Cell Lung Cancer Progression Research Articles

MicroRNA-4651 targets bromodomain-containing protein 4 to inhibit non-small cell lung cancer cell progression.

Bromodomain-containing protein 4 (BRD4) overexpression in non-small cell lung cancer (NSCLC) promotes cancer progression. Here, we show that miR-4651 selectively targets and negatively regulates BRD4 in A549 and primary human NSCLC cells. RNA pull-down experiments confirmed that miR-4651 directly binds to BRD4 mRNA. Further, ectopic overexpression of miR-4651 in A549cells and primary NSCLC cells decreased BRD4 3'-UTR luciferase reporter activity and its expression, whereas miR-4651 inhibition elevated both. Functional studies demonstrated that NSCLC cell growth, proliferation, and migration were suppressed with ectopic miR-4651 overexpression but enhanced with miR-4651 inhibition. BRD4 re-expression using a 3'-UTR mutant BRD4 reversed A549cell inhibition induced by miR-4651 overexpression. Further, miR-4651 overexpression or inhibition failed to alter the functions of BRD4-KO A549cells. In vivo, miR-4651-overexpressing A549 xenografts grew slowly than control A549 xenografts in severe combined immunodeficient mice. Finally, miR-4651 was downregulated in human NSCLC tissues, correlating with BRD4 elevation. Together, miR-4651 targets BRD4 to inhibit NSCLC cell growth in vitro and in vivo.

LncRNA HOXB-AS3 exacerbates proliferation, migration, and invasion of lung cancer via activating the PI3K-AKT pathway.

Lung cancer remains the leading cause of cancer-related death all over the world. In spite of the great advances made in surgery and chemotherapy, the prognosis of lung cancer patients is poor. A substantial fraction of long noncoding RNAs (lncRNAs) can regulate various cancers. A recent study has reported that lncRNA HOXB-AS3 plays a critical role in cancers. However, its biological function remains unclear in lung cancer progression. In the current research, we found HOXB-AS3 was obviously elevated in NSCLC tissues and cells. Functional assays showed that inhibition of HOXB-AS3 was able to repress A549 and H1975 cell proliferation, cell colony formation ability and meanwhile, triggered cell apoptosis. Furthermore, the lung cancer cell cycle was mostly blocked in the G1 phase whereas the cell ratio in the S phase was reduced. Also, A549 and H1975 cell migration and invasion capacity were significantly repressed by the loss of HOXB-AS3. The PI3K/AKT pathway has been implicated in the carcinogenesis of multiple cancers. Here, we displayed that inhibition of HOXB-AS3 suppressed lung cancer cell progression via inactivating the PI3K/AKT pathway. Subsequently, in vivo experiments were utilized in our study and it was demonstrated that HOXB-AS3 contributed to lung cancer tumor growth via modulating the PI3K/AKT pathway. Overall, we implied that HOXB-AS3 might provide a new perspective for lung cancer treatment via targeting PI3K/AKT.

Nm23-H1 inhibits lung cancer bone-specific metastasis by upregulating miR-660-5p targeted SMARCA5.

