Cancer Cellular Proliferation Research Articles

Perineural invasion in prostate cancer is associated with Schwann cells and disruption of circadian rhythm-related gene expression: A bioinformatics approach

Schwann cells in the tumor microenvironment are reportedly involved in the production of several factors that benefit cancer cell growth. During this process, Schwann cells are dedifferentiated and facilitate cancer cellular proliferation. The cells then migrate to the region close to the tumor tissue and assist the development of the neoplastic cells. Accordingly, the present study was designed to evaluate the perineural invasion in prostate cancer, in association with disrupted circadian rhythm-related gene expression in Schwann cells. Initially, we identified a database reporting gene expression in Schwann cells in a neoplastic context from the GEO Datasets platform in the GEO repository. The database contains the expression results from experiments in which two factors produced by tumor cells were added to cell cultures. Comparisons were made between samples from the first and third passages. Then, these data were used in differential gene expression analysis and combined with differential expression data of genes upregulated in perineural invasion-negative and -positive prostate cancers. We observed that the “axon guidance” pathway was upregulated in perineural invasion-negative prostate cancers. Meanwhile, upregulated mRNAs activated the “axon guidance” pathway and, together with ROBO1 and MPZ upregulation, inhibited perineural invasion pathways. Both genes are also associated with inhibition of Schwann cell migration. PER3, NR3C1, PPARGC1A, TIMP3, ID2, PDE6B, and CAVIN1 were upregulated in perineural invasion-negative tumors, while SLC25A10 was upregulated. We also observed upregulated expression in perineural invasion-positive tumors for genes such as PPARGC1A, TIMP3, S100A8, ID2, DEFB1, AQP3, ASS1, PDE6B, NEFH, and CAVIN1. AQP3 and NEFH were upregulated only in perineural invasion-positive tumors and PER3 and NR3C1 were upregulated only in perineural invasion-negative samples. The findings revealed that circadian rhythm and/or melatonin disruption are associated with dedifferentiation of Schwann cells, which consequently produce a set of factors that drive tumor progression. These processes may also be involved in tumor invasion into the perineural tissue in prostate cancer.

Sanggenol B, a plant bioactive, as a safer alternative to tackle cancer by antagonising human FGFR

Fibroblast Growth Receptor Factor (FGFR) are a family of proteins which are, in addition to their biological role, are involved in various pathological functions, such as cancer cellular proliferation, and metastasis. Deregulation of FGFRs at various points could result in malignancy. A conformational transition of the DFG (Asp–Phe–Gly) motif can switch the enzyme from a catalytically active (DFG-in) to an inactive (DFG-out) state. There are a few FDFR inhibitors which have received approval from the FDA, but these have adverse side effects. Hence, there is a demand for safer alternatives. With this aim, Ligand and Structure based virtual screening was carried to identify suitable lead molecule. In this process, Four Featured atom-based 3D Pharmacophore with quantitative structure–activity relationship analysis (3D-QSAR) was developed. The External validation of the hypothesis was carried invoking criteria such as Area under the ROC curve. Natural plant compound databases such as the Traditional Chinese medicine, NPACT and the ZINC Natural databases were chosen for pharmacophore filtering, which was followed by virtual screening against FGFR isoforms. The compound Sanggenol B was identified as the most suitable lead molecule. Structural stability of the protein-ligand complex and interactions of the ligand (Sanggenol B & the reference compound Ponatinib) with FGFR were analysed for 1000 ns (triplicate) by means of molecular simulation and the binding free energy was calculated using MMGBSA. Sanggenol B (PubChem CID: 15233694) binds effectively at the active site with favourable energies and is proposed as a safe alternative from a natural source. Communicated by Ramaswamy H. Sarma

Abstract 954: Combined treatment of γ-tocotrienol, and vitamin D3 synergistically inhibits breast cancer cell proliferation, migration, and epithelial-to-mesenchymal transition (EMT)

