BACKGROUND: Oral cancer has been estimated as the sixth most frequent solid cancer all over the world, in which tongue squamous cell carcinoma (TSCC) is the most common type of oral cancers. However, the mechanism of TSCC metastasizing to lymph node and distant sites has not been completely understood.
METHODS: In this study, RT-qPCR method was used to detect the mRNA level of Numb, PTEN and Notch1 genes, as well as EMT-associated genes. Western blot assay was utilized to detect protein level of these genes. In addition, we determined cell proliferation by MTT assay and employed transwell invasion assay and wound healing assay to probe the abilities of invasion and migration, respectively. To investigate the role of PTEN, its inhibitor VO-Ohpic trihydrate was used to treat SCC-4 and CAL27 cells.
RESULTS: We found that Numb expression was downregulated in SCC-9 and CAL-27 cells compared to NHOK cells. Instead, Notch1 level in SCC-9 and CAL-27 cells were higher than that in NHOK cells. Furthermore, the results showed that Numb overexpression significantly suppressed proliferation, migration and invasion of SCC-9 and CAL-27 cells via regulating Notch1 signaling and EMT-related genes expression. By contrast, we observed that RBP-Jκ knockdown had an inhibitory role in proliferation, migration and invasion of SCC-9 and CAL-27 cells. In cells with Numb overexpression or RBP-Jκ knockdown, p-FAK and EMT-related genes were remarkably regulated.
CONCLUSIONS: Our findings provide new mechanism of understanding the metastasis of TSCC and help develop therapeutic strategies for treating tongue cancer.

BACKGROUND: We have previously reported the functional anti-cancer effects of the products of enzymatic hydrolysis of Citrus unshiu peel (εCUP) and fermented extraction of Citrus unshiu peel (ƒCUP) in human pancreatic cancer. Despite their different characteristics and effects, the underlying mechanism remains elusive.
PURPOSE: In this study, we further demonstrate the impact of ingredient contents of Citrus unshiu peel on the cancer's natural features.
METHODS: Anti-pancreatic cancer activities following combined treatment of naringenin and hesperetin were demonstrated in vitro and in vivo experiments.
RESULTS: Combined treatment with naringenin and hesperetin inhibited the growth of human pancreatic cancer cells (εCUP mimic condition, p < 0.001 for Miapaca-2 cells) through induction of caspase-3 cleavage compared to separate treatment with naringenin or hesperetin. Combined treatment with naringenin and hesperetin also inhibited the migration (εCUP mimic condition, p < 0.001 for Panc-1 cells) of human pancreatic cancer cells. The εCUP mimic condition had the most effective anti-cancer features; in contrast, which had no inhibitory effect on growth and migration of normal cells (HUVECs and Detroit551 cells). In addition, εCUP mimic condition inhibited the phosphorylation of focal adhesion kinase (FAK) and p38 signaling compared with separate treatment with naringenin or hesperetin. Of note, εCUP mimic condition showed a prominent anti-growth effect (p < 0.001) compared with control or ƒCUP mimic condition in vivo xenograft models.
CONCLUSION: These results suggest that combined treatment with naringenin and hesperetin might be a promising anti-cancer strategy for pancreatic cancers without eliciting toxicity on normal cells.

OBJECTIVE: Tumor necrosis factor-beta (TNF-β), as an inflammatory mediator that has been shown to promote tumorigenesis, induces NF-κB. Natural multi-targeted agent resveratrol in turn shows anti-inflammatory and anti-cancer properties. Epithelial-to-mesenchymal transition (EMT) allows cancer cells to turn into a motile state with invasive capacities and is associated with metastasis and development of cancer stem cells (CSC). However, TNF-β-induced EMT and the anti-invasion mechanism of resveratrol on CRC are not yet completely understood.
METHODS: We investigated the underlying molecular mechanisms of resveratrol on TNF-β/TNF-βR-induced EMT and migration of CRC cells (HCT116, RKO, SW480) in monolayer or 3D alginate cultures.
RESULTS: TNF-β, similar to TNF-α, induced significant cell proliferation, morphological change, from an epithelial to a spindle-like mesenchymal shape with the formation of filopodia and lamellipodia associated with the expression of EMT parameters (elevated vimentin and slug, reduced E-cadherin), increased migration/invasion, and formation of CSC in all CRC cells. Interestingly, these effects were dramatically decreased in the presence of resveratrol or anti-TNF-βR with TNF-β co-treatment, inducing biochemical changes to the mesenchymal-epithelial transition (MET), with a planar cell surface and suppressed formation of CSC cells. This was associated with a significant increase in apoptosis. Furthermore, we found that resveratrol suppressed TNF-β-induced NF-κB and NF-κB-regulated gene biomarkers associated with growth, proliferation, and invasion. Finally, TNF-βR interacts directly with focal adhesion kinase (FAK) and NF-κB.
CONCLUSION: These results suggest that resveratrol down-regulates TNF-β/TNF-βR-induced EMT, at least in part via specific suppression of NF-κΒ and FAK in CRC cells.