BackgroundNm23‐H1 gene has been found to be an inhibitor of tumor metastasis in lung cancer. MicroRNAs (miRNAs) play key roles in tumor metastasis through multiple signaling pathways. This study explored whether the nm23‐H1 gene could inhibit invasion and metastasis of lung cancer cells by regulating miRNA‐660‐5p targets.MethodsQuantitative real‐time PCR (qRT‐PCR) and western blots were used to measure the expression of nm23‐H1 and miR‐660‐5p of various human lung cancer cell lines. Cell counting kit‐8 (CCK‐8), wound‐healing and transwell assay were carried out to assess cell proliferation, migration and invasion of each cell line. Xenograft were applied to determine in vivo effects of miR‐660‐5p among nude mice. Luciferase assay and western blot were performed to determine the target gene of miR‐660‐5p.ResultsWe found that high expression of nm23‐H1 correlated with decreased miRNA‐660‐5p expression. Inhibiting miR‐660‐5p suppressed lung cancer cells progression significantly in vitro, whereas overexpression of miR‐660‐5p facilitated tumor growth and bone metastasis in vivo. In addition, as the potential target gene of miR‐660‐5p, SMARCA5 overexpression in vitro suppressed tumor progression and osteolytic metastasis associated RANKL signaling, which is congruent with the effect of nm23‐H1 on the lung cancer cells.ConclusionNm23‐H1 inhibits tumor progression and bone‐specific metastasis of lung cancer by regulating miR‐660‐5p/SMARCA5/RANKL axis, which indicates the related genes may serve as potential targets for the treatment of human lung cancer.Key pointsSignificant findings of the studyHigh expression of nm23‐H1 correlated with decreased miRNA‐660‐5p expression. Further, downregulation of miR‐660‐5p significantly suppressed the tumor progression and bone‐specific metastasis of lung cancer cells.What this study addsThis is the first study to show an inverse association between nm23‐H1 and miR‐660‐5p, and confirm that nm23‐H1 inhibits tumor progression and bone‐specific metastasis of lung cancer by regulating miR‐660‐5p/SMARCA5/RANKL axis.

Effective targeting of the ubiquitin-like modifier NEDD8 for lung adenocarcinoma treatment.

Protein neddylation, a process of conjugating neural precursor cell expressed, developmentally downregulated 8 (NEDD8) to substrates, plays a tumor-promoting role in lung carcinogenesis. Our previous study showed MLN4924, an inhibitor of NEDD8 activating enzyme (E1), significantly inhibits the growth of multiple cancer cells. However, resistance can develop to MLN4924 by mutation. Therefore, it is important to further understand how NEDD8 acts in lung cancer. In the present study, we demonstrated NEDD8 is overactivated in lung cancers and confers a worse patient overall survival. Furthermore, we report that in lung adenocarcinoma cells, NEDD8 depletion significantly suppressed lung cancer cell growth and progression both in vitro and in vivo. Mechanistic studies revealed that NEDD8 depletion induced the accumulation of a panel of tumor-suppressive cullin-RING ubiquitin ligase substrates (e.g., p21, p27, and Wee1) via blocking their degradation, triggering cell cycle arrest at G2 phase, thus inducing apoptosis or senescence in a cell-line-dependent manner. The present study demonstrates the role of NEDD8 in regulating the malignant phenotypes of lung cancer cells and further validates NEDD8 as a potential therapeutic target in lung cancer.

MiR‐148b suppressed non‐small cell lung cancer progression via inhibiting ALCAM through the NF‐κB signaling pathway

BackgroundNon‐small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. MiRNAs are recognized as important molecules in cancer biology. The aim of the study was to identify a novel biomarker miR‐148b and its mechanism in the modulation of NSCLC progression.MethodsThe expressional level of miR‐148b was analyzed by RT‐PCR. The effect of miR‐4317 on proliferation was evaluated through 3‐(4,5‐Dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2Htetrazolium bromide (MTT) assay. The effect of miR‐148b on the metastasis of NSCLC was detected through transwell assays. The verification of the target of miR‐148b was assessed by TargetScan and dual‐luciferase reporter assay. The related proteins in this study were analyzed by western blot.ResultsOur findings confirmed that miR‐148b was decreased in NSCLC and NSCLC patients with lower expression exhibited poorer overall survival (OS). Increasing miR‐148b significantly repressed proliferation, invasion and migration. More importantly, activated leukocyte cell adhesion molecule (ALCAM) was determined as the direct target of miR‐148b, and reintroduction of ALCAM attenuated miR‐148b effect on the progress of NSCLC. In addition, NF‐κB signaling pathway was modulated by miR‐148b/ALCAM axis.ConclusionsOur results indicated that miR‐148b is able to suppress NSCLC growth and metastasis via targeting ALCAM through the NF‐κB pathway. These findings provided new evidence that miR‐148b serves as a potential biomarker and novel target for NSCLC treatment.

STK31 regulates the proliferation and cell cycle of lung cancer cells via the Wnt/β‑catenin pathway and feedback regulation by c‑myc.

