Neuron-derived neurotensin promotes pancreatic cancer invasiveness and gemcitabine resistance via the NTSR1/Akt pathway.

Perineural invasion and neurogenesis are frequently observed in pancreatic ductal adenocarcinoma (PDAC) and link to poor outcome. However, how neural factors affect PDAC prognosis and the underlying mechanism as well as counteracting therapeutic are still unclear. In silico systematic analysis was performed with PROGgene to identify potential neural factor and its receptor in pancreatic cancer. In vitro assays including migration, invasion, 3D recruitment, and gemcitabine resistance were performed to study the effect of neuron-derived neurotensin (NTS) on pancreatic cancer behavior. Orthotopic animal study was used to validate the in vitro findings. Gene set enrichment analysis (GSEA) was performed to confirm the results from in silico to in vivo. Expression of NTS and its receptor 1 (NTSR1) predicted poor prognosis in PDAC. NTS synthetic peptide or neuron-derived condition medium promoted pancreatic cancer invasiveness and recruitment in 2D and 3D assays. NTS-induced effects depended on NTSR1 and PI3K activation. GDC-0941, a clinically approved PI3K inhibitor, counteracted NTS-induced effects in vitro. Inhibition of NTSR1 in pancreatic cancer cells resulted in decreased tumor dissemination and diminished PI3K activation in vivo. NTS boosted gemcitabine resistance via NTSR1 in pancreatic cancer. Our results suggest that neural cell-secreted NTS plays an important role in promoting PDAC.

RNF43 Inactivation Enhances the B-RAF/MEK Signaling and Creates a Combinatory Therapeutic Target in Cancer Cells.

RING finger 43 (RNF43), a RING-type E3 ubiquitin ligase, is a key regulator of WNT signaling and is mutated in 6-10% of pancreatic tumors. However, RNF43-mediated effects remain unclear, as only a few in vivo substrates of RNF43 are identified. Here, it is found that RNF43-mutated pancreatic cancer cells exhibit elevated B-RAF/MEK activity and are highly sensitive to MEK inhibitors. The depletion of RNF43 in normal pancreatic ductal cells also enhances MEK activation, suggesting that it is a physiologically regulated process. It is confirmed that RNF43 ubiquitinates B-RAF at K499 to promote proteasome-dependent degradation, resulting in reduced MEK activity and proliferative ability in cancer cells. In addition, phosphorylation of B-RAF at T491 suppresses B-RAF ubiquitination by decreasing the interaction between RNF43 and B-RAF. Mutations at K499 in B-RAF are identified in various cancer types. MEK and WNT inhibitors synergistically suppress the growth of RNF43-mutated pancreatic cancer cells in vitro and in vivo. Collectively, the research reveals a novel mechanism by which RNF43 inhibits B-RAF/MEK signaling to suppress tumor growth and provide a new strategy for the treatment of RNF43-inactivated pancreatic cancer.

Pancreatic cancer cell-derived semaphorin 3A promotes neuron recruitment to accelerate tumor growth and dissemination.

Perineural invasion and neurogenesis are frequently observed in pancreatic ductal adenocarcinoma (PDAC), and they are associated with a poor prognosis. Axon guidance factor semaphorin 3A (SEMA3A) is upregulated in PDAC. However, it remains unclear whether cancer-derived SEMA3A influences nerve innervation and pancreatic tumorigenesis. In silico analyses were performed using PROGgene and NetworkAnalyst to clarify the importance of SEMA3A and its receptors, plexin A1 (PLXNA1) and neuropilin 2 (NRP2), in pancreatic cancer. In vitro assays, including migration, neurite outgrowth, and 3D recruitment, were performed to study the effects of SEMA3A on neuronal behaviors. Additionally, an orthotopic animal study using C57BL/6 mice was performed to validate the in vitro findings. Expression of SEMA3A and its receptors predicted worse prognosis for PDAC. Cancer-derived SEMA3A promoted neural migration, neurite outgrowth, and neural recruitment. Furthermore, SEMA3A-induced effects depended on PLXNA1, NRP2, and MAPK activation. Trametinib, an approved MAPK kinase (MEK) inhibitor, counteracted SEMA3A-enhanced neuronal activity in vitro. Inhibition of SEMA3A by shRNA in pancreatic cancer cells resulted in decreased neural recruitment, tumor growth, and dissemination in vivo. Our results suggested that cancer-secreted SEMA3A plays an important role in promoting neo-neurogenesis and progression of PDAC.

Upregulating sirtuin 6 ameliorates glycolysis, EMT and distant metastasis of pancreatic adenocarcinoma with krüppel-like factor 10 deficiency.

