P53 In Lung Cancer Cells Research Articles

Exosome-mediated delivery of RNA and DNA for gene therapy

Gene therapy promises to revolutionize biomedicine and personalized medicine by modulating or compensating the expression of abnormal genes. The biggest obstacle for clinical application is the lack of an effective, non-immunogenic delivery system. We show that bovine colostrum exosomes and polyethyleneimine matrix (EPM) delivers short interfering RNA (siRNA) or plasmid DNA (pDNA) for effective gene therapy. KRAS, a therapeutic focus for many cancers, was targeted by EPM-delivered KRAS siRNA (siKRAS) and inhibited lung tumor growth (>70%) and reduced KRAS expression (50%-80%). Aberrant p53 is another therapeutic focus for many cancers. EPM-mediated introduction of wild-type (WT) p53 pDNA (pcDNA-p53) resulted in p53 expression in p53-null H1299cells in culture, subcutaneous lung tumor, and tissues of p53-knockout mice. Additionally, chemo-sensitizing effects of paclitaxel were restored by exogenous WT p53 in lung cancer cells. Together, this novel EPM technology represents an effective 'platform' for delivery of therapeutic nucleic acids to treat human disease.

Abstract 3514: RANBP9 presence affects levels of Tip60 and activated p53 in lung cancer cells in response to DNA damage

Abstract Lung cancer is the leading cause of cancer-related deaths. Despite recent success with targeted and immune-therapies, DNA-damaging agents, such as platinum-based drugs, still represent the first-line of treatment. However, side effects and drug resistance limit their efficacy and use. We recently demonstrated that RAN Binding Protein 9 (RANBP9) plays a critical role in the DNA-damage response (DDR) of lung cancer cells. However, how it affects the mechanisms and outcome of the DDR is not known. In this regard, p53 activity is crucial for cell cycle arrest or programmed cell death. In particular, acetylation of p53 at lysine 120 by the Tat-Interacting Protein of 60 KDa (Tip60) is dispensable for p53-mediated growth arrest but is essential for p21 induction and p53-dependent apoptosis. It has been reported that RANBP9 and Tip60 co-localize. Here, we investigate whether RANBP9 affects Tip60-dependent activation of p53 and its relevance in deciding cell faith in response to DNA damage. For our study we used H1299, A549 and HeLa cells. By CRISPR/Cas9, we generated RANBP9 wild type (WT) and knockout (KO) A549 clones. We also established KO clones stably re-expressing RANBP9-FLAG. Cell response to DNA damage was assessed upon treatment with Ionizing Radiation (IR) and/or Cisplatin (CDDP). Interaction between RANBP9 with Tip60 was assessed by immunoprecipitation of transfected HeLa cell lysates. We show that RANBP9 and Tip60 co-immunoprecipitate either by pulling-down RANBP9 or Tip60. Using different deletion mutants of Tip60, we found that a region encompassing the Zinc Finger and part of the Histone Acetyl-Transferase domain is required for the interaction with RANBP9. Further, the enzymatic-dead Tip60 retained its ability to interact with RANBP9. Using p53 null H1299 cells, we established that this interaction is independent of p53. However, RANBP9 depletion affects both total and activated p53 (p-p53s15) levels after DNA damage. Re-expressing RANBP9 in stable KO clones rescued p53 levels. Upon CDDP treatment, p53 protein levels were higher in the clones stably re-expressing RANBP9 compared to the KO ones. Upon IR or CDDP treatment, p21 protein levels were higher in the presence of RANBP9. Finally, we found that Tip60 levels were higher in CDDP-treated and untreated WT cells comparing to the KO ones. Our results indicate that RANBP9 and Tip60 constitutively interact. The decrease of p53 in RANBP9 absence suggests that RANBP9 might favor p53 acetylation by Tip60 upon DNA damage. Further investigations will elucidate how RANBP9 affects the levels and the functions of Tip60. Also, we will measure the levels of p53-AcK120 both in the WT and KO clones. If p53-AcK120 is impaired when RANBP9 is absent, RANBP9 KO cells might undergo cell death instead of cell cycle arrest. Altogether, our results shed light on a potential RANBP9-Tip60-p53 axis at play during the DDR. Citation Format: Shimaa Soliman, Arturo Orlacchio, Anna Tessari, Marina Capece, Rosa Visone, Carlo Croce, Dario Palmieri, Vincenzo Coppola. RANBP9 presence affects levels of Tip60 and activated p53 in lung cancer cells in response to DNA damage [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 3514.

