensor to regulate AMP-activated protein kinase (AMPK) signaling and is commonly mutated in various cancers, including non-small cell lung cancer (NSCLC). Tumor cells deficient in LKB1 may be uniquely sensitized to metabolic stresses, which may offer a therapeutic window in oncology. To address this question we have explored how functional LKB1 impacts the metabolism of NSCLC cells using 13C metabolic flux analysis. Isogenic NSCLC cells expressing functional LKB1 exhibited higher flux through oxidative mitochondrial pathways compared to those deficient in LKB1. Re-expression of LKB1 also increased the capacity of cells to oxidize major mitochondrial substrates, including pyruvate, fatty acids, and glutamine. Furthermore, LKB1 expression promoted an adaptive response to energy stress induced by anchorage-independent growth. Finally, this diminished adaptability sensitized LKB1-deficient cells to combinatorial inhibition of mitochondrial complex I and glutaminase. Together, our data implicate LKB1 as a major regulator of adaptive metabolic reprogramming and suggest synergistic pharmacological strategies for mitigating LKB1-deficient NSCLC tumor growth.

MiR-3940-5p Functions as a Tumor Suppressor in Non–Small Cell Lung Cancer Cells by Targeting Cyclin D1 and Ubiquitin Specific Peptidase-28

miR-3940-5p level was lower in non–small cell lung cancer (NSCLC) tumor tissues than that in the matched tumor-adjacent tissues and correlated with clinicopathological features. Cyclin D1 (CCND1), a key driver of malignant transformation in NSCLC, was overexpressed in many cancers, including NSCLC. The ubiquitin specific peptidase-28 (USP28) was also overexpressed in NSCLC and associated with poor prognosis of NSCLC patients. We searched for miR-3940-5p targets by using TargetScan and miRanda online tools and found that CCND1 and USP28 were potential targets of miR-3940-5p. Based on these findings, we speculated that miR-3940-5p might target CCND1 and USP28 to inhibit NSCLC growth. We determined the expression of miR-3940-5p, CCND1, and USP28 by quantitative real-time polymerase chain reaction and Western blot assays, respectively, and found downregulation of miR-3940-5p and upregulation of CCND1 and USP28 in NSCLC tissues and cell lines. Cell proliferation and apoptosis assays showed that miR-3940-5p suppressed proliferation and promoted apoptosis in NSCLC cells, and silencing CCND1 and USP28 both recapitulated the effects of miR-3940-5p on NSCLC cells. Furthermore, we verified that CCND1 and USP28 were direct targets of miR-3940-5p and also found that the effects of NSCLC cell proliferation and apoptosis by miR-3940-5p were attenuated by overexpression of CCND1 or USP28. The animal experiments also showed that overexpression of miR-3940-5p inhibited the growth of NSCLC tumors in vivo. These results confirmed our speculation that miR-3940-5p inhibits proliferation and induces apoptosis in NSCLC cells by targeting CCND1 and USP28. These findings facilitate a better understanding of the molecular mechanisms underlying NSCLC initiation and progression and provide promising diagnostic markers and therapeutic targets for NSCLC.

MicroRNA-107 inhibits tumor growth and metastasis by targeting the BDNF-mediated PI3K/AKT pathway in human non-small lung cancer.

Abnormal expression of microRNA-107 (miR-107) was found in non-small cell lung cancer (NSCLC). However, little is known about its role and molecular mechanism in NSCLC progression and metastasis. Therefore, the aims of this study were to clarify the potential role of miR-107 and molecular mechanism in NSCLC progression and metastasis. Quantitative real-time polymerase chain reaction assay showed that miR-107 expression levels were significantly decreased in NSCLC tissue and cell lines. Low miR-107 levels in tumor tissue correlated with advanced TNM stage and lymph node metastasis. Function assays showed that overexpression of miR-107 suppressed cell proliferation, migration and invasion in A549 cells in vitro, and inhibited NSCLC tumor growth in vivo. Further mechanism assays suggested the brain-derived neurotrophic factor (BDNF) was identified as a target gene of miR-107 in NSCLC cells. In addition, BDNF expression was upregulated, and inversely correlated with miR-107 in NSCLC tissues. Enforced overexpression of BDNF effectively reversed the tumor suppressive functions of miR-107 on NSCLC proliferation, migration and invasion. miR-107 overexpression or downregulation of BDNF was able to inhibit activation of PI3K/AKT signaling pathway. Taken together, our findings present the first evidence that miR-107 could suppress NSCLC metastasis by targeting BDNF and indirectly regulating PI3K/AKT signaling pathway, which might lead to a potential therapeutic strategy focusing on miR-107 and BDNF for human NSCLC.

