Blocked Cell Proliferation Research Articles

Metformin induces apoptosis in TRAIL-resistant colorectal cancer cells

Resistance to chemotherapy drugs, which commonly occurs during the treatment of colorectal cancer (CRC), can lead to tumor recurrence and metastasis, so combinational treatment strategies according to the cancer cell type are urgently needed to overcome drug resistance and increase therapeutic efficiency. To this end, the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising anticancer strategy. Some CRC cell lines such as SW620 have low sensitivity to TRAIL, so additional sensitizers are required to make the strategy effective. Therefore, we focused on the apoptotic effect of combinational metformin and TRAIL treatment on TRAIL-resistant SW620 cells. Treatment with TRAIL alone did not induce apoptosis whereas combined treatment with metformin and TRAIL significantly increased it. TRAIL activated caspases through an extrinsic pathway but increased resistance to apoptosis through the protein kinase B or AKT (PKB/AKT)/mammalian target of rapamycin (mTOR) pathway. On the other hand, metformin reduced the inhibitory effect of X-linked inhibitor of apoptosis (XIAP) by blocking the AKT and nuclear factor kappa B (NF-κB) pathways and activated CCAAT-enhancer-binding protein homologous protein (CHOP) via endoplasmic reticulum (ER)-stress but without inducing apoptosis. In addition, metformin induced cell-cycle arrest, thereby blocking cell proliferation and growth. These results were also confirmed through an in vivo mouse xenograft CRC model, in which combined treatment with metformin and TRAIL induced tumor cell death, thus demonstrating the anticancer effect of their coadministration. Therefore, cotreatment of metformin and TRAIL could be an effective anticancer treatment strategy for TRAIL-resistant CRC.

Myostatin Changes in Females with UI after Magnetic Stimulation: A Quasi-Experimental Study.

Background and Objectives: Urinary incontinence (UI) is the involuntary loss of urine caused by a weakness in the pelvic floor muscles (PFMs) that affects urethral closure. Myostatin, which prevents the growth of muscles, is a protein expressed by human skeletal muscle cells. Indeed, it has been observed that myostatin concentration rises during skeletal muscle inactivity and that suppressing serum myostatin promotes muscle growth and strength. Furthermore, therapeutic interventions that reduce myostatin signalling may lessen the effects of aging on skeletal muscle mass and function. For this reason, the aim of the study was to assess if flat magnetic stimulation technology affects serum myostatin levels, as myostatin can block cell proliferation at the urethral sphincter level. Materials and Methods: A total of 19 women, 75% presenting stress urinary incontinence (SUI) and 25% urgency urinary incontinence (UUI), were enrolled. A non-invasive electromagnetic therapeutic system designed for deep pelvic floor area stimulation was used for eight sessions. Results: The ELISA (enzyme linked immunosorbent assay) test indicated that the myostatin levels in blood sera had significantly decreased. Patients' ultrasound measurements showed a significant genital hiatus length reduction at rest and in a stress condition. The Pelvic Floor Bother Questionnaire consistently revealed a decrease in mean scores when comparing the pre- and post-treatment data. Conclusions: Effective flat magnetic stimulation reduces myostatin concentration and genital hiatus length, minimizing the severity of urinary incontinence. The results of the study show that without causing any discomfort or unfavourable side effects, the treatment plan significantly improved the PFM tone and strength in patients with UI.

The Investigation of Nfκb Inhibitors to Block Cell Proliferation in OSCC Cells Lines.

Oral cancers, with oral squamous cell carcinoma (OSCC) as the predominant type, have a significant impact on morbidity and mortality rates. Therefore, targeting the NFκB pathway shows promise in cancer therapy. This study investigated the impact of two NFκB inhibitors, LY2409881 and MLN4924, on cell proliferation, apoptosis susceptibility, and in vivo tumorigenesis in OSCC cell lines CAL27 and SCC15. The results revealed that both LY2409881 and MLN4924 effectively suppressed cell proliferation, induced apoptosis, and arrested the cell cycle at the G2/M phase-a phenomenon likely associated with the NFκB pathway. Furthermore, MLN4924 demonstrated potent inhibitory effects on cell proliferation at low μM concentrations, surpassing the effectiveness of LY2409881 as an inhibitor. (All results: p<0.05) Conclusion: These findings highlight the potential of LY2409881 and MLN4924 as novel therapeutic agents for OSCC, thereby offering new insights for the clinical management of this condition.

