Targeting TLK2 inhibits the progression of gastric cancer by reprogramming amino acid metabolism through the mTOR/ASNS axis.

Several recent studies have suggested that TLKs are related to tumor progression. However, the function and mechanism of action of TLK2 in gastric cancer (GC) remain elusive. In this study, TLK2 was found to be significantly upregulated in patients with GC and was identified as an independent prognostic factor for GC. Consistently, TLK2 knockdown markedly reduced the aggressiveness of GC, whereas its overexpression had the opposite effect. IP-MS revealed that the effects of TLK2 on GC were mainly associated with metabolism reprogramming. TLK2 knockdown suppressed amino acid synthesis by downregulating the mTORC1 pathway and ASNS expression in GC cells. Mechanistically, mTORC1 directly interacts with the ASNS protein and inhibits its degradation. Further experiments validated that the ASNS protein was degraded via ubiquitination instead of autophagy. Inhibiting and activating the mTORC1 pathway can upregulate and downregulate ASNS ubiquitination, respectively, and the mTORC1 pathway can reverse the regulatory effects of TLK2 on ASNS. Furthermore, TLK2 was found to regulate the mRNA expression of ASNS. TLK2 directly interacted with ATF4, a transcription factor of ASNS, and promoted its expression. The kinase inhibitor fostamatinib significantly inhibited the proliferative, invasive, and migratory capabilities of GC cells by inhibiting TLK2 activity. Altogether, this study reveals a novel functional relationship between TLK2 and the mTORC1/ASNS axis in GC. Therefore, TLK2 may serve as a potential therapeutic target for GC.

Activated Pancreatic Stellate Cells Enhance the Warburg Effect to Cause the Malignant Development in Chronic Pancreatitis.

Chronic pancreatitis (CP) is a precancerous condition associated with pancreatic ductal adenocarcinoma (PDAC), but its evolutionary mechanism is unclear. Pancreatic stellate cells (PSCs) are closely related to the occurrence and development of CP and PDAC, but it is not clear whether PSCs play a key role in this "inflammation-cancer transition". Our research found that co-culture with activated PSCs promoted the proliferation, migration and invasion of normal pancreatic duct epithelial cells and pancreatic cancer cells. At the same time, activated PSCs had a significant effect on the expression of the glycolysis markers (pyruvate kinase M2, lactate dehydrogenase A, glucose transporter 1, hexokinase-II and monocarboxylate transporter 4; PKM2, LDHA, GLUT1, HK2 and MCT4) in normal pancreatic duct epithelial cells and pancreatic cancer cells and increased lactic acid production and glucose consumption in these two cells. In vivo experiments showed that the expression of the glycolysis markers in pancreatic duct epithelial cells and the marker protein (α-SMA) of activated PSCs in the pancreatic duct peripancreatic interstitium were higher in pancreatic cancer tissues and chronic pancreatitis tissues than in normal pancreatic tissues in both animals and humans. In addition, analysis of human tissue specimens showed that there is a correlation between the expression of glycolysis markers and α-SMA. These findings indicate that activated PSCs play an important role in the development and progression of chronic pancreatitis into pancreatic cancer by regulating and promoting aerobic glycolysis. Our research provides a new theoretical basis for further understanding the mechanism of CP malignancy and the selection of targets for reversing CP malignancy.

Overexpression of circRNA chr7:154954255-154998784+ in cancer-associated pancreatic stellate cells promotes the growth and metastasis of pancreatic cancer by targeting the miR-4459/KIAA0513 axis.

