NSCLC Growth Research Articles

Abstract 1537: CD63 acts as a key modulator of tumor progression and innate immune evasion in non-small cell lung cancer

Abstract Introduction: Lung cancer is a major reason for cancer-related mortality worldwide, causing more than two million cancer deaths per year. Over the last decade, new therapies have allowed long-term survival for patients even in advanced stages. However, only a minority of patients benefits from these therapies and patients often grow resistant over time. An important mechanism that contributes to cancer growth and the development of therapy resistances is the cross-talk between cancer cells and their tissue microenvironment, in particular fibroblasts and macrophages. In this context, we explored whether the tetraspanin CD63 - which is commonly found on intra- and extracellular membranes - stimulates cancer growth and mediates pro-tumorigenic functions of the tissue microenvironment in NSCLC. Methods: We analyzed patient tissue via immunohistochemistry and in-situ-hybridization. We deleted CD63 in cancer cell lines (A549, H810, LC19) and primary fibroblasts via CRISPR-Cas9. To assess phagocytosis, we used in vitro EdU assays and adaptive transfer models in vivo, and identified changes in cell signaling via mass spectrometry. We used an in vitro phagocytosis assay to determine the effect of CD63 on cancer cell phagocytosis through macrophages and analyzed the expression of M1 and M2-associated surface markers through flow cytometry upon co-culturing PBMC with cancer cells over one week. Results: In patient sections, cancer tissue did not only express more CD63 on the gene expression and protein level but a stronger CD63 expression also correlated with a denser macrophage infiltrate. Looking for pro-tumorigenic functions, deleting CD63 in three cancer cell lines reduced cancer cell growth both in vitro and in an adaptive transfer model in vivo. Additionally, deleting CD63 impaired the pro-tumorigenic focal adhesion signaling in primary fibroblasts. Concerning the interaction between cancer cells and macrophages, deleting CD63 reduced the macrophage-mediated phagocytosis of cancer cells and co-culturing primary monocytes with CD63-deficient lung cancer cells induced the expression of M1-associated surface markers on the PBMC. Conclusion: In conclusion, these results demonstrate that the tetraspanin CD63 drives NSCLC growth by stimulating cancer cell proliferation - in part by interfering with focal adhesion signaling - and facilitating immune evasion by preventing cancer cell phagocytosis through macrophages and inducing an immunosuppressive phenotype in macrophages. Citation Format: Tristan Lerbs, Lu Cui, Cristabelle De Souza, Reinhard Buettner, Gerlinde Wernig. CD63 acts as a key modulator of tumor progression and innate immune evasion in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1537.

Pyrogallol promotes growth arrest by activating the p53-mediated up-regulation of p21 and p62/SQSTM1-dependent degradation of β-catenin in nonsmall cell lung cancer cells.

Pyrogallol (1,2,3-trihydroxybenzene), a polyphenolic natural compound, has attracted considerable attention with regard to its potential anticancer activity. However, further study is needed to elucidate the underlying mechanism related to the antiNSCLC activity of pyrogallol and provide a comprehensive theoretical basis for better clinical utilization of pyrogallol. Our current study aims to investigate the effects and potential underlying mechanisms of pyrogallol on the inhibition of NSCLC growth. Our results showed that pyrogallol treatment induced cell cycle arrest at the G2/M phase and apoptosis in two different NSCLC cell lines. Mechanistically, we found that the induction of cell cycle arrest in NSCLC cells at the G2/M phase by pyrogallol was due to the upregulation of p21 in a p53-dependent manner. And blockade of p53 and p21 effectively abolished the cell cycle arrest at the G2/M phase. Meanwhile, p53 inhibition has been found to abrogate the pyrogallol-induced apoptosis of the two NSCLC cells. Moreover, we revealed that the inhibitory effects of pyrogallol on β-catenin signaling resulted from autophagy initiation depending on p53 activation, accompanied by an increase in p62/SQSTM1 expression, thus p62 subsequently interacting with ubiquitinated β-catenin and facilitating autophagic destruction of β-catenin. Furthermore, in vivo experiments demonstrated that pyrogallol exerted growth inhibition on NSCLC with low toxicity through the same molecular mechanism as observed in vitro. Our findings could contribute to the understanding of the mechanism by which pyrogallol negatively regulates NSCLC growth, which could be effective in treating NSCLC.

