Cell Lines HCC827 Research Articles

Genome-wide profiling of micro-RNA expression in gefitinib-resistant human lung adenocarcinoma using microarray for the identification of miR-149-5p modulation.

To understand the mechanism involved in gefitinib resistance, we established gefitinib-resistant human HCC827/GR-8-1 cell line from the parental HCC827 cell line. We compared the micro-RNA expression profiles of the HCC827 cells HCC827/GR-8-1 using Agilent micro-RNA microarrays. The micro-RNAs, such as the miR-149-5p, were up- or downregulated and associated with acquired gefitinib resistance. Quantitative real-time polymerase chain reaction was then performed to verify the expression patterns of different micro-RNAs. The result showed that miR-149-5p was upregulated in the HCC827/GR-8-1 cell line. To investigate the biological function of miR-149-5p in non-small cell lung cancer cells acquired gefitinib resistance, we examined cell proliferation using a cell counting kit-8 assay. Cell viability was evaluated after the miR-149-5p mimics, inhibitors, and negative control were separately transfected into the non-small cell lung cancer cells. The results showed that the non-small cell lung cancer cells transfected with miR-149-5p mimics exhibited reduced cell motility. The drug-sensitivity assay results revealed that the overexpression of miR-149-5p effectively evaluates the half maximal inhibitory concentration values of the cell in response to gefitinib, and the downregulation of miR-149-5p can attenuate the half maximal inhibitory concentration values of the cell lines in response to gefitinib. Furthermore, the levels of miR-149-5p in the HCC827 and HCC827/GR-8-1 cells were inversely correlated with caspase-3 expression. In conclusion, this study revealed that miR-149-5p is upregulated in the HCC827/GR-8-1 cells and involved in the acquired gefitinib resistance.

MicroRNA-124 regulates the radiosensitivity of non-small cell lung cancer cells by targeting TXNRD1.

Radiation treatment remains one of the major modalities in the treatment of lung cancer. Although the majority of patients initially respond to treatment with radiation, resistance inevitably develops and leads to treatment failure. Therefore, the identification of the underlying molecular mechanisms of radiation resistance may facilitate the development of novel approaches for overcoming resistance, and enhance the efficacy of treatment with radiation in lung and other types of cancer. In the present study we established three radiation-resistant sub-cell lines derived from the radiation-sensitive lung cancer cell line HCC827. Using a polymerase chain reaction microRNA (miRNA) array, multiple miRNAs were identified to be markedly downregulated in radiation-resistant cells, including miRNA (miR)-124, miR-191 and miR-205. It was observed that overexpression of miR-124 sensitized the resistant cells to treatment with radiation and that thioredoxin reductase 1 (TXNRD1) is a novel target of miR-124. Furthermore, it was demonstrated that knockdown of TXNRD1 using small interfering RNA increased the basal level of reactive oxygen species and sensitized the cells to radiation treatment. The results of the present study demonstrated that multiple miRNAs are downregulated in radiation-resistant lung cancer cells and that downregulation of miR-124 mediates radiation resistance through the targeting of TXNRD1 mRNA expression. The present study revealed a novel molecular mechanism of miRNA-mediated radiation resistance and identified miR-124-regulated TXNRD1 as a novel therapeutic target for overcoming radiation resistance in the treatment of lung cancer.

NF-kappaB Is Involved in the Regulation of EMT Genes in Breast Cancer Cells.

The metastatic process in breast cancer is related to the expression of the epithelial-to-mesenchymal transition transcription factors (EMT-TFs) SNAIL, SLUG, SIP1 and TWIST1. EMT-TFs and nuclear factor-κB (NF-κB) activation have been associated with aggressiveness and metastatic potential in carcinomas. Here, we sought to examine the role of NF-κB in the aggressive properties and regulation of EMT-TFs in human breast cancer cells. Blocking NF-κB/p65 activity by reducing its transcript and protein levels (through siRNA-strategy and dehydroxymethylepoxyquinomicin [DHMEQ] treatment) in the aggressive MDA-MB-231 and HCC-1954 cell lines resulted in decreased invasiveness and migration, a downregulation of SLUG, SIP1, TWIST1, MMP11 and N-cadherin transcripts and an upregulation of E-cadherin transcripts. No significant changes were observed in the less aggressive cell line MCF-7. Bioinformatics tools identified several NF-κB binding sites along the promoters of SNAIL, SLUG, SIP1 and TWIST1 genes. Through chromatin immunoprecipitation and luciferase reporter assays, the NF-κB/p65 binding on TWIST1, SLUG and SIP1 promoter regions was confirmed. Thus, we suggest that NF-κB directly regulates the transcription of EMT-TF genes in breast cancer. Our findings may contribute to a greater understanding of the metastatic process of this neoplasia and highlight NF-κB as a potential target for breast cancer treatment.

Expression of AdipoR1 and AdipoR2 Receptors as Leptin-Breast Cancer Regulation Mechanisms.

