Tumor Suppressor In Lung Cancer Research Articles

Long non-coding RNA BRE-AS1 inhibits the proliferation, migration, and invasion of cancer cells in triple-negative breast cancer and predicts patients\u2019 survival by downregulating miR-21

BackgroundBRE-AS1 is a recently identified tumor suppressor in non-small cell lung cancer. It role in other human diseases remains elusive.MethodsDifferential expression of BRE-AS1 in with triple-negative breast cancer (TNBC) patients (n = 74, patient group) and healthy volunteers (n = 58, control group) was studied with RT-qPCR. The direct interaction between BRE-AS1 and premature microRNA-21 (miR-21) was assessed by RNA pull-down assay. The interactions among BRE-AS1, miR-21 and PTEN were evaluated by overexpression assays. CCK-8 assay and Transwell assay were used to evaluate cell behaviors.ResultsBRE-AS1 was downregulated in TNBC, while miR-21 was highly expressed in TNBC. Low expression levels of lncRNA BRE-AS1 and high expression levels of miR-21 were significantly correlated with unfavorable survival outcomes. BRE-AS1 and miRNA-21 were inversely correlated across TNBC samples, not control samples. BRE-AS1 decreased miR-21 expression and increased PTEN expression while miR-21showed no role in BRE-AS1 expression. RNA pull-down assay illustrated that BRE-AS1 may sponge premature miR-21 to suppress it maturation. Overexpression of BRE-AS1 decreased cell behaviors, while overexpression of miR-21 promoted cell behaviors. MiR-21 suppressed the role of BRE-AS1 in cancer cell behaviors.ConclusionTherefore, BRE-AS1 may inhibit TNBC by downregulating miR-21.

GPC5 suppresses lung cancer progression and metastasis via intracellular CTDSP1/AhR/ARNT signaling axis and extracellular exosome secretion.

Lung cancer is the leading cause of cancer-related death worldwide. Glypican-5 (GPC5) is a member of heparan sulfate proteoglycans, and its biological importance in initiation and progression of lung cancer remains controversial. In the present study, we revealed that GPC5 transcriptionally enhanced the expression of CTDSP1 (miR-26b host gene) via AhR-ARNT pathway, and such up-regulation of CTDSP1 intracellularly contributed to the inhibited proliferation of lung cancer cells. Moreover, exosomes derived from GPC5-overexpressing human lung cancer cells (GPC5-OE-derived exosomes) had an extracellular repressive effect on human lymphatic endothelial cells (hLECs), leading to decreased tube formation and migration. Comparison between GPC5-WT- and GPC5-OE-derived exosomes showed that miR-26b (embedded within introns of CTDSP1 gene) was significantly up-regulated in GPC5-OE-derived exosomes and critical to the influence on hLECs. On the mechanism, we demonstrated that miR-26b transferred into hLECs directly targeted to PTK2 3'-UTR and led to PTK2 down-regulation, resulting in defects in tube formation and migration of hLECs. By uncovering the regulation network among GPC5, miR-26b, miR-26b host gene (CTDSP1), and target gene (PTK2), our findings demonstrated that GPC5 functioned as a tumor suppressor in human lung cancer.

SCAMP3 is a mutant EGFR phosphorylation target and a tumor suppressor in lung adenocarcinoma.

Mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase domain constitutively activate EGFR resulting in lung tumorigenesis. Activated EGFR modulates downstream signaling by altering phosphorylation-driven interactions that promote growth and survival. Secretory Carrier Membrane Proteins (SCAMPs) are a family of transmembrane proteins that regulate recycling of receptor proteins, including EGFR. The potential role of SCAMPs in mutant EGFR function and tumorigenesis has not been elucidated. Using quantitative mass-spectrometry based phosphoproteomics, we identified SCAMP3 as a target of mutant EGFRs in lung adenocarcinoma and sought to further investigate the role of SCAMP3 in the regulation of lung tumorigenesis. Here we show that activated EGFR, either directly or indirectly phosphorylates SCAMP3 at Y86 and this phosphorylation increases the interaction of SCAMP3 with both wild type and mutant EGFRs. SCAMP3 knockdown increases lung adenocarcinoma cell survival and increases xenograft tumor growth in vivo, demonstrating a tumor suppressor role of SCAMP3 in lung tumorigenesis. The tumor suppressor function is a result of SCAMP3 promoting EGFR degradation and attenuating MAP kinase signaling pathways. SCAMP3 knockdown also increases multinucleated cells in culture, suggesting that SCAMP3 is required for efficient cytokinesis. The enhanced growth, increased colony formation, reduced EGFR degradation and multinucleation phenotype of SCAMP3 depleted cells were reversed by re-expression of wild type SCAMP3, but not SCAMP3 Y86F, suggesting that Y86 phosphorylation is critical for SCAMP3 function. Taken together, the results of this study demonstrate that SCAMP3 functions as a novel tumor suppressor in lung cancer by modulating EGFR signaling and cytokinesis that is partly Y86 phosphorylation dependent.

