Inhibited Cell Colony Formation Research Articles

Unveiling the antibreast cancer mechanism of Euphorbia hirta ethanol extract: computational and experimental study

Breast cancer, the most prevalent cancer in women globally, is characterized by a low survival rate. Euphorbia hirta is predicted to exhibit promising antibreast cancer properties. This study aimed to explore the active compounds present in the ethanol extract of E. hirta and elucidate their mechanism for countering breast cancer using computational and experimental methodologies. The active components of the E. hirta ethanol extract were determined using Liquid chromatography high-resolution mass spectrometry. Furthermore, the identified active compounds were subjected to computational analysis involving drug-likeness screening, membrane permeability, bioactivity, toxicity assessment and protein target prediction using QSAR principles followed by molecular docking and molecular dynamics simulations. In vitro experimental investigations included toxicity, rhodamine, apoptosis, cell migration and colony formation assays. These findings indicated that six compounds with drug-like properties and physicochemical features exhibited bioactivity relevant to breast cancer. These compounds, namely, quercetin, luteolin, gallic acid, gentisic acid, quinic acid and caffeic acid, target proteins associated with breast cancer progression, including EGFR, KDR, SRC, FASN, CDK4 and ERBB2. According to molecular docking and dynamics studies, these active compounds bind securely, potentially inhibiting their target protein activity. The ethanol extract of E. hirta was toxic and selective for T47D cells. The extract induced apoptosis and loss of mitochondrial membrane potential in T47D cells, restraining their migration and colony formation. The research findings demonstrate that the extract of E. hirta effectively antagonizes breast cancer by inducing apoptosis, suppressing cellular migration, and inhibiting cell colony formation through the targeted modulation of associated proteins.

LIPH contributes to glycolytic phenotype in pancreatic ductal adenocarcinoma by activating LPA/LPAR axis and maintaining ALDOA stability

BackgroundLIPH, a membrane-associated phosphatidic acid-selective phospholipase A1a, can produce LPA (Lysophosphatidic acid) from PA (Phosphatidic acid) on the outer leaflet of the plasma membrane. It is well known that LIPH dysfunction contributes to lipid metabolism disorder. Previous study shows that LIPH was found to be a potential gene related to poor prognosis with pancreatic ductal adenocarcinoma (PDAC). However, the biological functions of LIPH in PDAC remain unclear.MethodsCell viability assays were used to evaluate whether LIPH affected cell proliferation. RNA sequencing and immunoprecipitation showed that LIPH participates in tumor glycolysis by stimulating LPA/LPAR axis and maintaining aldolase A (ALDOA) stability in the cytosol. Subcutaneous, orthotopic xenograft models and patient-derived xenograft PDAC model were used to evaluate a newly developed Gemcitabine-based therapy.ResultsLIPH was significantly upregulated in PDAC and was related to later pathological stage and poor prognosis. LIPH downregulation in PDAC cells inhibited colony formation and proliferation. Mechanistically, LIPH triggered PI3K/AKT/HIF1A signaling via LPA/LPAR axis. LIPH also promoted glycolysis and de novo synthesis of glycerolipids by maintaining ALDOA stability in the cytosol. Xenograft models show that PDAC with high LIPH expression levels was sensitive to gemcitabine/ki16425/aldometanib therapy without causing discernible side effects.ConclusionLIPH directly bridges PDAC cells and tumor microenvironment to facilitate aberrant aerobic glycolysis via activating LPA/LPAR axis and maintaining ALDOA stability, which provides an actionable gemcitabine-based combination therapy with limited side effects.Graphical

Bufadienolides from the Bufo viridis toad venom exert cytotoxic effects on cancer cells by inducing cell apoptosis and cell cycle arrest

A series of bufadienolides were isolated from the Bufo viridis toad venom, and their cytotoxic activities against three human cancer cell lines (HeLa, HT-29, MCF7) and a non-cancer cell line (L-O2) were explored using the MTT assay in vitro. All of nine compounds exhibited cytotoxic activities against the three cancer cell lines, with compound D4 exhibiting potent cytotoxic activity against HeLa cells and was better than positive control. Herein, we further evaluated the effect of compound D4 on HeLa cells. The results revealed that compound D4 has excellent cytotoxic effect on HeLa cells by inhibiting cell colony formation and migration, promoting cell apoptosis, increasing reactive oxygen species (ROS) levels and arresting of HeLa cells in S and G2/M phases. These findings encourage further work on the chemistry and bioactivity of the Bufo viridis toad venom.