The development of breast cancer is influenced by the adipose tissue through the proteins leptin and adiponectin. However, there is little research concerning AdipoR1 and AdipoR2 receptors and the influence of leptin over them. The objective of this work was to analyze the expression of AdipoR1 and AdipoR2, modulated by differential concentrations of leptin in an obesity model (10 ng/mL, 100 ng/mL, and 1000 ng/mL) associated with breast cancer in MCF-7 and HCC1937 cell lines. Each cell line was characterized through immunohistochemistry, and the expression of AdipoR1 and AdipoR2 was analyzed by PCR in real time using TaqMan® probes. Leptin induced an increase in cell population of MCF-7 (23.8%, 10 ng/mL, 48 h) and HCC1937 (17.24%, 1000 ng/mL, 72 h). In MCF-7, the expression of AdipoR1 decreased (3.81%, 1000 ng/mL) and the expression of AdipoR2 increased by 13.74 times (10 ng/mL) with regard to the control. In HCC1937, the expression of AdipoR1 decreased by 86.28% (10 ng/mL), as well as the expression of AdipoR2 (50.3%, 100 ng/mL). In regard to the results obtained, it could be concluded that leptin has an effect over the expression of AdipoR1 and AdipoR2 mRNA.

Variant CD44 expression is enriching for a cell population with cancer stem cell-like characteristics in human lung adenocarcinoma.

Background: Preliminary studies have identified cancer stem cells (CSCs) in various cancers and there are several ongoing clinical studies targeting these cells. CD44 (standard or variant isoforms) and/or aldehyde dehydrogenase (ALDH) expression is the most commonly used markers for the identification of CSCs. The goal of the current study was to examine the ability of CD44v, either alone or in combination with ALDH, to identify CSCs within human lung cancer cells lines.Methods: We examined several lung adenocarcinoma cell lines for the ability of CD44v and/or ALDH expression to enrich for cells with CSC characteristics such as in vitro differential proliferation rate, chemotherapeutic-resistance, tumorsphere formation, and in vivo tumorigenicity. We also compared their in vivo secondary tumor formation, and histological characteristics of their xenograft tumors, and examined their expression of PD-L1, EGFR, xCT, and reactive oxygen species (ROS).Results: Both CD44vhigh/ALDHhigh and CD44vhigh/ALDHlow cells were enriched in cells with CSC characteristics, with the CD44vhigh/ALDHlow cells being more proliferative and more resistant to chemotherapeutics, whereas CD44vhigh/ALDHhigh cells were more efficient in forming tumorspheres in vitro, in making primary xenograft tumors, and in propagating secondary tumors in vivo. Applying stricter sorting gates to select for cells with the highest CD44v/ALDH expression caused the CD44vhigh/ALDHlow cells to lose their high proliferation rates and chemotherapeutic resistance ability, but enriched for the tumorsphere-forming cells among the CD44vhigh/ALDHhigh and CD44vhigh/ALDHlow cells. CD44vhigh expression was associated with PD-L1 and xCT expression in both H1650 and HCC827 cells. This association was not modified by ALDH expression in the H1650 cell line. However, in the HCC827 cell line, ALDH expression was negatively associated with PD-L1 and positively associated with xCT expression.Conclusion: Lung adenocarcinoma cells with high CD44v expression are enriched for CSCs. Addition of ALDH as an enrichment marker bestowed some CSCs characteristics to CD44vhigh/ALDHlow cells and others to CD44vhigh/ALDHhigh cells. We propose that lung adenocarcinoma contains different CSCs, each of them endowed with different CSC characteristics.

Wnt3a Expression Is Associated with Epithelial-Mesenchymal Transition and Impacts Prognosis of Lung Adenocarcinoma Patients.

Background: Epithelial-mesenchymal transition (EMT) plays an important role in the invasion and migration during cancer metastasis. Wnt3a is one of the ligands in canonical Wnt/β-catenin signaling pathway, which contributes to the carcinogenesis and progression of lung cancer cell lines. The aim of this study was to evaluate the association between Wnt3a and EMT-related proteins (E-cadherin and N-cadherin), and to further investigate its impact on prognosis of lung adenocarcinoma patients.Methods: A total of 147 lung adenocarcinoma patients were included and their clinicopathological characteristics were collected in this retrospective study. The expression levels of Wnt3a, E-cadherin and N-cadherin in post-surgery cancerous and adjacent normal tissues were assessed by immunohistochemistry. The association between Wnt3a and EMT-related proteins and their prognostic values were systematically evaluated. HCC827 and PC9 cell lines were treated with Wnt3a to detect the expression of EMT-related and Wnt/β-catenin signaling-associated proteins, as well as the in vitro migration and invasion abilities.Results: High Wnt3a expression level was significantly associated with low E-cadherin (P<0.001) and high N-cadherin (P<0.001) expression levels in lung adenocarcinoma tissues. Besides, high Wnt3a level predicted poorer lung adenocarcinoma survival by univariate Cox analysis (P=0.001), while the multivariate result was not significant (P=0.355). Subgroup analysis suggested that the prognostic value of Wnt3a expression level was significant in stage T1-T2 (log rank P=0.003) and stage N0 (log rank P=0.031) patients. The multivariate Cox analysis suggested N-cadherin was an independent prognostic factor for lung adenocarcinoma patients (P=0.012). After including these markers into a nomogram, the Harrell's C-index of the nomogram was 0.755. The decision-curve analysis of our nomogram performed net benefit at the threshold probability from 21.6% to 82.0%, and the current model had a better prognostic value than TNM-classification with a lower Akaike information criterion (AIC) value of 166.54. In vitro experiments suggested that Wnt3a could regulate EMT-related proteins and promotes in vitro invasion and migration abilities.Conclusions: Wnt3a could regulate EMT-related proteins and promote the migration and invasion process of lung adenocarcinoma. Although its value as an independent prognostic factor was limited, the combined model suggested good prognostic performance for lung adenocarcinoma patients.