Abstract 209: The effect of heme influx on initiation and tumorigenesis of NSCLC

Abstract Heme is a central molecule for mitochondrial function and for all processes involved in oxygen utilization. Heme serves as a prosthetic group or as a cofactor for a number of oxidative phosphorylation enzymes and other oxygen-utilizing hemoproteins. Heme also directly regulates the synthesis, translocation and assembly of these enzyme complexes. Most, if not all, human cells can synthesize and uptake heme from the circulation. A number of epidemiological studies have shown that high heme intake is associated with increased risk of cancer, including lung cancer. Recent studies carried out in our lab showed intensified mitochondrial respiration and increased levels of heme and hemoproteins in non-small-cell lung cancer (NSCLC) cells. Together, these experimental and epidemiological studies strongly suggest that heme is an “oncometabolite.” To assess the status of heme metabolism in cancer cells, we performed a series of experiments in NSCLC cells and compared the results with an immortalized normal lung cell line, HBEC30KT. We used Zinc protoporphyrin, an analogue of heme to measure the level of heme uptake. We found that heme uptake as well as heme synthesis are significantly elevated in all NSCLC cells compared to HBEC. The rate of heme degradation was also measured in these cell lines. Previously, our lab demonstrated that lowering intracellular heme levels selectively decreases oxygen consumption in NSCLC cells and inhibits cell migration and colony formation. Experiments are underway to test ways to alter intracellular heme availability and characterize their effects on NSCLC tumor growth and metastasis. Citation Format: Sagar Sohoni, Md Maksudul Alam, Chantal Vidal, Jagmohan Hooda, Li Zhang. The effect of heme influx on initiation and tumorigenesis of 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 209.

Piperlongumine inhibits lung tumor growth via inhibition of nuclear factor kappa B signaling pathway.

Piperlongumine has anti-cancer activity in numerous cancer cell lines via various signaling pathways. But there has been no study regarding the mechanisms of PL on the lung cancer yet. Thus, we evaluated the anti-cancer effects and possible mechanisms of PL on non-small cell lung cancer (NSCLC) cells in vivo and in vitro. Our findings showed that PL induced apoptotic cell death and suppressed the DNA binding activity of NF-κB in a concentration dependent manner (0–15 μM) in NSCLC cells. Docking model and pull down assay showed that PL directly binds to the DNA binding site of nuclear factor-κB (NF-κB) p50 subunit, and surface plasmon resonance (SPR) analysis showed that PL binds to p50 concentration-dependently. Moreover, co-treatment of PL with NF-κB inhibitor phenylarsine oxide (0.1 μM) or p50 siRNA (100 nM) augmented PL-induced inhibitory effect on cell growth and activation of Fas and DR4. Notably, co-treatment of PL with p50 mutant plasmid (C62S) partially abolished PL-induced cell growth inhibition and decreased the enhanced expression of Fas and DR4. In xenograft mice model, PL (2.5–5 mg/kg) suppressed tumor growth of NSCLC dose-dependently. Therefore, these results indicated that PL could inhibit lung cancer cell growth via inhibition of NF-κB signaling pathway in vitro and in vivo.