Abstract Metastatic breast cancer is a leading cause of death of women in the United States. The metastatic process involves epithelial cells losing their cellular polarity, adhesion to the basement membrane, and acquiring migratory and invasive properties. Metastatic epithelial cells take on a mesenchymal phenotype, hence this process is termed epithelial-to-mesenchymal transition (EMT). EMT is characterized by a reduction in epithelial cell marker expression (E-cadherin, cytokeratin 8, and cytokeratin 18), and an increased expression of mesenchymal cell markers (N-cadherin, vimentin, and fibronectin). Previous studies have demonstrated that dietary intake of γ-tocotrienol and/or vitamin D3 may provide benefit in the preventing the growth and development of metastatic breast cancer. γ-Tocotrienol is a natural isoform of vitamin E that shows potent anticancer activity against a variety of cancer cell types at treatment doses that have little or no effect on normal cell growth or viability. Vitamin D3 has also been shown to display potent antiproliferative and antiangiogenic activity against human breast cancer cells, particularly triple negative breast cancers. Studies were conducted here to examine the effects of γ-tocotrienol and vitamin D3 given alone or in combination on MDA-MB-231 triple negative breast cancer (TNBC) cellular proliferation, migration, and epithelial to mesenchymal transition. Treatment with 10-16 µM γ-tocotrienol or 20-40 nM vitamin D3 induced a significant dose-responsive decrease in MDA-MB-231 growth after a 4-day culture period. Combined treatment with sub-effective doses of γ-tocotrienol (8 μM) and vitamin D3 (10 nM) induced a synergistically inhibition of MDA-MB-231 cell growth, as determined isobologram analysis. Studies using the matrigel invasion assay showed that similar combination treatment with subeffective doses of γ-tocotrienol and vitamin D3, significantly inhibited MDA-MB-231 motility. These effects appear to result from a reversal in EMT. Western blot analysis shows that combined treatment resulted in an increased expression of the epithelial cell markers E-cadherin, cytokeratins 8 and cytokeratins 18 and a corresponding reduction in mesenchymal cell markers N-cadherin and vimentin. Taken together, these findings indicate that combined treatment with subeffective doses of γ-tocotrienol and vitamin D3 synergistically inhibit TNBC MDA-MB-231 breast cancer cell growth, viability, motility, and induced a reversal of EMT. These findings strongly suggest that combined γ-tocotrienol and vitamin D3 treatment may provide significant benefits in the treatment of metastatic TNBC in women. This study was supported in part by funding from the Louisiana Cancer Foundation. Citation Format: Md. Rafi Anwar, Taylor A. Hurst, Paul W. Sylvester. Combined treatment of γ-tocotrienol, and vitamin D3 synergistically inhibits breast cancer cell proliferation, migration, and epithelial-to-mesenchymal transition (EMT) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 954.

Study on the Mechanism of Diosgenin Targeting STAT3 to Inhibit Colon Cancer Proliferation and Migration.

To elucidate regulatory effects and molecular mechanisms of diosgenin on colon cancer, this study administered diosgenin at concentrations of 10 (low), 50 (medium), and 100 μmol/L (high concentration group) at the cell level, respectively. EdU, colony formation, and Transwell assays were implemented to determine SW480 cellular proliferation and migration. Assays of flow cytometry and TUNEL were employed to estimate cell apoptosis. Additionally, nude mouse tumorigenesis assay was used to further verify the regulatory function of diosgenin on colon cancer. The target protein of diosgenin was predicted via molecular docking. The results showed that all three concentrations of diosgenin could reduce colon cancer cellular proliferation and migration, and after diosgenin treatment, colon cancer cellular apoptosis was markedly increased, and the 100 μmol/L diosgenin group produced the most satisfactory inhibition on colon cancer cell proliferation. Ki67 expression was markedly reduced whereas those of Bax and caspase3 were greatly increased after diosgenin treatment. The nude mouse tumorigenesis assay indicated that the parameters of tumorous volume and mass of diosgenin treatment group were greatly decreased as compared to control, and as the concentration of diosgenin increased, the inhibitory effect was more significant. Molecular docking indicated that STAT3 served as a target protein of diosgenin. Moreover, after diosgenin treatment on colon cancer cells, the STAT3 expression was markedly reduced. The STAT3 overexpression would counteract the inhibitory effect of 50 μmol/L diosgenin in both suppressing colon cancer cellular proliferation and migration and promoting apoptosis. Taken together, all our outcomes demonstrated the diosgenin effects in not only inhibiting colon cancer cellular proliferation and migration but also promoting cancerous cellular apoptosis. Diosgenin is a regulatory player in targeting and regulating STAT3.

