siRNA‐induced CD44 knockdown suppresses the proliferation and invasion of colorectal cancer stem cells through inhibiting epithelial–mesenchymal transition

CD44 has shown prognostic values and promising therapeutic potential in multiple human cancers; however, the effects of CD44 silencing on biological behaviors of cancer stem cells (CSCs) have not been fully understood in colorectal cancer. To examine the contribution of siRNA‐induced knockdown of CD44 to the biological features of colorectal CSCs, colorectal CSCs HCT116‐CSCs were generated, and CD44 was knocked down in HCT116‐CSCs using siRNA. The proliferation, migration and invasion of HCT116‐CSCs were measured, and apoptosis and cell‐cycle analyses were performed. The sensitivity of HCT116‐CSCs to oxaliplatin was tested, and xenograft tumor growth assay was performed to examine the role of CD44 in HCT116‐CSCs tumorigenesis in vivo. In addition, the expression of epithelial–mesenchymal transition (EMT) markers E‐cadherin, N‐cadherin and vimentin was quantified. siRNA‐induced knockdown of CD44 was found to inhibit the proliferation, migration and invasion, induce apoptosis, promote cell‐cycle arrest at the G1/G0 phase and increase the sensitivity of HCT116‐CSCs to oxaliplatin in HCT116‐CSCs, and knockdown of CD44 suppressed in vivo tumorigenesis and intrapulmonary metastasis of HCT116‐CSCs. Moreover, silencing CD44 resulted in EMT inhibition. Our findings demonstrate that siRNA‐induced CD44 knockdown suppresses the proliferation, invasion and in vivo tumorigenesis and metastasis of colorectal CSCs by inhibiting EMT.     

ASIC1a stimulates the resistance of human hepatocellular carcinoma by promoting EMT via the AKT/GSK3β/Snail pathway driven by TGFβ/Smad signals

Multidrug resistance is the main obstacle to curing hepatocellular carcinoma (HCC). Acid‐sensing ion channel 1a (ASIC1a) has critical roles in all stages of cancer progression, especially invasion and metastasis, and in resistance to therapy. Epithelial to mesenchymal transition (EMT) transforms epithelial cells into mesenchymal cells after being stimulated by extracellular factors and is closely related to tumour infiltration and resistance. We used Western blotting, immunofluorescence, qRT‐PCR, immunohistochemical staining, MTT, colony formation and scratch healing assay to determine ASIC1a levels and its relationship to cell proliferation, migration and invasion. ASIC1a is overexpressed in HCC tissues, and the amount increased in resistant HCC cells. EMT occurred more frequently in drug‐resistant cells than in parental cells. Inactivation of ASIC1a inhibited cell migration and invasion and increased the chemosensitivity of cells through EMT. Overexpression of ASIC1a upregulated EMT and increased the cells’ proliferation, migration and invasion and induced drug resistance; knocking down ASIC1a with shRNA had the opposite effects. ASIC1a increased cell migration and invasion through EMT by regulating α and β‐catenin, vimentin and fibronectin expression via the AKT/GSK‐3β/Snail pathway driven by TGFβ/Smad signals. ASIC1a mediates drug resistance of HCC through EMT via the AKT/GSK‐3β/Snail pathway.     

Neuron specific enolase promotes tumor metastasis by activating the Wnt/β-catenin pathway in small cell lung cancer

Neuron-specific enolase (NSE) has been used as a specific biomarker for small cell lung cancer (SCLC) patients. Nevertheless, the biological function and mechanism of NSE in SCLC are still unclear. In this study, we clarified the role of NSE in the progression of SCLC and found that NSE expression was positively correlated with distant metastasis. Functional analysis showed that overexpression of NSE promoted migration and invasion of SCLC cells. Mechanism analysis showed that NSE overexpression induced epithelial-mesenchymal transition (EMT) of SCLC cells. Moreover, overexpression of NSE increased the protein expression of β-catenin and its downstream target genes, and silencing β-catenin eliminated NSE-mediated cell migration, invasion and EMT process. Furthermore, NSE interacted with β-catenin and inhibited the degradation of β-catenin. Besides, the animal experiments also indicated that NSE could promote the EMT process and distant metastasis of SCLC cells in vivo. In summary, our results revealed that NSE could promote the EMT process of SCLC cells by activating the Wnt/β-catenin signaling pathway, thereby promoting cell migration, invasion and distant metastasis, which might serve as a potential target for the therapy of SCLC patients.     

