Non-small Cell Lung Cancer Xenograft Mouse Model Research Articles

M2 macrophage-derived lncRNA NORAD in EVs promotes NSCLC progression via miR-520g-3p/SMIM22/GALE axis

Non-small cell lung cancer (NSCLC) constitutes the majority of lung cancer cases, accounting for over 80%. RNAs in EVs play a pivotal role in various biological and pathological processes mediated by extracellular vesicle (EV). Long non-coding RNAs (lncRNAs) are widely associated with cancer-related functions, including cell proliferation, migration, invasion, and drug resistance. Tumor-associated macrophages are recognized as pivotal contributors to tumorigenesis. Given these insights, this study aims to uncover the impact of lncRNA NORAD in EVs derived from M2 macrophages in NSCLC cell lines and xenograft mouse models of NSCLC. EVs were meticulously isolated and verified based on their morphology and specific biomarkers. The interaction between lncRNA NORAD and SMIM22 was investigated using immunoprecipitation. The influence of SMIM22/GALE or lncRNA NORAD in EVs on glycolysis was assessed in NSCLC cell lines. Additionally, we evaluated the effects of M2 macrophage-derived lncRNA NORAD in EVs on cell proliferation and apoptosis through colony formation and flow cytometry assays. Furthermore, the impact of M2 macrophage-derived lncRNA NORAD in EVs on tumor growth was confirmed using xenograft tumor animal models. The results underscored the potential role of M2 macrophage-derived lncRNA NORAD in EVs in NSCLC. SMIM22/GALE promoted glycolysis and the proliferation of NSCLC cells. Furthermore, lncRNA NORAD in EVs targeted SMIM22 and miR-520g-3p in NSCLC cells. Notably, lncRNA NORAD in EVs promoted the proliferation of NSCLC cells and facilitated NSCLC tumor growth through the miR-520g-3p axis. In conclusion, M2 macrophage-derived lncRNA NORAD in EVs promotes NSCLC progression through the miR-520g-3p/SMIM22/GALE axis.

Aurora Kinase A Inhibition Potentiates Platinum and Radiation Cytotoxicity in Non-Small-Cell Lung Cancer Cells and Induces Expression of Alternative Immune Checkpoints.

Despite major advances in non-small-cell lung cancer (NSCLC) treatment, the five-year survival rates for patients with non-oncogene-driven tumors remain low, necessitating combinatory approaches to improve outcomes. Our prior high-throughput RNAi screening identified Aurora kinase A (AURKA) as a potential key player in cisplatin resistance. In this study, we investigated AURKA's role in platinum and radiation sensitivity in multiple NSCLC cell lines and xenograft mouse models, as well as its effect on immune checkpoints, including PD-L1, B7x, B7-H3, and HHLA2. Of 94 NSCLC patient tumor specimens, 91.5% tested positive for AURKA expression, with 34% showing moderate-to-high levels. AURKA expression was upregulated following cisplatin treatment in NSCLC cell lines PC9 and A549. Both AURKA inhibition by alisertib and inducible AURKA knockdown potentiated the cytotoxic effects of cisplatin and radiation, leading to tumor regression in doxycycline-inducible xenograft mice. Co-treated cells exhibited increased DNA double-strand breaks, apoptosis, and senescence. Additionally, AURKA inhibition alone by alisertib increased PD-L1 and B7-H3 expression. In conclusion, our study demonstrates that AURKA inhibition enhances the efficacy of platinum-based chemotherapy in NSCLC cells and modulates the expression of multiple immune checkpoints. Therefore, combinatory regimens with AURKA inhibitors should be strategically designed and further studied within the evolving landscape of chemo-immunotherapy.

Suppression of JAK2/STAT3 Pathway by Notoginsenoside R1 Reduces Epithelial-Mesenchymal Transition in Non-small Cell Lung Cancer.