Algorithm-Based Meta-Analysis Reveals the Mechanistic Interaction of the Tumor Suppressor LIMD1 With Non-Small-Cell Lung Carcinoma.

Non-small-cell lung carcinoma (NSCLC) is the major type of lung cancer, which is among the leading causes of cancer-related deaths worldwide. LIMD1 was previously identified as a tumor suppressor in lung cancer, but their detailed interaction in this setting remains unclear. In this study, we have carried out multiple genome-wide bioinformatic analyses for a comprehensive understanding of LIMD1 in NSCLC, using various online algorithm platforms that have been built for mega databases derived from both clinical and cell line samples. Our results indicate that LIMD1 expression level is significantly downregulated at both mRNA and protein levels in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), with a considerable contribution from its promoter methylation rather than its gene mutations. The Limd1 gene undergoes mutation only at a low rate in NSCLC (0.712%). We have further identified LIMD1-associated molecular signatures in NSCLC, including its natural antisense long non-coding RNA LIMD1-AS1 and a pool of membrane trafficking regulators. We have also identified a subgroup of tumor-infiltrating lymphocytes, especially neutrophils, whose tumor infiltration levels significantly correlate with LIMD1 level in both LUAD and LUSC. However, a significant correlation of LIMD1 with a subset of immune regulatory molecules, such as IL6R and TAP1, was only found in LUAD. Regarding the clinical outcomes, LIMD1 expression level only significantly correlates with the survival of LUAD (p<0.01) but not with that of LUSC (p>0.1) patients. These findings indicate that LIMD1 plays a survival role in LUAD patients at least by acting as an immune regulatory protein. To further understand the mechanisms underlying the tumor-suppressing function of LIMD1 in NSCLC, we show that LIMD1 downregulation remarkably correlates with the deregulation of multiple pathways that play decisive roles in the oncogenesis of NSCLC, especially those mediated by EGFR, KRAS, PIK3CA, Keap1, and p63, in both LUAD and LUSC, and those mediated by p53 and CDKN2A only in LUAD. This study has disclosed that LIMD1 can serve as a survival prognostic marker for LUAD patients and provides mechanistic insights into the interaction of LIMD1 with NSCLC, which provide valuable information for clinical applications.

MiR-30b-5p inhibits cancer progression and enhances cisplatin sensitivity in lung cancer through targeting LRP8.

Accumulated evidence has demonstrated that miRNAs are closely implicated in lung carcinogenesis. Herein, we explored the expression pattern of miR-30b-5p in lung cancer, and aimed to uncover miR-30b-5p roles in lung cancer progression and drug resistance. miR-30b-5p expression profiles in lung cancer tissues and the matched non-tumor tissues were determined by using qPCR. Cell viability, migration, invasion and in vivo tumorigenesis were determined by using the CCK-8, colony formation, wound healing, transwell chambers experiments and tumor xenograft models. RNA immunoprecipitation (RIP) and dual luciferase reporter experiments were applied to evaluate the relationship between miR-30b-5p and LRP8. The results demonstrated that miR-30b-5p showed a low expression profile in lung cancer tissues and cells, and closely linked to poor prognosis and malignant clinical process. Cell viability, migration, invasiveness and tumorigenesis were significantly weakened following miR-30b-5p overexpression in A549 and NCI-H1299 cells, while cell apoptosis rates were increased. In addition, miR-30b-5p was lowly expressed in A549/DDP (a cisplatin drug resistant cell line) as compared with A549 cells, and miR-30b-5p increased A549/DDP cell sensitivity to DDP. However, these above roles of miR-30b-5p were all significantly impaired following the overexpression of LRP8 which was overexpressed in lung cancer tissues. Collectively, this study demonstrated that miR-30b-5p functions as a tumor suppressor in lung cancer, and re-sensitizes lung cancer cells to DDP by targeting LRP8.

