Promoted Pancreatic Ductal Adenocarcinoma Cell Research Articles

CALB2 drives pancreatic cancer metastasis through inflammatory reprogramming of the tumor microenvironment

BackgroundEarly dissemination to distant organs accounts for the dismal prognosis of patients with pancreatic ductal adenocarcinoma (PDAC). Chronic, dysregulated, persistent and unresolved inflammation provides a preferred tumor microenvironment (TME) for tumorigenesis, development, and metastasis. A better understanding of the key regulators that maintain inflammatory TME and the development of predictive biomarkers to identify patients who are most likely to benefit from specific inflammatory-targeted therapies is crucial for advancing personalized cancer treatment.MethodsThis study identified cell-specific expression of CALB2 in human PDAC through single-cell RNA sequencing analysis and assessed its clinicopathological correlations in tissue microarray using multi-color immunofluorescence. Co-culture systems containing cancer-associated fibroblasts (CAFs) and patient-derived organoids (PDOs) in vitro and in vivo were employed to elucidate the effects of CALB2-activated CAFs on PDAC malignancy. Furthermore, CUT&RUN assays, luciferase reporter assays, RNA sequencing, and gain- or loss-of-function assays were used to unravel the molecular mechanisms of CALB2-mediated inflammatory reprogramming and metastasis. Additionally, immunocompetent KPC organoid allograft models were constructed to evaluate CALB2-induced immunosuppression and PDAC metastasis, as well as the efficacy of inflammation-targeted therapy.ResultsCALB2 was highly expressed both in CAFs and cancer cells and correlated with an unfavorable prognosis and immunosuppressive TME in PDAC patients. CALB2 collaborated with hypoxia to activate an inflammatory fibroblast phenotype, which promoted PDAC cell migration and PDO growth in vitro and in vivo. In turn, CALB2-activated CAFs upregulated CALB2 expression in cancer cells through IL6-STAT3 signaling-mediated direct transcription. In cancer cells, CALB2 further activated Ca2+-CXCL14 inflammatory axis to facilitate PDAC metastatic outgrowth and immunosuppression. Genetic or pharmaceutical inhibition of CXCL14 significantly suppressed CALB2-mediated metastatic colonization of PDAC cells in vivo and extended mouse survival.ConclusionsThese findings identify CALB2 as a key regulator of inflammatory reprogramming to promote PDAC metastatic progression. Combination therapy with αCXCL14 monoclonal antibody and gemcitabine emerges as a promising strategy to suppress distant metastasis and improve survival outcomes in PDAC with CALB2 overexpression.

Abstract C038: Cancer-associated fibroblasts maintain critical pancreatic cancer cell lipid homeostasis in the tumor microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy whose abundant cancer-associated fibroblasts (CAFs) create a hallmark “desmoplasia” that severely limits oxygen and nutrient delivery. Hypoxia inhibits oxygen-dependent metabolic processes. For example, unsaturated lipid biosynthesis relies on oxygen to maintain desaturase enzymatic activities. Here, we investigated the mechanisms by which PDAC cells maintain lipid homeostasis under these conditions. Exogenous unsaturated lipids, rather than de novo synthesis, sustain PDAC cell viability by relieving endoplasmic reticulum (ER) stress under nutrient scarcity. Furthermore, CAFs are less hypoxic than adjacent malignant cells in vivo due to proximal blood vessel accessibility, nominating them as a potential source of these obligatory species. CAF-conditioned medium promoted PDAC cell survival upon nutrient and oxygen deprivation, an effect reversed by delipidation. We determined that lysophosphatidylcholines (LPCs) are particularly enriched in CAF-conditioned medium and preferentially taken up by PDAC cells, where they are converted to phosphatidylcholines (PCs) to sustain membrane integrity. Inhibiting LPC to PC conversion reversed the ability of exogenous unsaturated LPCs to sustain cancer cell survival and restrain ER stress in vitro and in vivo. Collectively, these results reveal a critical lipid cross-feeding mechanism enabling PDAC cell survival and identify an innovative approach to targeting this metabolic dependency. Efforts to target lipid crosstalk between CAFs and PDAC cells will be discussed. Citation Format: Xu Han, Michelle Burrows, Laura C Kim, Jimmy Xu, Will Vostrejs, Tran Ngoc Van Le, Yanqing Jiang, Edna Cukierman, Reiss Binder Kim, Ben Z Stanger, Clementina Mesaros, Brian Keith, Celeste Simon. Cancer-associated fibroblasts maintain critical pancreatic cancer cell lipid homeostasis in the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C038.

