Cell Lines HCC827 Research Articles

Epigenetic activation of HORMAD1 in basal-like breast cancer: role in Rucaparib sensitivity.

Basal-like breast cancer (BLBC) is an aggressive breast cancer subtype with features similar to the basal cells surrounding the mammary ducts. Treatment of patients with BLBC has been challenging due to the lack of well-defined molecular targets. Due to the clinical and pathological similarities of BLBC with BRCA-deficient breast cancers, the effectiveness of Poly (ADP-ribose) polymerase inhibitors (PARPi) has been tested in early phase clinical trials for patients with advanced BLBC, with limited clinical responses. Recently, it was reported that HORMAD1 overexpression sensitizes BLBC to HR-targeting agents by suppressing homologous recombination. Our independent analysis suggests that HORMAD1 is aberrantly overexpressed in about 80% of BLBC, and its expression in normal tissues is restricted to testis. Our experimental data suggests that HORMAD1 overexpression correlates with focal hypomethylation in BLBC. On the other hand, investigation of the Genomics of Drug Sensitivity in Cancer dataset revealed significantly reduced sensitivity of HORMAD1-overexpressing BLBC cell lines to Rucaparib, a commonly used PARPi. To further assess the role of HORMAD1 in PARPi sensitivity, we generated three HORMAD1-overexpressing xenograft models using the HORMAD1-low BLBC cell lines HCC1954, HCC1806, and BT20; we then subjected these xenograft models to Rucaparib treatment. Ectopic expression of HORMAD1 enhances tumor formations in two of these models, and significantly reduces sensitivity to Rucaparib in the HCC1954 model. Taken together, our data suggest that epigenetic activation of HORMAD1 by hypomethylation in BLBC may endow reduced sensitivity to Rucaparib treatment in some tumor models.

Abstract 4884: OXPHOS: A novel target for cancer therapy in oncogene addicted tumor

Abstract Cancer cells are highly dependent on glycolysis over oxidative phosphorylation (OXPHOS). However, there is emerging evidence that (a) dysfunctional OXPHOS may be a major factor driving oncogene addiction and (b) feedback activation of the transcription factor STAT3 is a common surrogate survival pathway in oncogene addicted cancer cells. As such, invasive cancers exhibit enhanced oxidative phosphorylation, mitochondrial biogenesis and mitochondrial oxygen consumption rate. Therefore, inhibiting OXPHOS and STAT3 activity could have potential therapeutic implications for oncogene addicted and OXPHOS-dependent cancers. Here we report that oncogene addicted non-small cell lung carcinoma (NSCLC) cell line HCC827 and its gefitinib resistant clone and malignant melanoma cell line A375 and its vemurafanib resistant clone have significantly higher OXPHOS activity. In addition, these cells express higher level of the mitochondrial electron transport chain complex I protein (NDUFA9) and increased complex I activity as well as higher mitochondrial DNA copy number. Of note, significantly increased activity of STAT3 is detected in oncogene addicted cancer cells, and the increased mitochondrial oxygen consumption and complex I activity could be significantly inhibited by a novel small molecule inhibitor of STAT3, OPB-51602. The latter is shown to be an effect that might be independent of the STAT3 inhibitory activity of the small molecule compound. Most importantly, the novel STAT3 inhibitor showed strong activity in three different murine models of carcinogenesis as well as in patients treated with the STAT3 inhibitor. These data provide evidence that oncogene addicted cancer cells switch to OXPHOS over glycolysis and serve as proof of principle for identifying targeted new OXPHOS inhibitor in recalcitrant cancers. Citation Format: Jayshree L. Hirpara, Jie Eu Qing, Andrea Wong, Naoto Ohi, Tsunoda Takeshi, Boon Cher Goh, Shazib Pervaiz. OXPHOS: A novel target for cancer therapy in oncogene addicted tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4884.

Abstract LB-186: Evaluation of EGFR degradation as a mechanism of tumor evasion in EGFR-TKI resistant non-small cell lung cancer (NSCLC) cells compared to EGFR-TKI sensitive cells

Abstract The resistance mechanisms to EGFR-TKIs in Non-Small Cell Lung Cancer (NSCLC) are not well elucidated. Here we evaluate EGFR degradation as a mechanism of tumor evasion in EGFR-TKI resistant NSCLC cells (H1975) compared to EGFR-TKI sensitive cells (HCC827). After erlotinib treatment, EGFR protein half-life was quantified by cycloheximide chase. EGFR expression on cell membrane was measured by flow cytometry and transcription levels were measured by Real-Time PCR. EGFR half-life was shorter in H1975 compared to HCC827 cells after cycloheximide inhibition of protein synthesis. EGFR phosphorylation levels were not affected by erlotinib in H1975 while in HCC827 they were significantly reduced (p < 0.001). EGFR expression on cell membrane decreased similarly in both H1975 and HCC827 cell lines. Also the transcription levels of EGFR remained unaffected by erlotinib treatment in both cell lines. However, there was more apoptosis in HCC827 compared to H1975 with 64% vs. 22%. Our results suggest that H1975 cells present more EGFR degradation however, they show less cell death by apoptosis and this could represent a resistance mechanism to EGFR-TKIs. Citation Format: Hernandez-Pedro Y. Norma, Soca-Chafre Giovanny, Orozco-Morales Mario, Barrios-Bernal Pedro, Arrieta Oscar. Evaluation of EGFR degradation as a mechanism of tumor evasion in EGFR-TKI resistant non-small cell lung cancer (NSCLC) cells compared to EGFR-TKI sensitive cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-186.