Iridium(III) complexes inhibit the proliferation and migration of BEL-7402 cells through the PI3K/AKT/mTOR signaling pathway

Iridium(III) complexes are largely studied as anti-cancer complexes due to their excellent anti-cancer activity. In this article, two new iridium(III) complexes [Ir(piq)2(THPIP)]PF6 (THPIP = 2,4-di-tert-butyl-6-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)phenol, piq = deprotonated 1-phenylisoquinoline) (Ir1) and [Ir(bzq)2(THPIP)]PF6 (bzq = deprotonated benzo[h]quinolone) (Ir2) were synthesized. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays showed that complex Ir1 exhibits moderate activity (IC50 = 29.9 ± 4.6 μM) and Ir2 shows high cytotoxicity (IC50 = 9.8 ± 1.8 μM) against BEL-7402 cells. Further studies on the mechanism showed that Ir1 and Ir2 induced apoptosis by changing the mitochondrial membrane potential, Ca2+ release, ROS accumulation, and cell cycle arrest at the S phase. The complexes can effectively inhibit cell colony formation and migration. The expression of B-cell lymphoma-2 (Bcl-2) family proteins, PI3K (phosphatidylinositol 3-kinase), AKT (protein kinase B), mTOR (mammalian target of rapamycin), and p-mTOR was studied by immunoblotting. Complexes Ir1 and Ir2 downregulated the expression of anti-apoptotic protein Bcl-2 and increased the expression of autophagy-related proteins of Beclin-1 and LC3-II. Further experiments showed that the complexes inhibited the production of glutathione (GSH) and increased the amounts of malondialdehyde (MDA). Fluorescence of HMGB1 was significantly increased. We also investigated the effect of the complexes on the expression of genes using RNA-sequence analysis, we further calculated the lowest binding energies between the complexes and proteins using molecular docking. Taken together, the above results indicated that complexes Ir1 and Ir2 induce apoptosis in BEL-7402 cells through a ROS-mediated mitochondrial dysfunction and inhibition of the PI3K/AKT/mTOR signaling pathway.

Synthesis of nitrogen mustard-based fluorophores for cell imaging and cytotoxicity studies.

Nitrogen mustards are important alkylating anticancer drugs used for neoplasms treatment. However, little research about the integration of luminophore into nitrogen mustard-based compounds for both imaging and therapeutic application was reported. In this study, we report a series of novel nitrogen mustard-containing 1-furyl-2-en-1-one and 1-thienyl-2-en-1-one derivatives as intramolecular charge transfer-based luminophore for research in both imaging subcellular localization and antiproliferation toward lung cancer cells. The target products were prepared by Knoevenagel condensation and characterized by nuclear magnetic resonance and high-resolution mass spectrometer. The absorption and fluorescence studies were carried out by ultraviolet-visible and fluorescence spectrophotometers, respectively. Cell morphology was observed under an inverted microscope. Cytotoxicity test was detected by MTT assay. Cellular localization was observed by a confocal laser scanning microscope. Colony formation ability was carried out by colony formation assay. Cell migration ability was detected by transwell migration assay. Differences between the two groups were analyzed by two-tailed Student's t-test. The difference with P < 0.05 (*) was considered statistically significant. The compounds were synthesized in high yield. The λmax and Stokes shift of these compounds reach up to 567 and 150 nm, respectively. These compounds exhibited good antiproliferative activity against lung cancer cells, with compound 3h exhibiting the best IC50 of 13.1 ± 2.7 μM. Furthermore, the selected compound 3h is located preferentially in lysosomes and a small amount in nuclei, effectively inhibiting cell colony formation and migration abilities toward A549 cells. These findings suggested that nitrogen mustard-based fluorophores might be a potential effective chemotherapeutic agent in lung cancer therapy.