MiR-144-3p inhibits the proliferation and metastasis of lung cancer A549 cells via targeting HGF

AimMicroRNAs have been confirmed as vital regulators in gene expression, which could affect multiple cancer cell biological behaviors. This study aims to elucidate the molecular mechanism of miR-144-3p in lung cancer cellular proliferation and metastasis.MethodsMiR-144-3p expression in lung cancer tissues and cell lines was detected by qRT-PCR. HGF was predicted as the target gene of miR-144-3p using TargetScan and dual luciferase reporter assay. Immunohistochemistry and qRT-PCR were used to explore the impacts of HCF on lung cancer tissues and cell lines. Impacts of miR-144-3p and HGF on cancer cellular proliferation, migration and invasion were elucidated by CCK-8, Flow cytometry, Transwell invasion and Wound-healing assay. Moreover, nude mouse xenograft model was established to evaluate the effects of miR-144-3p on lung cancer cells.ResultsMiR-144-3p exhibited a reduction in both lung cancer tissues and cell lines. HGF was a direct target of miR-144-3p. In contrast to the miR-144-3p expression level, HGF showed a higher level in lung cancer tissues and cell lines. Overexpression miR-144-3p suppressed A549 and NCI-H1299 cell proliferation and metastasis, whereas this was reversed by HGF. MiR-144-3p exhibited an inhibitory effect on A549 cell-induced tumor growth of nude mice.ConclusionsThis study reveals miR-144-3p/HGF axis may be involved in the suppression of lung cancer cellular proliferation and development, and miR-144-3p may function as a potential therapeutic target in lung cancer treatment in the future.

E2 + norethisterone promotes the PI3K–AKT pathway via PGRMC1 to induce breast cancer cell proliferation

Objective This study aimed to find evidence that progesterone receptor membrane component 1 (PGRMC1) promotes estradiol (E2) + norethisterone (NET)-induced breast cancer proliferation through activation of the phosphatidylinositol-3-kinase (PI3K)–AKT pathway. Methods PGRMC1-mediated breast cancer cellular proliferation and phosphorylation of PGRMC1 were studied using wild-type (hemagglutinin [HA]-tagged) MCF-7 cells, which were stably transfected with expression vector containing HA (MCF-7-HA cells), PGRMC1 (MCF-7-PGRMC1 cells) and Ser181 point mutated PGRMC1 (MCF-7-PGRMC1-S181A cells). Bioinformatics, cell proliferation, western blot, isobaric tags for relative and absolute quantitation (iTRAQ)-based RNA sequencing, real-time quantitative polymerase chain reaction (RT-qPCR) and cell cycle in vitro assays were performed to indicate the function of PGRMC1 and its possible mechanisms in breast cancer. Results NET + E2 elicited a significant proliferation in MCF-7-Vec at 10−6 M and 10−10 M, respectively. MCF-7-PGRMC1 did increase the phosphorylation of AKT or ERK, which can be blocked by treatment with casein kinase 2 (CK2) inhibitor quinalizarin or in MCF-7-PGRMC1-S181A cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the PI3K–AKT pathway is upregulated in MCF-7-PGRMC1 cells. Importantly, upregulation of the PI3K–AKT pathway mainly through promotion of cell cycle regulation strongly promoted cell proliferation in MCF-7-PGRMC1 cells. Conclusions CK2 is involved in phosphorylation of PGRMC1 at S181. The mechanism for the action of PGRMC1 for mediating proliferative progestogen effects obviously starts with promotion cell cycle regulation, and then activation of the PI3K–AKT pathway.

IGHG1 upregulation promoted gastric cancer malignancy via AKT/GSK-3\u03b2/\u03b2-Catenin pathway

BackgroundDespite current advances in gastric cancer treatment, disease metastasis and chemo-resistance remain as major hurdles against better overall prognosis. Previous studies indicated that IGHG1 as well as -Catenin serve as important regulators of tumor cellular malignancy. Therefore, understanding detailed molecular mechanism and identifying druggable target will be of great potentials in future therapeutic development.MethodsSurgical tissues and gastric cancer cell lines were retrieved to evaluate IGHG1 expression for patients with or without lymph node/distal organ metastasis. Functional assays including CCK8 assay, Edu assay, sphere formation assay and transwell assay, wound healing assay, etc. were subsequently performed to evaluate the impact of IGHG1/-catenin axis on tumor cell proliferation, migration and chemo-resistance.ResultsGastric cancer tissues and tumor cell lines demonstrated significantly higher level of IGHG1. Functional study further demonstrated that IGHG1 promoted proliferative and migration as well as chemo-resistance of gastric cancer tumor cells. Further experiments indicated that IGHG1 activated AKT/GSK-3/-Catenin axis, which played crucial role in regulation of proliferative and chemo-resistance of gastric cancer cells.ConclusionThis study provided novel evidences that IGHG1 acted as oncogene by promotion of gastric cancer cellular proliferation, migration and chemo-resistance. Our research further suggested that IGHG1/AKT/GSK-3β/β-Catenin axis acted as novel pathway which regulated gastric cancer cellular malignant behavior. Our research might inspire future therapy development to promote overall prognosis of gastric cancer patients.