Background: Promoter methylation of tumor suppressor genes (TSGs) is a well-reported portent in carcinogenesis; hence, it is worthy to investigate this in high-risk Northeast population of India. The study was designed to investigate methylation status of 94 TSGs in esophageal squamous cell carcinoma (ESCC). Further, the effect of OPCML promoter methylation on gene expression was analyzed by immunohistochemistry. Moreover, in silico protein-protein interactions were examined among 8 TSGs identified in the present study and 23 epigenetically regulated genes reported previously by our group in ESCC.
Materials and Methods: Methylation profiling was carried out by polymerase chain reaction array and OPCML protein expression was examined by tissue microarray-based immunohistochemistry.
Results: OPCML, NEUROG1, TERT, and WT1 genes were found hypermethylated and SCGB3A1, CDH1, THBS1, and VEGFA were hypomethylated in Grade 2 tumor. No significant change in OPCML expression was observed among control, Grade 1, and Grade 2 tumor. Conclusively, hypermethylation of the studied OPCML promoter in Grade 2 tumor produced no effect on expression. Unexpectedly, OPCML expression was downregulated in Grade 3 tumor in comparison to other groups signifying that downregulation of OPCML expression may lead to higher grade of tumor formation at the time of diagnosis of ESCC in patients. Significant interactions at protein level were found as VEGFA:PTK2, CTNNB1:CDH1, CTNNB1:VEGFA, CTNNB1:NEUROG1, CTNND2:CDH1, and CTNNB1:TERT. These interactions are pertinent to Wnt/β-catenin and TGF-β-Smad pathways.
Conclusions: Deranged OPCML expression may lead to high-grade ESCC as well as epigenetically regulated genes, that is, CDH1, CTNNB1, CTNND2, THBS1, PTK2, WT1, OPCML, TGFB1, and SMAD4 may alter the Wnt/β-catenin and TGF-β-Smad pathways in ESCC. Further study of these genes could be useful to understand the molecular pathology of ESCC with respect to epithelial-mesenchymal transition (EMT) mediated by Wnt/β-catenin and TGF-β signaling pathways.

Glioblastoma (GBM) is the most aggressive type of brain tumor, with an overall survival of 17 months under the current standard of care therapy. CD99, an over-expressed transmembrane protein in several malignancies, has been considered a potential target for immunotherapy. To further understand this potentiality, we analyzed the differential expression of its two isoforms in human astrocytoma specimens, and the CD99 involved signaling pathways in glioma model U87MG cell line. CD99 was also analyzed in GBM molecular subtypes. Whole transcriptomes by RNA-Seq of

The aim of the present study was to investigate the effects of lysyl oxidase‑like 2 (LOXL2) on the invasion, migration and epithelial‑to‑mesenchymal transition (EMT) of renal cell carcinoma (RCC) cells through the steroid receptor coactivator (Src)/focal adhesion kinase (FAK) signaling pathway. RCC tissues and adjacent normal tissues were collected from 80 patients with RCC. Immunohistochemistry was used to determine the positive expression rate of the LOXL2 protein. The expression levels of LOXL2 in the HK‑2, 786‑O, ACHN, Caki1 and A498 cell lines were detected by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). The high LOXL2‑expressing 786‑O cells were selected for gene silencing experiments, whereas Caki1 cells, which exhibited low LOXL2 expression, were used for overexpression experiments. RT‑qPCR and western blot analysis were applied to determine the expression of LOXL2, FAK, Src, matrix metalloproteinase (MMP)‑9, epithelial (E)‑cadherin, neuronal (N)‑cadherin and vimentin. A MTT assay, a Transwell assay, a wound healing assay and flow cytometry were performed to detect cell proliferation, invasion, migration, cell cycle distribution and apoptosis, respectively. The protein expression rate of LOXL2 in RCC tissues was higher compared with that in adjacent normal tissues. Compared with adjacent normal tissues, the mRNA and protein expression levels of LOXL2, FAK, Src, MMP‑9, N‑cadherin and vimentin and the levels of FAK and Src phosphorylation were increased, while the mRNA and protein expression levels of E‑cadherin were decreased in RCC tissues. Following the transfection of 786‑O cells with small interfering (si) RNA against LOXL2, the mRNA and protein expression levels of FAK, Src, MMP‑9, N‑cadherin and vimentin and the levels of phosphorylated FAK and Src were notably decreased in the si‑LOXL2 and PP2 inhibitor treated groups, while that of E‑cadherin was substantially increased. Additionally, cell proliferation, invasion, migration and the percentage of RCC cells in the G1 phase were reduced, and cell apoptosis was increased. Additionally, Caki1 cells transfected with LOXL2 exhibited an opposite trend. In summary, these results indicate that LOXL2 silencing inhibits the invasion, migration and EMT in RCC cells through inhibition of the Src/FAK signaling pathway.

The globo-series glycosphingolipids (GSLs) SSEA3, SSEA4, and Globo-H specifically expressed on cancer cells are found to correlate with tumor progression and metastasis, but the functional roles of these GSLs and the key enzyme β1,3-galactosyltransferase V (β3GalT5) that converts Gb4 to SSEA3 remain largely unclear. Here we show that the expression of β3GalT5 significantly correlates with tumor progression and poor survival in patients, and the globo-series GSLs in breast cancer cells form a complex in membrane lipid raft with caveolin-1 (CAV1) and focal adhesion kinase (FAK) which then interact with AKT and receptor-interacting protein kinase (RIP), respectively. Knockdown of β3GalT5 disrupts the complex and induces apoptosis through dissociation of RIP from the complex to interact with the Fas death domain (FADD) and trigger the Fas-dependent pathway. This finding provides a link between SSEA3/SSEA4/Globo-H and the FAK/CAV1/AKT/RIP complex in tumor progression and apoptosis and suggests a direction for the treatment of breast cancer, as demonstrated by the combined use of antibodies against Globo-H and SSEA4.

