Inhibits Tumor Cell Invasion Research Articles

A novel 4,6-disubstituted-1,2,4-triazolo-1,3,4-thiadiazole derivative inhibits tumor cell invasion and potentiates the apoptotic effect of TNFα by abrogating NF-κB activation cascade.

Condensed-bicyclic 4,6-substituted1,2,4-triazolo-1,3,4-thiadiazole derivatives (CBTT) have been shown to possess a wide spectrum of pharmacological activities. In this study, several novel CBTT derivatives were synthesized and investigated for their possible role as anti-neoplastic agents. The anti-proliferative effect of various CBTT derivatives was analyzed against tumor cell lines by (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay. One of the potential CBTT derivative, 5-(3-(2,3-dichlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)flurobenzonitrile (DTTF) was found to be the most potent against cervical cancer SiHa cells and exhibited minimal effect against normal cells. Molecular docking analysis indicated that transcription factor NF-κB was one of the potential molecular targets modulated by DTTF. Specifically, the drug blocked the TNFα-induced phosphorylation of upstream IκBα kinase in a time-dependent manner leading to the suppression of NF-κB activation and nuclear translocation. DTTF also potentiated the apoptotic effect of TNFα, as well as significantly inhibited migration and invasion of tumor cells. Overall, these findings indicate a potential novel role and mechanism(s) of action of DTTF as an anticancer agent against diverse malignancies.

Draft Genome Sequence of Streptomyces sp. TP-A0874, a Catechoserine Producer

We report the draft genome sequence of Streptomyces sp. TP-A0874 isolated from compost. This strain produces catechoserine, a new catecholate-type inhibitor of tumor cell invasion. The genome harbors at least six gene clusters for polyketide and nonribosomal peptide biosyntheses. The biosynthetic gene cluster for catechoserines was identified by bioinformatic analysis.

Identification of Caspase-6 as a New Regulator of Alternatively Activated Macrophages

Alternatively activated macrophages (AAMs) play essential roles in the promotion of tissue remodeling, vasculogenesis, and tumor progression; however, the detailed mechanisms underlying the activation of AAMs remain largely unknown. Here, by using quantitative proteomic analysis, we identified 62 proteins that were up-regulated in IL-4-induced macrophages. Among these, Caspase-6 was increased significantly. Caspase-6 is important in the apoptotic signaling pathway; however, its role in non-apoptosis is also reported. Here, we first examined the non-apoptotic role of Caspase-6 in the alternative activation of macrophages after administration of IL-4, 4T1 tumor conditional medium, or co-culture with 4T1 cells. Both treatments promoted alternative activation of RAW264.7 cells and primary macrophages, whereas disruption of caspase-6 expression and activity could markedly suppress the biomarker levels of AAMs. Overexpression of Caspase-6 could significantly promote the activation of AAMs. Importantly, we further present evidence that caspase-6 could regulate breast cancer cell invasion by modulating MMP-2 and MMP-9 expression in 4T1 tumor-associated macrophages, as ablation of protein levels or activity of caspase-6 suppressed tumor cell invasion in vitro In conclusion, the observed results markedly expanded our views of the dynamic changes in protein composition during alternative activation of macrophages, and they revealed a critical new role of caspase-6 in regulating this cellular biological process, which suggested that caspase-6 might be a key nod molecule to regulate immunological steady-state and be a therapeutic candidate for tumor immunotherapy.

Macrophage PI3Kγ Drives Pancreatic Ductal Adenocarcinoma Progression

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a low 5-year survival rate, yet new immunotherapeutic modalities may offer hope for this and other intractable cancers. Here, we report that inhibitory targeting of PI3Kγ, a key macrophage lipid kinase, stimulates antitumor immune responses, leading to improved survival and responsiveness to standard-of-care chemotherapy in animal models of PDAC. PI3Kγ selectively drives immunosuppressive transcriptional programming in macrophages that inhibits adaptive immune responses and promotes tumor cell invasion and desmoplasia in PDAC. Blockade of PI3Kγ in PDAC-bearing mice reprograms tumor-associated macrophages to stimulate CD8(+) T-cell-mediated tumor suppression and to inhibit tumor cell invasion, metastasis, and desmoplasia. These data indicate the central role that macrophage PI3Kγ plays in PDAC progression and demonstrate that pharmacologic inhibition of PI3Kγ represents a new therapeutic modality for this devastating tumor type. We report here that PI3Kγ regulates macrophage transcriptional programming, leading to T-cell suppression, desmoplasia, and metastasis in pancreas adenocarcinoma. Genetic or pharmacologic inhibition of PI3Kγ restores antitumor immune responses and improves responsiveness to standard-of-care chemotherapy. PI3Kγ represents a new therapeutic immune target for pancreas cancer. Cancer Discov; 6(8); 870-85. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 803.

