1205Lu Cells Research Articles

Abstract 4592: Preclinical characterization of ATG-042-198, a novel MTAP null-selective PRMT5 inhibitor

Abstract Background: PRMT5 inhibitors are considered as a promising approach for cancer therapy due to their crucial role in the initiation and progression of many types of malignancies. Nevertheless, the crucial involvement of PRMT5 in controlling hematopoiesis, even in normal cells, resulted in significant on-target-off-tumor hematological toxicity when using first-generation, non-selective PRMT5 inhibitors in clinical settings, which hampered the clinical development of these drugs. The MTAP gene is deleted in around 10-15% of all human malignancies. As a result, the loss of MTAP leads to the buildup of MTA, which can bind to PRMT5 and partially inhibit PRMT5 activity. Inhibitors targeting the PRMT5-MTA complex become a promising strategy for treating MTAPnull cancer in a synthetically lethal manner. Herein, we report the preclinical feature of ATG-042-198, a novel MTAPnull-selective PRMT5 inhibitor. Methods: The in vitro activity and MTAP selectivity of ATG-042-198 on cell proliferation and cellular symmetric dimethylarginine (SDMA) expression were profiled using HCT116 MTAP wild type (wt) cells and HCT116 MTAP knock out (ko) cells, as well as in five MTAPnull cell lines. The in vivo efficacy was tested in cell derived xenograft (CDX) mouse models with HCT116 MTAP wt cells, HCT116 MTAP ko cells and LU99 cells (MTAPnull). The in vitro and in vivo pharmacokinetic properties were assessed with corresponding assay methods. Results: ATG-042-198 demonstrated potent and selectively inhibitory effect on HCT116 MTAP ko cells’ proliferation (IC50=44nM) and SDMA expression (IC50=0.146nM), but very weak effect on HCT116 MTAP wt cells’ proliferation (IC50=4550nM) and SDMA expression (IC50>500nM). Consistently, ATG-042-198 showed excellent anti-proliferation activities on multiple endogenous MTAPnull cell lines (A549, IC50=2nM; LU99, IC50=6nM; U87MG, IC50=11nM; NALM-6, IC50=37nM; MIA PaCa-2, IC50=43nM). ATG-042-198 demonstrated high permeability (Caco2 assay, ER=2), good metabolic stability (Human liver microsomes assay, T1/2>145min), and low risk of drug interaction (CYP inhibition assay, 3A4/2C19/2D6, all IC50>10μM). In vivo PK study showed that ATG-042-198 was well absorbed, with a dose-dependent increase in plasma distribution and high oral bioavailability (F=108%). Plasma free drug concentrations were continuously higher than the IC50 of cell proliferation inhibition within 24 hours after 30mpk administration. In three CDX models (HCT116 MTAP wt, HCT116 MTAP ko and LU99), ATG-042-198 at a dose of 30 mg/kg QD potently and selectively inhibited tumor growth (TGI=-1%, 89% and 106%) without inducing body weight loss. Conclusions: In summary, ATG-042-198 is an oral MTAP null-selective PRMT5 inhibitor with potent efficacy against MTAPnull tumor. It also demonstrated good tolerability and preclinical PK profiles. Citation Format: Ya Kong, Ming Zhang, Lulu Jiang, Ruiting Xu, Bin Jiang, Guoqiang Dai, Yulong He, Huiling Liu, Jay Mei, Bing Hou, Bo Shan. Preclinical characterization of ATG-042-198, a novel MTAP null-selective PRMT5 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4592.

Microrheological comparison of melanoma cells by atomic force microscopy

Melanoma is one of the most severe cancers due to its great potential to form metastasis. Recent studies showed the importance of mechanical property assessment in metastasis formation which depends on the cytoskeleton dynamics and cell migration. Although cells are considered purely elastic, they are viscoelastic entities. Microrheology atomic force microscopy (AFM) enables the assessment of elasticity and viscous properties, which are relevant to cell behavior regulation. The current work compares the mechanical properties of human neonatal primary melanocytes (HNPMs) with two melanoma cell lines (WM793B and 1205LU cells), using microrheology AFM. Immunocytochemistry of F-actin filaments and phosphorylated focal adhesion kinase (p-FAK) and cell migration assays were performed to understand the differences found in microrheology AFM regarding the tumor cell lines tested. AFM revealed that HNPMs and tumor cell lines had distinct mechanical properties. HNPMs were softer, less viscous, presenting a higher power-law than melanoma cells. Immunostaining showed that metastatic 1205LU cells expressed more p-FAK than WM793B cells. Melanoma cell migration assays showed that WM73B did not close the gap, in contrast to 1205LU cells, which closed the gap at the end of 23 h. These data seem to corroborate the high migratory behavior of 1205LU cells. Microrheology AFM applied to HNPMs and melanoma cells allowed the quantification of elasticity, viscous properties, glassy phase, and power-law properties, which have an impact in cell migration and metastasis formation. AFM study is important since it can be used as a biomarker of the different stages of the disease in melanoma.