Knockdown of Peroxiredoxin V increased the cytotoxicity of non-thermal plasma-treated culture medium to A549 cells.

Administration of non-thermal plasma therapy via the use of plasma-activated medium (PAM) might be a novel strategy for cancer treatment, as it induces apoptosis by increasing reactive oxygen species (ROS) levels. Peroxiredoxin V (PRDX5) scavenges ROS and reactive nitrogen species and is known to regulate several physiological and pathological reactions. However, its role in lung cancer cells exposed to PAM is unknown. Here, we investigated the effect of PRDX5 in PAM-treated A549 lung cancer cells and determined the mechanism underlying its cytotoxicity. Cell culture medium was treated with low temperature plasma at 16.4 kV for 0, 60, 120, or 180 s to develop PAM. PRDX5 was knocked down in A549 cells via transfection with short hairpin RNA targeting PRDX5. Colony formation and wound healing assays, flow cytometry, fluorescence microscopy, and western blotting were performed to detect the effect of PRDX5 knockdown on PAM-treated A549 cells. PAM showed higher cytotoxicity in lung cancer cells than in control cells, downregulated the mitogen-activated protein kinase signaling pathway, and induced apoptosis. PRDX5 knockdown significantly inhibited cell colony formation and migration, increased ROS accumulation, and reduced mitochondrial membrane potential in lung cancer cells. Hence, PRDX5 knockdown combined with PAM treatment represents an effective option for lung cancer treatment.

Lecanoric acid mediates anti-proliferative effects by an M phase arrest in colon cancer cells

Lichen extracts containing, among other compounds, depsides such as evernic acid, atranorin, and lecanoric acid possess anti-proliferative effects. We aimed to identify lichen metabolites that are responsible for the observed anti-proliferative effects. We performed cytotoxicity, cell colony, cell cycle and apoptosis assays in various cell lines or primary immune cells. We analyzed several cell cycle proteins and apoptosis-related proteins to gain insights into the underlying mechanism. All depsides reduced the viability of the tested cell lines (HCT-116, HEK293T, HeLa, NIH3T3, RAW246.7) in a cell line-dependent manner with lecanoric acid being the most effective. Atranorin did not influence the cell cycle or colony formation in HCT-116 cells, but induced apoptosis in HCT-116 cells. Evernic acid showed no anti-proliferative effects. Lecanoric acid inhibited cell colony formation already at 0.03 µg/ml in HCT-116 cells and induced a G2 cell cycle block in several cell lines. Moreover, lecanoric acid arrested the cell cycle, presumably in the M phase, since expression of cyclin B1 and phosphorylated histone H3 was upregulated, whereas the inactive cyclin-dependent kinase 1 (CDK1) was reduced in HCT-116 cells. Most importantly, cell death induced by lecanoric acid was more prominent in cancer cells than in primary human immune and endothelial cells. In conclusion, lecanoric acid seems to mediate its anti-proliferative effects via arrest of cells in the M phase. Our data suggest lecanoric acid may be a potential new candidate for anti-cancer therapy, because it has anti-proliferative effects on cancer cell lines, and does not affect primary immune cells.

Synthesis and evaluation of RNase L-binding 2-aminothiophenes as anticancer agents

Aminothiophene is a scaffold that is widely present in drugs and biologically active small molecules as chemical probes. In this study, 43 compounds sharing a 2-aminothiophenone-3-carboxylate (ATPC) scaffold, known to activate the ribonuclease L (RNase L), were synthesized and selected ATPCs showed enhancement of thermal stability of RNase L upon binding. Screening of antiproliferation activities against human cancer cell lines revealed that ATPCs represented by compounds 4l and 50 showed potent single-digit micromolar antiproliferation activity against human cancer cell lines. Compounds 4l and 50 exhibited time- and dose-dependent proliferation inhibition, induced cellular apoptosis measured by cleaved PARP and via flow cytometry, inhibited cell migration, and inhibited cell colony formation. Combining the results reported in this work, ATPCs were evaluated as potential anticancer agents mediated by RNase L-binding and apoptosis induction. The work contributes to the study on the polypharmacological properties of aminothiophene-containing small molecules.

