MAPK‐mediated upregulation of fibrinogen‐like protein 2 promotes proliferation,migration, and invasion of colorectal cancer cells

Fibrinogen‐like protein 2 (FGL2) has been reported to play a key role in the development of human cancers. However, it is still unmasked whether FGL2 plays a potential role in colorectal carcinogenesis. In this study, the messenger RNA and protein expression levels were measured by quantitative real‐time polymerase chain reaction and western blot. Cell counting kit‐8 assay, transwell migration, and invasion assay were carried out to evaluate the proliferation, migration, and invasion of LOVO and SW620 cells. FGL2 was upregulated in colorectal cancer (CRC) tissues, as well as cell lines. Mitogen‐activated protein kinase (MAPK) signaling was activated in CRC tissues and cell lines. FGL2 was confirmed to be downregulated by MAPK signaling inhibitor U0126. Further, we determined that knockdown of FGL2 caused a reduction of proliferation, migration, and invasion in LOVO and SW620 cells. Consistently, treatment of LOVO and SW620 cells with U0126 led to a decrease in cell proliferation, migration, and invasion. However, these changes initiated by U0126 were abolished by FGL2 overexpression. To conclude, MAPK‐mediated upregulation of FGL2 promotes the proliferation, migration, and invasion of CRC cells.     

Knockdown of DNA‐binding protein A enhances the chemotherapy sensitivity of colorectal cancer via suppressing the Wnt/β‐catenin/Chk1 pathway

DNA‐binding protein A (dbpA) is reported to be upregulated in many cancers and associated with tumor progress. The present study aimed to investigate the role of dbpA in 5‐fluorouracil (5‐FU)‐resistant and oxaliplatin (L‐OHP)‐resistant colorectal cancer (CRC) cells. We found that 5‐FU and L‐OPH treatment promoted the expression of dbpA. Enhanced dbpA promoted the drug resistance of SW620 cells to 5‐FU and L‐OHP. DbpA knockdown inhibited cell proliferation, induced cell apoptosis, and cell cycle arrested in SW620/5‐FU and SW620/L‐OHP cells. Besides, dbpA short hairpin RNA (shRNA) enhanced the cytotoxicity of 5‐FU and L‐OHP to SW620/5‐FU and SW620/L‐OHP cells. Meanwhile, dbpA shRNA inhibited the activation of the Wnt/β‐catenin pathway that induced by 5‐FU stimulation in SW620/5‐FU cells. Activation of the Wnt/β‐catenin pathway or overexpression of checkpoint kinase 1 (Chk1) abrogated the promoting effect of dbpA downregulation on 5‐FU sensitivity of CRC cells. Importantly, downregulation of dbpA suppressed tumor growth and promoted CRC cells sensitivity to 5‐FU in vivo. Our study indicated that the knockdown of dbpA enhanced the sensitivity of CRC cells to 5‐FU via Wnt/β‐catenin/Chk1 pathway, and DbpA may be a potential therapeutic target to sensitize drug resistance CRC to 5‐FU and L‐OHP.     

Long non-coding RNA-422 acts as a tumor suppressor in colorectal cancer

Colorectal cancer (CRC) is one of the most frequent cancer in numerous of countries worldwidely. The initiation and progression of CRC is an extremely complex process, and have been suggested a correlation with Long non-coding RNAs (lncRNAs). Our results showed that lncRNA-422(ENST00000415820) significantly downregulated in the tissues and serum of CRC patients, and is closely associated with the poor prognosis. Then gain or loss of lncRNA-422 models in SW480 and SW620cells were established. The results showed that lncRNA-422 overexpression inhibited cell proliferation, migration, and invasion. Knockdown of lncRNA-422 promoted tumorigensis. Western blot and qRT-PCR were performed to examine the activity of the PI3K/AKT/mTOR pathway in CRC cells after alternation of lncRNA-422. Results showed that lncRNA-422 acts as a tumor suppressor by PI3K/AKT/mTOR pathway in CRC.     