P3.02b-041 Genome-Wide Screen of DNA Methylation Changes Reveals GABBR2 as a Novel Potential Target for EGFR 19 Deletion Adenocarcinoma with Erlotinib: Topic: EGFR Biomarkers

Lung cancer is the leading cause of cancer related death around the world. The last decade has witnessed the rapid development of epidermal growth factor receptor (EGFR) directly targeted therapies that have significantly changed the treatment of non-small-cell lung cancer (NSCLC). However, the challenge of acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) has been an issue when talking with activating EGFR mutations, which makes it crucial to elucidate the mechanism of EGFR-TKI targeted drug resistance. Methyl-sensitive cut counting sequencing (MSCC), one of the most commonly used whole-genome DNA methylation sequencing technology, was applied to investigate the changes of paired tissue DNA methylation before and after TKI (erlotinib) treatment lasting two cycles with partial response (PR) for stage IIIa (N2) lung adenocarcinoma patients (N=2) with activating EGFR 19 deletion. Sequenom EpiTYPER assay method was further analyzed to double confirm the changed methylated candidate genes in these two patients, through comparing the methylated changes in paired tissues before and after TKI treatment. Western blotting, cell cycle and apoptosis analysis by the up/down regulation of a candidate gene (GABBR2) were performed in three lung adenocarcinoma cell lines, A549 (EGFR wild type), HCC4006 (EGFR 19 deletion, DelL747-E749) and HCC827 (EGFR 19 deletion, DelE746-A750), to elucidate the mechanism of GABBR2 gene in the regulation of EGFR-TKI treatment. Sixty aberrant methylated genes were firstly discovered using MSCC method in these two patients harboring EGFR 19 del mutation treated with erlotinib. Two aberrant methylated genes, CBFA2T3 and GABBR2, were clearly validated by the Sequenom EpiTYPER assay subsequently. GABBR2 was significantly down regulated in HCC4006 and HCC827 cells harboring EGFR 19 mutations but remained conservative in A549 cells with EGFR wild-type after erlotinib treatment by Western blotting. The phenomenon was in line with the obvious apoptosis of HCC4006 and HCC827 cell lines after erlotinib treatment, but not in A549 cells. GABBR2 was further induced down regulation after erlotinib exposure through apoptosis method silenced by siRNA using RNAi technology. Meanwhile, GABBR2 gene was demonstrated up regulation rescued the apoptosis significantly, when overexpressing GABBR2 in HCC827 cell lines along with erlotinib treatment. Genome-wide screen of DNA methylation changes demonstrated that GABBR2 gene might be as a novel potential treatment target for stage IIIa (N2) EGFR 19 deletion adenocarcinoma with erlotinib treatment. Our research provides a new theoretical basis for the epigenetic study of EGFR mutated lung adenocarcinoma treatment.

MEK inhibitors against MET-amplified non-small cell lung cancer.

Several receptor tyrosine kinases (RTKs) including EGFR, ALK, and MET have been identified as therapeutic targets in non-small cell lung cancer (NSCLC). Among the downstream pathways of RTKs, the MAPK pathway is particularly important for cancer cell proliferation, differentiation, and survival. In this study, the effects of MEK inhibitors (trametinib and PD0325901) in several NSCLC cell lines with driver gene alterations, especially RTK genes, were tested in vitro using an MTT assay, and a wide range of sensitivities was found. In particular, all the EGFR-mutated cell lines were resistant to MEK inhibitors, whereas all the MET-amplified cell lines were sensitive. A bioinformatics technique and western blot analyses showed that the PI3K/AKT pathway is more activated in EGFR-mutated NSCLC than in MET-amplified NSCLC, and a PI3K inhibitor enhanced the sensitivity to trametinib in the EGFR-mutated cell lines, suggesting that this pathway is associated with resistance to MEK inhibitors. Although the HCC827 cell line (EGFR mutation) was resistant to MEK inhibitors, the HCC827CNXR cell line, whose driver gene shifts from EGFR to MET, exhibited enhanced sensitivity to MEK inhibitors, indicating the biological importance of the MAPK pathway for MET-amplified NCSLC. Furthermore, a synergistic effect of crizotinib (a MET inhibitor) and trametinib was observed in MET-amplified NCLC cell lines. Our findings indicate that the MAPK pathway is biologically important for MET-amplified NSCLC and strongly encourage the development of combination therapy with a MET inhibitor and a MEK inhibitor against MET-amplified NSCLC.

MicroRNA-186 suppresses cell proliferation and metastasis through targeting MAP3K2 in non-small cell lung cancer.

MicroRNAs are a class of small endogenous non-coding RNAs that play crucial roles in the initiation and progression of human cancers. miR-186 was found decreased in various human malignancies and function as a tumor suppressor. However, the regulating mechanism of miR-186 in growth and metastasis of human non-small cell lung cancer (NSCLC) is still poorly understood. We investigated the role of miR-186 in the growth and metastasis of human NSCLC. In the present study, we found that miR-186 was significantly decreased in lung cancer tissues and cells. Furthermore, overexpression of miR-186 suppressed lung cancer cell proliferation, migration and invasion, and induced cell apoptosis. Moreover, we found that confirmed mitogen-activated protein kinase kinase kinase 2 (MAP3K2) protein was increased in lung cancer tissues and confirmed that MAP3K2 is a target gene of miR-186. In addition, knockdown of MAP3K2 by RNA interference inhibited lung cancer cell proliferation, migration and invasion, and promoted cell apoptosis invitro. Furthermore, we observed tthat the overexpression of MAP3K2 partially reversed the inhibitory effect of miR-186 on the proliferation and metastasis of A549 and HCC827 cell lines. Taken together, our data indicated that miR-186 regulates lung cancer growth and metastasis through suppressing MAP3K2 expression, at least partly. Therefore, miR-186-MAP3K2 may represent a new and useful potential clinical treatment and diagnosis target for NSCLC.

