Lung Tumor Cell Lines Research Articles

The Cationic Amphiphilic Drug Hexamethylene Amiloride Eradicates Bulk Breast Cancer Cells and Therapy-Resistant Subpopulations with Similar Efficiencies.

Simple SummaryA limitation to successful therapeutic outcomes for breast and other cancer patients is the ability of small subsets of tumor cells to resist the apoptotic cell death provoked by currently employed therapeutic agents. These therapy-resistant cancer stem cell populations can then seed recurrent tumors and metastatic lesions, compromising the efficacy of the treatment regimen. The aim of our study was to assess the hypothesis that cationic amphiphilic drugs (CADs), which induce tumor cell death via the unrelated programmed necrotic mechanism, exhibit efficacy toward cancer stem cell populations that are resistant to currently employed therapeutics. We found that the therapy-resistant stem-like subpopulation of cells from a variety of breast cancer models are as sensitive to CADs as the bulk population. Our observations imply that the incorporation of cationic amphiphilic anticancer agents into existing therapeutic regimens could ultimately improve breast cancer patient outcomes by minimizing tumor recurrence and metastatic outgrowth.The resistance of cancer cell subpopulations, including cancer stem cell (CSC) populations, to apoptosis-inducing chemotherapeutic agents is a key barrier to improved outcomes for cancer patients. The cationic amphiphilic drug hexamethylene amiloride (HMA) has been previously demonstrated to efficiently kill bulk breast cancer cells independent of tumor subtype or species but acts poorly toward non-transformed cells derived from multiple tissues. Here, we demonstrate that HMA is similarly cytotoxic toward breast CSC-related subpopulations that are resistant to conventional chemotherapeutic agents, but poorly cytotoxic toward normal mammary stem cells. HMA inhibits the sphere-forming capacity of FACS-sorted human and mouse mammary CSC-related cells in vitro, specifically kills tumor but not normal mammary organoids ex vivo, and inhibits metastatic outgrowth in vivo, consistent with CSC suppression. Moreover, HMA inhibits viability and sphere formation by lung, colon, pancreatic, brain, liver, prostate, and bladder tumor cell lines, suggesting that its effects may be applicable to multiple malignancies. Our observations expose a key vulnerability intrinsic to cancer stem cells and point to novel strategies for the exploitation of cationic amphiphilic drugs in cancer treatment.

Antiproliferative Activity of Two Unusual Dimeric Flavonoids, Brachydin E and Brachydin F, Isolated from Fridericia platyphylla (Cham.) L.G.Lohmann: In Vitro and Molecular Docking Evaluation.

Despite the breakthrough in the development of anticancer therapies, plant-derived chemotherapeutics continue to be the basis of treatment for most types of cancers. Fridericia platyphylla is a shrub found in Brazilian cerrado biome which has cytotoxic, anti-inflammatory, and analgesic properties. The aim of this study was to investigate the antiproliferative potential of the crude hydroethanolic extract, subfraction (containing 59.3% of unusual dimeric flavonoids Brachydin E and 40.7% Brachydin F), as well as Brachydin E and Brachydin F isolated from F. platyphylla roots. The cytotoxic activity was evaluated in glioblastoma, lung, prostate, and colorectal human tumor cell lines. The crude hydroethanolic extract did not present cytotoxic activity, but its subfraction presented lower IC50 values for glioblastoma (U-251) and prostate adenocarcinoma (PC-3) cell lines. Brachydins E and F significantly reduced cell viability, proliferation, and clonogenic potential of PC-3, inducing them to the process of regulated cell death. In silico studies have indicated nuclear receptors as targets for Brachydins E and F, and molecular docking has pointed out their binding into glucocorticoid receptor (GR) ligand pocket. Targeting GR pathway has been described as a therapeutic strategy, especially for prostate cancer. These results suggest that Brachydin E and Brachydin F are promising compounds to be further explored for their antitumor effects.

TRIM55 suppresses malignant biological behavior of lung adenocarcinoma cells by increasing protein degradation of Snail1

ABSTRACT Up until now, cancer refractoriness and distal organ metastatic disease remain as major obstacles for oncologists to achieve satisfactory therapeutic effects for lung adenocarcinoma patients. Previous studies indicated that TRIM55, which participates in the natural development of muscle and cardiovascular system, plays a protective role in hepatocellular carcinoma (HCC) pathogenesis. Therefore, in this study, we aimed to unveil the detailed molecular mechanism of TRIM55 and identify the potential target for lung adenocarcinoma patients. Surgical samples and lung cancer cell lines were collected to detect the TRIM55 expression for patients with or without lymph node/distal organ metastasis. Cellular functional assays including transwell assay, wound healing assay, cellular survivability assay, etc. as well as ubiquitination assay were performed to evaluate the impact of TRIM55/Snail1 regulatory network via the UPP pathway on lung cancer tumor cell migration and chemo-resistance. Lung cancer tissues and tumor cell lines exhibited significantly lower levels of TRIM55 expression. Functional study further indicated that TRIM55 inhibited chemo-resistance, migration, and cancer stem-cell like phenotype of tumor cells. Further detailed molecular experiments indicated that TRIM55 promoted degradation of Snail1 via the UPP pathway, which played an interesting role in the regulation of cancer cell malignancy. This study provided novel theory that TRIM55 acted as a potential tumor suppressor by inhibition of tumor cell malignancy through enhancement of Snail1 degradation via the UPP pathway. Our research will inspire further exploration on TRIM55 to promote therapeutic effects for lung adenocarcinoma patients.

