Non-small Cell Lung Carcinoma Proliferation Research Articles

RGS20 promotes non-small cell lung carcinoma proliferation via autophagy activation and inhibition of the PKA-Hippo signaling pathway

BackgroundNovel therapeutic targets are urgently needed for treating drug-resistant non-small cell lung cancer (NSCLC) and overcoming drug resistance to molecular-targeted therapies. Regulator of G protein signaling 20 (RGS20) is identified as an upregulated factor in many cancers, yet its specific role and the mechanism through which RGS20 functions in NSCLC remain unclear. Our study aimed to identify the role of RGS20 in NSCLC prognosis and delineate associated cellular and molecular pathways.MethodsImmunohistochemistry and lung cancer tissue microarray were used to verify the expression of RGS20 between NSCLC patients. CCK8 and cell cloning were conducted to determine the proliferation ability of H1299 and Anip973 cells in vitro. Furthermore, Transcriptome sequencing was performed to show enrichment genes and pathways. Immunofluorescence was used to detect the translocation changes of YAP to nucleus. Western blotting demonstrated different expressions of autophagy and the Hippo-PKA signal pathway. In vitro and in vivo experiments verified whether overexpression of RGS20 affect the proliferation and autophagy of NSCLC through regulating the Hippo pathway.ResultsThe higher RGS20 expression was found to be significantly correlated with a poorer five-year survival rate. Further, RGS20 accelerated cell proliferation by increasing autophagy. Transcriptomic sequencing suggested the involvement of the Hippo signaling pathway in the action of RGS20 in NSCLC. RGS20 activation reduced YAP phosphorylation and facilitated its nuclear translocation. Remarkably, inhibiting Hippo signaling with GA-017 promoted cell proliferation and activated autophagy in RGS20 knock-down cells. However, forskolin, a GPCR activator, increased YAP phosphorylation and reversed the promoting effect of RGS20 in RGS20-overexpressing cells. Lastly, in vivo experiments further confirmed role of RGS20 in aggravating tumorigenicity, as its overexpression increased NSCLC cell proliferation.ConclusionOur findings indicate that RGS20 drives NSCLC cell proliferation by triggering autophagy via the inhibition of PKA-Hippo signaling. These insights support the role of RGS20 as a promising novel molecular marker and a target for future targeted therapies in lung cancer treatment.

Disruption of LPA-LPAR1 pathway results in lung tumor growth inhibition by downregulating B7-H3 expression in fibroblasts.

Lysophosphatidic acids (LPAs) belong to a class of bioactive lysophospholipids with multiple functions including immunomodulatory roles in tumor microenvironment (TME). LPA exerts its biological effects via its receptors that are highly expressed in fibroblasts among other cell types. As cancer-associated fibroblasts (CAFs) are a key component of the TME, it is important to understand LPA signaling and regulation of receptors in fibroblasts or CAFs and associated regulatory roles on immunomodulation-related molecules. Cluster analysis, immunoblotting, real-time quantitative-PCR, CRISPR-Cas9 gene editing system, immunohistochemical staining, coculture model, and in vivo xenograft model were used to investigate the effects of LPA-LPAR1 on B7-H3 in tumor promotion of CAFs. In this study, we found that LPAR1 and CD276 (B7-H3) were generally highly expressed in fibroblasts with good expression correlation. LPA induced B7-H3 up-expression through LPAR1, and stimulated fibroblasts proliferation that could be inhibited by silencing LPAR1 or B7-H3 as well as small molecule LPAR1 antagonist (Ki16425). Using engineered fibroblasts and non-small cell lung carcinoma (NSCLC) cell lines, subsequent investigations demonstrated that CAFs promoted the proliferation of NSCLC in vitro and in vivo, and such effect could be inhibited by knocking out LPAR1 or B7-H3. The present study provided new insights for roles of LPA in CAFs, which could lead to the development of innovative therapies targeting CAFs in the TME. It is also reasonable to postulate a combinatory approach to treat malignant fibrous tumors (such as NSCLC) with LPAR1 antagonists and B7-H3 targeting therapies.

Chloroquine induces transitory attenuation of proliferation of human lung cancer cells through regulation of mutant P53 and YAP.

