Migration Of Pancreatic Cancer Cells Research Articles

TAp73 Inhibits EMT and Cell Migration in Pancreatic Cancer Cells through Promoting SMAD4 Expression and SMAD4-Dependent Inhibition of ERK Activation.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease due to early metastatic spread, late diagnosis and the lack of efficient therapies. A major driver of cancer progression and hurdle to successful treatment is transforming growth factor (TGF)-β. Recent data from pancreatic cancer mouse models showed that transcriptionally active p73 (TAp73), a p53 family member, inhibits tumor progression through promoting tumor suppressive canonical TGF-β/Smad signaling, while preventing non-canonical TGF-β signaling through extracellular signal-regulated kinases (ERK)1/2. Here, we studied whether this mechanism also operates in human PDAC. Using the PDAC-derived tumor cell lines PANC-1, HPAFII and L3.6pl, we showed that TAp73 induces the expression of the epithelial marker and invasion suppressor E-cadherin and the common-mediator Smad, SMAD4, while at the same time suppressing expression of the EMT master regulator SNAIL and basal and TGF-β1-induced activation of ERK1 and ERK2. Using dominant-negative and RNA interference-based inhibition of SMAD4 function, we went on to show that inhibition of ERK activation by TAp73 is mediated through SMAD4. Intriguingly, both SMAD4 and the α isoform of TAp73-but not the β isoform-interfered with cell migration, as shown by xCELLigence technology. Our findings highlighted the role of TAp73-SMAD4 signaling in tumor suppression of human PDAC and identified direct inhibition of basal and TGF-β-stimulated pro-invasive ERK activation as an underlying mechanism.

Pancreatic stellate cell-derived exosomal tRF-19-PNR8YPJZ promotes proliferation and mobility of pancreatic cancer through AXIN2.

The pancreatic stellate cells (PSCs) play an important role in the development of pancreatic cancer (PC) through mechanisms that remain unclear. Exosomes secreted from PSCs act as mediators for communication in PC. This study aimed to explore the role of PSC-derived exosomal small RNAs derived from tRNAs (tDRs) in PC cells. Exosomes from PSCs were extracted and used to detect their effects on PC cell proliferation, migration and invasion. Exosomal tDRs profiling was performed to identify PSC-derived exosomal tDRs. ISH and qRT-PCR were used to examine the tRF-19-PNR8YPJZ levels and clinical value in clinical samples. The biological function of exosomal tRF-19-PNR8YPJZ was determined using the CCK-8, clone formation, wound healing and transwell assays, subcutaneous tumour formation and lung metastatic models. The relationship between the selected exosomal tRF-19-PNR8YPJZ and AXIN2 was determined by RNA sequencing, luciferase reporter assay. PSC-derived exosomes promoted the proliferation, migration, and invasion of PC cells. Novel and abundant tDRs are found to be differentially expressed in PANC-1 cells after treatment with PSC-derived exosomes, such as tRF-19-PNR8YPJZ. PC tissue samples showed markedly higher levels of tRF-19-PNR8YPJZ than normal controls. Patients with PC exhibiting high tRF-19-PNR8YPJZ expression had a highly lymph node invasion, metastasis, perineural invasion, advanced clinical stage and poor overall survival. Exosomal tRF-19-PNR8YPJZ from PSCs targeted AXIN2 in PC cells and decreased its expression, thus activating the Wnt pathway and promoting proliferation and metastasis. Exosomal tRF-19-PNR8YPJZ from PSCs promoted proliferation and metastasis in PC cells via AXIN2.

Doublecortin-like kinase 1 activates Hippo pathway to promote migration, invasion and proliferation of pancreatic cancer cells