Abstract B16: Lymphatics in triple-negative breast cancer: Contribution to chemotherapeutic efficacy in vitro and in vivo

Abstract Background: Triple-negative breast cancer (TNBC) accounts for approximately 15% of breast cancers and is associated with a poorer prognosis and higher rate of distant recurrence compared to receptor positive breast cancer. Due to an absence of effective targeted therapies, cytotoxic chemotherapy remains the standard of care for TNBC patients. However, the aggressive nature of these tumors makes preventing progression to metastatic disease a priority in therapeutic strategy. Inhibition of lymphatic activation represents an attractive approach to reduce peritumoral lymphatic vessel recruitment and attenuate cancer cell invasion to lymph nodes and subsequent metastasis to distant sites. However, little is currently known regarding the role of lymphatics in TNBC and how common therapeutic strategies can influence lymphatic reactivity. We hypothesized that inhibiting lymphatic activation in combination with standard of care chemotherapy will enhance therapeutic response in TNBC. Methods: Our lab has developed a novel 3D tissue-engineered tri-culture system that recapitulates the tumor-lymphatic interface within the breast tumor microenvironment. It consists of a porous tissue culture insert on which we seed human lymphatic endothelial cells (LECs) to form a monolayer on the underside. Atop the insert, we place Collagen I gel with human mammary fibroblasts and human breast cancer cells labeled with fluorescent Cell Tracker dye. After gelation, flow is applied via a pressure head and the system equilibrates overnight (16-18h). Drugs are applied via flow and then flushed from the system with basal media. After 48h from first application, gels are removed, dissociated, and analyzed via flow cytometry for death [Live/Dead and apoptosis (Caspase 3/7)], while the inserts are analyzed via microscopy for invaded cancer cells. Animal experiments were performed according to guidelines set forth by the UVA Animal Care and Use Committee. Twenty female Balb/c mice were implanted with 250,000 4T1 cells and treated with either anti-VEGFR3 antibody or IgG control and docetaxel (n=5 per group) or saline control once tumors were palpable. Tumors were dissected and dissociated once they reached 150 mm3, and analyzed via flow cytometry for stromal cell populations. Lungs were analyzed for metastases by cryosectioning and H&amp;E staining. Results: We used our in vitro system to screen the common chemotherapeutics docetaxel, doxorubicin, 5-FU, and carboplatin across three common TNBC cell lines MDAMB231, HCC1806, and HCC38. We found that the presence of LECs significantly increases TNBC cell survival and invasiveness, shifting EC50s for cell death 10-1000 fold (EC50 shift nonlinear regression, p&amp;lt;0.05 for +/- LECs) and doubling to tripling invasion of cancer cells (analysis by two-way ANOVA, p&amp;lt;0.05). This poor response to therapy can be attenuated by treatment with the VEGFR3 inhibitor MAZ51. In vivo, we have seen that adjuvant anti- VEGFR3 therapy can reduce lymphatic endothelial cell populations in the tumor stroma with and without docetaxel chemotherapy when analyzed by flow cytometry (n=4 per group, p&amp;lt;0.05). Additionally, combination-treated mice showed reduced metastasis to the lung. Conclusion: Together, these data could indicate the effectiveness of anti-VEGFR3 therapy as an addition to chemotherapy to reduce metastatic progression of TNBC by limiting tumor-induced and therapy-induced lymphangiogenesis. Studies are ongoing to optimize and understand the ability of this adjuvant therapeutic strategy to increase therapeutic efficacy in TNBC. Citation Format: Alexandra H. Harris, Jennifer M. Munson. Lymphatics in triple-negative breast cancer: Contribution to chemotherapeutic efficacy in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B16.

Met gene amplification and protein hyperactivation is a mechanism of resistance to both first and third generation EGFR inhibitors in lung cancer treatment

The 3rd generation epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs; e.g., AZD9291), which selectively and irreversibly inhibit EGFR activating and T790M mutants, represent very promising therapeutic options for patients with non-small cell lung cancer (NSCLC) that has become resistant to 1st generation EGFR-TKIs due to T790M mutation. However, eventual resistance to the 3rd generation EGFR-TKIs has already been described in the clinic, resulting in disease progression. Therefore, there is a great challenge and urgent need to understand how this resistance occurs and to develop effective strategies to delay or overcome the resistance. The current study has demonstrated that Met amplification and hyperactivation is a resistance mechanism to both 1st and 3rd generation EGFR-TKIs since both erlotinib- and AZD9291-resistant HCC827 cell lines possessed amplified Met gene and hyperactivated Met, and were cross-resistant to AZD9291 or erlotinib. Met inhibition overcame the resistance of these cell lines to AZD9291 both in vitro and in vivo, including enhancement of apoptosis or G1 cell cycle arrest. Hence, we suggest that Met inhibition is also an effective strategy to overcome resistance of certain EGFR-mutated NSCLCs with Met amplification to AZD9291, warranting the further clinical validation of our findings.