Non-small cell lung carcinoma (NSCLC) is the most common cause of cancer-associated deaths worldwide. Though recent development in targeted therapy has improved NSCLC prognosis, yet there is an unmet need to identify novel causative factors and appropriate therapeutic regimen against NSCLCs. In this study, we identify key molecular factors de-regulated in NSCLCs. Analyze their expression by real-time PCR and immunoblot; map their localization by immuno-fluorescence microscopy. We further propose an FDA approved drug, chloroquine (CQ) that affects the function of the molecular factors and hence can be repurposed as a therapeutic strategy against NSCLCs. Available NSCLC mutation data reflects a high probabilistic chance of patients harboring a p53 mutation, especially a gain of function (GOF)-R273H mutation. The GOF-P53 mutation enables the P53 protein to potentially interact with non-canonical protein partners facilitating oncogenesis. In this context, analysis of existing transcriptomic data from R273H-P53 expressing cells shows a concomitant up-regulation of Yes-associated protein (YAP) transcriptional targets and its protein partner TEAD1 in NSCLCs, suggesting a possible link between R273H-P53 and YAP. We therefore explored the inter-dependence of R273H-P53 and YAP in NSCLC cells. They were found to co-operatively regulate NSCLC proliferation. Genetic or pharmacological inhibition of YAP and GOF-P53 resulted in sensitization of NSCLC cells. Further analysis of pathways controlled by GOF-P53 and YAP showed that they positively regulate the cellular homeostatic process- autophagy to mediate survival. We hence postulated that a modulation of autophagy might be a potent strategy to curb proliferation. In accordance to above, autophagy inhibition, especially with the FDA-approved drug- chloroquine (CQ) resulted in cytoplasmic accumulation and reduced transcriptional activity of GOF-P53 and YAP, leading to growth arrest of NSCLC cells. Our study highlights the importance of GOF-P53 and YAP in NSCLC proliferation and proposes autophagy inhibition as an efficient strategy to attenuate NSCLC tumorigenesis.

A Beclin 1-targeting stapled peptide synergizes with erlotinib to potently inhibit proliferation of non-small-cell lung cancer cells

Epidermal Growth Factor Receptor (EGFR) is a major drug target for non-small-cell lung carcinoma (NSCLC). Tyrosine Kinase Inhibitors (TKIs) like erlotinib are potent inhibitors of EGFR and have achieved impressive clinical success against NSCLC. However, NSCLC cells readily develop resistance to TKIs by acquiring mutations in EGFR or other oncogenes. Novel strategies to inhibit EGFR are needed to overcome this urgent problem of TKI resistance. Beclin 1 is an essential autophagy protein and is intimately involved in tumorigenesis and EGFR signaling. Here we present data to show that a Beclin 1-targeting stapled peptide Tat-SP4 can inhibit the EGFR signaling pathway by enhancing the Beclin 1-mediated endolysosomal degradation of EGFR. This inhibition mechanism is orthogonal to that employed by TKIs and is effective against either wild-type or mutant EGFR. Tat-SP4 alone showed moderate anti-proliferative efficacy in NSCLC cells but synergized with erlotinib to potently inhibit NSCLC proliferation. These results suggest that Beclin 1-targeting stapled peptides may be used in combination with TKIs to enhance their efficacy, particularly for NSCLC subtypes refractory to current regiments.

Role of Cytokines and Chemokines in NSCLC Immune Navigation and Proliferation.

With over a million deaths every year around the world, lung cancer is found to be the most recurrent cancer among all types. Nonsmall cell lung carcinoma (NSCLC) amounts to about 85% of the entire cases. The other 15% owes it to small cell lung carcinoma (SCLC). Despite decades of research, the prognosis for NSCLC patients is poorly understood with treatment options limited. First, this article emphasises on the part that tumour microenvironment (TME) and its constituents play in lung cancer progression. This review also highlights the inflammatory (pro- or anti-) roles of different cytokines (ILs, TGF-β, and TNF-α) and chemokine (CC, CXC, C, and CX3C) families in the lung TME, provoking tumour growth and subsequent metastasis. The write-up also pinpoints recent developments in the field of chemokine biology. Additionally, it covers the role of extracellular vesicles (EVs), as alternate carriers of cytokines and chemokines. This allows the cytokines/chemokines to modulate the EVs for their secretion, trafficking, and aid in cancer proliferation. In the end, this review also stresses on the role of these factors as prognostic biomarkers for lung immunotherapy, apart from focusing on inflammatory actions of these chemoattractants.