Abstract 4120: c-Met inhibition blocks MEK-induced tumor cell invasion in uveal melanoma

Abstract Uveal melanoma is the most common primary intraocular malignant tumor in adults. About half of patients with uveal melanoma will develop metastatic disease to the liver and the lung. These tumors are characterized by mutations in G-proteins (GNAQ and GNA11), activation of MAPK, and over-expression of c-Met. We have previously reported that c-Met inhibition inhibits tumor cell invasion and metastasis formation in uveal melanoma. Altiratinib is a selective inhibitor for c-Met as well as TIE2, VEGFR2 and TRK kinases. We found that this agent had no effect on inhibiting cell growth, consistent with our previous findings with crizotinib. However, it did inhibit invasion of uveal melanoma cells through matrigel in a concentration dependent fashion (25 nM to 250 nM) with inhibition of phospho-Met noted at concentrations as low as 25 nM. Selumetinib, a MEK inhibitor, is currently in clinical trial in patients with this disease. We found that treatment with 250 nM selumetinib inhibited cell proliferation but unexpectedly induced a marked increase in cell invasion of GNAQ and GNA11 mutant cell lines. This was associated with an increase in c-Met RNA and protein expression, as well as receptor phosphorylation, after 24 hours of selumetinib treatment. Combining selumetinib with altiratinib inhibited cell invasion to the level of altiratinib alone and continued to inhibit cell proliferation to the level of selumetinib alone. This effect was recapitulated by the knockdown of c-Met by siRNA prior to treatment with selumetinib. In a uveal melanoma xenograft model, the combination treatment of 15 mg/kg altiratinib and 25 mg/kg selumetinib significantly delayed tumor growth compared to vehicle control, altiratinib and selumetinib alone. Western blot analysis of tumor tissue confirmed target inhibition of p-Met and p-ERK in animals treated with altiratinib and selumetinib, respectively. Furthermore, tumor metastasis was inhibited by altiratinib, selumetinib and combination treatments in a uveal melanoma mouse model. These results indicate that the combined inhibition of MEK and c-Met by selumetinib and altiratinib, respectively, may be sufficient to suppress uveal melanoma tumor growth and metastasis. This strategy can potentially be used as therapy for patients with primary uveal melanoma who are at high risk for the development of metastatic disease. Citation Format: Oliver Surriga, Bryan D. Smith, Grazia Ambrosini, Daniel L. Flynn, Richard D. Carvajal, Gary K. Schwartz. c-Met inhibition blocks MEK-induced tumor cell invasion in uveal melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4120.

Hypoxia-inducible factor-1α mediates the toll-like receptor 4 signaling pathway leading to anti-tumor effects in human hepatocellular carcinoma cells under hypoxic conditions.

Hypoxia-inducible factor-1α (HIF-1α) and toll-like receptor 4 (TLR4) are involved in numerous mechanisms of cancer biology, including cell proliferation and survival; however the interaction of the two factors under hypoxic conditions remains unclear. The present study investigated the in vitro mechanism that results in the suppression of tumor cell growth and cellular functions when HIF-1α is silenced. In the present study, the human hepatocellular carcinoma HepG2 cell line was transfected with short hairpin RNA (shRNA) against HIF-1α and cultured under hypoxic conditions (1% O2 for 24 h). The expression of HIF-1α and various growth factors, including epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2), were examined using quantitative polymerase chain reaction and immunoblotting. Tumor growth was measured using a Cell Counting Kit-8 assay and tumor activity was measured using tumor cell invasion and migration assays. Lipopolysaccharide and TAK-242 were used to activate and inhibit TLR4, respectively, to observe the role of TLR4 in the HIF-1α silenced tumor cells. The expression of TLR4 signaling pathway associates, including myeloid differentiation primary response gene 88 (MyD88), apoptosis signal-regulating kinase 1 (ASK1), p38 mitogen-activated protein kinases and HIF-1α, were analyzed by western blot assay. Under hypoxic conditions, silencing of HIF-1α expression suppressed tumor cell growth and regulated the expression of tumor growth-associated genes, including EGF, HGF, VEGF and FG2. Suppression of tumor cell invasion and migration was also observed in the HIF-1α silenced HepG2 cell line. In addition, TLR4 was identified to be involved in HIF-1α and MyD88 accumulation, and activation of ASK1 and p38 were demonstrated to be critical for TLR4-mediated HIF-1α pathway. In conclusion, silencing of HIF-1α expression may induce anti-tumor effects under hypoxic conditions in HepG2 cells via the TLR4 mediated pathway, suggesting that the HIF-1α/TLR4 signaling cohort may act as a novel therapeutic target for the treatment of hepatocellular cancer.