Construction of a prognostic model with exosome biogenesis- and release-related genes and identification of RAB27B in immune infiltration of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and fatal disease. Exosomes are extracellular vesicles that plays a vital rule in the progression and metastasis of PDAC. However, the specific mechanism of exosome biogenesis and release in the tumorigenesis and development of pancreatic cancer remains elusive. The aim of this study is to develop novel biomarkers and construct a reliable prognostic signature to accurately stratify patients and optimize clinical decision-making. Gene expression and clinical data were acquired from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Univariate Cox regression analysis, random forest analysis, least absolute shrinkage and selection operator (LASSO) regression analysis, and multivariate Cox regression analysis were used to construct the risk signature. The effectiveness of the model was validated by survival point plot, Kaplan-Meier survival analysis, and receiver operating characteristic (ROC) curve in training, testing and entire cohorts. Meanwhile, single sample gene set enrichment analysis (ssGSEA), ESTIMATE and CIBERSORT algorithm were utilized to assess the association of the risk signature with the immune status in the PDAC tumor microenvironment. We also performed functional enrichment, tumor mutation analysis, and DNA methylation analyses based on the risk signature. The function of the core gene was further verified by polymerase chain reaction (PCR), western blot, bicinchoninic acid (BCA), immunohistochemistry (IHC) and in vitro experiments including cell proliferation, migration, and apoptosis experiments. We constructed an exosome biogenesis- and release-related risk model which could serve as an effective and independent prognosis predictor for PDAC patients. The immune infiltration analysis revealed that our signature was related to the PDAC immune microenvironment, mainly associated with a lower proportion of natural killer (NK) cells and CD8+ T cells. Tissue microarray IHC confirmed the association of RAB27B with poor prognosis in PDAC. Knockdown of RAB27B expression promoted PDAC cells' apoptosis, while decreased cellular proliferation and migration. Also, knockdown of RAB27B expression led to reduced exosome secretion, while RAB27B overexpression promoted exosome secretion. The predictive signature can predict overall survival, help elucidate the mechanism of exosome biogenesis and release, and provide immunotherapy guidance for PDAC patients.

PLEK2 promotes migration and invasion in pancreatic ductal adenocarcinoma by MMP1 through IL-17 pathway.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognosis primarily due to metastasis. Accumulating evidence suggests that PLEK2 acts as an oncogene in various tumors. This study aimed to investigate the effects of PLEK2 on PDAC. Expression analysis of PLEK2 was conducted using qRT-PCR, Western blot, and immunohistochemistry in PDAC. Wound healing and transwell assays were performed to evaluate the impact of PLEK2 on cell migration and invasion. A xenograft tumor model was employed to assess the in vivo proliferation of PLEK2. Additionally, the downstream pathway of PLEK2 was analyzed through RNA-seq and confirmed by Western blot analysis. The results demonstrated the upregulation of PLEK2 expression in tumor specimens. High PLEK2 expression was significantly associated with poor overall survival and advanced TNM stages. Correlation analyses revealed positive correlations between PLEK2 and TGF-β, EGFR, and MMP1. Wound healing and transwell assays demonstrated that PLEK2 promoted PDAC cell migration and invasion, potentially through the activation of the epithelial-to-mesenchymal transition process. The in vivo experiment further confirmed that PLEK2 knockdown suppressed tumor growth. RNA-seq analysis revealed PLEK2's regulation of MMP1 and activation of p-ERK and p-STAT3, which were verified by Western blot analysis. Overall, the present study suggests that PLEK2 may play a tumor-promoting role in PDAC. These findings provide valuable insights into the molecular mechanisms of pancreatic cancer and highlight the potential of PLEK2 as a therapeutic target.