Circular RNA circMAN2B2 facilitates glioma progression by regulating the miR-1205/S100A8 axis

The aim of this study was to research the mechanism of circMAN2B2 in the development of glioma. In our study, we found that circMAN2B2 has a higher expression in glioma tissues and cells, which was negatively related to the overall survival of glioma patients. The cell counting kit-8 assay, 5-ethynyl-2′-deoxyuridine labeling assay, transwell assay, and the nude mice assay indicated that knockdown of circMAN2B2 inhibited the cell proliferation, invasion, migration and decreased tumor size. In terms of mechanism, knockdown of circMAN2B2 increased the expression of miR-1205. Moreover, circMAN2B2 regulated S100A8 expression by inhibiting miR-1205. We also showed that knockdown of S100A8 inhibited cell proliferation, invasion, and migration. Increasing S100A8 expression rescued the effect of si-circMAN2B2. In conclusion, circMAN2B2 could improve cell proliferation, invasion, and migration of the glioma by inhibiting miR-1205 and promoting the expression of S100A8.     

20(S)‐Protopanaxadiol inhibits epithelial‐mesenchymal transition by promoting retinoid X receptor alpha in human colorectal carcinoma cells

Colorectal carcinoma (CRC) recurrence is often accompanied by metastasis. Most metastasis undergo through epithelial‐mesenchymal transition (EMT). Studies showed that retinol X receptor alpha (RXRα) and 20(S)‐Protopanaxadiol (PPD) have anti‐tumour effects. However, the anti‐metastasis effect of 20(S)‐PPD and the effect of RXRα on EMT‐induced metastasis are few studies on. Therefore, the role of RXRα and 20(S)‐PPD in CRC cell metastasis remains to be fully elucidated. RXRα with clinicopathological characteristics and EMT‐related expression in clinical samples were examined. Then, RXRα and EMT level in SW480 and SW620 cells, overexpressed and silenced RXRα in SW620 cells and SW480 cells, respectively, were evaluated. Finally, 20(S)‐PPD effect on SW620 and SW480 cells was evaluated. The results showed that a lower RXRα expression in cancer tissues, and a moderate negative correlation between RXRα and N stage, and tended to higher level of EMT. SW480 and SW620 cells had the highest and lowest RXRα expression among four CRC cell lines. SW480 had lower EMT level than SW620. Furthermore, 20(S)‐PPD increased RXRα and inhibited EMT level in SW620 cell. Finally, 20(S)‐PPD cannot restore SW480 cells EMT level to normal when RXRα silencing. These findings suggest that 20(S)‐PPD may inhibit EMT process in CRC cells by regulating RXRα expression.     

AEG‐1 induces gastric cancer metastasis by upregulation of eIF4E expression

Gastric cancer is the third leading cause of cancer‐related deaths worldwide, and patients with lymph node, peritoneal and distant metastasis have a poor prognosis. Overexpression of Astrocyte‐elevated gene‐1 (AEG‐1) has been reported to be correlated with the progression and metastasis of gastric cancer. However, its mechanisms are quite unclear. In this study, we found that elevated expression of AEG‐1 was correlated with metastasis in human gastric cancer tissues. Moreover, gain‐ or loss‐of‐function of AEG‐1, respectively, promoted or suppressed epithelial–mesenchymal transition (EMT), migration and invasion of gastric cancer cells. AEG‐1 positively regulated eIF4E, MMP‐9 and Twist expression. Manipulating eIF4E expression by transfection of overexpression constructs or siRNAs partially eliminated AEG‐1‐regulated EMT, cell migration and invasion. In addition, overexpression or knockdown of eIF4E promoted or suppressed EMT, cell migration and invasion in parallel with upregulation of MMP‐9 and Twist expression, while manipulating eIF4E expression partially abrogated AEG‐1‐induced MMP‐9 and Twist. Finally, silencing of AEG‐1 expression not only inhibited tumour growth in parallel with downregulation of eIF4E, MMP‐9 and Twist expression in a xenograft nude mouse model, but also suppressed lymph node and peritoneal metastasis of gastric cancer in an orthotopic nude mouse model. These findings suggest that AEG‐1 promotes gastric cancer metastasis through upregulation of eIF4E‐mediated MMP‐9 and Twist, which provides new diagnostic markers and therapeutic targets for cancer metastasis.     