Abstract LB211: Tumor-reactive and anti-PD-L1 co-stimulated killer cells (TRACK-NK) for immunotherapy of non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) remains one of the major causes of cancer related mortality worldwide, yet clinical advances with checkpoint inhibitors suggest that an anti-tumor immune response can be harnessed to prolong survival in NSCLC patients. We recently described the identification of a tumor reactive natural killer (NK) cell population by its in vivo upregulation of PD-L1 in the presence of tumor, referred to here as TRACK-NK cells. PD-L1(+) NK cells are more potent than PD-L1(-) NK cells against tumor cell targets. Further, the administration of anti-PD-L1 antibody (atezolizumab) functions as an immune agonist on TRACK-NK cells resulting in significantly enhanced degranulation (P < 0.001) and secretion of IFN-gamma (P < 0.001) in the presence of tumor. Here we investigated the functional activity and product safety of allogeneic, off-the-shelf TRACK-NK cells retrovirally transduced to express soluble (s) IL-15 to treat NSCLC without or with the addition of atezolizumab. TRACK-NK cells derived from umbilical cord blood NK cells were expanded over 1,000-fold ex-vivo in 17 days with ~50% of the final product engineered to express sIL15 and cytokine-activated to express PD-L1. The phenotype of the final TRACK-NK cell product showed a significant increase in the expression of PD-L1, CD25 and CD69 when compared to non-transduced (P < 0.01) or NK cells transduced to express sIL15 without cytokine activation (s15 NK cells) (P < 0.01). Following cryopreservation, our thawed TRACK-NK cell product demonstrated high recovery (>85%) and viability (>80%). In a Real Time Cell Analysis (RTCA) in vitro cytotoxicity assay against a human NSCLC cell line (A549), TRACK-NK cells were 10 times more potent than non-transduced (NT) NK cells (P < 0.001) and 2 times more potent than s15 NK cells (P < 0.01). To assess the efficacy of our frozen, allogeneic off-the-shelf TRACK-NK cells in vivo, we utilized immunodeficient mice bearing human A549 metastatic NSCLC. Intravenous infusion of TRACK-NK cells resulted in significantly more suppression of luciferase-A549 NSCLC growth in mice compared to mice treated with NT NK cells (P < 0.05) or s15 NK cells (P < 0.01). In terms of dose response, 1 × 107 TRACK-NK cells per dose delivered every other day for a total of 4 doses showed significantly better tumor control compared to a dose of 5 × 106 TRACK-NK cells (P < 0.001) but similar control compared to 2 × 107 TRACK-NK cells. The combination of TRACK-NK cells plus atezolizumab showed better control of NSCLC growth in vivo compared to TRACK-NK cells alone (P < 0.01). Finally, we evaluated the safety of our TRACK-NK product in immunodeficient mice without tumor engraftment and did not observe any significant change in body weight, body temperature, liver enzymes, kidney function, or blood counts compared to mice treated with vehicle control. No cytokine release syndrome was observed. In summary, our studies using human allogeneic off-the-shelf TRACK-NK cells proved to be safe and non-toxic, demonstrated enhanced cytotoxicity against NSCLC in vitro and enhanced control of tumor growth in vivo, further improved with atezolizumab. As the intravenous infusion of activated NK cells naturally traffic to lung, our TRACK-NK platform will move forward to the clinic for the treatment of relapsed and refractory NSCLC patients. Citation Format: Ting Lu, Anthony G. Mansour, Rui Ma, Christian Bustillos, Zhiyao Li, Shoubao Ma, Zhenlong Li, Hanyu Chen, Kun-Yu Teng, Jianying Zhang, Michael A. Caligiuri, Jianhua Yu. Tumor-reactive and anti-PD-L1 co-stimulated killer cells (TRACK-NK) for immunotherapy of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB211.

LncRNA HOXC-AS3 promotes non-small-cell lung cancer growth and metastasis through upregulation of YBX1

NSCLC is common and is the primary cause of cancer-related deaths due to a lack of early diagnosis and its propensity for metastasis. The pathogenesis of NSCLC is still unclear. Here, we explored the molecular mechanisms underlying NSCLC development, focusing on the HOXC-AS3/YBX1/HOXC8 axis. Human NSCLC specimens and cell lines were used. qRT-PCR and western blotting were utilised to examine the levels of HOXC-AS3/YBX1/HOXC8. CCK-8, colony formation, scratch wound healing and Transwell assays were performed to evaluate cancer cell proliferation, migration and invasion. A nude mouse xenograft model was used to examine tumour growth and metastasis in vivo. RNA pull-down, chromatin immunoprecipitation, coimmunoprecipitation and dual-luciferase assays were applied to validate the interactions of HOXC-AS3/YBX1, MDM2/YBX1 and the YBX1/HOXC8 promoter. The levels of HOXC-AS3 and HOXC8 were increased in human NSCLC specimens and cells. Knockdown of HOXC-AS3 suppressed NSCLC cell proliferation, migration and invasion, as well as tumour growth and metastasis in vivo. HOXC-AS3 directly bound to YBX1 to suppress its ubiquitination mediated by MDM2. YBX1 bound to the HOXC8 promoter and enhanced its transcription. Knockdown of HOXC8 inhibited the effects of HOXC-AS3 overexpression on NSCLC. HOXC-AS3 promotes NSCLC growth and metastasis by stabilising YBX1 and thus increasing HOXC8 transcription. Our study indicates that the HOXC-AS3/YBX1/HOXC8 axis could serve as a biomarker for NSCLC diagnosis or as a target for therapy development.