SPINT1-AS1 Drives Cervical Cancer Progression via Repressing miR-214 Biogenesis.

Accumulating evidences have revealed the dysregulated expressions and critical roles of non-coding RNAs in various malignancies, including cervical cancer. Nevertheless, our knowledge about the vast majority of non-coding RNAs is still lacking. Here we identified long non-coding RNA (lncRNA) SPINT1-AS1 as a novel cervical cancer-associated lncRNA. SPINT1-AS1 was increased in cervical cancer and correlated with advanced stage and poor prognosis. SPINT1-AS1 was a direct downstream target of miR-214, a well-known tumor suppressive microRNA (miRNA) in cervical cancer. Intriguingly, SPINT1-AS1 was also found to repress miR-214 biogenesis via binding DNM3OS, the primary transcript of miR-214. The interaction between SPINT1-AS1 and DNM3OS repressed the binding of DROSHA and DGCR8 to DNM3OS, blocked DNM3OS cleavage, and therefore repressed mature miR-214 biogenesis. The expression of SPINT1-AS1 was significantly negatively correlated with miR-214 in cervical cancer tissues, supporting the reciprocal repression between SPINT1-AS1 and miR-214 in vivo. Through downregulating mature miR-214 level, SPINT1-AS1 upregulated the expression of β-catenin, a target of miR-214. Thus, SPINT1-AS1 further activated Wnt/β-catenin signaling in cervical cancer. Functionally, SPINT1-AS1 drove cervical cancer cellular proliferation, migration, and invasion in vitro, and also tumorigenesis in vivo. Deletion of the region mediating the interaction between SPINT1-AS1 and DNM3OS, overexpression of miR-214, and inhibition of Wnt/β-catenin signaling all reversed the roles of SPINT1-AS1 in cervical cancer. Collectively, these findings identified SPINT1-AS1 as a novel cervical cancer-associated oncogenic lncRNA which represses miR-214 biogenesis and activates Wnt/β-catenin signaling, highlighting its potential as prognostic biomarker and therapeutic target for cervical cancer.

Abstract 2573: The anticancer effects of the vitamin E isoform, γ-tocotrienol, and vitamin D3 act synergistically to inhibit MDA-MB-231 triple negative breast cancer (TNBC) cell proliferation and viability in vitro

Abstract Epidemiological studies suggest that dietary intake of some natural products may be effective in reducing the risk of breast cancer. Furthermore, various phytochemicals have been shown to interfere with cell signaling pathways involved in regulating breast cancer cell proliferation and viability. γ-Tocotrienol is a natural isoform of vitamin E that displays potent anticancer activity against a variety of cancer cell types at treatment doses that have little or no effect on normal cell growth or viability. Vitamin D3 is an abundant phytochemical which also displays potent antiproliferative and antiangiogenic activity against human breast cancer cells. Studies were conducted to examine the effects of γ-tocotrienol and vitamin D3 given alone or in combination on MDA-MB-231 triple negative breast cancer (TNBC) cellular proliferation, migration, colony formation, invasion, cell cycle progression, and apoptosis. Results showed that combined treatment with subeffective doses of γ-tocotrienol (8 μM) and vitamin D3 (10 nM) synergistically inhibited the growth of MDA-MB-231 cells, as determined by the MTT assay and isobologram analysis. Additional studies indicated that this combination treatment resulted in a significant inhibition in cancer cell migration and a significant reduction in cancer cell colony formation. Studies using the Matrigel invasion assay showed that combined treatment with subeffective doses of γ-tocotrienol and vitamin D3, significantly inhibited MDA-MB-231 invasiveness. Flow cytometry analysis showed that similar combined treatment of γ-tocotrienol and vitamin D3 significantly increased the percentage of cells found in G0/G1 phase, as compared to cells in the vehicle-treated control group. After 4 days of combined treatment with subeffective doses of γ-tocotrienol and vitamin D3, the percentage of cells in the G0/G1 phase of the cell cycle increased to 55% (p<0.05, all p values compared to untreated cells), as compared to 37.48% for cells in the vehicle-treated control group. In addition, combination treatment with subeffective doses of γ-tocotrienol and vitamin D3, significantly increased the number of apoptotic and necrotic cells, as comparted to cells in the vehicle-treated control group. In summary, these findings indicate that combined treatment with subeffective doses of γ-tocotrienol and vitamin D3 act synergistically to inhibit MDA-MB-231 human breast cancer cell growth and viability, and strongly suggest that combined treatment with these agents may provide some benefits in the treatment of TNBC in women. This study was supported in part by funding from the Louisiana Cancer Foundation. Citation Format: Md. R. Anwar, A.K.M.N. Hossian, Georgios Matthaiolampakis, Paul W. Sylvester. The anticancer effects of the vitamin E isoform, γ-tocotrienol, and vitamin D3 act synergistically to inhibit MDA-MB-231 triple negative breast cancer (TNBC) cell proliferation and viability in vitro [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 2573.