Lung cancer, which is a leading cause of cancer-related deaths, is diagnosed at a male to female ratio of 2.1:1. Serine-threonine kinase 31 (STK31) is a novel cancer/testis (CT)-related gene that is highly expressed in several types of cancers, such as lung and colorectal cancer, and plays crucial roles in cancer. In the present study, increased expression of STK31 and β-catenin was observed in lung cancer tissues and cell lines. Downregulation of STK31 expression in lung cancer cells significantly inhibited their proliferation by arresting the cell cycle in the G1 phase concurrent with decreased β-catenin, c-myc and cyclin D1 protein levels, while upregulation of STK31 had the opposite effects. In addition, STK31-induced lung cancer cell viability, proliferation, cell cycle progression, and expression of related genes were completely attenuated by a Wnt/β-catenin inhibitor (XAV939). Similar to XAV939, a c-myc inhibitor (10058-F4) also significantly attenuated STK31-induced proliferation and cell cycle progression in lung cancer cells. Inhibiting c-myc and TRRAP significantly decreased the expression of STK31, and a chromatin immunoprecipitation (ChIP) assay confirmed that c-myc directly bound to the STK31 promoter. These results indicated that STK31 may act as an oncogene in lung cancer and that c-myc may be the transcription factor that promotes STK31 expression. Moreover, the results suggested that c-myc can also regulate STK31 expression in a positive feedback loop, and the downregulation of STK31 in lung cancer cells had an inhibitory effect on cell viability, cell proliferation and cell cycle progression, likely by inactivating the Wnt/β-catenin pathway and positive feedback regulation by c-myc.

Hsa_circ_0062389 promotes the progression of non-small cell lung cancer by sponging miR-103a-3p to mediate CCNE1 expression

Recently, increasing evidence showed that circular RNAs (circRNAs) play critical roles in tumor progression. However, the roles of hsa_circ_0062389 in non-small cell lung cancer (NSCLC) development remain unclear. In the present study, hsa_circ_0062389 expression was significantly increased in NSCLC tissues and cell lines. High hsa_circ_0062389 expression was associated with advanced TNM stage and lymph-node metastasis. Function assays showed that hsa_circ_0062389 suppression reduced NSCLC cells proliferation and arrested cell cycle in G0/G1 phase. In mechanism, hsa_circ_0062389 directly interacted with miR-103a-3p in NSCLC, and CCNE1 acted as a target of miR-103a-3p. Furthermore, rescue assays showed that miR-103a-3p suppression or CCNE1 overexpression abolished the effects of hsa_circ_0062389 suppression on lung cancer cells progression. Therefore, our results showed that the hsa_circ_0062389/miR-103a-3p/CCNE1 axis might contribute to the tumorigenesis of NSCLC, which provided a new strategy for cancer treatment.

Follistatin-like Protein 1 Inhibits Lung Cancer Metastasis by Preventing Proteolytic Activation of Osteopontin.

Follistatin-like protein 1 (FSTL1) plays a critical role in lung organogenesis, but is downregulated during lung cancer development and progression. The prognostic significance and functional consequences of FSTL1 downregulation in lung cancer are unclear. Here, reduced levels of FSTL1 were detected in various tumors compared with normal tissues and were associated with poor clinical outcome in patients with non-small cell lung cancer, particularly those with lung adenocarcinoma. FSTL1 expression negatively correlated with the metastatic potential of lung cancer cells. Antibody-based neutralization of extracellular FSTL1 increased cellular migration/invasion while addition of recombinant FSTL1 protein diminished the metastatic capacity of lung cancer cells in vitro and in vivo. Notably, treatment with FSTL1 effectively prevented the metastatic progression of lung cancer cells in an orthotopic animal model. Mechanistically, FSTL1 directly bound to the proform of secreted phosphoprotein 1 (SPP1)/osteopontin, restraining proteolytic activation of SPP1, which led to inactivation of integrin/CD44-associated signaling and rearrangement of the actin cytoskeleton. Combined low expression of FSTL1 and high expression of SPP1 predicted a poorer prognosis for patients with lung cancer. This study highlights the novel interaction between FSTL1 and SPP1 and new opportunities to effectively target SPP1-driven metastatic cancers characterized by FSTL1 downregulation. SIGNIFICANCE: These findings describe the novel interaction between FSTL1 and SPP1 and its role in the metastatic progression of lung adenocarcinoma.