Targeting Intrinsic and Extrinsic Pathways of Ferroptosis: A Novel Anticancer Strategy of Curcumin

Background The turmeric rhizome yields a medicinal compound called curcumin. Curcumin’s anticancer methods mostly involve blocking cell invasion and proliferation, controlling gene expression, preventing angiogenesis, triggering apoptosis, and inducing ferroptosis. A brand-new nonapoptotic kind of planned cell death is called ferroptosis. Ferroptosis can be initiated through two major pathways: the extrinsic or transporter-dependent pathway, and the intrinsic or enzyme-regulated pathway. It is unknown, therefore, how curcumin and its derivatives prevent cancer by controlling ferroptosis. Purpose Through this review, we concentrate on the possible anticancer mechanisms of curcumin by means of its effect on ferroptosis; the regulation of iron metabolism, the synthesis of reactive oxygen species, and the targeting of antioxidant pathways represent a few of these processes. Methods Retrieve English literature of curcumin and its derivatives in the treatment of tumor by ferroptosis from databases such as Web of Science, etc. Conclusion This review provides a rationale for the clinical use of curcumin and its derivatives as anticancer agents.

Transcriptional repression of the oncofetal LIN28B gene by the transcription factor SOX6

The identification of regulatory networks contributing to fetal/adult gene expression switches is a major challenge in developmental biology and key to understand the aberrant proliferation of cancer cells, which often reactivate fetal oncogenes. One key example is represented by the developmental gene LIN28B, whose aberrant reactivation in adult tissues promotes tumor initiation and progression. Despite the prominent role of LIN28B in development and cancer, the mechanisms of its transcriptional regulation are largely unknown. Here, by using quantitative RT-PCR and single cell RNA sequencing data, we show that in erythropoiesis the expression of the transcription factor SOX6 matched a sharp decline of LIN28B mRNA during human embryo/fetal to adult globin switching. SOX6 overexpression repressed LIN28B not only in a panel of fetal-like erythroid cells (K562, HEL and HUDEP1; ≈92% p < 0.0001, 54% p = 0.0009 and ≈60% p < 0.0001 reduction, respectively), but also in hepatoblastoma HepG2 and neuroblastoma SH-SY5H cells (≈99% p < 0.0001 and ≈59% p < 0.0001 reduction, respectively). SOX6-mediated repression caused downregulation of the LIN28B/Let-7 targets, including MYC and IGF2BP1, and rapidly blocks cell proliferation. Mechanistically, Lin28B repression is accompanied by SOX6 physical binding within its locus, suggesting a direct mechanism of LIN28B downregulation that might contribute to the fetal/adult erythropoietic transition and restrict cancer proliferation.

Sterigmatocystin induces autophagic and apoptotic cell death of liver cancer cells via downregulation of XIAP

XIAP, or the X-linked Inhibitor of Apoptosis Protein, is the most extensively studied member within the IAP gene family. It possesses the capability to impede apoptosis through direct inhibition of caspase activity. Various kinds of cancers overexpress XIAP to enable cancer cells to avoid apoptosis. Consequently, the inhibition of XIAP holds significant clinical implications for the development of anti-tumor medications and the treatment of cancer. In this study, sterigmatocystin, a natural compound obtained from the genus asperigillus, was demonstrated to be able to induce apoptotic and autophagic cell death in liver cancer cells. Mechanistically, sterigmatocystin induces apoptosis by downregulation of XIAP expression. Additionally, sterigmatocystin treatment induces cell cycle arrest, blocks cell proliferation, and slows down colony formation in liver cancer cells. Importantly, sterigmatocystin exhibits a remarkable therapeutic effect in a nude mice model. Our findings revealed a novel mechanism through which sterigmatocystin induces apoptotic and autophagic cell death of liver cancer cells by suppressing XIAP expression, this offers a promising therapeutic approach for treating liver cancer patients.

Abstract 2865: Efficacy of MDM2 and epigenetic inhibitors treatments for neuroblastoma