Oncogenic signaling inhibits c-FLIPL expression and its non-apoptotic function during ECM-detachment

Inhibition of programmed cell death pathways is frequently observed in cancer cells where it functions to facilitate tumor progression. However, some proteins involved in the regulation of cell death function dichotomously to both promote and inhibit cell death depending on the cellular context. As such, understanding how cell death proteins are regulated in a context-dependent fashion in cancer cells is of utmost importance. We have uncovered evidence that cellular FLICE-like Inhibitory Protein (c-FLIP), a well-known anti-apoptotic protein, is often downregulated in tumor tissue when compared to adjacent normal tissue. These data argue that c-FLIP may have activity distinct from its canonical role in antagonizing cell death. Interestingly, we have discovered that detachment from extracellular matrix (ECM) serves as a signal to elevate c-FLIP transcription and that oncogenic signaling blocks ECM-detachment-induced c-FLIP elevation. In addition, our data reveal that downregulation of c-FLIP promotes luminal filling in mammary acini and that c-FLIP overexpression in cancer cells inhibits colony formation in cells exposed to ECM-detachment. Taken together, our study reveals an unexpected, non-apoptotic role for c-FLIP during ECM-detachment and raises the possibility that c-FLIP may have context-dependent roles during tumorigenesis.

Evaluation of N-aryl-β-alanine derivatives as anticancer agents in triple-negative breast cancer and glioblastoma in vitro models

Synthesis of β-amino acid derivatives containing hydrazone and azole moieties is described. For this purpose, the appropriate hydrazide was treated with aromatic aldehydes, ketones and phenyl iso(thio)cyanates to obtain the desired outcome. The synthesized target compounds were evaluated for their anticancer properties. The assay displayed 3,3′-((2,6-diethylphenyl)azanediyl)bis(N′-(benzylidene)propanehydrazide) to possess the convincing anticancer effect against triple-negative breast cancer cells in vitro. To further study the anticancer properties of compounds containing a hydrazone moiety in breast cancer, series of previously and newly prepared dihydrazones were investigated. It was determined that derivatives with the bis(N′-(4-bromobenzylidene) fragment in the structure are exclusively cytotoxic to cancer cells. The most active compounds against both cell lines were those containing electron withdrawing 4-BrPh or 4-ClPh moieties, together with either chlorine, bromine or iodine groups in para position of phenyl ring. Selected two representative compounds showed migrastatic activity in MDA-MB-231 cell line, where both of them reduced the growth of breast cancer and glioblastoma cell 3D cultures and inhibited cell colony formation. 2009 Elsevier Ltd. All rights reserved.

Discovery and activity verification of reniformin A as an anti-tumor leading compound

Reverse prediction and molecular docking techniques were employed to evaluate the feasibility of reniformin A(RA) as an anti-tumor leading compound. Based on the reverse prediction, network pharmacology was used to construct a &quot;disease-compound-target-pathway&quot; network. Thirty-nine tumor-related targets of RA were predicted, which participated in the regulation of multiple cellular activities such as apoptosis, cell cycle, and tumor metastasis, and regulated estrogen signal transduction and inflammatory response. Discovery Studio 2020 was adopted for molecular docking and toxicity prediction(TOPKAT). As revealed by the results, the binding affinity of RA with the tumor-related targets ABL1, ESR1, SRC and BCL-XL was stronger than that of oridonin(OD), while its mutagenicity, rodent carcinogenesis, and oral LD_(50) in rats were all inferior to that of OD. Furthermore, in vitro experiments were performed to confirm the anti-tumor activity of RA, and the mechanism was preliminarily discussed. The results demonstrated that RA was superior to OD in cytotoxicity, inhibition of cell colony formation, and induction of apoptosis. RA, possessing potent anti-tumor activity, is expected to be a new anti-tumor leading compound.