Inhibition of metabotropic glutamate receptor 1 suppresses tumor growth and angiogenesis in experimental non-small cell lung cancer

Metabotropic glutamate receptor 1 (mGlu1 receptor) is expressed in many cancer cell types as compared to normal counterparts underscoring its potential role in tumor behavior. The aim of present study was to test the role of mGlu1 receptor in experimental non-small cell lung cancer (NSCLC). First, protein expression of mGlu1 receptor was higher in human NSCLC cell lines, including both adenocarcinoma and squamous carcinoma subtypes, when compared to normal bronchial epithelial cells. Inhibition of mGlu1 receptor by BAY36-7620 (an mGlu1 receptor-specific inhibitor) inhibited tumor growth and prolonged survival of mice with tumors of A549 or H1299. Treatment with BAY36-7620 suppressed AKT phosphorylation in A549 tumors and pre-treatment with BAY36-7620 blocked the L-quisqualate (a potent mGlu1 receptor agonist)-induced AKT phosphorylation in A549 cells. Treatment with BAY36-7620 reduced cellular proliferation of A549 cells. Treatment with BAY36-7620 enhanced cleaved PARP levels and reduced protein expression of bcl-2, HIF-1α, and VEGF. In contrast, treatment with L-quisqualate reduced cleaved PARP levels and enhanced protein expression of bcl-2, HIF-1α, VEGF, and IL-8, which was reversed by co-incubation with MK2206 (an AKT inhibitor). Pre-treatment with BAY36-7620 blocked the VEGF-induced AKT phosphorylation in HUVECs. Treatment of HUVECs with L-quisqualate resulted in enhancement of capillary tube formation, which was reversed by co-incubation with MK2206. Furthermore, mGlu1 receptor knockdown suppressed tumor growth and prolonged survival of mice with tumors of A549 or H1299. Collectively, inhibition of mGlu1 receptor suppressed tumor growth and angiogenesis in experimental NSCLC.

Tigecycline targets nonsmall cell lung cancer through inhibition of mitochondrial function.

Nonsmall cell lung cancer (NSCLC) is the most common type of lung cancer with a high mortality rate and still remains therapeutically a challenge. A strategy to target NSCLC is to identify agents that are effective against NSCLC cells while sparing normal cells. We show that tigecycline, an FDA-approved antibiotic drug, preferentially targets NSCLC cells. Tigecycline is effective in inhibiting proliferation and inducing apoptosis of multiple cell lines derived from two common NSCLC subtypes: adenocarcinoma and squamous cell carcinoma. Tigecycline also dose-dependently inhibits colony formation of NSCLC subpopulation of cells with highly proliferative and invasive properties. Compared to NSCLC cells, tigecycline affects proliferation and survival of normal fibroblast cells significantly to a less extent. More importantly, tigecycline significantly inhibits NSCLC tumor growth through decreasing proliferation and increasing apoptosis of tumor cells in vivo. Tigecycline significantly inhibits mitochondrial respiration, mitochondrial membrane potential, and ATP levels and increases reactive oxygen species (ROS), suggesting that tigecycline impairs mitochondrial functions. Our study suggests that tigecycline may be a useful therapeutic agent, and inhibiting mitochondrial functions may represent a new targeted therapy for NSCLC.

Zoledronic acid increases the antitumor effect of gefitinib treatment for non-small cell lung cancer with EGFR mutations.

Non-small cell lung cancer (NSCLC) patients with epithelial growth factor receptor (EGFR) mutations and bone metastases are often concurrently administered tyrosine kinase inhibitors (TKIs) and bisphosphonates. Yet, the effects and mechanisms of these agents are unclear. In the present study, we aimed to ascertain whether zoledronic acid (ZA) increases the antitumor effects of gefitinib treatment on NSCLC with EGFR mutations and the related mechanisms of action. The effects of ZA and gefitinib on NSCLC tumor cells with EGFR mutations (HCC827, HCC827 GR and H1975) in regards to proliferation, apoptosis, cell cycle and signaling pathways were detected. ZA increased the antitumor effects of gefitinib on NSCLC with EGFR activating mutations and TKI resistance invitro. Gefitinib caused cell cycle arrest in the G0/G1phase, ZA induced Sphase accumulation and the effect of the combined treatment was neutralization. Combined treatment obviously inhibited STAT3 and/or p‑STAT3 protein expression compared with treatment with each single drug invitroandinvivo, and it also significantly inhibited TKI resistance NSCLC tumor growth invivo. In conclusion, ZA increased the antitumor effects of gefitinib on NSCLC with EGFR activating mutations and TKI resistance by regulating the cell cycle, inducing caspase-3expression and inhibiting STAT3expression.