It has bene reported that a novel saponin-notoginsenoside R1 (NGR1) possesses strong anti-tumor activities. This study aimed to investigate the role and mechanism of NGR1 in non-small cell lung cancer (NSCLC). NSCLC cell viability, proliferation, migration, and invasiveness were assessed using the ex vivo assays. NSCLC xenograft mouse models were constructed to confirm the role of NGR1 in vivo. Epithelial-mesenchymal transition (EMT)-related proteins and key markers in the JAK2/STAT3 pathway were examined using immunoblotting and immunohistochemistry analyses. NGR1 treatment suppressed NSCLC cell growth ex vivo and in vivo. It also decreased the migratory and invasive capacities of NSCLC cells. Additionally, NGR1 increased E-cadherin expression and reduced N-cadherin, vimentin, and snail expression in TGF-β1-treated NSCLC cells and xenograft tumors. JAK2/STAT3 pathway was inhibited by NGR1. Moreover, a specific inhibitor of JAK2, AG490, or STAT3 silencing significantly enhanced the effects of NGR1 against the EMT process in NSCLC cells. NGR1 restrains EMT process in NSCLC by inactivating JAK2/STAT3 signaling, suggesting the potential of NGR1 in anti-NSCLC therapy.

Abstract 5815: Next-generation RNAi therapeutics: siRNA-loaded exosomes targeting KRAS G12C in non-small cell lung cancer (NSCLC)

Abstract Background: NSCLC represents the predominant subtype of lung cancer, boasting an overall 5-year survival rate of 26%, a figure that fluctuates based on the disease stage. Within NSCLC, certain cancer gene mutations, notably KRAS G12C, are currently classified as 'undruggable targets,' creating hurdles in achieving effective treatment. The development of innovative anticancer drugs aimed at these challenging targets is expected to present a viable solution to these difficulties. Methods: Exosomes have garnered attention as an inventive drug delivery system for in vivo gene delivery through systemic injection. We used a new post-loading approach by actively loading KRAS G12C siRNA into exosomes using the shock waves (SWEET: shock wave exosome engineering technology ) in this study. The effect of in vitro and in vivo were tested in NCI-H358 (KRAS G12C mt NSCLC) cells and its xenograft mouse model. The tumor size and weight were measured twice a week. Results: We developed an exosome-based therapeutic using siRNA to effectively inhibit KRAS G12C, targeting NSCLC. The SWEET platform's post-loading method showed a high efficiency in loading KRAS G12C siRNA into exosomes. The KRAS G12C siRNA-loaded exosomes effectively silenced oncogenic KRAS G12C expression in cancer cells; they inhibited tumor growth when administered intravenously in a NSCLC xenograft mouse model. Remarkably, the anticancer effect of KRAS G12C-loaded exosome was similar to that of Sotorasib, a KRAS G12C mutation targeting anti-cancer drug. Conclusion: Our study showed KRAS G12C siRNA loaded exosomes were effectively delivered to NSCLC tumor tissue via intravenous (IV) administration. And its anti-cancer effects were very similar to sotorasib, showing potential as a treatment for KRAS G12C mutation-expressing cancer. The effective delivery of siRNA to tumor tissues through exosomes highlights the potential of this technology as a promising approach for developing new RNAi therapeutics for cancer. Citation Format: Hyo Kyeong Kim, Yujeong Choi, Kyoung Hwa Kim, Yeongju Byun, Tae Hee Kim, Jae Hwan Kim, Shung Hyun An, DaeHo Bae, MyeongKwan Choi, Jihwa Chung, Kihwan Kwon. Next-generation RNAi therapeutics: siRNA-loaded exosomes targeting KRAS G12C in non-small cell lung cancer (NSCLC) [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 5815.

Abstract 4084: Potent anti-tumor activity of ABN202 (anti-EGFR antibody-interferon-beta mutein) through direct cytotoxicity and indirect immune activation against non-small cell lung cancer, regardless of EGFR mutation status