Role of Endogenous Serotonin Replica Mediated Through β-Catenin/Wnt Signaling in Lung Cancer Prevention and Prognosis - an in-vitro Study in A549 Cancer Cell Lines

Background: Lung cancer is still the most common cause of carcinoma-related death in the globe. Non-pharmacological treatments such as physical activity and exercise have been shown to improve quality of life and provide better results in lung cancer patients. Lung cancer patients usually lack adequate amounts of physical activity and exercise, both of which can improve quality of life.
 Aim: To analyse Chemotherapeutic potential of endogenous serotonin replica mediated through β-catenin/Wnt signaling in lung tumor cell lines (A549).
 Methods and Materials: The National Centre for Cell Sciences (NCCS), Pune, India, provided the human lung cancer cell line (A549). MTT assay was used to determine cell viability. Real-time PCR was used to examine -Catenin mRNA, Wnt mRNA, and GSK mRNA gene expression. The significance of the acquired data was determined using one-way analysis of variance and Duncan's multiple range test with Graph Pad Prism version 5. Duncan's test was used to determine significance at the 0.05 level.
 Results: The Results suggest that maximum inhibition of cell growth was at concentration (2-4 mM/ml) used in this study when compared to control. The cancer cells were significantly inhibited and it was found that there was notable reduction in GSK mRNA gene expression, β-Catenin, Wnt when compared to control at a dose of 2 mM/ml.
 Conclusion: It may be concluded, based on the analyses' findings and the study's limitations, that the role of exercise induced endogenous serotonin may act as the regulator of wnt/β-catenin signaling in lung cancer cells. The exercise may help in maintaining the equilibrium of the gene expression by modeling Wnt/β-catenin signaling pathway and act as a protective factor in prevention.

Bmi1 Resistance Pathway and Immune Checkpoint Blockade in Lung Cancer

Combined chemotherapy and radiation is a standard therapy for locally advanced lung cancers. However, the resistance mechanisms to these therapies and how they affect further adjuvant immunotherapy are not well understood. Our previous studies have shown that radiation led to the activation of Bmi1 as a mechanism of radiation resistance, which in turn upregulated the inhibitory immune checkpoint ligand PD-L1. Our hypothesis is that inhibition of Bmi1, a target for chemoradiotherapy resistance, with concurrent PD-L1 checkpoint blockade would have a synergistic enhanced immune response after chemoradiation therapy in a preclinical model of lung cancer.A small molecule inhibitor, PTC-596 (PTC Therapeutics, NJ), has been shown to inhibit the expression of Bmi1. For in vitro studies, we tested the effects of PTC-596 on the expression of both Bmi1 and PD-L1 in several lung tumor cell lines. To create a mouse model that simulates the PACIFIC trial treatment regimen, Lewis lung carcinoma cells (LLC1; 1 x 106 unsorted cells in 100 ul) derived in C57BL/6J strain were injected subcutaneously into the flank of immunocompetent C67BL/6J mice. When average tumor volume reached 100 mm3, the mice were treated with phase 1 treatment of radiation (dose 30 Gy in 10 fractions daily) delivered through SAARP platform and cisplatin (2mg/kg, every 3rd day for 10 days) and then randomized to receive phase 2 treatments: 1). Vehicle control every day; 2). Inhibitory anti-PD-L1 antibody every other day (200mg/kg, every 3 days for 4 cycles); 3). PTC-596 once a day (12.5 mg/kg, once a day); 4). Combined inhibitory anti-PD-1 antibody and PTC-596. Each treatment group had 10 mice.In both human and mouse lung tumor cell lines, fractionated radiation treatments upregulated Bmi1 expression and also increase the frequency of Bmi1-high, PD-L1-high expressing cells. Treatment with PTC-596 downregulated expression of Bmi1 and PD-L1 in these cells. In the PACIFIC study mouse model, cisplatin chemotherapy with fractionated radiation resulted significant growth delay than compared to no treatment. In the phase 2 of adjuvant treatment, the anti-PD-L1 antibody plus PTC-596 combination group, but not in groups receiving either treatment alone, had continued significant tumor size reduction. Immunological markers are being analyzed to determine if the immune response is enhanced.These results suggest that chemoradiation therapy leads to the activation of Bmi1 as a resistance mechanism, which in turn results in the activation of inhibitory immune checkpoint PD-L1 leading to immune escape and acquired resistance to immunotherapy. Treatment with PTC-596 demonstrated synergistic benefits to anti-PD-L1 immunotherapy after pre-chemoradiation treatment of preclinical mouse lung tumor model.