PURPOSE: Circular RNAs (circRNAs) have been reported to act as important regulators in pancreatic cancer. Abnormal expression of circRNAs in pancreatic cancer cells (PCCs) can promote the development of pancreatic cancer; however, the role of circRNAs in cancer-associated pancreatic stellate cells (CaPSCs) remains unclear. PATIENTS AND METHODS: In this study, we isolated CaPSCs from pancreatic cancer tissues from 5 pancreatic cancer patients and NaPSCs from normal pancreatic tissue from 5 patients with benign pancreatic disease. After the PSCs were co-cultured with the pancreatic cancer cell line PANC-1, a CCK-8 assay was used to detect PANC-1 proliferation ability, and CaPSCs1, which had the strongest promoting effect on PANC-1 proliferation, and NaPSCs1, which had the weakest effect, were screened. Then, the circRNA, microRNA (miRNA) and mRNA profiles between CaPSCs1 and NaPSCs1 were compared by RNA-seq. The candidate circRNA/miRNA/target protein axis was selected using bioinformatics analysis. circRNAs were silenced and miRNAs were overexpressed in CaPSCs1, and the expression of circRNAs, miRNAs and target proteins were detected by qRT-PCR and Western blot, respectively. At the same time, CCK8, wound healing, and Transwell assays were used to detect the proliferation, migration and invasion of PANC-1 cells in the different co-culture groups. Moreover, a tumour xenograft model was used to observe the tumorigenic ability of PANC-1 cells in different co-culture groups. Finally, immunohistochemistry was used to detect the expression of target proteins in PDAC tissues, and the clinicopathological features and prognosis were analysed. RESULTS: The expression of the differentially expressed RNAs identified by RNA-seq was verified by qRT-PCR, and the chr7:154954255-154998784+/miR-4459/KIAA0513 axis was selected from the candidate targets. Functional studies of PANC-1 cells after co-culture with chr7:154954255-154998784+-silenced CaPSCs1 showed that the proliferation, invasion and metastasis of PANC-1 cells decreased. Moreover, after chr7:154954255-154998784+ was silenced, the expression of miR-4459 in CaPSCs1 increased, and the expression of KIAA0513 decreased. When PANC-1 cells were co-cultured with CaPSCs1 with miR-4459 overexpression, they showed an increased ability to proliferate, invade and metastasize. Additionally, when miR-4459 was overexpressed in CaPSCs1, the expression of chr7:154954255-154998784+ and KIAA0513 decreased. Animal experiments revealed that silencing chr7:154954255-154998784+ in CaPSCs1 inhibited tumour growth in nude mice inoculated with CaPSCs1+PANC-1 cells. Finally, we performed immunohistochemistry and a prognostic analysis of KIAA0513 expression in paraffin tissue samples from patients with pancreatic cancer and found that high expression of KIAA0513 was associated with more aggressive clinicopathological factors. Furthermore, patients with high expression of KIAA0513 had worse disease-free survival (DFS) and overall survival (OS). CONCLUSION: Chr7:154954255-154998784+ may promote the development of pancreatic cancer through the miR-4459/KIAA0513 axis in CaPSCs and may be an important therapeutic target for patients with pancreatic cancer in the future.

A modified method for isolating human quiescent pancreatic stellate cells.

BACKGROUND: This study explored a simple, high-yield method for isolating quiescent human pancreatic stellate cells (PSCs) to provide sufficient and reliable raw materials for PSC-related studies. MATERIALS AND METHODS: Single-cell suspensions were prepared from normal human pancreatic tissue specimens using the gentleMACS™ tissue processor, which enhanced the yield and viability of the suspensions. Percoll density gradient centrifugation was then performed to isolate quiescent normal PSCs (NPSCs). Cell viability was determined by trypan blue staining, and the states of the NPSCs were determined by autofluorescence and oil red O staining. The purity of human activated PSCs (APSCs) was determined by immunofluorescence assays. RESULTS: The yield of NPSCs was ~(2.75±0.65)×106 cells/g. The maximum cell viability was 92%, whereas the maximum cell purity was 95%. CONCLUSION: The method employed in this study to isolate PSCs is a simple, high-yield and stable method that is worth popularizing.

Upregulated FFAR4 correlates with the epithelial-mesenchymal transition and an unfavorable prognosis in human cholangiocarcinoma.