Circ_0011292 knockdown mitigates progression and drug resistance in PTX-resistant non-small-cell lung cancer cells by regulating miR-433-3p/CHEK1 axis.

BackgroundNon‐small‐cell lung cancer (NSCLC) is one of the most common malignant tumors on earth. Circular RNAs have been disclosed to be vital regulators in the chemoresistance and development of diverse cancers, including NSCLC. Here, we attempted to explore the function of circ_0011292 in paclitaxel (PTX)‐resistant NSCLC cells.MethodsQuantitative real‐time polymerase chain reaction or western blot was performed to detect the expression of circ_0011292, microRNA‐433‐3p (miR‐433‐3p), and checkpoint kinase 1 (CHEK1). Ribonuclease R (RNase R) assay was performed to assess the stability of circ_0011292. Cell Counting Kit‐8 assay was conducted to evaluate the half maximal inhibitory concentration of PTX and cell viability. Cell proliferation was monitored by Edu incorporation and colony formation assays. Cell cycle and apoptosis were detected by flow cytometry. Transwell assay was implemented to assess cell migration and invasion. Western blot assay was utilized to determine the protein levels. Dual‐luciferase reporter assay was carried out to verify the targeted interaction between miR‐433‐3p and circ_0011292 or CHEK1. Xenograft tumor model was constructed for determining the effect of circ_0011292 in NSCLC growth in vivo.ResultsCirc_0011292 was upregulated in PTX‐resistant NSCLC tissues and cells. Circ_0011292 or CHEK1 knockdown enhanced PTX sensitivity and cell apoptosis, and repressed cell proliferation, migration, and invasion in PTX‐resistant NSCLC cells. Mechanistically, circ_0011292 was a sponge of miR‐433‐3p and miR‐433‐3p directly targeted CHEK1. Meanwhile, silencing miR‐433‐3p or overexpressing CHEK1 respectively abrogated the impacts of circ_0011292 deletion or miR‐433‐3p introduction on PTX resistance and cell progression in PTX‐resistant NSCLC cells in vitro. Moreover, circ_0011292 could positively modulate CHEK1 expression through sponging miR‐433‐3p. In addition, circ_0011292 knockdown retarded tumor growth of NSCLC in vivo.ConclusionCirc_0011292 could accelerate PTX resistance and cell malignant progression of NSCLC cells partially through the regulation of circ_0011292/miR‐433‐3p/CHEK1 axis.

Cholesterol promotes EGFR-TKIs resistance in NSCLC by inducing EGFR/Src/Erk/SP1 signaling-mediated ERR\u03b1 re-expression

BackgroundThe use of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) brings remarkable benefits for the survival of patients with advanced NSCLC harboring EGFR mutations. Unfortunately, acquired resistance seems to be inevitable and limits the application of EGFR-TKIs in clinical practice. This study reported a common molecular mechanism sustaining resistance and potential treatment options to overcome EGFR-TKIs resistance.MethodsEGFR-TKIs resistant NSCLC cells were established and confirmed by MTT assay. Cholesterol content was detected and the promotional function of cholesterol on NSCLC growth was determined in vivo. Then, we identified ERRα expression as the downstream factor of cholesterol-mediated drug resistance. To dissect the regulatory mechanism, we conducted experiments, including immunofluorescence, co-immunoprecipitation, luciferase reporter assay and chromatin immunoprecipitation assay.ResultsLong-term exposure to EGFR-TKIs generate drug resistance with the characteristic of cholesterol accumulation in lipid rafts, which promotes EGFR and Src to interact and lead EGFR/Src/Erk signaling reactivation-mediated SP1 nuclear translocation and ERRα re-expression. Further investigation identifies ERRα as a target gene of SP1. Functionally, re-expression of ERRα sustains cell proliferation by regulating ROS detoxification process. Lovastatin, a drug used to decrease cholesterol level, and XCT790, an inverse agonist of ERRα, overcome gefitinib and osimertinib resistance both in vitro and in vivo.ConclusionsOur study indicates that cholesterol/EGFR/Src/Erk/SP1 axis-induced ERRα re-expression promotes survival of gefitinib and osimertinib-resistant cancer cells. Besides, we demonstrate the potential of lowing cholesterol and downregulation of ERRα as effective adjuvant treatment of NSCLC.