Circulating miR-320a Acts as a Tumor Suppressor and Prognostic Factor in Non-small Cell Lung Cancer.

Dysregulated expression profiles of microRNAs (miRNAs) have been observed in several types of cancer, including non-small cell lung cancer (NSCLC); however, the diagnostic and prognostic potential of circulating miRNAs in NSCLC remains largely undefined. Here we found that circulating miR-320a was significantly down-regulated (~5.87-fold; p < 0.0001) in NSCLC patients (n = 80) compared to matched control plasma samples from healthy subjects (n = 80). Kaplan-Meier survival analysis revealed that NSCLC patients with lower levels of circulating miR-320a had overall poorer prognosis and survival rates compared to patients with higher levels (p < 0.0001). Moreover, the diagnostic and prognostic potential of miR-320a correlated with clinicopathological characteristics such as tumor size, tumor node metastasis (TNM) stage, and lymph node metastasis. Functionally, depletion of miR-320a in human A549 lung adenocarcinoma cells induced their metastatic potential and reduced apoptosis, which was reversed by exogenous re-expression of miR-320a mimics, indicating that miR-320a has a tumor-suppressive role in NSCLC. These results were further supported by high levels of epithelial-mesenchymal transition (EMT) marker proteins (e.g., Beta-catenin, MMP9, and E-cadherin) in lung cancer cells and tissues via immunoblot and immunohistochemistry experiments. Moreover, through bioinformatics and dual-luciferase reporter assays, we demonstrated that AKT3 was a direct target of miR-320a. In addition, AKT3-associated PI3K/AKT/mTOR protein-signaling pathways were elevated with down-regulated miR-320a levels in NSCLC. These composite data indicate that circulating miR-320a may function as a tumor-suppressor miRNA with potential as a prognostic marker for NSCLC patients.

A novel circular RNA, hsa_circ_0030998 suppresses lung cancer tumorigenesis and Taxol resistance by sponging miR-558.

Circular RNAs (circRNAs) are single-stranded RNAs which form a covalently closed continuous loop. Although originally shown to be non-protein-coding, some circRNAs can give rise to micropeptides. circRNAs have also been shown to play essential regulatory roles in a variety of developmental and disease processes. In a previous study, hsa_circ_0030998 was identified as a circRNA downregulated in lung cancer, but its potential implications and mechanisms in lung cancer were not addressed. Here, we showed that overexpressing circ_0030998 decreased proliferation, migration, and invasion of lung cancer cells, while also dampening resistance to Taxol, a classical antitumor drug. Depleting circ_0030998 reversed these phenotypic effects. A high circ_0030998 expression was correlated with a high survival rate in lung cancer patients. Additionally, we found circ_0030998 could downregulate miR-558 expression, serving as a microRNA sponge. In conclusion, our data support that hsa_circ_0030998 can slow down the progression of lung cancer by targeting miR-558 and suppress malignant phenotypes such as proliferation, migration, and invasion progression of lung cancer cells. Therefore, we highlight that circ_0030998 could be a novel tumor suppressor of lung cancer.

MiR-369 inhibits Liver Cancer progression by targeting ZEB1 pathway and predicts the prognosis of HCC patients.

Increasing evidences show that microRNAs (miRNAs) are involved in the regulation of tumorigenesis, progression, recurrence and drug resistance of hepatocellular carcinoma (HCC). miR-369 works as a tumor suppressor in both lung cancer and thyroid cancer. However, the potential biological function of miR-369 in HCC is unknown. Herein, we for first found that miR-369 expression was downregulated in HCC tissues and predicted the poor prognosis of HCC patients. Forced miR-369 expression inhibited the proliferation and metastasis of HCC cells in vitro and in vivo. Mechanically, bioinformatics and luciferase reporter analysis identified Zinc finger E-box binding homeobox 1 (ZEB1) as a direct target of miR-369 in HCC cells. miR-369 overexpressing downregulated the ZEB1 mRNA and protein expression in HCC cells. miR-369 expression was negatively associated with ZEB1 expression in human HCC tissues. More importantly, the ZEB1 siRNA diminished the discrepancy of growth and metastasis capacity between miR-369 overexpression HCC cells and control cells.