Anti-cancer effects of Piper nigrum via inducing multiple molecular signaling in vivo and in vitro

Ethnopharmacological relevancePiper nigrum is widely used as a folk medicine including usage for pain relief, fevers, as well as an anti-cancer agent. However the crude extract of piperine free P. nigrum (PFPE), which inhibits breast cancer, and its mechanisms are still being kept secret. This research aims to elucidate the anti-cancer effects of PFPE and its mechanisms. Materials and methodsAnti-cancer effects of PFPE were investigated in N-nitroso-N-methylurea (NMU)-induced mammary tumorigenesis rats and breast cancer cell lines MCF-7 and ZR-75-1. Furthermore, the cancer prevention effects of PFPE were investigated in rats. Western blotting was employed to study protein levels induced by PFPE. ResultsPFPE was found to up-regulate p53, and down-regulate estrogen receptor (ER), E-cadherin (E-cad), matrix metalloproteinase 9 (MMP-9), matrix metalloproteinase 2 (MMP-2), c-Myc, and vascular endothelial growth factor (VEGF) levels in breast cancer rats. Moreover, PFPE decreased protein levels of E-cad, c-Myc, and VEGF in MCF-7 cells. These results suggest that PFPE can enhance breast cancer cell response to phytochemicals, then induce cell cycle arrest, and inhibit cancer cell proliferation resulting in tumor size decrease in the PFPE treated group. It further suggests that PFPE may suppress tumor cell invasion, migration, and angiogenesis. In addition, PFPE possessed cancer prevention effects through generation of reactive oxygen species (ROS) to higher cancer cell cellular stress. ConclusionsPFPE may possess anti-cancer and cancer prevention effects; hence, it deserves further investigation as a novel candidate for breast cancer treatment.

MicroRNA-126 inhibits tumor cell invasion and metastasis by downregulating ROCK1 in renal cell carcinoma.

MicroRNAs (miRNAs) are involved in cancer development and progression. Renal cell carcinoma (RCC) frequently undergoes metastasis and has a high mortality rate. The current study measured miRNA-126 (miR-126) expression levels in 128 pairs of clear cell RCC and adjacent normal kidney tissue samples by reverse transcription-quantitative polymerase chain reaction, and analyzed the association between miR-126 and various clinicopathological parameters. In addition, cell proliferation, wound healing and cell invasion assays were conducted using RCC cells overexpressing miR-126. Potential miR-126 target genes and the signaling pathways that may be regulated by miR-126 were then examined. miR-126 expression was significantly reduced in patients with metastatic RCC compared with patients without metastasis. Consistently, overexpression of miR-126 in RCC cells significantly inhibited cell proliferation, migration and invasion in vitro compared with negative control miRNA. A luciferase reporter assay demonstrated that miR-126 targets Rho associated coiled-coil containing protein kinase 1 (ROCK1) by directly binding the 3′-untranslated region. Furthermore, western blotting identified miR-126 as an important regulator of the AKT and extracellular signal-regulated 1/2 signaling pathways. The results of the present study indicate that miR-126 inhibits RCC cell proliferation, migration and invasion by downregulating ROCK1. These findings suggest that miR-126 may be valuable as a potential target for therapeutic intervention in RCC.