Proteinase-activated receptor-2 enhances Bcl2-like protein-12 expression in lung cancer cells to suppress p53 expression.

IntroductionThe pathogenesis of lung cancer is unclear. Less expression of p53 or p53 mutation was identified in lung cancer cells, which plays a role in the development of lung cancer. Recent reports indicate that Bcl2-like protein-12 (Bcl2L12) can inhibit the expression of p53. Lung cancer cells express proteinase-activated receptor-2 (PAR2). This study tests the hypothesis that activation of PAR2 inhibits the expression of p53 in lung cancer cells.Material and methodsLung cancer cells were collected from patients with non-small cell lung cancer (NSCLC). The cells were exposed to active peptides or trypsin in the culture for 48 h. The expression of p53 was assessed by RT-qPCR and Western blotting.ResultsWe observed that lung cancer cells express Bcl2L12. Activation of PAR2 increases expression of Bcl2L12 in lung cancer cells. Bcl2L12 mediates PAR2-suppressed p53 expression in lung cancer cells. IgE-activated mast cell suppression of p53 expression in lung cancer cells can be prevented by knocking down Bcl2L12. The Bcl2L12 bound Mdm2, the transcription factor of p53, to prevent the Mdm2 from binding to the promoter of p53 and thus inhibited p53 expression in lung cancer cells. PAR2 could attenuate lung cancer cell apoptosis via inducing Bcl2L12.ConclusionsLung cancer cells express Bcl2L12, which mediates the effects of activation of PAR2 on suppressing the expression of p53 in lung cancer cells, implying that Bcl2L12 may be a novel therapeutic target for the treatment of lung cancer.

P1.03-14 Interaction Between Celecoxib and Cisplatin is Dependent on The p53 Status in Human NSCLC Cell Lines

As a cyclooxygenase-2 inhibitor, the potential of celecoxib, to enhance the efficacy of chemotherapy is being investigated in clinical trials related to cancer treatment. In this study, we investigated whether celecoxib would increase the anti-proliferative effects of cisplatin in human lung cancer cell lines. The results demonstrated the synergistic effects of celecoxib with cisplatin in wild-type p53 cells and their antagonistic effects in mutated or deleted p53 cells. Combination indices of 0.82–0.93 reflected a synergistic effect between celecoxib and cisplatin in lung cancer cells with wild-type p53. Combination indices of 1.63–3.00 reflected antagonism between celecoxib and cisplatin in lung cancer cells with mutated or deleted p53. Apoptosis was shown to increase with the addition of celecoxib and cisplatin. Apoptosis was accomplished by the downregulation of slug expression. Celecoxib increased the nuclear localization of active p53 in lung cancer cells of wild-type p53. Taken together, our data demonstrated clear synergistic effects in wild-type p53 cells and antagonistic effects in mutated or deleted p53 cells when celecoxib and cisplatin are combined.

MiR-641 Functions as a Tumor Suppressor by Targeting MDM2 in Human Lung Cancer.

Lung cancer is the leading cause of deaths due to cancer. Studies suggest an important role of microRNAs (miRNAs) in a variety of cancers, including lung cancer. In the present study, we evaluated the role of miR-641 in human lung cancer A549 cells. Quantitative RT-PCR and Western blot were used to measure mRNA and protein expression, respectively. Cell viability and cell apoptosis were respectively measured by MTT assay and flow cytometry. In addition, luciferase activity assay was used to identify the target of miR-641. The expression of miR-641 was downregulated in lung cancer tissues and lung cancer cell lines (p < 0.05 or p < 0.01). Overexpression of miR-641 significantly inhibited proliferation and induced apoptosis of lung cancer cells (p < 0.05, p < 0.01, or p < 0.001). MDM2 was identified as a direct target of miR-641. Overexpression of miR-641 decreased the expression of MDM2 and increased the expression of p53 in lung cancer cells.