Objective: To explore the mechanism of Doublecortin-like kinase 1 (DCLK1) in promoting cell migration, invasion and proliferation in pancreatic cancer. Methods: The correlation between DCLK1 and Hippo pathway was analyzed using TCGA and GTEx databases and confirmed by fluorescence staining of pancreatic cancer tissue microarrays. At the cellular level, immunofluorescence staining of cell crawls and western blot assays were performed to clarify whether DCLK1 regulates yes associated protein1 (YAP1), a downstream effector of the Hippo pathway. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to analyze the expressions of YAP1 binding transcription factor TEA-DNA binding proteins (TEAD) and downstream malignant behavior-promoting molecules CYR61, EDN1, AREG, and CTGF. Transwell test of the DCLK1-overexpressing cells treated with the Hippo pathway inhibitor Verteporfin was used to examine whether the malignant behavior-promoting ability was blocked. Analysis of changes in the proliferation index of experimental cells used real-time label-free cells. Results: TCGA combined with GTEx data analysis showed that the expressions of DCLK1 and YAP1 molecules in pancreatic cancer tissues were significantly higher than those in adjacent tissues (P<0.05). Moreover, DCLK1was positively correlated with the expressions of many effectors in the Hippo pathway, including LATS1 (r=0.53, P<0.001), LATS2 (r=0.34, P<0.001), MOB1B (r=0.40, P<0.001). In addition, the tissue microarray of pancreatic cancer patients was stained with multicolor fluorescence, indicated that the high expression of DCLK1 in pancreatic cancer patients was accompanied by the up-regulated expression of YAP1. The expression of DCLK1 in pancreatic cancer cell lines was analyzed by the CCLE database. The results showed that the expression of DCLK1 in AsPC-1 and PANC-1 cells was low. Thus, we overexpressed DCLK1 in AsPC-1 and PANC-1 cell lines and found that DCLK1 overexpression in pancreatic cancer cell lines promoted YAP1 expression and accessible to the nucleus. In addition, DCLK1 up-regulated the expression of YAP1 binding transcription factor TEAD and increased the mRNA expression levels of downstream malignant behavior-promoting molecules. Finally, Verteporfin, an inhibitor of the Hippo pathway, could antagonize the cell's malignant behavior-promoting ability mediated by high expression of DCLK1. We found that the number of migrated cells with DCLK1 overexpressing AsPC-1 group was 68.33±7.09, which was significantly higher than 22.00±4.58 of DCLK1 overexpressing cells treated with Verteporfin (P<0.05). Similarly, the migration number of PANC-1 cells overexpressing DCLK1 was 65.66±8.73, which was significantly higher than 37.00±6.00 of the control group and 32.33±9.61 of Hippo pathway inhibitor-treated group (P<0.05). Meanwhile, the number of invasive cells in the DCLK1-overexpressed group was significantly higher than that in the DCLK1 wild-type group cells, while the Verteporfin-treated DCLK1-overexpressed cells showed a significant decrease. In addition, we monitored the cell proliferation index using the real-time cellular analysis (RTCA) assay, and the proliferation index of DCLK1-overexpressed AsPC-1 cells was 0.66±0.04, which was significantly higher than 0.38±0.01 of DCLK1 wild-type AsPC-1 cells (P<0.05) as well as 0.05±0.03 of DCLK1-overexpressed AsPC1 cells treated with Verteporfin (P<0.05). PANC-1 cells showed the same pattern, with a proliferation index of 0.77±0.04 for DCLK1-overexpressed PANC-1 cells, significantly higher than DCLK1-overexpressed PANC1 cells after Verteporfin treatment (0.14±0.05, P<0.05). Conclusion: The expression of DCLK1 is remarkably associated with the Hippo pathway, it promotes the migration, invasion, and proliferation of pancreatic cancer cells by activating the Hippo pathway.

TMPOP2 Inhibition Suppresses Pancreatic Cancer Cell Migration and Development by Repressing The JNK/STAT3 Pathway.

Pancreatic cancer is a malignancy with a poor prognosis and high mortality. The lincRNA TMPOP2 is highly expressed in gynecological cancers and may exhibit tumor-promoting functions. However, the function of TMPOP2 in pancreatic cancer is limited. TMPOP2 expression in pancreatic cancer and adjacent tissues is analyzed from The Cancer Genome Atlas (TCGA) and GTEx database. It shows the high expression of TMPOP2 in pancreatic cancer tissues. Similar results are observed in resected pancreatic adenocarcinoma tumors and adjacent tissues from 20 patients and the relative cell lines. When the pancreatic cell lines aretransfected with si-TMPOP2, it shows that TMPOP2 downregulation inhibits the cells migration and EMT. Furthermore, the potential mechanism isexplored by detecting the expression of c-Jun N-terminal kinase (JNK), phosphorylated JNK, signal transducer and activator of transcription 3 (STAT3), and phosphorylated STAT3. It suggests that TMPOP2 knockdown inactivates JNK and STAT3 phosphorylation. When a JNK activator (anisomycin) is added to the cells with si-NC or si-TMPOP2, it canpartially reverse the migration and EMT inhibition of the cells with inhibited TMPOP2. TMPOP2 inhibition suppresses the migration and EMT of pancreatic cancer by repressing the JNK/STAT3 pathway. Thus, this may be a novel target for pancreatic cancer therapy.

DTX3L Accelerates Pancreatic cancer Progression via FAK/PI3K/AKT Axis.

DTX3L (Deltex E3 ubiquitin ligase 3L) is an E3 ubiquitin ligase, a member of the deltex family. It is also known as B-lymphoma and BAL-associated protein (BBAP). DTX3L has been proven to play an important role in various tumor development; however, its role in pancreatic cancer remains unknown. So, we analyzed the DTX3L expression in pancreatic cancer based on the TCGA database and verified it in our samples by qRT‑PCR and western blot. We identified that DTX3L was highly expressed in pancreatic cancer, and its expression level was significantly negatively correlated with patients' survival. Using CCK8, colony formation, transwell, and wound healing assays, we found that upregulated DTX3L promotes pancreatic cancer cell proliferation, invasion, and migration. Mechanically, DTX3L combined with EGFR (epidermal growth factor receptor) and prevented the ubiquitination degradation of it. Upregulated EGFR activated the FAK/PI3K/Akt pathway and promoted the progression of pancreatic cancer. Moreover, we found that DTX3L can weaken pancreatic cancer cells' sensitivity to chemotherapy using the orthotopic implant tumor model. In conclusion, DTX3L accelerates pancreatic cancer progression by EGFR dependent FAK/PI3K/Akt pathway activation and may become a potential target for pancreatic cancer treatment.