Abstract B39: PI3K therapy-induced adaptive mitochondria reprogramming fuels metastatic traits

Abstract Tumors rewire cell metabolism and autophagy to adapt to environmental stress conditions, including hypoxia, starvation and oxidative stress. Such adaptive reprogramming includes a switch from oxidative phosphorylation to glycolysis, use of glutamine as fuel instead of glucose and increased biosynthesis of lipids and nucleotides. Although these processes promote resistance to apoptosis and support biomass expansion, they are also energetically inefficient, making it unclear how transformed cells accomplish highly energy-demanding tasks, including invasion and metastasis. Scattered evidences suggest that mitochondrial oxidative phosphorylation may be important for cancer traits, including stemmness, tumor repopulation and drug resistance. We recently demonstrated that targeting of the universal cancer node phosphatidylinositol-3 kinase (PI3K) induces extensive bioenergetics and transcriptional reprogramming in tumor cells, paradoxically culminating with preservation of homeostatic mitochondrial bioenergetics, opposing permeability transition and tumor cell death. Here we sought to understand how tumors adapt to molecular therapy as it pertains to tumor progression. Our results show that PI3K inhibitors used in the clinic induce global changes in activation of growth factor receptor kinases, reactivation of Akt and mTOR signaling, and dramatically increased tumor cell invasion. This involves the trafficking of energetically active mitochondria to the cortical cytoskeleton of tumor cells, supporting membrane lamellipodia dynamics, heightened turnover of focal adhesion complexes and increased velocity and distance of random cell migration. Furthermore, a single dose of PI3Ki increased liver metastasis after intrasplenic-injection of tumor cells in mice. Mechanistic studies pinpoint the role of Akt, mTOR, mitochondrial Hsp90s, the autophagy initiating complex ULK1/FIP200 and focal adhesion kinase (FAK) in the adaptive response to PI3Ki. Blocking oxidative phosphorylation by targeting mtHsp90s prevented the repositioning of mitochondria to sites of cell movements, impaired membrane dynamics and suppressed tumor cell invasion. Our results suggest a potential drawback for PI3K therapy, namely the increased risk of metastasis in patients. Targeting mitochondrial reprogramming is feasible, and could provide a novel therapeutic strategy to limit disease dissemination. Note: This abstract was not presented at the conference. Citation Format: M. Cecilia Caino, Dario C. Altieri. PI3K therapy-induced adaptive mitochondria reprogramming fuels metastatic traits. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B39.

Development of a specific affinity-matured exosite inhibitor to MT1-MMP that efficiently inhibits tumor cell invasionin vitroand metastasisin vivo

The membrane-associated matrix metalloproteinase-14, MT1-MMP, has been implicated in pericellular proteolysis with an important role in cellular invasion of collagenous tissues. It is substantially upregulated in various cancers and rheumatoid arthritis, and has been considered as a potential therapeutic target. Here, we report the identification of antibody fragments to MT1-MMP that potently and specifically inhibit its cell surface functions. Lead antibody clones displayed inhibitory activity towards pro-MMP-2 activation, collagen-film degradation and gelatin-film degradation, and were shown to bind to the MT1-MMP catalytic domain outside the active site cleft, inhibiting binding to triple helical collagen. Affinity maturation using CDR3 randomization created a second generation of antibody fragments with dissociation constants down to 0.11 nM, corresponding to an improved affinity of 332-fold with the ability to interfere with cell-surface MT1-MMP functions, displaying IC50 values down to 5 nM. Importantly, the new inhibitors were able to inhibit collagen invasion by tumor-cells in vitro and in vivo primary tumor growth and metastasis of MDA-MB-231 cells in a mouse orthotopic xenograft model. Herein is the first demonstration that an inhibitory antibody targeting sites outside the catalytic cleft of MT1-MMP can effectively abrogate its in vivo activity during tumorigenesis and metastasis.

Epb41l3 suppresses esophageal squamous cell carcinoma invasion and inhibits MMP2 and MMP9 expression.