Overcoming Resistance to Anti-PD-1 With Tumor Agnostic NBTXR3: From Bench to Bedside

Immune checkpoint inhibitors (ICI) can improve outcomes in patients who respond to treatment, however most patients exhibit resistance. Overcoming this resistance is the main challenge in immune-oncology and recent studies suggest radiotherapy (RT) may improve ICI response rates. NBTXR3, composed of functionalized hafnium oxide nanoparticles, is injected intratumorally and activated by RT. NBTXR3 increases RT energy deposit inside tumor cells and subsequent tumor cell death, without adding toxicity to healthy tissues. Here we present evidence that NBTXR3 activated by RT primes the immune system, producing an anti-tumor immune response, including activation of the cGAS-STING pathway, that overcomes anti-PD-1 resistance both in murine models and patients.Abscopal assays were conducted in immunocompetent mice. Anti-PD-1 sensitive or resistant lung tumor cell lines were injected in both flanks. Intratumoral injection of NBTXR3 (or vehicle) followed by RT was performed in right flank (primary) tumors only. Some mice also received anti-PD-1 injections. Tumor growth was monitored, and tumor immune cell infiltrates analyzed by immunohistochemistry (IHC). Separately, in the phase II/III randomized Act.in.Sarc [NCT02379845] trial patients with locally advanced soft tissue sarcoma (STS) received either NBTXR3+RT or RT alone followed by tumor resection. Pre- and post-treatment tumor samples from patients in both groups were analyzed by IHC and Digital Pathology for immune biomarkers. The safety and efficacy of NBTXR3 plus stereotactic body radiotherapy (SBRT) in combination with anti-PD-1 is being evaluated in three cohorts of patients with advanced cancers in the Phase I 1100 [NCT03589339] trial.Pre-clinical studies demonstrated that NBTXR3+RT induces an immune response not observed with RT alone and enhances systemic control. IHC showed significant increase of CD8+ T-cell infiltrates in both NBTXR3+RT treated and untreated tumors compared to RT alone. Increased CD8+ T-cell and decreased FOXP3+ Treg density (pre- vs post-treatment) was also observed in tumors from STS patients treated with NBTXR3+RT. Furthermore, NBTXR3+RT in combination with anti-PD-1 improved local and systemic control in mice bearing anti-PD-1 resistant lung tumors, produced long-term memory, and reduced spontaneous lung metastases. Preliminary efficacy data from the 1100 trial showed tumor regression in 8/9 patients. Of note, tumor regression was observed in 6/7 patients who had progressed on prior anti-PD-1.The clinical efficacy of NBTXR3+RT has been demonstrated as a single agent in STS. Here we demonstrate that it overcomes resistance to anti-PD-1 treatment mechanisms in mice and led to tumor regression in patients having progressed on anti-PD-1 therapy. These results highlight the potential of NBTXR3+RT to positively impact the immuno-oncology field.

LncRNA XIST Contributes to Cisplatin Resistance of Lung Cancer Cells by Promoting Cellular Glycolysis through Sponging miR-101-3p

Background: Non-small-cell lung carcinoma is one of the most frequently diagnosed cancers. Cisplatin (CDDP) is a currently applied standard anticancer agent for advanced lung cancers. Although effectively clinical response was achieved initially, a large fraction of lung cancer patients developed cisplatin resistance. Therefore, understanding the molecular mechanisms of chemoresistance is crucial for anti-lung cancer therapy. Long non-coding RNA (lncRNA)-X-inactive-specific transcript (XIST) has been reported to be positively associated with multiple cancers. Currently, the precise role and mechanism of XIST in cisplatin resistance of lung cancer have not been elucidated. Methods: The expression levels of miR-101-3p and lncRNA XIST were detected by qRT-PCR. Cisplatin-resistant lung cancer cell line was established by selecting the survival cells under gradually increased cisplatin treatments. The cell proliferation was detected by MTT assay, and the cellular glucose metabolism rate was evaluated by Seahorse metabolic flux analysis and glucose uptake and lactate product assays. Glycolysis-related protein expression levels were detected by Western blot. Dual luciferase reporter was constructed to determine the lncRNA-miRNA interaction. Results: Here, we report XIST is significantly upregulated in lung cancer tissues compared with normal lung tissues. In addition, cisplatin-resistant lung cancer cells displayed remarkably elevated XIST expression. We demonstrated that miR-101-3p functioned as a tumor suppressor in lung cancer and sensitized lung cancer cells to cisplatin. Bioinformatics analysis predicted miR-101-3p could be a potential target of XIST through direct binding with it as a competing endogenous RNA, which was further validated from lung tumor tissues and cell lines by luciferase assay. Intriguingly, XIST significantly promoted cellular glycolysis rate of lung cancer cells. The extracellular acidification rate, glucose uptake, and lactate product were elevated by XIST overexpression. On the contrary, miR-101-3p effectively suppressed glycolysis rate. Finally, we demonstrated silencing XIST significantly recovered miR-101-3p expression and downregulated expression of glycolysis key enzymes, a phenotype could be further overridden by miR-101-3p inhibition. Conclusions: This study reveals a new molecular mechanism for the lncRNA-XIST-promoted cisplatin resistance via sponging miR-101-3p, leading to de-repression of cellular glycolysis. Moreover, these findings warrant further in vivo investigations to study XIST as a potential target to overcome cisplatin resistance.

ASN004, A 5T4-targeting scFv-Fc Antibody-Drug Conjugate with High Drug-to-Antibody Ratio, Induces Complete and Durable Tumor Regressions in Preclinical Models.

