Positive Crosstalk Between Hedgehog and NF-κB Pathways Is Dependent on KRAS Mutation in Pancreatic Ductal Adenocarcinoma

It has been shown that aberrant activation of the Hedgehog (Hh) and nuclear factor-kappa B (NF-κB) signaling pathways plays an important role in the pancreatic carcinogenesis, and KRAS mutation is a hallmark of pancreatic ductal adenocarcinoma (PDAC). Until now, the role of KRAS mutation in the context of crosstalk between Hh and NF-κB signaling pathways in PDAC has not been investigated. This study was to determine whether the crosstalk between the Hh and NF-κB pathways is dependent on KRAS mutation in PDAC. The correlation between Gli1, Shh, NF-κB p65 expression and KRAS mutation in PDAC tissues was firstly examined by immunohistochemistry. Next, Western blotting, qPCR, and immunofluorescence were conducted to examine the biological effects of interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α) as NF-κB signaling agonists, Shh as an Hh ligand alone or in combination with KRAS small interfering RNA (si-KRAS) in KRAS-mutant PDAC cells (MT-KRAS; SW1990 and Panc-1), wild-type KRAS PDAC cells (WT-KRAS; BxPC-3) and mutant KRAS knock-in BxPC-3 cells in vitro as well as tumor growth in vivo. KRAS mutation-dependent crosstalk between Hh and NF-κB in PDAC cells was further assessed by Ras activity and luciferase reporter assays. The aberrant Hh and NF-κB pathway activation was found in PDAC tissues with KRAS mutation. The same findings were confirmed in MT-KRAS PDAC cells and MT-KRAS knock-in BxPC-3 cells, whereas this activation was not observed in WT-KRAS PDAC cells. However, the activation was significantly down-regulated by KRAS silencing in MT-KRAS PDAC cells. Furthermore, MT-KRAS cancer cell proliferation and survival in vitro and tumor growth after inoculation with MT-KRAS cells in vivo were promoted by NF-κB and Hh signaling activation. The pivotal factor for co-activation of NF-κB and Hh signaling is MT-KRAS protein upregulation, showing that positive crosstalk between Hh and NF-κB pathways is dependent upon KRAS mutation in PDAC.

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CircNEIL3 regulatory loop promotes pancreatic ductal adenocarcinoma progression via miRNA sponging and A-to-I RNA-editing

Molecular Cancer ◽

10.1186/s12943-021-01333-7 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Peng Shen ◽

Taoyue Yang ◽

Qun Chen ◽

Hao Yuan ◽

Pengfei Wu ◽

...

Keyword(s):

Cell Cycle ◽

Pancreatic Ductal Adenocarcinoma ◽

Rna Editing ◽

Biological Effects ◽

Clinical Stage ◽

Ductal Adenocarcinoma ◽

Luciferase Reporter ◽

Mesenchymal Transition

Abstract Background A growing number of studies have focused on investigating circRNAs as crucial regulators in the progression of multiple cancer types. Nevertheless, the biological effects and underlying mechanisms of circRNAs in pancreatic ductal adenocarcinoma (PDAC) remain unclear. Methods Differentially expressed circRNAs between cancerous tissue and adjacent normal tissues were identified by RNA sequencing in PDAC. Subsequently, in vitro and in vivo functional experiments were performed to investigate the functional roles of circNEIL3 in PDAC tumour growth and metastasis. Furthermore, RNA pull-down, dual-luciferase reporter assays, RNA immunoprecipitation (RIP) assays, fluorescent in situ hybridization (FISH) and Sanger sequencing assays were performed to examine the circular interaction among circNEIL3, miR-432-5p and adenosine deaminases acting on RNA 1 (ADAR1). Results CircNEIL3 was upregulated in PDAC and promoted the progression of PDAC cells both in vitro and in vivo. Mechanistically, circNEIL3 was shown to regulate the expression of ADAR1 by sponging miR-432-5p to induce RNA editing of glioma-associated oncogene 1 (GLI1), ultimately influencing cell cycle progression and promoting epithelial-to-mesenchymal transition (EMT) in PDAC cells. Moreover, we discovered that the circNEIL3/miR-432-5p/ADAR1 axis was correlated with the PDAC clinical stage and overall survival of PDAC patients, while ADAR1 may reduce the biogenesis of circNEIL3. Conclusions Our findings reveal that circNEIL3 facilitates the proliferation and metastasis of PDAC through the circNEIL3/miR-432-5p/ADAR1/GLI1/cell cycle and EMT axis and that its expression is regulated by ADAR1 through a negative feedback loop. Therefore, circNEIL3 may serve as a prognostic marker and a therapeutic target for PDAC.