Abstract Pediatric Neuroblastoma (NB) is one of the most prevalent solid tumors in children that arise from embryonic neural crest cells and poses a significant challenge in the field of oncology. There are about 700 to 800 new cases of neuroblastoma diagnosed each year in the United States, and accounts for approximately 15% of all childhood cancer mortalities. Since this malignancy predominantly affects infants and young children, it is imperative to decipher the underlying molecular factors driving its pathogenesis. The novel molecular therapeutic approach includes the use of epigenetic modifiers as drugs. This approach aims to reverse significant epigenetic events underlying cancer pathogenesis, particularly abnormalities in histone modifications and DNA methylation. The reduction of both DNA and histone methylation levels leads to the reactivation of tumor suppressor genes and inhibits cancer cell proliferation and cell growth. In this study, we used selective and reversible small molecule agents such as MDM2 inhibitor (RG7388) and epigenetic modifiers (CM272 and SAHA) to block cell proliferation by inducing cell cycle arrest via enforcing Epigenetic alterations. Our initial experiments were designed to understand the effects of RG7388, CM272, and SAHA treatments on NB and their impact on cell cycle progression. It was hypothesized that the decrease in DNA methylation and increase in histone modification- augment p21 expression via the use of MDM2 inhibitors and epigenetic modifiers. To test this hypothesis, the NB cells (SK-N-SK and IMR-32) were treated with RG7388, CM272, and SAHA for 24 hours. The extent of Cell death was determined for NB cells by using the Trypan Blue Dye Exclusion (TBDE) method, and CellTiter-Glo® Luminescent Cell Viability Assay. Induction of apoptosis was determined by using the Immunofluorescence staining method with DEVD-amc substrate. The results of our study suggested that RG7388, CM272, and SAHA were able to induce cell death in the SK-N-SH cells following 24-hour treatment. Furthermore, we analyzed the levels of Tumor suppressor (p53), cell cycle arrest (p21, p27, AurkB, CDC25A, CyclinD) cell death (Bax, Bcl2, LC3, Beclin, PARP, Cleaved PARP, RIP1, RIP3, MLKL, p-AKT), and Epigenetic biomarker (DNMT1, DNMT3A, DNMT3B, acetyl histoneH3, H4, and H2b) protein expression levels using western blot analysis. The results showed significant elevation of p53, p21, p27, and acetylation of histone family biomarkers after treatment when compared with the control group. In summary, our results indicate that RG7388, CM272, and SAHA treatments can effectively induce cellular responses and prevent cell growth and progression by enforcing epigenetic changes in the NB cells. (This research was supported by the National Pediatric Cancer Foundation, Tampa, Florida, and the Royal Dames of Cancer Research Inc. of Ft. Lauderdale, Florida) Citation Format: Tahani Momenah, Umamaheswari Natarajan, Shyam Jaganathan, Appu Rathinavelu. Efficacy of MDM2 and epigenetic inhibitors treatments for neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2865.

Abstract 1985: Ablation of adenylosuccinate lyase retarded the cell proliferation and induces mitochondrial dysfunction in endocrine therapy resistant breast cancer cells

Abstract Approximately 75% of breast tumors express estrogen receptors (ER+); recurrent ER+ breast cancer is generally incurable. Endocrine therapy (ET) is the mainstay of treatment for ER+ breast cancer and works by targeting either ER activity (Tamoxifen or Fulvestrant) or availability of its ligand estrogen (Letrozole, Exemestane). Unfortunately, endocrine therapies are limited by the emergence of resistance; resistance to endocrine therapies (ET) remains a significant clinical challenge. We have explored the role of adenylosuccinate lyase (ADSL), an enzyme of the de-novo purine biosynthesis pathway, in ET-resistant breast cancer cells. Protein expression of ADSL was significantly higher in two different ET-resistant cells (LCC9 and T47D-4HT) when compared with their respective parental ET-sensitive cells (MCF-7 and T47D). siRNA mediated ablation of ADSL in LCC9 and T47D-4HT cells blocked cell proliferation and both colony and spheroid formation. Depletion of ADSL in LCC9 cells obstructed cell cycle progression from G1 to S phase and elevated the protein expression of cyclin D1/D3, CDK2/4, and cyclin E. In T47D-4HT cells, ADSL depletion led to an accumulation of cells in S-phase with reduced protein expression of cyclin D1. Notably, ADSL ablation in LCC9 cells lead to DNA replication stress, resulting in activation (phosphorylation) of ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR) and its key downstream effector checkpoint kinase 1 (Chk1), which further limited the phosphatase activity of cdc25A leading to deactivation (phosphorylation) of Cyclin-Dependent Kinase 2 (CDK2). ADSL knockdown reduced the mitochondrial membrane potential and impaired the oxygen consumption (OCR) and extracellular acidification (ECAR) rates in both LCC9 and T47D-4HT cells. Of note, addition of AICAR to ADSL-depleted LCC9 cells significantly increased cell proliferation, colony formation, and restored mitochondrial membrane potential. RNA sequencing data revealed the effect of ADSL depletion on enrichment of different pathways in LCC9 and TH47D-4HT cells. Importantly, p53 signaling was significantly enriched in LCC9 cells but not in T47D-4HT cells after ADSL knockdown (T47D cells have a mutated p53 gene). ER+ breast cancer patients treated with ET showed high ADSL expression that is associated with poor recurrence-free survival. In summary, our findings indicate that ADSL-mediated de novo purine synthesis is crucial for cell proliferation. Therefore, targeting ADSL may be a novel therapeutic approach for ER, ET-resistant breast cancer. Citation Format: ANIL KUMAR Yadav, Lu Jin, Robert Clarke, Surojeet Sengupta. Ablation of adenylosuccinate lyase retarded the cell proliferation and induces mitochondrial dysfunction in endocrine therapy resistant breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1985.