The 5T4 oncofetal antigen (trophoblast glycoprotein) is expressed in a wide range of malignant tumors but shows very limited expression in normal adult tissues. ASN004 is a 5T4-targeted antibody-drug conjugate (ADC) that incorporates a novel single-chain Fv-Fc antibody and Dolaflexin drug-linker technology, with an Auristatin F hydroxypropylamide payload drug-to-antibody ratio of approximately 10-12. The pharmacology, toxicology, and pharmacokinetic properties of ASN004 and its components were investigated in vitro and in vivo ASN004 showed high affinity for the 5T4 antigen and was selectively bound to and internalized into 5T4-expressing tumor cells, and potent cytotoxicity was demonstrated for a diverse panel of solid tumor cell lines. ASN004 induced complete and durable tumor regression in multiple tumor xenograft models, derived from human lung, breast, cervical, and gastric tumor cell lines having a wide range of 5T4 expression levels. A single dose of ASN004, as low as 1 mg/kg i.v., achieved complete tumor regression leading to tumor-free survivors in the A431 cervical cancer model. In head-to-head studies, superior activity of ASN004 was demonstrated against trastuzumab-DM1, in a low-5T4/high-HER2 expressing gastric tumor model, and 10-fold greater potency was found for ASN004 against the 5T4-targeted ADC PF-06263507 in a lung tumor model. In marmoset monkeys, ASN004 was well tolerated at doses up to 1.5 mg/kg Q3W i.v., and showed dose-dependent exposure, linear pharmacokinetics, and markedly low exposure of free payload drug. Taken together, these findings identify ASN004 as a promising new ADC therapeutic for clinical evaluation in a broad range of solid tumor types.

The RanBP2/RanGAP1-SUMO complex gates β-arrestin2 nuclear entry to regulate the Mdm2-p53 signaling axis.

Mdm2 antagonizes the tumor suppressor p53. Targeting the Mdm2-p53 interaction represents an attractive approach for the treatment of cancers with functional p53. Investigating mechanisms underlying Mdm2-p53 regulation is therefore important. The scaffold protein β-arrestin2 (β-arr2) regulates tumor suppressor p53 by counteracting Mdm2. β-arr2 nucleocytoplasmic shuttling displaces Mdm2 from the nucleus to the cytoplasm resulting in enhanced p53 signaling. β-arr2 is constitutively exported from the nucleus, via a nuclear export signal, but mechanisms regulating its nuclear entry are not completely elucidated. β-arr2 can be SUMOylated, but no information is available on how SUMO may regulate β-arr2 nucleocytoplasmic shuttling. While we found β-arr2 SUMOylation to be dispensable for nuclear import, we identified a non-covalent interaction between SUMO and β-arr2, via a SUMO interaction motif (SIM), that is required for β-arr2 cytonuclear trafficking. This SIM promotes association of β-arr2 with the multimolecular RanBP2/RanGAP1-SUMO nucleocytoplasmic transport hub that resides on the cytoplasmic filaments of the nuclear pore complex. Depletion of RanBP2/RanGAP1-SUMO levels result in defective β-arr2 nuclear entry. Mutation of the SIM inhibits β-arr2 nuclear import, its ability to delocalize Mdm2 from the nucleus to the cytoplasm and enhanced p53 signaling in lung and breast tumor cell lines. Thus, a β-arr2 SIM nuclear entry checkpoint, coupled with active β-arr2 nuclear export, regulates its cytonuclear trafficking function to control the Mdm2-p53 signaling axis.

Jinfukang regulates integrin/Src pathway and anoikis mediating circulating lung cancer cells migration

Ethnopharmacological relevanceMetastasis is the main cause of death in lung cancer patients. Circulating tumor cells (CTCs) may be an important target of metastasis intervention. Previous studies have shown that Jinfukang could prevent the recurrence and metastasis of lung cancer, and we have established a circulating lung tumor cell line CTC-TJH-01. However, whether Jinfukang inhibition of lung cancer metastasis is related to CTCs is still unknown. Aim of the studyTo further explore the mechanism of Jinfukang in anti-metastasis of lung cancer from the perspective of intervention of CTCs. Materials and methodsCTC-TJH-01 and H1975 cells were treated with Jinfukang. Cell viability was detected by CCK8, and the cell apoptosis was detected by flow cytometry. Transwell was used to detected cell migration and invasion. Cell anoikis was detected by anoikis detection kit. Protein expression was analysis by Western blot. ResultsJinfukang could inhibit the proliferation, migration and invasion of CTC-TJH-01 and H1975 cells. Besides, Jinfukang could also induce anoikis in CTC-TJH-01 and H1975 cells. Analysis of the mRNA expression profile showed ECM-receptor interaction and focal adhesion were regulated by Jinfukang. Moreover, it was also find that Jinfukang significantly inhibited integrin/Src pathway in CTC-TJH-01 and H1975 cells. When suppress the expression of integrin with ATN-161, it could promote Jinfukang to inhibit migration and induce anoikis in CTC-TJH-01 and H1975 cells. ConclusionsOur results indicate that the migration and invasion of CTCs are inhibited by Jinfukang, and the mechanism may involve the suppression of integrin/Src axis to induce anoikis. These data suggest that Jinfukang exerts anti-metastatic effects in lung cancer may through anoikis.

PLCγ1‑dependent invasion and migration of cells expressing NSCLC‑associated EGFR mutants.