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Modulation of FoxO1 Expression by miR-21 to Promote Growth of Pancreatic Ductal Adenocarcinoma

Cellular Physiology and Biochemistry ◽

10.1159/000369686 ◽

2015 ◽

Vol 35 (1) ◽

pp. 184-190 ◽

Cited By ~ 27

Author(s):

Weifeng Song ◽

Qi Li ◽

Lei Wang ◽

Liwei Wang

Keyword(s):

Pancreatic Ductal Adenocarcinoma ◽

Underlying Mechanism ◽

Ductal Adenocarcinoma ◽

Luciferase Reporter ◽

Reporter Assay ◽

Primary Tumors ◽

Protein Levels ◽

Infusion System

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal primary tumors in humans, with undetermined tumorigenesis. Although previous work by us, and by others, has clearly demonstrated an involvement of miR-21 in the growth of PDAC, the underlying mechanism has not been clarified. Methods: Here we analyzed the regulation of FoxO1 by miR-21 in vitro and in vivo, using luciferase-reporter assay and pancreatic intraductal infusion of antisense of miR-21, respectively. Results: We found that overexpression of miR-21 in PDAC cells decreased FoxO1 protein levels, whereas inhibition of miR-21 increased FoxO1 levels. Further, miR-21 bound to FoxO1 mRNA to prevent its translation through its 3'UTR. Moreover, administration of antisense of miR-21 through an intraductal infusion system significantly decreased miR-21 levels and increased FoxO1 levels in implanted PDAC, resulting in a significant decrease in PDAC growth. Conclusion: Taken together, our data highlight miR-21/FoxO1 axis as a novel therapeutic target for inhibiting the growth of PDAC.

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FUS-induced circRHOBTB3 facilitates cell proliferation via miR-600/NACC1 mediated autophagy response in pancreatic ductal adenocarcinoma

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-02063-w ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Taoyue Yang ◽

Peng Shen ◽

Qun Chen ◽

Pengfei Wu ◽

Hao Yuan ◽

...

Keyword(s):

Cell Proliferation ◽

Pancreatic Ductal Adenocarcinoma ◽

Cell Lines ◽

Rna Binding ◽

Ductal Adenocarcinoma ◽

Luciferase Reporter ◽

Circular Rnas ◽

Pdac Cell

Abstract Background Circular RNAs (circRNAs) are becoming a unique member of non-coding RNAs (ncRNAs) with emerging evidence of their regulatory roles in various cancers. However, with regards to pancreatic ductal adenocarcinoma (PDAC), circRNAs biological functions remain largely unknown and worth investigation for potential therapeutic innovation. Methods In our previous study, next-generation sequencing was used to identify differentially expressed circRNAs in 3 pairs of PDAC and adjacent normal tissues. Further validation of circRHOBTB3 expression in PDAC tissues and cell lines and gain-and-loss function experiments verified the oncogenic role of circRHOBTB3. The mechanism of circRHOBTB3 regulatory role was validated by pull-down assays, RIP, luciferase reporter assays. The autophagy response of PANC-1 and MiaPaca-2 cells were detected by mCherry-GFP-LC3B labeling and confocal microscopy, transmission electron microscopy and protein levels of LC3B or p62 via Western blot. Results circRHOBTB3 is highly expressed in PDAC cell lines and tissues, which also promotes PDAC autophagy and then progression in vitro and in vivo. Mechanistically, circRHOBTB3 directly binds to miR-600 and subsequently acts as a miRNA-sponge to maintain the expression level of miR-600-targeted gene NACC1, which facilitates the autophagy response of PDAC cells for adaptation of proliferation via Akt/mTOR pathway. Moreover, the RNA-binding protein FUS (FUS) directly binds to pre-RHOBTB3 mRNA to mediate the biogenesis of circRHOBTB3. Clinically, circRHOBTB3, miR-600 and NACC1 expression levels are correlated with the prognosis of PDAC patients and serve as independent risk factors for PDAC patients. Conclusions FUS-mediated circRHOBTB3 functions as a tumor activator to promote PDAC cell proliferation by modulating miR-600/NACC1/Akt/mTOR axis regulated autophagy.