EPB41L3 may play a role as a metastasis suppressor by supporting regular arrangements of actin stress fibres and alleviating the increase in cell motility associated with enhanced metastatic potential. Downregulation of epb41l3 has been observed in many cancers, but the role of this gene in esophageal squamous cell carcinoma (ESCC) remains unclear. Our study aimed to determine the effect of epb41l3 on ESCC cell migration and invasion. We investigated epb41l3 protein expression in tumour and non-tumour tissues by immunohistochemical staining. Expression in the non-neoplastic human esophageal cell line Het-1a and four ESCC cell lines - Kyse150, Kyse510, Kyse450 and Caes17 - was assessed by quantitative Polymerase Chain Reaction (qPCR) and Western blotting. Furthermore, an EPB41L3 overexpression plasmid and EPB41L3-specific small interfering RNA were used to upregulate EPB41L3 expression in Kyse150 cells and to downregulate EPB41L3 expression in Kyse450 cells, respectively. Cell migration and invasion were evaluated by wound healing and transwell assays, respectively. The expression levels of p-AKT, matrix metalloproteinase (MMP)2 and MMP9 were evaluated. Expression of epb41l3 was significantly lower in tumour tissues than in non-tumour tissues and in ESCC cell lines compared with the Het-1a cell line. Kyse450 and Caes17 cells exhibited higher expression of epb41l3 than Kyse150 and Kyse510 cells. Overexpressing epb41l3 decreased Kyse150 cell migration and invasion, whereas EPB41L3-specific small interfering RNA silencing increased these functions in Kyse450 cells. Furthermore, overexpressing epb41l3 led to downregulation of MMP2 and MMP9 in Kyse150 and Kyse510 cells. Our findings reveal that EPB41L3 suppresses tumour cell invasion and inhibits MMP2 and MMP9 expression in ESCC cells.

A Novel High Content Imaging-Based Screen Identifies the Anti-Helminthic Niclosamide as an Inhibitor of Lysosome Anterograde Trafficking and Prostate Cancer Cell Invasion.

Lysosome trafficking plays a significant role in tumor invasion, a key event for the development of metastasis. Previous studies from our laboratory have demonstrated that the anterograde (outward) movement of lysosomes to the cell surface in response to certain tumor microenvironment stimulus, such as hepatocyte growth factor (HGF) or acidic extracellular pH (pHe), increases cathepsin B secretion and tumor cell invasion. Anterograde lysosome trafficking depends on sodium-proton exchanger activity and can be reversed by blocking these ion pumps with Troglitazone or EIPA. Since these drugs cannot be advanced into the clinic due to toxicity, we have designed a high-content assay to discover drugs that block peripheral lysosome trafficking with the goal of identifying novel drugs that inhibit tumor cell invasion. An automated high-content imaging system (Cellomics) was used to measure the position of lysosomes relative to the nucleus. Among a total of 2210 repurposed and natural product drugs screened, 18 “hits” were identified. One of the compounds identified as an anterograde lysosome trafficking inhibitor was niclosamide, a marketed human anti-helminthic drug. Further studies revealed that niclosamide blocked acidic pHe, HGF, and epidermal growth factor (EGF)-induced anterograde lysosome redistribution, protease secretion, motility, and invasion of DU145 castrate resistant prostate cancer cells at clinically relevant concentrations. In an effort to identify the mechanism by which niclosamide prevented anterograde lysosome movement, we found that this drug exhibited no significant effect on the level of ATP, microtubules or actin filaments, and had minimal effect on the PI3K and MAPK pathways. Niclosamide collapsed intralysosomal pH without disruption of the lysosome membrane, while bafilomycin, an agent that impairs lysosome acidification, was also found to induce JLA in our model. Taken together, these data suggest that niclosamide promotes juxtanuclear lysosome aggregation (JLA) via modulation of pathways involved in lysosome acidification. In conclusion, we have designed a validated reproducible high-content assay to screen for drugs that inhibit lysosome trafficking and reduce tumor invasion and we summarize the action of one of these drugs.

MicroRNA-340 inhibits the migration, invasion, and metastasis of breast cancer cells by targeting Wnt pathway.