The increased tyrosine kinase activity of non‑small cell lung cancer (NSCLC)‑associated epidermal growth factor receptor (EGFR) mutants results in deregulated pathways that contribute to malignant cell survival, tumor progression and metastasis. Previous studies investigating lung cancer‑associated EGFR have focused on the prognostic implications of receptor kinase mutations in patients with NSCLC; however, the role of EGFR mutations in tumor cell invasion and migration remains undetermined. The present study was designed to investigate the role of NSCLC‑associated mutant EGFR‑driven signaling pathways in cell proliferation and invasion. Non‑endogenous EGFR‑expressing 293cells stably expressing EGFR mutants that are sensitive or resistant to Food and Drug Administration(FDA)‑approved EGFR‑targeted tyrosine kinase inhibitors(TKIs) were used in the present study. The experiments demonstrated an increased phosphorylation of phospholipase (PLC)γ1, c‑Cbl, signal transducer and activator of transcription(Stat), extracellular regulated kinase (Erk)1/2, Akt, Shc and Gab1 proteins in cells expressing a mutant form, rather than the wild‑type receptor. As PLCγ1 is a known regulator of metastatic development, mutant receptor‑mediated PLCγ1 activation was further evaluated. To examine the effects of EGFR and PLCγ1 phosphorylation, the metastatic potential of cells expressing mutants was investigated using wound healing, Transwell cell migration and invasion assays. The inhibition of receptor phosphorylation with the 1st, 2nd and 3rdgeneration TKIs, gefitinib, afatinib, osimertinib, respectively, reduced PLCγ1 phosphorylation, and reduced the invasive and migratory potential of 293cells, confirming PLCγ1 as one of the probable downstream effectors of mutant EGFR signaling. However, the PLC inhibitor, U73122, inhibited cell migration and invasion without affecting EGFR signaling and PLCγ1 phosphorylation. Notably, U73122 reduced Akt and Erk1/2 phosphorylation within 25min of its application; however, 100%cell viability was recorded even after 48h. Upon further investigation, proliferative signaling pathways remained active at 48h, in accordance with cell viability. Therefore, the present study concludes that mutant receptor‑mediated PLCγ1 activation may play a significant role in the migration and invasion of NSCLC tumors; however, its regulatory role in tumor cell proliferation warrants further investigation and validation in lung tumor cell lines harboring EGFR mutations.

Abstract 4578: Development of MEM-288, a dual-transgene armed and conditionally replication-enhanced oncolytic adenovirus with potent systemic antitumor immunity

Abstract MEM-288 is a conditionally replication-enhanced and dual-transgene armed oncolytic adenovirus designed for the treatment of both solid and liquid tumor indications as either a standalone product or in combination with immune checkpoint inhibitors (ICI). Multiple modifications to the adenovirus backbone have been incorporated to confer tumor-selective replication that includes delta-24 (Δ24) E1, E1b 55kb and E3 viral genome deletions. Furthermore, MEM-288 is armed with two potent immune agonist transgenes: MEM40, a novel chimeric CD40 ligand (CD40L) designed for stable cell surface expression of CD40L that functions as a potent activator of CD40 expressing dendritic cells (DCs), and the DC and T cell maturation and activating cytokine IFNβ. MEM-288 is rationally designed to overcome limitations of other OV by conferring enhanced tumor selectivity, oncolytic activity, and ability to directly enhance dendritic cell (DC) and T cell functionality. MEM-288 exhibited ~100-fold greater replication and oncolytic activity in a broad range of human solid tumors and blood tumor types, including a variety of human lung tumor cell lines, in comparison with control Δ24, ΔE1b 55kb and ΔE3 backbone adenoviruses. MEM-288 also exhibited ~1,000-fold greater viral replication in tumor cells compared to normal cell types, including immature dendritic cells and cancer associated human lung fibroblasts cells. These positive in vitro results were expanded upon in multiple in vivo immunocompetent mouse tumor models. Intratumoral administration of MEM-288 in the syngeneic mouse lung metastatic model 344SQ demonstrated potent anti-metastatic activity of MEM-288 compared to control OVs. MEM-288 also generated a highly efficacious response against both intratumoral injected and distant non-injected contralateral tumors in the B16-F10 melanoma model, including significantly greater inhibition of tumor growth with MEM-288 compared to the combination of anti-CTLA4 + PD-1. In addition, the combination of MEM-288 with anti-CTLA4 + PD-1 significantly enhanced anti-tumor activity with tumor regressions and 100% mouse survival at day 30 compared to 56% and 0% survival with individual treatments of either MEM-288 or anti-CTLA4 + PD-1, respectively. MEM-288 generated significantly better T cell tumor antigen clonal expansion following intratumoral administration in the B16-OVA melanoma model compared to a matched control OV that only expresses MEM40, suggesting IFNβ is a critical mediator of the T cell clonal expansion in combination with MEM40. These positive preclinical data suggest MEM-288 is a potent tumor-selective oncolytic virus with desirable mechanistic features that can be ideally combined with ICI, even in the ICI refractory setting. These studies warrant clinical evaluation for lung cancer and other tumor types that are currently under development. Citation Format: Hong Zheng, Wenjie Dai, Scott Antonia, Mark J. Cantwell, Amer A. Beg. Development of MEM-288, a dual-transgene armed and conditionally replication-enhanced oncolytic adenovirus with potent systemic antitumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4578.