Effects of MTX-23, a Novel PROTAC of Androgen Receptor Splice Variant-7 and Androgen Receptor, on CRPC Resistant to Second-Line Antiandrogen Therapy

Although second-line antiandrogen therapy (SAT) is the standard of care in men with castration-resistant prostate cancer (CRPC), resistance inevitably occurs. One major proposed mechanism of resistance to SAT involves the emergence of androgen receptor (AR) splice variant-7, AR-V7. Recently, we developed MTX-23 using the principle of proteolysis targeting chimera (PROTAC) to target both AR-V7 and AR-full length (AR-FL). MTX-23 has been designed to simultaneously bind AR's DNA binding domain (DBD) and the Von Hippel-Lindau (VHL) E3 ubiquitin ligase. Immunoblots demonstrated that MTX-23's degradation concentration 50% (DC50) for AR-V7 and AR-FL was 0.37 and 2 μmol/L, respectively. Further studies revealed that MTX-23 inhibited prostate cancer cellular proliferation and increased apoptosis only in androgen-responsive prostate cancer cells. The antiproliferative effect of MTX-23 was partially reversed when either AR-V7 or AR-FL was overexpressed and was completely abrogated when both were overexpressed. To assess the potential therapeutic value of MTX-23, we next generated 12 human prostate cancer cell lines that are resistant to the four FDA-approved SAT agents-abiraterone, enzalutamide, apalutamide, and darolutamide. When resistant cells were treated with MTX-23, decreased cellular proliferation and reduced tumor growth were observed both in vitro and in mice. These results collectively suggest that MTX-23 is a novel PROTAC small molecule that may be effective against SAT-resistant CRPC by degrading both AR-V7 and AR-FL.

H2S‐Scavenged and Activated Iron Oxide‐Hydroxide Nanospindles for MRI‐Guided Photothermal Therapy and Ferroptosis in Colon Cancer

Overproduced hydrogen sulfide (H2 S) is of vital importance for the progress of colon cancer and promotes cancer cellular proliferation. Devising pharmacological nanomaterials for tumor-specific H2 S activation will be significant for precise colon cancer treatment. Herein, a biocompatible fusiform iron oxide-hydroxide nanospindles (FeOOH NSs) nanosystem for magnetic resonance imaging (MRI), ferroptosis, and H2 S based cascade reaction-enhanced combinational colon cancer treatment is developed. The FeOOH NSs can effectively scavenge endogenous H2 S via the reduction reaction to prohibit the growth of CT26 colon cancer. The cascade produced FeS driven by overexpressed H2 S exhibits near-infrared-triggered photothermal therapy capability and Fe2+ -mediated ferroptosis functionality. Meanwhile, the as-prepared FeOOH NSs can light up tumor tissues as a potent MRI contrast agent. Additionally, FeOOH NSs present desirable biosafety in a murine model for up to three months and avoid any long-term toxicity. Furthermore, it is found that these H2 S-responsible nanotheranostics do not cause any cure effects on other cancer types, such as 4T1 breast cancer. Overall, the findings illustrate that the biocompatible FeOOH NSs can be successfully employed as a theranostic for specifically treating colon cancer, which may promote the clinical translation and development of H2 S-responsive nanoplatforms.

