Larotrectinib induces autophagic cell death through AMPK/mTOR signalling in colon cancer

Larotrectinib (Lar) is a highly selective and potent small‐molecule inhibitor used in patients with tropomyosin receptor kinase (TRK) fusion‐positive cancers, including colon cancer. However, the underlying molecular mechanisms specifically in patients with colon cancer have not yet been explored. Our data showed that Lar significantly suppressed proliferation and migration of colon cancer cells. In addition, Lar suppressed the epithelial–mesenchymal transition (EMT) process, as evidenced by elevation in E‐cadherin (E‐cad), and downregulation of vimentin and matrix metalloproteinase (MMP) 2/9 expression. Furthermore, Lar was found to activate autophagic flux, in which Lar increased the ratio between LC3II/LC3I and decreased the expression of p62 in colon cancer cells. More importantly, Lar also increased AMPK phosphorylation and suppressed mTOR phosphorylation in colon cancer cells. However, when we silenced AMPK in colon cancer cells, Lar‐induced accumulation of autolysomes as well as Lar‐induced suppression of the EMT process were significantly diminished. An in vivo assay also confirmed that tumour volume and weight decreased in Lar‐treated mice than in control mice. Taken together, this study suggests that Lar significantly suppresses colon cancer proliferation and migration by activating AMPK/mTOR‐mediated autophagic cell death.     

IGF2BP3 promotes cell metastasis and is associated with poor patient survival in nasopharyngeal carcinoma

Metastasis contributes to treatment failure in nasopharyngeal carcinoma (NPC) patients. Our study aimed at elucidating the role of insulin‐like growth factor 2 mRNA binding protein 3 (IGF2BP3) in NPC metastasis and the underlying mechanism involved. IGF2BP3 expression in NPC was determined by bioinformatics, quantitative polymerase chain reaction and immunohistochemistry analyses. The biological function of IGF2BP3 was investigated by using in vitro and in vivo studies. In this study, IGF2BP3 mRNA and protein levels were elevated in NPC tissues. In addition, IGF2BP3 exerted an oncogenic role by promoting epithelial‐mesenchymal transition (EMT), thereby inducing NPC cell migration and invasion. Further studies revealed that IGF2BP3 regulated the expression of key regulators of EMT by activating AKT/mTOR signalling, thus stimulating NPC cell migration and invasion. Remarkably, targeting IGF2BP3 delayed NPC metastasis through attenuating p‐AKT and vimentin expression and inducing E‐cadherin expression in vivo. Moreover, IGF2BP3 protein levels positively correlated with distant metastasis after initial treatment. Importantly, IGF2BP3 expression served as an independent prognostic factor in predicting the overall survival and distant metastasis‐free survival of NPC patients. This work identifies IGF2BP3 as a novel prognostic marker and a new target for NPC treatment.     

Polydatin inhibits ZEB1‐invoked epithelial‐mesenchymal transition in fructose‐induced liver fibrosis

High fructose intake is a risk factor for liver fibrosis. Polydatin is a main constituent of the rhizome of Polygonum cuspidatum, which has been used in traditional Chinese medicine to treat liver fibrosis. However, the underlying mechanisms of fructose‐driven liver fibrosis as well as the actions of polydatin are not fully understood. In this study, fructose was found to promote zinc finger E‐box binding homeobox 1 (ZEB1) nuclear translocation, decrease microRNA‐203 (miR‐203) expression, increase survivin, activate transforming growth factor β1 (TGF‐β1)/Smad signalling, down‐regulate E‐cadherin, and up‐regulate fibroblast specific protein 1 (FSP1), vimentin, N‐cadherin and collagen I (COL1A1) in rat livers and BRL‐3A cells, in parallel with fructose‐induced liver fibrosis. Furthermore, ZEB1 nuclear translocation‐mediated miR‐203 low‐expression was found to target survivin to activate TGF‐β1/Smad signalling, causing the EMT in fructose‐exposed BRL‐3A cells. Polydatin antagonized ZEB1 nuclear translocation to up‐regulate miR‐203, subsequently blocked survivin‐activated TGF‐β1/Smad signalling, which were consistent with its protection against fructose‐induced EMT and liver fibrosis. These results suggest that ZEB1 nuclear translocation may play an essential role in fructose‐induced EMT in liver fibrosis by targeting survivin to activate TGF‐β1/Smad signalling. The suppression of ZEB1 nuclear translocation by polydatin may be a novel strategy for attenuating the EMT in liver fibrosis associated with high fructose diet.     