Abstract 4780: Comparison of safety and efficacy outcomes with nonthermal plasma and tirapazamine in porcine skin and mouse melanoma models

Abstract A novel preclinical experimental therapy for various cancer types including melanoma is the combination of nonthermal plasma (NTP) and tirapazamine (TPZ) delivered in situ. This patented combination therapy delivers reactive oxygen species (ROS) and reactive nitrogen species (RNS) from the atmosphere that are activated and ionized with helium gas to create the state of matter known as plasma. The combination with the prodrug, TPZ, that only activates to become a ROS in hypoxic environments, shows synergistic targeting of cancer cells. In this study, we assayed the effects of the dual therapy on porcine skin, which is the closest model to represent human skin and is commonly used for testing drug therapy. The results demonstrated that porcine skin showed no significant cell killing with the therapy. We also compared the combination therapy in normal skin keratinocytes and 1205Lu cells derived from a melanoma metastasis to the lung. The cells were treated with NTP and TPZ and maintained in a hypoxic chamber. Four days following therapy the keratinocytes continued to grow in culture, whereas the melanoma cells expressing endogenous gap junctions, or overexpressed Cx43 gap junctions showed decreased viability. However, the melanoma cells with a dominant negative Cx43 mutant did not show this loss in viability. This confirms our previous report that gap junctions mediate the passage of derivatives of ROS to increase the extent of cell death. To determine the effects of NTP on regulating the lethal dose of TPZ, we performed IC50 curves for TPZ in the presence or absence of NTP in B16-F10 murine metastatic melanoma cells. We demonstrated a decrease in the IC50 from 28 μM without NTP to 17.4 μM with NTP. In further analysis, the effects of treating with NTP in the presence or absence of tissue culture media were compared. The results showed a highly significant increased efficacy of NTP in the absence of media. The combination therapy was more effective than each independent treatment. To further confirm the experimental analysis, we induced tumor formation with the B16-F10 cells in a syngeneic C57BL/6 mouse model. The tumors from mice that were untreated or treated with NTP or TPZ alone, showed a significant increase in tumor volume as compared to the tumors from mice receiving the combination treatment in situ. In comparison of the tumor growth rates, the control, NTP-, or TPZ-treated tumors had increased by approximately 350% by day 10, whereas the tumors in the mice that received the dual therapy only increased by an average of 70%. The results from these studies collectively implicate the combination of NTP and TPZ as a potential synergistic treatment for in situ melanomas that offer selectivity as compared to normal tissue. Citation Format: Matthew Yehl, Dominik Kucharski, Michelle Eubank, Xavier Ramadan, Ahmad Dianat, Bianca Pham, Timothy C. Hutcherson, Sandra Sexton, Shoshanna N. Zucker. Comparison of safety and efficacy outcomes with nonthermal plasma and tirapazamine in porcine skin and mouse melanoma models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4780.

Dinactin: A New Antitumor Antibiotic with Cell Cycle Progression and Cancer Stemness Inhibiting Activities in Lung Cancer.

Lung cancer, especially non-small cell lung cancer (NSCLC), is one of the most complex diseases, despite the existence of effective treatments such as chemotherapy and immunotherapy. Since cancer stem cells (CSCs) are responsible for chemo- and radio-resistance, metastasis, and cancer recurrence, finding new therapeutic targets for CSCs is critical. Dinactin is a natural secondary metabolite produced by microorganisms. Recently, dinactin has been revealed as a promising antitumor antibiotic via various mechanisms. However, the evidence relating to cell cycle progression regulation is constrained, and effects on cancer stemness have not been elucidated. Therefore, the aim of this study is to evaluate the new function of dinactin in anti-NSCLC proliferation, focusing on cell cycle progression and cancer stemness properties in Lu99 and A549 cells. Flow cytometry and immunoblotting analyses revealed that 0.1-1 µM of dinactin suppresses cell growth through induction of the G0/G1 phase associated with down-regulation of cyclins A, B, and D3, and cdk2 protein expression. The tumor-sphere forming capacity was used to assess the effect of dinactin on the cancer stemness potential in NSCLC cells. At a concentration of 1 nM, dinactin reduced both the number and size of the tumor-spheres. The quantitative RT-PCR analyses indicated that dinactin suppressed sphere formation by significantly reducing expression of CSC markers (i.e., ALDH1A1, Nanog, Oct4, and Sox2) in Lu99 cells. Consequently, dinactin could be a promising strategy for NSCLC therapy targeting CSCs.

Sphingosine-1 Phosphate Receptor Modulators Increase In Vitro Melanoma Cell Line Proliferation at Therapeutic Doses Used in Patients with Multiple Sclerosis.