Microbiome in Oral Squamous Cell Carcinoma: Mechanisms and Signaling Pathways

Oral squamous cell carcinoma is part of head and neck squamous cell carcinoma which is the ultimate cause of morbidity and mortality in cancer. The alteration of microbial community in the saliva might act as a helpful marker for the prediction, detection and prognosis oral cancer, particularly the transition of cancer precursor lesion. There are three mechanisms of action of oral microbiota in cancer pathogenesis, chronic inflammation of bacterial stimulation, carcinogenesis by cytoskeletal rearrangements, and carcinogenic substances that produced by microorganisms. Changes in the composition of microbiota could therefore have the potential to be used as a significant oral biomarker to predict the pathological transition from oral epithelial precursor lesion to cancer.Keywords: microbiome, oral cancer cellular proliferation, microorganism, oral cancer, oral squamous cell carcinoma

MicroRNA-205-5p inhibits three-dimensional spheroid proliferation of ErbB2-overexpressing breast epithelial cells through direct targeting of CLCN3.

We previously reported that microRNA-205-5p (miR-205-5p) is significantly decreased in the ErbB2-overexpressing breast epithelial cell line MCF10A-ErbB2 compared with control cells. In this study, we identified a direct target of miR-205-5p, chloride voltage-gated channel 3 (CLCN3). CLCN3 expression was induced by ErbB2 overexpression; this induced expression was then reduced to control levels by the transfection of the miR-205-5p precursor. In RNA-binding protein immunoprecipitation with Ago1/2/3 antibody, CLCN3 was significantly enriched in 293T embryonic kidney cells with miR-205-5p mimic transfection compared with negative control mimic transfection. In luciferase reporter assays using CLCN3 3′-UTR constructs, the miR-205-5p mimic significantly decreased reporter activity of both wild-type and partial mutant constructs in MCF10A-ErbB2 cells. In contrast, no inhibitory effects of the miR-205-5p mimic were detected using the complete mutant constructs. Since miR-205-5p expression in exosomes derived from MCF10A-neo cells was substantially higher than in exosomes derived from MCF10A-ErbB2 cells, we next investigated whether an exosome-mediated miR-205-5p transfer could control CLCN3 expression. To this end, exosomal miR-205-5p derived from MCF10A-neo cells was functionally transferred to MCF10A-ErbB2 cells, which served to decrease the expression of CLCN3. To assess the roles of CLCN3 in breast cancer, we next performed three-dimensional (3D) spheroid proliferation analyses using MCF10A-ErbB2 cells treated with MCF10A-neo-derived exosomes or CLCN3 shRNA stably expressing SKBR3 and MDA-MB-453 breast cancer cells. Our results showed that both treatment with MCF10A-neo-derived exosome and CLCN3 shRNA expression suppressed 3D spheroid proliferation. Collectively, these novel findings suggest that CLCN3 may be a novel direct target of miR-205-5p and this CLCN3/miR-205-5p interaction may serve a pivotal role in regulating breast cancer cellular proliferation under physiological conditions.

A randomized, double-blind, window of opportunity trial evaluating the effects of chloroquine in breast cancer patients

Chloroquine has demonstrated anti-tumor activities through autophagy inhibition and cell cycle disruption. This study aimed to assess the effect of single-agent chloroquine on breast tumor cellular proliferation in a randomized, phase II, double-blind, placebo-controlled, pre-surgical window of opportunity trial. Patients with newly diagnosed breast cancer were randomized 2:1 to chloroquine 500mg daily or placebo for 2- to 6-weeks prior to their breast surgery. The primary outcome was the relative change in measures of proliferation (Ki67) in primary breast cancer cells pre- and post-treatment. Adverse events and toxicity profiles were also evaluated. From September 2015 to December 2016, 70 patients were randomized [46 (66%) chloroquine and 24 (34%) placebo]. Ten patients who were randomized to chloroquine withdrew from study due to adverse events. Mean duration of drug intake was 15days (range 14-29days). There were no significant differences between the chloroquine or placebo arms with respect to either the percentage change (- 0.4 vs. - 1.2, p = 0.088) or absolute change (- 2.0% vs. - 5.2%, p = 0.066) in Ki67 index pre- and post-drug treatment. Although adverse effects were minimal and all classified as grade 1, the effects were significant enough to cause nearly 15% of patients to discontinue therapy. Treatment with single-agent chloroquine 500mg daily in the preoperative setting was not associated with any significant effects on breast cancer cellular proliferation. It was, however, associated with toxicity that may affect its broader use in oncology.