BACKGROUND: Free fatty acid receptor 4 (FFAR4) is associated with the epithelial mesenchymal transition (EMT) and is involved in the progression of several types of cancer. However, the role of FFAR4 in cholangiocarcinoma (CCA) remains unclear. OBJECTIVE: The present study evaluated the diagnosis and prognosis of CCA using FFAR4 as a biomarker. METHODS: Immunohistochemistry was employed to detect expression of FFAR4 in 98 samples of CCA tissues and adjacent tissues. In addition, expression of E-cadherin, vimentin, Snail-1, CK7 and CK19 in the 98 samples of CCA tissues was detected, and relationships with FFAR4 were analyzed. Correlation between FFAR4 and clinical pathological factors and prognosis was also analyzed. RESULTS: FFAR4 was highly expressed in 72.4% (71/98) of CCA tissues and 29.6% (29/98) of adjacent tissues, with a statistically significant difference between the two tissue types (P< 0.05). A negative correlation between high expression FFAR4 and E-cadherin expression in CCA tissues was also observed (r=-0.445, P< 0.001), and high expression of FFAR4 was positively correlated with vimentin (r= 0.354, P< 0.001), Snail-1(r= 0.496, P< 0.001), CK7(r= 0.494, P< 0.001) and CK19 (r= 0.532, P< 0.001). Moreover, the degree of FFAR4 expression was associated with aggressive clinicopathological characteristics, such as histological grade, perineural invasion (PNI), lymph node metastasis (LNM), advanced TNM stage and preoperative serum CA19-9 level (P< 0.05). In terms of prognosis, CCA patients with high FFAR4 expression showed shorter disease-free survival (DFS) (P< 0.05) and overall survival (OS) (P< 0.05) than did patients with low FFAR4 expression. CONCLUSIONS: FFAR4 overexpression may mediate the process of CCA EMT. In addition, FFAR4 is promising as a new diagnostic molecule and therapeutic target for CCA.

Circular RNA Signature Predicts Gemcitabine Resistance of Pancreatic Ductal Adenocarcinoma.

Gemcitabine resistance is currently the main problem of chemotherapy for advanced pancreatic cancer patients. The resistance is thought to be caused by altered drug metabolism or reduced apoptosis of cancer cells. However, the underlying mechanism of Gemcitabine resistance in pancreatic cancer remains unclear. In this study, we established Gemcitabine resistant PANC-1 (PANC-1-GR) cell lines and compared the circular RNAs (circRNAs) profiles between PANC-1 cells and PANC-1-GR cells by RNA sequencing. Differentially expressed circRNAs were demonstrated using scatter plot and cluster heatmap analysis. Gene ontology and pathway analysis were performed to systemically map the genes which are functionally associated to those differentially expressed circRNAs identified from our data. The expression of the differentially expressed circRNAs picked up by RNAseq in PANC-1-GR cells was further validated by qRT-PCR and two circRNAs were eventually identified as the most distinct targets. Consistently, by analyzing plasma samples form pancreatic ductal adenocarcinoma (PDAC) patients, the two circRNAs showed more significant expression in the Gemcitabine non-responsive patients than the responsive ones. In addition, we found that silencing of the two circRNAs could restore the sensitivity of PANC-1-GR cells to Gemcitabine treatment, while over-expression of them could increase the resistance of normal PANC-1 and MIA PACA-2 cells, suggesting that they might serve as drug targets for Gemcitabine resistance. Furthermore, the miRNA interaction networks were also explored based on the correlation analysis of the target microRNAs of these two circRNAs. In conclusion, we successfully established new PANC-1-GR cells, systemically characterized the circRNA and miRNA profiles, and identified two circRNAs as novel biomarkers and potential therapeutic targets for Gemcitabine non-responsive PDAC patients.

The BET inhibitor I-BET762 inhibits pancreatic ductal adenocarcinoma cell proliferation and enhances the therapeutic effect of gemcitabine.

As one of the most fatal malignancies, pancreatic ductal adenocarcinoma (PDAC) has significant resistance to the currently available treatment approaches. Gemcitabine, the standard chemotherapeutic agent for locally advanced and metastatic PDAC, has limited efficacy, which is attributed to innate/acquired resistance and the activation of prosurvival pathways. Here, we investigated the in vitro efficacy of I-BET762, an inhibitor of the bromodomain and extraterminal (BET) family of proteins, in treating PDAC cell lines alone and in combination with gemcitabine (GEM). The effect of these two agents was also examined in xenograft PDAC tumors in mice. We found that I-BET762 induced cell cycle arrest in the G0/G1 phase and cell death and suppressed cell proliferation and metastatic stem cell factors in PDAC cells. In addition, the BH3-only protein Bim, which is related to chemotherapy resistance, was upregulated by I-BET762, which increased the cell death triggered by GEM in PDAC cells. Moreover, GEM and I-BET762 exerted a synergistic effect on cytotoxicity both in vitro and in vivo. Furthermore, Bim is necessary for I-BET762 activity and modulates the synergistic effect of GEM and I-BET762 in PDAC. In conclusion, we investigated the effect of I-BET762 on PDAC and suggest an innovative strategy for PDAC treatment.