PMI-controlled mannose metabolism and glycosylation determines tissue tolerance and virus fitness

Host survival depends on the elimination of virus and mitigation of tissue damage. Herein, we report the modulation of D-mannose flux rewires the virus-triggered immunometabolic response cascade and reduces tissue damage. Safe and inexpensive D-mannose can compete with glucose for the same transporter and hexokinase. Such competitions suppress glycolysis, reduce mitochondrial reactive-oxygen-species and succinate-mediated hypoxia-inducible factor-1α, and thus reduce virus-induced proinflammatory cytokine production. The combinatorial treatment by D-mannose and antiviral monotherapy exhibits in vivo synergy despite delayed antiviral treatment in mouse model of virus infections. Phosphomannose isomerase (PMI) knockout cells are viable, whereas addition of D-mannose to the PMI knockout cells blocks cell proliferation, indicating that PMI activity determines the beneficial effect of D-mannose. PMI inhibition suppress a panel of virus replication via affecting host and viral surface protein glycosylation. However, D-mannose does not suppress PMI activity or virus fitness. Taken together, PMI-centered therapeutic strategy clears virus infection while D-mannose treatment reprograms glycolysis for control of collateral damage.

Hesperetin Inhibits Bladder Cancer Cell Proliferation and Promotes Apoptosis and Cycle Arrest by PI3K/AKT/FoxO3a and ER Stress-mitochondria Pathways.

Hesperetin (HSE) is a natural flavonoid derived from the hydrolysis of Hesperidin, which is mainly found in traditional natural Chinese herbs, such as Chenpi and Hovenia caryophyllus. HSE displays anti-inflammatory and antioxidant activities. However, its potential mechanism of action on bladder cancer (BLCA) remains unknown. The aim of this study was to investigate the potential mechanism of action of HSE on BLCA cells. Network pharmacology analysis was used to construct a composite target network, combined with Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to identify HSE-induced cell death patterns and signaling pathway alterations. Cytotoxicity evaluation was determined by CCK-8 assay. A clone formation assay was performed to assess cell proliferative capacity. Scratch and Transwell assays were performed to evaluate cell migration and invasion ability. Hoechst 33342 staining was visualized to observe morphological features of apoptosis. Apoptosis, cycle distribution, reactive oxygen species (ROS) generation, and mitochondrial membrane potential (MMP) changes were examined by flow cytometry. Western blot analysis was performed to analyze the expression of key proteins associated with cell proliferation, apoptosis, cycle block, PI3K/AKT/FoxO3a and endoplasmic reticulum (ER) stress-mitochondrial pathways. Network pharmacology analysis was performed to identify 155 potential candidate targets of HSE-BLCA, and further topological analysis was performed to obtain 34 hub-gene. Enrichment analysis yielded patterns of death and key pathways, revealing that the anti-BLCA effect of HSE may be related to the positive regulation of PI3K/AKT/FoxO3a and ER stress-mitochondrial pathways. in vitro results showed that HSE blocked cell proliferation, migration, and invasion in a concentration-dependent manner and triggered apoptosis, G0/G1 phase blockade, ROS production, and MMP depolarization. In addition, Western blot results showed that HSE downregulated phosphorylated (p)-3-phosphoinositide-dependent kinase-1 (PI3K), phosphorylated (p)-AKT serine/threonine kinase 1 (AKT), phosphorylated (p)-Forkhead box O 3a (FoxO3a), anti-apoptotic proteins, proliferation-associated proteins, and cell cycle promoters, whereas the levels of proteins related to the expression of cell cycle regulators, pro-apoptotic proteins, and ER stress-mitochondrial pathway were up-regulated in BLCA cells by the intervention of HSE. PI3K agonist (YS-49) and ER stress inhibitor (4-PBA) partially or completely reversed HSE-mediated proliferation, apoptosis, and cycle blockade in BLCA cells. The anticancer effects of HSE in BLCA may be attributed to its coordination of actions, inhibiting cell proliferation, migration, and invasion, inducing apoptosis, G0/G1 phase arrest, generating reactive oxygen species, causing MMP loss, and engaging the caspase protein family. These actions are likely mediated through the PI3K/AKT/FoxO3a and ER stress-mitochondrial pathways. Thus, our findings suggest that HSE is a promising novel therapeutic candidate for the prevention and treatment of BLCA.