Abstract 4788: Preclinical evaluation of E-191, a highly selective EGFR T790M inhibitor

Abstract Objective: Patients of non-small cell lung cancer (NSCLC) with activating EGFR mutations initially respond to the first generation reversible EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib. However, most of the patients acquire resistance within 10~16 months. The most common mechanism of resistance is the EGFR T790M mutation. In addition, selectivity of the EGFR inhibitors is important, as inhibition of EGFR wild type can lead to dose limiting toxicities, including rash and diarrhea. This may be the reason why the second generation irreversible EGFR inhibitors, such as afatinib, which also inhibit EGFRWT, is not widely used. Here, we disclose our clinical candidate compound E-191 as a novel irreversible inhibitor that potently inhibits the resistance mutant forms of EGFR with a good selectivity sparing EGFRWT. Method: The anti-proliferative activity of E-191 was evaluated in NCI-H1975 (EGFRT790M/L58R), HCC827 (EGFRd746-750), and A431 (EGFRWT) cell lines. Daily oral administration of E-191 at 3 doses was used to evaluate the in vivo anti-tumor activity in three cell-derived tumor xenograft (CDX) models, NCI-H1975, HCC827, and Ba/F3 (EGFRT790M /d746-750). The in vivo selectivity was evaluated in the A431 (EGFRWT) CDX model. Effects on EGFR signaling pathway were assessed by western blot analysis of the downstream effector proteins p-EGFR (Y1068), p-AKT (Ser473) and p-ERK (Thr202/Tyr204). Result: E-191 displayed potent anti-proliferative activity in the EGFR mutant cell lines NCI-H1975 (IC50 = 22 nM) and HCC827 (IC50=1.9 nM), and a much less potent activity in the EGFRWT A431 cell line (IC50 = 531 nM). E-191 has about 24 fold selectivity for EGFRT790M over EGFRWT. E-191 potently inhibited tumor growth at all doses tested (P<0.001) in the NCI-H1975, HCC827 and Ba/F3 (EGFRT790M /d746-750) models, while significantly less so in the A431 model. Western blot analysis of the tumor samples in the NCI-H1975 model study showed that E-191 inhibited p-EGFR (Y1068), p-AKT (Ser473), and p-ERK (Thr202/Tyr204), indicating that the tumor growth inhibition was through inhibition of the resistant mutant EGFR. Conclusion: We have demonstrated with in vitro and in vivo preclinical models that E-191 is a selective inhibitor of the resistance mutant forms of EGFR, potently inhibiting EGFRT790M but not EGFRWT. These results make us believe that E-191 has a good potential in the clinic for the treatment of resistant EGFR driven NSCLC. Citation Format: Liu Xile, Lihong Hu, Charles Z Ding, Weifeng Mao, Liqing Wang, Xiaohe Shi, Chaofeng Long, Xiaoxin Chen, Haijun Li, Xing Liu, Zhuowei Liu. Preclinical evaluation of E-191, a highly selective EGFR T790M inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4788.

Abstract 4903: AXL inhibition overcomes erlotinib resistance in AXL-activated non-small cell lung cancer cells

Abstract Mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) are present in 10-35% patients with NSCLC. In patients with EGFR-mutated NSCLC, EGFR tyrosine kinase inhibitors, including erlotinib, have demonstrated excellent initial responses. However, despite improved outcomes observed in patients treated with EGFR TKIs, most patients eventually develop drug resistance. We and others recently demonstrated that the activation of oncogene AXL is necessary to confers erlotinib resistance in both cell and mouse models as well as in human tumor tissues. While genetic aberrations affecting AXL appear rare, we recently identified two patients with EGFR-mutated lung cancer who simultaneously harbor AXL A273V or AXL V289M mutation. Both patients were resistant to erlotinib treatment (one case of primary and one case of acquired resistance). In this study, we first evaluated whether these AXL mutations conferred resistance to erlotinib treatment. Using HCC827 and PC-9 NSCLC cell lines stably expressing the AXL A273V or V289M mutation, we confirmed that AXL A273V significantly increased resistance to erlotinib treatment. Subsequently we also conducted a preclinical study to test if a new AXL inhibitor MGCD265 could overcome such erlotinib resistance in NSCLC cells. Using an in vitro MTS assay, we first validated that administration of MGCD265 in combination with erlotinib significantly inhibited the viability of erlotinib-resistant HCC827-ER cells, compared to erlotinib- or MGCD265-alone treatment. Through conducting an array of cell functional studies, we found that combination of MGCD265 and erlotinib significantly decreased cell cycle progression in S phase, enhanced apoptosis, and repressed cell migration relative to single-agent erlotinib or MGCD265 treatment in HCC827-ER cells. We further performed RNA-sequencing to evaluate the gene expression profile and the cellular pathways altered in cells treated with erlotinib alone, MGCD265 alone or a combination of erlotinib and MGCD265. RNA-sequencing analysis identified that several groups of genes involved in cell survival inhibition or apoptosis promotion were upregulated, whereas genes involved in cell cycle process, DNA replication or cancer metabolism were downregulated in cells treated with the combination of erlotinib and MGCD265. Taken together, our results indicated that AXL mutations may confer resistance to EGFR TKI treatment, and effective blockade of the AXL pathway may represent a novel strategy to overcome erlotinib resistance for the treatment of NSCLC patients. Citation Format: Feng Wang. AXL inhibition overcomes erlotinib resistance in AXL-activated non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4903.