S100P dissociates myosin IIA filaments and focal adhesion sites to reduce cell adhesion and enhance cell migration

S100 proteins promote cancer cell migration and metastasis. To investigate their roles in the process of migration we have constructed inducible systems for S100P in rat mammary and human HeLa cells that show a linear relationship between its intracellular levels and cell migration. S100P, like S100A4, differentially interacts with the isoforms of nonmuscle myosin II (NMIIA, K(d) = 0.5 μM; IIB, K(d) = 8 μM; IIC, K(d) = 1.0 μM). Accordingly, S100P dissociates NMIIA and IIC filaments but not IIB in vitro. NMIIA knockdown increases migration in non-induced cells and there is no further increase upon induction of S100P, whereas NMIIB knockdown reduces cell migration whether or not S100P is induced. NMIIC knockdown does not affect S100P-enhanced cell migration. Further study shows that NMIIA physically interacts with S100P in living cells. In the cytoplasm, S100P occurs in discrete nodules along NMIIA-containing filaments. Induction of S100P causes more peripheral distribution of NMIIA filaments. This change is paralleled by a significant drop in vinculin-containing, actin-terminating focal adhesion sites (FAS) per cell. The induction of S100P, consequently, causes significant reduction in cellular adhesion. Addition of a focal adhesion kinase (FAK) inhibitor reduces disassembly of FAS and thereby suppresses S100P-enhanced cell migration. In conclusion, this work has demonstrated a mechanism whereby the S100P-induced dissociation of NMIIA filaments leads to a weakening of FAS, reduced cell adhesion, and enhanced cell migration, the first major step in the metastatic cascade.     

Interaction of extracellular S100A4 with RAGE prompts prometastatic activation of A375 melanoma cells

S100A4, a member of the S100 protein family of EF‐hand calcium‐binding proteins, is overexpressed in various tumour entities, including melanoma, and plays an important role in tumour progression. Several studies in epithelial and mesenchymal tumours revealed a correlation between extracellular S100A4 and metastasis. However, exact mechanisms how S100A4 stimulates metastasis in melanoma are still unknown. From a pilot experiment on baseline synthesis and secretion of S100A4 in human melanoma cell lines, which are in broad laboratory use, A375 wild‐type cells and, additionally, newly generated A375 cell lines stably transfected with human S100A4 (A375‐hS100A4) or human receptor for advanced glycation endproducts (A375‐hRAGE), were selected to investigate the influence of extracellular S100A4 on cell motility, adhesion, migration and invasion in more detail. We demonstrated that A375 cells actively secrete S100A4 in the extracellular space via an endoplasmic reticulum‐Golgi‐dependent pathway. S100A4 overexpression and secretion resulted in prometastatic activation of A375 cells. Moreover, we determined the influence of S100A4‐RAGE interaction and its blockade on A375, A375‐hS100A4, A375‐hRAGE cells, and showed that interaction of RAGE with extracellular S100A4 contributes to the observed activation of A375 cells. This investigation reveals additional molecular targets for therapeutic approaches aiming at blockade of ligand binding to RAGE or RAGE signalling to inhibit melanoma metastasis.     

CPT1A‐mediated succinylation of S100A10 increases human gastric cancer invasion

Gastric cancer (GC) is a malignancy of the lining of the stomach and is prone to distant metastasis, which involves a variety of complex molecules. The S100 proteins are a family of calcium‐binding cytosolic proteins that possess a wide range of intracellular and extracellular functions and play pivotal roles in the invasion and migration of tumour cells. Among these, S100A10 is known to be overexpressed in GC. Lysine succinylation, a recently identified form of protein post‐translational modification, is an important regulator of cellular processes. Here, we demonstrated that S100A10 was succinylated at lysine residue 47 (K47), and levels of succinylated S100A10 were increased in human GC. Moreover, K47 succinylation of S100A10 was stabilized by suppression of ubiquitylation and subsequent proteasomal degradation. Furthermore, carnitine palmitoyltransferase 1A (CPT1A) was found to function as a lysine succinyltransferase that interacts with S100A10. Succinylation of S100A10 is regulated by CPT1A, while desuccinylation is regulated by SIRT5. Overexpression of a succinylation mimetic mutant, K47E S100A10, increased cell invasion and migration. Taken together, this study reveals a novel mechanism of S100A10 accumulation mediated by succinylation in GC, which promotes GC progression and is regulated by the succinyltransferase CPT1A and SIRT5‐mediated desuccinylation.     

microRNA-211 promotes invasion and migration of colorectal cancer cells by targeting FABP4 via PPARγ

Fatty acid binding protein 4 (FABP4) is a novel tumor regulator that is abnormally expressed in many human cancers. In our study, upregulated microRNA-211 (miR-211) and reduced FABP4 expression were detected in colorectal cancer (CRC) patients and CRC cells. Mimic miR-211 or anti-miR-211 were transfected to investigate the effects of miR-211 on SW480 cells. The results showed that miR-211 promoted but anti-miR-211 inhibited cell migration, invasion, and epithelial–mesenchymal transition (EMT) of SW480 cells. Luciferase activity was decreased after cotransfection with miR-211 and WT-FABP4-UTR in SW480 cells. And reduced FABP4 protein expression by miR-211 indicated that FABP4 was the targeted gene of miR-211. miR-211 inhibited the activation of peroxisome proliferator-activated receptor (PPAR) γ, whereas overexpression of FABP4 reversed that effect. Finally, FABP4 inhibited the migration, invasion, and EMT of SW480 cells, whereas PPARγ agonist reversed the effects of FABP4. Thus, the miR-211/FABP4/PPARγ axis may be a novel target for CRC therapy.     