TUSC3 inhibits cell proliferation and invasion in cervical squamous cell carcinoma via suppression of the AKT signalling pathway

The decreased expression of tumour suppressor candidate 3 (TUSC3) is associated with proliferation in several types of cancer, leading to an unfavourable prognosis. The present study aimed to assess the cellular and molecular function of TUSC3 in patients with cervical squamous cell carcinoma (CSCC). Levels of mRNA expressions of TUSC3 were analysed in CSCC tissues and six cell lines using qRT‐PCR. Immunohistochemistry(IHC) was used to evaluate the protein expression level of TUSC3 in four paired specimens, 220 paraffin‐embedded CSCC specimens and 60 cases of normal cervical tissues(NCTs), respectively. Short hairpin RNA interference was employed for TUSC3 knockdown. Cell proliferation, migration and invasion were evaluated using growth curve, MTT assay, wound healing, transwell assay and xenograft tumour model, respectively. The results demonstrated that TUSC3 mRNA and protein expression levels were downregulated in CSCC samples. Multivariate and univariate analyses indicated that TUSC3 was an independent prognostic factor for patients with CSCC. Decreased TUSC3 expression levels were significantly associated with proliferation and an aggressive phenotype of cervical cancer cells both in vitro and in vivo. Moreover, the knockdown of TUSC3 promoted migration and invasion of cancer cells, while the increased expression of TUSC3 exhibited the opposite effects. The downregulation of TUSC3 facilitated proliferation and invasion of CSCC cells through the activation of the AKT signalling pathway. Our data demonstrated that the downregulation of TUSC3 promoted CSCC cell metastasis via the AKT signalling pathway. Therefore, TUSC3 may serve as a novel prognostic marker and potential target for CSCC.     

FGF9/FGFR1 promotes cell proliferation,epithelial-mesenchymal transition,M2 macrophage infiltration and liver metastasis of lung cancer

Fibroblast growth factors 9 (FGF9) modulates cell proliferation, differentiation and motility for development and repair in normal cells. Abnormal activation of FGF9 signaling is associated with tumor progression in many cancers. Also, FGF9 may be an unfavorable prognostic indicator for non-small cell lung cancer patients. However, the effects and mechanisms of FGF9 in lung cancer remain elusive. In this study, we investigated the FGF9-induced effects and signal activation profiles in mouse Lewis lung carcinoma (LLC) in vitro and in vivo. Our results demonstrated that FGF9 significantly induced cell proliferation and epithelial-to-mesenchymal transition (EMT) phenomena (migration and invasion) in LLC cells. Mechanism-wise, FGF9 interacted with FGFR1 and activated FAK, AKT, and ERK/MAPK signal pathways, induced the expression of EMT key proteins (N-cadherin, vimentin, snail, MMP2, MMP3 and MMP13), and reduced the expression of E-cadherin. Moreover, in the allograft mouse model, intratumor injection of FGF9 to LLC-tumor bearing C57BL/6 mice enhanced LLC tumor growth which were the results of increased Ki67 expression and decreased cleaved caspase-3 expression compared to control groups. Furthermore, we have a novel finding that FGF9 promoted liver metastasis of subcutaneous inoculated LLC tumor with angiogenesis, EMT and M2-macrophage infiltration in the tumor microenvironment. In conclusion, FGF9 activated FAK, AKT, and ERK signaling through FGFR1 with induction of EMT to stimulate LLC tumorigenesis and hepatic metastasis. This novel FGF9/LLC allograft animal model may therefore be useful to study the mechanism of liver metastasis which is the worst prognostic factor for lung cancer patients with distant organ metastasis.     