Abstract 2036: Alternative activation of TGFβ signaling pathways in breast cancer tumors with different subtype is related to a differential expression of transcription factors that induce EMT

Abstract Breast cancer is the leading cause of cancer death among women worldwide being distant metastases the main cause of disease. Patients with triple negative breast cancer tumors develop distant metastases earlier compared to luminal tumors. Epithelial-mesenchymal transition (EMT) induced by TGFβ signaling pathways, is the main mechanism to promote metastasis through the expression of the transcription factors TWIST, SNAIL, SLUG and ZEB1. Expression of EMT transcription factors has been studied in cancer cell lines however studies in breast cancer tumors with different subtype have not been reported. In addition, the molecular mechanism that induce EMT program is not known yet. In this study we evaluated the expression of the four transcription factors as well as the state of TGFβ/SMAD, ERK/MAPK and PI3K/AKT pathways in breast cancer cell lines HCC1937 (triple negative) and T47D (luminal) after TGFβ treatment. The same analysis was done by immunohistochemistry in 100 breast cancer tumors with different subtype. After TGFβ treatment T47D showed activation of ERK/MAPK and PI3K/AKT pathways and expression of only two transcription factors: SNAIL and SLUG. HCC1937 cells showed activation of TGFβ/SMAD and ERK/MAPK pathways and expression of TWIST, SLUG and ZEB1 but not SNAIL. Similar results were observed in breast cancer tumors, being TGFβ/SMAD pathway active in the majority of triple negative tumors, whereas an active state of PI3K/AKT was observed in luminal tumors. Active ERK/MAPK pathway was observed in both triple negative and luminal tumors. In relation to expression of transcription factors triple negative tumors with an active state of the TGFβ/SMAD pathway showed expression of transcription factors TWIST, SNAIL and SLUG, whereas those tumors with an active state of ERK/MAPK pathway showed only ZEB1 expression. Luminal tumors with an active PI3K/AKT pathway frequently showed ZEB1 expression. Our results showed that expression of a specific EMT transcription factor induced by TGFβ is related to the tumor subtype and depends on the activated signaling pathway. These results suggest different mechanisms of EMT induction by TGFβ in relation to breast cancer tumor subtype. Citation Format: Victoria Ortega-Hernández, Wanda Fernandez, Pilar Carvallo. Alternative activation of TGFβ signaling pathways in breast cancer tumors with different subtype is related to a differential expression of transcription factors that induce EMT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2036.

Abstract 4319: Epigenetic reprogramming associated with BRCA1 loss of function in ovarian cancer induces enhanced interferon gamma signaling

Abstract Inactivating mutations of the breast cancer type 1 susceptibility protein (BRCA1) have been implicated in breast and ovarian cancer (OC) initiation. Having observed differential response of OC cells to histone deacetylase inhibitors (HDACi) dependent on the presence of a functional BRCA1, we mapped promoters marked by acetylation of lysine 9 or lysine 27 of histone H3 (H3K9ac, H3K27ac) by ChIP-sequencing, and measured gene expression by RNA-sequencing in the isogenic OC cell lines UWB1.289 carrying an inactivating mutation of BRCA1 (BRCA1-null) and UWB1.289 cells with restored BRCA1 gene (BRCA1+). Significant differences in promoter levels of H3K9 and H3K27 acetylation were associated with marked alteration of gene expression levels between BRCA-null and BRCA1+ cells and in response to the HDACi, entinostat. Pathway analyses revealed cellular movement, cellular development, cellular growth and proliferation, TGFβ1, TNF, and INF-γ among the top cellular/molecular functions and upstream regulators enriched by the absence of BRCA1 among H3K9ac differentially marked and differentially expressed genes. Importantly, the same pathways and regulators were enriched in response to entinostat in BRCA1+ cells, indicating that changes in gene expression affecting these functions and regulators in BRCA1-null cells depend on promoter-associated histone acetylation. Integrated analyses revealed that TGFβ1 and IFN-γ were also activated in BRCA1-null/or low vs. BRCA1-normal ovarian tumors in the TCGA database. IFN-γ target genes were upregulated at baseline and responded less to IFN-γ stimulation in BRCA1-null vs. BRCA1+ cells. Binding of HDAC1 to promoters of IFN-γ target genes was increased in BRCA1-null cells. Similarly, the BRCA1-null breast cancer cell line HCC1937 responded less to IFN-γ stimulation compared with HCC1937 cells in which BRCA1 was restored. The knockdown of STAT1, the main IFN-γ signaling transduction molecule, inhibited BRCA1+ cell proliferation, but did not affect BRCA1-null cells. We conclude that the transcriptome of BRCA1-null cells and tumors is altered through changes in histone acetylation affecting cancer-related pathways, including IFN-γ cellular responses. These changes could alter tumor progression in BRCA-null OC and impact response to immunotherapy. Citation Format: Horacio Cardenas, Jessica Thomes Pepin, Guanglong Jiang, Salvatore Condello, Kenneth P. Nephew, Yunlong Liu, Daniela Matei. Epigenetic reprogramming associated with BRCA1 loss of function in ovarian cancer induces enhanced interferon gamma signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4319.