Adaptive Protein Translation by the Integrated Stress Response Maintains the Proliferative and Migratory Capacity of Lung Adenocarcinoma Cells.

The integrated stress response (ISR) is a conserved pathway that is activated by cells that are exposed to stress. In lung adenocarcinoma, activation of the ATF4 branch of the ISR by certain oncogenic mutations has been linked to the regulation of amino acid metabolism. In the present study, we provide evidence for ATF4 activation across multiple stages and molecular subtypes of human lung adenocarcinoma. In response to extracellular amino acid limitation, lung adenocarcinoma cells with diverse genotypes commonly induce ATF4 in an eIF2α-dependent manner, which can be blocked pharmacologically using an ISR inhibitor. Although suppressing eIF2α or ATF4 can trigger different biological consequences, adaptive cell-cycle progression and cell migration are particularly sensitive to inhibition of the ISR. These phenotypes require the ATF4 target gene asparagine synthetase (ASNS), which maintains protein translation independently of the mTOR/PI3K pathway. Moreover, NRF2 protein levels and oxidative stress can be modulated by the ISR downstream of ASNS. Finally, we demonstrate that ASNS controls the biosynthesis of select proteins, including the cell-cycle regulator cyclin B1, which are associated with poor lung adenocarcinoma patient outcome. Our findings uncover new regulatory layers of the ISR pathway and its control of proteostasis in lung cancer cells. IMPLICATIONS: We reveal novel regulatory mechanisms by which the ISR controls selective protein translation and is required for cell-cycle progression and migration of lung cancer cells.

Huaier extract suppresses non-small cell lung cancer progression through activating NLRP3-dependent pyroptosis.

Recent studies have reported the anticancer activity of huaier extract in various human malignancies. However, little is known about the effect of huaier extract in non-small cell lung cancer (NSCLC) and its underlying mechanism. The current study aimed to investigate whether huaier extract affects the progression of NSCLC. mRNA and proteins expression of pyroptotic-related genes (NLRP3, caspase-1, IL-1β, and IL-18) in NSCLC tissues and cells were, respectively, detected by qRT-PCR and western blot. The effects of huaier extract on NSCLC cell viability and cytotoxicity were evaluated by CCK-8 assay, colony formation assay, and LDH detection kit. Besides, we established a xenograft model to assess the antitumor effect of huaier extract on tumor growth in vivo. Our results showed that the expression of pyroptotic-related genes was downregulated in NSCLC tissues and cell lines. Huaier extract pretreatment inhibited cell viability and the percentage of colony formation of H520 and H358 cells, and upregulated the expression of pyroptotic-related genes. Mechanistically, huaier extract exhibited antitumor effect in NSCLC via inducing NLRP3-dependent pyroptosis in vitro and in vivo. In conclusion, our finding confirmed that huaier extract played an antitumor role in NSCLC progression through promoting pyroptotic cell death, which provided a new potential strategy for NSCLC clinical treatment.

A long non-coding RNA LNBC3 facilitates non-small cell lung cancer progression by stabilizing BCL6.

