Proliferation Of Cancer Cells In Culture Research Articles

Glycosphingolipid synthesis mediates immune evasion in KRAS-driven cancer.

Cancer cells frequently alter their lipids to grow and adapt to their environment1-3. Despite the critical functions of lipid metabolism in membrane physiology, signalling and energy production, how specific lipids contribute to tumorigenesis remains incompletely understood. Here, using functional genomics and lipidomic approaches, we identified de novo sphingolipid synthesis as an essential pathway for cancer immune evasion. Synthesis of sphingolipids is surprisingly dispensable for cancer cell proliferation in culture or in immunodeficient mice but required for tumour growth in multiple syngeneic models. Blocking sphingolipid production in cancer cells enhances the anti-proliferative effects of natural killer and CD8+ T cells partly via interferon-γ (IFNγ) signalling. Mechanistically, depletion of glycosphingolipids increases surface levels of IFNγ receptor subunit 1 (IFNGR1), which mediates IFNγ-induced growth arrest and pro-inflammatory signalling. Finally, pharmacological inhibition of glycosphingolipid synthesis synergizes with checkpoint blockade therapy to enhance anti-tumour immune response. Altogether, our work identifies glycosphingolipids as necessary and limiting metabolites for cancer immune evasion.

EEF1A2 promotes PTEN-GSK3β-SCF complex-dependent degradation of Aurora kinase A and is inactivated in breast cancer.

The translation elongation factor eEF1A promotes protein synthesis. Its methylation by METTL13 increases its activity, supporting tumor growth. However, in some cancers, a high abundance of eEF1A isoforms is associated with a good prognosis. Here, we found that eEF1A2 exhibited oncogenic or tumor-suppressor functions depending on its interaction with METTL13 or the phosphatase PTEN, respectively. METTL13 and PTEN competed for interaction with eEF1A2 in the same structural domain. PTEN-bound eEF1A2 promoted the ubiquitination and degradation of the mitosis-promoting Aurora kinase A in the S and G2 phases of the cell cycle. eEF1A2 bridged the interactions between the SKP1-CUL1-FBXW7 (SCF) ubiquitin ligase complex, the kinase GSK3β, and Aurora-A, thereby facilitating the phosphorylation of Aurora-A in a degron site that was recognized by FBXW7. Genetic ablation of Eef1a2 or Pten in mice resulted in a greater abundance of Aurora-A and increased cell cycling in mammary tumors, which was corroborated in breast cancer tissues from patients. Reactivating this pathway using fimepinostat, which relieves inhibitory signaling directed at PTEN and increases FBXW7 expression, combined with inhibiting Aurora-A with alisertib, suppressed breast cancer cell proliferation in culture and tumor growth in vivo. The findings demonstrate a therapeutically exploitable, tumor-suppressive role for eEF1A2 in breast cancer.

Targeting the Oncofetal Transcription Factor Protein SALL4 in Cancer By a Non-IMiD Degrader

Sal-like protein 4 (SALL4) is a C2H2 zinc finger transcription factor (TF) with two naturally occurring isoforms; SALL4A and SALL4B. It is typically detected in fetal tissues and silenced in most normal adult tissues. It is therefore intriguing that SALL4 is aberrantly re-expressed in about one-third of almost all primary human malignancies, presumably by demethylation dependent mechanisms. Direct evidence of the causative role of SALL4 in cancer has been demonstrated in SALL4 transgenic mice, which developed myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and/or liver tumors. Loss-of-function studies by SALL4 knock-down using shRNA showed cell growth inhibition and death in leukemias and solid tumors in culture and in in vivo xenotransplants. TF's, such as SALL4, which are critical for cancer development and survival, have historically been viewed as “undruggable”. However, rather than blocking activity, an alternative approach to target TFs is to induce protein degradation. Immunomodulatory imide drugs (IMiDs), including thalidomide, lenalidomide and pomalidomide, are used in treating patients with multiple myeloma (MM), MDS with 5q deletion, mantle cell lymphoma (MCL), and other hematological malignancies. Recently, several groups have reported that IMiDs can degrade SALL4 in a proteasome-dependent manner. To that end, we sought to investigate whether IMiDS could be utilized to treat SALL4-positive/expressing cancers. Initially, our studies observed that IMiDs had no effect on SALL4-positive cancer cells. Additional investigations thereafter demonstrated that IMiDs could only degrade SALL4A. These findings suggested that SALL4B may not be affected by IMiDs, and may be essential for SALL4-mediated cancer cell survival. Further investigation revealed that SALL4B knockdown led to an increase in apoptosis and inhibition of cancer cell growth. Moreover, through high-throughput screening, we identified a new non-IMiD SALL4 degrader that targets SALL4B via proteasomal degradation and which exhibited potent anti-cancer activity, inhibiting cancer cell proliferation in culture and in vivotumor growth by 70%. Our observation therefore suggest that protein degraders could possess isoform specific effects. Additionally, our results exemplify the importance of delineating drug action and oncogenesis at the isoform level to develop more effective cancer therapeutics.