Abstract 4893: The use of glycyrrhetinic acid derivatives to inhibit blebbishield emergency program pathogenesis overcoming erlotinib resistance in non-small cell lung cancer

Abstract Background: Bleb transformation is an initial induction of apoptosis leading to cellular compartmentalization into vesicles, increased glycolysis/mitochondrial activity combined with dynamin mediated endocytosis allowing the cell to tether vesicles to form stem like spheroids protecting exposed genetic material. Our laboratory has found that not only do both Erlotinib sensitive and resistant cell lines undergo blebbishield transformation in response to treatment, but the addition of Glycyrrhetinic acid derivative (CDODA-Me) overwhelms this pathway leading to cellular apoptosis. Methods: NSCLC cell lines HCC827 (both erlotinib resistant and sensitive), were treated with CDODA-Me and Erlotinib (combination and single treatment). MicroRNA expression profiling was used to identify and validate reference genes involved with the synergistic effects comparing combination treatment to erlotinib single treatment groups. Spheroids were collected after 24hr treatment using blebbishield extraction media (BEM) consisting of 12µM erlotinib alone and in combination with 2µM CDODA-Me. Western Blot analysis was used to confirm identified pathways as well as to quantify the effects of CDODA-Me on VEGF and TGF-β cellular release levels during treatment as well as MET and EGFR kinase activation. Results: Genomic studies showed an increase in microRNA has-miR-711 (approximately 4-fold) which is responsible for inhibiting dynamin mediated trafficking, various growth factor expression levels (including VEGF and TGF-β), as well as the expression of receptors associated with these growth factors (EGFR, MET, VEGFR). This inhibition was confirmed through western blot analysis showing a decrease in receptor activation but no effects on receptor expression (6 fold and 8 fold decrease when compared to erlotinib single treatment for EGFR and MET respectively). Sphere reattachment was found to be dependent on the presence of growth factors and other unknown cell signaling molecules through the fact that spheres did not attach in fresh media, but attached in as little as 3hrs in media obtained after 24hr incubation in parental cells. Media obtained after incubation in parental cells provided higher levels of VEGF and TGF-β when compared to media obtained after 24hr treatment with (resistant to non-resistant expression ratios of 3.94 and 4.06 for HCC827 (4µM erlotinib resistant) and HCC827 Cl4 respectively). Actin fluorescent staining 48hrs after reseeding spheroids showed that cells grown in untreated parental media regained normal branch like morphology while those grown in CDODA-Me treated parental media remained rounded and died. Conclusions: Based on the results of these studies, CDODA-Me has been shown to inhibit NSCLC cell types from overcoming apoptosis thereby inhibiting increased drug resistance as well as tumorgenicity. Citation Format: Ebony Nottingham, Elizabeth Mazzio, Arindam Mondal, Mandip Sachdeva. The use of glycyrrhetinic acid derivatives to inhibit blebbishield emergency program pathogenesis overcoming erlotinib resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4893.

Abstract 4274: Identifying the regulatory network structure of the genes involved in signaling pathways underlying ER-PR-HER2+ breast cancer using Bayesian modeling of genome-wide NRF1 DNA motif sequence-enriched genes