Overexpression of a cell adhesion molecule, TSLC1, as a possible molecular marker for acute-type adult T-cell leukemia

AbstractAdult T-cell leukemia (ATL) caused by human T-cell leukemia virus type 1 (HTLV-1) infection, occurs in 2% to 4% of the HTLV-1 carriers with a long latent period, suggesting that additional alterations participate in the development of ATL. To characterize and identify novel markers of ATL, we examined the expression profiles of more than 12 000 genes in 8 cases of acute-type ATL using microarray. One hundred ninety-two genes containing interleukin 2 (IL-2) receptor α were up-regulated more than 2-fold compared with CD4+ and CD4+CD45RO+ T cells, and tumor suppressor in lung cancer 1 (TSLC1), caveolin 1, and prostaglandin D2 synthase showed increased expression of more than 30-fold. TSLC1 is a cell adhesion molecule originally identified as a tumor suppressor in the lung but lacks its expression in normal or activated T cells. We confirmed ectopic expression of the TSLC1 in all acute-type ATL cells and in 7 of 10 ATL- or HTLV-1–infected T-cell lines. Introduction of TSLC1 into a human ATL cell line ED enhanced both self-aggregation and adhesion ability to vascular endothelial cells. These results suggested that the ectopic expression of TSLC1 could provide a novel marker for acute-type ATL and may participate in tissue invasion, a characteristic feature of the malignant ATL cells.

MITF functions as a tumor suppressor in non-small cell lung cancer beyond the canonically oncogenic role.

Microphthalamia-associated transcription factor (MITF) is a critical mediator in melanocyte differentiation and exerts oncogenic functions in melanoma progression. However, the role of MITF in non-small cell lung cancer (NSCLC) is still unknown. We found that MITF is dominantly expressed in the low-invasive CL1-0 lung adenocarcinoma cells and paired adjacent normal lung tissues. MITF expression is significantly associated with better overall survival and disease-free survival in NSCLC and serves as an independent prognostic marker. Silencing MITF promotes tumor cell migration, invasion and colony formation in lung adenocarcinoma cells. In xenograft mouse model, MITF knockdown enhances metastasis and tumorigenesis, but decreases angiogenesis in the Matrigel plug assay. Whole transcriptome profiling of the landscape of MITF regulation in lung adenocarcinoma indicates that MITF is involved in cell development, cell cycle, inflammation and WNT signaling pathways. Chromatin immunoprecipitation assays revealed that MITF targets the promoters of FZD7, PTGR1 and ANXA1. Moreover, silencing FZD7 reduces the invasiveness that is promoted by silencing MITF. Strikingly, MITF has significantly inverse correlations with the expression of its downstream genes in lung adenocarcinoma. In summary, we demonstrate the suppressive role of MITF in lung cancer progression, which is opposite to the canonical oncogenic function of MITF in melanoma.

SH3GL3 functions as a potent tumor suppressor in lung cancer in a SH3 domain dependent manner

Lung cancer is one of the most common and aggressive cancer and a leading cause of cancer related deaths worldwide. Few studies have prospectively examined the functions of SH3GL3 in cancer progression, however, the biological functions of SH3GL3 in lung cancer initiation and progression remains largely unknown. Here, we show that both mRNA and protein levels of SH3GL3 are weakly expressed in lung cancer tissues and cell lines and its expression negatively correlates with the survival of lung cancer patients. Overexpression of SH3GL3 dramatically inhibits lung cancer cells malignancy behaviors, including proliferation and migration. Additionally, SH3GL3 curbs cell cycle at G0/G1 phase and induces cellular apoptosis of lung cancer cells and inhibits lung cancer stem cell self-renewal dependents on its SH3 domain. Moreover, we report for the first time that SH3GL3 inhibits lung cancer progression partially through p21 and up-regulates p21 in transcriptional level. Collectively, our work suggests that SH3GL3 acts as a potent tumor suppressor in lung cancer progression and have a potential to serve as diagnostic and prognostic biomarker of lung cancer.

In vivo miRNA knockout screening identifies miR-190b as a novel tumor suppressor.