Role of LGMN in tumor development and its progression and connection with the tumor microenvironment.

Legumain (LGMN) has been demonstrated to be overexpressed not just in breast, prostatic, and liver tumor cells, but also in the macrophages that compose the tumor microenvironment. This supports the idea that LGMN is a pivotal protein in regulating tumor development, invasion, and dissemination. Targeting LGMN with siRNA or chemotherapeutic medicines and peptides can suppress cancer cell proliferation in culture and reduce tumor growth in vivo. Furthermore, legumain can be used as a marker for cancer detection and targeting due to its expression being significantly lower in normal cells compared to tumors or tumor-associated macrophages (TAMs). Tumor formation is influenced by aberrant expression of proteins and alterations in cellular architecture, but the tumor microenvironment is a crucial deciding factor. Legumain (LGMN) is an in vivo-active cysteine protease that catalyzes the degradation of numerous proteins. Its precise biological mechanism encompasses a number of routes, including effects on tumor-associated macrophage and neovascular endothelium in the tumor microenvironment. The purpose of this work is to establish a rationale for thoroughly investigating the function of LGMN in the tumor microenvironment and discovering novel tumor early diagnosis markers and therapeutic targets by reviewing the function of LGMN in tumor genesis and progression and its relationship with tumor milieu.

Cellular Senescence Promotes Skin Carcinogenesis through p38MAPK and p44/42MAPK Signaling.

Cellular senescence entails an irreversible growth arrest that evolved in part to prevent cancer. Paradoxically, senescent cells secrete proinflammatory and growth-stimulatory molecules, termed the senescence-associated secretory phenotype (SASP), which is correlated with cancer cell proliferation in culture and xenograft models. However, at what tumor stage and how senescence and the SASP act on endogenous tumor growth in vivo is unknown. To understand the role of senescence in cancer etiology, we subjected p16-3MR transgenic mice, which permit the identification and selective elimination of senescent cells in vivo, to the well-established two-step protocol of squamous cell skin carcinoma, in which tumorigenesis is initiated by a carcinogen 7,12-dimethylbenz[α]anthracene, and then promoted by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We show that TPA promotes skin carcinogenesis by inducing senescence and a SASP. Systemic induction of senescence in nontumor-bearing p16-3MR mice using a chemotherapy followed by the two-step carcinogenesis protocol potentiated the conversion of benign papillomas to carcinomas by elevating p38MAPK and MAPK/ERK signaling. Ablation of senescent cells reduced p38MAPK and MAPK/ERK signaling, thereby preventing the progression of benign papillomas to carcinomas. Thus, we show for the first time that senescent cells are tumor promoters, not tumor initiators, and that they stimulate skin carcinogenesis by elevating p38MAPK and MAPK/ERK signaling. These findings pave the way for developing novel therapeutics against senescence-fueled cancers. SIGNIFICANCE: These findings identify chemotherapy-induced senescence as a culprit behind tumor promotion, suggesting that elimination of senescent cells after chemotherapy may reduce occurrence of second cancers decades later. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/17/3606/F1.large.jpg.

Abstract 3568: Ferroptosis induced by xCT abrogation is dependent on GPX4 protein expression regulated by L-cystine/cysteine metabolism