MicroRNAs (miRNAs) play a key role in tumor metastasis based on their capacity to regulate the expression of tumor-related genes. Over-expression of key genes such as c-MYC and CTNNB1 (encoding β-catenin) in Wnt/β-catenin-dependent and ROCK1 in Wnt/β-catenin-independent signaling pathways (Rho/Rho-associated kinase (ROCK) signaling pathway) has already been identified as the hallmarks of many tumors, and their role in breast cancer has also been investigated and confirmed. miR-340 characterization as an onco-suppressor miRNA has been previously reported. However, the mechanism by which it inhibits metastasis has not been completely elucidated. Quantitative real-time PCR (qPCR), Western blot, and luciferase assays were used to confirm the effect of miR-340 on the 3'-untranslated region (UTR) of the target genes. Lentiviral particles containing miR-340 were also used to evaluate the effect of miR-340 restoration on cell proliferation, migration, and invasion in vitro in the invasive MDA-MB-231 cell line. By applying bioinformatic approaches for the prediction of miRNAs targeting 3'-UTRs of CTNNB1, c-MYC, and ROCK1, we found out that miR-340 could dramatically down-regulate metastasis by targeting Wnt signaling in breast cancer cells. In the current study, analyzing miR-340 by reverse transcription quantitative PCR (RT-qPCR) in MDA-MB-231 showed that it was remarkably down-regulated in the metastatic breast cancer cell line. We found that restoration of miR-340 in the invasive breast cancer cell line, MDA-MB-231, suppresses the expression of the target genes' messenger RNA (mRNA) and protein and, as a result, inhibits tumor cell invasion and metastasis. Our findings highlight the ability of bioinformatic approaches to find miRNAs targeting specific genes. By bioinformatic analysis, we confirmed the important role of miR-340 as a pivotal regulator of breast cancer metastasis in targeting previously validated (ROCK1) and potentially novel genes, i.e., (CTNNB1 and c-MYC).

Upregulation of microRNA-31 targeting integrin α5 suppresses tumor cell invasion and metastasis by indirectly regulating PI3K/AKT pathway in human gastric cancer SGC7901 cells.

To verify the hypothesis that upregulation of microRNA-31 (miR-31) targeting integrin α5 (ITGA5) suppresses tumor cell invasion and metastasis by indirectly regulating phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in human SGC7901 gastric cancer (GC) cells. The miRTarBase was used to predict whether ITGA5 is the target gene of miR-31, which was further confirmed by luciferase reporter gene assay. The SGC7901 GC cells were divided into five groups including the blank, miR-31 mimic, miR-31 mimic control, miR-31 inhibitor, and miR-31 inhibitor control groups. Reverse transcriptase-polymerase chain reaction (RT-PCR), western blotting, cell scratch test, and transwell assays were respectively performed in our study. TGA5 was found as the target gene of miR-31. The RT-PCR detection revealed that, compared with the blank group, ITGA5 messenger RNA (mRNA) expression decreased in the miR-31 mimic group, but increased in the miR-31 inhibitor group. The western blotting examination suggested that the expressions of ITGA5, PI3K, and AKT proteins reduced in the miR-31 mimic group, but enhanced in the miR-31 inhibitor group when compared to the blank group, respectively. The cell scratch and transwell assays indicated that the miR-31 expressions were negatively associated with GC cell migration and invasion. Besides, RT-PCR combined with western blotting demonstrated that the miR-31 expressions were higher in the normal tissues than those in the GC tissues, while the ITGA5 mRNA and protein showed lower expression in the normal tissues than they did in the GC tissues. Our study concluded that upregulation of miR-31 targeting ITGA5 may suppress tumor cell invasion and metastasis by indirectly regulating PI3K/AKT signaling pathway in human SGC7901 GC cells.

MiR-940 Suppresses Tumor Cell Invasion and Migration via Regulation of CXCR2 in Hepatocellular Carcinoma.

Aim. To investigate the expression of miR-940 in the hepatocellular carcinoma (HCC) and its impact on function and biological mechanism in the HCC cells. Methods. Quantitative RT-PCR analysis was used to quantify miR-940 expression in 46 cases of tissues and cells. Transfection of HCC cell lines was performed by miR-940 mimics; the abilities of invasion and migration were assessed through Transwell array. Western blot represents the alteration in expression of CXCR2 by miR-940 mimics. Results. miR-940 expression was decreased significantly in the HCC tissues and the relevant cell lines. miR-940 upregulation suppressed the invasion and migration of HCC cells in vitro. Furthermore, the CXCR2 was downregulated to suppress invasion and migration after miR-940 mimics. Moreover, decreased miR-940 expression was negatively correlated with Edmondson grade (P = 0.008), tumor microsatellite or multiple tumors (P = 0.04), vascular invasion (P = 0.035), and recurrence and metastasis (P = 0.038). Kaplan-Meier analysis demonstrated that decreased miR-940 expression contributed to poor overall survival (P < 0.05). Conclusions. Our findings present that miR-940 acts as a pivotal adaptor of CXCR2 and its transcription downregulated CXCR2 expression to decrease HCC invasion and migration in vitro. Our study suggests that miR-940 may be a novel poor prognostic biomarker for HCC.