All-trans-retinoic acid (ATRA) is a potent inducer of cellular differentiation, growth arrest, and apoptosis as well as a front-line therapy for acute promyelocytic leukemia (APL). The present study provides evidence that induction of autophagy is required for ATRA to induce differentiation of APL (NB4) cells into granulocytes. ATRA treatment causes ~12-fold increase in the number of acidic vesicular organelles and induces marked up-regulation of LC3-II, autophagy-related 5 (ATG5), and Beclin-1. Transmission electron microscopy (TEM) revealed a decrease in mitochondria and ATRA-induced differentiation. To determine the role of autophagy in the differentiation of APL, we knocked down ATG5 in NB4 cells to find that ATRA-induced differentiation is significantly inhibited during ATG5 knock down in cells, indicating the role of autophagy in differentiation of APL. Further experiments revealed restriction of autophagy during ATRA-induced differentiation and inhibition of tissue transglutaminase 2 (TG2) and phospho-focal adhesion kinase (p-FAK), which are known to have roles in differentiation and cell attachment. We examined expression of Beclin-1 and B-cell lymphoma-2 (Bcl-2) and levels of mechanistic target of rapamycin (mTOR) after ATRA treatment. ATRA inhibits Bcl-2, up-regulates Beclin-1 expression, and reduces induction of mTOR activation/phosphorylation in NB4 cells. Our results reveal that autophagy has roles in regulation of differentiation, mitochondria elimination, and cell attachment during ATRA-induced APL differentiation.

BACKGROUND: Targeting the c-Met signaling pathway has become a therapeutic strategy in multiple types of cancer. We unveiled a novel c-Met regulating mechanism that could be applied as a modality for oral squamous cell carcinoma (OSCC) therapy.
METHODS: Upregulation of keratin 16 (KRT16) was found by comparing isogenic pairs of low and high invasive human OSCC lines via microarray analysis. OSCC cells with ectopic expression or silencing of KRT16 were used to scrutinize functional roles and associated molecular mechanisms.
RESULTS: We observed that high KRT16 expression significantly correlated with poorer pathological differentiation, advanced stages, increased lymph nodes metastasis, and decreased survival rate from several Taiwanese OSCC patient cohorts. We further revealed that miR-365-3p could target ETS homologous factor (EHF), a KRT16 transcription factor, to decrease migration, invasion, metastasis and chemoresistance in OSCC cells via inhibition of KRT16. Under confocal microscopic examination, c-Met was found possibly partially associates with KRT16 through β5-integrin. Colocalization of these three proteins may facilitate c-Met and β5-integrin-mediated signaling in OSCC cells. Depletion of KRT16 led to increased protein degradation of β5-integrin and c-Met through a lysosomal pathway leading to inhibition of their downstream Src/STAT3/FAK/ERK signaling in OSCC cells. Knockdown of KRT16 enhanced chemosensitivity of OSCC towards 5-fluorouracil (5-FU). Various combination of c-Met inhibitor (foretinib), protein tyrosine kinase inhibitor (genistein), β5-integrin antibody, and 5-FU markedly augmented cytotoxic effects in OSCC cells as well as tumor killing effects in vitro and in vivo.
CONCLUSIONS: Our data indicate that targeting a novel miR-365-3p/EHF/KRT16/β5-integrin/c-Met signaling pathway could improve treatment efficacy in OSCC.

Almost 55% to 80% of patients with breast cancer have an unfavorable pathological complete response to chemotherapy. MicroRNAs are small noncoding RNAs involved in cancer progression; however, their utility as predictors of pathological complete response to neoadjuvant chemotherapy is unclear. Here, we investigated if miR-143 could discriminate between pathological complete response and no-polymerase chain reaction of patients with locally advanced triple negative breast cancer that have received a fluorouracil-cisplatin/paclitaxel-based neoadjuvant treatment. Data showed that miR-143 exhibited a significant low expression ( P < .0006) in patients that achieved pathological complete response in comparison to nonresponder group. Receiver operating characteristic curve analysis suggested that miR-143 could be a good predictor of pathological complete response (area under curve = 0.849, P < .0006). Moreover, Kaplan-Meier analysis indicated that before neoadjuvant therapy low levels of miR-143 were associated to increased disease free survival. To gain insights into cellular functions of miR-143, we firstly showed that miR-143 was severely repressed in breast cancer cell lines and tumors in comparison to normal mammary cells and tissues. Ectopic restoration of miR-143 using RNA mimics inhibited both cell proliferation and migration and sensitized breast cancer cells to cisplatin therapy in vitro. To decipher the signaling networks regulated by miR-143, we used a high-throughput enzyme-linked immunosorbent assay-based phosphorylation antibody array. Phospho-proteomic profiling revealed that miR-143 coordinately reduced the protein levels and phosphorylation status of multiple oncoproteins involved in AKT, WNT/β-catenin, SAPK/JNK, FAK, and JAK/STAT signaling pathways. Moreover, low miR-143 and high GSK3-β, RAF1, paxillin, and p21CIP1 expression levels in a large cohort of patients with breast cancer were associated with worst outcome. In summary, miR-143 could be a potential predictor of response to neoadjuvant therapy and it may function as a divergent regulator of diverse signaling networks to suppress cell proliferation and migration in breast cancer.