Abstract Nutrients in the tumor microenvironment play an essential role in tumor development and proudly impact cancer metabolism. The xCT antiporter, which mediates cellular uptake of L-cystine coupled with glutamate export, has been considered as a promising therapeutic target due in part to its high expression in cancer cells to promote L-cystine uptake as a precursor for glutathione (GSH) synthesis to maintain redox homeostasis. Although inhibition of xCT has been shown to have anticancer effect in multiple models, emerging evidence also reveals that blocking xCT function could confer survival advantages to cancer cells. These opposite observations suggest that xCT inhibition could be a double-edged sword, and its impact on the overall outcome (cancer-inhibitory or tumor-promoting) is likely context-dependent via yet unknown mechanisms. Here, we show that the expression of glutathione peroxidase 4 (GPX4) is a key molecular determinant that decides the cell fate when xCT is suppressed. In cells with low or no expression of GPX4, inhibition of xCT causes severe ROS stress leading to massive cell death via ferroptosis. In contrast, high expression of GPX4 confers cancer cells the ability to escape ROS-induced ferroptosis. Mechanistic study suggests that the intracellular L-cysteine plays a major role in regulating GPX4 protein level, likely via direct binding to GPX4 and stabilize the protein. Pharmacological and genetic disruption of xCT in cancer cells induces dramatic ferroptosis in culture conditions lacking L-cysteine, whereas the presence of L-cysteine in culture or in vivo renders cancer cells resistance to xCT abrogation. Importantly, genetic knockout of xCT by CRISPR/cas9 reduces cancer cell proliferation in culture but promotes tumor growth in vivo, suggesting that the metabolic changes induced by xCT disruption favor cell growth in vivo. Biochemical and molecular analyses show that the in vivo microenvironment containing L-cysteine enables the xCT-disrupted cells to maintain a high GPX4 level, and thus prevents ferroptosis. Furthermore, metabolic flux using U-13C5 glutamine reveals that xCT abrogation leads to intracellular accumulation of more glutamate, which promotes cancer cell survival and proliferation in the low glucose tissue microenvironment in vivo. Our study has identified GPX4 as a key molecule that determines cell fate under metabolic stress induced by xCT suppression, and suggests that modulation of L-cystine/cysteine metabolism might be an effective strategy to overcome ferroptosis-resistant cancer cells. Citation Format: Yanyu Zhang, Peng Huang. Ferroptosis induced by xCT abrogation is dependent on GPX4 protein expression regulated by L-cystine/cysteine metabolism [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 3568.

Inhibiting effect of oleocanthal on neuroblastoma cancer cell proliferation in culture

ABSTRACTWe investigated the potential anticancer effects of oleocanthal (OC) on neuroblastoma cells. Cells were divided into four groups: group 1, neuroblastoma cells were treated with OC; group 2, neurons that differentiated from neuroblastoma cells were treated with phosphate-buffered saline(PBS); group 3, bone marrow derived neuronal (BMDN) cells that were differentiated from bone marrow derived mesenchymal stem cells (BMSCs) were treated with OC; group 4, BMDN cells that were differentiated from BMSCs were treated with PBS. Groups 2 and 4 were control groups. The effects of OC on cell viability, oxidative stress, neurite inhibition and apoptosis at IC50 dose were investigated using MTT analysis, i-NOS and e-NOS measurement, neurotoxicity screening test (NST) and TUNEL staining, respectively. MTT analysis demonstrated that cells were significantly less viable in group 1 than in group 3. i-NOS and e-NOS staining intensity was significantly greater in group 1 than in group 3. NST revealed that OC inhibited neurite growth in both neuroblastoma and BMND cells; inhibition was significantly less in group 3 than in group 1. Significantly more TUNEL labeled cells were found in group 1 than in group 3. We found that OC prevented growth and proliferation of neuroblastoma cells in culture by increasing oxidative stress and apoptosis. We also found that the cytotoxicity of OC is negligible in BMDN cells.

Artificial Circular RNA Sponges Targeting MicroRNAs as a Novel Tool in Molecular Biology

Artificial Circular RNA Sponges Targeting MicroRNAs as a Novel Tool in Molecular Biology

Citrus reticulata peel oil inhibits non-small cell lung cancer cell proliferation in culture and implanted in nude mice.

Non-small cell lung cancer (NSCLC) accounts for most cases of lung cancer. The peel oil of mandarin Citrus reticulata Blanco cv. Dancy (MPO) is a natural source of essential oils and carotenoids. Volatile and non-volatile lipid compounds were characterized by chromatographic methods. We demonstrate that MPO causes a dose-dependent growth inhibition of NSCLC model cells (A549) in culture and tumour growth in vivo of the same cells implanted in nude mice fed with MPO-supplemented diets. MPO induced cell cycle arrest mainly at the G0/G1 phase and reduced the amount of membrane-bound Ras protein along with apoptosis induction. No toxicological effect was found in liver parameters analysed in treated mice and histopathological analyses of their organs did not show any morphological changes. In conclusion, the data suggest that MPO possesses significant antitumor activity without causing systemic toxicity, proposing it as a dietary supplement that may be helpful in cancer prevention.