S100 Calcium-Binding Protein A6 Promotes Epithelial-Mesenchymal Transition through β-Catenin in Pancreatic Cancer Cell Line

The pathogenesis of pancreatic ductal adenocarcinoma (PDAC) remains poorly understood. S100 calcium-binding protein A6 (S100A6) has been associated with PDAC; however, the effect of S100A6 on PDAC migration and invasion has not yet been explored. In this study, Panc-1 cells were transfected with a plasmid to induce overexpression of S100A6, and β-catenin was knocked down using a specific short hairpin RNA (shRNA). The wound-healing and Transwell assays demonstrated that S100A6 promoted PDAC cell migration and invasion. Furthermore, β-catenin shRNA inhibited the migration and invasion of PDAC cells. We confirmed that S100A6 induces PDAC cell migration and invasion via activation of β-catenin in vitro. Assessment of mRNA and protein levels revealed that S100A6 induces increased expression of β-catenin, N-cadherin and vimentin, and decreased expression of E-cadherin in PDAC cells. β-catenin shRNA also altered the expression of epithelial-mesenchymal transition (EMT)-related markers in PDAC cells. Specifically, expression of E-cadherin was increased, whereas expression of N-cadherin and vimentin was decreased. Finally, we demonstrated that S100A6 alters the expression of EMT-related markers via β-catenin activation. In conclusion, S100A6 induces EMT and promotes cell migration and invasion in a β-catenin-dependent manner. S100A6 may therefore represent a novel potential therapeutic target for the treatment of pancreatic cancer.     

siRNA抑制S100A4表达对卵巢癌细胞CP70生物学特性的影响

目的：探讨人卵巢癌顺铂耐药细胞株CP70沉默S100A4基因后,CP70细胞对顺铂敏感性、凋亡及细胞迁移的影响。方法：设计并合成S100A4基因特异性的siRNA并转染入卵巢癌细胞CP70,48 h后应用RT-PCR和Western Blot检测在mRNA和蛋白水平siRNA对S100A4的影响,MTT法检测转染siRNA后卵巢癌细胞CP70对顺铂敏感性的变化。用流式细胞术检测顺铂（40μM）对转染S100A4 siRNA后对卵巢癌细胞CP70凋亡的影响,Transwell法观察siRNA抑制S100A4后对卵巢癌CP70迁移能力的影响。结果：与空白对照组、阴性对照组相比,S100A4siRNA转染组CP70细胞的S100A4基因和蛋白表达降低（P〈0.01）。MTT法检测顺铂敏感性发现S100A4 siRNA转染组CP70细胞顺铂敏感性增强。在顺铂刺激下,siRNA转染组细胞凋亡率高于其他各组,差异具有统计学意义（P〈0.05）。Transwell发现CP70细胞迁移能力明显下降（P〈0.05）。结论：S100A4 siRNA能够明显抑制CP70细胞S100A4的表达,从而增强细胞对顺铂的敏感性,促进细胞凋亡,减弱细胞的迁移能力。S100A4有望成为逆转卵巢癌铂类耐药的治疗靶点。     

Downregulation of AKT3 Increases Migration and Metastasis in Triple Negative Breast Cancer Cells by Upregulating S100A4

Background

Treatment of breast cancer patients with distant metastases represents one of the biggest challenges in today’s gynecological oncology. Therefore, a better understanding of mechanisms promoting the development of metastases is of paramount importance. The serine/threonine kinase AKT was shown to drive cancer progression and metastasis. However, there is emerging data that single AKT isoforms (i.e. AKT1, AKT2 and AKT3) have different or even opposing functions in the regulation of cancer cell migration in vitro, giving rise to the hypothesis that inhibition of distinct AKT isoforms might have undesirable effects on cancer dissemination in vivo.