Abstract 1477: Co-targeting c-Met and c-Src pathways to inhibit metastatic cancer cell migration and invasion

Abstract Overexpression of tyrosine kinases c-Src and c-Met has been shown to be highly associated with disease progression and poor clinical outcomes in many types of cancers. Although small molecule Src or Met inhibitors are being individually evaluated in clinical trials, patient data suggest that single treatment with any of these inhibitors only yield limited effects. One possibility is that redundant signaling pathways in tumor cells may allow tumor cell functions less interrupted upon inhibition of only one pathway. We therefore propose that c-Src and c-Met are two compensatory pathways that may cooperate to promote cancer metastasis. In order to delineate this hypothesis, we studied treatment effects of combining inhibitors targeting c-Src (dasatinib) and c-Met (ASLAN002) on multiple functional behaviors associated with tumor metastasis in c-Src and c-Met expressing metastatic cancer cells. Combination of Src- and Met-inhibitors was found to strongly inhibit cell colony formation by clonogenic survival assay. In a wound-healing assay, compared to either treatment alone, the combinatorial treatment exhibited greater suppression on cell motility. In addition, using a Matrigel-based Transwell assay, dasatinib plus ASLAN002 resulted in significant reduction of cell invasion in the presence or absence of HGF. Western blot analysis demonstrated that co-targeting Met and Src kinases lead to not only inhibition on c-Met and c-Src autophosphorylation but also multiple downstream kinases such as focal adhesion kinase (FAK), Akt and extracellular signal-regulated kinase (ERK). These data indicate that combined application of small molecule Src and Met inhibitors cause significant impairment on functional behaviors associated with cancer metastasis in vitro including survival, migration and invasion, suggesting that this combination intervention modality may serve as a potential anti-metastatic strategy that could be further tested in metastatic models in vivo. Citation Format: Yao Dai, Dietmar W. Siemann. Co-targeting c-Met and c-Src pathways to inhibit metastatic cancer cell migration and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1477.

Discovery of N-aryl sulphonamide-quinazoline derivatives as anti-gastric cancer agents in vitro and in vivo via activating the Hippo signalling pathway

Hippo signalling pathway plays a crucial role in tumorigenesis and cancer progression. In this work, we identified an N-aryl sulphonamide-quinazoline derivative, compound 9i as an anti-gastric cancer agent, which exhibited potent antiproliferative ability with IC50 values of 0.36 μM (MGC-803 cells), 0.70 μM (HCT-116 cells), 1.04 μM (PC-3 cells), and 0.81 μM (MCF-7 cells), respectively and inhibited YAP activity by the activation of p-LATS. Compound 9i was effective in suppressing MGC-803 xenograft tumour growth in nude mice without obvious toxicity and significantly down-regulated the expression of YAP in vivo. Compound 9i arrested cells in the G2/M phase, induced intrinsic apoptosis, and inhibited cell colony formation in MGC-803 and SGC-7901 cells. Therefore, compound 9i is to be reported as an anti-gastric cancer agent via activating the Hippo signalling pathway and might help foster a new strategy for the cancer treatment by activating the Hippo signalling pathway regulatory function to inhibit the activity of YAP.

In Vitro Antitumor Potential of Sulfated Polysaccharides from Seaweed Caulerpa cupressoides var. flabellata.