Abstract 170: Loss of the ubiquitin-specific peptidase 18 (USP18) reduces lung cancer metastasis

Abstract Metastasis is a major cause of cancer-related mortality. Yet, current antineoplastic strategies do not effectively repress metastasis. Thus, there is a pressing need to explore targets that can reduce metastasis, especially in lung cancer that is the most common cause of cancer mortality. Our prior work reported that expression of the ubiquitin-specific peptidase 18 (USP18) was augmented in diverse cancers (including lung cancers) and USP18 knock-down suppressed in vitro and in vivo growth of murine and human lung cancers. This deubiquitinase stabilizes target proteins by removing interferon-stimulated gene 15 (ISG15) from substrate proteins. We explored here whether engineered loss of USP18 decreased lung cancer metastasis. Stable USP18 knockdown was independently achieved in human (A549 and H1299) and murine (344SQ and KC2) lung cancer cells by use of small hairpin RNAs (shRNAs). Notably, knockdown of USP18 expression reduced lung cancer cellular proliferation, migration and invasion versus control shRNA transfectants (p < 0.001). Lung cancer cells selected for high metastatic potential (344SQ and KC2) were independently injected via tail vein or subcutaneously to establish metastatic mouse models. USP18 knockdown statistically-significantly decreased lung cancer metastases as compared to control shRNA transfectants. To elucidate engaged mechanisms, Reverse Phase Protein Array (RPPA) analysis was performed to interrogate over 300 growth-regulatory proteins in human (A549 and H1299) and murine (344SQ and KC2) lung cancer cells having engineered USP18 knockdown. Results were compared to control shRNA transfectants. Ingenuity pathway analysis highlighted fatty acid synthase (FASN) as a potential USP18 target that regulated lung cancer metastasis. Independent immunoblot analysis confirmed FASN expression was reduced in cancers once USP18 knockdown was achieved. Functional validation of FASN in lung metastasis is underway. These and other findings that will be presented provide a strong rationale for targeting the deubiquitinase USP18 to reduce lung cancer metastasis and combat lung cancers. Citation Format: Zibo Chen, Lin Zheng, Yulong Chen, Masanori Kawakami, Lisa Mustachio, Jason Roszik, Xin Liu, Jonathan Kurie, Xi Liu, Ethan Dmitrovsky. Loss of the ubiquitin-specific peptidase 18 (USP18) reduces lung cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 170.

β-Elemene Synergizes With Gefitinib to Inhibit Stem-Like Phenotypes and Progression of Lung Cancer via Down-Regulating EZH2.

The inhibitors for EGF receptor tyrosine kinase (EGFR-TKIs) such as gefitinib have been used as a standard treatment for non-small cell lung cancer (NSCLC), but the increasingly occurrence of drug resistance, the associated adverse effects and the enrichment of cancer stem cells significantly impedes its clinical application. β-elemene is a natural sesquiterpene with potent anti-cancer ability, and also it is renowned for its plant-origin, safety and the additive effect with traditional therapies, which prompt us to explore its potential to co-operate with TKIs to achieve greater therapeutic efficacy. Impressively, our study demonstrates that, elemene, in combination of gefitinib, displayed a significantly higher activity in inhibiting lung cancer cellular proliferation, migration and invasion. More importantly, combinative treatment profoundly impaired the epithelial to mesenchymal transition (EMT), the stem-like properties and the self-renewal capacity of lung cancer cells, and hence impeded the in vivo tumor development. We also reveal that the synergistic anti-tumor effect of elemene and gefitinib was largely mediated their regulation of enhancer of zeste homolog 2 (EZH2), an oncogenic histone methyltransferase and gene transcriptional regulator. Thus, our data indicate that combinative treatment of elemene and gefitinib has greater anti-neoplastic activity and greater efficacies in targeting cancer stem-like properties, mainly through regulating the malignant gene modifier and hence the subsequent effector molecules required for cancer progression. The findings may have potential implications for treating aggressive and resistant lung cancers.

MiR-20b promotes cellular proliferation and migration by directly regulating phosphatase and tensin homolog in prostate cancer.

MicroRNAs are small non-coding RNAs, which are critical regulators of carcinogenesis and tumor progression. Previous studies have identified that microRNA-20b (miR-20b) acts as an oncogene in numerous cancers. However, the role of miR-20b in prostate cancer remains unclear. The present study aimed to investigate the expression of miR-20b in prostate cancer and to examine whether modulating miR-20b expression impacts prostate cancer cellular proliferation and migration. It was revealed that miR-20b was strongly expressed in prostate cancer tissues compared with adjacent normal prostate tissues (P<0.05). Knockdown of miR-20b expression by miR-20b inhibitor inhibited VCaP and PC-3 cell growth and migration. Through bioinformatics analysis, phosphatase and tensin homolog (PTEN) was predicted as a target gene of miR-20b in prostate cancer cells, which was validated by dual-luciferase reporter assay and western blot analysis. In addition, restoration of PTEN expression levels did not affect endogenous miR-20b expression in prostate cancer cells. In conclusion, the present study indicated that miR-20b promotes cellular proliferation and migration by directly regulating PTEN in prostate cancer.