SGA is a sulfated glucan from Antrodia cinnamomea. In this study, we showed that SGA suppressed tumor growth in vitro and in vivo. SGA also potentiated cisplatin-induced cytotoxicity in lung cancer cells. TGFβ signaling and overexpression of Slug are regarded as the critical events in lung tumor malignancy. Functional studies revealed that SGA inhibited the TGFβ/FAK/AKT axis by inducing lipid-raft-mediated lysosome-dependent TGFβ receptor degradation, resulting in suppressing cancer cell viability and migration. Moreover, SGA elimination of TGFβ-mediated intracellular signaling promoted Slug degradation in H1975 cells. Mechanistically, we demonstrated that proteasome-dependent Slug degradation was controlled by TGFβ-mediated downstream signaling pathways; however, inhibitors of AKT and GSK3 abolished Slug degradation. Our findings suggested that SGA targets of the TGFβ/AKT/GSK3β axis played a key role in enhancing Slug degradation and suppressing lung cancer cells. In addition, SGA may be a potential therapeutic supplement for lung cancer.

BACKGROUND: Focal adhesion kinase (FAK) is a cytoplasmatic protein tyrosine kinase that associates with both integrins and growth factor receptors toward the adhesion, migration and invasion of cancer cells. The G-protein coupled estrogen receptor (GPER) has been involved in the stimulatory action of estrogens in breast tumor. In this study, we have investigated the engagement of FAK by GPER signaling in triple negative breast cancer (TNBC) cells.
METHODS: Publicly available large-scale database and patient data sets derived from "The Cancer Genome Atlas" (TCGA; www.cbioportal.org ) were used to assess FAK expression in TNBC, non-TNBC tumors and normal breast tissues. MDA-MB 231 and SUM159 TNBC cells were used as model system. The levels of phosphorylated FAK, other transduction mediators and target genes were detected by western blotting analysis. Focal adhesion assay was carried out in order to determine the focal adhesion points and the formation of focal adhesions (FAs). Luciferase assays were performed to evaluate the promoters activity of c-FOS, EGR1 and CTGF upon GPER activation. The mRNA expression of the aforementioned genes was measured by real time-PCR. Boyden chamber and wound healing assays were used in order to evaluate cell migration. The statistical analysis was performed by ANOVA.
RESULTS: We first determined by bioinformatic analysis that the mRNA expression levels of the gene encoding FAK, namely PTK2, is higher in TNBC respect to non-TNBC and normal breast tissues. Next, we found that estrogenic GPER signaling triggers Y397 FAK phosphorylation as well as the increase of focal adhesion points (FAs) in TNBC cells. Besides, we ascertained that GPER and FAK activation are involved in the STAT3 nuclear accumulation and gene expression changes. As biological counterpart, we show that FAK inhibition prevents the migration of TNBC cells upon GPER activation.
CONCLUSIONS: The present data provide novel insights regarding the action of FAK in TNBC. Moreover, on the basis of our findings estrogenic GPER signaling may be considered among the transduction mechanisms engaging FAK toward breast cancer progression.

BACKGROUND: Lysyl oxidase-like 4 (LOXL4) has been found to be dysregulated in several human malignancies, including hepatocellular carcinoma (HCC). However, the role of LOXL4 in HCC progression remains largely unclear. In this study, we investigated the clinical significance and biological involvement of LOXL4 in the progression of HCC.
METHODS: LOXL4 expression was measured in HCC tissues and cell lines. Overexpression, shRNA-mediated knockdown, recombinant human LOXL4 (rhLOXL4), and deletion mutants were applied to study the function of LOXL4 in HCC. Exosomes derived from HCC cell lines were assessed for the ability to promote cancer progression in standard assays. The effects of LOXL4 on the FAK/Src pathway were examined by western blotting.
RESULTS: LOXL4 was commonly upregulated in HCC tissues and predicted a poor prognosis. Elevated LOXL4 was associated with tumor differentiation, vascular invasion, and tumor-node-metastasis (TNM) stage. Overexpression of LOXL4 promoted, whereas knockdown of LOXL4 inhibited cell migration and invasion of HCC in vitro, and overexpressed LOXL4 promoted intrahepatic and pulmonary metastases of HCC in vivo. Most interestingly, we found that HCC-derived exosomes transferred LOXL4 between HCC cells, and intracellular but not extracellular LOXL4 promoted cell migration by activating the FAK/Src pathway dependent on its amine oxidase activity through a hydrogen peroxide-mediated mechanism. In addition, HCC-derived exosomes transferred LOXL4 to human umbilical vein endothelial cells (HUVECs) though a paracrine mechanism to promote angiogenesis.
CONCLUSIONS: Taken together, our data demonstrate a novel function of LOXL4 in tumor metastasis mediated by exosomes through regulation of the FAK/Src pathway and angiogenesis in HCC.