Seaweeds are important source of bioactive compounds, including sulfated polysaccharides (SP). Because of their structural heterogeneity, these compounds are promising sources of anticancer compounds. SP from brown and red seaweeds have shown antimelanoma activity in different in vitro and in vivo models. However, SP from green seaweed are still poorly evaluated. Therefore, SP were extracted from the green alga Caulerpa cupressoides var. flabellata, and their antiproliferative, anti-migratory, and inhibitory effect on melanin production on B16-F10 melanoma cells was evaluated. Cell assays, including flow cytometry, demonstrated that SP (100-1000μgmL-1) are non-cytotoxic, do not induce apoptosis or necrosis, and do not interfere with cell cycle. However, SP (1000μgmL-1) were found to significantly inhibit cell colony formation (80-90%), cell migration (40-75%), and melanin production (~ 20%). In summary, these results showed that SP inhibited important melanoma development events without cytotoxicity effects, suggesting that C. cupressoides may be an important source of SP with antitumor properties.

Acteoside as a potential therapeutic option for primary hepatocellular carcinoma: a preclinical study

BackgroundHepatocellular carcinoma (HCC) is a common malignant tumor with characteristics of poor prognosis, high morbidity and mortality worldwide. In particular, only a few systemic treatment options are available for advanced HCC patients, and include sorafenib and the recently described atezolizumab plus bevacizumab regimen as possible first-line treatments. We here propose acteoside, a phenylethanoid glycoside widely distributed in many medicinal plants as a potential candidate against advanced HCC.MethodsCell proliferation, colony formation and migration were analyzed in the three human HCC cell lines BEL7404, HLF and JHH-7. Angiogenesis assay was performed using HUVESs. The BEL7404 or JHH-7 xenograft nude mice model was established to analyze the possible antitumor effects of acteoside. qRT-PCR and western blotting were used to reveal the potential antitumor mechanisms of acteoside.ResultsActeoside inhibited cell proliferation, colony formation and migration in all the three human HCC cell lines BEL7404, HLF and JHH-7. The prohibition of angiogenesis by acteoside was revealed by the inhibition of tube formation and cell migration of HUVECs. The combination of acteoside and sorafenib produced stronger inhibition of cell colony formation and migration of the HCC cells as well as of angiogenesis of HUVECs. The in vivo antitumor efficacy of acteoside was further demonstrated in BEL7404 or JHH-7 xenograft nude mice model, with an enhancement when combined with sorafenib in inhibiting the growth of JHH-7 xenograft. Further treatment of JHH-7 cells with acteoside revealed an increase in the level of tumor suppressor protein p53 as well as a decrease of kallikrein-related peptidase (KLK1, 2, 4, 9 and 10) gene level with no significant changes of the rest of KLK1–15 genes.ConclusionsActeoside exerts an antitumor effect possibly through its up-regulation of p53 levels as well as inhibition of KLK expression and angiogenesis. Acteoside could be useful as an adjunct in the treatment of advanced HCC in the clinic.

Abstract 1854: Oridonin inhibits cell growth of esophageal carcinoma cells via induction of apoptosis

Abstract Esophageal Cancer estimated leading deaths are more than 400,000 every year in worldwide, while 80% due to esophageal squamous cell carcinoma. In worldwide, esophageal cancer leading death ranks in sixth among cancer leading causes of death, while the 5-year survival rate of esophageal cancer is 19%. Oridonin, a tetracyclic diterpenes compound, has been indicated the anti-tumor activity for digestive system tumors. To assess the anticancer activity of oridonin on esophageal cancer, we performed cell proliferation experiment of oridonin on esophageal squamous cell carcinoma cell line TE-8 using SRB assay. We found that IC50 of oridonin on TE-8 treated for 72h is 3.0μM, which is better than positive control Gemcitabine treatment on TE-8 for 72h (5.7 μM), as well as TE-8 cells shrunk and floated with increasing concentration on oridonin. At the same time, oridonin induced a dose-dependent inhibition of cell colony formation of esophageal cancer cell line. Colony numbers of TE-8 cell line treated with 1μM and 2μM oridonin are 111 and 54, respectively, which are significantly different (p&amp;lt;0.05). Accompanying with marked morphological changes, flow cytometer results showed that oridonin triggered sub-G0/G1 cell cycle arrest in a dose-dependent manner. After treatment by 10μM and 20μM oridonin for 24h, percentage of sub-G0/G1 cells were 25.5 and 55.3%, respectively, which are significantly different (p&amp;lt;0.05). The apoptosis results showed that oridonin treatment led to the accumulation of cells in early-(Annexin V+/PI-) and late-stage (Annexin V+/PI+) apoptosis in a dose-dependent manner. Citation Format: Xiaojun Zhang, Mengtao Xing, Yangcheng Ma, Xiao Wang, Jianying Zhang. Oridonin inhibits cell growth of esophageal carcinoma cells via induction of apoptosis [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 1854.