A disintegrin and metalloproteinase 8 induced epithelial‐mesenchymal transition to promote the invasion of colon cancer cells via TGF‐β/Smad2/3 signalling pathway

A disintegrin and metalloproteinase 8 (ADAM8) protein is a multi‐domain transmembrane glycoprotein which involves in extracellular matrix remodelling, cell adhesion, invasion and migration. ADAM8 and epithelial‐mesenchymal transition (EMT) play an important role in tumour invasion has been well established. However, the interaction between ADAM8 and EMT has remained unclear. The data of colon cancer patients obtained from TCGA (The Cancer Genome Atlas) and GTEx (Genotype‐Tissue Expression Project) were analysed by the bioinformatics research method. The expression of ADAM8 in colon cancer cells was up‐regulated and down‐regulated by transfecting with the expression plasmid and small interfering RNA, respectively. Transwell invasion assay, immunohistochemistry, immunocytochemistry, Western blotting and qRT‐PCR were utilized to study the effect of ADAM8 on colon cancer cell''s EMT and its related mechanisms. Analysis of TCGA and GTEx data revealed that ADAM8 was linked to poor overall survival in colon cancer patients. Besides, ADAM8 was correlated with multiple EMT biomarkers (E‐cadherin, N‐cadherin, Vimentin, Snail2 and ZEB2). In vitro, we also proved that the up‐regulation of ADAM8 could promote EMT effect and enhance the invasive ability of colon cancer cells. On the contrary, the down‐regulation of ADAM8 in colon cancer cells attenuated these effects above. Further studies suggested that ADAM8 modulated EMT on colon cancer cells through TGF‐β/Smad2/3 signalling pathway. Our research suggested that ADAM8 could be a potential biomarker for the prognosis of colon cancer and induced EMT to promote the invasion of colon cancer cells via activating TGF‐β/Smad2/3 signalling 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.     

MIF/SCL3A2 depletion inhibits the proliferation and metastasis of colorectal cancer cells via the AKT/GSK‐3β pathway and cell iron death

This study investigated the mechanisms of migration inhibitory factor (MIF) and solute carrier family 3 member 2 (SLC3A2) in colorectal cancer progression. The levels of MIF and SLC3A2 expression in cells were measured by RT‐qPCR. SW480 and SW620 cells were transfected with sh‐MIF and sh‐SLC3A2, respectively. MIF, SLC3A2, GPX4, E‐cadherin and N‐cadherin expression were detected by immunofluorescence (IF). CCK8 and Transwell assays were performed to detect cell proliferation and migration. Co‐immunoprecipitation (CoIP) was used to measure the binding activity of MIF and SLC3A2. Finally, a nude mouse tumorigenicity assay was used to confirm the functions of MIF and SLC3A2 in colorectal cancer. Results showed that the levels of MIF and SLC3A2 expression were up‐regulated in colorectal cancer cells. Inhibition of MIF or SLC3A2 expression prevented cell proliferation, migration, epithelial‐mesenchymal transition (EMT) and invasion. In addition, knockdown of MIF and SLC3A2 promoted iron death in SW480 and SW620 cells. CoIP results showed that MIF and SLC3A2 directly interact with each other. Knockdown of both MIF and SLC3A2 inhibited tumour growth and metastasis via the AKT/GSK‐3β pathway in vivo. The Akt/GSK‐3β pathway was found to participate in regulating MIF and SLC3A2 both in vivo and in vitro. MIF and SLC3A2 might be potential biomarkers for monitoring the treatment of colorectal cancer.     