NSUN2 stimulates tumor progression via enhancing TIAM2 mRNA stability in pancreatic cancer

NSUN2 is a nuclear RNA methyltransferase which catalyzes 5-methylcytosine (m5C), a posttranscriptional RNA modification. Aberrant m5C modification has been implicated in the development of multiple malignancies. However, its function in pancreatic cancer (PC) needs to be elucidated. Herein, we determined that NSUN2 was overexpressed in PC tissues and related to aggressive clinical features. Silence of NSUN2 by lentivirus weakened the capability of proliferation, migration and invasion of PC cells in vitro and inhibited the growth and metastasis of xenograft tumors in vivo. Contrarily, overexpression of NSUN2 stimulated PC growth and metastasis. Mechanistically, m5C-sequencing (m5C-seq) and RNA-sequencing (RNA-seq) were carried out to identify downstream targets of NSUN2 and results showed that loss of NSUN2 led to decreased m5C modification level concomitant with reduced TIAM2 mRNA expression. Further validation experiments proved that NSUN2 silence accelerated the decay of TIAM2 mRNA in a YBX1-dependent manner. Additionally, NSUN2 exerted its oncogenic function partially through enhancing TIAM2 transcription. More importantly, disruption of the NSUN2/TIAM2 axis repressed the malignant phenotype of PC cells through blocking epithelial-mesenchymal transition (EMT). Collectively, our study highlighted the critical function of NSUN2 in PC and provided novel mechanistic insights into NSUN2/TIAM2 axis as promising therapeutic targets against PC.

Multiple Omics Analysis of the Role of RBM10 Gene Instability in Immune Regulation and Drug Sensitivity in Patients with Lung Adenocarcinoma (LUAD).

The RNA-binding protein RBM10 can regulate apoptosis during the proliferation and migration of pancreatic cancer, endometrial cancer, and osteosarcoma cells; however, the molecular mechanism underlying lung adenocarcinoma is rarely reported. Recent studies have detected multiple truncated and missense mutations in RBM10 in lung adenocarcinoma, but the role of RBM10 in lung adenocarcinoma is unclear. This study mainly explored the immune regulation mechanism of RBM10 in the development of lung adenocarcinoma and its influence on sensitivity to targeted therapy drugs. The transcriptome data of CGAP were used to analyze the RNA-seq data of lung adenocarcinoma patients from different subgroups by using the CIBERSORT algorithm to infer the relative proportion of various immune infiltrating cells, and Spearman correlation analysis was performed to determine the gene expression and immune cell content. In addition, this study utilized drug trial data from the GDSC database. The IC50 estimates for each specific targeted therapy were obtained by using a regression method, and the regression and prediction accuracy were tested via ten cross-validations with the GDSC training set. An immunohistochemical test was performed on the samples of 20 patients with lung adenocarcinoma in the subcomponent analysis of immune cells, and the protein expression of RBM10 in lung adenocarcinoma tissues was verified by cellular immunofluorescence assays. Nucleic acids were extracted at low temperatures, and qRT-PCR was used to verify the expression levels of the mRNA of RBM10 in lung adenocarcinoma tissues and normal tissues (p < 0.05). After screening and inclusion using a machine language, the results showed that RBM10 was significantly highly expressed in the lung adenocarcinoma tissues. The related signaling pathways were mainly concentrated in ncRNA processing, rRNA metabolic processes, ribosome biogenesis, and the regulation of translation. The qRT-PCR for 20 lung adenocarcinoma tissues showed that the expression of RBM10 in these tissues was significantly different from that in normal tissues (p = 0.0255). Immunohistochemistry analysis and cell immunofluorescence staining also confirmed that RBM10 was involved in the immune regulation of lung adenocarcinoma tissues, and the number of immune cell aggregations was significantly higher than that of the control group. RBM10 regulates B cell memory-CIBERSORT (p = 0.042) and B cell memory-CIBERSOTRT-abs (p = 0.027), cancer-associated fibroblast-EPIC (p = 0.001), cancer-associated fibroblast- MCPCounter (p = 0.0037), etc. The risk score was significantly associated with the sensitivity of patients to lapatinib (p = 0.049), nilotinib (p = 0.015), pazopanib (p = 0.001), and sorafenib (p = 0.048). RBM10 can inhibit the proliferation and invasion of lung adenocarcinoma cells through negative regulation and promote the apoptosis of lung adenocarcinoma cells through immunomodulatory mechanisms. The expression level of RBM10 affects the efficacy of targeted drug therapy and the survival prognosis of lung adenocarcinoma patients, which has a certain guiding significance for the clinical treatment of these patients.