Regulation of the Mechanism of TWIST1 Transcription by BHLHE40 and BHLHE41 in Cancer Cells.

BHLHE40 and BHLHE41 (BHLHE40/41) are basic helix-loop-helix type transcription factors that play key roles in multiple cell behaviors. BHLHE40/41 were recently shown to be involved in an epithelial-to-mesenchymal transition (EMT). However, the precise mechanism of EMT control by BHLHE40/41 remains unclear. In the present study, we demonstrated that BHLHE40/41 expression was controlled in a pathological stage-dependent manner in human endometrial cancer (HEC). Our in vitro assays showed that BHLHE40/41 suppressed tumor cell invasion. BHLHE40/41 also suppressed the transcription of the EMT effectors SNAI1, SNAI2, and TWIST1. We identified the critical promoter regions of TWIST1 for its basal transcriptional activity. We elucidated that the transcription factor SP1 was involved in the basal transcriptional activity of TWIST1 and that BHLHE40/41 competed with SP1 for DNA binding to regulate gene transcription. This study is the first to report the detailed functions of BHLHE40 and BHLHE41 in the suppression of EMT effectors in vitro. Our results suggest that BHLHE40/41 suppress tumor cell invasion by inhibiting EMT in tumor cells. We propose that BHLHE40/41 are promising markers to predict the aggressiveness of each HEC case and that molecular targeting strategies involving BHLHE40/41 and SP1 may effectively regulate HEC progression.

Evodiamine Attenuates PDGF-BB-Induced Migration of Rat Vascular Smooth Muscle Cells through Activating PPARγ.

The uncontrolled migration of vascular smooth muscle cells (VSMCs) into the intima is a critical process in the development of atherosclerosis. Evodiamine, an indole alkaloid extracted from the Chinese medicine evodia, has been shown to inhibit tumor cell invasion and protect the cardiovascular system, but its effects on VSMCs remain unknown. In the present study, we investigated the inhibitory effects of evodiamine on the platelet-derived growth factor-BB (PDGF-BB)-induced VSMC migration using wound healing and transwell assays, and assessed its role in decreasing the protein levels of matrix metalloproteinases and cell adhesion molecules. More importantly, we found that evodiamine activated the expression and nuclear translocation of peroxisome proliferator-activated receptor γ (PPARγ). Inhibition of PPARγ activity by using its antagonist T0070907 and its specific siRNA oligonucleotides significantly attenuated the inhibitory effects of evodiamine on VSMC migration. Taken together, our results indicate a promising anti-atherogenic effect of evodiamine through attenuation of VSMC migration by activating PPARγ.

SOX4 expression is associated with treatment failure and chemoradioresistance in oral squamous cell carcinoma.

BackgroundIn humans, sex-determining region-Y (SRY) related high-mobility-group box 4 (SOX4) is linked to development and tumorigenesis. SOX4 is over-expressed in several cancers and has prognostic significance. This study evaluated whether SOX4 affects oncogenic behavior and chemoradiotherapy response in head and neck squamous cell carcinoma (HNSCC) cells, and documented the relationship between its expression and prognosis in oral squamous cell carcinoma (OSCC).MethodsWe used small interfering RNA in HNSCC cells to evaluate the effect of SOX4 on cell proliferation, apoptosis, chemoradiation-induced apoptosis, invasion, and migration. SOX4 expression in OSCC tissues was investigated by immunohistochemistry.ResultsSOX4 knockdown (KO) decreased cell proliferation and induced apoptosis by activating caspases-3 and −7, and poly-ADP ribose polymerase and suppressing X-linked inhibitor of apoptosis protein in HNSCC cells; it also enhanced radiation/cisplatin-induced apoptosis; and suppressed tumor cell invasion and migration. Immunostaining showed SOX4 protein was significantly increased in OSCC tissues compared with adjacent normal mucosa. SOX4 expression was observed in 51.8 % of 85 OSCC tissues, and was significantly correlated with treatment failure (P = 0.032) and shorter overall survival (P = 0.036) in patients with OSCC.ConclusionsSOX4 may contribute to oncogenic phenotypes of HNSCC cells by promoting cell survival and causing chemoradioresistance. It could be a potential prognostic marker for OSCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1875-8) contains supplementary material, which is available to authorized users.