Abstract Epidermal growth factor receptor (EGFR) is overexpressed or mutated in many patients with non-small cell lung cancer (NSCLC). Targeting EGFR mutation by tyrosine kinase inhibitors (TKIs) has significantly improved overall survival (OS) in patients with EGFR mutations. However, many patients experience tumor recurrence due to various resistance mechanisms. Therefore, there is an unmet medical need for new treatment strategies for patients with EGFR-positive NSCLC, regardless of EGFR mutation status. ABN202 is an antibody-cytokine fusion protein (ACFP) that serves as a platform technology compromising the IFN-β mutein (ABN102) fused to various antibodies for specific tumor targeting. In a previous study, we designed ABN202 (αEGFR) to comprise an EGFR targeting antibody (Cetuximab) and IFN-β mutein (ABN102) and we confirmed anti-cancer activities of ABN202 against EGFR-positive NSCLC. In this study, the direct cytotoxicity of ABN202 (αEGFR) was evaluated in human NSCLC cell lines with various driving mutations and EGFR expression levels. The indirect immune activation was tested in human peripheral blood mononuclear cells (PBMCs) and human IFNAR1/2 knock-in (KI) mice. The in vivo efficacy was evaluated in human NSCLC xenograft mouse models and human IFNAR1/2 KI mouse models. ABN202 (αEGFR) demonstrates potent anti-tumor activity through direct cytotoxicity and indirect immune activation in NSCLC, regardless of EGFR mutation status. Taken together, we suggest that ABN202 (αEGFR) is a promising drug candidate against NSCLC, regardless of EGFR mutation status. Citation Format: Hee Geon Park, Sung Hun Cho, Ha Yeon Park, Mi Gyoung Jo, Ji Hyun Park, Myeung Ryun Seo, Ji Hyun Kim, Sung Youl Hong, Kyoung Song, Chan Gyu Lee, Hae Min Jeong, Sae Hyung Lee, Na Young Kim, Jun Young Choi, Young Kee Shin. Potent anti-tumor activity of ABN202 (anti-EGFR antibody-interferon-beta mutein) through direct cytotoxicity and indirect immune activation against non-small cell lung cancer, regardless of EGFR mutation status [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 4084.

Aerobic Exercise Improves Radiation Therapy Efficacy in Non-Small Cell Lung Cancer: Preclinical Study Using a Xenograft Mouse Model.

The "oxygen effect" improves radiation efficacy; thus, tumor cell oxygen concentration is a crucial factor for improving lung cancer treatment. In the current study, we aimed to identify aerobic exercise-induced changes in oxygen concentrations in non-small cell lung cancer (NSCLC) cells. To this end, an NSCLC xenograft mouse model was established using human A549 cells. Animals were subsequently subjected to aerobic exercise and radiation three times per week for 2 weeks. Aerobic exercise was performed at a speed of 8.0 m/m for 30 min, and the tumor was irradiated with 2 Gy of 6 MV X-rays (total radiation dose 12 Gy). Combined aerobic exercise and radiation reduced NSCLC cell growth. In addition, the positive effect of aerobic exercise on radiation efficacy through oxygenation of tumor cells was confirmed based on hypoxia-inducible factor-1 and carbonic anhydrase IX expression. Finally, whole-transcriptome analysis revealed the key factors that induce oxygenation in NSCLC cells when aerobic exercise was combined with radiation. Taken together, these results indicate that aerobic exercise improves the effectiveness of radiation in the treatment of NSCLC. This preclinical study provides a basis for the clinical application of aerobic exercise to patients with NSCLC undergoing radiation therapy.

A CD276-Targeted Antibody-Drug Conjugate to Treat Non-Small Lung Cancer (NSCLC).

Non-small cell lung cancer (NSCLC) patients, accounting for approximately 85% of lung cancer cases, are usually diagnosed in advanced stages. Traditional surgical resection and radiotherapy have very limited clinical benefits. The objective of this study was to develop and evaluate a targeted therapy, antibody-drug conjugate (ADC), for NSCLC treatment. Specifically, the CD276 receptor was evaluated and confirmed as an ideal surface target of NSCLC in the immunohistochemistry (IHC) staining of seventy-three patient tumor microarrays and western blotting analysis of eight cell lines. Our anti-CD276 monoclonal antibody (mAb) with cross-activity to both human and mouse receptors showed high surface binding, effective drug delivery and tumor-specific targeting in flow cytometry, confocal microscopy, and in vivo imaging system analysis. The ADC constructed with our CD276 mAb and payload monomethyl auristatin F (MMAF) showed high anti-NSCLC cytotoxicity to multiple lines and effective anti-tumor efficacy in both immunocompromised and immunocompetent NSCLC xenograft mouse models. The brief mechanism study revealed the integration of cell proliferation inhibition and immune cell reactivation in tumor microenvironments. The toxicity study did not detect off-target immune toxicity or peripheral toxicity. Altogether, this study suggested that anti-CD276 ADC could be a promising candidate for NSCLC treatment.