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Biological Effect and Mechanism of the miR-23b-3p/ANXA2 Axis in Pancreatic Ductal Adenocarcinoma

Cellular Physiology and Biochemistry ◽

10.1159/000494468 ◽

2018 ◽

Vol 50 (3) ◽

pp. 823-840 ◽

Cited By ~ 8

Author(s):

Dan-ming Wei ◽

Yi-wu Dang ◽

Zhen-bo Feng ◽

Lu Liang ◽

Lu Zhang ◽

...

Keyword(s):

Pancreatic Ductal Adenocarcinoma ◽

Chorioallantoic Membrane ◽

Tumor Formation ◽

Ductal Adenocarcinoma ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Chick Chorioallantoic Membrane ◽

Pancreatic Cancer Cells

Background/Aims: Accumulating evidence strongly suggests that microRNAs (miRNAs) modulate the expression of known tumor suppressor genes and oncogenes. In the present study, we found that the proliferation and invasion ability of pancreatic ductal adenocarcinoma (PDAC) cells were significantly suppressed by the overexpression of miR-23b-3p. In addition, there are miR-23b-3p binding sites in annexin A2 (ANXA2). Here, we investigated whether miR-23b-3p had an impact on the progression and metastasis of PDAC by targeting ANXA2. Methods: Cell proliferation, migration, and invasion, and cell cycle assays were performed to explore the effect of miR-23b-3p on various malignant phenotypes of pancreatic cancer cells. The size of tumors was observed following miR-23b-3p overexpression in an in vivo chick chorioallantoic membrane assay. Dual-luciferase reporter, quantitative real-time PCR, western blot, and immunohistochemical analyses were used to validate the relationship between miR-23b-3p and ANXA2 in vitro. Results: We observed that miR-23b-3p could bind specifically to the 3′ untranslated region of ANXA2 and inhibit its expression. MiR-23b-3p overexpression downregulated the expression of ANXA2 mRNA in PDAC cells and limited the size of tumors or even prevented tumor formation. In addition, there was a negative correlation between miR-23b-3p expression and ANXA2 protein expression in clinical specimens. Conclusion: MiR-23b-3p inhibits the development and progression of PDAC by regulating ANXA2 directly.

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Circular RNA CircEYA3 induces energy production to promote pancreatic ductal adenocarcinoma progression through the miR-1294/c-Myc axis

Molecular Cancer ◽

10.1186/s12943-021-01400-z ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Zeyin Rong ◽

Si Shi ◽

Zhen Tan ◽

Jin Xu ◽

Qingcai Meng ◽

...