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BACKGROUND: A growing body of studies have demonstrated the aberrant expression of microRNAs (miRNAs) contributes to human tumor metastasis. MicroRNA-124-3p (miR-124-3p), which is down-regulated in various cancers, has been found to be involved in several signaling pathways relevant to tumor cell migration and invasion. However, the roles of miR-124-3p in human bladder cancer remain unclear. This study aims to investigate the functional significance of miR-124-3p and to understand how it targets the integrin receptor, and thus affects the progression of human bladder cancer.
METHODS: Clinical specimens from 36 patients and three human bladder cancer cell lines were analyzed for miR-124-3p and integrin α3 (ITGA3) . To investigate the effects of miR-124-3p and ITGA3 on proliferation of bladder cancer cells, the MTT assay, colon-formation assay and flow cytometry were performed. In addition, wound healing assay and transwell assay were carried out to examine the migration and invasion of the bladder cancer cells transfected with miR-124-3p mimics or si-ITGA3. The luciferase reporter assay, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were applied to validate the miR-124-3p directly binding with ITGA3. Finally, western blot was used to examine the expression level of the proteins involved in FAK/PI3K/AKT and FAK/Src signal pathway as well as epithelial-mesenchymal transition (EMT) process.
RESULTS: The down-regulation of miR-124-3p and up-regulation of ITGA3 were observed in clinical specimens and bladder cancer cell lines. Overexpression of miR-124-3p or silencing ITGA3 inhibited tumor cell migration and invasion. Luciferase assay confirmed miR-124-3p directly targets ITGA3, and western blot suggested that miR-124-3p plays a crucial role in the EMT and metastasis of human bladder cancer through FAK/PI3K/AKT and FAK/Src signaling mechanism. Also, by targeting ITGA3, miR-124-3p can modulate the expression of N- and E-cadherin, and thus inhibit the EMT.
CONCLUSIONS: By targeting ITGA3 and downstream FAK/PI3K/AKT and FAK/Src signaling pathways, miR-124-3p suppresses cell migration and invasion in bladder cancer. Our study reasonably speculates that miR-124-3p can be potentially developed as a therapeutic target and prognostic biomarker for bladder cancer.

Thyroid cancer is the most abundant tumor of the endocrine organs. Poorly differentiated thyroid cancer is still difficult to treat. Human cells exposed to long-term real (r-) and simulated (s-) microgravity (µ

PURPOSE: The interaction between malignant cells and surrounding healthy tissues is a critical factor in the metastatic progression of breast cancer (BC). Extracellular vesicles, especially exosomes, are known to be involved in inter-cellular communication during cancer progression. In the study presented herein, we aimed to evaluate the role of circulating plasma exosomes in the metastatic dissemination of BC and to investigate the underlying molecular mechanisms of this phenomenon.
METHODS: Exosomes isolated from plasma of healthy female donors were applied in various concentrations into the medium of MDA-MB-231 and MCF-7 cell lines. Motility and invasive properties of BC cells were examined by random migration and Transwell invasion assays, and the effect of plasma exosomes on the metastatic dissemination of BC cells was demonstrated in an in vivo zebrafish model. To reveal the molecular mechanism of interaction between plasma exosomes and BC cells, a comparison between un-treated and enzymatically modified exosomes was performed, followed by mass spectrometry, gene ontology, and pathway analysis.
RESULTS: Plasma exosomes stimulated the adhesive properties, two-dimensional random migration, and transwell invasion of BC cells in vitro as well as their in vivo metastatic dissemination in a dose-dependent manner. This stimulatory effect was mediated by interactions of surface exosome proteins with BC cells and consequent activation of focal adhesion kinase (FAK) signaling in the tumor cells.
CONCLUSIONS: Plasma exosomes have a potency to stimulate the metastasis-promoting properties of BC cells. This pro-metastatic property of normal plasma exosomes may have impact on the course of the disease and on its prognosis.

Osteosarcoma is a highly malignant bone tumor. However, due to the high complexity of the occurrence and metastasis of osteosarcoma, the exact mechanism promoting its development and progression remains to be elucidated. This study highlights the causal link between solute carrier family 25 member 22 (SLC25A22) and the development, progression, and metastasis of osteosarcoma. SLC25A22 is upregulated in human osteosarcoma and predicts a poor prognosis. The upregulation of SLC25A22 in osteosarcoma tissues was significantly associated with cell proliferation, invasion, and metastasis. Studies of functional gain (overexpression) and loss (knockdown) showed that SLC25A22 significantly increases the ability of osteosarcoma cells to proliferate, as well as invade and metastasize in vitro. At the same time, the expression of SLC25A22 promoted the progression of the cellcycle of osteosarcoma cell lines and inhibited the apoptosis of osteosarcoma cells. Analysis using a mouse xenograft model showed that xenografts of SLC25A22 stable overexpressing osteosarcoma cells had a significant increase in tumor volume and weight compared to the control group. Lung metastasis models in mice showed that expression of SLC25A22 promoted lung metastasis of osteosarcoma in vivo. Furthermore, SLC25A22 inhibited phosphatase and tensin homolog expression and increased phosphorylation of protein kinase b (Akt) and Focal Adhesion Kinase (FAK) in the phosphatase and tensin homolog signaling pathway. In summary, SLC25A22 is highly expressed in osteosarcoma, promoting osteosarcoma cell proliferation and invasion by inhibiting the phosphatase and tensin homolog signaling pathway.

BACKGROUND: Increasing evidence has revealed that circular RNAs (circRNAs) play crucial roles in cancer biology. However, the role and underlying regulatory mechanisms of circFNDC3B in bladder cancer (BC) remain unknown.
METHODS: A cell invasion model was established by repeated transwell assays, and invasion-related circRNAs in BC were identified through an invasion model. The expression of circFNDC3B was detected in 82 BC tissues and cell lines by quantitative real-time PCR. Functional assays were performed to evaluate the effects of circFNDC3B on proliferation, migration and invasion in vitro-, and on tumorigenesis and metastasis in vivo. The relationship between circFNDC3B and miR-1178-3p was confirmed by fluorescence in situ hybridization, pull-down assay and luciferase reporter assay.
RESULTS: In the present study, we identified a novel circRNA (circFNDC3B) through our established BC cell invasion model. We found that circFNDC3B was dramatically downregulated in BC tissues and correlated with pathological T stage, grade, lymphatic invasion and patients' overall survival rate. Functionally, overexpression of circFNDC3B significantly inhibited proliferation, migration and invasion both in vitro and in vivo. Mechanistically, circFNDC3B could directly bind to miR-1178-3p, which targeted the 5'UTR of the oncogene G3BP2. Moreover, circFNDC3B acted as a miR-1178-3p sponge to suppress G3BP2, thereby inhibiting the downstream SRC/FAK signaling pathway.
CONCLUSIONS: CircFNDC3B may serve as a novel tumor suppressive factor and potential target for new therapies in human BC.