MicroRNAs (miRNAs) play important roles in the development of various cancers including lung cancer which is one of the devastating diseases worldwide. How miRNAs function in de novo lung tumorigenesis remains largely unknown. We here developed a CRISPR/Cas9-mediated dual guide RNA (dgRNA) system to knockout miRNAs in genetically engineered mouse model (GEMM). Through bioinformatic analyses of human lung cancer miRNA database, we identified 16 downregulated miRNAs associated with malignant progression and performed individual knockout with dgRNA system in KrasG12D/Trp53L/L (KP) mouse model. Using this in vivo knockout screening, we identified miR-30b and miR-146a, which has been previously reported as tumor suppressors and miR-190b, a new tumor-suppressive miRNA in lung cancer development. Over-expression of miR-190b in KP model as well as human lung cancer cell lines significantly suppressed malignant progression. We further found that miR-190b targeted the Hus1 gene and knockout of Hus1 in KP model dramatically suppressed lung tumorigenesis. Collectively, our study developed an in vivo miRNA knockout platform for functionally screening in GEMM and identified miR-190b as a new tumor suppressor in lung cancer.

MiR‑7/SP1/TP53BP1 axis may play a pivotal role in NSCLC radiosensitivity.

MicroRNA-7 (miR-7) has been identified as a tumor suppressor in non-small cell lung cancer (NSCLC) and a radiosensitivity regulator. Numerous studies have revealed that specific protein 1 (SP1) plays a critical role in the tumorigenesis of various types of cancers and regulates radiosensitivity and tumor suppressor p53-binding protein 1 (TP53BP1), which plays an essential role in DNA repair. However, it is not clear whether miR-7 has a regulatory effect on SP1 and TP53BP1 in NSCLC. In the present study it was revealed that miR-7 directly binds to the 3′UTR of SP1, thereby suppressing SP1 expression to regulate radiosensitivity. Overexpression of miR-7 and SP1 and knockdown of miR-7 and SP1 were performed using lentiviral transfection. Protein and mRNA abundance of SP1 and TP53BP1 were determined using western blotting and RT-qPCR, respectively, while miR-7 binding to SP1 was validated using a luciferase reporter assay. Biological function analysis indicated that miR-7 negatively regulated SP1 and inhibited cell proliferation, migration, and invasion when combined with radiation. It was also revealed that the expression of TP53BP1 was positively regulated by SP1 or negatively regulated by miR-7. In conclusion, SP1 was a target of miR-7, and the decreased expression of SP1 resulting from miR-7 overexpression in NSCLC was vital for improving radiosensitivity in NSCLC cells. Moreover, SP1 expression was detected in 95 paired NSCLC and adjacent normal tissues, and it was determined that SP1 was significantly upregulated in NSCLC tissues and that its upregulation was correlated with the degree of tissue differentiation. Thus, SP1 and/or miR-7 may be potential molecular targets in NSCLC radiotherapy.

UNC5A, an epigenetically silenced gene, functions as a tumor suppressor in non-small cell lung cancer.

UNC5A has been reported to be related with human cancers. However, the function and mechanism in non-small cell lung carcinoma (NSCLC) remains unknown. We analyzed two NSCLC cell lines (A549 and H157), one normal human bronchial epithelial cell line (BEAS-2B) and the tissues of NSCLC. We used quantitative real-time PCR (qRT-PCR), western blot and immunohistochemical (IHC) staining to examine the expression of UNC5A. Methylation status of the UNC5A promoter was analyzed using methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP). We used western blot to analyzed protein levels of PI3K/Akt pathway. We found that the mRNA expression of UNCA5 was significantly downregulated in NSCLC cells and tissues. The promoter of UNC5A was hypermethylated in NSCLC cells compared to normal control cells. The expression of UNC5A could be reversed by demethylation agent in NSCLC cells. The expression of UNC5A was decreased in NSCLC samples and significantly associated with the advanced types of NSCLC. Functionally, knockdown of UNC5A promoted cell proliferation, migration, invasion and induced apoptosis in NSCLC, overexpression of UNC5A yielded the opposite result. Moreover, we found that UNC5A negatively regulated PI3K/Akt signaling pathway in NSCLC. UNC5A is a novel epigenetically silenced gene in NSCLC and consequent under-expression of UNC5A may contribute to NSCLC tumorigenesis through regulating PI3K/Akt pathway.