S1P1 receptor modulators (S1P1-RM) are oral disease-modifying therapies (DMTs) for multiple sclerosis (MS). Several authorities have raised doubts that S1P1-RM are responsible for an increased risk of melanoma in patients with MS. We studied the in vitro effects of S1P1-RM on different melanoma cell lines to compare the effect of available S1P1-RM on the proliferation of human melanoma cells. Four S1P1-RM were studied which are currently approved for managing MS, namely fingolimod (Gilenya®), siponimod (Mayzent®), ozanimod (Zeposia®), and ponesimod (Ponvory®). We tested these four drugs at different concentrations, including therapeutic doses (0.5, 1.6, 5.5, 18, and 60µM), on human melanoma cell lines (501Mel cells, 1205LU cells, and M249R cells) to analyze in vitro cell proliferation monitored with the IncuCyte ZOOM live cell microscope (Essen Bioscience). At therapeutic doses, median confluence increased overall for all lineages: + 122% for ozanimod (p < 0.001), + 71% for ponesimod (p < 0.001), + 67% for siponimod (NS), and + 41% for fingolimod (p = 0.094). Ozanimod- and ponesimod-treated cells increased confluency in 501Mel, 1205LU, and M249R cell lines (p < 0.001). These data suggest an increased proliferation of various melanoma cell lines with S1P1-RM treatments used at therapeutic concentrations for patients with MS and should raise the question of increased dermatologic surveillance.

ODP555 In-vitro Combo Treatments of Human Melanoma (1205Lu, WM238) Cell Lines with Curcumin, Vitamins and Steroids

Abstract Previous combo treatment experiments on BLM human melanoma cell line identified two combos vit-A + RU-486 and Cur+P+RU as the best combinations to decrease melanoma cell growth as well as IL-8 secretion in -vitro. In order to confirm this finding, experiments were repeated on two other human melanoma cell lines 1205Lu and WM238. Initial single or solo compound treatment on 1205Lu cells resulted in cell growth in the range of 37–59% compared to untreated control 1205Lu cell growth at 100%. A double combo treatment with combinations such as D+P, D+RU, A+P, A+RU, A+D resulted in cell growth in the range of 17 to 37% on the 1205Lu cell line. Whereas on WM238 cell line resulted in cell growth in between 18–22%. A triple combo such as combinations of three compounds (Cur+P+RU, Cur+A+P, Cur+D+RU) resulted in cell growth in the range of 19-30% on 1205Lu cells, whereas on WM238 cells resulted in between 19–20% cell growth. Elisarray of the supernatants from the combos which showed a significant decrease in cell growth revealed the regulation only in IL-8 secretion. IL-8 secretion in the supernatants of combos treated with A+RU and Cur+P+RU was17.5 and 8. 0 pg/ml respectively on the 1205Lu cell line. Whereas, IL-8 secretion was 0.515 and 0.926 pg/ml respectively in the supernatants of A+RU and Cur+P+RU combo treatments on the WM238 cell line. Conclusion Based on cell growth, Elisarray and IL-8 quantitation, two combos A+RU and Cur+P+RU also fared well on the two other cell lines 1205Lu and WM238. Presentation: No date and time listed

SERPINB8 and furin regulate ITGAX expression and affect the proliferation and invasion of melanoma cells.

In the past 10 years, the systemic treatment of advanced melanoma has undergone tremendous changes through the development of targeted therapy. However, there is still a long way to go. This study aims to characterize the function and interaction of ITGAX, SERPINB8 and furin in BRAF V600E mutant melanoma. Differentially expressed genes related to BRAF V600E mutation and BRAFi treatment were obtained by analysing GSE141484 and GSE22838. two kinds of BRAFi (Vemurafenib, 10μM; Dabrafenib, 1μM) were used to treat A375 and 1205Lu cells, respectively. The expression of ITGAX, SERPINB8 and Furin in A375 and 1205Lu cells was down-regulated by specific siRNAs, and cell proliferation, clone formation and invasion were detected by CCK-8, colony formation and transwell assays. The physical binding of furin and SERPINB8 was detected by immunoprecipitation. BRAFi treatment down-regulated the ITGAX and SERPINB8 expression and did not change furin expression. Knockdown of ITGAX and SERPINB8 both inhibited the proliferation and invasion of A375 and 1205Lu cells. Knocking down SERPINB8 down-regulated the expression of ITGAX. Furin knockdown and inhibitors all up-regulated the protein level of ITGAX. SERPINB8 can physically bind to furin. In summary, SERPINB8 and furin regulate the expression of ITGAX in melanoma cells, and ITGAX significantly promotes the proliferation and invasion of melanoma cells.

Abstract 1841: MRTX849 inhibits P-gp and demonstrates CNS exposure in mouse models and cancer patients and demonstrates antitumor activity in intracranial mouse models of lung cancer brain metastasis