Abstract 876: Genome-scale genetic knockout screen identifies modifiers of EGFR dependence in non-small cell lung cancer cells

Abstract Non-small cell lung cancers with epidermal growth factor receptor (EGFR) gene mutations can exhibit a strong dependence on its signaling for growth and survival. They are also sensitive to EGFR tyrosine kinase inhibitors (TKIs), which provide superior clinical benefits to conventional chemotherapy. However, despite initial response, most patients experience relapse with resistant tumors within a year. This study aims to identify modifiers of dependence on mutant EGFR signaling and the mechanisms by which they do so in order to improve therapeutic strategies and outcomes. We first performed a genome-scale CRISPR-Cas9 genetic knockout screen. In this system, gene silencing occurs by site-specific double-strand-breaks induced by the Cas9 nuclease that is localized to its target by a single-guide RNA (sgRNA). The error-prone repair pathway introduces insertion/deletion mutations leading to frameshifts or premature stop codons. We constructed a pooled sgRNA library targeting more than 18,000 protein-coding human genes with multiple sgRNA vectors. The cell line HCC827 was used in our screen as it is EGFR-mutant and sensitive to EGFR TKIs. We transduced HCC827 lung cancer cells with the pooled sgRNA library and cultured them in the presence of Erlotinib, an EGFR TKI, or DMSO control for 17 days. At the end of the treatment period we identified sgRNAs that were enriched in Erlotinib-treated groups over control groups, indicating genes whose loss-of-function confer TKI resistance. We applied the RNAi gene enrichment ranking (RIGER) algorithm to identify gene hits with enrichment of multiple sgRNAs. Top-ranked candidates include previously confirmed genes PTEN, NF1, NF2, TSC1, and TSC2, validating this system as a means to identify modifiers of EGFR dependence in HCC827 cells. A novel candidate gene is the E3 ubiquitin ligase HUWE1. We showed that suppression of HUWE1 by sgRNAs or inducible short hairpin RNA (shRNA) in HCC827 cells re-activated AKT and ERK1/2 signaling pathways and increased cell proliferation and survival in response to EGFR inhibition. These findings were confirmed in vivo by implanting mouse xenografts of HCC827 cells expressing shHUWE1 and monitoring tumor development in response to EGFR-TKI therapy. Tumors expressing HUWE1 led to durable tumor regression throughout the treatment period but tumors with suppressed HUWE1 continued to grow into large tumors. Immunohistochemistry analysis of excised tumors also revealed increased AKT and ERK1/2 signaling as well as increased proliferation and decreased apoptosis. We have shown that dependence on EGFR signaling can be decreased in EGFR-mutant lung cancer cells through mechanisms that involve the activation of AKT and ERK1/2 signaling pathways. Ongoing studies involve identifying HUWE1 substrates/interactions that participate in tumor cell response to EGFR inhibitors, revealing a novel mechanism of resistance to EGFR-targeted therapy. Citation Format: Jon DiMaina, Chris Duckworth, Hiu Wing Cheung. Genome-scale genetic knockout screen identifies modifiers of EGFR dependence in non-small cell lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 876.

Abstract 2947: MicroRNA 10b, 27a and 27b are involved in the resistance to treatment with epidermal growth factor tyrosine inhibitor (EGFR-TKI) in EGFR mutant lung cancer cell line

Abstract EGFR TKIs are now standard of care in lung cancer patients with EGFR mutation. Invariably, these patients develop resistance and require treatment with another EGFR TKI or chemotherapy. The gate-keeper mutation T790M is responsible for about 50% of resistance. Rocelitinib(R) is a third generation EGFR TKI active in lung cancer cells with T790M mutation. Other mechanisms may also confer resistance in these patients. High expression of microRNA (miR)-10 is associated with worse prognosis in resected lung cancer patients with EGFR mutation. Both miR-27a and 27b are associated with increased expression of c-met which is a potential mechanism of resistance to EGFR TKI. We hypothesized that miR 10b and 27a and 27b modulate resistance in E-resistant lung cancer cells. Material and Methods Lung cancer cell lines with EGFR mutation HCC827 and HCC4006 were treated with 5 nM of Erlotinib (E) for 72 hours. E-resistant cells were then treated with R at 21.5 nM for 72h. The cells were harvested and microRNA profiling was performed by real time PCR. HTB177 cell line without EGFR mutation was used as control. Results We observed increased expression of miR-10b and miR-27a in HCC4006 compared to control cells HTB 177 cells; miR-27b is upregulated in HCC827 cell line. The increased expression of miR-10b and miR 27a were higher in CRL-2871 than the control cells 9-fold and 8-fold, respectively. MicroRNA- 27b was increased 300-fold in HCC827 compared with control. Treatment with R reduced the proliferation in HCC827 to 30% and in HCC4006 to 50% compared to control. It did not inhibit the proliferation of HTB177 cells. R increase miR-10b in HCC4006 by 3-fold, miR-27a by 1.5-fold; in HCC827 it reduced miR-10b to 0.4, miR-27a to 0.4 and miR-27B 0.5. Conclusion We observed upregulation of miR-10b, 27a and 27b in lung cancer cells lines with EGFR mutation that were resistant to E treatment. Treatment of E resistant cells with R reduced proliferation and changed the expression of miR-10b, 27a and 27b. Our results suggest an epigenetic mechanism of resistance in addition to the T790M mutation. MicroRNAs may be studied as targets for developing novel approach for to treating patients with EGFR TKI resistance. Citation Format: Hannah Montes, Emma B D Reyes, Mukut Sharma, Chao H. Huang. MicroRNA 10b, 27a and 27b are involved in the resistance to treatment with epidermal growth factor tyrosine inhibitor (EGFR-TKI) in EGFR mutant lung cancer cell line. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2947.