Epigenetic reprogramming-induced guanidinoacetic acid synthesis promotes pancreatic cancer metastasis and transcription-activating histone modifications

BackgroundPancreatic ductal adenocarcinoma (PDAC) tends to undergo distant metastasis, especially liver metastasis, leading to a poor prognosis. Metabolic remodelling and epigenetic reprogramming are two important hallmarks of malignant tumours and participate in regulating PDAC tumorigenesis and metastasis. However, the interaction between these two processes during PDAC metastasis has not been fully elucidated.MethodsWe performed metabolomics analysis to identify the critical metabolites associated with PDAC liver metastasis and focused on guanidinoacetic acid (GAA). Intracellular GAA content was significantly increased in liver metastatic PDAC cells compared to primary cancer cells in mouse xenograft tumour models. The effects of GAA supplementation and glycine amidinotransferase (GATM) knockdown on PDAC metastasis were assessed by analysing cell migration, filopodia formation, epithelial-mesenchymal transition (EMT), and in vivo metastasis in different cell and animal models. Next, ChIP‒qPCR, 3C‒qPCR, and CRISPRi/dCas9-KRAB experiments were used to validate the “epigenome-metabolome" mechanism. Finally, the results of in vitro approaches, including RNA-seq, CUT&RUN, RT‒qPCR, and western blot analyses, as well as luciferase reporter gene assay and transwell assay, revealed the GAA-c-Myc-HMGA axis and transcription-activating histone modifications reprogramming.ResultsA high level of intracellular GAA was associated with PDAC liver metastasis. GAA could promote the migration, EMT, and liver metastasis of pancreatic cancer cells in vitro and in vivo. Next, we explored the role of GATM-mediated de novo GAA synthesis in pancreatic cancer metastasis. High expression of GATM was positively correlated with advanced N stage in PDAC. Knockdown of GATM significantly reduced the intracellular level of GAA, suppressed EMT, and inhibited PDAC liver metastasis, and these effects were attenuated by GAA supplementation. Mechanistically, we identified the active enhancers looped to the Gatm gene locus that promoted GATM expression and PDAC liver metastasis. Furthermore, we found that GAA promoted cell migration and EMT by regulating c-Myc-mediated high mobility group AT-hook protein expression. Moreover, GAA increased the H3K4me3 modification level by upregulating histone methyltransferases, which induced the transcription of metastasis-related genes, including Myc.ConclusionsThese findings revealed the critical role of the epigenome-metabolome interaction in regulating PDAC liver metastasis and suggested potential therapeutic strategies targeting GAA metabolism and epigenetic regulatory mechanisms.

PITX2 in pancreatic stellate cells promotes EMT in pancreatic cancer cells via the Wnt/β-catenin pathway.

Since the prognosis of patients with pancreatic cancer is very poor and there is a lack of treatment methods, this study is performed to investigate the function of PITX2 in pancreatic stellate cells (PSCs) in the progression of pancreatic cancer. Scientific hypotheses are proposed according to bioinformatics analysis and tissue microarray analysis. Stable knockdown of PITX2 in PSCs is achieved through lentiviral infection. The relative expressions of PITX2, α-SMA, vimentin, CTNNB1, AXIN1 and LEF1 are measured in wild-type PSCs and PITX2-knockdown PSCs. Proliferative capacity is measured by EdU assay. After coculture with PSCs, the proliferation, invasion and migration capacity of pancreatic cancer cells are tested. EMT and Wnt/β-catenin downstream genes of pancreatic cancer cells are investigated to reveal the potential mechanism. Bioinformatics analysis reveals that the PITX2 gene is highly expressed in stromal cells in pancreatic cancer and is correlated with squamous-type PDAC. Analysis of PDAC tissue microarray further demonstrates that high PITX2 level in stromal cells is correlated with poor prognosis in PDAC. After stable knockdown of PITX2 in PSCs, the relative protein levels of α-SMA, vimentin, CTNNB1, AXIN1 and LEF1 are decreased, and the proliferative capacity of PSCs is also decreased. After coculture with PSCs, in which PITX2 expression is downregulated, the proliferation, invasion and migration capacities of pancreatic cancer cells are inhibited. Thus, our results show that PITX2-silenced PSCs inhibit the growth, migration and invasion of pancreatic cancer cells via reduced EMT and Wnt/β-catenin signaling.

USP33 promotes pancreatic cancer malignant phenotype through the regulation of TGFBR2/TGFβ signaling pathway

Pancreatic cancer (PC) ranked fourth among cancer-related death worldwide with a survival rate less than 5%. The abnormal proliferation and distant metastasis are major obstacles for the diagnosis and treatment of pancreatic cancer, therefore, it is urgent for researchers to uncover the molecular mechanisms underlying the PC proliferation and metastasis. In current study, we found that USP33, a member of deubiquitinating enzyme family, was upregulated among PC samples and cells, meanwhile, the high expression of USP33 correlated with poor prognosis of patients. Function experiments revealed that USP33 overexpression promoted the proliferation, migration and invasion of PC cells while the inhibition of USP33 expression in PC cells exhibited the opposite effect. The mass spectrum and luciferase complementation assay screened TGFBR2 as the potential binding protein of USP33. Mechanistically, USP33 triggered the deubiquitination of TGFBR2 and prevented its degradation by lysosome, therefore promoted TGFBR2 accumulation in cell membrane and eventually contributed to the sustained activation of TGF-β signaling. Moreover, our results revealed that the activation of TGF-β targeted gene ZEB1 promoted the transcription of USP33. In conclusion, our study found that USP33 contributed to the proliferation and metastasis of pancreatic cancer through a positive feedback loop with TGF-β signaling pathway. Moreover, this study suggested that USP33 may serve as a potential prognostic and therapeutic target in PC.