LncRNA EPB41L4A-AS2 represses Nasopharyngeal Carcinoma Metastasis by binding to YBX1 in the Nucleus and Sponging MiR-107 in the Cytoplasm

Nasopharyngeal carcinoma (NPC) is known for its potential to progress to the lymph nodes and distant metastases at an early stage. As an important regulator in tumorigenesis biological processes, the functions of lncRNA in NPC tumor development remain largely unclear. In this research, the expression of EPB41L4A-AS2 in NPC tissues and cells was analyzed via real-time quantitative polymerase chain reaction (qRT-PCR). CCK8, colony formation, and EDU experiments were used to determine the viability of NPC cells. Transwell and wound healing assays were performed to test NPC cell migration and invasion. RNA pull-down and mass spectrometry analysis were used to identify potential binding proteins. Then, a popliteal lymph node metastasis model was established to test NPC metastasis. EPB41L4A-AS2 is repressed by transforming growth factor-beta, which is downregulated in NPC cells and tissue. It is associated with the presence of distant metastasis and adverse outcomes. The univariate and multivariate survival assays confirmed that EPB41L4A-AS2 expression was an independent predictor of progression-free survival (PFS) in patients with NPC. Biological analyses showed that overexpression of EPB41L4A-AS2 reduced the metastasis and invasion of NPC in vitro and in vivo, but had no significant effect on cell proliferation. Mechanistically, in the nucleus we identified that EPB41L4A-AS2 relies on binding to YBX1 to reduce the stability of Snail mRNA to enhance the expression of E-cadherin and reverse the progression of epithelial-to-mesenchymal transition (EMT). In the cytoplasm, we found that EPB41L4A-AS2 blocked the invasion and migration of NPC cells by promoting LATS2 expression via sponging miR-107. In a whole, the findings of this study help to further understand the metastasis mechanism of NPC and could help in the prevention and treatment of NPC metastasis.     

Enhanced nuclear localization of YAP1‐2 contributes to EGF‐induced EMT in NSCLC

YAP1, a key mediator of the Hippo pathway, plays an important role in tumorigenesis. Alternative splicing of human YAP1 mRNA results in two major isoforms: YAP1‐1, which contains a single WW domain, and YAP1‐2, which contains two WW domains, respectively. We here investigated the functions and the underlying regulatory mechanisms of the two YAP1 isoforms in the context of EGF‐induced epithelial‐mesenchymal transition (EMT) in non‐small cell lung cancer (NSCLC). Human NSCLC cell lines express both YAP1‐1 and YAP1‐2 isoforms—although when compared to YAP1‐1, YAP1‐2 mRNA levels are higher while its protein expression levels are lower. EGF treatment significantly promoted YAP1 expression as well as EMT process in NSCLCs, whereas EGF‐induced EMT phenotype was significantly alleviated upon YAP1 knockdown. Under normal culture condition, YAP1‐1 stable expression cells exhibited a stronger migration ability than YAP1‐2 expressing cells. However, upon EGF treatment, YAP1‐2 stable cells showed more robust migration than YAP1‐1 expressing cells. The protein stability and nuclear localization of YAP1‐2 were preferentially enhanced with EGF treatment. Moreover, EGF‐induced EMT and YAP1‐2 activity were suppressed by inhibitor of AKT. Our results suggest that YAP1‐2 is the main isoform that is functionally relevant in promoting EGF‐induced EMT and ultimately NSCLC progression.     