Transcriptome Profiling of A549 Xenografts of Nonsmall-cell Lung Cancer Treated with Qing-Re-Huo-Xue Formula.

Lung cancer is one of the most common malignant tumors, and non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cases. Chinese herbal formula Qing-Re-Huo-Xue (QRHXF) has shown antitumor effects in the NSCLC xenograft mouse model of Lewis cells. However, the molecular mechanisms underlying the antitumor effects of QRHXF remain unknown. In this study, an A549 xenograft mouse model was established, and the mice were then treated with QRHXF or vehicle through oral gavage. Tumor growth was monitored. Tumors were subsequently harvested, and RNA sequencing was performed. Compared with the control group, mice treated with QRHXF showed smaller tumor size and slower tumor growth. RNA sequencing results indicated 36 differentially expressed genes between QRHXF treated and control groups. 16 upregulated and 20 downregulated genes were identified. Enrichment analysis showed four differential expression genes (DEGs) related to tumor growth pathways RASAL2, SerpinB5, UTG1A4, and PDE3A. In conclusion, this study revealed that QRHXF could inhibit tumor growth in an A549 xenograft mouse model, and the target genes of QRHXF may include PDE3A, RASAL2, SERPIB5, and UTG1A4.

Long noncoding RNA regulatory factor X3- antisense RNA 1 promotes non-small cell lung cancer via the microRNA-577/signal transducer and activator of transcription 3 axis

ABSTRACT Lung cancer is the most frequent malignancy, and non-small cell lung cancer (NSCLC) is its most common pathological type. Molecular targeted therapy has been testified to be effective in intervening in the occurrence and development of malignancies. This study investigates the effect of lncRNA Regulatory Factor X3- antisense RNA 1 (RFX3-AS1) in NSCLC progression. The RFX3-AS1 profile in NSCLC tissues and cells was measured by quantitative reverse transcription PCR (qRT-PCR). The RFX3-AS1 overexpression model was constructed. The cell counting kit-8 (CCK-8) experiment and cell colony formation assay were adopted to test cell viability. The cell apoptosis was determined by flow cytometry (FCM). Cell migration and invasion were monitored by the Transwell assay, and Western blot was implemented to verify the protein profiles of signal transducer and activator of transcription 3 (STAT3), E-cadherin, Vimentin and N-cadherin. In vivo, we validated the impact of RFX3-AS1 overexpression on the NSCLC xenograft mouse model. The targeting relationships between RFX3-AS1 and miR-577, miR-577 and STAT3 were confirmed by the dual-luciferase reporter assay. The results manifested that overexpressing RFX3-AS1 markedly facilitated NSCLC cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT), and suppressed cell apoptosis. In contrast, miR-577, which was a downstream target of RFX3-AS1, dramatically impeded the malignant biological behaviors of NSCLC cells. STAT3 was a direct target of miR-577, and it was negatively regulated by the latter. STAT3 activation reversed miR-577-mediated anti-tumor roles. In brief, RFX3-AS1 aggravated NSCLC progression by regulating the miR-577/STAT3 axis.

Anti-PD-L1 and anti-CD73 combination therapy promotes T cell response to EGFR-mutated NSCLC.

Treatment with anti–PD-1 and anti-PD-L1 therapies has shown durable clinical benefit in non–small cell lung cancer (NSCLC). However, patients with NSCLC with epidermal growth factor receptor (EGFR) mutations do not respond as well to treatment as patients without an EGFR mutation. We show that EGFR-mutated NSCLC expressed higher levels of CD73 compared with EGFR WT tumors and that CD73 expression was regulated by EGFR signaling. EGFR-mutated cell lines were significantly more resistant to T cell killing compared with WT cell lines through suppression of T cell proliferation and function. In a xenograft mouse model of EGFR-mutated NSCLC, neither anti–PD-L1 nor anti-CD73 antibody alone inhibited tumor growth compared with the isotype control. In contrast, the combination of both antibodies significantly inhibited tumor growth, increased the number of tumor-infiltrating CD8+ T cells, and enhanced IFN-γ and TNF-α production of these T cells. Consistently, there were increases in gene expression that corresponded to inflammation and T cell function in tumors treated with the combination of anti–PD-L1 and anti-CD73. Together, these results further support the combination of anti-CD73 and anti–PD-L1 therapies in treating EGFR-mutated NSCLC, while suggesting that increased T cell activity may play a role in response to therapy.