HUWE1 controls the development of non-small cell lung cancer through down-regulation of p53.

Lung cancer is the most frequent cancer type and the leading cause of tumor-associated deaths worldwide. TP53 is an important tumor suppressor gene and is frequently inactivated in lung cancer. E3 ligases targeting p53, such as MDM2, are involved in the development of lung cancer. The E3 ligase HUWE1, which targets many tumor-associated proteins including p53, has been reported to be highly expressed in lung cancer; however, its role in lung tumorigenesis is unclear.Methods: The expression of HUWE1 and p53 in lung cancer cells was modulated and the phenotypes were assessed by performing soft agar colony forming assays, cell cycle analysis, BrdU incorporation assays, and xenograft tumor growth assays. The effect on tumorigenesis in genetically-engineered mice was also analyzed. The mechanism through which HUWE1 sustained lung cancer cell malignancy was confirmed by western blotting. HUWE1 expression in clinical lung cancer was identified by immunohistochemistry and validated by analyzing lung adenocarcinoma and lung squamous carcinoma samples from the Cancer Genome Atlas (TCGA) database. Finally, we assessed the association between HUWE1 expression and patient outcome using online survival analysis software including survival information from the caBIG, GEO, and TCGA database.Results: Inactivation of HUWE1 in a human lung cancer cell line inhibited proliferation, colony-forming capacity, and tumorigenicity. Mechanistically, this phenotype was driven by increased p53, which was due to attenuated proteasomal degradation by HUWE1. Up-regulation of p53 inhibited cancer cell malignancy, mainly through the induction of p21 expression and the down-regulation of HIF1α. Huwe1 deletion completely abolished the development of EGFRVIII-induced lung cancer in Huwe1 conditional knockout mice. Furthermore, survival analysis of lung cancer patients showed that increased HUWE1 expression is significantly associated with worse prognosis.Conclusion: Our data suggest that HUWE1 plays a critical role in lung cancer and that the HUWE1-p53 axis might be a potential target for lung cancer therapy.

Addiction of lung cancer cells to GOF p53 is promoted by up-regulation of epidermal growth factor receptor through multiple contacts with p53 transactivation domain and promoter

Human lung cancers harboring gain-of-function (GOF) p53 alleles express higher levels of the epidermal growth factor receptor (EGFR). We demonstrate that a number of GOF p53 alleles directly upregulate EGFR. Knock-down of p53 in lung cancer cells lowers EGFR expression and reduces tumorigenicity and other GOF p53 properties. However, addiction of lung cancer cells to GOF p53 can be compensated by overexpressing EGFR, suggesting that EGFR plays a critical role in addiction. Chromatin immunoprecipitation (ChIP) using lung cancer cells expressing GOF p53 alleles showed that GOF p53 localized to the EGFR promoter. The sequence where GOF p53 is found to interact by ChIP seq can act as a GOF p53 response element. The presence of GOF p53 on the EGFR promoter increased histone H3 acetylation, indicating a mechanism whereby GOF p53 enhances chromatin opening for improved access to transcription factors (TFs). ChIP and ChIP-re-ChIP with p53, Sp1 and CBP histone acetylase (HAT) antibodies revealed docking of GOF p53 on Sp1, leading to increased binding of Sp1 and CBP to the EGFR promoter. Up-regulation of EGFR can occur via GOF p53 contact at other novel sites in the EGFR promoter even when TAD-I is inactivated; these sites are used by both intact and TAD-I mutated GOF p53 and might reflect redundancy in GOF p53 mechanisms for EGFR transactivation. Thus, the oncogenic action of GOF p53 in lung cancer is highly dependent on transactivation of the EGFR promoter via a novel transcriptional mechanism involving coordinated interactions of TFs, HATs and GOF p53.