Abstract MRTX849 is a covalent, mutant selective, KRAS G12C inhibitor in development for cancer patients harboring this mutation. KRAS G12C is mutated in ~14% of lung adenocarcinoma and KRAS G12C inhibitors adagrasib and sotorasib have demonstrated clinical activity. While early data are encouraging, approximately one-third of KRAS-mutant non-small cell lung cancer (NSCLC) patients develop brain metastases and this remains a significant unmet medical need. P-glycoprotein 1 (P-gp)-mediated efflux is a major mechanism for the active transport of small molecules out of the CNS. MRTX849 is a P-gp inhibitor and inhibits its own efflux at plasma exposure levels achieved in humans at the 600 mg BID dose level resulting in achieving appreciable drug levels in cerebrospinal fluid (CSF). Total and free-fraction adjusted plasma concentrations of MRTX849 were 8.6 μM and 43 nM; respectively, 8 hours post administration of a clinically relevant oral dose of 200 mg/kg to mice. In addition, the CSF concentration, a measure of the free brain concentration, was 52 nM, which is above the cellular pERK1/2 IC50 value measured at 24 hours in cancer cell lines (~5 nM). Administration of 100 mg/kg MRTX849 to mice also resulted in CSF exposure above the cellular IC50 and a CSF/plasma (free-fraction adjusted) partition coefficient (Kp,uu) value of 0.4. To evaluate the tumor growth inhibition following oral administration of MRTX849 in an orthotopic model of lung cancer brain metastasis, immunocompromised mice were intracranially implanted with luciferase-labeled human NSCLC LU99 cells. Lower bioluminescence imaging (BLI)-based tumor flux was observed for the 100 mg/kg BID MRTX849-treated mice compared to vehicle and an 88% reduction in bioluminescence signal compared to baseline was observed suggesting strong tumor regression and consistent with significantly longer survival in treated mice. In addition, MRTX849 treated brain tumors demonstrated 85% reduced ERK phosphorylation. Similarly, treatment with MRTX849 resulted in potent tumor regression and significant survival extension in another intracranial KRAS G12C model of NSCLC using LU65 cells. CSF levels were determined at steady-state in two patients in the MRTX849-001 Phase1/2 clinical trial. The CSF/plasma (free fraction adjusted) Kp,uu value was 0.47 with patient CSF levels achieved consistent with CSF levels observed in responding mouse models (24-35 nM). These data demonstrate MRTX849 crosses the blood brain barrier in preclinical models and cancer patients. In addition, antitumor activity observed in mouse models of brain metastases provides rationale for exploring the utility of MRTX849 for the treatment of patients harboring KRAS G12C mutant lung cancer with brain metastases. Citation Format: Kazuhide Shimizu, Jill Hallin, Mohini Singh, Naema Nayyar, Matthew R. Strickland, Aaron C. Burns, Cornelius Cilliers, Lauren Hargis, Peter A. Olson, Matthew A. Marx, Priscilla K. Brastianos, Hiroaki Wakimoto, James G. Christensen. MRTX849 inhibits P-gp and demonstrates CNS exposure in mouse models and cancer patients and demonstrates antitumor activity in intracranial mouse models of lung cancer brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1841.

In-Vitro Effects of Active Vitamin-D3 and Vitamin-A on Human Melanoma (BLM and1205Lu) Cell Growth

Though UV ray in the Sun light is essential for vitamin-D3 formation, yet UV rays can lead to skin cancer. Lack of vitamin-D3 also can lead to cancer. Hence, we decided to study the effect of vitamin-D3 on human melanoma cell models. Our aims were to study the in-vitro effects of vitamin-D3 and vitamin-A on human melanoma (BLM, 1205Lu) cell growth, as vitamins D3 and A are important for a healthy skin. Initially dose-curve (100 nM to 100 μM concentration) study was carried out with vitamin-D3 and vitamin-A (retinoic acid) on BLM cells to determine the optimal concentrations of vitamins-D3 and A for co-incubation with progesterone (50 μM) and RU-486 (50 μM). Supernatants from the treated cells were subjected to Elisarray. Vitamin-D3 and vitamin-A showed a dose-dependent decrease in cell growth. Based on the dose-curve, it was decided to use 25 μM (at 57% cell growth) of vitamin-D3 and 50 μM (at 55% cell growth) of vitamin-A for co-incubation studies. Co-incubation of vitamin-D3 with vitamin-A showed an additive effect on the decrease of BLM cell growth (20%). Similarly co-incubation of vitamin-D3 with progesterone (33% cell growth) and with RU-486 (28% cell growth) as well as co-incubation of vitamin-A with progesterone (31% cell growth) and RU-486 (18% cell growth) showed an additive effect on the decrease of BLM cell growth. Based on the BLM co-incubation studies, we decided to repeat the studies on 1205Lu cells. So, 25 μM (at 34% cell growth) of vitamin-D3 and 50 μM (at 44% cell growth) of vitamin-A were used for co-incubation studies. Co-incubation of vitamin-D3 with vitamin-A showed an additive effect on the decrease of cell growth (22%). Though co-incubation of vitamin-D3 with progesterone did not show any difference in cell growth (51%), yet co-incubation with RU-486 showed a decrease in 1205Lu cell growth (21%). Similarly co-incubation of vitamin-A with progesterone (25% cell growth) and with RU-486 (23% cell growth) showed a synergistic effect on the decrease of 1205Lu cell growth. Conclusion: These studies suggested that combination of vitamins and steroids might be effective in decreasing melanoma cell growth. Hence, various combination of vitamins and steroids will be tested for their effect on melanoma cell growth in vitro in order to develop a combo drug treatment for melanoma.

CIC Is a Mediator of the ERK1/2-DUSP6 Negative Feedback Loop.