Keyword(s):

Pancreatic Ductal Adenocarcinoma ◽

Energy Production ◽

Atp Synthesis ◽

Circular Rna ◽

Gene Expression Omnibus ◽

Ductal Adenocarcinoma ◽

Luciferase Reporter ◽

Circular Rnas

Abstract Background Extensive studies have demonstrated the pivotal roles of circular RNAs (circRNAs) in the occurrence and development of different human cancers. However, the expression and regulatory roles of circRNAs in pancreatic ductal adenocarcinoma (PDAC) are unclear. Methods CircEYA3 was explored based on Gene Expression Omnibus (GEO) dataset analysis. qRT-PCR was applied to determine the expression of circRNAs, miRNAs and mRNAs in PDAC cells and tissues. The biological roles of circEYA3 in vitro and in vivo were determined by performing a series of functional experiments. Further, dual luciferase reporter, fluorescence in situ hybridization (FISH), RNA pull-down assays, and RNA immunoprecipitation (RIP) assays were used to confirm the interaction of circEYA3 with miR-1294. Results CircEYA3 was elevated in PDAC tissues and cells, and a higher level of circEYA3 was significantly associated with a poorer prognosis in patients with PDAC. Functionally, circEYA3 increased energy production via ATP synthesis to promote PDAC progression in vitro and in vivo. Mechanistically, circEYA3 functions as an endogenous miR-1294 sponge to elevate c-Myc expression, thus exerting its oncogenic functions. Conclusion CircEYA3 promotes the progression of PDAC through the miR-1294/c-Myc signalling axis, and circEYA3 may be an efficient molecular therapeutic target in PDAC.

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Role of PLEXIND1/TGFβ Signaling Axis in Pancreatic Ductal Adenocarcinoma Progression Correlates with the Mutational Status of KRAS

Cancers ◽

10.3390/cancers13164048 ◽

2021 ◽

Vol 13 (16) ◽

pp. 4048

Author(s):

Sneha Vivekanandhan ◽

Vijay S. Madamsetty ◽

Ramcharan Singh Angom ◽

Shamit Kumar Dutta ◽

Enfeng Wang ◽

...

Keyword(s):

Tumor Growth ◽

Cell Viability ◽

Pancreatic Ductal Adenocarcinoma ◽

Cell Lines ◽

Pdz Domain ◽

Ductal Adenocarcinoma ◽

Binding Motif ◽

Pdac Cell

PLEXIND1 is upregulated in several cancers, including pancreatic ductal adenocarcinoma (PDAC). It is an established mediator of semaphorin signaling, and neuropilins are its known coreceptors. Herein, we report data to support the proposal that PLEXIND1 acts as a transforming growth factor beta (TGFβ) coreceptor, modulating cell growth through SMAD3 signaling. Our findings demonstrate that PLEXIND1 plays a pro-tumorigenic role in PDAC cells with oncogenic KRAS (KRASmut). We show in KRASmut PDAC cell lines (PANC-1, AsPC-1,4535) PLEXIND1 downregulation results in decreased cell viability (in vitro) and reduced tumor growth (in vivo). Conversely, PLEXIND1 acts as a tumor suppressor in the PDAC cell line (BxPC-3) with wild-type KRAS (KRASwt), as its reduced expression results in higher cell viability (in-vitro) and tumor growth (in vivo). Additionally, we demonstrate that PLEXIND1-mediated interactions can be selectively disrupted using a peptide based on its C-terminal sequence (a PDZ domain-binding motif), an outcome that may possess significant therapeutic implications. To our knowledge, this is the first report showing that (1) PLEXIND1 acts as a TGFβ coreceptor and mediates SMAD3 signaling, and (2) differential roles of PLEXIND1 in PDAC cell lines correlate with KRASmut and KRASwt status.

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KLF7/VPS35 axis contributes to hepatocellular carcinoma progression through CCDC85C-activated β-catenin pathway

Cell & Bioscience ◽

10.1186/s13578-021-00585-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yarong Guo ◽