Differential regulation of rho GTPases during lung adenocarcinoma migration and invasion reveals a novel role of the tumor suppressor StarD13 in invadopodia regulation

BackgroundLung cancer is the second most commonly occurring cancer. The ability to metastasize and spread to distant locations renders the tumor more aggressive. Members of the Rho subfamily of small GTP-binding proteins (GTPases) play a central role in the regulation of the actin cytoskeleton and in cancer cell migration and metastasis. In this study we investigated the role of the RhoA/Cdc42 GAP, StarD13, a previously described tumor suppressor, in malignancy, migration and invasion of the lung cancer cells A549.MethodsWe knocked down StarD13 expression in A549 lung cancer cells and tested the effect on cell migration and invadopodia formation using time lapse imaging and invasion assays. We also performed rescue experiments to determine the signaling pathways downstream of StarD13 and transfected the cells with FRET biosensors for RhoGTPases to identify the proteins involved in invadopodia formation.ResultsWe observed a decrease in the level of expression of StarD13 in lung tumor tissues compared to normal lung tissues through immunohistochemistry. StarD13 also showed a lower expression in the lung adenocarcinoma cell line A549 compared to normal lung cells, WI38. In addition, the depletion of StarD13 increased cell proliferation and viability in WI38 and A549 cells, suggesting that StarD13 might potentially be a tumor suppressor in lung cancer. The depletion of StarD13, however, inhibited cell motility, conversely demonstrating a positive regulatory role in cell migration. This was potentially due to the constitutive activation of RhoA detected by pull down and FRET assays. Surprisingly, StarD13 suppressed cell invasion by inhibiting Cdc42-mediated invadopodia formation. Indeed, TKS4 staining and invadopodia assay revealed that StarD13 depletion increased Cdc42 activation as well as invadopodia formation and matrix degradation. Normal lung cells depleted of StarD13 also produced invadopodia, otherwise a unique hallmark of invasive cancer cells. Cdc42 knock down mimicked the effects of StarD13, while overexpression of a constitutively active Cdc42 mimicked the effects of its depletion. Finally, immunostaining and FRET analysis revealed the absence of StarD13 in invadopodia as compared to Cdc42, which was activated in invadopodia at the sites of matrix degradation.ConclusionIn conclusion, StarD13 plays distinct roles in lung cancer cell migration and invasion through its differential regulation of Rho GTPases.92FP97T_AMtXGPZ_u9jGL2Video abstract.

Long Non-Coding RNA GATA6-AS1 Sponges miR-324-5p to Inhibit Lung Cancer Cell Proliferation and Invasion.

BackgroundLong non-coding RNAs (lncRNAs), which are key regulators of gene expression, are involved in lung cancer progression. Although numerous differentially expressed lncRNAs have been reported, merely a limited number of studies have been performed to verify their functions in lung cancer.MethodsRNA sequencing data were re-analyzed to investigate the GATA6-AS1 expression in lung cancer. RT-qPCR was performed to verify the expression of GATA6-AS1 in collected tissue samples and cell lines. CCK-8 and transwell assays were carried out to evaluate the role of GATA6-AS1 in lung cancer cells. Dual-luciferase reporter assay and bioinformatic analysis were used to explore the miRNA which can be sponged by GATA6-AS1 in lung cancer cells.ResultsCurrently, we focused on exploring the role and mechanisms of GATA6-AS1 in lung cancer. Expression of GATA6-AS1 was decreased in lung cancer based on the analysis of RNA sequencing dataset, TCGA data and RT-qPCR of clinical tissue samples. Via overexpression of GATA6-AS1, it was revealed that GATA6-AS1 inhibited lung cancer cell proliferation and invasion. Oncogene miR-324-5p was predicted to interact with GATA6-AS1. RT-qPCR and dual-luciferase reporter assay verified the regulation of miR-324-5p by GATG6-AS1 in lung cancer cells. Overexpression of GATA6-AS1 increased the expression of FBXO11 and SP1, two target genes of miR-324-5p. We further showed that miR-324-5p mimic reversed the effect of GATA6-AS1 overexpression in lung cancer cells.ConclusionOverall, our findings demonstrated GATA6-AS1 as a novel tumor suppressor in lung cancer.