SummaryDUSP6 functions as an important negative feedback component of the MAPK/ERK signaling pathway. Although DUSP6 expression is tightly regulated by ERK1/2 signaling, the molecular mechanism of this regulation remains partially understood. In this work, we show that the transcriptional repressor CIC functions downstream of the ERK1/2 signaling to negatively regulate DUSP6 expression. CIC directly represses DUSP6 transcription by binding to three cis-regulatory elements (CREs) in DUSP6 promoter. p90RSK, a downstream target of ERK1/2, phosphorylates CIC at S173 and S301 sites, which creates a 14-3-3 recognition motif, resulting in 14-3-3-mediated nuclear export of CIC and derepression of DUSP6. Finally, we demonstrate that the oncogenic CIC-DUX4 fusion protein acts as a transcriptional activator of DUSP6 and its nuclear/cytoplasmic distribution remains regulated by ERK1/2 signaling. These results complete an ERK1/2/p90RSK/CIC/DUSP6 negative feedback circuit and elucidate the molecular mechanism of how RTK/MAPK signaling harnesses the transcriptional repressor activity of CIC in mammalian cells.

Mitochondrial Fatty Acid Oxidation Is Not Vital for Melanoma Cell Proliferation and Migration

Abstract Objectives Melanoma, the 5th most common cancer in the US, is the most aggressive form of skin cancer. Excess adiposity is associated with an increased risk of melanoma in males, and a high-fat diet promotes melanoma tumor growth in mice. Our group found that lipid droplet (LD) content increases with melanoma cell aggressiveness and that fatty acid uptake inhibition reduces cell proliferation and migration; however, the function of these lipids in melanoma is unknown. Since lipids can be used to produce energy by fatty acid oxidation (FAO), we sought to determine whether melanoma cells were using FAO to maintain aggressiveness. Methods Gene expression microarray was used to identify differences in gene expression, which was confirmed by RT-qPCR. WM983A, WM983B, 1205Lu and A375 human melanoma cells were treated with 5 µM Etomoxir and cell proliferation, migration, and respiration were quantified using the Quant-iT™ PicoGreen™ dsDNA Assay Kit, Boyden Chamber transwell migration, and Seahorse XFe96 Flux Analyzer, respectively. Results The most significantly altered genes expressed between four invasive, lipid-rich and four non-invasive, lipid-poor melanoma cells pertained to fatty acid metabolism. Carnitine palmitoyltransferase 1A (CPT1a), the rate limiting enzyme in mitochondrial FAO (mFAO), mRNA was increased in lipid-rich cells compared to the lipid-poor cells (p &amp;lt; 0.05, n = 6–7), indicating that melanoma cells may use LD lipids for mFAO. To determine the importance of mFAO to melanoma cells, we inhibited mFAO with etomoxir, an irreversible CPT1 inhibitor. Treatment of lipid-rich 1205Lu and A375 cells or lipid-poor WM983A and WM983B cells with etomoxir for 4 days had no effect on cell proliferation. Extracellular flux analysis of cells with or without etomoxir showed no difference in ATP production, indicating that melanoma cells do not use mFAO to generate energy under normal conditions. In motile cells, etomoxir reduced lipid-rich A375 cell migration by 8.8% (p = 0.05, n = 2). Conclusions Lipids play a role in melanoma aggressiveness; however, our results indicate that mFAO of lipids is not vital to melanoma cell proliferation or migration. Further studies are required to understand the implication of excess adiposity and circulating lipids in melanoma development and progression. Funding Sources Institutional Department Fund

HDGF enhances VEGF‑dependent angiogenesis and FGF‑2 is a VEGF‑independent angiogenic factor in non‑small cell lung cancer.

Non-small cell lung cancer (NSCLC) accounts for over 80% of all diagnosed lung cancer cases. Lung cancer is the leading cause of cancer-related deaths worldwide. Most NSCLC cells overexpress vascular endothelial growth factor-A (VEGF-A) which plays a pivotal role in tumour angiogenesis. Anti-angiogenic therapies including VEGF-A neutralisation have significantly improved the response rates, progression-free survival and overall survival of patients with NSCLC. However, the median survival of these patients is shorter than 18 months, suggesting that NSCLC cells secrete VEGF-independent angiogenic factors, which remain unknown. We aimed to explore these factors in human NSCLC cell lines, A549, Lu99 and EBC-1 using serum-free culture, to which only EBC-1 cells could adapt. By mass spectrometry, we identified 1,007 proteins in the culture supernatant derived from EBC-1 cells. Among the identified proteins, interleukin-8 (IL-8), macrophage migration inhibitory factor (MIF), galectin-1, midkine (MK), IL-18, galectin-3, VEGF-A, hepatoma-derived growth factor (HDGF), osteopontin (OPN), connective tissue growth factor (CTGF) and granulin (GRN) are known to be involved in angiogenesis. Tube formation, neutralisation and RNA interference assays revealed that VEGF-A and HDGF function as angiogenic factors in EBC-1 cells. To confirm whether VEGF-A and HDGF also regulate angiogenesis in the other NSCLC cell lines, we established a novel culture method. NSCLC cells were embedded in collagen gel and cultured three-dimensionally. Tube formation, neutralisation and RNA interference assays using the three-dimensional (3D) culture supernatant showed that VEGF-A and HDGF were not angiogenic factors in Lu99 cells. By gene microarray in EBC-1 and Lu99 cells, we identified 61 mRNAs expressed only in Lu99 cells. Among these mRNAs, brain-derived neurotrophic factor (BDNF), fibroblast growth factor-2 (FGF-2) and FGF-5 are known to be involved in angiogenesis. Tube formation and neutralisation assays clarified that FGF-2 functions as an angiogenic factor in Lu99 cells. These results indicate that HDGF enhances VEGF-dependent angiogenesis and that FGF-2 is a VEGF-independent angiogenic factor in human NSCLC cells.