Abstract 809: Neurotensin increases tyrosine phosphorylation of HER3

Abstract Neurotensin (NTS) is an autocrine growth factor for non-small cell lung cancer cells. NTS stimulates and the NTSR1 antagonist SR48692 inhibits NSCLC growth (Moody et al., Life Sci 2014; 100: 25). NSCLC patients, whose tumors have high NTSR1, have significantly lower relapse-free survival than those with low NTSR1 (Younes et al., Oncotarget 2014; 5: 8252). NTS addition to NSCLC cells increases the tyrosine phosphorylation of the EGFR and HER2 (Moody et al., Eur J Pharmacol 2019; 865:172735). Activation of HER3 in NSCLC mediates aggressive growth and drug resistance. Therefore the ability of NTSR1 to regulate transactivation of HER3 was investigated. By RT-PCR, HER3 and NTSR1 but not NTSR2 mRNA was present in NSCLC cells. NTS addition to NCI-H358 or H441 cells increased HER3 and ERK tyrosine phosphorylation by 210% and 240%, respectively and the increase caused by NTS was antagonized by SR48692 or mAb3481 (HER3 mAb). The increase is phosphorylation of Tyr1289-HER3 caused by NTS or neuregulin-1 addition to NSCLC cells was due to the formation of HER2-HER3 and EGFR-HER3 heterodimers. NTSR1 regulation of HER3 or Akt phosphorylation was impaired by PP2 (Src inhibitor) or GM6001 (MMP inhibitor). Because N-acetylcysteine (antioxidant) or Tiron (superoxide scavenger) inhibited the ability of NTS to increase HER3 phosphorylation, reactive oxygen species (ROS) are essential. Using a clonogenic assay, NTS increased NSCLC colony number whereas SR48692 or mAb3481 reduced colony number. The results indicate that NTS increased HER3 tyrosine phosphorylation in a ROS-/Src-/MMP-dependent manner. In summary, NTSR1 transactivates HER3 resulting in ERK and Akt phosphorylation increasing the proliferation and survival of NSCLC cells. Citation Format: Terry W. Moody, Irene Ramosalvarez, Robert T. Jensen. Neurotensin increases tyrosine phosphorylation of HER3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 809.

Abstract 731: Neurotensin causes tyrosine phosphorylation of HER3

Abstract Neurotensin (NTS) is an autocrine growth factor for non-small cell lung cancer cells. NTS stimulates and the NTSR1 antagonist SR48692 inhibits NSCLC growth (Moody et al., Life Sci 2014; 100: 25). NSCLC patients, whose tumors have high NTSR1, have significantly lower relapse-free survival than those with low NTSR1 (Younes et al., Oncotarget 2014; 5: 8252). NTS addition to NSCLC cells increases the tyrosine phosphorylation of the EGFR and HER2 (Moody et al., Eur J Pharmacol 2019; 865:172735). Activation of HER3 in NSCLC mediates aggressive growth and drug resistance. Therefore the ability of NTSR1 to regulate transactivation of HER3 was investigated. By RT-PCR, HER3 and NTSR1 but not NTSR2 mRNA was present in NSCLC cells. NTS addition to NCI-H358 or H441 cells increased HER3 and ERK tyrosine phosphorylation by 210% and 240%, respectively and the increase caused by NTS was antagonized by SR48692 or mAb3481 (HER3 mAb). The increase is phosphorylation of Tyr1289-HER3 caused by NTS or neuregulin-1 addition to NSCLC cells was due to the formation of HER2-HER3 and EGFR-HER3 heterodimers. NTSR1 regulation of HER3 or Akt phosphorylation was impaired by PP2 (Src inhibitor) or GM6001 (MMP inhibitor). Because N-acetylcysteine (antioxidant) or Tiron (superoxide scavenger) inhibited the ability of NTS to increase HER3 phosphorylation, reactive oxygen species (ROS) are essential. Using a clonogenic assay, NTS increased NSCLC colony number whereas SR48692 or mAb3481 reduced colony number. The results indicate that NTS increased HER3 tyrosine phosphorylation in a ROS-/Src-/MMP-dependent manner. In summary, NTSR1 transactivates HER3 resulting in ERK and Akt phosphorylation increasing the proliferation and survival of NSCLC cells. Citation Format: Terry W. Moody, Irene Ramosalvarez, Robert T. Jensen. Neurotensin causes tyrosine phosphorylation of HER3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 731.

MicroRNA‑564 inhibits the progression of non‑small cell lung cancer via targeting plexin A4