GRECO-1: Phase 1/2 Study of Stereotactic Body Radiation Therapy (SBRT) With or Without GC4711 for Early Stage, Centrally Located or Large Non-Small Cell Lung Cancer (NSCLC)

<h3>Purpose/Objective(s)</h3> The number of patients diagnosed with early-stage NSCLC has been increasing with screening programs and more frequent thoracic imaging. Many patients are not ideal surgical candidates and SBRT offers an effective alternative. SBRT for larger and/or centrally located tumors is described with higher risk of toxicity and poorer local control (Timmerman 2006). Improving the safety and efficacy of SBRT with GC4711 may be of significant value in today's NSCLC management for all patients. GC4711 converts superoxide to hydrogen peroxide and has the potential to act as a radiation response modifier, increasing both tumor control and safety of SBRT. In preclinical models, the addition of selective dismutase mimetics to SBRT regimens enhanced tumor control while also protecting normal tissues from radiation damage (Sishc, AACR 2018). Consistent with this, GC4711 augmented the anti-tumor activity of SBRT in NSCLC xenograft mouse models. <h3>Materials/Methods</h3> This Phase 1/2 trial is designed to test the safety of GC4711 and its potential to reduce lung injury due to SBRT and improve tumor control for early stage cT1c-3N0M0 peripheral or centrally located (within 2cm of the proximal bronchial tree) NSCLC. An open-label, Phase 1 cohort of 5 subjects receiving GC4711, 100 mg IV (15 min), prior to each of 5 consecutive daily (M-F) SBRT fractions of 10-12Gy (Bezjak 2019) will be followed by a placebo-controlled, double-blind Phase 2 cohort of approximately 66 subjects randomized to either GC4711 or placebo concurrent with SBRT. For the primary endpoint, decline in diffusion capacity of carbon monoxide at 6 months will be compared to baseline (Stone 2015). All subjects will be followed for 12 months to report clinical (CTCAE 5.0) and radiographic pneumonitis, measured at the same time-points as quarterly lung function tests, other adverse effects, tumor control (RECIST 1.1), survival and quality of life (FACT-L). Exploratory correlative studies include ctDNA and immune profiling. <h3>Results</h3> Trial-in-progress NCT04476797 <h3>Conclusion</h3> GC4711 may improve the benefit-risk ratio of 5-fraction SBRT in early-stage NSCLC; improving tumor control while reducing the risk of lung injury.

Depletion of circ-BIRC6, a circular RNA, suppresses non-small cell lung cancer progression by targeting miR-4491.

Circular RNAs (circRNAs) are non-coding RNAs molecules consisting of a covalently closed continuous loop which have no 5'-3' polarity and contain no polyA tail. Accumulating evidence demonstrates that circRNAs are involved in the initiation and progression of human malignancies. In this study, we explored the expression profile and regulatory role of circ-baculoviral IAP repeat-containing 6 (circ-BIRC6), a circular RNA, in malignant behaviors in non-small cell lung cancer (NSCLC). Expression levels of circ-BIRC6 and miR-4491 were examined in NSCLC patient samples and cell lines using quantitative real time PCR (qRT-PCR). In vitro roles of circ-BIRC6 knockdown on cell viability, colony formation, and apoptosis were assessed using the CCK-8, colony formation assay, and flow cytometry, respectively. The interactions between circ-BIRC6 and miR-449 were assessed using luciferase reporter and qRT-PCR assays. The in vivo role of circ-BIRC6 knockdown on tumor growth and apoptosis was evaluated in a xenograft mouse model of NSCLC. We found that expression levels of circ-BIRC6 in NSCLC patient samples and cell lines were elevated. Small interfering RNA (siRNA)-mediated circ-BIRC6 knockdown suppressed cell proliferation, colony formation, migration and invasion, and promoted apoptosis in NCI-H460 and A549 cells. In addition, miR-4491 was identified as a tumor-suppressor miRNA in NSCLC and circ-BIRC6 functions as a molecular sponge for miR-4491. Furthermore, circ-BIRC6 knockdown suppressed Wnt2B/β-catenin pathway. In vivo assay showed that depletion of circ-BIRC6 suppressed tumor growth, enhanced apoptosis, and decreased miR-4491 levels in a mouse xenograft model. These findings demonstrate that circ-BIRC6 functions as a critical regulator of proliferation and apoptosis via binding to and negatively regulating miR-4491, suggesting that circ-BIRC6 might be a potential target for treatment of NSCLC.