CA 19-9 but Not IGF-1/IGFBP-2 Is a Useful Biomarker for Pancreatic Ductal Adenocarcinoma (PDAC) and Chronic Pancreatitis (CP) Differentiation.

There are still no effective diagnostic and prognostic biomarkers in pancreatic ductal adenocarcinoma (PDAC). The differentiation between PDAC and chronic pancreatitis (CP) is often challenging. The inflammatory mass in the course of CP causes diagnostic difficulties in differentiating them from neoplastic lesions and, thus, delays the initiation of radical treatment. Insulin-like growth factor 1 (IGF-1) and insulin-like growth factor-binding protein 2 (IGFBP-2) form a network involved in PDAC development. The role of IGFs in promoting pancreatic cancer cell proliferation, survival, and migration is well established, and their ability to stimulate tumor growth and metastasis is well documented. The aim of the study was to evaluate the usability of IGF-1, IGFBP-2, and IGF-1/IGFBP-2 ratio in PDAC and CP differentiation. The study included 137 patients: 89 patients with PDAC and 48 patients with CP. All subjects were tested for the levels of IGF-1 and IGFBP-2 using the ELISA method (Corgenix UK Ltd. R&D Systems), along with the level of CA 19-9 in serum. Additionally, the IGF-1/IGFBP-2 ratio was calculated. Further analyses used logit and probit models with varying determinants in order to discern between PDAC and CP patients. The models served as a basis for AUROC calculation. The mean IGF-1 serum level was equal to 52.12 ± 33.13 ng/mL in PDAC vs. 74.23 ± 48.98 ng/mL in CP (p = 0.0053). The mean level of IGFBP-2 was equal to 305.95 ± 194.58 ng/mL in PDAC vs. 485.43 ± 299 ng/mL in CP (p = 0.0002). The mean CA 19-9 serum concentration was 434.95 ± 419.98 U/mL in PDAC vs. 78.07 ± 182.36 U/mL in CP (p = 0.0000). The mean IGF-1/IGFBP-2 ratio was 0.213 ± 0.14 in PDAC vs. 0.277 ± 0.33 in CP (p = 0.1914). The diagnostic usefulness of indicators for the purpose of PDAC and CP differentiation was assessed by means of AUROC comparison. The AUROCs of IGF-1, IGFBP-2, and IGF-1/IGFBP-2 ratio ranged below 0.7, being lower than the AUROC of CA 19-9 (0.7953; 0.719 within 95% CI). Together, the CA 19-9 and IGFBP-2 AUROCs also ranged below 0.8. When age was included, the AUROC increased to 0.8632, and its 95% confidence interval held above the 0.8 limit. The sensitivity of the used markers was not correlated to the stage of pancreatic PDAC. The presented results indicate that CA 19-9 is a marker demonstrating high potential for PDAC and CP differentiation. The inclusion of additional variables into the model, such as the serum level of IGF-1 or IGFBP-2, slightly increased the sensitivity in differentiating CP from PDAC. The IGF-1/IGFBP-2 ratio turned out to be a good marker of pancreatic diseases, but insufficient for the purpose of CP and PDAC differentiation.

Neuropeptide Y Peptide Family and Cancer: Antitumor Therapeutic Strategies.

Currently available data on the involvement of neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) and their receptors (YRs) in cancer are updated. The structure and dynamics of YRs and their intracellular signaling pathways are also studied. The roles played by these peptides in 22 different cancer types are reviewed (e.g., breast cancer, colorectal cancer, Ewing sarcoma, liver cancer, melanoma, neuroblastoma, pancreatic cancer, pheochromocytoma, and prostate cancer). YRs could be used as cancer diagnostic markers and therapeutic targets. A high Y1R expression has been correlated with lymph node metastasis, advanced stages, and perineural invasion; an increased Y5R expression with survival and tumor growth; and a high serum NPY level with relapse, metastasis, and poor survival. YRs mediate tumor cell proliferation, migration, invasion, metastasis, and angiogenesis; YR antagonists block the previous actions and promote the death of cancer cells. NPY favors tumor cell growth, migration, and metastasis and promotes angiogenesis in some tumors (e.g., breast cancer, colorectal cancer, neuroblastoma, pancreatic cancer), whereas in others it exerts an antitumor effect (e.g., cholangiocarcinoma, Ewing sarcoma, liver cancer). PYY or its fragments block tumor cell growth, migration, and invasion in breast, colorectal, esophageal, liver, pancreatic, and prostate cancer. Current data show the peptidergic system's high potential for cancer diagnosis, treatment, and support using Y2R/Y5R antagonists and NPY or PYY agonists as promising antitumor therapeutic strategies. Some important research lines to be developed in the future will also be suggested.

A bioinformatics analysis and an experimental validation of the hypoxia-related prognostic model.