PHF14 Promotes Cell Proliferation and Migration through the AKT and ERK1/2 Pathways in Gastric Cancer Cells.

PHF14 is a new member belonging to PHD finger proteins. PHF14 is involved in multiple biologic processes including Dandy–Walker syndrome, mesenchyme growth, lung fibrosis, renal fibrosis, persistent pulmonary hypertension, and tumor development. This study aims to explore whether PHF14 plays an important role in gastric cancer. Here, PHF14 is indicated as a tumor promoter. The expression of PHF14 enhances no matter in clinical samples or in gastric cancer cells. High expression of PHF14 impairs survival of patients. Attenuation of PHF14 inhibits cell proliferation in gastric cancer cells. PHF14 downregulation inhibits the expression of cell cycle-related proteins, CDK6 and cyclin D1. Furthermore, silencing of PHF14 reduces the level of phosphorylated AKT as well as phosphorylated ERK1/2. Finally, downregulation of PHF14 in gastric cancer cells inhibits colony formation in vitro and tumorigenesis in vivo. These results indicate that PHF14 promotes tumor development in gastric cancer, so PHF14 thereby acts as a potential target for gastric cancer therapy.

Abstract B14: CRAF-mediated inactivation of epigenetic repressor complexes promotes KRAS-driven tumorigenesis

Abstract Oncogenic mutations in KRAS serve as the initiating event in many human cancers, most notably pancreatic and lung cancers. While KRAS can signal through multiple downstream effector pathways, the MAPK pathway functions as one of the most critical downstream effectors promoting tumorigenesis. Despite its prominent role, inhibitors targeting the MAPK pathway have limited efficacy in KRAS mutant tumors. In the case of BRAF inhibitors, paradoxical activation is observed in KRAS-mutant tumors, while MEK and ERK inhibitors disable negative feedback loops resulting in pathway reactivation. Combinations of MEK and ERK inhibitors have been explored clinically, but a safe combination dose has not yet been established. Given this, identifying novel vulnerabilities in KRAS mutant tumors represents a significant unmet clinical need. Hence, we analyzed the dependency of KRAS-mutant tumors in the Achilles dataset and found a significant subset of KRAS and NRAS-mutant tumors that appear to depend on CRAF. Importantly, KRAS-mutant tumors were not dependent on BRAF or ARAF, the other 2 RAF isoforms. This is consistent with the studies in KRAS-mutant lung GEMMs, where CRAF and not BRAF depletion has been shown to reduce tumor incidence, suggesting that CRAF serves as the main signaling hub downstream of mutant KRAS. In this work, we investigate the kinase-dependent and -independent roles of CRAF in KRAS-mutant tumors. We demonstrate that CRAF depletion in KRAS-mutant human cancer cells inhibits colony formation on soft agar assay and mediates tumor growth inhibition in mouse xenograft models. CRAF loss resulted in activation of cell death and cell cycle arrest pathways without any alterations to downstream MAPK signaling. Interestingly, this suppression of growth upon CRAF loss in KRAS mutant cancer cells was rescued by expression of kinase-dead variants of CRAF, suggesting CRAF kinase function is not required to promote KRAS-driven tumorigenesis. In KRAS-mutant tumor cells, we observe that CRAF preferentially heterodimerizes with ARAF and upon treatment with a MEK inhibitor, the dimer preference on CRAF switches to BRAF, resulting in elevated CRAF kinase activity. To explore molecular determinants of CRAF kinase independent functions, we performed an enrichment screen to identify effectors whose loss confers a growth advantage in the CRAF-null KRAS mutant cells. We identified a class of epigenetic repressor complexes that were strongly depleted in the CRAF-null KRAS mutant cells and are in the process of validating these hits. Further, to dissect CRAF kinase-dependent versus -independent functions, we are establishing a CRAF gene signature using KRAS-mutant cancer cells and also, identifying CRAF-interacting partners that promote CRAF kinase-independent functions. Thus far, this study elucidates novel kinase-independent roles of cRAF that could be harnessed to effectively target KRAS-mutant human cancers. Citation Format: Avinashnarayan Venkatanarayan, Christiaan Klijn, Shiva Malek. CRAF-mediated inactivation of epigenetic repressor complexes promotes KRAS-driven tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B14.