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

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

MiR-101-3p sensitizes non-small cell lung cancer cells to irradiation.

Recent studies have revealed that microRNAs regulate radiosensitivity of non-small cell lung cancer (NSCLC). The aim of this study was to investigate whether miR-101-3p is correlated with radiosensitivity of NSCLC. According to our results, miR-101-3p was downregulated in NSCLC tissues and cell lines. Moreover, miR-101-3p was decreased in A549 cells’ response to irradiation in a dose-dependent manner. Upregulation of miR-101-3p decreased survival fraction and colony formation rate and increased irradiation-induced apoptosis in irradiation-resistant cells, while miR-101-3p depletion had the opposite effects in irradiation-sensitive cells. Furthermore, mechanistic target of rapamycin (mTOR) is a target gene of miR-101-3p. The expressions of mTOR, p-mTOR, and p-S6 were curbed by overexpression of miR-101-3p in A549R cells, which was enhanced by repression of miR-101-3p in A549 cells. Intriguingly, elevation in mTOR abated miR-101-3p upregulation-induced increase in irradiation sensitivity in irradiation-resistant cell line. In contrast, rapamycin undermined miR-101-3p inhibitor-mediated reduction of irradiation sensitivity in irradiation-sensitive cell line. Besides, miR-101-3p overexpression enhanced the efficacy of radiation in an NSCLC xenograft mouse model. In conclusion, miR-101-3p sensitized A549 cells to irradiation via inhibition of mTOR-signaling pathway.

MIER3 suppresses the progression of non-small cell lung cancer by inhibiting Wnt/β-Catenin pathway and histone acetyltransferase activity.

BackgroundThe mesoderm induction early response 1, family member 3 (MIER3) gene has been recognized as potentially being associated with cancer. However, in relation to the development of non-small cell lung cancer (NSCLC), the expression pattern and the role of MIER3 are yet to be reported. The aim of this research was to investigate the rate of expression of MIER3 in NSCLC cells and tissues and to investigate the role of MIER3 in NSCLC.MethodsSeventeen patients received NSCLC tissues and corresponding healthy tissues. MTT assay was used to determine cell proliferation. For detecting mRNA and protein expression, we used both quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot method. To measure cell apoptosis and cell cycle distribution, we applied the flow cytometry technique. We used a wound-healing assay and a Transwell invasion assay to study cell migration and invasion.ResultsIn comparison with adjacent normal tissues, the expression of MIER3 was down-regulated in NSCLC tissues. In addition, the level of MIER3 in NSCLC cell lines was also lower than in pulmonary epithelial cell BEAS-2B. Moreover, when MIER3 was overexpressed, cell proliferation, migration, and invasion were significantly inhibited, apoptosis increased, and cell cycle arrest was induced in A549 and H460 cells. MIER3 overexpression also suppressed tumor growth in NSCLC xenograft mouse models. Furthermore, our study demonstrated that MIER3 down-regulated the Wnt/β-catenin signaling pathway in NSCLC cells. More importantly, MIER3 decreased the activity of histone acetyltransferase (HAT) p300, which may have contributed to its regulation on β-catenin and tumorigenesis.ConclusionsThe data suggests MIER3 takes on the tumor-suppressor role in the progression of NSCLC and, therefore, could prove to be a valuable clinical marker in the prognosis of the disease.