Hypoxia plays an important role in the development of pancreatic cancer (PCA). However, there is few research on the application of hypoxia molecules in predicting the prognosis of PCA. We aimed to establish a prognostic model based on hypoxia-related genes (HRGs) for PCA to discover new biomarkers, and to reveal the potential of this prognostic model for evaluating the tumor microenvironment (TME). Univariate Cox regression analysis was used to identify HRGs associated with overall survival (OS) of PCA samples. A hypoxia-related prognostic model was established based on least absolute shrinkage and selection operator (LASSO) regression analysis in The Cancer Genome Atlas (TCGA) cohort. The model was validated in the Gene Expression Omnibus (GEO) datasets. The Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) algorithm was used to estimate the infiltration of immune cells. A wound healing assay and transwell invasion assay were used to explore the biological functions of target genes in PCA. A total of 18 HRGs were differentially expressed between the tumor and normal pancreatic tissue, 4 (BHLHE40, ENO1, SDC4, and TGM2) of which were selected to construct a prognostic model. According to this model, patients in the high-risk group had a less favorable prognosis. Furthermore, the proportion of M0 macrophages was significantly higher in high-risk tissue-type patients, whereas naïve B cells, plasma cells, CD8+ T cells, and activated CD4+ memory T cells were significantly lower. The expression of BHLHE40 in PCA cells was significantly up-regulated under hypoxic conditions. Moreover, BHLHE40 was shown to regulate the transcription and expression of the downstream target gene TLR3. The wound healing assay and transwell invasion assay indicated that BHLHE40 mediated PCA cell migration and invasion by targeting the downstream gene TLR3. The hypoxia-related prognostic model established by the expression pattern of 4 HRGs can be used to predict the prognosis and assess the TME of PCA patients. Mechanically, activation of the BHLHE40/TLR3 axis is responsible for the promoted invasion and migration of PCA cells in a hypoxic environment.

Thymoquinone affects the gemcitabine sensitivity of pancreatic cancer by regulating collagen via hypoxia inducible factor-1α.

Objective: To clarify the potential therapeutic effects of thymoquinone (TQ) on pancreatic cancer and its gemcitabine (GEM) sensitivity. Methods: The expression levels of hypoxia inducible factor-1α (HIF-1α), collagens (COL1A1, COL3A1, and COL5A1), and transforming growth factor-β1 (TGFβ1) in pancreatic cancer and para-carcinoma tissues were compared using immunohistochemical methods, and their relationships with TNM staging were analyzed. The effects of TQ on apoptosis, migration, invasion, and GEM sensitivity of pancreatic cancer cells were assessed using in vitro and in vivo experiments. Western blot and immunohistochemistry were used to detect the expression levels of HIF-1α, extracellular matrix (ECM) production pathway-related proteins, and TGFβ/Smad signaling pathway-related proteins. Results: The expression levels of HIF-1α, COL1A1, COL3A1, COL5A1, and TGFβ1 in pancreatic cancer tissues were significantly higher than those in para-carcinoma tissues and correlated with TNM staging (p < 0.05). TQ and GEM administration inhibited the migration and invasion of the human pancreatic cancer cell line PANC-1 and promoted the apoptosis of PANC-1 cells. The combination of TQ and GEM was more effective than GEM alone. Western blot analysis showed that the expression levels of HIF-1α, ECM production pathway-related proteins, and TGFβ/Smad signaling pathway-related proteins were significantly decreased when TQ was used to treat PANC-1 cells (p < 0.05), and the expression levels of these proteins in the TQ + GEM group were significantly more decreased than those in the GEM group. Overexpression or knockdown of HIF-1α in PANC-1 cells showed the same effects as those induced by TQ administration. In vivo experiments showed that in PANC-1 tumor-bearing mice, tumor volume and tumor weight in mice treated with GEM and TQ were significantly lower than those in control or GEM-treated mice, whereas cell apoptosis was significantly increased (p < 0.05). Western blot and immunohistochemistry results showed that the levels of HIF-1α, ECM production pathway-related proteins, and TGFβ/Smad signaling pathway-related proteins in the GEM + TQ treatment group were further decreased compared to the control group or the GEM treatment group (p < 0.05). Conclusion: In pancreatic cancer cells, TQ can promote apoptosis, inhibit migration, invasion, and metastasis, and enhance the sensitivity to GEM. The underlying mechanism may involve the regulation of ECM production through the TGFβ/Smad pathway, in which HIF-1α plays a key role.