Abstract Nuclear respiratory factor 1 (NRF1) [alpha-palindromic binding protein (α-PAL)] is a redox-sensitive transcription factor. NRF1 localizes to several thousand sites in the genome. Role of NRF1 has remained largely unexplored in breast cancer (BCa). We have recently shown that reactive oxygen species (ROS) contribute to estrogen-induced growth of BCa cells through an NRF1 signaling pathway. In this study, we used a systematic approach integrating ChIP DNA-seq computational analysis, NRF1 protein-DNA motif binding analysis, breast cancer signaling pathway analysis, and Bayesian modeling to discover genes involved in HER2+ (HER2+ER−PR−) breast cancer. First, we used ChIP-seq of normal human mammary epithelial cells (HMEC) and three breast cancer cell lines, HCC1954, MCF7 and T47D, to identify NRF1 regulated genes in breast cancer. We then identified the genome-wide NRF1-enriched DNA regions in estrogen- and progesterone receptors-negative and amplified HER2 (ER-/PR-/HER2+) - HCC1954 breast cancer cell line compared to normal human mammary epithelial cells (HMEC) to discover the NRF1 regulatory network. Analysis of NRF1 binding activity in breast cancer cell line HCC1954 compared to normal mammary epithelial cells (HMEC) shows that more than 7,000 genes have NRF1 binding activity in the region located +/- 2,000 bp from TSS and the top 2,000 correlated with breast cancer showed a significant difference in the average amount of binding protein. The majority (83%) of NRF1 target genes that may participate in the process of cells acquiring characteristics of malignancy present NRF1 binding sites in the regulatory region located +/- 2,000 bp from TSS. Finally we used RNA microarray data from TCGA to detect changes in NRF1 expression levels and to develop a probabilistic graphic model using Bayesian network analysis to investigate how the putative causal genes may interact in breast cancer. NRF1 expression was significantly higher in HER+ breast cancer samples compared to normal breast tissues. This is consistent with our experimental data, which also showed higher expression of NRF1 in HER2+ breast cancer brain metastasis samples. NRF1 binding sites are present in the regulatory region located +/- 2,000 bp from TSS in the majority of target genes (83%). NRF1 binding activity in the region located +/- 2,000 bp from TSS significantly correlated with breast cancer. Bayesian structural modeling revealed 27 NRF1 target genes are part of the hallmarks of breast cancer. NRF1 is a parent of GADD45A, WNT9A and SHC2. Likewise, SHC2 is parent of CDK4, which in turn is parent of BRCA1. In summary, these findings revealed novel roles of NRF1 and its regulatory network presumably involved in the development of HER2+ER−PR− breast tumor, one of the two most aggressive breast cancer subtypes with poor prognosis. Citation Format: Jairo D. Ramos, Jayanta Das, Changwon Yoo, Deoudutta Roy. Identifying the regulatory network structure of the genes involved in signaling pathways underlying ER-PR-HER2+ breast cancer using Bayesian modeling of genome-wide NRF1 DNA motif sequence-enriched genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4274.

Abstract LB-271: Antitumor activity of new epidermal growth factor receptor inhibitors in non-small cell lung and breast cancer

Abstract Non-small cell lung cancer (NSCLC) and breast cancer (BrCa) are two of the most common forms of cancer. Since EGFR and HER2 are proven to be key targets in cancer therapy, we have designed and synthesized new molecules, belonging to a distinct chemical category of the existing drugs. Lapatinib was selected as an ideal prototype compound for the design of new analogues with potential action against EGFR and HER2 due to its ability to target and inhibit both receptors with great selectivity and efficiency. Structural analogues of lapatinib were synthesized by replacing the central ring of quinazoline by purine or bioisosteric rings aiming to investigate and potentially ameliorate the biological and pharmacological action. For the preparation of the target molecules, synthetic pathways have been designed and developed, which allow their high yielding preparation. The potency of the various analogues was evaluated in vitro using the following methodologies: MTT cytotoxic assay, Western Blot and ELISA assays to evaluate the ability of the analogues to inhibit EGFR phosphorylation in comparison with the activity of lapatinib and cell uptake studies based on mass spectrometry techniques. From a pool of 12 different analogues, two compounds, an imidazopyridine and an imidazopyrimidine derivative had the highest potencies, which were comparable to lapatinib in the NSCLC cell line A549 and in the BrCa cell line HCC-1954 (using MTT cytotoxic assays average IC50 values of 3 μM & 30 μM, respectively versus 11 μM for lapatinib). The same compounds were also tested in the NSCLC cell line NCI-H1975, which harbours the L858R + T790M mutations responsible for resistance (average IC50 values of 10 μM & 30 μM, respectively versus 9 μM for lapatinib). In addition, by using Western Blot and ELISA assays the two agents were the most potent compared to lapatinib, as they caused 50% inhibition of pEGFR at a 10 μM concentration. We have shown that the imidazopyridine derivative enters the A549 cells at a relatively slower rate in comparison to lapatinib. Presumably the physicochemical properties of the analogue are responsible for its higher accumulation (at 48 h incubation intracellular concentration of 17 ng/100.000 cells versus 10 ng/100.000 cells for lapatinib). Encouraged by the preliminary results two key molecules with similar potency to lapatinib will undergo further evaluation in vivo to determine their pharmacokinetic characteristics using advanced mass spectrometric approaches and their efficacy in clinically relevant mouse models. Citation Format: ARIS DOUKATAS, Efthymios-Spyridon Gavriil, Theodoros Karampelas, Panagiotis Marakos, Nicole Pouli, Constantin Tamvakopoulos. Antitumor activity of new epidermal growth factor receptor inhibitors in non-small cell lung and breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-271.