Effects of MicroRNA-22 (miR-222) on Non-Small-Cell Lung Carcinoma (NSCLC) Cells Apoptosis and Proliferation Through Protein Kinase B/Mammalian Target of Rapamycin (AKT/mTOR) Signaling Pathway

Our study investigated miR-222’s effect on NSCLC apoptosis and proliferation. H1650 cells were assigned into NSCLC group, SI group and IN group followed by analysis of Akt-mTOR protein content, cell apoptosis and proliferation, and miR-222 expression by Western blot, Hoechst 33258 fluorescence staining, qRT-PCR, cck-8, and immunohistochemistry miR-222 expression was lowest in IN group and highest in SI group (P < 0.05). The nucleus shrinkage rate in IN group and NSCLC group was significantly higher than that in SI group (P < 0.05) with NSCLC group showed more cells apoptosis than IN group (P < 0.05). The SI group had significantly higher OD value than IN group and NSCLC group (P < 0.05) with NSCLC group having significantly higher OD value than IN group (P < 0.05). The Akt-mTOR and p-Akt/p-mTOR expression was lowest in IN group and highest in SI group (P < 0.05) with lower level in IN group than NSCLC group (P < 0.05). The number of positive Akt-mTOR of H1650 cells was highest in SI group while lowest in IN group (P < 0.05). The decreased miR-222 expression in NSCLC can promote cancer cell apoptosis and inhibit lung cancer development, which may be via down-regulating Akt-mTOR signaling.

MALAT1 modulated FOXP3 ubiquitination then affected GINS1 transcription and drived NSCLC proliferation.

An increasing number of studies have shown that long-noncoding RNAs (lncRNAs) are involved in the post-translational modifications (PTMs) of protein in a variety of tumors. However, little is known about the exact regulation mechanism of lncRNAs in regulating PTMs in non-small-cell lung carcinoma (NSCLC) proliferation. Metastasis-associated lung adenocarcinoma transcript1 (MALAT1) and GINS complex subunit 1(GINS1) both were upregulated and promoted proliferation progression in NSCLC. In this study, the clinicopathologic significance of MALAT1 and GINS1 in NSCLC was investigated, a positive correlation in their expression was found. The silencing of MALAT1 decreased GINS1 expression and inhibited NSCLC proliferation in vitro and in vivo. The upregulation of GINS1 reversed NSCLC proliferation inhibited by MALAT1 knockdown. FOXP3 (forkhead box protein 3) was identified as the critical transcription factor for GINS1 transcription. In addition, MALAT1 could stabilize FOXP3 by binding to zinc finger (ZF) domain and leucine zipper (LZ) domain of FOXP3. Interestingly, these two domains were also interaction domains for FOXP3 binding with E3 ligase STUB1 (STIP1 homology and U-box containing protein 1). In this way, MALAT1 masked the protein-interacting domain, and inhibited FOXP3 ubiquitination by STUB1. Together, our results identified a novel regulatory axis of MALAT1-FOXP3-GINS1, and demonstrated that MALAT1 played an important modulatory role in PTM of FOXP3 which affects GINS1 transcription and drives proliferation character in NSCLC.

CircRNF20 aggravates the progression of non-small-cell lung carcinoma by activating MAPK9.