Abstract 1820: Art1, an extracellular mono-ADP-ribosyltransferase, is upregulated in response to cellular stress and promotes lung cancer growth

Abstract Cell surface mono-ADP ribosyltransferases (ARTs) transfer the ADP-ribose moiety from NAD to amino acid residues on target proteins and post-translationally regulate their function. Most mammalian ARTs are extracellular with glycosylphosphatidylinositol (GPI) anchors. ARTs can target cell surface proteins on bystander cells or soluble proteins in the tumor microenvironment, particularly P2RX7 on T cells1. We hypothesized that an evolutionarily conserved parallel protective role may be provided by ART1 expression in lung cancer cells, particularly during times of cellular stress2. A mouse KP1 lung tumor cell line with ART1 expression was established from KRASG12D/P53-/- mice and transduced with shRNA targeting ART1 (KP1-sh) or an empty vector (KP1-ev). For in vitro studies, KP1 WT cells were treated with an ER stress inducer thapsigargin, with radiation, or with chemotherapy. Using human materials, we evaluated ART1 expression in NSCLC tumors by whole tumor RT-PCR/RNAseq, immunofluorescence (IF), and immunohistochemistry (IHC). For evaluation of tumor growth in immune competent models, KP1 cells were injected into syngeneic C57BL/6 mice into the flank or intravenously to generate orthotopic lung tumors. Following in vitro treatment with thapsigargin, expression of ART1 increased 11.8-fold in KP1 WT cells (24 hrs). Similarly, following radiation (8Gy x 3,) ART1 expression increased 1.93-fold by RT-PCR and surface protein expression increased by 1.87 fold (p<0.0005) by IF in KP1 cells. Similar increases were seen following cisplatin (10uM, 24 hrs) treatment (1.87-fold). Radiation (2.26-fold) and cisplatin (25uM, 3.30-fold) also induced ART1 expression in human A549 cells (24 hrs). In a human tissue microarray of patients who received neoadjuvant chemotherapy or radiation therapy (n=200), all tumors expressed ART1 and 38% of strongly expressed ART1. We also compared pre-treatment biopsy and post-treatment surgical resection tumor samples (n=21) in an ongoing clinical trial (NCT02904954) of neoadjuvant durvalumab +/- stereotactic radiation (8Gy x 3). ART1 was expressed at low levels by RNAseq, but trended upward (4-fold) after treatment, particularly in those patients who received radiation therapy (6-fold). Expression trended higher in nonresponders. Of these tumors without major pathologic response (n=13), 44% had moderate-strong ART1 expression by IHC. In murine flank tumor models, KP1-ev cells grew significantly faster than KP1-sh cells (688 mm3 vs. 331mm3 at 4 weeks, p=0.004). Similarly, following tail vein injection, mice injected with KP1-ev cells had heavier tumor burdens by micro-CT scan and significantly greater total lung weights (p=0.036) at sacrifice than did mice injected with KP1-sh cells. In conclusion, ART1 is broadly expressed in human lung tumors and is upregulated by cellular stress invoked by common treatments such as radiation and chemotherapy. Knockdown of ART1 abrogates tumor growth and may be a potential therapeutic target for combination therapy in lung cancer patients. 1. PMID: 7930612 2. PMID: 25292337 Citation Format: Sumit Mukherjee, Erik Wennerberg, Clarey Hung, Najla Saadallah, Shashi Kariyawasam, Christopher Agrusa, Amanda Valeta, Nasser Altorki, Timothy McGraw, Sandra Demaria, Brendon Stiles. Art1, an extracellular mono-ADP-ribosyltransferase, is upregulated in response to cellular stress and promotes lung cancer growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1820.

Abstract 2158: ART1 tumor expression mediates immune resistance in non-small cell lung cancer by elimination of P2 × 7R+ CD8 tissue resident memory T cells and conventional type I dendritic cells