Overcoming acquired resistance of EGFR-mutant NSCLC cells to the third generation EGFR inhibitor, osimertinib, with the natural product honokiol.

The development of acquired resistance to osimertinib (Osim) (AZD9291 or TAGRISSOTM), an FDA‐approved third‐generation epidermal growth factor receptor (EGFR) inhibitor for the treatment of EGFR‐mutant nonsmall cell lung cancer (NSCLC), limits the long‐term benefits for patients. Thus, effective treatment options are urgently needed. To this end, we explored whether honokiol (HNK), a natural product with potential antitumor activity, may be used to overcome Osim resistance. The combination of HNK and Osim synergistically decreased the survival of several Osim ‐resistant cell lines with enhanced effects on inhibiting cell colony formation and growth and on inducing apoptosis. This combination also showed greater growth suppression of Osim‐resistant xenograft tumors including those with 19del, T790M, and C797S triple mutations in nude mice. Mechanistically, the augmented induction of apoptosis by the combination is largely due to enhanced Mcl‐1 reduction through facilitating its degradation. A synthetic HNK derivative exerted similar effects with greater efficacy. Our findings warrant further study of HNK and its derivatives in overcoming Osim resistance in the clinic.

Overcoming acquired resistance of epidermal growth factor receptor-mutant non-small cell lung cancer cells to osimertinib by combining osimertinib with the histone deacetylase inhibitor panobinostat (LBH589).

The major clinical obstacle that limits the long-term benefits of treatment with osimertinib (AZD9291) in patients with epidermal growth factor receptor-mutant non-small cell lung cancer is the development of acquired resistance. Therefore, effective strategies that can overcome acquired resistance to osimertinib are urgently needed. The authors' current efforts in this direction have identified LBH589 (panobinostat), a clinically used histone deacetylase inhibitor, as a potential agent in overcoming osimertinib resistance. Cell growth and apoptosis in vitro were evaluated by measuring cell numbers and colony formation and by detecting annexin V-positive cells and protein cleavage, respectively. Drug effects on tumor growth in vivo were assessed with xenografts in nude mice. Alterations of tested proteins in cells were monitored with Western blot analysis. Gene knockout was achieved using the CRISPR/Cas9 technique. The combination of LBH589 and osimertinib synergistically decreased the survival of different osimertinib-resistant cell lines, including those harboring C797S mutations, with greater inhibition of cell colony formation and growth. The combination enhanced the induction of apoptosis in osimertinib-resistant cells. Importantly, the combination effectively inhibited the growth of osimertinib-resistant xenograft tumors in nude mice. Mechanistically, the combination of LBH589 and osimertinib enhanced the elevation of Bim in osimertinib-resistant cells. Knockout of Bim in osimertinib-resistant cells substantially attenuated or abolished apoptosis enhanced by the LBH589 and osimertinib combination. These results collectively support a critical role of Bim elevation in the induction of apoptosis of osimertinib-resistant cells for this combination. The current findings provide strong preclinical evidence in support of the potential for LBH589 to overcome osimertinib resistance in the clinic.