Discovery and synthesis of novel magnolol derivatives with potent anticancer activity in non-small cell lung cancer

EGFR T790 M accounts for 50% to 60% of cases of non-small-cell lung carcinoma (NSCLC) resistance to the first-generation EGFR tyrosine kinase inhibitors (TKIs). Hence, identifying novel compounds with activity against TKIs resistant is of great value. In this study, twenty honokiol and magnolol derivatives were isolated from the EtOH extract of Magnolia officinalis and the antiproliferative activity was evaluated on HCC827 (19del EGFR mutation), H1975 (L858 R/T790 M EGFR mutation), and H460 (KRAS mutation) cell lines. Among the isolated compounds, piperitylmagnolol (a 3-substituted magnolol derivative) showed the best antiproliferative activity against those three cell lines with the IC50 values of 15.85, 15.60 and 18.60 μM, respectively, which provided a direction for the structural modification of magnolol. Further structural modification led to the synthesis of thirty-one magnolol derivatives, and compounds A13, C1, and C2 exhibited significant and broad-spectrum antiproliferative activity with the IC50 values ranging from 4.81 to 13.54 μM, which were approximately 4- and 8-fold more potent than those of honokiol and magnolol, respectively. Moreover, their aqueous solubility was remarkably improved with 12-, 400- and 105 fold greater than those of honokiol and magnolol. Anti-tumor mechanism research revealed that these three compounds were able to induce cell cycle arrest at G0/G1 phase, cause efficient apoptosis in H1975 cells, and also prevent the migration of HUVECs in a dose-dependent manner through Cdk2, Cdk4, Cyclin E, and Cyclin D1 inhibition as well as up-regulation of cleaved-PARP and cleaved-caspase 3 levels. In in vivo antitumor activity, C2 (10, 30 and 100 mg/kg, po) dose-dependently inhibited the tumor growth in H1975 xenograft model with the tumor inhibition rate of 46.3%, 59.3% and 61.2% respectively, suggesting that C2 is a potential oral anticancer agent deserving further investigation.

Design, Synthesis and Evaluation of the Biological Activities of Some New Carbohydrazide and Urea Derivatives.

Urea and carbohydrazide derivatives are important compounds exhibiting cytotoxic activities. In this study, a series of new urea and carbohydrazide derivatives containing an pyridine ring were synthesized and evaluated for cytotoxic activity. The proposed structures of the synthesized compounds were confirmed using elemental analysis, IR, and 1H-NMR spectroscopic techniques. The cytotoxic potencies of synthesized compounds were determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) on BRCA mutant-carrying HCC1937 and Capan-1 cell lines, as well as on MCF7, HeLa, and MRC5 cells. 3a, 3b, 3c and 3d showed cytotoxic activity against all cancer cell lines. Our data indicate that compounds 3a-d are more selective to cancer cells compared with nontumoral fibroblasts; however, these compounds are not more potent on HR defective cells with BRCA mutants.

Synthesis and biological evaluation of irreversible EGFR tyrosine kinase inhibitors containing pyrido[3,4-d]pyrimidine scaffold

In the present study, a new class of compounds containing pyrido[3,4-d]pyrimidine scaffold with an acrylamide moiety was designed as irreversible EGFR-TKIs to overcome acquired EGFR-T790M resistance. The most promising compound 25h inhibited HCC827 and H1975 cells growth with the IC50 values of 0.025 μM and 0.49 μM, respectively. Meanwhile, 25h displayed potent inhibitory activity against the EGFRL858R (IC50 = 1.7 nM) and EGFRL858R/T790M (IC50 = 23.3 nM). 25h could suppress EGFR phosphorylation in HCC827 and H1975 cell lines and significantly induce the apoptosis of HCC827 cells. Additionally, compound 25h could remarkably inhibit cancer growth in established HCC827 xenograft mouse model at 50 mg/kg in vivo. These results indicated that the 2,4-disubstituted 6-(5-substituted pyridin-2-amino)pyrido[3,4-d]pyrimidine derivatives can serve as effective EGFR inhibitors and potent anticancer agents.

Loss of EGFR confers acquired resistance to AZD9291 in an EGFR-mutant non-small cell lung cancer cell line with an epithelial-mesenchymal transition phenotype.

AZD9291 is an irreversible, small-molecule inhibitor which has potency against mutant EGFR- and T790M-resistant mutation. Despite the encouraging efficacy in clinical, the acquired resistance will finally occur. Further study will need to be done to identify the acquired resistance mechanisms and determine the next treatment. We established an AZD9291-resistant cell line (HCC827/AZDR) from parental HCC827 cell line through stepwise pulsed selection of AZD9291. The expression of EGFR and its downstream pathways were determined by western blot analysis or immunofluorescence assay. The sensitivity to indicated agents were evaluated by MTS. Compared with parental HCC827 cells, the HCC827/AZDR cells showed high resistance to AZD9291 and other EGFR-TKIs, and exhibited a mesenchymal-like phenotype. Almost complete loss of EGFR expression was observed in HCC827/AZDR cells. But the activation of downstream pathway, MAPK signaling, was found in HCC827/AZDR cells even in the presence of AZD9291. Inhibition of MAPK signaling had no effect on cell viability of HCC827/AZDR and could not reverse AZD9291 resistance because of the subsequent activation of AKT signaling. When treated with the combination of AKT and MAPK inhibitor, HCC827/AZDR showed remarkable growth inhibition. Loss of EGFR could be proposed as a potential acquired resistance mechanism of AZD9291 in EGFR-mutant NSCLC cells with an EMT phenotype. Despite the loss of EGFR, the activation of MAPK pathway which had crosstalk with AKT pathway could maintain the proliferation and survival of resistant cells. Blocking MAPK and AKT signaling may be a potential therapeutic strategy following AZD9291 resistance.