BackgroundThe non‐small cell lung cancer (NSCLC) is a common malignancy worldwide. Numerous reports have shown the critical role of long non‐coding RNAs (lncRNAs) in NSCLC. However, the role of a novel lncRNA named LNBC3 is still unknown.MethodsBy lncRNA profiling, novel lncRNAs related to NSCLC were identified. LNBC3 expression was quantified by qRT‐PCR. Migration and viability assays were performed to evaluate the function of LNBC3 in vitro. In vivo xenograft model was conducted to determine the oncogenic functions of LNBC3. RNA immunoprecipitation (RIP) followed by mass spectrometry (MS) was utilized to identify BCL6 as LNBC3 binding target.ResultsLNBC3 is markedly overexpressed in tumor tissues and NSCLC cell lines. Higher LNBC3 levels correlated with advanced TNM stages, larger tumor size, and metastasis. LNBC3 promoted NSCLC migration and viability. The in vivo experiments demonstrated that xenograft tumor growth and proliferation were facilitated with increasing LNBC3 levels. The antisense oligonucleotides (ASOs) targeting LNBC3 substantially inhibited lung cancer progression. Mechanistic studies showed that LNBC3 could interact with BCL6 leading to BCL6 stabilization through reduced proteasomal degradation.ConclusionsCollectively, our data have identified a novel lncRNA LNBC3 in NSCLC progression. The LNBC3‐BCL6 axis might be a potential target for pharmaceutical intervention.

YY1-mediated overexpression of long noncoding RNA MCM3AP-AS1 accelerates angiogenesis and progression in lung cancer by targeting miR-340-5p/KPNA4 axis.

Lung cancer is famous as an aggressive malignant tumor and is the main cause of cancer-associated mortality globally. Tumor angiogenesis is a vital part in cancer, which influences cell proliferation and metastasis. Increasing studies have claimed that long noncoding RNAs (lncRNAs) were involved in the progression of several cancers. Based on previous studies, this study focused on the role and mechanism of lncRNA MCM3AP antisense RNA 1 (MCM3AP-AS1) in lung cancer. At first, MCM3AP-AS1 expression was found to be elevated in lung cancer cells. Depletion of MCM3AP-AS1 repressed cell proliferation, migration, and angiogenesis in lung cancer cells. YY1 was confirmed to mediate MCM3AP-AS1 transcription in lung cancer cells. Moreover, the molecular mechanism investigation revealed that MCM3AP-AS1 could sponge miR-340-5p and elevate KPNA4 expression. On the basis of rescue assays, we identified that the overexpression of KPNA4 partly counteracted the suppressed effect of MCM3AP-AS1 knockdown on angiogenesis and progression in lung cancer cells. Conclusively, the YY1-mediated overexpression of MCM3AP-AS1 accelerated angiogenesis and progression in lung cancer by targeting miR-340-5p/KPNA4 axis, which highlighted the possibility of MCM3AP-AS1 as a promising therapeutic target for lung cancer.

RRM2 Regulated By LINC00667/miR-143-3p Signal Is Responsible For Non-Small Cell Lung Cancer Cell Progression.

BackgroundNon-small cell lung cancer (NSCLC) is a common and fatal cancer worldwide with a very low 5-year overall survival rate. Ribonucleotide reductase M2 subunit (RRM2), a small subunit of the ribonucleotide reductase complex, has been found to be an oncogenic role in a variety of tumors including NSCLC. However, the regulatory mechanism of RRM2 in NSCLC is not clear. Increasing evidence suggests that non-coding RNAs (ncRNAs) including miRNAs and lincRNAs may promote or inhibit tumor initiation and development through regulating the expression of oncogenic genes. It is interesting to find ncRNAs which play important role in regulating RRM2 expression.Materials and methodsThe expression levels of RRM2, LINC0066 and miR-143-3p in NSCLC tumor tissues and cell lines were detected using qRT-PCR. The regulatory relationships among RRM2, LINC0066 and miR-143-3p were predicted using database analysis and verified by luciferase reporter assay and RIP analysis. The proliferation ability of NSCLC cells was assessed using CCK8 and colony formation assays. The expression of related proteins was determined by Western blot. In vivo effect of RRM2, LINC0066 and miR-143-3p to NSCLC were detected through xenograft experiments.ResultsIn this study, we found RRM2 was upregulated in NSCLC tumor and cell lines, and the aberrant upregulation predicted a poor prognosis. Then, we predicted and confirmed that RRM2 was negatively regulated by miR-143-3p. Further study implied that LINC00667 acted as a ceRNA by sponging miR-143-3p and regulated RRM2 expression indirectly. Moreover, we found that the growth of NSCLC was regulated by LINC00667/miR-143-3p/RRM2 signal pathway both in vitro and in vivo. LINC00667 and RRM2 promoted the tumor growth while miR-143-3p inhibited it.ConclusionOur study revealed a LINC00667/miR-143-3p/RRM2 signal pathway that played an important role in the progress of NSCLC, which might be potential therapeutic targets for NSCLC.