846 Resistance to apoptosis and PD-L1 induction in melanoma is associated with suppressed aryl hydrocarbon receptor signaling

Metastatic melanoma is an aggressive form of skin cancer with a 5-year survival rate of 15% to 20%. Unlike primary melanoma, metastatic disease is difficult to treat due to acquisition of chemoresistance and immune evasion through PD-L1 expression. Highlighting the intrinsic differences in signaling networks that are overactive or suppressed between primary and metastatic melanoma will provide essential cues to understanding mechanisms of therapeutic resistance and immune evasion. We detected reduced cytochrome p450 enzymes, Cyp1a1 and Cyp1b1, in vertical growth phase melanoma cell line, WM793, and its metastatic variant, 1205Lu, compared to primary melanoma cells WM1552c. Since their expression is directly controlled by aryl hydrocarbon receptor (AhR), whose expression was also decreased in 1205Lu and WM793 cells, we tested whether reduced AhR signaling in melanoma cells confers chemoresistance and immune evasion properties. We used paclitaxel (ptx)-mediated apoptosis and interferon gamma (IFNg)-mediated PD-L1 induction as end points to evaluate chemotherapeutic resistance and immune evasion, respectively. 1205Lu and WM793 cells were resistant to ptx-mediated apoptosis (<10%) and PD-L1 expression increased by 3 to 6-fold following IFNg treatment. WM1552c cells, however, were sensitive to ptx-mediated apoptosis (>25%) with minimal induction of PD-L1 (1.5-fold) following IFNg treatment. Treatment with AhR antagonist, CH223191, prior to ptx decreased the percentage of apoptotic cells to <10% in WM1552 cells. IFNg-induced PD-L1 expression also increased by 3-folds in WM1552c cells following AhR inhibition although the mean fluorescence intensity was still lower than 1205Lu cells. Our data indicates that a suppressed AhR signaling in vertical phase and metastatic melanoma cells may have been evolved to resist apoptosis to chemotherapy and promote immune evasion via enhanced PD-L1 induction. Elucidating components of AhR pathway that are affected can be of high therapeutic value for metastatic melanoma.

Green Tea Catechin Is an Alternative Immune Checkpoint Inhibitor that Inhibits PD-L1 Expression and Lung Tumor Growth.

The anticancer activity of immune checkpoint inhibitors is attracting attention in various clinical sites. Since green tea catechin has cancer-preventive activity in humans, whether green tea catechin supports the role of immune checkpoint inhibitors was studied. We here report that (−)-epigallocatechin gallate (EGCG) inhibited programmed cell death ligand 1 (PD-L1) expression in non–small-cell lung cancer cells, induced by both interferon (IFN)-γ and epidermal growth factor (EGF). The mRNA and protein levels of IFN-γ–induced PD-L1 were reduced 40–80% after pretreatment with EGCG and green tea extract (GTE) in A549 cells, via inhibition of JAK2/STAT1 signaling. Similarly, EGF-induced PD-L1 expression was reduced about 37–50% in EGCG-pretreated Lu99 cells through inhibition of EGF receptor/Akt signaling. Furthermore, 0.3% GTE in drinking water reduced the average number of tumors per mouse from 4.1 ± 0.5 to 2.6 ± 0.4 and the percentage of PD-L1 positive cells from 9.6% to 2.9%, a decrease of 70%, in lung tumors of A/J mice given a single intraperitoneal injection of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In co-culture experiments using F10-OVA melanoma cells and tumor-specific CD3+ T cells, EGCG reduced PD-L1 mRNA expression about 30% in F10-OVA cells and restored interleukin-2 mRNA expression in tumor-specific CD3+ T cells. The results show that green tea catechin is an immune checkpoint inhibitor.

Abstract 2665: Green tea catechin, EGCG, enhances antitumor immunity by down-regulation of PD-L1 expression in non-small human lung cancer cell lines