SIAH2 antagonizes TYK2-STAT3 signaling in lung carcinoma cells

The Janus tyrosine kinases JAK1-3 and tyrosine kinase-2 (TYK2) are frequently hyperactivated in tumors. In lung cancers JAK1 and JAK2 induce oncogenic signaling through STAT3. A putative role of TYK2 in these tumors has not been reported. Here, we show a previously not recognized TYK2-STAT3 signaling node in lung cancer cells. We reveal that the E3 ubiquitin ligase seven-in-absentia-2 (SIAH2) accelerates the proteasomal degradation of TYK2. This mechanism consequently suppresses the activation of STAT3. In agreement with these data the analysis of primary non-small-cell lung cancer (NSCLC) samples from three patient cohorts revealed that compared to lung adenocarcinoma (ADC), lung squamous cell carcinoma (SCC) show significantly higher levels of SIAH2 and reduced STAT3 phosphorylation levels. Thus, SIAH2 is a novel molecular marker for SCC. We further demonstrate that an activation of the oncologically relevant transcription factor p53 in lung cancer cells induces SIAH2, depletes TYK2, and abrogates the tyrosine phosphorylation of STAT1 and STAT3. This mechanism appears to be different from the inhibition of phosphorylated JAKs through the suppressor of cytokine signaling (SOCS) proteins. Our study may help to identify molecular mechanisms affecting lung carcinogenesis and potential therapeutic targets.

Cleistanthoside A tetraacetate-induced DNA damage leading to cell cycle arrest and apoptosis with the involvement of p53 in lung cancer cells

Lung cancer is the leading cause of cancer-related death worldwide and resistance to chemotherapeutic drugs is the major obstacle for effective treatment. The present study investigated the anticancer potential of cleistanthoside A tetraacetate (CAT), a derivative of cleistanthoside A from Phyllanthus taxodiifolius Beille on human lung cancer cells, LU-1. Multiple molecular approaches were used in this study and include measuring the anti-proliferative effect of CAT in LU-1 cells using flow cytometry; evaluating the induction of apoptosis by monitoring DNA fragmentation, phosphatidylserine externalization and activation of caspase-3 activity; and assaying the expression of regulatory proteins involved in cell cycle arrest and apoptosis using immunoblots. CAT potently inhibited LU-1 proliferation through an early G1 arrest with down-regulation of cdk4/6 and cyclin D1 proteins. CAT also inhibited DNA topoisomerase IIα activity resulting in DNA damage and increased the expression of the p53 protein with the subsequent induction of apoptosis. A decrease in the Bcl-2/Bax ratio, activation of caspase-3 activity and cleavage of PARP accompanied apoptosis. CAT is highly toxic to lung cancer and its primary targets are the inhibition of topoisomerase IIα activity and inducing apoptosis through a G1 arrest. These properties indicate that CAT is a promising anticancer agent for treatment of lung cancer.

MicroRNA HSA-miR-125a-5p induces apoptosis by activating p53 in lung cancer cells

ABSTRACTThe mature microRNA hsa-miR-125a-5p is derived from the 5′ end of pre-miR-125a. Although hsa-miR-125a-5p is dysregulated in some tumors, its specific roles in lung cancer cell apoptosis is still unknown. To study its function, the authors examined the effects of hsa-miR-125a-5p on apoptosis in lung cancer cells and investigated its probable regulatory mechanism. The authors showed that hsa-miR-125a-5p expression was lower in different lung cancer cell lines than in Human bronchial epithelial (HBE) cells by quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR). In gain-of-function experiments, the authors found that hsa-miR-125a-5p suppressed proliferation and induced apoptosis in A549 cells by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and flow cytometry, respectively. In addition, wild-type p53 mRNA and protein expression was increased by hsa-miR-125a-5p overexpression. Moreover, blocking wild-type p53 attenuated the effect of hsa-miR-125a-5p on apoptosis. In loss-of-function experiments, wild-type p53 mRNA and protein expression was decreased by blocking hsa-miR-125a-5p. The effect of hsa-miR-125a-5p inhibitor on apoptosis was also weakened by blocking wild-type p53. Taken together, these data suggest that hsa-miR-125a-5p induces apoptosis via a p53-dependent pathway in human lung cancer cells. These results provide new insight into the roles of the miR-125a family in lung cancer.