MNSFβ regulates placental development by conjugating IGF2BP2 to enhance trophoblast cell invasiveness

ObjectivesSuccess in pregnancy in mammals predominantly depends on a well‐developed placenta. The differentiation of invasive trophoblasts is a fundamental process of placentation, the abnormalities of which are tightly associated with pregnancy disorders including preeclampsia (PE). Monoclonal nonspecific suppressor factor beta (MNSFβ) is an immunosuppressive factor. Its conventional knockout in mice induced embryonic lethality, whereas the underlying mechanism of MNSFβ in regulating placentation and pregnancy maintenance remains to be elucidated.MethodsTrophoblast‐specific knockout of MNSFβ was generated using Cyp19‐Cre mice. In situ hybridization (ISH), haematoxylin and eosin (HE), immunohistochemistry (IHC) and immunofluorescence (IF) were performed to examine the distribution of MNSFβ and insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2) at the foeto‐maternal interface. The interaction and expression of MNSFβ, IGF2BP2 and invasion‐related molecules were detected by immunoprecipitation (IP), immunoblotting and quantitative real‐time polymerase chain reaction (qRT‐PCR). The cell invasion ability was measured by the Transwell insert assay.ResultsWe found that deficiency of MNSFβ in trophoblasts led to embryonic growth retardation by mid‐gestation and subsequent foetal loss, primarily shown as apparently limited trophoblast invasion. In vitro experiments in human trophoblasts demonstrated that the conjugation of MNSFβ with IGF2BP2 and thus the stabilization of IGF2BP2 essentially mediated the invasion‐promoting effect of MNSFβ. In the placentas from MNSFβ‐deficient mice and severe preeclamptic (PE) patients, downregulation of MNSFβ was evidently associated with the repressed IGF2BP2 expression.ConclusionsThe findings reveal the crucial role of MNSFβ in governing the trophoblast invasion and therefore foetal development, and add novel hints to reveal the placental pathology of PE.     

AKT2 contributes to increase ovarian cancer cell migration and invasion through the AKT2-PKM2-STAT3/NF-κB axis

Multiple studies have shown that protein kinase Bβ (AKT2) is involved in the development and progression of ovarian cancer, however, its precise role remains unclear. Here we explored the underlying molecular mechanisms how AKT2 promotes ovarian cancer progression. We examined the effects of AKT2 in vitro in two ovarian cancer cell lines (SKOV3 and HEY), and in vivo by metastasis assay in nude mice. The migration and invasion ability of SKOV3 and HEY cells was determined by transwell assay. Overexpression and knockdown (with shRNA) experiments were carried out to unravel the underlying signaling mechanisms induced by AKT2. Overexpression of AKT2 led to increased expression of pyruvate kinase (PKM2) in ovarian cancer cells and in lung metastatic foci from nude mice. Elevated AKT2/PKM2 expression induced cell migration and invasion in vitro, as well as lung metastasis in vivo; silencing AKT2 blocked these effects. Meanwhile, PKM2 overexpression was unable to increase AKT2 expression. The expressions of p-PI3K, p-AKT2, and PKM2 were increased when stimulated by epidermal growth factor (EGF); however, these expressions were blocked when inhibited the PI3K by LY294002. STAT3 expression was elevated and NF-κB p65 nuclear translocation was activated both in vitro and in vivo when either AKT2 or PKM2 was overexpressed; and these effects were inhibited when silencing AKT2 expression. Taken together, AKT2 increases the migration and invasion of ovarian cancer cells in vitro and promotes lung metastasis in nude mice in vivo through PKM2-mediated elevation of STAT3 expression and NF-κB activation. In conclusion, we highlight a novel mechanism of the AKT2-PKM2-STAT3/NF-κB axis in the regulation of ovarian cancer progression, and our work suggested that both AKT2 and PKM2 may be potential targets for the treatment of ovarian cancer.     

Elevated MicroRNA-31 Expression Regulates Colorectal Cancer Progression by Repressing Its Target Gene SATB2