Downstream of tyrosine kinase 7 transcript variant 1 (DOK7V1) is a docking protein mediating signal transduction between receptors and intracellular downstream molecules. Our previous study indicated that DOK7V1 was decreased in lung cancer and its lower expression was associated with a decreased survival rate. The 5‑year overall survival rate for patients with lung cancer was 20.2 and 18.6% for high and low DOK7 expression, respectively; the 5‑year disease‑free survival rate for patients with lung cancer was 14.3 and 16.9% for high and low DOK7 expression, respectively. DOK7V1 inhibited proliferation and migration, but enhanced adhesion, of lung cancer cells. In the present study, the effect of DOK7V1 and its domains [pleckstrin homology (PH) and phosphotyrosine‑binding (PTB) domain] on the malignant phenotype and associated signaling pathway in lung cancer cells was investigated. The results indicated that truncation of DOK7V1 domains (DOK7V1Δ‑PH and DOK7V1Δ‑PTB) inhibited the proliferation and migration of lung cancer cells which exhibited the same trend as DOK7V1, whereas DOK7V1Δ‑PH and DOK7V1Δ‑PTB exhibited different functions from those of DOK7V1 in cell matrix adhesion. Consistently, DOK7V1 overexpression in lung cancer cells suppressed the phosphoinositide 3‑kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathways, but activated the focal adhesion kinase (FAK)/paxillin signaling pathway. Taken together, these results indicate that DOK7V1 may inhibit proliferation and migration via negatively regulating the PI3K/AKT/mTOR signaling pathway, and increase adhesion by upregulating the FAK/paxillin signaling pathway in lung cancer cells.

Vasculogenic mimicry (VM) refers to the fluid-conducting channels formed by aggressive tumor cells rather than endothelial cells (EC) with elevated expression of genes associated with vascularization. VM has been considered as one of the reasons that glioblastoma becomes resistant to anti-VEGF therapy. However, the molecular basis underlying VM formation remains unclear. Here we report that the insulin-like growth factor-binding protein 2 (IGFBP2) acts as a potent factor to enhance VM formation in glioma. Evidence showed that elevated IGFBP2 expression was positively related with VM formation in patients with glioma. Enforced expression of IGFBP2 increased network formation of glioma cells in vitro by activating CD144 and MMP2 (Matrix Metalloproteinase 2). U251 cells with stable knockdown of IGFBP2 led to decreased VM formation and tumor progression in orthotopic mouse model. Mechanistically, IGFBP2 interacts with integrin α5 and β1 subunits and augments CD144 expression in a FAK/ERK pathway-dependent manner. Luciferase reporter and ChIP assay suggested that IGFBP2 activated the transcription factor SP1, which could bind to CD144 promoter. Thus, IGFBP2 acts as a stimulator of VM formation in glioma cells via enhancing CD144 and MMP2 expression.

Glioblastoma (GBM), the most aggressive brain tumor, has a poor prognosis due to the ease of migration to surrounding healthy brain tissue. Recent studies have shown that bradykinin receptors are involved in the progression of various cancers. However, the molecular mechanism and pathological role of bradykinin receptors remains unclear. We observed the expressions of two major bradykinin receptors, B1R and B2R, in two different human GBM cell lines, U87 and GBM8901. Cytokine array analysis showed that bradykinin increases the production of interleukin (IL)-8 in GBM via B1R. Higher B1R levels correlate with IL-8 expression in U87 and GBM8901. We observed increased levels of phosphorylated STAT3 and SP-1 in the nucleus as well. Using chromatin immunoprecipitation assay, we found that STAT3 and SP-1 mediate IL-8 expression, which gets abrogated by the inhibition of FAK and STAT3. We further demonstrated that IL-8 expression and cell migration are also regulated by the SP-1. In addition, expression levels of STAT3 and SP-1 positively correlate with clinicopathological grades of gliomas. Interestingly, our results found that inhibition of HDAC increases IL-8 expression. Moreover, stimulation with bradykinin caused increases in acetylated SP-1 and p300 complex formation, which are abrogated by inhibition of FAK and STAT3. Meanwhile, knockdown of SP-1 and p300 decreased the augmentation of bradykinin-induced IL-8 expression. These results indicate that bradykinin-induced IL-8 expression is dependent on B1R which causes phosphorylated STAT3 and acetylated SP-1 to translocate to the nucleus, hence resulting in GBM migration.