Exosome-mediated gefitinib resistance in lung cancer HCC827 cells via delivery of miR-21.

Acquired resistance to gefitinib remains a major challenge in cancer treatment. In the present study, the effect of exosomes on the transmission of gefitinib resistance from gefitinib-resistant HCC827 lung cancer cells (H827R) to their gefitinib-sensitive counterparts and the potential underlying mechanisms by which this occurs was investigated. Exosomes were obtained from the cell supernatant using ultracentrifugation and the ExoQuick-TC exosome precipitation solution. Drug resistance was assessed by flow cytometry, apoptosis assays and cell counting kit-8 assays. The expression of microRNA (miR)-21 was analyzed by reverse transcription-quantitative polymerase chain reaction. Exosomes released by H827R cells (R/exo) may decrease the sensitivity of the human NSCLC HCC827 cell line to gefitinib. The results indicated that miR-21 expression was increased in R/exo and R/exo-treated H827S cells. However, miR-21 inhibition abrogated exosome-mediated drug resistance. Phosphorylated-protein kinase B (p-Akt), which is downstream of miR-21, was downregulated following gefitinib treatment; however, R/exo pretreatment elevated p-Akt levels and promoted the activation of Akt. By contrast, miR-21 inhibition reduced p-Akt expression. Therefore, the induction of miR-21 via exosomes and the activation of Akt may be mechanisms by which exosomes mediate the transfer of drug resistance.

Determination of Apoptotic Stage of HCC827 Lung Adenocarcinoma Cell Line, Following Calcium Sulfide Nanoclusters Treatment

Lung cancer is the number one cause of death independently of gender. By the time of the initial diagnosis, more than 50% of all lung cancer patients have already developed secondary tumors due to metastases. Despite current treatments such as surgery, chemotherapy, and radiotherapy, the prognosis for these patients still poor. Subsequently, there exists the need for more efficient treatments in order to increase the survival rate of patients suffering this disease. Previously, in‐vitro experiments in our lab demonstrated that Calcium Sulfide (CaS) nanoclusters induced apoptosis and necrosis in the non‐small lung cell adenocarcinoma (NSCLC) cell line HCC827. Using the Flow Cytometry technology, in this project we aimed to determine the effect of CaS in these cells evaluating apoptotic markers including, Caspase‐3/7 activity, Bcl‐2 phosphorylation, mitochondria potential, DNA‐damage, and oxidative stress. We hypothesized that CaS will show significant differences in these assays when compared to the Dimethyl sulfoxide (DMSO) control. We added a single dose of CaS [3.8μM] (experimental treatment), DMSO (control vehicle) or Etoposide [10 nM] (positive control) at time 0 and then collected the data after 24, 48, and 72 hours. As our results, we did not observe any statistically significant effect on apoptotic markers, Caspase‐3/7 and Bcl‐2. Analysis of the Mitopotential assay showed an statistical increase (p≤0.01) of dead cells population at 48 hours and of depolarized/lives cells at 72 hours compared to DMSO, in HCC827 cells treated with CaS. However, at 48 hours cells treated with CaS decrease the numbers of non‐affected cells (p≤0.05), increase DNA double‐strand breaks (p≤0.001), decrease the activation of Ataxia‐telangiectasia mutated kinase (ATM) and the phosphorylation of H2A.X significantly (p≤0.01). These results suggested that the detrimental effect of CaS on malignant HCC827 cell line is influenced by the accumulation of oxidative stress damage also affecting the cell cycle as our lab previously reported. Additional studies are ongoing to validate these results. Within the next month we plan to study the lung non‐malignant cells MRC‐5 using the same assays.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Synthesis and evaluation of 2,9-disubstituted 8-phenylthio/phenylsulfinyl-9H-purine as new EGFR inhibitors

In present study, we described the synthesis and biological evaluation of a new class of EGFR inhibitors containing 2,9-disubstituted 8-phenylthio/phenylsulfinyl-9H-purine scaffold. Thirty-one compounds were synthesized. Among them, compound C9 displayed the IC50 of 29.4 nM against HCC827 cell line and 1.9 nM against EGFRL858R. Compound C12 showed moderate inhibitory activity against EGFRL858R/T790M/C797S (IC50 = 114 nM). Western bolt assay suggested that compound C9 significantly inhibited EGFR phosphorylation. In vivo test, compound C9 remarkably exhibited inhibitory effect on tumor growth at 5.0 mg/kg by oral administration in established nude mouse HCC827 xenograft model. These results indicate that the 2,9-disubstituted 8-phenylsulfinyl/phenylsulfinyl-9H-purine derivatives can act as potent EGFR(L858R) inhibitors and effective anticancer agents. Additionally, optimization of compound C12 may result in discovering the fourth-generation EGFR-TKIs.