Abstract Extracellular NAD+ (eNAD) is released from stressed and dying cells where it acts as a proinflammatory mediator. However, in the presence of mono-ADP-ribosyltransferases (ARTs) eNAD serves as a substrate for ADP-ribosylation of the P2 × 7 receptor (P2RX7), resulting in NAD-induced cell death (NICD). P2RX7 is expressed on several immune cells including CD103+ conventional type I dendritic cells (cDC1s) which are essential for initiation of tumor-specific immune responses as well as on tissue resident memory T cells (TRMs), of which high tumor infiltration has been associated with improved survival in non-small cell lung cancer (NSCLC) [1]. NICD of P2 × 7R+ TRMs was recently identified as a regulator of TRM tissue homeostasis [2]. CD38 is expressed on activated immune cells and can reduce eNAD levels by converting eNAD into ADPR. We tested the hypothesis that ART1 expression in NSCLC constitutes a mechanism of immune resistance by mediating NICD of P2 × 7R+ TRMs and P2 × 7R+ cDC1s. ART1 expression in human NSCLC samples was determined by immunohistochemistry scoring of NSCLC tumors using a tissue microarray (TMA) (n=493). In addition, matched tumor and adjacent normal lung tissue samples were analyzed for ART1 expression by qPCR (n=40) and for infiltration of TRM and cDC1s by flow cytometry. A mouse KP1 lung tumor cell line with high ART1 expression was established from KRASG12D/P53−/− mice and transduced with a doxycycline-inducible shRNA targeting ART1 (ART1KD). KP1 cells and their derivatives were intravenously injected into syngeneic C57BL/6 mice to generate orthotopic lung tumors. Lung tumor nodules were enumerated two weeks after tumor inoculation using H&E-stained FFPE lung sections and lung-derived cell suspensions were analyzed by flow cytometry for infiltration of T cell and DC subsets. Increased expression of ART1 in tumor compared to normal lung was observed in 55% of NSCLC samples by TMA analysis, and confirmed by qPCR. The frequency of P2RX7+ CD8 TRMs (p<0.05) and P2 × 7R+ cDC1s (p<0.01) were reduced in ART1+ tumors compared to matched normal lung tissue. In line with the hypothesis that CD38 protects P2 × 7R+ cells from ART1-mediated NICD, P2 × 7R+ CD8 TRMs and P2 × 7R+ cDC1s present within ART1+ tumors had elevated expression of CD38. In mice, knockdown of ART1 in KP1 tumors resulted in significantly delayed growth in immunocompetent mice (p<0.001) but not in nude or CD8-depeleted mice. In an orthotopic lung model, ART1KD resulted in a 75% decrease in lung metastatic nodules (p<0.01) and was associated with increased infiltration of CD8 T cells (P<0.0001) and cDC1s (p<0.01). In vitro experiments confirmed that CD38- P2RX7+ CD8 T cells were susceptible to ADP-ribosylation and NICD when co-cultured with ART1high lung tumor cells, while P2 × 7R+ CD8 T cells that co-expressed CD38 were resistant to NICD. In conclusion, we show that ART1 is overexpressed in NSCLC and identify ART1-mediated NICD of P2 × 7R+ TRMs and cDC1s as a possible novel immune escape mechanism in NSCLC. Furthermore, we describe a novel role of CD38 expression on immune cells in its protection against NICD. 1. Nizard, M., et al., Induction of resident memory T cells enhances the efficacy of cancer vaccine. Nat Commun, 2017. 8: p. 15221. 2. Stark, R., et al., T RM maintenance is regulated by tissue damage via P2RX7. Sci Immunol, 2018. 3(30). Citation Format: Erik Wennerberg, Clarey Hung, Amanda Valeta, Timothy McGraw, Sandra Demaria, Brendon Stiles. ART1 tumor expression mediates immune resistance in non-small cell lung cancer by elimination of P2 × 7R+ CD8 tissue resident memory T cells and conventional type I dendritic cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2158.

FGL1 regulates acquired resistance to Gefitinib by inhibiting apoptosis in non-small cell lung cancer

BackgroundThis study investigated the role of fibrinogen-like protein 1 (FGL1) in regulating gefitinib resistance of PC9/GR non-small cell lung cancer (NSCLC).MethodsThe effect of different concentrations of gefitinib on cell proliferation were evaluated using the CCK-8 assay. FGL1 expression in the normal human bronchial epithelial cell line Beas-2B, as well as four lung tumor cell lines, H1975, A549, PC9, and PC9/GR, was investigated by using western blotting and qRT-PCR. FGL1 was knocked down using small interfering RNA to evaluate the effects of FGL1 on PC9 and PC9/GR. The correlation between FGL1 expression and gefitinib resistance was determined in vitro via CCK-8 and colony formation assays, and flow cytometry and in vivo via flow cytometry and immunohistochemistry.ResultsFGL1 expression was significantly upregulated in non-small cell lung cancer cells with EGFR mutation and higher in the gefitinib-resistant NSCLC cell line PC9/GR than in the gefitinib-sensitive NSCLC cell line PC9. Further, FGL1 expression in PC9 and PC9/GR cells increased in response to gefitinib treatment in a dose-dependent manner. Knockdown of FGL1 suppressed cell viability, reduced the gefitinib IC50 value, and enhanced apoptosis in PC9 and PC9/GR cells upon gefitinib treatment. Mouse xenograft experiments showed that FGL1 knockdown in PC9/GR tumor cells enhanced the inhibitory and apoptosis-inducing actions of gefitinib. The potential mechanism of gefitinib in inducing apoptosis of PC9/GR cells involves inhibition of PARP1 and caspase 3 expression via suppression of FGL1.ConclusionsFGL1 confers gefitinib resistance in the NSCLC cell line PC9/GR by regulating the PARP1/caspase 3 pathway. Hence, FGL1 is a potential therapeutic target to improve the treatment response of NSCLC patients with acquired resistance to gefitinib.

Cytotoxicity of ruthenium-N,N-disubstituted-N′-acylthioureas complexes

Five new complexes with general formula [Ru(Ln)(PP)(bipy)]PF6, where Ln=N,N'-dimethyl-N-Acyl thiourea, and P-P: 1,2-bis(diphenylphosphino)ethane (dppe) or 1,4-bis(diphenylphosphino)butane (dppb)) were synthesized and characterized by elemental analysis, molar conductivity, cyclic voltammetry, IR, NMR (1H, 13C{1H} and 31P{1H}), and single crystal X-ray diffractometry. The cytotoxicity of compounds against lung and breast tumor cell lines was significant, where two complexes, [Ru(L3)(bipy)(dppe)]PF6 (3) and [Ru(L3)(bipy)(dppb)]PF6 (6), were selected to evaluate changes in morphology, inhibition of migration and cell death in the MDA-MB-231 lineage. The complexes caused alterations in the cell morphology and were able to inhibit cell migration at the concentrations evaluated, induce the cell cycle arrested in the Sub-G1 phase, and induced cell death by apoptosis. All the complexes presented interaction with HSA, and the interaction studies with DNA suggested weak interactions, probably by the minor groove.