Dexmedetomidine enhances hypoxia-induced cancer cell progression.

Dexmedetomidine (DEX) is widely used in perioperative settings for analgesia and sedation; however, little is known about its effects on the hypoxia-induced progression of tumor cells. In the present study, the effects of DEX on hypoxia-induced growth and metastasis of lung cancer cells and colorectal cancer cells was examined. A549 cells and HCT116 cells were treated with normoxia, hypoxia, co-treatment of hypoxia and DEX, and atipamezole (an α2 adrenoceptor antagonist) for 4 h. The proliferation rate of cells was determined by MTT assays. Cell metastatic potential was evaluated by Transwell assays. Survivin and hypoxia inducible factor (HIF)-1α were detected by western blotting. Matrix metalloproteinase (MMP)-2 and MMP-9 were measured using reverse transcription-quantitative PCR. It was demonstrated that hypoxia treatment promoted the proliferation and may promote the metastasis of the two cancer cell lines. DEX substantially contributed to the survival and aggressiveness of the two cancer cell lines following hypoxia. Furthermore, DEX upregulated the expression of survivin, MMP-2, MMP-9 and HIF-1α in the two cancer cell lines in response to hypoxia. Finally, the effects of DEX on the hypoxia-induced growth and metastatic potential of cancer cells were reversed by atipamezole. Collectively, DEX enhances the hypoxia-induced progression of lung cancer cells and colorectal cancer cells by regulating HIF-1α signaling, which may be associated with the α2 adrenoceptor pathway.

Downregulation of miR‑138‑5p promotes non‑small cell lung cancer progression by regulating CDK8.

Dysregulation of microRNAs (miRNAs) is frequently observed during cancer development. Aberrant expression of miRNA‑138‑5p (miR‑138‑5p) has been found in many types of cancer. However, the role and the mechanisms underlying miR‑138‑5p function in non‑small cell lung cancer (NSCLC) progression remain unknown. In the present study, miR‑138‑5p expression was identified to be decreased in tumor tissues compared with matched normal tissues from patients with NSCLC. In addition, low expression of miR‑138‑5p was detected in three NSCLC cell lines compared with a normal lung epithelium cell line. Moreover, CDK8 mRNA expression was increased in tumor tissues compared with matched normal tissues from patients with NSCLC, and an inverse association between miR‑138‑5p and CDK8 was observed. Furthermore, CDK8 was predicted to be a target of miR‑138‑5p. Dual luciferase assay confirmed that miR‑138‑5p could directly bind to the 3' untranslated region of CDK8 mRNA. In A549 cells, overexpression of miR‑138‑5p inhibited cell growth and significantly increased cell apoptosis rates and the number of cells in G0/G1 phase. Moreover, forced overexpression of CDK8 partially reversed miR‑138‑5p mimic‑induced cell growth arrest and alteration of cell apoptosis and cell cycle. In conclusion, the present results suggested that miR‑138‑5p may be a tumor suppressor in NSCLC cells and a promising therapeutic target for treating patients with NSCLC.

Long noncoding RNA LINC00657 induced by SP1 contributes to the non-small cell lung cancer progression through targeting miR-26b-5p/COMMD8 axis.