BACKGROUND: Adhesion-mediated activation of FAK/ERK signalling pathway, enabled by the formation of filopodial protrusions (FLP), has been shown to be an important event for triggering of dormancy-to-proliferation switch and metastatic outgrowth of breast cancer cells (BCC). We studied the role of actin-binding protein profilin1 (Pfn1) in these processes.
METHODS: Quantitative immunohistochemistry (IHC) of BC tissue microarray (TMA) and survival analyses of curated transcriptome datasets of BC patients were performed to examine Pfn1's association with certain clinicopathological features. FLP formation and single cell outgrowth of BCC were assessed using a 3D matrigel culture that accurately predicts dormant vs metastatic outgrowth phenotypes of BCC in certain microenvironment. Gene expression studies were performed to identify potential biological pathways that are perturbed under Pfn1-depleted condition.
RESULTS: Lower Pfn1 expression is correlated with lower nuclear grade of breast tumours and longer relapse-free survival of BC patients. Pfn1 depletion leads to defects in FLP and outgrowth of BCC but without impairing either FAK or ERK activation. Guided by transcriptome analyses, we further showed that Pfn1 depletion is associated with prominent SMAD3 upregulation. Although knockdown and overexpression experiments revealed that SMAD3 has an inhibitory effect on the outgrowth of breast cancer cells, SMAD3 knockdown alone was not sufficient to enhance the outgrowth potential of Pfn1-depleted BCC suggesting that other proliferation-regulatory pathways in conjunction with SMAD3 upregulation may underlie the outgrowth-deficient phenotype of BCC cells upon depletion of Pfn1.
CONCLUSION: Overall, these data suggest that Pfn1 may be a novel biomarker for BC recurrence and a possible target to reduce metastatic outgrowth of BCC.

The p53-inducible gene 3 (PIG3) is one of the p53-induced genes at the onset of apoptosis, which plays an important role in cell apoptosis and DNA damage response. Our previous study reported an oncogenic role of PIG3 associated with tumor progression and metastasis in non-small cell lung cancer (NSCLC). In this study, we further analyzed PIG3 mRNA expression in 504 lung adenocarcinoma (LUAD) and 501 lung squamous cell carcinoma (LUSC) tissues from The Cancer Genome Atlas database and we found that PIG3 expression was significantly higher in LUAD with lymph node metastasis than those without, while no difference was observed between samples with and without lymph node metastasis in LUSC. Gain and loss of function experiments were performed to confirm the metastatic role of PIG3 in vitro and to explore the mechanism involved in its oncogenic role in NSCLC metastasis. The results showed that PIG3 knockdown significantly inhibited the migration and invasion ability of NSCLC cells, and decreased paxillin, phospho-focal adhesion kinase (FAK) and phospho-Src kinase expression, while its overexpression resulted in the opposite effects. Blocking FAK with its inhibitor reverses PIG3 overexpression-induced cell motility in NSCLC cells, indicating that PIG3 increased cell metastasis through the FAK/Src/paxillin pathway. Furthermore, PIG3 silencing sensitized NSCLC cells to FAK inhibitor. In conclusion, our data revealed a role for PIG3 in inducing LUAD metastasis, and its role as a new FAK regulator, suggesting that it could be considered as a novel prognostic biomarker or therapeutic target in the treatment of LUAD metastasis.

The present study aimed to examine the role of microRNA‑433 in the growth and death of cervical cancer cells. RNA isolation, reverse transcription‑quantitative polymerase chain reaction analysis, an MTT assay, flow cytometry, and western blot analysis were used for this investigation. The results showed that the expression of microRNA‑433 was downregulated in patients with cervical cancer. The disease‑free survival and overall survival rates of patients with low expression levels of microRNA‑433 were lower, compared with those in patients with high expression levels of microRNA‑433. The expression levels of microRNA‑433 were downregulated in cervical cancer in vitro. It was found that the downregulation of microRNA‑433 promoted the growth and inhibited the apoptosis of cervical cancer cells through activating focal adhesion kinase (FAK)/phosphoinositide 3‑kinase (PI3K)/AKT signaling. However, the upregulation of microRNA‑433 induced apoptosis and suppressed the growth of cervical cancer cells through inhibiting the FAK/PI3K/AKT signaling pathway. In addition, FAK or PI3K inhibitors promoted the death of cervical cancer cells following the downregulation of microRNA‑433. These results revealed that microRNA‑433 suppressed the growth of cervical cancer cells via the FAK/PI3K/AKT signaling pathway.

Colon cancer is one of the most lethal and common malignancies worldwide. STK899704, a novel synthetic agent, has been reported to exhibit anticancer effects towards numerous cancer cells. However, the effect of STK899704 on the biological properties of colon cancer, including cancer cell migration and cancer stem cells (CSCs), remains unknown. Here, we examined the inhibitory effect of STK899704 on cell migration and CSC stemness. In the wound healing assay, STK899704 significantly inhibited the motility of colon cancer cells. Furthermore, STK899704 downregulated the mRNA expression levels of the cell migration mediator focal adhesion kinase (FAK). STK899704 also suppressed mitogen-activated protein kinase kinase and extracellular signal-regulated kinase, which are downstream signaling molecules of FAK. Additionally, STK899704 inhibited stemness gene expression and sphere formation in colon cancer stem cells. These results suggest that STK899704 can be used to treat human colon cancer. [BMB Reports 2018; 51(11): 596-601].