To explore the clinical significance of circRNF20 in non-small-cell lung carcinoma (NSCLC), and its regulatory effects on NSCLC cell functions by activating MAPK9. Relative levels of circRNF20 and MAPK9 in NSCLC tissues were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The relationship between circRNF20, MAPK9 and pathological factors in NSCLC patients was analyzed. Prognostic potentials of circRNF20 and MAPK9 in NSCLC were assessed by Kaplan-Meier method. The interaction between circRNF20 and MAPK9 was tested by Dual-Luciferase reporter assay. Regulatory effects of circRNF20 and MAPK9 on proliferative abilities in H358 and SPC-A1 cells were examined by Cell Counting Kit-8 (CCK-8) and colony formation assay. CircRNF20 and MAPK9 were upregulated in NSCLC tissues than normal ones. They were correlated to T stage and poor prognosis in NSCLC patients, while their levels were unrelated to gender, age, and incidences of lymphatic and distant metastasis. Knockdown of circRNF20 attenuated proliferative abilities in H358 and SPC-A1 cells. On the contrary, the overexpression of MAPK9 yielded the opposite results. MAPK9 was the target gene binding circRNF20, which was able to reverse the regulatory effect of circRNF20 on NSCLC proliferation. CircRNF20 and MAPK9 are upregulated in NSCLC cases, which are closely linked to T stage in NSCLC patients. They are independent prognostic factors for NSCLC. By activating MAPK9, circRNF20 stimulates NSCLC proliferation.

The p53/miR-193a/EGFR feedback loop function as a driving force for non-small cell lung carcinoma tumorigenesis

Background:Non-small cell lung carcinoma (NSCLC) is a major worldwide health threat due to its low cure rate and high lethality. Emerging evidence suggests that epidermal growth factor receptor (EGFR) plays vital roles in cancer initiation and progression, and is considered an important cancer-driving protein. However, how EGFR expression is regulated during NSCLC development remains to be fully elucidated.Methods:In NSCLC clinical samples, EGFR protein levels were measured by western blotting and qRT-PCR, respectively. Combining microRNA (miRNA) target prediction software and the pulldown assay, we predicted microRNAs (miRNAs) that targeted EGFR. Next, three NSCLC cell lines, A549 (p53 WT), H322 (p53 mutant), and H1299 (p53 null), were used to demonstrate the direct targeting of EGFR by miR-193a. In addition, we investigated the biological effects of EGFR inhibition by miR-193a in vitro using Cell Counting Kit-8, 5-Ethynyl-2′-deoxyuridine (EdU), transwell, and apoptosis assays. Then, using ChIP and luciferase assays, we demonstrated that miR-193a was directly activated by p53 at the transcriptional level and that p53-induced-miR-193a and EGFR form a double-negative feedback loop.Results:We found that EGFR mRNA and protein were upregulated in NSCLC. We predicted that EGFR was a target of miR-193a and validated that miR-193a bound directly to the 3′-UTR of the EGFR mRNA. Moreover, miR-193a inhibited NSCLC proliferation and invasion, and promotes NSCLC apoptosis by directly downregulating EGFR. Then, we demonstrated that p53 directly activated miR-193a transcription, whereas EGFR functioned as a transcriptional repressor to negatively control miR-193a expression, forming a feedback loop. The loop promoted NSCLC cell proliferation and migration and accelerated tumor growth in xenograft mice.Conclusions:This study highlights a double-negative feedback loop in NSCLC. The feedback loop is crucial because overexpressing EGFR strongly accelerated tumor growth, while miR-193a restoration blocked tumor growth in vivo. Our findings are in line with the emerging opinion that miRNAs and protein regulators form regulatory networks in critical biological processes and that their dysregulation can lead to cellular dysfunction. In conclusion, this study provides important insights into the molecular mechanisms of NSCLC progression and may help inform the development of new therapeutics for managing NSCLC.

Long non-coding RNA-CCAT2 promotes the occurrence of non-small cell lung cancer by regulating the Wnt/β-catenin signaling pathway.