LncRNA FAM83A-AS1 promotes epithelial–mesenchymal transition of pancreatic cancer cells via Hippo pathway

ABSTRACT Background Long non-coding RNAs (lncRNAs) have been proved to play a vital role in pancreatic cancer (PC). However, the role of lncRNA FAM83A-AS1 in PC remains unclear. In this study, we explored the biological function and underlying mechanism of FAM83A-AS1 in PC cells. Methods The FAM83A-AS1 expression was assessed via public databases and validated by qRT-PCR. The biofunction and immune cell infiltration of FAM83A-AS1 were analyzed through GO, KEGG, GESA and ssGSEA. The migration, invasion and proliferation abilities of PC cells were examined by Transwell, wound healing, CCK8 and colony formation. The EMT and Hippo pathway markers were evaluated by western blot. Results FAM83A-AS1 expression was higher in PC tissues and cells than normal. Additionally, FAM83A-AS1 was associated with poor prognosis of PC and involved in cadherin binding and immune infiltration. Subsequently, we proved FAM83A-AS1 overexpression enhanced the migration, invasion and proliferation abilities of PC cells, whereas FAM83A-AS1 downregulation inhibited those. Moreover, western blot results showed that FAM83A-AS1 knockdown increased the E-cadherin expression and decreased the expression of N-cadherin, β-catenin, Vimentin, Snail and Slug. On the contrary, FAM83A-AS1 upregulation results in the opposite effects. Besides, FAM83A-AS1 overexpression inhibited the expression of p-YAP, p-MOB1, p-Lats1, SAV1, MST1 and MST2 as well as the results of FAM83A-AS1 knockdown were opposite. Conclusion FAM83A-AS1 promoted EMT of PC cells via Hippo signaling inactivation and may be a potential diagnosis and prognosis target.

EZH2 regulates pancreatic cancer cells through E2F1, GLI1, CDK3, and Mcm4

Pancreatic cancer (PC) is one of the most common malignant tumors in digestive tract. To explore the role of epigenetic factor EZH2 in the malignant proliferation of PC, so as to provide effective medical help in PC. Sixty paraffin sections of PC were collected and the expression of EZH2 in PC tissues was detected by immunohistochemical assay. Three normal pancreas tissue samples were used as controls. The regulation of EZH2 gene on proliferation and migration of normal pancreatic cell and PC cell were determined by MTS, colony forming, Ki-67 antibody, scratch and Transwell assays. Through differential gene annotation and differential gene signaling pathway analysis, differentially expressed genes related to cell proliferation were selected and verified by RT-qPCR. EZH2 is mainly expressed in the nuclei of pancreatic tumor cells, but not in normal pancreatic cells. The results of cell function experiments showed that EZH2 overexpression could enhance the proliferation and migration ability of PC cell BXPC-3. Cell proliferation ability increased by 38% compared to the control group. EZH2 knockdown resulted in reduced proliferation and migration ability of cells. Compared with control, proliferation ability of cells reduced by 16%-40%. The results of bioinformatics analysis of transcriptome data and RT-qPCR demonstrated that EZH2 could regulate the expression of E2F1, GLI1, CDK3 and Mcm4 in normal and PC cells. The results revealed that EZH2 might regulate the proliferation of normal pancreatic cell and PC cell through E2F1, GLI1, CDK3 and Mcm4.

Hsa_circRNA_001859 regulates pancreatic cancer progression and epithelial-mesenchymal transition through the miR-21-5p/SLC38A2 pathway.

This study attempts to investigate whether hsa_circRNA_001859 (circ_001859) could regulate the proliferation and invasion of pancreatic cancer through the miR-21-5p/SLC38A2 pathway. GSE79634 microarray was analyzed with R package. The expression of circ_001859 in pancreatic cancer tissues and cells was verified by qRT-PCR. After the overexpression of circ_001859, cell proliferation, cell migration and invasion were verified by colony formation and transwell assay. The targeting relationship between miR-21-5p and circ_001859 was predicted by TargetScan and was verified by dual luciferase reporter assay, RNA pull down and qRT-PCR. The effect of miR-21-5p on cell proliferation, migration and invasion were investigated by colony formation and transwell assay respectively. Similarly, the targeting relationship between miR-21-5p and SLC38A2 was predicted by TargetScan and was verified by dual luciferase reporter assay, western blot and qRT-PCR. The effect of SLC38A2 on cell proliferation was investigated by colony formation. Circ_001859 was lowly expressed in pancreatic cancer tissues and cells. In vitro assays showed that overexpression of circ_001859 could inhibit the proliferation, migration and invasion of pancreatic cancer. In addition, this effect was also confirmed in xenograft transplantation model. Circ_001859 could be bind to miR-21-5p and sponge its expression in pancreatic cancer cells. Overexpression of miR-21-5p enhanced the proliferation, migration and invasion ability of pancreatic cancer cells, while the inhibition of miR-21-5p expression suppressed these abilities. Moreover, miR-21-5p directly targeted at SLC38A2 and inhibited SLC38A2 expression levels while circ_001859 up-regulated SLC38A2 levels. SLC38A2 expression knockdown enhanced cell proliferation but SLC38A2 overexpression resulted in decreased proliferation, and effects of SLC38A2 could be rescued by miR-21-5p and circ_001859. In addition, both QRT-PCR and immunofluorescence confirmed that circ_001859 could regulate tumor epithelial-mesenchymal transition (EMT) through the miR-21-5p/SLC38A2 pathway. This study suggests that circ_001859 may inhibit the proliferation, invasion and EMT of pancreatic cancer through the miR-21-5p/SLC38A2 pathway.

Identification of m7G-Related LncRNA Signature for Predicting Prognosis and Evaluating Tumor Immune Infiltration in Pancreatic Adenocarcinoma.