Co-targeting poly(ADP-ribose) polymerase (PARP) and histone deacetylase (HDAC) in triple-negative breast cancer: Higher synergism in BRCA mutated cells

PurposeDespite similarities with BRCA-mutated breast cancers, triple-negative breast cancers (TNBC) remain resistant to poly(ADP-ribose) polymerase (PARP) inhibitors as single agents. Histone deacetylase inhibitors (HDACi) can decrease expression of proteins involved in DNA repair. We thus hypothesized that a HDACi (suberoylanilide hydroxamic acid (SAHA) or belinostat) could sensitize TNBC to the PARP inhibitor olaparib. MethodsHuman TNBC cells were co-treated with olaparib and either SAHA or belinostat, and their effects on survival, proliferation, cell cycle, apoptosis and DNA repair pathways were evaluated. Subcutaneous xenografts were used to determine the effect of the combination treatment in vivo. ResultsHDACi and olaparib synergistically inhibited proliferation of a panel of 8 TNBC cell lines in vitro and in nude mice harboring TNBC xenografts in vivo. We noted a weaker synergism in PTEN-deficient TNBC cells and a stronger synergism in BRCA1-mutated TNBC cells. In the BRCA1-mutated cell line HCC-1937, we observed a drastic decrease in the expression of proteins involved in homologous recombination (HR), leading to a large imbalance of the ratio P-H2AX/RAD51. In BRCA1 wild type (wt) cell lines, effect of the combination treatment relied on DNA damage-induced cell cycle arrest followed by induction of apoptosis. ConclusionIn summary, these results provide a preclinical rationale to combine a HDACi with a PARP inhibitor to reduce HR efficiency in TNBC and sensitize these aggressive tumors to PARP inhibition.

Discovery of 2,4,6-trisubstitued pyrido[3,4-d]pyrimidine derivatives as new EGFR-TKIs

Targeting acquired drug resistance is the major challenge in the treatment of EGFR-driven non-small cell lung cancer (NSCLC). In this study, a novel class of compounds containing pyrido[3,4-d]pyrimidine scaffold was designed as new generation EGFR-TKIs to overcome this challenge. The most promising compound B30 inhibited HCC827 and H1975 cells growth with the IC50 values of 0.044 μM and 0.40 μM, respectively. Meanwhile, B30 displayed potent inhibitory activity against the EGFRL858R (IC50 = 1.1 nM) and EGFRL858R/T790M/C797S (IC50 = 7.2 nM). B30 could suppress EGFR phosphorylation in a dose-dependent manner in HCC827 cell line and significantly induce the apoptosis of HCC827 cells. Molecular docking indicated that the hydroxyl in B30 could form additional hydrogen bond with mutant Ser797. These findings strongly support our assumption that 2,4,6-trisubstitued pyrido[3,4-d] pyrimidine derivatives can serve as EGFR-TKIs. The predicted hydrogen bond interaction formed by a small molecule inhibitor with mutant Ser797 is available to design the fourth-generation EGFR-TKIs.

Estrogen receptor β1 activation accelerates resistance to epidermal growth factor receptor-tyrosine kinase inhibitors in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related deaths worldwide. Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR‑TKIs) have revolutionized the treatment of patients with advanced EGFR-mutant NSCLC. However, drug resistance eventually develops in the majority of patients despite an excellent initial response. The present study aimed to investigate the mechanism of acquired resistance to EGFR-TKIs and to explore strategies to overcome the resistance to EGFR-TKIs from a gender perspective. PC9 and Hcc827 cell lines, sensitized to EGFR-TKI, and secondary TKI-resistant PC9-ER (erlotinib resistant) and Hcc827-ER cell lines were evaluated for the expression of ERβ1. The proliferative ability of both cell lines was analyzed after transfection of siRNA-ERβ1 using Cell Counting Kit-8 and colony formation assays. Extracellular signal-regulated protein kinases1 and 2 (ERK1/2) and Akt activation were detected. The co-inhibition efficiency of erlotinib and fulvestrant was analyzed in PC9-ER xenografts. The expression of ERβ1 was investigated in tumor tissues of EGFR-TKI-treated patients, and its correlation with clinicopathological factors and progression-free survival (PFS) was assessed. The expression of ERβ1 was upregulated secondary to EGFR-TKIs in PC9 and Hcc827 cell lines, with β-estradiol dependence. Both PC9-ER and Hcc827-ER cell lines were re-sensitized to erlotinib after downregulation of the expression of ERβ1. ERK1/2 and Akt pathways were activated following the silencing of the expression of ERβ1 in PC9-ER and Hcc827 cell lines. The co-treatment of erlotinib and fulvestrant exhibited better growth inhibitory efficiency compared with the treatment of each agent alone in PC9-ER-derived xenografts. Primary NSCLC samples of 53patients treated with EGFR-TKIs were analyzed. ERβ1 was highly expressed, and the strong expression of cytoplasmic ERβ1 was related to a shorter PFS. In conclusion, ERβ1 was activated in EGFR-TKI secondary resistance. The downregulation of ERβ1 sensitized the cells to EGFR-TKIs. ERβ1 may be a key molecule in EGFR-TKI therapy. In addition, anti-ERβ1 treatment may reverse TKI resistance.