Preliminary study on the active substances and cellular pathways of lactic acid bacteria for colorectal cancer treatment.

Colorectal cancer (CRC) is a common malignant tumor and is one of the three most common cancers worldwide. Traditional surgical treatment, supplemented by chemotherapy and radiotherapy, has obvious side effects on patients. Immunotherapy may lead to some unpredictable complications. Low introduction rate and high cost are some of the problems of gene therapy, so finding a safe, reliable and least toxic treatment method became the main research direction for this study. Lactic acid bacteria and their metabolites are widely used in functional foods or as adjuvant therapies for various diseases because they are safe to eat and have no adverse reactions. Research has shown that lactic acid bacteria and their metabolites play an auxiliary therapeutic role in colorectal cancer mainly by improving the intestinal flora composition, inhibiting the growth of pathogenic bacteria and inhibiting the proliferation of cancer cells. It is now widely believed that the substances that probiotics such as lactic acid bacteria exert anti-cancer effects are mainly secondary metabolites such as butyric acid. Lb. plantarum AY01 isolated from fermented food has good anti-cancer ability, and its main anti-cancer substance is 2'-deoxyinosine. Through flow cytometry detection, it was found that Lb. plantarum AY01 can block cell proliferation in the S phase. In addition, Lb. plantarum AY01 culture reduces the sensitivity of mice to colitis-associated CRC induced by azoxymethane (AOM)/dextran sulfate sodium salt (DSS) and exhibits the occurrence and promotion of tumors. According to transcriptome analysis, Lb. plantarum AY01 may induce apoptosis of colorectal cancer cells by activating the p38 MAPK pathway. This experiment provided possibilities for the treatment of CRC.

DMSA-coated IONPs trigger oxidative stress, mitochondrial metabolic reprograming and changes in mitochondrial disposition, hindering cell cycle progression of cancer cells

There is increasing interest in modulating the redox homeostasis of tumors since high levels of reactive oxygen species (ROS) make them more vulnerable to changes in these species. Nanomedicine offers promise in this context as such applications may provoke biological responses that induce ROS production. Indeed, iron oxide nanoparticles (IONPs) can induce ROS accumulation through the so-called Fenton reaction of iron, further augmenting the ROS in tumors and overloading the antioxidant system beyond its capacity, thereby driving oxidative stress to a level that is incompatible with cell survival. Here, three different coatings for IONPs were compared to assess their intrinsic capacity to induce ROS production in cells. Of these coatings, dimercaptosuccinic acid-coated IONPs (DMSA-NPs) provoked the strongest ROS production, which was associated with the ability to reprogram the metabolism of cancer cells. This latter phenomenon involved shutting-down oxidative phosphorylation (OXPHOS), shifting mitochondrial morphology towards a more elongated phenotype, reducing the total mitochondrial mass and ultimately, blocking cell proliferation by inducing G0/G1 cell cycle arrest. Consequently, the data obtained highlights the importance of studying the chemical properties of IONPs, presenting DMSA-NPs as a novel tool to induce oxidative stress in cancer cells and alter their cell fate.

Comprehensive metabolite and biological profile of “Sulmona Red Garlic” ecotype’s aerial bulbils