Circular RNA circ-SLC7A6 acts as a tumor suppressor in non-small cell lung cancer through abundantly sponging miR-21

ABSTRACT Circular RNA (circRNA) is currently considered to be a key regulatory molecule in cancer biology. In the present study, we aimed to explore the functional and clinical roles of circ-SLC7A6 (a circRNA derived from SLC7A6 gene) in non-small cell lung cancer (NSCLC). Circ-SLC7A6 was significantly downregulated in NSCLC tissues in comparison to para-carcinoma tissues. Low circ-SLC7A6 was closely associated with larger tumor size, lymph node metastasis, advanced clinical stage and adverse outcome. Exogenous expression of circ-SLC7A6 evidently inhibited the proliferation and invasion of NSCLC cells. Further investigations revealed that miR-21 was the direct functional target of circ-SLC7A6, in which circ-SLC7A6 abundantly sponged miR-21 and elevated a cohort of tumor suppressors, thus inhibiting NSCLC progression. Interestingly, QKI, elevated by circ-SLC7A6, could directly bind to the introns flanking circ-SLC7A6 to facilitate circ-SLC7A6 production. Importantly, in vivo xenograft tumor experiments showed that reintroduction of circ-SLC7A6 retarded tumor growth as well as decreased lung metastatic nodules. Overall, our study demonstrates that circ-SLC7A6 is a novel tumor suppressor in NSCLC, targeting circ-SLC7A6/miR-21 axis may be a promising treatment for NSCLC patients.

Abstract 4717: Ly6-neurotoxin1 knockout in PDAC cells inhibits their growth in vitro and in vivo

Abstract Ly6/neurotoxin1 (Lynx1) functions as a brake for nicotinic receptors and was defined as a tumor suppressor in lung cancer. As pancreatic cancer development may be slowed down by cholinergic signaling, we investigated the role of Lynx1 in pancreatic ductal adenocarcinoma (PDAC) cell lines, both in vitro and in vivo. Lynx1 knockout cell clones were generated by transfecting the CRISPRCas9 plasmid - (pSpCas9 (BB)-2A-Puro) into Miapacaluci and BXPC3luci PDAC cells using jet Pei and jet Prime as transfecting agents, respectively. The annealed gRNA was directed towards exon 3 of the Lynx1 gene. Control clones were transfected with the plasmid alone. Clones from both cell lines were tested by qRT-PCR and Western blot for knockout efficiency as well as by proliferation assay. In addition, the expression levels of p-MEK, p-MAPK, p-mTOR and p-Rictor were verified in two BXPC3 cell clones in relation to Lynx1 expression. Furthermore, 4x106 cells of four BXPC3 cell clones (one control, 3 knockout clones) were injected, respectively, into the portal vein of nude rats to control for a possibly reduced tumor growth in the liver. The knockdown of Lynx1 was incomplete at mRNA level and ranged from 20 to 60% in BXPC3 clones and from 0 to 30% in Miapaca clones. At protein level, the respective values ranged from 0 to 90% in BXPC3 and from 22 to 50% in Miapaca clones. However, Lynx1 protein levels increased at later time points. All BXPC3 clones proliferated less quickly than the respective control when tested by MTT assay. Miapaca clones, however, didn't show a significant difference from the respective control, although their growth was clearly disturbed shortly after transfection. Concomitantly with reduced Lynx1 protein levels, there was reduction of p-mTOR (90%), p-Rictor (30%) and p-MEK (40%) in BXPC3 cells. In vivo, the BXPC3 clones showed a lag period of 1 to 2 weeks till the appearance of a first bioluminescence signal indicating tumor growth. Rats injected with cells from the control clone showed a steady increase in the bioluminescence signal (n=7 of 10) as compared to the most sensitive knockdown clone, which didn't show any signal in any of 4 injected rats (p=0.05). Cells of 2 other BXPC3 clones showed a reduced growth rate at best in 2 of 2 rats used for each clone, respectively. In conclusion, knockout of Lynx1 was incomplete at both mRNA and protein levels. Nevertheless, the respective BXPC3 clones exhibited reduced proliferation in vitro, which was associated with diminished p-mTOR, p-Rictor and p-MEK levels. In addition, they failed to establish a tumor in vivo or showed a reduced tumor growth rate. These findings suggest that Lynx1 is a vital gene and may play an important role in the growth and establishment of PDAC cells. Citation Format: Doaa Ali, Michael Zepp, Matthias Bozza, Maria Nikolova, Richard Harbottle, Martin R. Berger. Ly6-neurotoxin1 knockout in PDAC cells inhibits their growth in vitro and in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4717.