Non-small-cell lung cancer (NSCLC) is a kind of lung cancer with high incidence and poor outcomes all over the world. Studies have validated that the upregulation of long noncoding RNA LINC00657 is related to several cancers. Nevertheless, the underlying regulatory mechanism of LINC00657 in NSCLC has not been well elucidated. In the present study, quantitative reverse-transcription polymerase chain reaction (RT-qPCR) revealed that LINC00657 level was apparently elevated in NSCLC cells. Loss-of function assays demonstrated that LINC00657 silence retarded cell proliferation and migration in NSCLC cells. Moreover, the chromatin immunoprecipitation result identified the transcription factor SP1 could bind with LINC00657 promoter, and RT-qPCR proved SP1 positively regulated LINC00657 expression in NSCLC cells. In addition, the mechanistic investigations unveiled that LINC00657 was an endogenous sponge of miR-26b-5p and therefore boosted the expression of copper metabolism MURR1 domain-containing 8 (COMMD8), one of the targets of miR-26b-5p. Besides, miR-26b-5p could negatively regulate LINC00657 or COMMD8 in NSCLC cells. With the application of rescue assays, we uncovered that overexpression of COMMD8 partly mitigated the impairment of LINC00657 repression on NSCLC cell proliferation and migration. Together, our study illustrated that SP1-stimulated LINC00657 promoted NSCLC progression through targeting miR-26b-5p/COMMD8 axis, offering a novel potential therapeutic target for NSCLC.

AMMECR1 Inhibits Apoptosis and Promotes Cell-cycle Progression and Proliferation of the A549 Human Lung Cancer Cell Line.

The aim of this study was to characterize the role of Alport syndrome, mental retardation, midface hypoplasia, and elliptocytosis chromosomal region gene 1 (AMMECR1) in human lung cancer cell lines. AMMECR1 gene expression was evaluated in four lung cell lines, with A549 then selected for further in-depth examination. To characterize the role of AMMECR1, silencing was achieved utilizing lentivirus-mediated RNA interference, and confirmed by quantitative real-time polymerase chain reaction and western blotting. The impact of AMMECR1 silencing on cellular proliferation was assessed using Celigo-based and MTT assays. Apoptosis was determined using the annexin V-allophycocyanin single staining method. Cell-cycle arrest was assessed by flow cytometry. Finally, colony formation was assessed using Giemsa staining. In A549 cells, AMMECR1 silencing was found to significantly suppress cell proliferation, reduce colony formation, promote apoptosis, and arrest cells in the S and G2/M phases. AMMECR1 plays a critical role in cell proliferation, cell-cycle progression, and apoptosis of human lung cancer cells, and may serve as a potential therapeutic target for non-small-cell lung cancer.

Overexpression of SLFN5 induced the epithelial-mesenchymal transition in human lung cancer cell line A549 through β-catenin/Snail/E-cadherin pathway

Lung cancer is a disease with increasing morbidity worldwide in recent years. Approaches such as chemotherapy and biological targeting for its treatment are urgently needed. Epithelial-mesenchymal transition (EMT) is an important initiation stage for tumor cells to acquire invasive and metastatic abilities. Increasing findings have shown that human schlafen family member 5 (SLFN5) plays a key role in malignant tumors. However, the role of SLFN5 in lung cancer cells is not completely elucidated yet. In this study, overexpression or knockdown of SLFN5 gene were induced by lentiviral transfection in human lung cancer cell line A549, then the EMT of A549 was detected by green fluorescent protein labeling method, the migrative and invasive abilities were evaluated via transwell and wound-healing tests in vitro and chick chorioallantoic membrane inoculation in vivo, and the possible mechanism was studied by quantitative real-time PCR and Western blotting. Our results demonstrated that overexpression of SLFN5 promoted the morphology transformation of A549 from epithelial to mesenchymal, as well as migration and invasion. However, knockdown of SLFN5 resulted in the opposite results. Moreover, with the development of EMT after SLFN5 was overexpressed, A549 exhibited enhanced translocation of β-catenin from membrane to cytoplasm or nucleus, with higher level of EMT-related transcription factor Snail, and lower expression of adhesin E-cadherin. Together these results suggest that SLFN5 may act as a synergist in lung cancer cell tumorigenesis and progression, providing a potential target for developing drugs for lung cancer therapy in future.