“Sulmona Red Garlic” is a well-known Italian traditional product. Bulbs, used for culinary purposes, have been largely investigated for their medicinal properties whereas aerial bulbils are usually removed as waste material. Here, for the first time, chemical composition and biological properties of the hydroalcoholic extract from aerial bulbils were investigated. Complementary information on metabolite composition were obtained using both NMR based untargeted and HPLC-DAD targeted methodologies. The NMR analysis revealed the presence of sugars, organic acids, amino acids, organosulphur compounds (methiin, alliin, allicin and cycloalliin), and other secondary metabolites. In particular, methiin and alliin were identified for the first time in the NMR spectra of aerial bulbil garlic extracts. Polyphenol content was determined by HPLC-DAD analysis: catechin, chlorogenic acid, and gallic acid turned out to be the most abundant phenolics.Hydroalcoholic extract blocked cell proliferation of colon cancer cell line HCT116 with an IC50 of 352.07 µg/mL, while it was non-toxic to myoblast cell line C2C12. In addition, it caused seedling germination reduction of two edible and herbaceous dicotyledon species, namely Cichorium intybus and C. endivia. Moreover, the same extract reduced the gene expression of TNF-α (tumor necrosis factor), HIF1-α (hypoxia-inducible factor), VEGFA (vascular endothelial growth factor), and transient receptor potential (TRP) M8 (TRPM8) indicating the ability to contrast cancer development through the angiogenic pathway. Final, in silico experiments were also carried out supporting the biological effects of organosulphur compounds, particularly alliin, which may directly interact with TRPM8.The results here reported suggest the potential use of garlic aerial bulbils often considered a waste product as a source in phytotherapeutic remedies.

Dual network analysis of transcriptome data for discovery of new therapeutic targets in non-small cell lung cancer

The drug therapy for non-small cell lung cancer (NSCLC) have always been issues of poisonous side effect, acquired drug resistance and narrow applicable population. In this study, we built a novel network analysis method (difference- correlation- enrichment- causality- node), which was based on the difference analysis, Spearman correlation network analysis, biological function analysis and Bayesian causality network analysis to discover new therapeutic target of NSCLC in the sequencing data of BEAS-2B and 7 NSCLC cell lines. Our results showed that, as a proteasome subunit coding gene in the central of cell cycle network, PSMD2 was associated with prognosis and was an independent prognostic factor for NSCLC patients. Knockout of PSMD2 inhibited the proliferation of NSCLC cells by inducing cell cycle arrest, and exhibited marked increase of cell cycle blocking protein p21, p27 and decrease of cell cycle driven protein CDK4, CDK6, CCND1 and CCNE1. IPA and molecular docking suggested bortezomib has stronger affinity to PSMD2 compared with reported targets PSMB1 and PSMB5. In vitro and In vivo experiments demonstrated the inhibitory effect of bortezomib in NSCLC with different driven mutations or with tyrosine kinase inhibitors resistance. Taken together, bortezomib could target PSMD2, PSMB1 and PSMB5 to inhibit the proteasome degradation of cell cycle check points, to block cell proliferation of NSCLC, which was potential optional drug for NSCLC patients.

Intrinsic resistance to ROS1 inhibition in a patient with CD74-ROS1 mediated by AXL overexpression.

The vast majority of patients with ROS1 positive non-small cell lung cancer (NSCLC) derive clinical benefit from currently approved ROS1 therapies, including crizotinib and entrectinib. However, a small proportion of patients treated with ROS1 inhibitors fail to derive any clinical benefit and demonstrate rapid disease progression. The biological mechanisms underpinning intrinsic resistance remain poorly understood for oncogene-driven cancers. We generated a patient-derived cell line, CUTO33, from a ROS1 therapy naive patient with CD74-ROS1+ NSCLC, who ultimately did not respond to a ROS1 inhibitor. We evaluated a panel of ROS1+ patient-derived NSCLC cell lines and used cell-based assays to determine the mechanism of intrinsic resistance to ROS1 therapy. The CUTO33 cell line expressed the CD74-ROS1 gene fusion at the RNA and protein level. The ROS1 fusion protein was phosphorylated at baseline consistent with the known intrinsic activity of this oncogene. ROS1 phosphorylation could be inhibited using a wide array of ROS1 inhibitors, however these inhibitors did not block cell proliferation, confirming intrinsic resistance in this model and consistent with the patient's lack of response to a ROS1 inhibitor. CUTO33 expressed high levels of AXL, which has been associated with drug resistance. Combination of an AXL inhibitor or AXL knockdown with a ROS1 inhibitor partially reversed resistance. In summary, we demonstrate that AXL overexpression is a mechanism of intrinsic resistance to ROS1 inhibitors.

Thymidylate synthase promotes esophageal squamous cell carcinoma growth by relieving oxidative stress through activating nuclear factor erythroid 2-related factor 2 expression.