Νon-small cell lung cancer (NSCLC) is the most frequently diagnosed type of cancer, and the most prevalent cause of cancer-associated mortality. The present study aimed to investigate whether microRNA (miR)-564 influences NSCLC progression by regulating NSCLC cell growth and migration, via targeting plexin A4. Therefore, the expression levels of miR-564 and plexin A4 were evaluated in NSCLC specimens or cells using reverse transcription-quantitative PCR. Furthermore, colony formation and Cell Counting Kit-8 assays were performed to determine the proliferative ability of NSCLC cells. The cell migration capacity was assessed using a Transwell assay. In addition, to examine the binding ability of miR-564 on the plexin A4 3'-untranslated region (3'UTR), a dual-luciferase reporter assay was performed. A mouse xenograft model was established to evaluate the effect of miR-564 knockdown on tumor growth in vivo, whereas the expression of plexin A4 and Ki67 in NSCLC tissues was detected using immunohistochemistry. Notably, miR-564 was downregulated in both NSCLC cell lines and tissues, while its overexpression, following transfection with miR-564 mimics, attenuated the proliferation and proliferation, migration and invasion of NSCLC cells. By contrast, silencing of miR-564 using a miR-564 inhibitor promoted NSCLC cell proliferation, migration and invasion. The luciferase assay revealed that miR-564 directly targeted the plexin A4 3'UTR in A549 and H460 cells. Additionally, the overexpression of plexin A4 rescued the effect of miR-564 on NSCLC cell proliferation, migration and invasion abilities. Further in vivo studies demonstrated that miR-564 knockdown promoted NSCLC growth, while miR-564 overexpression resulted in the opposite effect in nude mice. Overall, the results of the present study revealed that miR-564 promotes the proliferation and migration of NSCLC cells, both in vitro and in vivo, via targeting plexin A4. Therefore, miR-564 may be considered as a possible therapeutic target for NSCLC.

Combining Nelfinavir With Chloroquine InhibitsIn VivoGrowth of Human Lung Cancer Xenograft Tumors

Nelfinavir is a human immunodeficiency virus protease inhibitor that is currently being repositioned as an anticancer drug. Chloroquine, an anti-malarial lysosomotropic drug, inhibits autophagy. It has been reported that the combination of nelfinavir and chloroquine significantly enhances endoplasmic reticulum (ER) stress and induces selective cell death in multiple cell line models (in vitro). We assessed the effects of the combination of these drugs on human NSCLC cell lines in vitro using cell proliferation assay and performed preclinical treatment studies using cell line-derived xenograft mouse models in vivo. In vitro, this combination enhanced inhibition of NSCLC cell proliferation with increased proteotoxicity, including ER stress, and apoptosis. In vivo, the growth of human NSCLC xenograft tumors was inhibited, which correlated with increased apoptosis and induction of ER stress as well as NSCLC growth in vitro. Our findings suggest that the induction of proteotoxicity provides a promising new target for developing anticancer drugs.

LINC01503/miR-342-3p facilitates malignancy in non-small-cell lung cancer cells via regulating LASP1

BackgroundNon-small cell lung cancer (NSCLC) is one of the major types of lung cancer, which is a prevalent human disease all over the world. LncRNA LINC01503 is a super-enhancer-driven long non-coding RNA that is dysregulated in several types of human cancer. However, its role in NSCLC remains unknown.MethodsThirty NSCLC patients were recruited between April 2012 and April 2016. Luciferase reporter assay, qRT-PCR, Cell Counting Kit-8 (CCK-8), Transwell migration assay, RNA pull-down assay, western blotting, 5-ethynyl-29-deoxyuridine (EdU) assays, and flow cytometry were utilized to characterize the roles and relationships among LINC01503, miR-342-3p, and LASP1 in NSCLC. The transplanted mouse model was built to examine their biological functions in vivo.ResultsWe demonstrated that the expression of lncRNA LINC01503 and LIM and SH3 domain protein 1 (LASP1) were upregulated and miR-342-3p was downregulated in NSCLC samples and cell lines. Functional experiments revealed that inhibiting the expression of LINC01503 or over-expression of miR-342-3p inhibited NSCLC growth and metastasis both in vitro and in vivo. In addition, LINC01503 could bind to miR-342-3p and affect the expression of LASP1.ConclusionThese results provide a comprehensive analysis of the roles of LINC01503 as a competing endogenous RNA (ceRNA) in NSCLC progression.

Niclosamide, an antihelmintic drug, enhances efficacy of PD-1/PD-L1 immune checkpoint blockade in non-small cell lung cancer

BackgroundPD-1/PD-L1 blockade has received approval for clinical application due to its encouraging benefit with improving prognosis in selected populations. Unfortunately, the response to immunotherapy for many patients remains unsatisfactory. It remains a great challenge to generate potential combinations that will outperform single agents alone with regard to anti-tumor activity.MethodsUsing NSCLC cell lines and mouse models, we explored the effects of combined niclosamide and PD-L1 blockade on tumor growth and T cell function. Furthermore, we investigated the relationship between PD-L1 and p-STAT3 expression in tumor samples from patients with NSCLC using IHC, as well as their relationship to patient survival.ResultsIn vitro, niclosamide, an antihelmintic drug, enhanced the cancer cell lysis mediated by T cells in the presence of PD-L1 blockade. Accordingly, mice treated with niclosamide and PD-L1 antibody showed significant delay in tumor growth and increased survival which were associated with the increase of tumor infiltrating T cells and granzyme B release. Importantly, we found niclosamide could decrease the expression of PD-L1 in both a concentration- and time-dependent manner in NSCLC cells, which was linked to the blockage of p-STAT3 binding to the promoter of PD-L1.ConclusionsAn enhancement of PD-L1 antibody by niclosamide was observed in inhibition of NSCLC growth in vitro and in vivo, which was involved in blockage of p-STAT3 binding to promoter of PD-L1 and finally downregulation of PD-L1 expression. These encourage the combination therapy of niclosamide and PD-1/PD-L1 blockade to be further studied in clinic.