Abstract 4461: Epigenetics in doxorubicin and docetaxel-resistant triple negative breast cancer

Abstract Triple negative breast cancers (TNBC) do not express estrogen receptor, progesterone receptor, or Her2/neu hence there are no targeted therapies for this type of breast cancer. Chemotherapy is the systemic treatment option with doxorubicin and docetaxel being the two most commonly used drugs. It is well known drug resistance can lead to treatment failure in these patients. It is therefore essential to uncover information on the mechanism of these resistance pathways, and identify novel molecular markers for developing potential pathway driven treatment. Our previous studies have shown that genes, ataxia telangiectasia mutated (ATM), TGF-β, Bcl-2, NDRG1 and NDRG2 are closely related to cancer stem cell marker CD24 expression, and drug resistance toward doxorubicin and docetaxel in TNBC. In this study, two TNBC cell lines HCC1806 and HCC1937 were epigenetically examined by ChIP-qPCR. The gene promoters of the above five genes, within a 1kb range were analyzed. Results showed that both drugs significantly decreased total Histone 3 quantity, which is representative of total nucleosome density, at ATM and TGFB1 promoters, and additionally doxorubicin at NDRG1 promoter. Doxorubicin also decreased histone acetylation and methylation of H3K9 significantly at NDRG1 and consistently at the other gene promoters, while docetaxel showed consistent increase of methylation at H3K9. Results suggest that treatment with the two drugs affects an epigenetic pathway involving one or several epigenetic cofactors (methyltransferases, acetyltransferases, nucleosome remodeling complexes, etc.). Elucidating this pathway may provide valuable information for the treatment of TNBC. Citation Format: Terra Sztain, Helena Chang. Epigenetics in doxorubicin and docetaxel-resistant triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4461.

Abstract 4665: The combination of polo-like kinase 1 inhibition and erlotinib overcomes T790M-mediated drug resistance in vitro and in vivo in non-small cell lung cancer

Abstract Introduction: Activation of epidermal growth factor receptor (EGFR) leads to the development and progression of human epithelial cancers, including non-small cell lung cancer (NSCLC). EGFR tyrosine kinase inhibitors (EGFR-TKI) are effective therapy for NSCLC with activating EGFR mutations. However, acquired resistance to EGFR-TKIs is unavoidable and occurs through various molecular mechanisms including the development of T790M secondary EGFR mutations, MET amplification, epithelial-mesenchymal transition (EMT) and other mechanisms. One of potential strategy to overcome EGFR-TKIs is through inhibition of polo-like kinase 1 (PLK1). PLK1 is one of the key regulators of mitotic progression and a DNA damage recovery checkpoint. PLK1 inhibitors are at various stages of clinical development. Our previous studies showed that mesenchymal NSCLC cell lines are more sensitive to PLK1 inhibitor than epithelial ones. This study examines the efficacy of PLK1 inhibition in NSCLC cell lines with acquired resistance to the EGFR-TKI erlotinib. Methods: Erlotinib resistant cell lines were developed in EGFR mutant, erlotinib sensitive cell lines PC9, HCC4006 and HCC827 by chronic exposure to stepwise increased concentrations of erlotinib. The effects of the PLK1 inhibitor volasertib alone or combined with erlotinib on proliferation, apoptosis, cell cycle, EGFR-dependent signaling, DNA damage and DNA damage response signaling were evaluated using CellTiter-Glo assay, TUNEL assay, BrDU incorporation assay, comet assay, western blot and immunofluorescence. Sensitivity of volasertib alone or in combination with erlotinib was also detected in the human tumor xenograft model. The combination index (CI) was calculated by Calcusyn software. Results: Acquired erlotinib resistant NSCLC cell lines with endogenous EGFR mutations (PC9, HCC4006 and HCC827) showed diverse resistance mechanisms: T790M EGFR mutation, MET amplification and EMT. Four of the 6 ER clones that underwent EMT became more sensitive to volasertib. Volasertib and erlotinib demonstrated synergistic effects only in cells bearing T790M EGFR mutations where we observed more pronounced G2M arrest and increased apoptosis. Volasertib alone or in combination showed enhanced DNA damage, activation of CHK1/ATR and CHK2/ATM pathways and an increase in γH2AX foci but only a modest effect on canonical signaling downstream of EGFR. The combination treatment exhibited a pronounced reduction in the size of tumor compared to single agent treatment in subcutaneous xenograft model generated with T790M mutant EGFR TKI-resistant PC9 cell line. Conclusions: These data demonstrate that PLK1 inhibition alone induces apoptosis and DNA damage in EGFR-TKI resistant cell lines with EMT. The combination of volasertib and erlotinib overcomes T790M-mediated drug resistance in vitro and in vivo. Citation Format: Ratnakar Singh, Yuehong Wang, Liguang Wang, Monique Nilsson, Ruchitha Goonatilake, Pan Tong, Lerong Li, Uma Giri, Jing Wang, John V. Heymach, Faye M. Johnson. The combination of polo-like kinase 1 inhibition and erlotinib overcomes T790M-mediated drug resistance in vitro and in vivo in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4665.