Retraction Note to: Aberrant promoter methylation of the CHD1 gene may contribute to the pathogenesis of breast cancer: a meta-analysis.

Cadherin-1 (CHD1), as an invasion suppressor gene, could suppress tumor cell invasion and metastasis in various tumors, but reduced CHD1 levels, resulting from epigenetic silencing, are common in poorly differentiated, advanced stage carcinomas. This meta-analysis was performed to evaluate the relationships between promoter methylation of CHD1 and breast cancer. Relevant studies were retrieved from the Web of Science (1945 ~ 2013), the Cochrane Library (Issue 12, 2013), PubMed (1966 ~ 2013), EMBASE (1980 ~ 2013), CINAHL (1982 ~ 2013), and the Chinese Biomedical Database (CBM) (1982 ~ 2013) using a systematic literature search. Results were summarized by meta-analyses, conducted using the STATA software (version 12.0, Stata Corporation, College Station, TX, USA). Odds ratios (ORs) and 95 % confidence intervals (95 % CIs) were calculated. In the present meta-analysis, 9 cohort studies with a total of 425 patients with breast cancer were included. Our meta-analysis results demonstrated that the frequency of CHD1 promoter methylation in cancer tissues was significantly higher than that in normal tissues, adjacent tissues, and benign tissues (cancer tissue vs. normal tissue OR = 30.87, 95 % CI = 16.76 ~ 56.86, P < 0.001; cancer tissue vs. adjacent tissue OR = 23.30, 95 % CI = 12.85 ~ 42.26, P < 0.001; cancer tissue vs. benign tissue OR = 2.94, 95 % CI = 1.60 ~ 5.40, P < 0.001; respectively). Ethnicity-stratified analysis indicated that aberrant CHD1 promoter methylation was strongly correlated with breast cancer among both Asians and Caucasians in the majority of subgroups. Our results suggest that aberrant promoter methylation of the CHD1 gene may have a high frequency in breast cancer tissues. Thus, CHD1 methylation could be correlated with the pathogenesis of breast cancer.

Abstract 1703: A 3D tissue culture-based utra-high content screening platform that uses phenotypic profiling of cancer tissues to identify selective inhibitors of receptor tyrosine kinases

Abstract 3D tissue cultures provide a more physiologically relevant context for the screening of compounds, compared to 2D cell cultures. Cells cultured in 3D hydrogels also show complex phenotypes, increasing the scope for phenotypic profiling. Here we describe a high-content screening platform that uses human micro-tissues cultured in 3D in standard 384 well assay plates to study the activity of biologically active molecules. Image analysis tools were developed to process 3D image data to measure 854 phenotype-derived parameters. Multiparametric analysis was used to evaluate the effect of compounds on tissue morphology. We applied this screening platform to measure the activity and selectivity of inhibitors of the c-Met and EGF receptor (EGFR) tyrosine kinases in 3D cultured prostate carcinoma cells. c-Met and EGFR activity was quantified based on the invasive phenotypic profiles induced by their respective ligands, HGF and EGF, which were added in parallel assays. c-Met and EGFR inhibitors generally showed the expected inhibitory profile equivalent to that obtained in the absence of cognate ligand. However, one phase-III c-Met inhibitor, displayed an unexpected phenotypic profile characterised by potent inhibition of tumour cell invasion and off-target effects consistent with a previously reported polypharmacology of this compound. The screening method was applied to a novel collection of 70 bi-specific compounds designed to target both c-Met and EGFR. Compounds were identified that induced phenotypic profiles indicative of selective inhibition of either c-Met, EGFR or both targets. In conclusion, this poster describes a fully scalable high-content screening platform that uses phenotypic profiling to evaluate receptor tyrosine kinase inhibitor selectivity and activity in a physiologically relevant setting. Citation Format: Leo S. Price, Tijmen Booij, Maarten Klop. A 3D tissue culture-based utra-high content screening platform that uses phenotypic profiling of cancer tissues to identify selective inhibitors of receptor tyrosine kinases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1703. doi:10.1158/1538-7445.AM2015-1703