Methods

The triple negative breast cancer cell line MDA-MB-231 was used to investigate the functional roles of AKT in migration and metastasis. AKT single and double knockdown cells were generated using isoform specific shRNAs. Migration was analyzed using live cell imaging, chemotaxis and transwell assays. The metastatic potential of AKT isoform knockdown cells was evaluated in a subcutaneous xenograft mouse model in vivo.

Results

Depletion of AKT3, but not AKT1 or AKT2, resulted in increased migration in vitro. This effect was even more prominent in AKT2,3 double knockdown cells. Furthermore, combined downregulation of AKT2 and AKT3, as well as AKT1 and AKT3 significantly increased metastasis formation in vivo. Screening for promigratory proteins revealed that downregulation of AKT3 increases the expression of S100A4 protein. In accordance, depletion of S100A4 by siRNA approach reverses the increased migration induced by knockdown of AKT3.

Conclusions

We demonstrated that knockdown of AKT3 can increase the metastatic potential of triple negative breast cancer cells. Therefore, our results provide a rationale for the development of AKT isoform specific inhibitors.     

TUFT1 Facilitates Metastasis,Stemness, and Vincristine Resistance in Colorectal Cancer via Activation of PI3K/AKT Pathway

Since the incidence and mortality of colorectal cancer (CRC) are increasing in recent years, the research on the pathogenesis of colorectal cancer has attracted more and more attention. Here, our results confirmed that the mRNA expression level and proteins accumulation of TUFT1 were significantly increased in CRC tissues from late-stage CRC patients (III?+?IV) (p?<?0.001), indicated by qPCR and IHC assay. The TUFT1 expression was positively correlated with tumor stage by analyzing 126 specimens from CRC patients. Next, we found that up-regulation of TUFT1 enhanced the migration and invasion of LoVo cells, whereas the down-regulation of TUFT1 observably weakened the migration and invasion of SW837 cells, indicating that TUFT1 promotes the metastasis of CRC cells. In addition, TUFT1 overexpression increased the number of mammary spheres and vincristine resistance of LoVo cells by sphere formation assay and measuring the IC50 value, suggesting the TUFT1 promotes stemness and the vincristine resistance of CRC cells. Finally, we found that TUFT1 overexpression increased p-AKT in LoVo cells, while down-regulation of TUFT1 decreased the p-AKT levels in SW837 cells. Therefore, we determined that the function of TUFT1 in CRC depends on PI3K/AKT pathway. Taken together, these data demonstrated that TUFI1 facilitates metastasis, stemness, and vincristine resistance of colorectal cancer cells via activation of PI3K/AKT pathway, which might act as a promising therapeutic target for CRC.

     

Direct inhibitory and indirect stimulatory effects of RAGE ligand S100 on sRANKL‐induced osteoclastogenesis

Diabetes results in increased fracture risk, and advance glycation endproducts (AGEs) have been implicated in this pathophysiology. S100 proteins are ligands for the receptor of AGEs (RAGE). An intracellular role of the S100 family member S100A4 (Mts1) to suppress mineralization has been described in pre‐osteoblastic MC3T3‐E1 cells. However, S100 proteins could have additional effects on bone. The goal of the current study was to determine effects of increased extracellular S100 on osteoclastogenesis. We first determined the direct effects of S100 on pre‐osteoclast proliferation and osteoclastic differentiation. RANKL‐treated RAW 264.7 cell proliferation and TRAP activity were significantly inhibited by S100, and the number and size of TRAP‐positive multinucleated cells were decreased. We then determined whether S100 could affect osteoclastogenesis by an indirect process by examining effects of conditioned media from S100‐treated MC3T3‐E1 cells on osteoclastogenesis. In contrast to the direct inhibitory effect of S100, the conditioned media promoted RAW 264.7 cell proliferation and TRAP activity, with a trend toward increased TRAP‐positive multinucleated cells. S100 treatment of the MC3T3‐E1 cells for 14 days did not significantly affect alkaline phosphatase, M‐CSF, or OPG gene expression. RANKL was undetectable in both untreated and treated cells. The treatment slightly decreased MC3T3‐E1 cell proliferation. Interestingly, S100 treatment increased expression of RAGE by the MC3T3‐E1 cells. This suggested the possibility that S100 could increase soluble RAGE, which acts as a decoy receptor for S100. This decrease in availability of S100, an inhibitor of pre‐osteoclast proliferation, could contribute to osteoclastogenesis, ultimately resulting in increased bone resorption. J. Cell. Biochem. 107: 917–925, 2009. © 2009 Wiley‐Liss, Inc.