PAT4 levels control amino-acid sensitivity of rapamycin-resistant mTORC1 from the Golgi and affect clinical outcome in colorectal cancer.

Tumour cells can use strategies that make them resistant to nutrient deprivation to outcompete their neighbours. A key integrator of the cell's responses to starvation and other stresses is amino-acid-dependent mechanistic target of rapamycin complex 1 (mTORC1). Activation of mTORC1 on late endosomes and lysosomes is facilitated by amino-acid transporters within the solute-linked carrier 36 (SLC36) and SLC38 families. Here, we analyse the functions of SLC36 family member, SLC36A4, otherwise known as proton-assisted amino-acid transporter 4 (PAT4), in colorectal cancer. We show that independent of other major pathological factors, high PAT4 expression is associated with reduced relapse-free survival after colorectal cancer surgery. Consistent with this, PAT4 promotes HCT116 human colorectal cancer cell proliferation in culture and tumour growth in xenograft models. Inducible knockdown in HCT116 cells reveals that PAT4 regulates a form of mTORC1 with two distinct properties: first, it preferentially targets eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and second, it is resistant to rapamycin treatment. Furthermore, in HCT116 cells two non-essential amino acids, glutamine and serine, which are often rapidly metabolised by tumour cells, regulate rapamycin-resistant mTORC1 in a PAT4-dependent manner. Overexpressed PAT4 is also able to promote rapamycin resistance in human embryonic kidney-293 cells. PAT4 is predominantly associated with the Golgi apparatus in a range of cell types, and in situ proximity ligation analysis shows that PAT4 interacts with both mTORC1 and its regulator Rab1A on the Golgi. These findings, together with other studies, suggest that differentially localised intracellular amino-acid transporters contribute to the activation of alternate forms of mTORC1. Furthermore, our data predict that colorectal cancer cells with high PAT4 expression will be more resistant to depletion of serine and glutamine, allowing them to survive and outgrow neighbouring normal and tumorigenic cells, and potentially providing a new route for pharmacological intervention.

Cellular mechanisms involved in the melatonin inhibition of HT‐29 human colon cancer cell proliferation in culture

The antiproliferative and proapoptotic properties of melatonin in human colon cancer cells in culture were recently reported. To address the mechanisms involved in these actions, HT-29 human colon cancer cells were cultured in RPMI 1640 medium supplemented with fetal bovine serum at 37 degrees C. Cell proliferation was assessed by the incorporation of [(3)H]-thymidine into DNA. Cyclic nucleotide levels, nitrite concentration, glutathione peroxidase and reductase activities, and glutathione levels were assessed after the incubation of these cells with the following drugs: melatonin membrane receptor agonists 2-iodo-melatonin, 2-iodo-N-butanoyl-5-methoxytryptamine, 5-methoxycarbonylamino-N-acetyltryptamine (GR-135,531), and the antagonists luzindole, 4-phenyl-2-propionamidotetralin, and prazosin; the melatonin nuclear receptor agonist CGP 52608, and four synthetic kynurenines analogs to melatonin 2-acetamide-4-(3-methoxyphenyl)-4-oxobutyric acid, 2-acetamide-4-(2-amino-5-methoxyphenyl)-4-oxobutyric acid, 2-butyramide-4-(3-methoxyphenyl)-4-oxobutyric acid and 2-butyramide-4-(2-amino-5-methoxyphenyl)-4-oxobutyric acid. The results show that the membrane receptors are not necessary for the antiproliferative effect of melatonin and the participation of the nuclear receptor in this effect is suggested. Moreover, the antioxidative and anti-inflammatory actions of melatonin, counteracting the oxidative status and reducing the production of nitric oxide by cultured HT-29 cells seem to be directly involved in the oncostatic properties of melatonin. Some of the synthetic kynurenines exert higher antiproliferative effects than melatonin. The results reinforce the clinical interest of melatonin due to the different mechanisms involved in its oncostatic role, and suggest a new synthetic pathway to obtain melatonin agonists with clinical applications to oncology.