The molecular mechanism underlying KRAS regulation on STK31 expression in pancreatic ductal adenocarcinoma.

KRAS gene mutations are common in pancreatic ductal adenocarcinoma (PDAC), but targeting mutant KRAS is still challenging. Here, an endoribonuclease-prepared small interfering RNA (esiRNA) library was used to screen new kinases that play critical roles in PDAC driven by KRAS gene mutations, and serine/threonine kinase 31 (STK31) was identified and characterized as a potential therapeutic target for KRAS-mutant PDAC. Our results showed that STK31 was upregulated in KRAS-mutant PDAC patients with poor survival and highly expressed in PDAC cell lines with KRASG12D mutation. Inhibition of STK31 in KRAS-mutant cell lines significantly reduced PDAC cell growth invitro and hindered tumor growth invivo. Gain and loss of function experiments revealed that STK31 is a downstream target of KRAS in PDAC. A pharmacological inhibition assay showed MAPK/ERK signaling involved in STK31 regulation. The further mechanistic study validated that c-Jun, regulated by KRAS/MAPK signaling, directly modulates the transcription level of STK31 by binding to its promoter region. Through RNA sequencing, we found that the cell cycle regulators CCNB1 and CDC25C are downstream targets of STK31. Taken together, our results indicate that STK31, which is the downstream target of the KRAS/MAPK/ERK/c-Jun signaling pathway in KRAS-mutant PDAC, promotes PDAC cell growth by modulating the expression of the cell cycle regulators CCNB1 and CDC25C.

TNFR2 promotes pancreatic cancer proliferation, migration, and invasion via the NF-κB signaling pathway.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignant disease with low overall survival; chemotherapy and immunotherapy have limited efficacy. Tumor necrosis factor receptor 2 (TNFR2), a type II transmembrane protein, contributes to the development and progression of several tumors. In this study, we elucidated the effect and molecular mechanisms of TNFR2. We used The Cancer Genome Atlas and the Genotype-Tissue Expression database to compare the expression of the TNFR2 gene between normal and malignant pancreatic tissue. Using immunohistochemical staining, we divided the patients into high and low-expression groups, then investigated clinicopathologic data and survival curves of pancreatic cancer patients. We measured TNFR2 protein expression in PANC-1 and ASPC-1 pancreatic cancer cells subjected to TNFR2 small interfering RNA or negative control treatment. We performed proliferation, invasion, and migration assays to study the biological effects of TNFR2 in PDAC. The molecular mechanisms were validated using western blotting. TNFR2 was more highly expressed in PDAC cells and tissues than controls. Abundant expression of TNFR2 was associated with aggressive clinicopathologic characteristics and poor outcomes. Overexpression of TNFR2 promoted PDAC cell proliferation, migration, and invasion in vitro. Mechanistically, TNFR2 binds to TNF-α and activates the NF-κB signaling pathway. TNFR2 is a prognostic marker that facilitates the proliferation, migration, and invasion of PDAC via the NF-κB signaling pathway. TNFR2 may become a therapeutic target.

Fibronectin fragments generated by pancreatic trypsin act as endogenous inhibitors of pancreatic tumor growth

BackgroundThe pancreatic microenvironment has a defensive role against cancer but it can acquire tumor-promoting properties triggered by multiple mechanisms including alterations in the equilibrium between proteases and their inhibitors. The identification of proteolytic events, targets and pathways would set the basis for the design of new therapeutic approaches.Methods and resultsHere we demonstrate that spheroids isolated from human and murine healthy pancreas and co-transplanted orthotopically with pancreatic ductal adenocarcinoma (PDAC) in mouse pancreas inhibited tumor growth. The effect was mediated by trypsin-generated fibronectin (FN) fragments released by pancreatic spheroids. Tumor inhibition was observed also in a model of acute pancreatitis associated with trypsin activation. Mass spectrometry proteomic analysis of fragments and mAb against different FN epitopes identified the FN type III domain as responsible for the activity. By inhibiting integrin α5β1, FAK and FGFR1 signaling, the fragments induced tumor cell detachment and reduced cell proliferation. Consistent with the mutual relationship between the two pathways, FGF2 restored both FGFR1 and FAK signaling and promoted PDAC cell adhesion and proliferation. FAK and FGFR inhibitors additively inhibited PDAC growth in vitro and in orthotopic in vivo models.ConclusionsThis study identifies a novel role for pancreatic trypsin and fibronectin cleavage as a mechanism of protection against cancer by the pancreatic microenvironment. The finding of a FAK-FGFR cross-talk in PDAC support the combination of FAK and FGFR inhibitors for PDAC treatment to emulate the protective effect of the normal pancreas against cancer.