The present study aimed to investigate the biological function of colon cancer-associated transcript 2 (CCAT2) in the occurrence and progression of non-small cell lung carcinoma (NSCLC) and its potential use in the early diagnosis and molecular-targeted therapy of NSCLC. The tumor tissues, para-carcinoma tissues and associated clinical data of 36 patients with NSCLC were collected in order to detect the expression of CCAT2 and assess the impact of factors including histopathological type, Tumor-Node-Metastasis stage and lymph node metastasis on CCAT2 expression. The lung cancer NCI-H1975 cell line was transfected with a small interfering RNA (siRNA) plasmid to determine the effect of si-CCAT2 on NSCLC proliferation, invasion and metastasis. The effect of si-CCAT2 on the expression of nuclear and cytoplasmic β-catenin protein in the lung cancer NCI-H1975 cell line was detected using western blot analysis. The expression levels of CCAT2 in the tumor tissues of patients with NSCLC were significantly higher than those in the normal para-carcinoma tissues (t=8.580, P<0.01). Subsequent to CCAT2 silencing, the proliferation and invasive abilities of NCI-H1975 cells were significantly decreased compared with control cells (P<0.05). In the si-CCAT2 group, the level of nuclear and cytoplasmic β-catenin proteins was decreased, and the activity of the Wnt signaling pathway was significantly inhibited compared with the control cells (P<0.01), and a synergistic effect was exerted with the Wnt signaling inhibitor FH535. CCAT2 may therefore promote the occurrence of NSCLC by regulating the Wnt/β-catenin signaling pathway.

CXCR4 Accelerates Osteoclastogenesis Induced by Non-Small Cell Lung Carcinoma Cells Through Self-Potentiation and VCAM1 Secretion

Background/Aims: Non-small cell lung carcinoma (NSCLC) metastasis to bone leads to skeletal-related events and a poor quality of life. Unravelling the mechanism of metastasis is crucial for improving survival. Previous work has implicated the role of CXCR4 in bone metastasis; however, the underlying mechanisms are unknown. Methods: The human bone metastasis tissue samples were obtained from lung cancer patients during surgery with consents. The patients were followed up and the overall survival curve was analysed. The expression of CXCR4, VCAM1, and ADAM17 was measured with real-time PCR, western blot and immunochemistry staining in human tissue or NSCLC cell lines. The effects of CXCR4, soluble VCAM1 and ADAM17 on NSCLC proliferation, migration and invasion were measured with CCK-8, monolayer scratch assay and transwell chamber, respectively. The amount of soluble VCAM1 in the conditioned medium was detected with ELISA. Tartrate-resistant acid phosphatasethe (TRAP) staining was performed to stain the multinucleated cells regarded as osteoclasts. Results: In this study, CXCR4 was found to be highly expressed in bone destruction area of metastatic NSCLC samples and related to poor survival in NSCLC patients with bone metastasis. CXCR4 potentiated NSCLC with enhanced proliferation and invasion abilities, while CXCR4 knockdown significantly suppressed the growth and invasion. Furthermore, CXCR4 promoted lung cancer-induced osteoclast differentiation with increased osteoclast formation. We also found that soluble VCAM1 (sVCAM1) secreted in NSCLC contributed to the osteoclastogenesis induced by CXCR4. The overexpression of CXCR4 increased sVCAM1, and the sVCAM1 secreted from CXCR4-overexpressing NSCLC cells recruited and arrested additional osteoclast progenitors to promote osteoclastogenesis. ADAM17 was confirmed to act as a downstream mediator of CXCR4. The chemical inhibition of ADAM17 with TAPI-2 decreased sVCAM1 secretion and the number of TRAP+ osteoclasts. Conclusion: Taken together, these results indicated that CXCR4 potentiated NSCLC and promoted osteoclastogenesis through sVCAM1, which was cleaved by ADAM17. These data support the pivotal role of the cross talk between CXCR4 and ADAM17-VCAM1 in NSCLC-induced bone metastasis.

MiR-124 Inhibits Growth and Enhances Radiation-Induced Apoptosis in Non-Small Cell Lung Cancer by Inhibiting STAT3

Background/Aims: A growing body of evidence indicates that the abnormal expression of microRNAs (miRNAs) play an important role in sensitizing the cellular response to ionizing radiation (IR). The aim of this study was to investigate whether the expression of miR-124 correlated with radiosensitivity in the context of non-small-cell lung carcinoma (NSCLC). Methods: Quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to quantify miR-124 expression in NSCLC tissues and cell lines. The role of miR-124 in NSCLC proliferation and radiosensitivity was analyzed using CCK-8 and flow cytometry apoptosis assays. Luciferase activity assays, RT-PCR, and Western blot assays were performed to confirm the target gene of miR-124. Results: In this study, we found that miR-124 was downregulated both in clinical NSCLC samples and in cell lines. miR-124 inhibited the proliferation of NSCLC cells and enhanced the apoptosis of NSCLC cells exposed to ionizing radiation. We identified signal transducer and activator of transcription 3 (STAT3) as a direct target of miR-124 by using target prediction algorithms and luciferase assays. Overexpression of STAT3 in A549 cell lines restored the enhanced radiosensitivity induced by miR-124. Conclusion: Taking these observations into consideration, we illustrated that miR-124 is a potential target for enhancing the radiosensitivity of NSCLC cells by targeting STAT3.