Abstract 3063: Discovery of a third-generation EGFR inhibitor, which is highly selective and potent against both resistant and activating EGFR mutations for NSCLC therapy

Abstract EGFR T790M mutation which renders resistance to the first-generation EGFR inhibitors,accounts for about 50% of all originally responsive non-small cell lung cancer patients. In order to overcome this type of resistance, we have developed a small molecular drug candidate with unique structural features that offer differentiated pharmacological and safety profiles pre-clinically when compared with other leading third-generation EGFR inhibitors. The compound selectively inhibits T790M as well as activating EGFR mutations, with much less inhibition to the wild type EGFR. Enzymatic inhibition IC50 against EGFR T790M,T790M/Del19 and T790M/L858R is less than 1nM. In addition, enzymatic inhibition IC50 against EGFR activating mutations Del19 and L858R is around 1nM, with much less inhibition to the wild type EGFR, a 10-time selection ratio. In cell based assays, the compound potently inhibits proliferation of EGFR T790M mutated NSCLC cell lines H1975(T790M/L858R)and PC9-GR(T790M/Del19)with an IC50 around 3nM. Similarly, it potently inhibits EGFR activating mutant NSCLC cell lines HCC827(Del19)and PC9(Del19). However, the compound displays about 200-fold less sensitivity to the EGFR wild type cancer cell line A431, with an IC50 around 600nM. When dosed to tumor-bearing animals, the compound dose-dependently induces profound regression of xenografts derived from both H1975 and primary human lung cancer LU1868, both of which carry an EGFR T790M mutation. It also induces regression of HCC827 xenografts potently, similarly to the first- and second-generation EGFR inhibitors; whereas it displays much less inhibition to the EGFR wild type A431 xenografts, demonstrating a higher specificity to the mutated EGFR forms when compared with the EGFR inhibitors on market. The drug candidate displays favorable PK and safety profiles. These properties warrant further development of this compound as first- as well as second-line therapy for NSCLC. Citation Format: Rudi Bao, Peng Gao, Fujun Zhang, Zhaolong Tong, Hongping Yu, Yaochang Xu. Discovery of a third-generation EGFR inhibitor, which is highly selective and potent against both resistant and activating EGFR mutations for NSCLC therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3063.

Abstract 2940: The p53 mutation R273H contributed to drug resistance of EGFR tyrosine kinase inhibitors

Abstract Tyrosine kinase inhibitors (TKIs) of epidermal growth factor receptor (EGFR) were the standard first-line treatments for lung cancers with activating EGFR mutations. Emergent drug resistance is the major problem in the patients who received treatment of EGFR TKIs. Second point mutation T790M mutations could be found in 50-60% patients who had drug resistance to EGFR TKIs. Alternative pathway of Met signaling, transformation to small cell lung cancer, over-expression of other HER family receptors or epithelial mesenchymal transit were reported in these patients who had drug resistance to EGFR TKIs. NFKB pathway played a role of early resistance to EGFR TKIs in the patients. The p53 mutation could be found in 70-80% lung adenocarcinoma patients. These mutations could be found in lung cancer patients who had wild type or mutated EGFR. Lung adenocarcinomas with mutated p53 lose tumor suppressor function of wild type p53 and have new function from mutated p53. We thought p53 mutation may have gain of function and result in drug resistance to EGFR TKIs in lung cancer patients. We knocked down p53 in three cancer cell line, MDA-MB-468 (p53 R273H), BT-549 (p53 R249S) and H322 (p53 R248L). We found knockdown of p53 R273H in MDA-MB-468 increased sensitivity to gefitinib. Knockdown of p53 R249S and p53 R248L showed the same response to gefitinib in cancer cell lines BT-549 and H322. Lung cancer cell line HCC827 had EGFR exon 19 deletion and was sensitive to gefitinib. We overexpressed p53 R273H, p53 R157H and p53 R249S in HCC827 lung cancer cell line. Overexpression of p53 R273H increased resistance to gefitinib in HCC827 lung cancer cell line. Overexpression of p53 R157H and p53 R249S did not increased the resistance to gefitinib. Conclusion: Gain of function of p53 R273H increased resistance to EGFR TKIs in cancer cell lines. Further immunohistochemistry staining and gene analysis of mutated p53 will be done in lung adenocarcinoma from lung cancer patients who received EGFR TKIs. Citation Format: Yueh-Fu Fang, Chun-Yu Lin, Peichih Lee. The p53 mutation R273H contributed to drug resistance of EGFR tyrosine kinase inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2940.