Pancreatic cancer-derived small extracellular vesical ezrin activates fibroblasts to exacerbate cancer metastasis through STAT3 and YAP-1 signaling pathways.

Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC), play an important role in tumorigenesis, metastasis, and chemoresistance. Tumor-derived small extracellular vesicles (sEVs), which mediate cell-to-cell communication between cancer cells and fibroblasts, are also critical for cancer progression and metastasis. However, it remains unclear how PDAC cell-derived sEVs activate fibroblasts, which contributes to tumor progression. Here, we report that ezrin (EZR) expression in PDAC cell-derived sEVs (sEV-EZR) can activate fibroblasts, resulting in increased migration ability and high expression of α-SMA, PDGFRB, and high production of extracellular matrix in fibroblasts. Reciprocally, sEV-EZR-activated fibroblasts enhanced PDAC cell proliferation, invasion, and metastasis to the liver in animal models. Conversely, fibroblasts treated with PDAC cell-derived sEVs with EZR knockdown resulted in the reduced metastatic ability of PDAC. Mechanistically, we demonstrated that PDAC cell-derived sEV-EZR increases the STAT3 and YAP-1 signaling pathways to induce fibroblast activation, and the activated fibroblasts promote PDAC cell proliferation, invasion, and liver metastasis. Inhibition of the STAT3 and YAP-1 signaling pathways by gene knockdown can abrogate sEV-EZR-induced effects. These findings suggest that targeting the interaction between PDAC cell-derived sEV-EZR and fibroblasts is a potential therapeutic strategy for PDAC.

Abstract 2218: The molecular mechanism underlying KRAS regulation on STK31 expression in pancreatic ductal adenocarcinoma

Abstract KRAS is frequently mutated in pancreatic ductal adenocarcinoma (PDAC). However, targeting mutant KRAS is still challenging. Kinase inhibitors are ideal targeted therapeutics for mutant KRAS-driven cancer. In our previous study, an esiRNA screening was prepared to identify some vital kinases that took part in KRAS mutant-driven pancreatic cancer. The STK31 was identified as a potential therapeutic target in KRAS mutant PDAC. This study aimed to investigate the underlying mechanism of STK31 in KRAS mutant PDAC as well as its therapeutic potential. The results demonstrated that STK31 was upregulated in KRAS mutant PDAC patients with poor survival. STK31 was also highly expressed in PDAC cell lines with KRAS mutant background. Inhibition of STK31 in KRAS mutant cell lines significantly reduced PDAC cell proliferation, colony formation, and migration. Gain and loss of function experiments revealed that STK31 was a downstream target of KRAS in PDAC. Pharmacological inhibition of several signaling pathways involved in KRAS showed MAPK/ERK signaling involved in STK31 regulation. Then, further mechanistic study validated that c-Jun, regulated by KRAS/MAPK/ERK signaling, directly regulated the transcription level of STK31 by binding to its promoter region. In summary, our results show that STK31 promotes PDAC cell proliferation under the control of the KRAS/MAPK/ERK/c-Jun transcriptional axis in PDAC. STK31 may act as a potential target in PDAC treatment. Citation Format: Yuting Liu, Shing Chun Tang, Yangchao Chen. The molecular mechanism underlying KRAS regulation on STK31 expression in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2218.