Musashi1 Promotes Non-Small Cell Lung Carcinoma Malignancy and Chemoresistance via Activating the Akt Signaling Pathway

Background/Aims: Lung cancer is one of the leading causes for cancer mortality. The poor therapeutic outcome of non-small cell lung carcinoma (NSCLC) is mainly due to late diagnosis and chemoresistance. In this study, we investigated the role of Musashi1 (MSI1) in NSCLC malignancy and chemoresistance. Methods: Colony formation, MTT, glucose uptake and lactate production assays were employed to study lung cancer cell malignancy and chemoresistance. RT-PCR and Western blotting were performed to detect mRNA and protein expressions of genes. We used immunohistochemistry and Pearson correlation analysis to study the relationship of gene expression. Results: We demonstrated that MSI1 was able to promote the proliferation and glucose metabolism of NSCLC cells, and to mediate the sensitivity to chemotherapy drugs in NSCLC cells. Importantly, we found that MSI1 could regulate the activity of Akt signaling. The regulation of NSCLC proliferation, glucose metabolism and chemoresistance by MSI1 was dependent on the modulation of the activity of the Akt signaling pathway. We also found that MSI1 was a target of miR-181a-5p, a microRNA involved in the regulation of cancer development. The expression levels of MSI1 and miR-181a-5p were negatively correlated in NSCLC. Conclusion: MSI1 promotes non-small cell lung carcinoma malignancy and chemoresistance via activating the Akt signaling pathway, which provides a new strategy for the therapy of NSCLC.

Expression of G protein-coupled receptor 56 is associated with tumor progression in non-small-cell lung carcinoma patients.

BackgroundG protein-coupled receptor 56 (GPR56) is an adhesion G protein-coupled receptor with essential functions for cell physiology and survival, and its expression correlates with prognosis in a number of malignancies. The aim of this study is to determine the relationship of GPR56 expression with clinicopathological parameters and prognosis in non-small-cell lung carcinoma (NSCLC).MethodsThe levels of GPR56 were evaluated by immunohistochemistry in 157 NSCLC tissue samples. The association between GPR56 and clinicopathological parameters was evaluated by χ2 test. Univariate and multivariate analyses were performed to demonstrate the prognosis role of GPR56. The function of GPR56 in NSCLC cell lines was also explored through overexpression and knockdown studies.ResultsThe expression level of GPR56 in tumor tissues was significantly correlated with the TNM stage of NSCLC (P=0.005). Univariate and multivariate analyses revealed that GPR56 can act as an independent prognostic factor for overall survival. Furthermore, through overexpression and knockdown experiments, we confirmed that GPR56 can promote the proliferation and invasion of NSCLC cells.ConclusionGPR56 plays an important role in tumor development and may serve as a promising target for prognostic prediction in NSCLC.

Expression of Ceramide Galactosyltransferase (UGT8) in Primary and Metastatic Lung Tissues of Non-Small-Cell Lung Cancer.

Ceramide galactosyltransferase (UGT8) is an enzyme that regulates the synthesis of sphingolipids of the myelin sheath in nervous systems. The protein raises an increasing research interest as a potential marker of cancer progression in various organs. In the present study we seek to determine whether UGT8 could play a role of a therapeutic marker in non-small cell lung carcinoma (NSCLC). We addressed the issue by examining the intensity of UGT8 expression in tissue specimens of primary and corresponding metastatic lung tumors in 19 NSCLC patients undergoing surgery. The methodology was one of immunohistochemical tissue staining using light microscopy. The findings were that the majority of both lung primary and metastatic tumor tissues were positive in UGT8 signals. The cytoplasmic expression of UGT8 was found in 68.4 % of cases of primary tumors and 82.2 % of metastases, with a positive correlation between the UGT8 expression in both tumor tissues. The normal tissue adjacent to tumors showed no positive UGT8 staining. However, we failed to find any appreciable difference in UGT8 expression depending on the clinical stage of NSCLC or lymph node involvement. Nor was there any association between UGT8 expression in tumor tissues and patients' survival time. We conclude that it is unlikely that therapeutic targeting of UGT8 could inhibit cell proliferation and invasion of NSCLC. UGT8, although enhanced in NSCLC tissues, does not meet the criteria of a lung tumor marker. Thus, UGT8 cannot be considered as having diagnostic or therapeutic utility in NSCLC. The pathophysiological meaning of enhanced expression of UGT8 in lung cancer remains to be explored in further studies.