Abstract 1973: HER2 promotes super enhancer formation in breast cancer

Abstract Background. HER2 positive (HER2+) breast cancer (BCa) occurs in 25-30% of cases and is associated with an aggressive phenotype. Multiple HER2-stimulated pathways are known but the genes that are specifically controlled by HER2 are poorly understood. We previously used RNA polymerase II (POL II) immunoprecipitation (ChIP) to identify 737 genes that bind POL II in HER2+ but not in HER2- BCa cell lines. 51 of these genes were differentially expressed in a HER2+ dependent manner and 113 genes were differentially expressed in a HER2-dependent manner in breast tumors from 812 patients. The 113 genes are not differentially expressed in cell lines but expressed in BCa cases can be considered “poised” for expression, and activated when in the context of a breast tumor. Methods. The 737 genes were examined using pathway finding tools and the MIT database for Super-enhancers and the dbSUPER database (2). The expression of certain genes in attached cell cultures and in mammosphere cultures were examined by qPCR. Results. Some of the 113 genes that are “poised” for expression in cell lines in a HER2-dependent manner, and then expressed in breast cancers in a HER2-dependent manner, encode proteins that commonly occur in Super-enhancers, which are a variety of DNA regulatory structures formed by looping that associate multiple bound transcription and DNA-modifying factors in proximity to target promoters. Examples include Mediator12 (MED12) and the Bromdomain protein 2 (BRD2), and DNA binding factors such as HDAC and CREB1-cofactors. Moreover, many pluripotency genes are also found in the list of 113 genes; NANOG, OCT3/4, and SOX2 (NOS genes). These genes commonly interact with SEs as associated factors and as targets. We confirmed by qPCR that the three NOS genes exhibit increased expression in mammosphere of HER2+ MCF7 cells but not in control cells. Using the MIT and dbSUPER (2) database we determined that 70 genes of the 113 genes (62%) that are located in or near SEs, including some present in the HER2+ BCa cell line HCC1954. Similarly, 33 (65%) of the 51 differentially expressed genes in HER2+ cell lines were associated with SEs in many diverse cell types. For comparison, the class of 573 genes that are bound to POL II in HER2+ cell lines, but are not differentially expressed in cell lines or breast cancer tissues, have a much lower fraction of genes, 213 or 37%, that encode potential members or targets of SE. The difference is highly significant, p = 0.008. Pathway analyses of the 113 genes indicates that major pathways enriched in these genes (p &amp;lt; 0.001) include EGFR and Map Kinase signaling, notch pathway signaling, hedgehog, Wnt, Integrin-mediated cell and cytokine signaling Interleukin 1 and Interleukin 14. Conclusion. These results suggest that the pathways associated with HER2 over-expression in BCa include genes associated with Super-enhancer assembly, as well as many genes encoded close to the location of Super-enhancers. 1.Rahmatpanah et al,. (2015) Oncotarget 2. Aziz Khan, Nucleic Acids Res., 2015 Citation Format: Farah Rahmatpanah, Zhenyu Jia, Yuanjie Hu, Frank Jones, Michael McClelland, Dan Mercola. HER2 promotes super enhancer formation in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1973.

Abstract 2105: c-Met hyperactivation is an universal resistance mechanism to both first and third generation EGFR inhibitors

Abstract c-Met amplification and acquisition of a second T790M mutation are key mechanisms accounting for majority of resistant cases to first generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs; i.e., erlotinib). The third generation EGFR-TKIs (e.g., AZD9291), which selectively and irreversibly inhibit EGFR activating and T790M mutants while sparing wild-type EGFR, represent very promising therapeutic options for NSCLC patients who have become resistant to 1st generation EGFR-TKIs due to T790M mutation. However, eventual resistance to the 3rd generation EGFR-TKIs has already been described in the clinic, resulting in disease progression. Therefore, there is a great challenge and urgent need to understand how this resistance occurs and to develop effective strategies to delay or overcome the resistance. We show that c-Met amplification and hyperactivation is an universal mechanism to both 1st and 3rd generation EGFR-TKIs since both erlotinib- and AZD9291-resistant HCC827 cell lines possessed elevated levels of c-Met (due to gene amplification) and p-c-Met and were cross-resistant to AZD9291 or erlotinib. Both chemical and genetic inhibition of c-Met overcame the resistance of these cell lines to AZD9291 including enhancement of apoptosis or G1 cell cycle arrest. Consistently the combination of AZD9291 and c-Met inhibition effectively inhibited the growth of both erlotinib- and AZD9291-resistant HCC827 xenografts in nude mice. Hence, we suggest that inhibition of c-Met is also an effective strategy to overcome resistance of EGFR-mutated NSCLCs with c-Met amplification or hyperactivation to AZD9291, providing the rationale for clinical development of this novel combination strategy. (SSR, TKO and SYS are Georgia Research Alliance Distinguished Cancer Scientists) Citation Format: Puyu Shi, You-Take Oh, Guojing Zhang, Weilong Yao, Ping Yue, Rajani Kanteti, Jacob Riehm, Ravi Salgia, Taofeek Owonikoko, Suresh S. Ramalingam, Mingwei Chen, Shi-Yong Sun. c-Met hyperactivation is an universal resistance mechanism to both first and third generation EGFR inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2105.

YAP promotes erlotinib resistance in human non-small cell lung cancer cells.

Yes-associated protein (YAP) is a main mediator of the Hippo pathway, which promotes cancer development. Here we show that YAP promotes resistance to erlotinib in human non-small cell lung cancer (NSCLC) cells. We found that forced YAP overexpression through YAP plasmid transfection promotes erlotinib resistance in HCC827 (exon 19 deletion) cells. In YAP plasmid-transfected HCC827 cells, GTIIC reporter activity and Hippo downstream gene expression of AREG and CTGF increased significantly (P<0.05), as did ERBB3 mRNA expression (P<0.05). GTIIC reporter activity, ERBB3 protein and mRNA expression all increased in HCC827 erlotinib-resistance (ER) cells compared to parental HCC827 cells. Inhibition of YAP by small interfering RNA (siRNA) increased the cytotoxicity of erlotinib to H1975 (L858R+T790M) cells. In YAP siRNA-transfected H1975 cells, GTIIC reporter activity and downstream gene expression of AREG and CTGF decreased significantly (P<0.05). Verteporfin, YAP inhibitor had an effect similar to that of YAP siRNA; it increased sensitivity of H1975 cells to erlotinib and in combination with erlotinib, synergistically reduced migration, invasion and tumor sphere formation abilities in H1975 cells. Our results indicate that YAP promotes erlotinib resistance in the erlotinib-sensitive NSCLC cell line HCC827. Inhibition of YAP by siRNA increases sensitivity of erlotinib-resistant NSCLC cell line H1975 to erlotinib.