Abstract 3870: TNF-MK2 signaling drives protective autophagy following MAPK pathway inhibition in pancreatic cancer

Abstract Targeting the oncogenic KRAS-RAF-MEK-ERK (MAPK) pathway has remain unsuccessful in the clinic for patients with pancreatic ductal adenocarcinoma (PDAC), a highly lethal cancer with few effective treatment options. To identify novel, effective therapeutic strategies, we investigated the impact of MEK and ERK inhibitors on production of inflammatory cytokines in PDAC cells. We found that both MEK and ERK inhibitors dramatically upregulated production of TNF, leading to activation of multiple TNF signaling pathways which include the pro-death and pro-survival NF-kB and p38.MK2 cascades. Silencing of TNF receptor 1 (TNFR1) abrogated the pro-apoptotic effect of MAPK inhibitors, suggesting that pro-death effect driven by TNF signaling is required for the therapeutic effect of MAPK inhibitors. However, targeting the NF-kB and MK2 pathways greatly augment the suppressive effect of MAPK inhibitors. Notably, the mechanisms via which MK2 promotes PDAC cell survival are mediated through phospho-activation of Heat shock protein 27 (Hsp27) and Beclin1, a critical mediator of autophagy. Intriguingly, silencing of TNFR1 abrogated induction of proactive autophagy following MAPK inhibition, strongly suggesting autocrine signaling as the inciting event of autophagy. Mechanistically, we showed that TNF signaling upregulate unfolded protein response (UPR) in PDAC cells, which is required for autophagy induction. Targeting MK2 abrogated transcription of key UPR genes and blocks subsequent autophagy. The combination of MK2 inhibitor ATI-450 and ERK inhibitor ulixertinib was more effective in curbing the growth of PDAC patient-derived xenograft in vivo and prolonged the survival of autochthonous PDAC mice (KPC model). Overall, our study provided novel insights on the mechanisms that drive protective autophagy following MAPK pathway inhibition and a rationale and feasible therapeutic combination that can be tested in clinical trials for PDAC patients. Citation Format: Iftikhar Ali Khawar, Qing Wei, Timothy Hung-Po Chen, Lin Lin, Patrick M. Grierson, Kian-Huat Lim. TNF-MK2 signaling drives protective autophagy following MAPK pathway inhibition in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3870.

KIF2C Facilitates Tumor Growth and Metastasis in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with a poor prognosis. For PDAC, an increase in the survival time of patients and a reduction mortality have not yet successfully been achieved. In many research works, Kinesin family member 2C (KIF2C) is highly expressed in several tumors. Nevertheless, the role of KIF2C in pancreatic cancer is unknown. In this study, we found that KIF2C expression is significantly upregulated in human PDAC tissues and cell lines such as ASPC-1 and MIA-PaCa2. Moreover, KIF2C upregulation is associated with a poor prognosis when combining the expression of KIF2C with clinical information. Through cell functional assays and the construction of animal models, we showed that KIF2C promotes PDAC cell proliferation, migration, invasion, and metastasis, both in vitro and in vivo. Finally, the results of sequencing showed that the overexpression of KIF2C causes a decrease in some proinflammatory factors and chemokines. The cell cycle detection indicated that the pancreatic cancer cells in the overexpressed group had abnormal proliferation in the G2 and S phases. These results revealed the potential of KIF2C as a therapeutic target for the treatment of PDAC.

PPDPF Promotes the Development of Mutant KRAS-Driven Pancreatic Ductal Adenocarcinoma by Regulating the GEF Activity of SOS1.

The guanine nucleotide exchange factor (GEF) SOS1 catalyzes the exchange of GDP for GTP on RAS. However, regulation of the GEF activity remains elusive. Here, the authors report that PPDPF functions as an important regulator of SOS1. The expression of PPDPF is significantly increased in pancreatic ductal adenocarcinoma (PDAC), associated with poor prognosis and recurrence of PDAC patients. Overexpression of PPDPF promotes PDAC cell growth in vitro and in vivo, while PPDPF knockout exerts opposite effects. Pancreatic-specific deletion of PPDPF profoundly inhibits tumor development in KRASG12D -driven genetic mouse models of PDAC. PPDPF can bind GTP and transfer GTP to SOS1. Mutations of the GTP-binding sites severely impair the tumor-promoting effect of PPDPF. Consistently, mutations of the critical amino acids mediating SOS1-PPDPF interaction significantly impair the GEF activity of SOS1. Therefore, this study demonstrates a novel model of KRAS activation via PPDPF-SOS1 axis, and provides a promising therapeutic target for PDAC.