BACKGROUND: TGF-β promotes tumor invasion and metastasis through inducing epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC). Circular RNAs (circRNAs) are recognized as functional non-coding RNAs involved in human cancers. However, whether and how circRNAs contribute to TGF-β-induced EMT and metastasis in NSCLC remain vague. Here, we investigated the regulation and function of Circular RNA hsa_circ_0008305 (circPTK2) in TGF-β-induced EMT and tumor metastasis, as well as a link between circPTK2 and transcriptional intermediary factor 1 γ (TIF1γ) in NSCLC.
METHODS: Circular RNAs were determined by human circRNA Array analysis, real-time quantitative reverse transcriptase PCR and northern blot. Luciferase reporter, RNA-binding protein immunoprecipitation (RIP), RNA pull-down and fluorescence in situ hybridization (FISH) assays were employed to test the interaction between circPTK2 and miR-429/miR-200b-3p. Ectopic overexpression and siRNA-mediated knockdown of circPTK2, TGF-β-induced EMT, Transwell migration and invasion in vitro, and in vivo experiment of metastasis were used to evaluate the function of circPTK2. Transcription and prognosis analyses were done in public databases.
RESULTS: CircPTK2 and TIF1γ were significantly down-regulated in NSCLC cells undergoing EMT induced by TGF-β. CircPTK2 overexpression augmented TIF1γ expression, inhibited TGF-β-induced EMT and NSCLC cell invasion, whereas circPTK2 knockdown had the opposite effects. CircPTK2 functions as a sponge of miR-429/miR-200b-3p, and miR-429/miR-200b-3p promote TGF-β-induced EMT and NSCLC cell invasion by targeting TIF1γ. CircPTK2 overexpression inhibited the invasion-promoting phenotype of endogenous miR-429/miR-200b-3p in NSCLC cells in response to TGF-β. CircPTK2 overexpression significantly decreased the expression of Snail, an important downstream transcriptional activator of TGF-β/Smad signaling. In an in vivo experiment of metastasis, circPTK2 overexpression suppressed NSCLC cell metastasis. Moreover, circPTK2 expression was dramatically down-regulated and positively correlated with TIF1γ expression in human NSCLC tissues. Especially, circPTK2 was significantly lower in metastatic NSCLC tissues than non-metastatic counterparts.
CONCLUSION: Our findings show that circPTK2 (hsa_circ_0008305) inhibits TGF-β-induced EMT and metastasis by controlling TIF1γ in NSCLC, revealing a novel mechanism by which circRNA regulates TGF-β-induced EMT and tumor metastasis, and suggesting that circPTK2 overexpression could provide a therapeutic strategy for advanced NSCLC.

Phenotypic plasticity and subsequent generation of intratumoral heterogeneity underly key traits in malignant melanoma such as drug resistance and metastasis. Melanoma plasticity promotes a switch between proliferative and invasive phenotypes characterized by different transcriptional programs of which MITF is a critical regulator. Here, we show that the acid ceramidase ASAH1, which controls sphingolipid metabolism, acted as a rheostat of the phenotypic switch in melanoma cells. Low ASAH1 expression was associated with an invasive behavior mediated by activation of the integrin alphavbeta5-FAK signaling cascade. In line with that, human melanoma biopsies revealed heterogeneous staining of ASAH1 and low ASAH1 expression at the melanoma invasive front. We also identified ASAH1 as a new target of MITF, thereby involving MITF in the regulation of sphingolipid metabolism. Together, our findings provide new cues to the mechanisms underlying the phenotypic plasticity of melanoma cells and identify new anti-metastatic targets.

Pancreatic cancer is a leading cause of mortality and morbidity worldwide. Due to drug resistance, and the high toxicity and adverse side effects of existing chemotherapeutic drugs, the current treatment of highly aggressive pancreatic cancer is considered inadequate. Allergen‑removed Rhus verniciflua Stokes (aRVS) has a strong antiproliferative effect in various cancer cells, and due to its low toxicity, it has emerged as an attractive candidate for cancer treatment. However, the potential use of aRVS as a treatment for pancreatic cancer is relatively unexplored. The present study examined the effects of aRVS on the invasion and migration of pancreatic cancer cells, and identified the molecular mechanisms underlying its anticancer effects. aRVS inhibited the Janus kinase/signal transducer and activator of transcription pathway in pancreatic cancer cells, and decreased the protein expression of mucin 4. In addition, it inhibited the activation of focal adhesion kinase and Src signaling, and decreased the expression of matrix metalloproteinase 9, which may reduce the migration and invasion of pancreatic cancer cells. In conclusion, the present study suggested that aRVS may be a potential treatment for aggressive pancreatic cancer.

OBJECTIVES: This study aimed to investigate potential gene and signal pathway associated with tumour progression.
METHODS: Related microarray data set of breast cancer was obtained from Gene Expression Omnibus database, and differential-expressed genes (DEGs) between two control samples and two treated samples were analysed using statistical software R. We collected 50 epigallocatechin-3-gallate(EGCG)-related genes and 119 breast cancer-related genes to create a knowledge base for following pathway analysis.
KEY FINDINGS: A total of 502 mRNAs were identified as DEGs based on microarray analysis. Upregulated DEGs mainly enriched in nuclear nucleosome, cell adhesion, DNA packaging complex, Wnt-activated receptor activity, etc., while the downregulated DEGs significantly enriched in ncRNA processing, mitotic nuclear division, DNA helicase activity, etc. DEGs mostly enriched in gap junction, cell cycle, oxidative phosphorylation, focal adhesion, etc. EGCG suppressed FAK signalling pathway. Furthermore, EGCG could inhibit breast cancer cell proliferation and promote apoptosis by modulating CCND1.
CONCLUSIONS: Epigallocatechin 3-gallate might exert influence on breast cancer progression through inhibiting focal adhesion kinase (FAK) signalling pathway.