Thymidylate synthase (TYMS) is involved in the malignant process of multiple cancers, and has gained much attention as a cancer treatment target. However, the mechanism in carcinogenesis of esophageal squamous cell cancer (ESCC) is little reported. The present study was to clear the biological roles and carcinogenic mechanism of TYMS in ESCC, and explored the possibility to use TYMS as a tumor marker in diagnosis and a drug target for the treatment of ESCC. Stably TYMS-overexpression cells established by lentivirus transduction were used for the analysis of cell proliferation. RNA sequencing was performed to explore the possible carcinogenic mechanisms. GEPIA databases analysis showed that TYMS expression in esophageal cancer tissues was higher than that in normal tissues. The MTT assay, colony formation assay, and nude mouse subcutaneous tumor model found that the overexpression of TYMS increased cell proliferation. Transcriptome sequencing analysis revealed that the promoted cell proliferation in TYMS-overexpression ESCC cells were mediated through activating genes expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and Nrf2 dependent antioxidant enzymes to relieve oxidative stress, which was confirmed by increased glutathione (GSH), glutathione peroxidase (GPX) activities, and reduced reactive oxygen species. Nrf2 active inhibitors (ML385) used in TYMS-overexpression cells inhibited the expression of Nrf2-dependent antioxidant enzyme genes, thereby increasing oxidative stress and blocking cell proliferation. Our study indicated a novel and effective regulatory capacity of TYMS in the cell proliferation of ESCC by relieving oxidative stress through activating expression of Nrf2 and Nrf2-dependent antioxidant enzymes genes. These properties make TYMS and Nrf2 as appealing targets for ESCC clinical chemotherapy.

Circular RNA sequencing identified circARNTL2 as a pathogenic factor in psoriasis by facilitating proliferation and cell cycle progression of keratinocytes

Psoriasis is a chronic recurrent skin disease, with excessive proliferation of keratinocytes. Recent studies indicated the pathogenic roles of circular RNA (circRNA) in psoriasis. Here, we screened the circRNA profiles from five psoriatic skin lesions and five normal skin tissues by circRNA sequencing and identified 1118 differentially expressed circRNAs (DECs) between psoriatic and normal groups. Among these DECs, high abundant circARNTL2 has been proven upregulated in psoriatic skin lesions by RT-qPCR assay. Then, the head-to-tail structure of circARNTL2 was validated by Sanger sequencing and RNase R digestion assay. Moreover, we determined cytoplastic location of circARNTL2 by RT-qPCR assay of nuclear/cytoplasmic RNA and FISH analysis. Further experiments demonstrated that silencing circARNTL2 expression could block cell proliferation and cell cycle progression of keratinocytes. Mechanistically, circARNTL2 can bind to and regulate Serpin B4 which also affects the proliferation of keratinocytes. These findings provide evidence for the role of circARNTL2 in psoriasis.

AKIP1 accelerates glioblastoma progression through stabilizing EGFR expression.

A Kinase Interacting Protein 1 (AKIP1) is found to be overexpressed in a variety of human cancers and associated with patients' worse prognosis. Several studies have established AKIP1's malignant functions in tumor metastasis, angiogenesis, and chemoradiotherapy resistance. However, the mechanism of AKIP1 involved in accelerating glioblastoma (GBM) progression remains unknown. Here, we showed that the expression of AKIP1 was positively correlated with the glioma pathological grades. Down-regulating AKIP1 greatly impaired the proliferation, colony formation, and tumorigenicity of GBM cells. In terms of the mechanism, AKIP1 cooperates with transcriptional factor Yin Yang 1 (YY1)-mediated Heat Shock Protein 90 Alpha Family Class A Member 1 (HSP90AA1) transcriptional activation, enhancing the stability of Epidermal Growth Factor Receptor (EGFR). YY1 was identified as a potential transcriptional factor of HSP90AA1 and directly interacts with AKIP1. The overexpression of HSP90α significantly reversed AKIP1 depletion incurred EGFR instability and the blocked cell proliferation. Moreover, we further investigated the interacted pattern between EGFR and HSP90α. These findings established that AKIP1 acted as a critical oncogenic factor in GBM and uncovered a novel regulatory mechanism in EGFR aberrant expression.

ZNF703 mRNA-Targeting Antisense Oligonucleotide Blocks Cell Proliferation and Induces Apoptosis in Breast Cancer Cell Lines.

The luminal B molecular subtype of breast cancers (BC) accounts for more than a third of BCs and is associated with aggressive clinical behavior and poor prognosis. The use of endocrine therapy in BC treatment has significantly contributed to the decrease in the number of deaths in recent years. However, most BC patients with prolonged exposure to estrogen receptor (ER) selective modulators such as tamoxifen develop resistance and become non-responsive over time. Recent studies have implicated overexpression of the ZNF703 gene in BC resistance to endocrine drugs, thereby highlighting ZNF703 inhibition as an attractive modality in BC treatment, especially luminal B BCs. However, there is no known inhibitor of ZNF703 due to its nuclear association and non-enzymatic activity. Here, we have developed an antisense oligonucleotide (ASO) against ZNF703 mRNA and shown that it downregulates ZNF703 protein expression. ZNF703 inhibition decreased cell proliferation and induced apoptosis. Combined with cisplatin, the anti-cancer effects of ZNF703-ASO9 were improved. Moreover, our work shows that ASO technology may be used to increase the number of targetable cancer genes.