Nicotine Stimulates Human Lung Cancer Cell Growth by Inducing Fibronectin Expression

The mechanisms by which tobacco promotes lung cancer remain incompletely understood. Herein, we report that nicotine, a major component of tobacco, promotes the proliferation of cultured non-small cell lung carcinoma (NSCLC) cells; this effect was most noticeable at 5 days. However, nicotine had no effect on apoptosis of NSCLC cells. In experiments designed to unveil the mechanisms for this effect, we found that nicotine also stimulated mRNA and protein expression of fibronectin. Fibronectin is a matrix glycoprotein that regulates important cellular processes (e.g., adhesion, proliferation, and differentiation) and is highly expressed in tobacco-related lung disorders. Of note, reagents against the integrin alpha5beta1 (antibodies, RGD peptides, alpha5 shRNA) blocked the mitogenic effects of nicotine. Thus, nicotine stimulated NSCLC cell proliferation indirectly via fibronectin induction. We then focused on the mechanisms responsible for nicotine-induced fibronectin expression in NSCLC cells and found that nicotine stimulated the surface expression of alpha7 nicotinic acetylcholine receptor (alpha7 nAChR), and that alpha-bungarotoxin, an inhibitor of alpha7 nAChR, abolished the nicotine-induced fibronectin response. The fibronectin-inducing effects of nicotine were associated with activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3-K)/mammalian target of rapamycin (mTOR) signaling pathways, and were abrogated by inhibitors of ERK (PD98059), PI3-K (LY294002), and mTOR (rapamycin), but not by inhibitors of protein kinase (PK)C (calphostin C) and PKA (H89). These observations suggest that nicotine stimulates NSCLC proliferation through induction of fibronectin, and that these events are mediated through nAChR-mediated signals that include ERK and PI3-K/mTOR pathways. This work highlights the role of fibronectin and alpha5beta1 integrins as potential targets for anti-lung cancer therapies.

Gastrin-Releasing Peptide Receptor Mediates Activation of the Epidermal Growth Factor Receptor in Lung Cancer Cells

Gastrin-releasing peptide receptor (GRPR) and the epidermal growth factor receptor (EGFR) are expressed in several cancers including non-small cell lung carcinoma (NSCLC). Here we demonstrate the activation of EGFR by the GRPR ligand, gastrin-releasing peptide (GRP), in NSCLC cells. GRP induced rapid activation of p44/42 MAPK in lung cancer cells through EGFR. GRP-mediated activation of MAPK in NSCLC cells was abrogated by pretreatment with the anti-EGFR-neutralizing antibody, C225. Pretreatment of NSCLC cells with neutralizing antibodies to the EGFR ligands, TGF-α or HB-EGF, also decreased GRP-mediated MAPK activation. On matrix metalloproteinase (MMP) inhibition, GRP failed to activate MAPK in NSCLC cells. EGF and GRP both stimulated NSCLC proliferation, and inhibition of either EGFR or GRPR resulted in cell death. Combining a GRPR antagonist with the EGFR tyrosine kinase inhibitor, gefitinib, resulted in additive cytotoxic effects. Additive effects were seen at gefitinib concentrations from 1 to 18μM, encompassing the ID50 values of both gefitinib-sensitive and gefitinib-resistant NSCLC cell lines. Because a major effect of GRPR appears to be promoting the release of EGFR ligand, this study suggests that a greater inhibition of cell proliferation may occur by abrogating EGFR ligand release in consort with inhibition of EGFR.