Several studies have brought about increasing evidence to support the hypothesis that miRNAs play a pivotal role in multiple processes of carcinogenesis, including cell growth, apoptosis, differentiation, and metastasis. In this study, we investigated the potential role of miR-31 in colorectal cancer (CRC) aggressiveness and its underlying mechanisms. We found that miR-31 increased in CRC cells originated from metastatic foci and human primary CRC tissues with lymph node metastases. Furthermore, the high-level expression of miR-31 was significantly associated with a more aggressive and poor prognostic phenotype of patients with CRC (p < 0.05). The stable over-expression of miR-31 in CRC cells was sufficient to promote cell proliferation, invasion, and migration in vitro. It facilitated tumor growth and metastasis in vivo too. Further studies showed that miR-31 can directly bind to the 3’untranslated region (3’UTR) of SATB2 mRNA and subsequently repress both the mRNA and protein expressions of SATB2. Ectopic expression of SATB2 by transiently transfected with pCAG-SATB2 vector encoding the entire SATB2 coding sequence could reverse the effects of miR-31 on CRC tumorigenesis and progression. In addition, ectopic over-expression of miR-31 in CRC cells induced epithelial-mesenchymal transition (EMT). Our results illustrated that the up-regulation of miR-31 played an important role in CRC cell proliferation, invasion, and metastasis in vitro and in vivo through direct repressing SATB2, suggesting a potential application of miR-31 in prognosis prediction and therapeutic application in CRC.     

MTHFD2 promotes tumorigenesis and metastasis in lung adenocarcinoma by regulating AKT/GSK‐3β/β‐catenin signalling

Recent studies have demonstrated that one‐carbon metabolism plays a significant role in cancer development. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), a mitochondrial enzyme of one‐carbon metabolism, has been reported to be dysregulated in many cancers. However, the specific role and mechanism of MTHFD2 in lung adenocarcinoma (LUAD) still remains unclear. In this study, we evaluated the clinicopathological and prognostic values of MTHFD2 in LUAD patients. We conducted a series of functional experiments in vivo and in vitro to explore novel mechanism of MTHFD2 in LUAD. The results showed that MTHFD2 was significantly up‐regulated in LUAD tissues and predicted poor prognosis of LUAD patients. Knockdown of MTHFD2 dramatically inhibited cell proliferation and migration by blocking the cell cycle and inducing the epithelial‐mesenchymal transition (EMT). In addition, MTHFD2 knockdown suppressed LUAD growth and metastasis in cell‐derived xenografts. Mechanically, we found that MTHFD2 promoted LUAD cell growth and metastasis via AKT/GSK‐3β/β‐catenin signalling. Finally, we identified miR‐30a‐3p as a novel regulator of MTHFD2 in LUAD. Collectively, MTHFD2 plays an oncogenic role in LUAD progression and is a promising target for LUAD diagnosis and therapy.     

Role of IGF2BP3 in trophoblast cell invasion and migration

The insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3) is a member of a highly conserved protein family that is expressed specifically in placenta, testis and various cancers, but is hardly detectable in normal adult tissues. IGF2BP3 has important roles in RNA stabilization and translation, especially during early stages of both human and mouse embryogenesis. Placenta is an indispensable organ in mammalian reproduction that connects developing fetus to the uterine wall, and is responsible for nutrient uptake, waste elimination and gas exchange. Fetus development in the maternal uterine cavity depends on the specialized functional trophoblast. Whether IGF2BP3 plays a role in trophoblast differentiation during placental development has never been examined. The data obtained in this study revealed that IGF2BP3 was highly expressed in human placental villi during early pregnancy, especially in cytotrophoblast cells (CTBs) and trophoblast column, but a much lower level of IGF2BP3 was detected in the third trimester placental villi. Furthermore, the expression level of IGF2BP3 in pre-eclamptic (PE) placentas was significantly lower than the gestational age-matched normal placentas. The role of IGF2BP3 in human trophoblast differentiation was shown by in vitro cell invasion and migration assays and an ex vivo explant culture model. Our data support a role of IGF2BP3 in promoting trophoblast invasion and suggest that abnormal expression of IGF2BP3 might be associated with the etiology of PE.     