IGFBP7 is a p53 target gene inactivated in human lung cancer by DNA hypermethylation

Insulin-like growth factor binding protein 7 (IGFBP7) was considered a tumor suppressor gene in lung cancer. However, the mechanism responsible for the downregulation of this gene has not yet been fully understood. In this study, we analyzed the epigenetic inactivation of IGFBP7 expression in human lung cancer. We found that 14 out of 16 lung cancer cell lines showed decreased expression of IGFBP7 compared to control cells by real-time RT-PCR, and 42 out of 90 patients (46.7%) with primary lung tumor exhibited negative staining of IGFBP7 by immunohistochemistry analysis. The IGFBP7 expression could be restored by demethylation agent 5-aza-2′-deoxycytidine (DAC) in 7 cancer cell lines. Methylation status of IGFBP7 was further evaluated by bisulfite sequencing (BS) and methylation-specific-PCR (MSP). It turned out that low expression of IGFBP7 was associated with DNA methylation in lung cancer cell lines and in primary lung tumors (P=0.019). To explore the regulatory role of p53 on IGFBP7, we transfected a wild type p53 expression vector into lung cancer cell lines H1299, H2228, and H82. Forced expression of p53 increased IGFBP7 expression only in H82 harboring no IGFBP7 methylation, while transfection in combination with DAC induced the expression of IGFBP7 in H1299 and H2228, in which IGFBP7 was methylated. Additionally, treatment with p53 inducer adriamycin (ADR) alone or in combination with DAC increased the expression of IGFBP7 in the 3 cell lines. Our data suggest that IGFBP7 is inactivated in lung cancer by DNA hypermethylation in both lung cancer cell lines and primary lung tumors, and IGFBP7 might be regulated by p53 in lung cancer cells.

Upregulation of BNIP3 promotes apoptosis of lung cancer cells that were induced by p53

It has been shown that p53 induces cell apoptosis and the Bcl-2 family plays key roles in this process. However, the molecular mechanism of p53 apoptotic pathway is still unclear. Here, we show that overexpression of exogenous wild-type p53 induced apoptosis in lung cancer cells and high metastasis potential cells had a faster rate of apoptosis than low metastasis potential cells. The expression of pro-apoptotic gene BNIP3 was increased significantly both in Anip973 and 95D cell lines which have high metastasis ability, but not AGZY83-a or little increased in 95C cell lines which possess low metastasis ability. Overexpression of BNIP3 increases apoptotic rate induced by p53 in AGZY83-a cells. Blocking the expression of BNIP3 by siRNA in Anip973 cells decreased apoptotic rate mediated by p53. Taken together, these data suggest that high level expression of BNIP3 mediated rapid apoptosis that was triggered by p53 in lung cancer cells.

The Molecular Biology of Lung Cancer Pathogenesis

Lung cancers exhibit multiple genetic lesions including mutations activating the dominant cellular proto-oncogenes as well as those inactivating the recessive or "tumor suppressor" genes. Candidate tumor suppressor genes include those on chromosomes 1p, 1q, 3p14, 3p21.3, 3p25 (VHL gene), 5q21 (APC/MCC gene cluster), 9p21-22 (interferon gene cluster), 11p, 13q (rb gene), 16p24, and 17p (p53 gene). Mutations in p53 inactivate its transcriptional activity, while replacement of a wild-type p53 in lung cancer cells inhibits growth and tumorigenicity suggesting that p53 acts as a master growth regulatory switch. Lung cancer cells exhibit several positive autocrine growth factor loops and express nicotine receptors which could function as tumor promoting systems. In addition, they express a negative autocrine loop involving opioids and their receptors which is reversed by nicotine acting through nicotinic acetylcholine receptors. The presence of nicotine receptors suggests nicotine or its metabolites may play a direct role in lung cancer pathogenesis.