Msps governs acentrosomal microtubule assembly and reactivation of quiescent neural stem cells

The ability of stem cells to switch between quiescence and proliferation is crucial for tissue homeostasis and regeneration. Drosophila quiescent neural stem cells (NSCs) extend a primary cellular protrusion from the cell body prior to their reactivation. However, the structure and function of this protrusion are not well established. Here, we show that in the protrusion of quiescent NSCs, microtubules are predominantly acentrosomal and oriented plus‐end‐out toward the tip of the primary protrusion. We have identified Mini Spindles (Msps)/XMAP215 as a key microtubule regulator in quiescent NSCs that governs NSC reactivation via regulating acentrosomal microtubule growth and orientation. We show that quiescent NSCs form membrane contact with the neuropil and E‐cadherin, a cell adhesion molecule, localizes to these NSC‐neuropil junctions. Msps and a plus‐end directed motor protein Kinesin‐2 promote NSC cell cycle re‐entry and target E‐cadherin to NSC‐neuropil contact during NSC reactivation. Together, this work establishes acentrosomal microtubule organization in the primary protrusion of quiescent NSCs and the Msps‐Kinesin‐2 pathway that governs NSC reactivation, in part, by targeting E‐cad to NSC‐neuropil contact sites.     

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.     

Vascular endothelial growth factor‐C in activating vascular endothelial growth factor receptor‐3 and chemokine receptor‐4 in melanoma adhesion

Vascular endothelial growth factor‐C (VEGF‐C) binds to receptor vascular endothelial growth factor receptor‐3 (VEGFR‐3) expressed on lymphatic endothelial and melanoma cells. Binding of VEGF‐C to VEGFR‐3 enhances receptor phosphorylation that activates mitogen‐activated protein kinase (MAP‐K) and phosphatidylinositol‐3‐kinase (PI3K). These signalling pathways regulate cell migration and adhesion in response to internal or external changes.In addition, the overexpression of VEGF‐C upregulates chemokine receptor CXCR‐4 in tumours (melanoma). CXCR‐4 is expressed on cells of the immune system (natural killer cells) and facilitates the migration of leukocytes in response to the CXCL12 ligand. The latter is expressed by lymphatic endothelial cells and by stromal cells in the tumour microenvironment (TME). The gradient established between CXCR‐4 expressed on tumour cells and CXCL12 produced by stromal and lymphatic endothelial cells enhances tumour cell metastasis.3‐(4‐Dimethylamino‐naphthalen‐1‐ylmethylene)‐1, 3‐dihydroindol‐2‐one, MAZ‐51, is an indolinone‐based synthetic molecule that inhibits the phosphorylation of the tyrosine kinase receptor VEGFR‐3. CTCE‐9908, a CXCR‐4 antagonist derived from human CXCL12, hinders receptor phosphorylation and the subsequent signalling pathways that would be activated.VEGF‐C is stimulated by transforming growth factor‐beta 1 (TGF‐β1), which facilitates cell–cell and cell‐matrix adhesion by regulating cadherins through the activation of focal adhesion kinase (FAK) and mediates paxillin upregulation.Increased VEGF‐C protein levels stimulated by TGF‐β bound to VEGFR‐3 impact on intracellular pathways that promote tumour cell adhesion. In addition, increased VEGF‐C protein levels lead to enhanced CXCR‐4 protein expression. Therefore, effective blocking of VEGR‐3 and CXCR‐4 may inhibit tumour cell metastasis by hampering intracellular proteins promoting adhesion.     

DEK‐targeting aptamer DTA‐64 attenuates bronchial EMT‐mediated airway remodelling by suppressing TGF‐β1/Smad,MAPK and PI3K signalling pathway in asthma