Cancer/testis antigen LDHC promotes proliferation and metastasis by activating the PI3K/Akt/GSK-3β-signaling pathway and the in lung adenocarcinoma

The cancer/testis antigen lactate dehydrogenase-C4 (LDHC) is a specific isoenzyme of the LDH family that regulates invasion and metastasis in some malignancies; however, little is known regarding its role in progression of lung adenocarcinoma (LUAD). Thus, we investigated LDHC expression by immunohistochemistry, and analyzed its clinical significance in 88 LUAD specimens. The role and molecular mechanisms subserving LDHC in cellular proliferation, migration, and invasion were explored both in vitro and in vivo. As a result, we found that high LDHC expression was significantly correlated with clinicopathological features of aggressive LUAD and a poor prognosis. Overexpression of LDHC induced LUAD cells to produce lactate and ATP, increased their metastatic and invasive potential—, and accelerated xenograft tumor growth. We further demonstrated that overexpression of LDHC affected the expression of cell proliferation-related proteins (cyclin D1 and c-Myc) and epithelial-mesenchymal transition (EMT)-related proteins (MMP-2, MMP-9, E-cadherin, Vimentin, Twist, Slug, and Snail) both in vitro and in vivo. Finally, excessive activation of LDHC enhanced the phosphorylation levels of AKT and GSK-3β, revealing activation of the PI3K/Akt/GSK-3β oncogenic-signaling pathways. Treatment with a PI3K inhibitor reversed the effects of LDHC overexpression by inhibiting cellular proliferation, migration, and invasion, with diminished levels of p-Akt and p-GSK3β. PI3K inhibition also reversed cell proliferation-related and EMT-related proteins in LDHC-overexpressing A549 cells. In conclusion, LDHC promotes proliferation, migration, invasion, and EMT in LUAD cells via activation of the PI3K/Akt/GSK-3β pathway.     

Calcium signaling induces a partial EMT

Epithelial plasticity, or epithelial‐to‐mesenchymal transition (EMT), is a well‐recognized form of cellular plasticity, which endows tumor cells with invasive properties and alters their sensitivity to various agents, thus representing a major challenge to cancer therapy. It is increasingly accepted that carcinoma cells exist along a continuum of hybrid epithelial–mesenchymal (E‐M) states and that cells exhibiting such partial EMT (P‐EMT) states have greater metastatic competence than those characterized by either extreme (E or M). We described recently a P‐EMT program operating in vivo by which carcinoma cells lose their epithelial state through post‐translational programs. Here, we investigate the underlying mechanisms and report that prolonged calcium signaling induces a P‐EMT characterized by the internalization of membrane‐associated E‐cadherin (ECAD) and other epithelial proteins as well as an increase in cellular migration and invasion. Signaling through Gαq‐associated G‐protein‐coupled receptors (GPCRs) recapitulates these effects, which operate through the downstream activation of calmodulin‐Camk2b signaling. These results implicate calcium signaling as a trigger for the acquisition of hybrid/partial epithelial–mesenchymal states in carcinoma cells.     

Naa10p and IKKα interaction regulates EMT in oral squamous cell carcinoma via TGF‐β1/Smad pathway

Epithelial‐mesenchymal transition (EMT) has been contributed to increase migration and invasion of cancer cells. However, the correlate of Naa10p and IKKα with EMT in oral squamous cell carcinoma (OSCC) is not yet fully understood. In our present study, we found N‐α‐acetyltransferase 10 protein (Naa10p) and IκB kinase α (IKKα) were abnormally abundant in oral squamous cell carcinoma (OSCC). Bioinformatic results indicate that the expression of Naa10p and IKKα is correlated with TGF‐β1/Smad and EMT‐related molecules. The Transwell migration, invasion, qRT‐PCR and Western blot assay indicated that Naa10p repressed OSCC cell migration, invasion and EMT, whereas IKKα promoted TGF‐β1–mediated OSCC cell migration, invasion and EMT. Mechanistically, Naa10p inhibited IKKα activation of Smad3 through the interaction with IKKα directly in OSCC cells after TGF‐β1 stimulation. Notably, knockdown of Naa10p reversed the IKKα‐induced change in the migration, invasion and EMT‐related molecules in OSCC cells after TGF‐β1 stimulation. These findings suggest that Naa10p interacted with IKKα mediates EMT in OSCC cells through TGF‐β1/Smad, a novel pathway for preventing OSCC.