PPARα regulates tumor cell proliferation and senescence via a novel target gene carnitine palmitoyltransferase 1C.

Carnitine palmitoyltransferase 1C (CPT1C), an enzyme located in the outer mitochondria membrane, has a crucial role in fatty acid transport and oxidation. It is also involved in cell proliferation and is a potential driver for cancer cell senescence. However, its upstream regulatory mechanism is unknown. Peroxisome proliferator activated receptor α (PPARα) is a ligand-activated transcription factor that regulates lipid metabolism and tumor progression. The current study aimed to elucidate whether and how PPARα regulates CPT1C and then affects cancer cell proliferation and senescence. Here, for the first time we report that PPARα directly activated CPT1C transcription and CPT1C was a novel target gene of PPARα, as revealed by dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays. Moreover, regulation of CPT1C by PPARα was p53-independent. We further confirmed that depletion of PPARα resulted in low CPT1C expression and then inhibited proliferation and induced senescence of MDA-MB-231 and PANC-1 tumor cell lines in a CPT1C-dependent manner, while forced PPARα overexpression promoted cell proliferation and reversed cellular senescence. Taken together, these results indicate that CPT1C is a novel PPARα target gene that regulates cancer cell proliferation and senescence. The PPARα-CPT1C axis may be a new target for the intervention of cancer cellular proliferation and senescence.

Bisphenol A Induces Sox2 in ER+ Breast Cancer Stem-Like Cells.

Bisphenol A (BPA) is an endocrine disrupting compound used in food and beverage plastic containers and has been shown to increase breast cancer cellular proliferation. However, the concentrations of BPA used in these experiments are far higher than the physiological levels of BPA detected in the human body. We observed in vitro that exposure of MCF-7 cells to physiological concentrations of BPA failed to increase cell proliferation or to induce canonical estrogen-responsive genes (pS2 and progesterone receptor (PR)), in contrast to 17β-estradiol (E2) treatment. However, MCF-7 cells treated with 10nM BPA induced ALDH1 expression, a marker of human mammary stem cells. When treated with 10nM BPA, mammospheres derived either from MCF-7 cells, a patient-derived xenograft, or the normal mouse mammary gland exhibited increased size; however, these effects were not observed in MDA-MB-231 mammospheres. Mechanistically, BPA induced SOX2 mRNA and protein in MCF-7 mammospheres, resulting from enhanced CREB phosphorylation, and subsequent binding of pCREB to a SOX2 downstream enhancer. These findings suggest that physiological levels of BPA increase estrogen receptor-positive breast cancer tumor maintenance through enhanced cancer stem-like cell activity via direct regulation of SOX2 transcription.

A preliminary study of screening bile tumor markers for extrahepatic cholangiocarcinoma with proteomics technology

Objective To analyze differentially expressed proteins in bile of extrahepatic cholangiocarcinoma (EHCC)by proteomics technology, and to detect the potential tumor markers for diagnosis of bile duct malignancy. Methods Bile samples from 16 patients with EHCC and 16 patients with sphincter of oddi dysfunction (control) were collected for proteomic analysis by isobaric tags for relative and absolute quantitation (iTRAQ). The differentially expressed proteins were identified by mass spectrometry and retrieved by protein database. Results Protein maps of both EHCC group and control group were established successfully by iTRAQ. Analysis software identified an average of 275 proteins in EHCC group and 218 proteins in control group. Statistical filter screened 12 proteins of more than double difference, 4 of which were of diagnostic value, identified by mass spectrometry, which were MUC3a, Annexin A3, REG3A and Tumor-associated trypsin inhibitor (TATI). Functional analysis revealed that these proteins were associated with cancer cellular oncogenesis, proliferation, differentiation and metastasis. Conclusion Proteomic analysis by iTRAQ can identify the protein variance in bile of patients with EHCC, and identify four new potential biomarkers correlated with biological behavior of cholangiocarcinoma. Key words: Cholangiocarcinoma; Bile ducts, extrahepatic; Proteomics; Bile; Tumor markers