Fluzoparib increases radiation sensitivity of non-small cell lung cancer (NSCLC) cells without BRCA1/2 mutation, a novel PARP1 inhibitor undergoing clinical trials

Poly (ADP-ribose) polymerase 1 inhibitors were originally investigated as anti-cancer therapeutics with BRCA1/2 genes mutation. Here, we investigate the effectiveness of a novel PARP1 inhibitor fluzoparib, for enhancing the radiation sensitivity of NSCLC cells lacking BRCA1/2 mutation. We used MTS assays, western blotting, colony formation assays, immunofluorescence staining, and flow cytometry to evaluate the radiosensitization of NSCLC cells to fluzoparib and explore the underlying mechanisms in vitro. Through BRCA1 and RAD50 genes knockdown, we established dysfunctional homologous recombination (HR) DNA repair pathway models in NSCLC cells. We next investigated the radiosensitization effect of fluzoparib in vivo using human NSCLC xenograft models in mice. The expression of PARP1 and BRCA1 in human NSCLC tumor samples was measured by immunohistochemistry. Furthermore, we sequenced HR-related gene mutations and analyzed their frequencies in advanced NSCLC. In vitro experiments in NSCLC cell lines along with in vivo experiments using an NSCLC xenograft mouse model demonstrated the radiosensitization effect of fluzoparib. The underlying mechanisms involved increased apoptosis, cell-cycle arrest, enhanced irradiation-induced DNA damage, and delayed DNA-damage repair. Immunohistochemical staining showed no correlation between the expression of PARP1 and BRCA1. Moreover, our sequencing results revealed high mutation frequencies for the BRCA1/2, CHEK2, ATR, and RAD50 genes. The potential therapeutic value of fluzoparib for increasing the radiation sensitivity of NSCLC is well confirmed. Moreover, our findings of high mutation frequencies among HR genes suggest that PARP1 inhibition may be an effective treatment strategy for advanced non-small cell lung cancer patients.

DS-1205b, a novel selective inhibitor of AXL kinase, blocks resistance to EGFR-tyrosine kinase inhibitors in a non-small cell lung cancer xenograft model

The AXL receptor tyrosine kinase is involved in signal transduction in malignant cells. Recent studies have shown that the AXL upregulation underlies epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) resistance in EGFR-mutant non-small cell lung cancer (NSCLC). In this study, we investigated the effect of DS-1205b, a novel and selective inhibitor of AXL, on tumor growth and resistance to EGFR TKIs. In AXL-overexpressing NIH3T3 cells, DS-1205b potently inhibited hGAS6 ligand-induced migration in vitro and exerted significant antitumor activity in vivo. AXL was upregulated by long-term erlotinib or osimertinib treatment in HCC827 EGFR-mutant NSCLC cells, and DS-1205b treatment in combination with osimertinib or erlotinib effectively inhibited signaling downstream of EGFR in a cell-based assay. In an HCC827 EGFR-mutant NSCLC xenograft mouse model, combination treatment with DS-1205b and erlotinib significantly delayed the onset of tumor resistance compared to erlotinib monotherapy, and DS-1205b restored the antitumor activity of erlotinib in erlotinib-resistant tumors. DS-1205b also delayed the onset of resistance when used in combination with osimertinib in the model. These findings strongly suggest that DS-1205b can prolong the therapeutic benefit of EGFR TKIs in nonclinical as well as clinical settings.

Arsenic circumvents the gefitinib resistance by binding to P62 and mediating autophagic degradation of EGFR in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is characterized by hyperexpression and/or gain-of-function mutations of the epidermal growth factor receptor (EGFR), resulting in an elevated overall kinase activity. Gefitinib is remarkably effective in patients with the L858R or ΔE746-A750-mutated of EGFR. However, drug resistance tends to develop because of the emergence of T790M mutation on EGFR. New strategies other than repressing kinase activity are thus required to treat NSCLC, thereby circumventing the resistance. In this study, arsenic trioxide (ATO) at 2 μM significantly inhibited the proliferation of the gefitinib-resistant NCI-H1975 cells of the EGFR L858R/T790M mutant compared with a modest inhibition in the gefitinib-sensitive HCC827 cells of ΔE746-A750 mutant and A549 cells of wild-type EGFR. Moreover, ATO significantly inhibited the overall kinase activity of EGFR primarily through quantitatively diminishing the EGFR in NCI-H1975 cells to an extent comparable with that reached by gefitinib in HCC827 cells. Furthermore, ATO promoted autophagic degradation of EGFR in NSCLC cells by directly binding to P62, which interacted with EGFR, preferentially the L858R/T790M mutant providing a plausible explanation for a more favorable effect of ATO on NCI-H1975 cells. Accordingly, the effect of ATO was further confirmed in the NSCLC xenograft mouse models. Our results reveal a new target for ATO with a unique molecular mechanism, i.e., ATO suppresses the overall catalytic potential of EGFR, significantly those with the L858R/T790M mutant in NCI-H1975 cells, through an autophagic degradation by interacting with P62. This study potentially offers an innovative therapeutic avenue for the NSCLC with L858R/T790M-mutated EGFR.