A Potent Derivative of Indolizino[6,7-b]Indole for Treatment of Human Non–Small Cell Lung Cancer Cells

The therapeutic effect in non–small cell lung cancer (NSCLC) patients is limited because of intrinsic and acquired resistance. Thus, an unmet need exists for the development of new drugs to improve the therapeutic efficacy in NSCLC patients. In this study, the novel small molecule indolizino[6,7-b]indole derivative BO-1978 was selected to evaluate its therapeutic effects on NSCLC and its preclinical toxicity in animal models. An in vitro cytotoxicity assay revealed that BO-1978 significantly suppressed the growth of various NSCLC cell lines with or without mutations in epidermal growth factor receptor (EGFR). Mechanistically, we demonstrated that BO-1978 exhibited multiple modes of action, including inhibition of topoisomerase I/II and induction of DNA cross-linking. Treatment of NSCLC cells with BO-1978 caused DNA damage, disturbed cell cycle progression, and triggered apoptotic cell death. Furthermore, BO-1978 significantly suppressed the growth of EGFR wild-type and mutant NSCLC tumors in xenograft tumor and orthotopic lung tumor models with negligible body weight loss. The combination of BO-1978 with gefitinib further suppressed EGFR mutant NSCLC cell growth in xenograft tumor and orthotopic lung tumor models. Preclinical toxicity studies showed that BO-1978 administration did not cause apparent toxicity in mice. Based on its significant therapeutic efficacy and low drug toxicity, BO-1978 is a potential therapeutic agent for treatment of NSCLC.

HER2-siRNA delivered by EGFR-specific single chain antibody inhibits NSCLC cell proliferation and tumor growth.

Overexpression of human epidermal growth factor receptor type2 (HER2) is closely associated with aggressive progression and poor prognosis in non-small cell lung cancer (NSCLC). Here, we generated an EGFR-scFv-arginine nonamer peptide fusion protein (scFv-9R) as a cargo to deliver HER2 specific siRNA into HER2-positive NSCLC cells both in vitro and in vivo. HER2-siRNAs delivered by scFv-9R effeciently silenced HER2 expression in EGFR-positive NSCLC cells, and consequently resulted in G1 arrest and cell growth inhibition. Importantly, intravenous injection of scFv-9R/HER2-siRNA complex markedly suppressed growth of EGFR-positive NSCLC xenograft in nude mice, resulting from downregulated HER2 expression, reduced cell proliferation and enhanced cell apoptosis. Collectively, our study provides a novel therapeutic strategy for the treatment of EGFR-positive, HER2-overexpressed NSCLC.

MiR-32 inhibits proliferation, epithelial-mesenchymal transition, and metastasis by targeting TWIST1 in non-small-cell lung cancer cells.

BackgroundBy analyzing published microRNA microarray studies, miR-32 was found to be markedly reduced in non-small-cell lung cancer (NSCLC) tissues compared with that in nontumor tissues. However, little is known about its role and molecular mechanism involved in NSCLC development and progression. Here, we report the effect of miR-32 on NSCLC cell proliferation, epithelial–mesenchymal transition (EMT), and metastasis.MethodsQuantitative real-time PCR was performed to detect the expression level of miR-32 in primary NSCLC cases and cell lines. miR-32-overexpressing H1299 and A549 cells were constructed by lipofection transfection. MTT, transwell chamber, and Western blot assays were used to assess the effect of miR-32 on proliferation, EMT, and metastasis of NSCLC cells, respectively. Target prediction and luciferase reporter assays were performed to investigate the targets of miR-32. Tumor formation assay in vivo was performed to investigate the antitumor effect of miR-32.ResultsAn inverse correlation existed between miR-32 expression level and NSCLC cell proliferation, EMT, and metastasis, and upregulation of miR-32 repressed NSCLC cell proliferation, EMT, and metastasis. Moreover, we identified and validated that TWIST1 was a direct target of miR-32, and miR-32 regulated NSCLC cell proliferation, EMT, and metastasis, at least in part via modulation of TWIST1. The animal experiments showed that overexpression of miR-32 inhibited the growth of NSCLC tumors in vivo.