Senescent Human Fibroblasts Increase the Early Growth of Xenograft Tumors via Matrix Metalloproteinase Secretion

Although cellular senescence is believed to have a tumor suppressor function, senescent cells have been shown to increase the potential for growth of adjacent cancer cells in animal models. Replicatively senescent human fibroblasts increase the growth of cotransplanted cancer cells in vivo, but the role of cells that have undergone damage-mediated stress-induced premature senescence (SIPS) has not been studied in mouse transplant models. Here, we show that human fibroblasts that have undergone SIPS by exposure to the DNA-damaging agent bleomycin increase the growth of cotransplanted cancer cells (MDA-MB-231) in immunodeficient mice. Xenografts containing SIPS fibroblasts (SIPSF) exhibited early tissue damage as evidenced by fluid accumulation (edema). Cancer cells adjacent to the fluid showed increased DNA synthesis. Fluid accumulation, increased xenograft size, and increased cell proliferation were all reduced by the matrix metalloproteinase (MMP) inhibitor GM6001. MMPs and other genes characteristic of inflammation/tissue injury were overexpressed in SIPSF. Inhibition of MMP activity did not affect SIPSF stimulation of cancer cell proliferation in culture. However, another overexpressed product (hepatocyte growth factor) did have a direct mitogenic action on cancer cells. Based on the present results, we propose that senescent cells may promote cancer growth both by a direct mitogenic effect and by an indirect effect via tissue damage. Senescent stromal cells may cause an MMP-mediated increase in permeability of adjacent capillaries, thereby exposing incipient cancer cells to increased levels of mitogens, cytokines, and other plasma products. This exposure may increase cancer cell proliferation and result in promotion of preneoplastic cells.

Discovery and Development of Pyrazole-Scaffold Hsp90 Inhibitors

This review explains why the chaperone Hsp90 is an exciting protein target for the discovery of new drugs to treat cancer in the clinic, and summarises the properties of natural product derived inhibitors before relating the discovery and current state of development of synthetic pyrazole compounds. Blockade of Hsp90 results in reduced cellular levels of several proteins implicated in cancer including CDK4, ERBB2 and C-RAF, and causes simultaneous inhibition of cancer cell proliferation in culture and of tumor xenograft growth in vivo. Hsp90 has an ATPase domain that is necessary for its Hsp chaperone function, and X-ray crystallography has shown that natural product inhibitors (geldanamycin, radicicol) of Hsp90 function bind to this domain. High throughput assays focusing on the ATPase activity of Hsp90 were developed and used to discover novel chemical starting points for cancer drug discovery. The discovery, synthesis and SAR of 3,4-diaryl pyrazoles is described. X-Ray crystallography of protein-inhibitor complexes revealed important interactions involving the resorcinol substituent at C-3, and these X-ray structures strongly influenced subsequent medicinal chemistry research that has resulted in highly potent inhibitors with sub-micromolar activity in cells. SAR and X-ray data are summarised for analogues in which the 4-phenyl substituent is replaced by amides or piperazine derivatives. Prospects for the pyrazoles as they progress towards clinical development are discussed in relation to current Phase I trials with derivatives of geldanamycin.

Green tea extracts decrease carcinogen-induced mammary tumor burden in rats and rate of breast cancer cell proliferation in culture.

Epidemiological evidence suggests tea (Camellia sinensis L.) has chemopreventive effects against various tumors. Green tea contains many polyphenols, including epigallocatechin-3 gallate (EGCG), which possess anti-oxidant qualities. Reduction of chemically induced mammary gland carcinogenesis by green tea in a carcinogen-induced rat model has been suggested previously, but the results reported were not statistically significant. Here we have tested the effects of green tea on mammary tumorigenesis using the 7,12-dimethylbenz(a)anthracene (DMBA) Sprague-Dawley (S-D) rat model. We report that green tea significantly increased mean latency to first tumor, and reduced tumor burden and number of invasive tumors per tumor-bearing animal; although, it did not affect tumor number in the female rats. Furthermore, we show that proliferation and/or viability of cultured Hs578T and MDA-MB-231 estrogen receptor-negative breast cancer cell lines was reduced by EGCG treatment. Similar negative effects on proliferation were observed with the DMBA-transformed D3-1 cell line. Growth inhibition of Hs578T cells correlated with induction of p27(Kip1) cyclin-dependent kinase inhibitor (CKI) expression. Hs578T cells expressing elevated levels of p27(Kip1) protein due to stable ectopic expression displayed increased G1 arrest. Thus, green tea had significant chemopreventive effects on carcinogen-induced mammary tumorigenesis in female S-D rats. In culture, inhibition of human breast cancer cell proliferation by EGCG was mediated in part via induction of the p27(Kip1) CKI.