Combination of Gentiana rhodantha and Gerbera anandria in the BL02 formula as therapeutics to non-small cell lung carcinoma acting via Rap1/cdc42 signaling: A transcriptomics/ bio-informatics biological validation approach

BackgroundNon-small cell lung cancer (NSCLC) ranks the most commonly diagnosed and highest mortality-leading cancer worldwide despite a variety of treatment strategies are available. The highly heterogeneous and aggressive property of NSCLC as well as its poor prognosis indicates the need for novel therapeutic targets identification. The objective of this study is to identify potential targets from the adjuvant herbal formula BL02 using a combined approach of high throughput transcriptomics and network pharmacology. MethodsThe quality and stability of BL02 were assessed by UHPLC analysis. The inhibitory effect of BL02 on NSCLC was measured by in vivo orthotopic intrathoracic mouse model and in vitro cellular models. EGFR-mutant HCC827 and wild type A549 cell lines were employed. Transcriptomics analysis was introduced to profile the gene expression of NSCLC cells treated with BL02; Network pharmacology and molecular docking analyses predicted the interaction of compounds and NSCLC targets. Immuno-blotting and pull-down assays verified the putative targets. ResultsThe UHPLC analysis revealed that BL02 was relatively stable between batches of production and for 24 months of storage. Orally administration of BL02 was safe and effective to inhibit pulmonary NSCLC growth in mice implanted with A549 and HCC827-generated tumors. BL02 exhibited relatively low cytotoxicity to NSCLC cells in vitro, but potently suppressed NSCLC cell motility. The transcriptomic analysis illustrated that EGFR and cellular adhesion-related signaling is involved in BL02 action. Further bioinformatics analysis validated BL02 activity is mediated by cdc42-regulated signaling. BL02 depolymerized the actin cytoskeleton through suppressing cdc42 and deactivating its upstream molecule Rap1. These effects may be primarily mediated by the direct binding of 5-methylcoumarin-4-cellobioside and mangiferin from BL02 to Rap1 protein. ConclusionOur study proposes an integration model of experimental, transcriptomic and bioinformatics analyses in the identification of novel therapeutic target of NSCLC from an adjuvant herbal formula BL02. Our findings revealed that inhibition of Rap1/cdc42 signaling by active compounds 5-methylcoumarin-4-cellobioside and mangiferin from BL02 might be potentially effective therapy for NSCLC.

MicroRNA 615-3p Inhibits the Tumor Growth and Metastasis of NSCLC via Inhibiting IGF2.

MicroRNAs are essential regulators of cancer-associated genes at the posttranscriptional level, and their expression is altered in cancer tissues. Herein we sought to identify the regulation of miR-615-3p in NSCLC progression and its mechanism. miR-615-3p expression was significantly downregulated in NSCLC tissue compared to control normal tissue. Exogenous overexpression of miR-615-3p inhibited the growth and metastasis of NSCLC cells. In addition, the in vivo mouse xenograft model showed that overexpression of miR-615-3p inhibited NSCLC growth and lung metastasis, whereas decreased expression of miR-615-3p caused an opposite outcome. Furthermore, we revealed that insulin-like growth factor 2 (IGF2) expression was negatively correlated with the miR-615-3p level in NSCLC specimens, and IGF2 knockdown mimicked the effect of miR-615-3p inhibition on NSCLC cell proliferation, migration, and invasion. In addition, overexpression of IGF2 rescued the inhibition of miR-615-3p in NSCLC cells. Together, our results indicated that miR-615-3p played important roles in the regulation of NSCLC growth and metastasis by targeting IGF2.

Guajadial inhibits NSCLC growth and migration following activation of the VEGF receptor-2

Guajadial, one of natural dialdehyde meroterpenoids, demonstrated significant antineoplasmic activity. The present research was to investigate the inhibitory effects of guajadial by using two NSCLC cells (A549 and H1650) proliferation and migration. Western blotting was employed to explore the underlying mechanisms of VEGF receptor (VEGFR)2-mediated. This research indicated that guajadial not only inhibited endothelial cell proliferation and migration but also suppress tumor growth in human NSCLC xenograft mouse models. It is also suggested that guajadial inhibited A549 proliferation via blocking the Ras/MAPK pathway.