Abstract (-)-Epigallocatechin gallate (EGCG) is now widely accepted as a non-toxic, effective cancer preventive compound. EGCG also acts as a synergist with anti-cancer drugs for cancer treatment. Considering the wide beneficial effects of EGCG, we think EGCG might have antitumor immunity. Programmed death-ligand 1 (PD-L1) expression on tumor cells is involved in PD-L1/PD-1 pathway. PD-L1 protein levels varied among 6 non-small human lung cancer cell lines (NSCLCs): LC-AI and Lu99 cell lines showed the highest PD-L1 expression, A549 and H322 cell lines were medium, and H1703 and H1299 were very low, independent on cancer cell lines. Since PD-L1 expression is induced by various cytokines and growth factors, we studied the effects of IFN-γ and EGF on cell-surface PD-L1 level using flow cytometry, and its gene expression using RT-PCR. Treatment with IFN-γ dose-dependently stimulated PD-L1 expression in both A549 and H1299 cells, and not effective on Lu99 cells, whereas treatment with EGF stimulated PD-L1 in Lu99 cells only. Pretreatment with EGCG for 3 h, dose-dependently inhibited INF-γ-induced PD-L1 gene expression and protein level on cell-surface in both A549 and H1299. It is important to note that EGCG also inhibited phosphorylation of STAT1. In addition, EGCG inhibited EGF-induced PD-L1 gene expression and protein level in Lu99 cells via inhibition of phosphorylation of Akt. In order to study the effects of EGCG in between rodent carcinogenesis experiment and antitumor immunity, green tea extract (GTE) was given to NNK-induced lung tumors in A/J mice, which showed significant reduction of PD-L1 protein level, determined by immunohistochemical analysis using anti-PD-L1 antibody. Furthermore, the group treated with NNK + GTE significantly showed reduced number of tumors per mouse from 3.2 to 2.2, and decreased tumor size. Treatment with Wortmannin, a PI3K inhibitor, as a control, did not inhibit PD-L1 expression in A549 and H1299 cells. The results strongly suggest that EGCG enhances antitumor immunity via inhibition of PD-L1 expression in cancer cells, resulting in significant cancer preventive activity. Citation Format: Anchalee Rawangkan, Keisuke Iida, Ryo Sakai, Hirota Fujiki, Masami Suganuma. Green tea catechin, EGCG, enhances antitumor immunity by down-regulation of PD-L1 expression in non-small human lung cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2665. doi:10.1158/1538-7445.AM2017-2665

CD271 Down-Regulation Promotes Melanoma Progression and Invasion in Three-Dimensional Models and in Zebrafish

CD271 is a neurotrophin receptor variably expressed in melanoma. Although contradictory data are reported on its role as a marker of tumor-initiating cells, little is known about its function in tumor progression. CD271 expression was higher in spheroids derived from freshly isolated cells of primary melanomas and in primary WM115 and WM793-B cell lines, and it decreased during progression to advanced stages in cells isolated from metastatic melanomas and in metastatic WM266-4 and 1205Lu cell lines. Moreover, CD271 was scarcely detected in the highly invasive spheroids (SKMEL28 and 1205Lu). CD271, originally expressed in the epidermis of skin reconstructs, disappeared when melanoma started to invade the dermis. SKMEL8 CD271(-) cells showed greater proliferation and invasiveness invitro and were associated with a higher number of metastases in zebrafish compared with CD271(+) cells. CD271 silencing in WM115 induced a more aggressive phenotype invitro and invivo. On the contrary, CD271 overexpression in SKMEL28 cells reduced invasion invitro, and CD271 overexpressing 1205Lu cells was associated with a lower percentage of metastases in zebrafish. A reduced cell-cell adhesion was also observed in the absence of CD271. Taken together, these results indicate that CD271 loss is critical for melanoma progression and metastasis.

A simple engineered platform reveals different modes of tumor-microenvironmental cell interaction

How metastatic cancer lesions survive and grow in secondary locations is not fully understood. There is a growing appreciation for the importance of tumor components, i.e. microenvironmental cells, in this process. Here, we used a simple microfabricated dual cell culture platform with a 500 μm gap to assess interactions between two different metastatic melanoma cell lines (1205Lu isolated from a lung lesion established through a mouse xenograft; and WM852 derived from a stage III metastatic lesion of skin) and microenvironmental cells derived from either skin (fibroblasts), lung (epithelial cells) or liver (hepatocytes). We observed differential bi-directional migration between microenvironmental cells and melanoma, depending on the melanoma cell line. Lung epithelial cells and skin fibroblasts, but not hepatocytes, stimulated higher 1205Lu migration than without microenvironmental cells; in the opposite direction, 1205Lu cells induced hepatocytes to migrate, but had no effect on skin fibroblasts and slightly inhibited lung epithelial cells. In contrast, none of the microenvironments had a significant effect on WM852; in this case, skin fibroblasts and hepatocytes—but not lung epithelial cells— exhibited directed migration toward WM852. These observations reveal significant effects a given microenvironmental cell line has on the two different melanoma lines, as well as how melanoma effects different microenvironmental cell lines. Our simple platform thus has potential to provide complex insights into different strategies used by cancerous cells to survive in and colonize metastatic sites.