Bao Chai ◽

Junmei Jia ◽

Mudan Yang ◽

Yanjun Li ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Proliferation ◽

Tumor Growth ◽

Luciferase Reporter ◽

Aberrant Expression ◽

Proliferation And Invasion ◽

Hcc Cells

Abstract Objective Dysregulation of KLF7 participates in the development of various cancers, but it is unclear whether there is a link between HCC and aberrant expression of KLF7. The aim of this study was to investigate the role of KLF7 in proliferation and migration of hepatocellular carcinoma (HCC) cells. Methods CCK8, colony growth, transwell, cell cycle analysis and apoptosis detection were performed to explore the effect of KLF7, VPS35 and Ccdc85c on cell function in vitro. Xenografted tumor growth was used to assess in vivo role of KLF7. Chip-qPCR and luciferase reporter assays were applied to check whether KLF7 regulated VPS35 at transcriptional manner. Co-IP assay was performed to detect the interaction between VPS35 and Ccdc85c. Immunohistochemical staining and qRT-PCR analysis were performed in human HCC sampels to study the clinical significance of KLF7, VPS35 and β-catenin. Results Firstly, KLF7 was highly expressed in human HCC samples and correlated with patients’ differentiation and metastasis status. KLF7 overexpression contributed to cell proliferation and invasion of HCC cells in vitro and in vivo. KLF7 transcriptional activation of VPS35 was necessary for HCC tumor growth and metastasis. Further, co-IP studies revealed that VPS35 could interact with Ccdc85c in HCC cells. Rescue assay confirmed that overexpression of VPS35 and knockdown of Ccdc85c abolished the VPS35-medicated promotion effect on cell proliferation and invasion. Finally, KLF7/VPS35 axis regulated Ccdc85c, which involved in activation of β-catenin signaling pathway, confirmed using β-catenin inhibitor, GK974. Functional studies suggested that downregulation of Ccdc85c partly reversed the capacity of cell proliferation and invasion in HCC cells, which was regulated by VPS35 upregulation. Lastly, there was a positive correlation among KLF7, VPS35 and active-β-catenin in human HCC patients. Conclusion We demonstrated that KLF7/VPS35 axis promoted HCC cell progression by activating Ccdc85c-medicated β-catenin pathway. Targeting this signal axis might be a potential treatment strategy for HCC.

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Homeobox-A13 acts as a functional prognostic and diagnostic biomarker via regulating P53 and Wnt signaling pathways in lung cancer

Cancer Biomarkers ◽

10.3233/cbm-200540 ◽

2021 ◽

pp. 1-16

Author(s):

Yang Wang ◽

Bo He ◽

Yan Dong ◽

Gong-Jin He ◽

Xiao-Wei Qi ◽

...

Keyword(s):

Lung Cancer ◽

Signaling Pathways ◽

Cox Regression ◽

Prognostic Significance ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Clinical Feature ◽

Diagnostic Biomarker

BACKGROUND: The prognosis of lung cancer patients is poor without useful prognostic and diagnostic biomarker. To search for novel prognostic and diagnostic markers, we previously found homeobox-A13 (HOXA13) as a promising candidate in lung cancer. OBJECTIVE: To determine the precisely clinical feature, prognostic and diagnostic value, possible role and mechanism of HOXA13. METHODS: Gene-expression was explored by real-time quantitative-PCR, western-blot and tissue-microarray. The associations were analyzed by Chi-square test, Kaplan-Meier and Cox-regression. The roles and mechanisms were evaluated by MTS, EdU, transwell, xenograft tumor and luciferase-reporter assays. RESULTS: HOXA13 expression is increased in tumors, and correlated with age of patients. HOXA13 expression is associated with unfavorable overall survival and relapse-free survival of patients in four cohorts. Interestingly, HOXA13 has different prognostic significance in adenocarcinoma (ADC) and squamous-cell carcinoma (SCC), and is a sex- and smoke-related prognostic factor only in ADC. Importantly, HOXA13 can serve as a diagnostic biomarker for lung cancer, especially for SCC. HOXA13 can promote cancer-cell proliferation, migration and invasion in vitro, and facilitate tumorigenicity and tumor metastasis in vivo. HOXA13 acts the oncogenic roles on tumor growth and metastasis by regulating P53 and Wnt/β-catenin signaling activities in lung cancer. CONCLUSIONS: HOXA13 is a new prognostic and diagnostic biomarker associated with P53 and Wnt/β-catenin signaling pathways.

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