YY1 promotes pancreatic cancer cell proliferation by enhancing mitochondrial respiration

KRAS-driven metabolic reprogramming is a known peculiarity features of pancreatic ductal adenocarcinoma (PDAC) cells. However, the metabolic roles of other oncogenic genes, such as YY1, in PDAC development are still unclear. In this study, we observed significantly elevated expression of YY1 in human PDAC tissues, which positively correlated with a poor disease progression. Furthermore, in vitro studies confirmed that YY1 deletion inhibited PDAC cell proliferation and tumorigenicity. Moreover, YY1 deletion led to impaired mitochondrial RNA expression, which further inhibited mitochondrial oxidative phosphorylation (OXPHOS) complex assembly and altered cellular nucleotide homeostasis. Mechanistically, the impairment of mitochondrial OXPHOS function reduced the generation of aspartate, an output of the tricarboxylic acid cycle (TCA), and resulted in the inhibition of cell proliferation owing to unavailability of aspartate-associated nucleotides. Conversely, exogenous supplementation with aspartate fully restored PDAC cell proliferation. Our findings suggest that YY1 promotes PDAC cell proliferation by enhancing mitochondrial respiration and the TCA, which favors aspartate-associated nucleotide synthesis. Thus, targeting nucleotide biosynthesis is a promising strategy for PDAC treatment.

RIOK3 promotes pancreatic ductal adenocarcinoma cell invasion and metastasis by stabilizing FAK

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive cancer, characterized by a high metastatic burden. RIO Kinase 3 (RIOK3) has been shown to promote invasion and metastasis of PDAC by cytoskeleton remodeling, but the exact mechanism is still unknown. In this study, we analyzed transcriptome sequencing data from RIOK3 stable knockdown PANC-1 cells and TCGA-PDAC data and discovered that RIOK3 was substantially related to focal adhesion signaling in PDAC. Additionally, silencing RIOK3 dramatically decreased Focal Adhesion Kinase (FAK) protein expression and phosphorylation (Tyr397 and Tyr925 sites). Immunoprecipitation assay verified the interaction of RIOK3 and FAK. Furthermore, RIOK3 considerably increased the protein stability of FAK protein but not FAK-Y925F protein. The biological function of RIOK3 in increasing PDAC cell invasion and migration was shown to be dependent on FAK activation. Moreover, we discovered that RIOK3 mutations were mainly characterized by amplification. RIOK3 mRNA was found to be significantly elevated in PDAC tissues and was associated with a poor prognosis. Furthermore, RIOK3 mRNA was significantly upregulated in later T-stage, pre-existing lymph node metastases, and later pathological stage samples. Overall, our study found that RIOK3 promotes PDAC cell invasion and metastasis by stabilizing FAK protein expression and upregulating its phosphorylation. This also provides a new target for therapeutic modalities targeting FAK.

HOXA10 promote pancreatic cancer progression via directly activating canonical NF-κB signaling pathway.

Although transcription factor homeobox A10 (HOXA10) plays an important role in regulating the development of the pancreas, a pathway of HOXA10 participates in pancreatic ductal adenocarcinoma (PDAC) progression has not been revealed. Immunohistochemistry assays were applied to demonstrate the relationship between HOXA10 expression and PDAC progression. Functional assays were used to illustrate the oncogenic role of HOXA10 in PDAC progression. Regulatory mechanisms of HOXA10 induced IKKβ gene transcription and the nuclear transcription factor kappa B (NF-κB) signal pathways activation were also investigated in PDAC cells. In the current study, we show that HOXA10 expression increased in PDAC with higher tumor stage and poor patient survival in public RNA-seq data suggesting HOXA10 is associated with PDAC progression. HOXA10 promotes PDAC cell proliferation, anchorage colony formation, and xenograft growth by activating canonical NF-κB signaling both in vitro and in vivo. Mechanically, HOXA10 up-regulates IKKβ gene transcription directly and subsequently sustain the activation of NF-κB independent of tumor necrosis factor-alpha in PDAC cells. Collectively, up-regulation of HOXA10 gene expression promote cell growth and tumor progression through directly activating canonical NF-κB signaling in PDAC.