Abstract B18: Non-thermal plasma, tirapazamine, and gap junctions: A novel approach to melanoma therapy through ROS induction

Abstract Melanoma cells typically have high levels of superoxide anion which may increase their susceptibility to cell death by reactive oxygen species (ROS). We have used this information to establish a system whereby melanoma cells are saturated with ROS through treatment with non-thermal plasma (NTP). NTP is formed from a mixture of highly charged helium and atmospheric gases to emit a total of 45 molecular species, many of which are ROS. We have recently demonstrated that NTP causes selective apoptotic cell death of melanoma cells as compared to normal skin keratinocytes when plated in co-culture. Since tumors flourish under low oxygen (hypoxic) conditions, we tested the effects of NTP under hypoxia. In order to enhance the effects of NTP, we combined this therapy with the experimental anticancer drug, tirapazamine, which promotes cell toxicity through DNA damage only under hypoxic conditions. The question then remains as to how to increase the target area of melanoma therapy. Our previous studies have demonstrated that gap junctions act via the bystander effect to enable the passage of cell-death promoting signals by NTP. We utilized stably transfected 1205Lu metastatic melanoma cells that express three different levels of gap junctions: endogenous low levels, increased levels through expression of connexin 43, or an absence of gap junctions due to expression of a dominant negative mutant connexin 43. We have shown that the cells with the mutant connexin 43 showed increased invasive potential as well as enhanced expression of HIF1α. The combination therapy with NTP and tirapazamine under hypoxic conditions caused a synergistic effect which increased cell death in all cells, yet was most apparent in the 1205Lu cells overexpressing the functional gap junctions. In addition, the target area of the NTP torch was dramatically increased under these conditions. This suggests that the ROS generated by NTP and tirapazamine induced the bystander effect and promoted the spreading of cell death signals well beyond the area of the plasma treatment. These results are indicative a novel future approach to melanoma therapy through selective ROS induction and modification of gap junction phenotype. Citation Format: Shoshanna N. Zucker, Cory Higley, Zethan Koch, Haneesha Goli, Peter Casey, Akeem Francis, Kevin Burke, Jennifer Zirnheld. Non-thermal plasma, tirapazamine, and gap junctions: A novel approach to melanoma therapy through ROS induction. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B18.

Regulation of viability, differentiation and death of human melanoma cells carrying neural stem cell biomarkers: a possibility for neural trans-differentiation.

During embryonic development, melanoblasts, the precursors of melanocytes, emerge from a subpopulation of the neural crest stem cells and migrate to colonize skin. Melanomas arise during melanoblast differentiation into melanocytes and from young proliferating melanocytes through somatic mutagenesis and epigenetic regulations. In the present study, we used several human melanoma cell lines from the sequential phases of melanoma development (radial growth phase, vertical growth phase and metastatic phase) to compare: (i) the frequency and efficiency of the induction of cell death via apoptosis and necroptosis; (ii) the presence of neural and cancer stem cell biomarkers as well as death receptors, DR5 and FAS, in both adherent and spheroid cultures of melanoma cells; (iii) anti-apoptotic effects of the endogenous production of cytokines and (iv) the ability of melanoma cells to perform neural trans-differentiation. We demonstrated that programed necrosis or necroptosis, could be induced in two metastatic melanoma lines, FEMX and OM431, while the mitochondrial pathway of apoptosis was prevalent in a vast majority of melanoma lines. All melanoma lines used in the current study expressed substantial levels of pluripotency markers, SOX2 and NANOG. There was a trend for increasing expression of Nestin, an early neuroprogenitor marker, during melanoma progression. Most of the melanoma lines, including WM35, FEMX and A375, can grow as a spheroid culture in serum-free media with supplements. It was possible to induce neural trans-differentiation of 1205Lu and OM431 melanoma cells in serum-free media supplemented with insulin. This was confirmed by the expression of neuronal markers, doublecortin and β3-Tubulin, by significant growth of neurites and by the negative regulation of this process by a dominant-negative Rac1N17. These results suggest a relative plasticity of differentiated melanoma cells and a possibility for their neural trans-differentiation without the necessity for preliminary dedifferentiation.

Inhibitor of differentiation-4 (Id4) stimulates pigmentation in melanoma leading to histiocyte infiltration.

TGF-β and the inhibitors of differentiation (Id) are linked. Smad7 and other TGF-β inhibitors can potently suppress melanomagenesis; however, little work examining Ids has been reported in melanoma, particularly for Id4. Here, we report that Id4, but not Id2 or Id3 expression, surprisingly, activated robust melanin production in xenografts of previously amelanotic (lacking pigment) 1205Lu/Smad7 (S7) cells. Fontana-Masson stain and de-novo expression of MART-1 and tyrosinase proteins confirmed melanin production. Additionally, pigment-laden CD163+ mouse histiocytes with areas of extensive necrosis were found throughout S7/Id4 tumors, but not in parental 1205Lu, S7/Id2 or S7Id3-derived tumors. Mechanistic investigation revealed increased nuclear M-microphthalmia-associated transcription factor (MITF) and MART-1 promoter activation following Id4 expression in 1205Lu and WM852 melanoma cells, suggesting broader implications for Id4 in melanin synthesis. In human tumors, melanin colocalized with Id4 expression establishing a correlation. Current chemotherapeutics for melanoma are only marginally effective. Immunotherapy provides the most promise, yet the role of innate immunity is poorly understood. Here, TGF-β suppression followed by Id4 expression results in extensive melanin synthesis and robust histiocyte recruitment following tumorigenesis, a novel role for Id4. Our results suggest that TGF-β suppression coupled with pigment overproduction triggers an innate immune response resulting in tumor necrosis.