P-049 Verteporfin suppresses cell proliferation, survival and migration of TCam-2 Human Seminoma Cells via inhibits the YAP-TEAD complex

Abstract Study question Does verteporfin administration effect on proliferation, survival and migration of TCam-2 human seminoma cells? Summary answer Verteporfin may be a promising anticancer agent for TCam-2 human seminoma cells via inhibition of cell viability, proliferation, migration and stimulation of apoptosis. What is known already Seminoma is germ cell tumor of the testis and TCam-2 cells is the first seminoma-derived cell line that retains many of the characteristic traits of seminoma. The Hippo signalling is a evolutionarily highly conserved pathway that controls organ size, tissue homeostasis, and cancer development. In mammals, the Hippo pathway consists of the serine/threonine kinases Mammalian Sterile 20-protein Kinase 1 and 2 (MST1/2), Large Tumor Suppressor Homologues 1 and 2 (LATS1/2), Yes-Associated Protein (YAP), and its transcriptional cofactor TAZ. Verteporfin inhibits YAP–TEAD complex, blocking cell proliferation and survival. Study design, size, duration TCam-2 cells were treated with different concentrations of verteporfin (1, 5, 10, 20 and 40 µM) for 24, 48 and 72 hours (h). Participants/materials, setting, methods TCam-2 cells were treated with verteporfin at different doses and incubation times. Afterwards, protein expression of Hippo pathway proteins was determined by western blot, mRNA levels were analyzed by qRT-PCR and protein localizations were evaluated by immunoflourescence staining. Cell migration was determinated with wound-healing scratch assay; cell viability, early-late apoptosis and necrosis rates were evaluated by flow cytometry. Main results and the role of chance We detected that 40 μM verteporfin decreased LATS1/2 and p-LATS1/2 protein expression after 48h and 72h treatment (p &amp;lt; 0.05, **p&amp;lt;0.01). MST1/2 and p-MST1/2 was significantly decreased in groups treated with 20 and 40 µM verteporfin after 48 and 72 h of treatment (p &amp;lt; 0.05, ****p&amp;lt;0.0001). YAP expression decreased at 5, 20 and 40 μM verteporfin treatment after 48h and all verteporfin concentrations after 72h (**p&amp;lt;0.01, ***p&amp;lt;0.001, ****p&amp;lt;0.0001). p-YAP expression significantly decreased at all verteporfin concentrations after 48h and only at 40 μM concentrations after 72h (**p&amp;lt;0.01). TEAD4 expression decreased at all verteporfin concentrations after 48 and 72h (****p&amp;lt;0.0001). After 48 and 72h of verteporfin treatment, no significant difference was observed in the expression of the MST1, MST2, LATS1, LATS2 and YAP1 mRNA levels in TCam-2 cells, while TEAD4 mRNA expression level was significantly decreased at 20 and 40 µM compared to control (**p&amp;lt;0.01, ***p&amp;lt;0.001). We also showed that Hippo signaling pathway proteins localized in the cell nucleus especially YAP1 and TEAD4, were translocated to cytoplasm depending on the increasing dose of verteporfin in TCam-2 cells. Verteporfin decreased cell proliferation (****p&amp;lt;0.0001), migration and cell viability (****p&amp;lt;0.0001), as well as increased the early-late apoptosis (**p&amp;lt;0.01, ***p&amp;lt;0.001, ****p&amp;lt;0.0001) in TCam-2 human seminoma cells dose-dependent manner. Limitations, reasons for caution The TCam-2 cell line was gifted by Prof. Dr. Hubert Schorle (Department of Developmental Pathology and Institute of Molecular Diagnostic Pathology, Faculty of Medicine, Germany, Bonn) to our laboratory. There is no limitation in the scope of the study. Wider implications of the findings We determined for the first time the effect of verteporfin treatment on the protein expression and mRNA levels of Hippo signaling pathway in TCam-2 human seminoma cells. We suggested that Hippo pathway and verteporfin relationship may be a therapeutic target for seminoma by affecting cell proliferation, survival, migration and apoptosis. Trial registration number not applicable