This study is to investigate the inhibitory effects and mechanisms of DEK‐targeting aptamer (DTA‐64) on epithelial mesenchymaltransition (EMT)‐mediated airway remodelling in mice and human bronchial epithelial cell line BEAS‐2B. In the ovalbumin (OVA)‐induced asthmatic mice, DTA‐64 significantly reduced the infiltration of eosinophils and neutrophils in lung tissue, attenuated the airway resistance and the proliferation of goblet cells. In addition, DTA‐64 reduced collagen deposition, transforming growth factor 1 (TGF‐β1) level in BALF and IgE levels in serum, balanced Th1/Th2/Th17 ratio, and decreased mesenchymal proteins (vimentin and α‐SMA), as well as weekend matrix metalloproteinases (MMP‐2 and MMP‐9) and NF‐κB p65 activity. In the in vitro experiments, we used TGF‐β1 to induce EMT in the human epithelial cell line BEAS‐2B. DEK overexpression (ovDEK) or silencing (shDEK) up‐regulated or down‐regulated TGF‐β1 expression, respectively, on the contrary, TGF‐β1 exposure had no effect on DEK expression. Furthermore, ovDEK and TGF‐β1 synergistically promoted EMT, whereas shDEK significantly reduced mesenchymal markers and increased epithelial markers, thus inhibiting EMT. Additionally, shDEK inhibited key proteins in TGF‐β1‐mediated signalling pathways, including Smad2/3, Smad4, p38 MAPK, ERK1/2, JNK and PI3K/AKT/mTOR. In conclusion, the effects of DTA‐64 against EMT of asthmatic mice and BEAS‐2B might partially be achieved through suppressing TGF‐β1/Smad, MAPK and PI3K signalling pathways. DTA‐64 may be a new therapeutic option for the management of airway remodelling in asthma patients.     

Naringenin Decreases Invasiveness and Metastasis by Inhibiting TGF-β-Induced Epithelial to Mesenchymal Transition in Pancreatic Cancer Cells

Epithelial to mesenchymal transition (EMT) promotes cellular motility, invasiveness and metastasis during embryonic development and tumorigenesis. Transforming growth factor-β (TGF-β) signaling pathway is a key regulator of EMT. A lot of evidences suggest that this process is Smad3-dependent. Herein we showed that exposure of aspc-1 and panc-1 pancreatic cancer cells to TGF-β1 resulted in characteristic morphological alterations of EMT, and enhancement of cell motility and gemcitabine (Gem) resistance along with an up-regulation of EMT markers genes such as vimentin, N-cadherin, MMP2 and MMP9. Naringenin (Nar) down-regulated EMT markers expression in both mRNA and protein levels by inhibiting TGF-β1/Smad3 signal pathway in the pancreatic cancer cells. Consequently, Nar suppressed the cells migration and invasion and reversed their resistance to Gem.     

Nucleobindin‐2/nesfatin‐1 enhances the cell proliferation,migration, invasion and epithelial‐mesenchymal transition in gastric carcinoma

Nesfatin‐1, a newly discovered adipokine derived from nucleobindin‐2 (NUCB2), has been described as a new prognostic marker in cancers. This study aimed to explore the functional role of NUCB2/nesfatin‐1 in the cell proliferation, migration and invasion in gastric carcinoma (GC). The expressions of NUCB2/nesfatin‐1 in GC tissues and normal adjacent tissues (NATs) were compared, and the effect of inhibition of NUCB2/nesfatin‐1 on the cell proliferation, migration, invasion and epithelial‐mesenchymal transition (EMT) in GC cell line SGC‐7901 was investigated. Cell transfection was conducted to inhibit NUCB2/nesfatin‐1 by short hairpin RNA. Cell proliferation, migration and invasion abilities were determined using cell counting kit‐8 (CCK‐8), 5‐ethynyl‐2′‐deoxyuridine (EdU), wound healing and transwell assays, respectively. The expressions of EMT markers E‐Cadherin and N‐Cadherin were determined using western blotting. The expression of NUCB2/nesfatin‐1 protein in GC tissues was significantly increased compared with that in NATs. Consistently, the serum concentrations of NUCB2/nesfatin‐1 were significantly higher in patients with GC as compared with those in the control group. Moreover, the results of CCK‐8 assay and EdU assay indicated that knockdown of NUCB2/nesfatin‐1 could markedly decrease SGC‐7901 proliferation. Furthermore, the results of wound healing assay and transwell assay demonstrated that knockdown of NUCB2/nesfatin‐1 significantly suppressed SGC‐7901 migration and invasion abilities. Additionally, knockdown of NUCB2/nesfatin‐1 decreased the expressions of N‐Cadherin and increased the expressions of E‐Cadherin in SGC‐7901 cells. These findings suggest that knockdown of NUCB2/nesfatin‐1 suppressed the proliferation, migration, invasion and EMT of SGC‐7901 cells, suggesting a potentially promising therapeutic target for GC.     