FHITlow /pHER2high signature in non-small cell lung cancer is predictive of anti-HER2 molecule efficacy.

Despite its efficacy in solid tumours, in particular HER2+ breast cancer, HER2-targeted therapy has given rise to disappointing results in non-small cell lung cancer (NSCLC). With the aim of refining the target population for anti-HER2 therapies in NSCLC, we investigated the relationships between HER2 and the tumour suppressor fragile histidine triad (FHIT) in lung tumour cells. First, we observed a negative correlation between FHIT expression and the activated form of HER2 (pHER2) in NSCLC samples and in lung tumour cell lines. Moreover, the silencing or overexpression of FHIT in lung cell lines led to an increase or decrease of HER2 activity, respectively. We also demonstrated that two anti-HER2 drugs, irbinitinib and trastuzumab, restore a more epithelial phenotype and counteract cell invasiveness and growth of FHIT-silenced tumour cell lines. Finally, we showed that the FHITlow /pHER2high phenotype predicts sensitivity to an anti-HER2 therapy in primary tumour cells from NSCLC patients. Our results show that FHIT regulates the activity of HER2 in lung tumour cells and that FHIT-inactivated tumour cells are sensitive to HER2 inhibitors. A new subclass of patients with NSCLC may be eligible for an anti-HER2 therapy. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Overcoming hypoxia-induced functional suppression of NK cells

BackgroundNatural killer (NK) cells are immune cells capable of killing virally infected cells and tumor cells without the need for antigen stimulation. Tumors, however, can create a suppressive microenvironment that...

3′‐(4‐(Benzyloxy)phenyl)‐1′‐phenyl‐5‐(heteroaryl/aryl)‐3,4‐dihydro‐1′H,2H‐[3,4′‐bipyrazole]‐2‐carboxamides as EGFR kinase inhibitors: Synthesis, anticancer evaluation, and molecular docking studies

Pyrazoline-linked carboxamide derivatives were designed, synthesized, and evaluated for potential epidermal growth factor receptor (EGFR) kinase inhibition, anticancer activity, and apoptotic and cardiomyopathy toxicity. Compounds 6m and 6n inhibit EGFR kinase at a concentration of 6.5 ± 2.91 and 3.65 ± 0.54 µM, respectively. Some of these compounds showed effects on proliferation, which were also then evaluated against four different human cancer cell lines, that is, MCF-7 (breast cancer), A549 (non-small-cell lung tumor), HCT-116 (colon cancer), and SiHa cells (cancerous tissues of the cervix uteri). The results showedthat certainsynthetic compounds showed significant inhibitor activity; compounds 6m and 6n were more cytotoxic than doxorubicin against A549 cancer cells, with IC50 values of 10.3 ± 1.07 and 4.6 ± 0.57 µM, respectively. Additionally, compounds 6m and 6n induced apoptosis in A549 cancer cells, as evidenced by 4',6-diamidino-2-phenylindole (DAPI) staining and phase-contrast microscopy. Potency to induce apoptosis by compound 6n was further confirmed by fluorescence-activated cell sorting using Annexin V-FITC and propidium iodide labeling. Compound 6n showed normal cardiomyocytes with no marked sign of pyknotic nuclei in cardiomyopathy and also normal histological appearance of the renal cortex when compared with that of control. Results of molecular docking studies suggested that compounds 6m and 6n can bind to the hinge region of the adenosine triphosphate-binding site of EGFR kinase, like the standard drug erlotinib. Therefore, the present study suggests that compounds 6m and 6n have potent in vitro antitumor activities against the human non-small-cell lung tumor cell line A549, which can be further explored in other cancer cell lines and in animal studies.

Simultaneous measurement of perillyl alcohol and its metabolite perillic acid in plasma and lung after inhalational administration in Wistar rats.

The inhalational administration of drugs is a practical and non-invasive approach with the potential to reduce side effects and with a quick onset of therapeutic activity. Perillyl alcohol (POH) is a monoterpene with antitumor activity that currently is undergoing clinical evaluation as an inhalational anticancer agent. A detection method was developed that will be applicable to pharmacokinetic studies of not only POH, but also its longer-lived main metabolite, perillic acid (PA), in lung tissue and plasma after inhalational delivery. The anticancer activity of POH was investigated in vitro with the use of various lung cancer cell lines. Toxicity was established by a standard MTT assay, and apoptosis markers were analyzed by Western blot. For the detection of POH and PA in lungs and plasma, albino Wistar rats were used that were exposed to POH inhalation. Tissues were subjected to chromatographic separation on an Agilent Zorbax Eclipse XDB C18 column, followed by detection of absorption in the ultraviolet (UV) range. In vitro, POH exerted cytotoxic activity against six different lung tumor cell lines, and apoptotic cell death was indicated by induction of active caspase 3 and cleavage of poly (ADP-ribose) polymerase 1 (PARP1). These results demonstrate that inhalational delivery of POH results in effective biodistribution and metabolism of POH in the systemic circulation. In addition, our study introduces a simple, rapid HPLC-UV method with high accuracy for simultaneous detection of POH and its metabolite PA in plasma, and for sensitive detection of PA in lung tissue, which should prove useful for applications in clinical studies.