Long non-coding RNA LINC00968 acts as oncogene in NSCLC by activating the Wnt signaling pathway.

Long non-coding RNAs (lncRNAs) have played critical roles in a variety of cancers, including non-small cell lung cancer (N SCLC). In our study, we focused on the biological function and clinical significance of lncRNA LINC00968 in NSCLC. It was indicated that LINC00968 was significantly increased in LUAD tissues, LUSC tissues and NSCLC cells compared to their corresponding controls. Inhibition of LINC00968 was able to repress NSCLC growth, migration, and invasion in vitro while upregulation of LINC00968 reversed this process. Additionally, downregulation of LINC00968 induced apoptosis capacity of A549 cell. Apoptosis-related proteins BCL-2 were decreased and BAX was increased by knockdown of LINC00968, respectively. Meanwhile we observed that Wnt signaling pathway was involved in the LINC00968-induced NSCLC progression. Finally, in vivo tumor xenografts were established using A549 cells to detect the function of LINC00968 in NSCLC tumorigenesis. Silencing LINC00968 greatly inhibited NSCLC tumor progression, which was consistent with the in vitro tests. In conclusion, we have uncovered that LINC00968 could be regarded as a novel prognostic biomarker and therapeutic target in NSCLC diagnosis and treatment.

Abstract C131: ‘KRAS addiction’ an artifact of 2D culture? Inhibitors of the mut-KRAS NSCLC 3D growth

Abstract Introduction: KRAS, the predominant form of mutated RAS (mut-KRAS), is found in ∼25% of patient tumors across many cancer types and plays a critical role in driving tumor growth and resistance to therapy. We identified CNKSR1 (connector enhancer of kinase suppressor of Ras 1) to be critical for mut-KRAS but not wild type (wt)-KRAS signaling and cell proliferation. Its product CNK1 is a multi-domain organizer protein found as part of the Ras membrane signaling nanocluster where it binds to mut-KRas although not to wt-KRas, and is necessary for mut-KRas cell growth and signaling. We have exploited the pleckstrin homology (PH)-domain of CNK1 as a target for drug discovery to inhibit mut-KRas. To understand the role of CNK1 as a regulator of KRas cell growth, evaluation of siKRAS and siCNK1 on 2D and 3D growth were evaluated. We also studied the effects of KRAS cell growth conditions on response to selective inhibitors the PH-domain of CNK1 identified using a computational modeling approach. Results: Using a panel of twelve NSCLC lines and siRNA knockdown of KRAS we found that the reported growth addiction of some cancer cell lines for mut-KRAS, and the resistance of others, is most likely an artifact of 2D culture. Mut-KRAS NSCLC cell lines showing addiction in 2D growth did not show addiction in 3D anchorage independent growth in agarose, where all cell lines were more sensitive. Wt-KRAS cell lines were largely unaffected by siKRAS knockdown. Additionally, the CNK1 inhibitor PHT-7390 IC50s for 2D growth inhibition of a panel of 8 mut-KRAS NSCLC lines ranged from 0.60 to 100 μM (ave 18.7 μM) yet was found to be considerably more potent in 3D culture with IC50s from 0.03 to 2.99 μM (ave 0.69 μM). The most pronounced difference was seen in H2009 and Calu1 mut-KRAS cells where PHT-7390 IC50s in 2D were >100 μM, while 0.093 and 0.35 μM in 3D. Wt-KRas NSCLC growth inhibition (3 lines) was largely unaffected by PHT-7390 under either condition with average IC50s of 68 and 69 μM in 2D and 3D conditions, respectively. Target engagement in 2D has shown these CNK1 inhibitors block mut-KRAS signaling but not that of wt-KRAS. Conclusion: Variable enhanced growth dependence on mut-KRAS (“addiction”) is seen in 2D but not 3D. Potent and specific inhibition of mut-KRAS cell line growth by CNK1 PH-domain inhibitors is considerably more robust in 3D culture suggesting a novel approach to inhibit mut-KRAS effect on cancer growth. Citation Format: D. Lynn Kirkpatrick, Roisin Delaney, Geoff Grandjean, Assael Madrigal, Martin Indarte, Mike Scott, Garth Powis. ‘KRAS addiction’ an artifact of 2D culture? Inhibitors of the mut-KRAS NSCLC 3D growth. [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 C131.