IRAK4 Signaling Drives Resistance to Checkpoint Immunotherapy in Pancreatic Ductal Adenocarcinoma

Checkpoint immunotherapy is largely ineffective in pancreatic ductal adenocarcinoma (PDAC). The innate immune nuclear factor (NF)-κB pathway promotes PDAC cell survival and stromal fibrosis, and is driven by Interleukin-1 Receptor Associated Kinase-4 (IRAK4), but its impact on tumor immunity has not been directly investigated. We interrogated The Cancer Genome Atlas data to identify the correlation between NF-κB and T cell signature, and a PDAC tissue microarray (TMA) to correlate IRAK4 activity with CD8+ T cell abundance. We performed RNA sequencing (RNA-seq) on IRAK4-deleted PDAC cells, and single-cell RNA-seq on autochthonous KPC (p48-Cre/TP53f/f/LSL-KRASG12D) mice treated with an IRAK4 inhibitor. We generated conditional IRAK4-deleted KPC mice and complementarily used IRAK4 inhibitors to determine the impact of IRAK4 on T cell immunity. We found positive correlation between NF-κB activity, IRAK4 and T cell exhaustion from The Cancer Genome Atlas. We observed inverse correlation between phosphorylated IRAK4 and CD8+ T cell abundance in a PDAC tissue microarray. Loss of IRAK4 abrogates NF-κB activity, several immunosuppressive factors, checkpoint ligands, and hyaluronan synthase 2, all of which drive T cell dysfunction. Accordingly, conditional deletion or pharmacologic inhibition of IRAK4 markedly decreased tumor desmoplasia and increased the abundance and activity of infiltrative CD4+ and CD8+ T cells in KPC tumors. Single-cell RNA-seq showed myeloid and fibroblast reprogramming toward acute inflammatory responses following IRAK4 inhibition. These changes set the stage for successful combination of IRAK4 inhibitors with checkpoint immunotherapy, resulting in excellent tumor control and markedly prolonged survival of KPC mice. IRAK4 drives T cell dysfunction in PDAC and is a novel, promising immunotherapeutic target.

A low amino acid environment promotes cell macropinocytosis through the YY1-FGD6 axis in Ras-mutant pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC), cancer with a high mortality rate and the highest rate of KRAS mutation, reportedly internalizes proteins via macropinocytosis to adapt to low amino acid levels in the tumor microenvironment. Here, we aimed to identify a key regulator of macropinocytosis for the survival of tumor cells in a low amino acid environment in PDAC. FYVE, RhoGEF, and PH domain-containing protein 6 (FGD6) were identified as key regulators of macropinocytosis. FGD6 promoted PDAC cell proliferation, macropinocytosis, and tumor growth both in vitro and in vivo. The macropinocytosis level was decreased with FGD6 knockdown in PDAC cell lines. Moreover, FGD6 promoted macropinocytosis by participating in the trans-Golgi network and enhancing the membrane localization of growth factor receptors, especially the TGF-beta receptor. TGF-beta enhanced macropinocytosis in PDAC cells. Additionally, YAP nuclear translocation induced by a low amino acid tumor environment initiated FGD6 expression by coactivation with YY1. Clinical data analysis based on TCGA and GEO datasets showed that FGD6 expression was upregulated in PDAC tissue, and high FGD6 expression was correlated with poor prognosis in patients with PDAC. In tumor tissue from KrasG12D/+/Trp53R172H/-/Pdx1-Cre (KPC) mice, FGD6 expression escalated during PDAC development. Our results uncover a previously unappreciated mechanism of macropinocytosis in PDAC. Strategies to target FGD6 and growth factors membrane localization might be developed for the treatment of PDAC.

Oxysterol-Binding Protein 2 Promotes Pancreatic Ductal Adenocarcinoma Progression Through Epithelial-Mesenchymal Transition.