N7-Methylguanosine (m7G) modification holds significant importance in regulating posttranscriptional gene expression in epigenetics. Long non-coding RNAs (lncRNAs) have been demonstrated to play a crucial role in cancer progression. m7G-related lncRNA may be involved in the progression of pancreatic cancer (PC), although the underlying mechanism of regulation remains obscure. We obtained RNA sequence transcriptome data and relevant clinical information from the TCGA and GTEx databases. Univariate and multivariate Cox proportional risk analyses were performed to build a twelve-m7G-associated lncRNA risk model with prognostic value. The model was verified using receiver operating characteristic curve analysis and Kaplan-Meier analysis. The expression level of m7G-related lncRNAs in vitro was validated. Knockdown of SNHG8 increased the proliferation and migration of PC cells. Differentially expressed genes between high- and low-risk groups were identified for gene set enrichment analysis, immune infiltration, and potential drug exploration. We conducted an m7G-related lncRNA predictive risk model for PC patients. The model had independent prognostic significance and offered an exact survival prediction. The research provided us with better knowledge of the regulation of tumor-infiltrating lymphocytes in PC. The m7G-related lncRNA risk model may serve as a precise prognostic tool and indicate prospective therapeutic targets for PC patients.

M6A methylation regulates hypoxia-induced pancreatic cancer glycolytic metabolism through ALKBH5-HDAC4-HIF1α positive feedback loop

Pancreatic cancer (PC) is the most hypoxic cancer type among solid tumors. The dynamic changes of RNA N6-methyl-adenosine (m6A) contribute to tumor cells adaption to hypoxic microenvironmental. However, the regulatory mechanisms of hypoxia response in PC remains elusive. Here, we reported that the m6A demethylase ALKBH5 mediated a decrease of total mRNA m6A modification during hypoxia. Subsequently, methylated RNA immunoprecipitation sequencing (MeRIP-seq) combined with RNA sequencing (RNA-seq) revealed transcriptome-wide gene expression alteration and identified histone deacetylase type 4 (HDAC4) as a key target gene of m6A modification under hypoxic conditionds. Mechanistically, m6A methylation recognized by m6A reader-YTHDF2 enhanced the stability of HDAC4, and then promoted glycolytic metabolism and migration of PC cells. Our assays also demonstrated that hypoxia-induced HDAC4 enhanced HIF1a protein stability, and overexpressed HIF1a promoted transcription of ALKBH5 in hypoxic pancreatic cancer cells. Together, these results found a ALKBH5/HDAC4/HIF1α positive feedback loop for cellular response to hypoxia in pancreatic cancer. Our studies uncover the crosstalk between histone acetylation and RNA methylation modification on layer of epigenetic regulation.

ZEB2 Regulates the Migration and Invasion of PANC-1 Pancreatic Cancer Cells: An Experimental Study

To investigate the effects and mechanisms of zinc finger E-box binding homeobox transcription factor-2 ( ZEB2) on the proliferation, colony formation, migration, and invasion abilities and the epithelial-mesenchymal transition (EMT) of PANC-1 cells, a human pancreatic cancer cell line. Data on the expression of ZEB2 in pancreatic cancer tissues and paracancerous tissues from The Cancer Genome Atlas (TCGA) database were analyzed. PANC-1 pancreatic cancer cells were divided into si-NC group, si- ZEB2 group, pcDNA3.1 group, and pcDNA3.1- ZEB2 group. qRT-PCR and Western blot were conducted to confirm the effectiveness of ZEB2 knockdown or overexpression. CCK-8, colony formation, wound healing, and Transwell assays were conducted to examine the effects of ZEB2 on the proliferation, colony formation, migration, and invasion of PANC-1 cells. qRT-PCR and immunofluorescence assays were performed to examine the expression of E-cadherin and vimentin, the EMT markers, in the cells. Prediction of proteins interacting with ZEB2 was made through the STRING database. TCGA database analysis showed that the expression level of ZEB2 in pancreatic cancer tissues was significantly higher than that in adjacent tissues ( P<0.05). Compared with those of cells in the control group, the proliferation, colony formation, migration, and invasion of cells in the si- ZEB2 group were decreased ( P<0.05). Compared with those of cells in the pcDNA3.1 group, the proliferation, colony formation, migration and invasion of cells in the pcDNA3.1- ZEB2 group were increased (all P<0.05). According to the results of qRT-PCR and immunofluorescence assays, compared with those of the si-NC group, the expression of E-cadherin mRNA, an epithelial marker, in the si- ZEB2 group increased, while the expression of vimentin mRNA, an mesenchymal marker, and the protein decreased. Compared with those of the pcDNA3.1 group, the expression of E-cadherin mRNA in the PANC-1 cells of the pcDNA3.1- ZEB2 group decreased, while the expression of vimentin mRNA and the protein increased (all P<0.05). Analysis with the STRING database predicted that 10 proteins had close interaction with ZEB2. Overexpression of ZEB2 promotes the migration, invasion, and the EMT process of PANC-1 pancreatic cancer cells.