244-MPT overcomes gefitinib resistance in non-small cell lung cancer cells.

The epidermal growth factor receptor (EGFR) is known to play a critical role in non-small cell lung cancer(NSCLC). Several EGFR tyrosine kinase inhibitors(TKIs), such as gefitinib, have been used as effective clinical therapies for patients with NSCLC. Unfortunately, acquired resistance to gefitinib commonly occurs after 6-12 months of treatment. The resistance is associated with the appearance of the L858R/T790M double mutation of the EGFR. In our present study, we discovered a compound,referred to as 244-MPT, which could suppress either gefitinib-sensitive or -resistant lung cancer cell growth and colony formation, and also suppressed the kinase activity of both wildtype and double mutant (L858R/T790M) EGFR. The underlying mechanism reveals that 244-MPT could interact with either the wildtype or double-mutant EGFR in an ATP-competitive manner and inhibit activity. Treatment with 244-MPT could substantially reduce the phosphorylation of EGFR and its downstream signaling pathways, including Akt and ERK1/2 in gefitinib-sensitive and -resistant cell lines. It was equally effective in suppressing EGFR phosphorylation and downstream signaling in NL20 cells transfected with wildtype, single-mutant (L858R) or mutant (L858R/T790M) EGFR. 244-MPT could also induce apoptosis in a gefitinib-resistant cell line and strongly suppress gefitinib-resistant NSCLC tumor growth in a xenograft mouse model. In addition, 244-MPT could effectively reduce the size of tumors in a gefitinib-resistant NSCLC patient-derived xenograft (PDX) SCID mouse model. Overall, 244-MPT could overcome gefitinib-resistance by directly targeting the EGFR.

Small-molecule targeting of signal transducer and activator of transcription (STAT) 3 to treat non-small cell lung cancer.

Lung cancer is the leading cause of cancer death in both men and women. Non-small cell lung cancer (NSCLC) has an overall 5-year survival rate of 15%. While aberrant STAT3 activation has previously been observed in NSCLC, the scope of its contribution is uncertain and agents that target STAT3 for treatment are not available clinically. We determined levels of activated STAT3 (STAT3 phosphorylated on Y705, pSTAT3) and the two major isoforms of STAT3 (α and β) in protein extracts of 8 NSCLC cell lines, as well as the effects of targeting STAT3 in vitro and in vivo in NSCLC cells using short hairpin (sh) RNA and two novel small-molecule STAT3 inhibitors, C188-9 and piperlongumine (PL). Levels of pSTAT3, STAT3α, and STATβ were increased in 7 of 8 NSCLC cell lines. Of note, levels of pSTAT3 were tightly correlated with levels of STAT3β, but not STAT3α. Targeting of STAT3 in A549 cells using shRNA decreased tSTAT3 by 75%; this was accompanied by a 47-78% reduction in anchorage-dependent and anchorage-independent growth and a 28-45% reduction in mRNA levels for anti-apoptotic STAT3 gene targets. C188-9 and PL (@30 μM) each reduced pSTAT3 levels in all NSCLC cell lines tested by ≥50%, reduced anti-apoptotic protein mRNA levels by 25-60%, and reduced both anchorage-dependent and anchorage-independent growth of NSCLC cell lines with IC50 values ranging from 3.06 to 52.44 μM and 0.86 to 11.66 μM, respectively. Treatment of nude mice bearing A549 tumor xenografts with C188-9 or PL blocked tumor growth and reduced levels of pSTAT3 and mRNA encoding anti-apoptotic proteins. STAT3 is essential for growth of NSCLC cell lines and tumors and its targeting using C188-9 or PL may be a useful strategy for treatment.