Oxysterol-binding protein 2 (OSBP2) is crucial for promoting the growth and development of cancers; however, its effects on pancreatic ductal adenocarcinoma (PDAC) are still unclear. Here, we report that OSBP2 is an efficient tumor-associated protein to lead to extremely malignant characteristics in PDAC. We discovered that increased OSBP2 expression in primary tumors was associated with shorter survival in PDAC patients. Therefore, we used immunohistochemistry (IHC) to analyze the levels of OSBP2 expression in PDAC tissues and adjacent paracancerous tissues. We used wound healing and Transwell assays to evaluate the effects of OSBP2 on PDAC cell (ASPC-1 and BXPC-3) migration and invasion, respectively, and CCK-8 and Annexin V/PI double staining to evaluate the effects of OSBP2 on PDAC cell proliferation and apoptosis, respectively. Western blotting was used to analyze the effect of OSBP2 on the PDAC cell phenotype. We also explored the effect of OSBP2 on chemosensitivity to gemcitabine (GEM) and 5-fluorouracil (5-FU). We validated these findings in an in vivo mouse model. The data show that OSBP2 overexpression promoted PDAC cell migration, invasion, proliferation and chemotherapy resistance, and decreased apoptosis. OSBP2 overexpression downregulated E-cadherin expression and upregulated N-cadherin, vimentin, Snail, Slug, ZEB1, and β-catenin expression. Taken together, our findings indicated that OSBP2 was overexpressed in PDAC and that upregulation of OSBP2 may promote PDAC progression. Therefore, OSBP2 may have potential diagnostic and therapeutic value in PDAC.

The long non-coding RNA DKFZp434J0226 regulates the alternative splicing process through phosphorylation of SF3B6 in PDAC

BackgroundLong noncoding RNAs (lncRNAs), a type of pervasive genes that regulates various biological processes, are differentially expressed in different types of malignant tumors. The role of lncRNAs in the carcinogenesis of pancreatic ductal adenocarcinoma (PDAC) remains unclear. Here, we investigated the role of the lncRNA DKFZp434J0226 in PDAC.MethodsAberrantly expressed mRNAs and lncRNAs among six PDAC and paired non-tumorous tissues were profiled using microarray analysis. Quantitative real-time polymerase chain reaction was used to evaluate DKFZp434J0226 expression in PDAC tissues. CCK-8 assay, wound-healing assay, soft agar colony formation assay, and transwell assay were performed to assess the invasiveness and proliferation of PDAC cells. Furthermore, RNA pull-down, immunofluorescence, RNA immunoprecipitation, and western blotting assays were performed to investigate the association between DKFZp434J0226 and SF3B6. Tumor xenografts in mice were used to test for tumor formation in vivo.ResultsIn our study, 222 mRNAs and 128 lncRNAs were aberrantly expressed (≥ twofold change). Of these, 66 mRNAs and 53 lncRNAs were upregulated, while 75 lncRNAs and 156 mRNAs were downregulated. KEGG pathway analysis and the Gene ontology category indicated that these genes were associated with the regulation of mRNA alternative splicing and metabolic balance. Clinical analyses revealed that overexpression of DKFZp434J0226 was associated with worse tumor grading, frequent perineural invasion, advanced tumor-node-metastasis stage, and decreased overall survival and time to progression. Functional assays demonstrated that DKFZp434J0226 promoted PDAC cell migration, invasion, and growth in vitro and accelerated tumor proliferation in vivo. Mechanistically, DKFZp434J0226 interacted with the splicing factor SF3B6 and promoted its phosphorylation, which further regulated the alternative splicing of pre-mRNA.ConclusionsThis study indicates that DKFZp434J0226 regulates alternative splicing through phosphorylation of SF3B6 in PDAC and leads to an oncogenic phenotype in PDAC.

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

BackgroundCircular 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.MethodsIn 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.ResultscircRHOBTB3 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.ConclusionsFUS-mediated circRHOBTB3 functions as a tumor activator to promote PDAC cell proliferation by modulating miR-600/NACC1/Akt/mTOR axis regulated autophagy.