Newly designed Protein Transduction Domain (PTD)‐mediated BMP‐7 is a potential therapeutic for peritoneal fibrosis

While the bone morphogenetic protein‐7 (BMP‐7) is a well‐known therapeutic growth factor reverting many fibrotic diseases, including peritoneal fibrosis by peritoneal dialysis (PD), soluble growth factors are largely limited in clinical applications owing to their short half‐life in clinical settings. Recently, we developed a novel drug delivery model using protein transduction domains (PTD) overcoming limitation of soluble recombinant proteins, including bone morphogenetic protein‐7 (BMP‐7). This study aims at evaluating the therapeutic effects of PTD‐BMP‐7 consisted of PTD and full‐length BMP‐7 on epithelial‐mesenchymal transition (EMT)‐related fibrosis. Human peritoneal mesothelial cells (HPMCs) were then treated with TGF‐β1 or TGF‐β1 + PTD‐BMP‐7. Peritoneal dialysis (PD) catheters were inserted into Sprague‐Dawley rats, and these rats were infused intra‐peritoneally with saline, peritoneal dialysis fluid (PDF) or PDF + PTD‐BMP‐7. In vitro, TGF‐β1 treatment significantly increased fibronectin, type I collagen, α‐SMA and Snail expression, while reducing E‐cadherin expression in HPMCs (P < .001). PTD‐BMP‐7 treatment ameliorated TGF‐β1‐induced fibronectin, type I collagen, α‐SMA and Snail expression, and restored E‐cadherin expression in HPMCs (P < .001). In vivo, the expressions of EMT‐related molecules and the thickness of the sub‐mesothelial layer were significantly increased in the peritoneum of rats treated with PDF, and these changes were significantly abrogated by the intra‐peritoneal administration of PTD‐BMP‐7. PTD‐BMP‐7 treatment significantly inhibited the progression of established PD fibrosis. These findings suggest that PTD‐BMP‐7, as a prodrug of BMP‐7, can be an effective therapeutic agent for peritoneal fibrosis in PD patients.     

The Effect of TIGAR Knockdown on Apoptotic and Epithelial‐Mesenchymal Markers Expression in Doxorubicin‐Resistant Non‐Small Cell Lung Cancer A549 Cell Lines

Resistance to chemotherapeutic drugs is a critical problem in cancer therapy, but the underlying mechanism has not been fully elucidated. TP53‐induced glycolysis regulatory phosphatase (TIGAR), an important glycolysis and apoptosis regulator, plays a crucial role in cancer cell survival by protecting cells against oxidative stress‐induced apoptosis. In the present study, we investigated whether TIGAR is involved in epithelial‐mesenchymal transition (EMT) in doxorubicin (DOX)‐resistant human non‐small cell lung cancer (NSCLC), A549/DOX cells. We found that the expression of TIGAR was significantly higher in A549/DOX cells than in the parent A549 cell lines. siRNA‐mediated TIGAR knockdown reduced migration, viability and colony survival of doxorubicin‐resistant lung cancer cells. Also, TIGAR knockdown decreased pro‐survival protein Bcl‐2 and increased pro‐apoptotic Bax and cleaved poly (ADP‐ribose) polymerase (PARP). Moreover, TIGAR depletion significantly up‐regulated both caspase‐3 and caspase‐9 expression. Furthermore, TIGAR depletion up‐regulated the expression of E‐cadherin and down‐regulated the expression of vimentin. These results indicate that TIGAR knockdown may inhibit EMT in doxorubicin (DOX)‐resistant human NSCLC and may represent a therapeutic target for a non‐small lung cancer cells chemoresistance.