MiR-99a Enhances the Radiation Sensitivity of Non-Small Cell Lung Cancer by Targeting mTOR

Background/Aims: Radiation therapy is an important and effective modality for the treatment of non-small cell lung cancer (NSCLC). MicroRNAs (miRNAs) are crucial post-transcriptional regulators that are involved in numerous important biologic processes. However, their potential involvement in radiation sensitivity remains unknown. Materials: We performed integrated analysis of miRNA expression in NSCLC using The Cancer Genome Atlas datasets. miR-99a was found to be significantly upregulated in cancer tissue and regulated cell survival. Cell culture was used to assess the role of miR-99a in radiation sensitivity. We then used flow cytometry to examine the effects of miR-99a on the cell cycle and apoptosis in cells exposed to radiation. To identify gene targets of miR-99a, a bioinformatics approach was adopted, and the findings of this analysis were verified using luciferase reporter assays. Finally, an in vivo study was conducted to examine the effect of miR-99a on tumor volume in an NSCLC mouse model undergoing radiation therapy. Results: miR-99a was significantly upregulated in radiation-sensitive A549 cells compared with radiation-resistant A549 cells. miR-99a overexpression was shown to enhance radiosensitivity, while inhibition of miR-99a resulted in radioresistance of NSCLC cell lines in vitro and in vivo. In addition, by bioinformatics software analysis and luciferase assays, mammalian target of rapamycin (mTOR) was identified as a direct target of miR-99a. Furthermore, AZD2014, an inhibitor of mTOR, enhanced radiosensitivity and apoptosis in NSCLC cell lines, while mTOR overexpression resulted in radioresistance and cell survival from miR-99a-induced cell apoptosis. Moreover, miR-99a overexpression further increased the efficacy of radiation therapy in an NSCLC xenograft mouse model, and miR-99a and mTOR expression was significantly inversely correlated. Conclusions: Altogether, these data suggested miR-99a functions as a tumor suppressor that has a critical role in regulating radiosensitivity of NSCLC by targeting the mTOR signaling pathway.

Inhibition of histone deacetylases sensitizes EGF receptor-TK inhibitor-resistant non-small-cell lung cancer cells to erlotinib in vitro and in vivo.

Intrinsic and/or acquired resistance of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) commonly occurs in patients with non-small-cell lung cancer (NSCLC). Here, we developed a combined therapy of histone deacetylase inhibition by a novel HDAC inhibitor, YF454A, with erlotinib to overcome EGFR-TKI resistance in NSCLC. The sensitization of the effects of erlotinib by YF454A was examined in a panel of EGFR-TKI-resistant NSCLC cell lines in vitro and two different erlotinib-resistant NSCLC xenograft mouse models in vivo. Western blotting and Affymetrix GeneChip expression analysis were further performed to determine the underlying mechanisms for the effects of the combination of erlotinib and YF454A. YF454A and erlotinib showed a strong synergy in the suppression of cell growth by blocking the cell cycle and triggering cell apoptosis in EGFR-TKI-resistant NSCLC cells. The combined treatment led to a significant decrease in tumour growth and tumour weight compared with single agents alone. Mechanistically, this combination therapy dramatically down-regulated the expression of several crucial EGFR-TKI resistance-related receptor tyrosine kinases, such as Her2, c-Met, IGF1R and AXL, at both the transcriptional and protein levels and consequently blocked the activation of downstream molecules Akt and ERK. Transcriptomic profiling analysis further revealed that YF454A and erlotinib synergistically suppressed the cell cycle pathway and decreased the transcription of cell-cycle related genes, such as MSH6 and MCM7. Our preclinical study of YF454A provides a rationale for combining erlotinib with a histone deacetylase inhibitor to treat NSCLC with EGFR-TKI resistance.