Abstract 1108: Intestine-specific homeobox recruits p300/CBP-associated factor and bromodomain-containing protein 4 to promote epithelial-mesenchymal transition

Abstract The epithelial-mesenchymal transition is an important process in the cancer progression, but its occurrence and the regulatory mechanism are not fully understood. Intestine specific homeobox acted both as a proto-oncogene and upstream regulator of EMT markers, by which modulates tumorigenic initiation and progression in lung cancer cells. ISX acetylated by PCAF recruits bromodomain-containing protein 4, to initiate chromatin remodeling, and up-regulated EMT downstream regulators in tumors cells. Ectopic expression of ISX was shown to enhance TWIST1, Snail1 expression by recruiting BRD4 to enhance Pol II dependent transcription, lead in to remodeling of the tumor microenvironment. In NSCLC carcinoma, increased ISX expression was noted correlating with distinct clinical metastatic features and poor prognosis. These results suggest that theISX-BRD4 axis mediates EMT signaling and exerts significant regulatory effects on tumor initiation and metastasis. Citation Format: Li-Ting Wang, Shyh-Shin Chiou, Shih-Hsien Hsu. Intestine-specific homeobox recruits p300/CBP-associated factor and bromodomain-containing protein 4 to promote epithelial-mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1108.

Abstract 1027: Inhibiting pathways predicted from a steroid hormone gene signature yields synergistic antitumor effects in lung cancer

Abstract Mounting preclinical and clinical evidence suggest an involvement of estrogen signaling in promoting non-small cell lung cancer (NSCLC) progression. In estrogen receptor β-positive (ERβ) cases, we previously found seven genes within the prediction analysis of microarrays 50 gene (PAM50) panel that showed strong association in predicting prognosis of NSCLC patients, with Myc, MIA, FGFR4, CXXC5, Grb-7 and FOXC1 being upregulated while PR was downregulated in highly aggressive lung tumors. These genes described one network containing ERβ and human epidermal growth factor receptor 2/3 (HER2/HER3) signaling, which suggests that these two interacting pathways define lung tumors with more aggressive biology. Here, we sought to evaluate the therapeutic potential of combining the pan-HER tyrosine kinase inhibitor, Dacomitinib, and the ER antagonist, Fulvestrant in NSCLC. Cell viability assay in three different human NSCLC cell lines 201T (EGFR wild-type), A549 (K-ras mutant) and HCC827 (EGFR exon 19 deletion) showed strong synergy between Dacomitinib and Fulvestrant, as determined by a combination index < 1. The combination strongly inhibited p-EGFR, p-HER2, p-HER3, p-HER4, and the downstream signaling p-AKT and p-MAPK compared to single treatments. Mechanistically, the transcription activity of activator protein-1 (AP-1) family members Fos and Jun were significantly reduced, as detected by the ability of AP-1 to bind to DNA. Expression of c-Myc and CyclinD1 were also significantly inhibited by the combination. In NSCLC cell lines, the combination reversed the gene signature associated with poor prognosis with c-Myc, MIA, CXXC5, FGFR4, Grb-7, FOXC1 being downregulated while PR was upregulated. Similarly, the AP-1 inhibitor t-5224 mimicked the effects of the combination in reversing the PAM50 gene signature, suggesting that the combination’s effect is largely mediated by AP-1 downregulation. In vivo, the combination also revealed a synergistic effect, measured by the combination ratio method (> 1), and induced tumor regression in 201T and A549 xenografts, with average tumor volume significantly lower than the single treatment groups. The immunohistochemical analysis of the xenografts revealed significant downregulation in Ki67. Western blot analysis of the tumor lysates from the combination treatment groups showed an increase in cleaved caspase-3 and a decrease in both ER and HER signaling. The PAM50 gene signature was also reversed in both xenograft studies detected by real-time-qPCR. Similar effects were seen for c-Myc and PR at the protein levels detected by immunohistochemistry staining. These observations encouragingly support the use of this combination clinically in NSCLC, considering its ability to suppress tumor growth and produce a gene signature that predicts better clinical outcomes, as assessed by the PAM50 genes. Citation Format: Abdulaziz A. Almotlak, Mariya Farooqui, Jill M. Siegfried. Inhibiting pathways predicted from a steroid hormone gene signature yields synergistic antitumor effects in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1027.