PPARα Activation Can Help Prevent and Treat Non–Small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) not amenable to surgical resection has a high mortality rate, due to the ineffectiveness and toxicity of chemotherapy. Thus, there remains an urgent need of efficacious drugs that can combat this disease. In this study, we show that targeting the formation of proangiogenic epoxyeicosatrienoic acids (EET) by the cytochrome P450 arachidonic acid epoxygenases (Cyp2c) represents a new and safe mechanism to treat NSCLC growth and progression. In the transgenic murine K-Ras model and human orthotopic models of NSCLC, we found that Cyp2c44 could be downregulated by activating the transcription factor PPARα with the ligands bezafibrate and Wyeth-14,643. Notably, both treatments reduced primary and metastatic NSCLC growth, tumor angiogenesis, endothelial Cyp2c44 expression, and circulating EET levels. These beneficial effects were independent of the time of administration, whether before or after the onset of primary NSCLC, and they persisted after drug withdrawal, suggesting the benefits were durable. Our findings suggest that strategies to downregulate Cyp2c expression and/or its enzymatic activity may provide a safer and effective strategy to treat NSCLC. Moreover, as bezafibrate is a well-tolerated clinically approved drug used for managing lipidemia, our findings provide an immediate cue for clinical studies to evaluate the utility of PPARα ligands as safe agents for the treatment of lung cancer in humans.

Abstract 4183: MicroRNA-7 targeting PSME3 inhibits the growth of non-small cell lung cancer.

Abstract MicroRNAs (miRNAs) are a family of small non-coding RNAs that function as important regulators of multiple target genes and participate in diverse biological roles in carcinogenesis..In this study, we found that the expression of miR-7 was downregulated not only in cell lines, but also in human NSCLC tissues compared to the matched adjacent tissues, which exhibited positive correlation with tumor volume.Restoration of its expression through miR-7 mimics in NSCLC cell lines including A549 and H1299 cells inhibited cell proliferation, colony formation, and cell cycle progression in vitro. More importantly, the tumorigenicity in nude mice was reduced after administration of miR-7 in vivo, strongly indicating that miR-7 is a main tumor suppressor miRNA in NSCLC. In advance, through a series of analysis derived from bioinformatics prediction, western blot and luciferase assay, we identified a novel target of miR-7, PSME3 (a proteasome activator) to be enrolled in the regulation with tumor. Expression of PSME3 in NSCLC tumor samples was significantly upregulated compared to adjacent non-tumor tissues, exhibiting a strong inverse relation with that of miR-7. In addition, knockdown of PSME3 gene induced similar effects to overexpression of miR-7 in NSCLC cells. All these findings strongly imply that the overexpression of PSME3 resulted from miR-7 downexpression in NSCLC plays an important role in promoting cancer cell progress, and consequently results in NSCLC growth. Thus, strategies targeting PSME3 and/or miR-7 may be the promising molecular therapy in NSCLC treatment. Citation Format: Yiwei Chu. MicroRNA-7 targeting PSME3 inhibits the growth of non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4183. doi:10.1158/1538-7445.AM2013-4183

Abstract 5346: MiR-331-5p targets receptor EphA2 and attenuates tumor growth and migration in NSCLC.

Abstract Introduction: Receptor EphA2 is over expressed in NSCLC and is associated with tumor progression and poor survival. MicroRNAs (miRs) are short, non-coding RNAs that post-transcriptionally regulate gene expression. Dysregulated expression of miRs has been observed in variety of malignancies. However, the role of miR-331-5p in NSCLC growth and migration is not known. We hypothesized that EphrinA1 treatment promotes miR-331-5p expression in NSCLC and miR-331 may inhibit tumor growth and invasion by targeting receptor EphA2. Methods: The A-549 and H-23 NSCLC cells were treated with Ephrin-A1 for 24 hr and the miR-331-5p expression was measured by real-time PCR analysis. EphA2 protein expression was studied through Western blotting analysis. The cell proliferation was studied by WST-1 assay. The tumor formation and invasion was determined by 3-D matrigel and Wound healing assays respectively. Luciferase assay was performed to confirm miR-331-5p as one of the direct and specific target for EphA2. Results: The miR-331-5p expression was inversely correlated with EphA2 expression. Transfection of NSCLC with precursor miR-331-5p inhibited proliferation, and migration of NSCLC cells. In addition the ability of tumor colony formation was significantly inhibited when the cells were transfected with miR-331-5p precursors. We have identified predicted target binding site of EphA2 in its 3’ UTR region by miR-331-5p. This was confirmed by Luciferase assay. Over-expression of miR-331-5p in NSCLC cells repressed the endogenous levels of EphA2 protein. Conclusion: This study for the first time reports, that miR-331-5p directly regulates EphA2 mRNA and protein expression in NSCLC. MiR-331 directly binds to 3’ -UTR region of EphA2 and inhibits tumor growth and migration. Our findings also provide an insight into the regulation of EphA2 expression and suggest that miR-331-5p has the capacity to regulate signalling pathways critical for the development and progression of NSCLC. Funding: RC1 from Florida Department Health, and VA Merit Citation Format: Bhagyalaxmi Sukka-Ganesh, Kamal A. Mohammed, Frederic Kaye, Najmunnisa Nasreen. MiR-331-5p targets receptor EphA2 and attenuates tumor growth and migration in NSCLC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5346. doi:10.1158/1538-7445.AM2013-5346