MiR129-1 regulates protein phosphatase 1D protein expression under hypoxic conditions in non-small cell lung cancer cells harboring a TP53 mutation.

Protein phosphatase 1D (PPM1D), which functions as an oncogene, is a known target of the tumor suppressor p53 and is involved in p53-regulated genomic surveillance mechanisms. PPM1D dephosphorylates both p53 and its ubiquitin ligase mouse double minute 2 homolog, as well as the RNA-binding protein (RBM)38, which turns RBM38 from an inducer to inhibitor of TP53 translation. In addition, RBM38 induces PPM1D translation. Hence, the PPM1D-RBM38-p53 axis is important in maintaining genomic integrity and is often altered during tumorigenesis. TP53, which encodes p53, is deleted or mutated in >50% of cancer types, including lung cancer. Mutant p53 has been revealed to complex with hypoxia-inducible factor 1α (HIF1α) and upregulate transcription of pro-metastatic genes. However, the mechanism underlying the action of the PPM1D-RBM38-p53 axis in the context of mutant p53 under normoxic and hypoxic conditions is yet to be elucidated. In the present study, using non-small cell lung cancer (NSCLC) cell lines harboring wild-type (A549 cells) or hot-spot mutant (NCI-H1770 and R249WΔ-TP53-A549 cells) TP53, it was demonstrated that in cells harboring mutant p53, RBM38 was not the primary regulator of PPM1D translation under hypoxic conditions. Knockdown of RBM38 in TP53 mutant cells did not affect the PPM1D protein expression under hypoxic conditions. Instead, in NCI-H1770 cells maintained under normoxic conditions, PPM1D was revealed as a target of micro RNA (miR)-129-1-3p, a known tumor suppressor in lung cancer. Hypoxia resulted in the downregulation of miR-129-1-3p expression, and thus, in the downregulation of PPM1D messenger RNA (mRNA) translation. In NCI-H1770 cells grown under hypoxic conditions, the transient transfection of miR-129-1-3p mimic, and not control mimic, repressed the expression of a reporter containing wild-type, but not miR-129-1-3p binding mutant, of the PPM1D 3′-untranslated region (UTR). Analysis of NSCLC cell lines from the Broad Institute Cancer Cell Encyclopedia and patients with NSCLC from The Cancer Genome Atlas dataset revealed significant co-occurrence of PPM1D/RBM38 and PPM1D/HIF1A mutations. However, there was no significant difference in the overall survival of patients with NSCLC with or without genomic alterations in TP53, RBM38, PPM1D and HIF1A. In summary, the current study demonstrated hypoxia-dependent miR-129-1-3p-mediated regulation of PPM1D protein expression in NSCLC cell line harboring mutant TP53.

Multifaceted p21 in carcinogenesis, stemness of tumor and tumor therapy.

Cancer cells possess metabolic properties that are different from those of benign cells. p21, encoded by CDKN1A gene, also named p21Cip1/WAF1, was first identified as a cyclin-dependent kinase regulator that suppresses cell cycle G1/S phase and retinoblastoma protein phosphorylation. CDKN1A (p21) acts as the downstream target gene of TP53 (p53), and its expression is induced by wild-type p53 and it is not associated with mutant p53. p21 has been characterized as a vital regulator that involves multiple cell functions, including G1/S cell cycle progression, cell growth, DNA damage, and cell stemness. In 1994, p21 was found as a tumor suppressor in brain, lung and colon cancer by targeting p53 and was associated with tumorigenesis and metastasis. Notably, p21 plays a significant role in tumor development through p53-dependent and p53-independent pathways. In addition, expression of p21 is closely related to the resting state or terminal differentiation of cells. p21 is also associated with cancer stem cells and acts as a biomarker for such cells. In cancer therapy, given the importance of p21 in regulating the G1/S and G2 check points, it is not surprising that p21 is implicated in response to many cancer treatments and p21 promotes the effect of oncolytic virotherapy.