Krüppel-like factor 10 (KLF10) is a tumor suppressor in multiple cancers. In a murine model of spontaneous pancreatic adenocarcinoma (PDAC), additional KLF10 depletion accelerated distant metastasis. However, Klf10 knockout mice, which suffer from metabolic disorders, do not develop malignancy. The mechanisms of KLF10 in PDAC progression deserve further exploration. KLF10-depleted and KLF10-overexpressing PDAC cells were established to measure epithelial-mesenchymal transition (EMT), glycolysis, and migration ability. A murine model was established to evaluate the benefit of genetic or pharmacological manipulation in KLF10-depleted PDAC cells (PDACshKLF10). Correlations of KLF10 deficiency with rapid metastasis, elevated EMT, and glycolysis were demonstrated in resected PDAC tissues, in vitro assays, and murine models. We identified sirtuin 6 (SIRT6) as an essential mediator of KLF10 that modulates EMT and glucose homeostasis. Overexpressing SIRT6 reversed the migratory and glycolytic phenotypes of PDACshKLF10 cells. Linoleic acid, a polyunsaturated essential fatty acid, upregulated SIRT6 and prolonged the survival of mice injected with PDACshKLF10. Modulating HIF1α and NFκB revealed that EMT and glycolysis in PDAC cells were coordinately regulated upstream by KLF10/SIRT6 signaling. Our study demonstrated a novel KLF10/SIRT6 pathway that modulated EMT and glycolysis coordinately via NFκB and HIF1α. Activation of KLF10/SIRT6 signaling ameliorated the distant progression of PDAC.Clinical Trial Registration: ClinicalTrials.gov. identifier: NCT01666184.

Protein arginine methyltransferase 3-induced metabolic reprogramming is a vulnerable target of pancreatic cancer.

BACKGROUND: The biological function of protein arginine methyltransferase 3 (PRMT3) is not well known because very few physiological substrates of this methyltransferase have been identified to date. METHODS: The clinical significance of PRMT3 in pancreatic cancer was studied by database analysis. The PRMT3 protein level of human pancreatic tumors was detected by immunoblotting and immunohistochemical staining. PRMT3-associated proteins and the methylation sites on the proteins were investigated using mass spectrometry. Seahorse Bioscience analyzed the metabolic reprogramming. Combination index analysis and xenograft animal model were conducted to explore the effects of combination of inhibitors of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and oxidative phosphorylation on tumor growth. RESULTS: We found that the expression of PRMT3 is upregulated in pancreatic cancer, and its expression is associated with poor survival. We identified GAPDH as a PRMT3-binding protein and demonstrated that GAPDH is methylated at R248 by PRMT3 in vivo. The methylation of GAPDH by PRMT3 enhanced its catalytic activity while the mutation of R248 abolished the effect. In cells, PRMT3 overexpression triggered metabolic reprogramming and enhanced glycolysis and mitochondrial respiration simultaneously in a GAPDH-dependent manner. PRMT3-overexpressing cancer cells were addicted to GAPDH-mediated metabolism and sensitive to the inhibition of GAPDH and mitochondrial respiration. The combination of inhibitors of GAPDH and oxidative phosphorylation induced a synergistic inhibition on cellular growth in vitro and in vivo. CONCLUSION: Our results suggest that PRMT3 mediates metabolic reprogramming and cellular proliferation through methylating R248 of GAPDH, and double blockade of GAPDH and mitochondrial respiration could be a novel strategy for the treatment of PRMT3-overexpressing pancreatic cancer.

Protein Arginine Methyltransferase 3 Enhances Chemoresistance in Pancreatic Cancer by Methylating hnRNPA1 to Increase ABCG2 Expression.

Pancreatic cancer is poorly responsive to chemotherapy due to intrinsic or acquired resistance. Our previous study showed that epigenetic modifying enzymes including protein arginine methyltransferase 3 (PRMT3) are dysregulated in gemcitabine (GEM)-resistant pancreatic cancer cells. Here, we attempt to elucidate the role of PRMT3 in chemoresistance. Overexpression of PRMT3 led to increased resistance to GEM in pancreatic cancer cells, whereas reduction of PRMT3 restored GEM sensitivity in resistant cells. We identified a novel PRMT3 target, ATP-binding cassette subfamily G member 2 (ABCG2), which is known to play a critical role in drug resistance. PRMT3 overexpression upregulated ABCG2 expression by increasing its mRNA stability. Mass spectrometric analysis identified hnRNPA1 as a PRMT3 interacting protein, and methylation of hnRNPA1 at R31 by PRMT3 in vivo and in vitro. The expression of methylation-deficient hnRNPA1-R31K mutant reduced the RNA binding activity of hnRNPA1 and the expression of ABCG2 mRNA. Taken together, this provides the first evidence that PRMT3 methylates the RNA recognition motif (RRM) of hnRNPA1 and promotes the binding between hnRNPA1 and ABCG2 to enhance drug resistance. Inhibition of PRMT3 could be a novel strategy for the treatment of GEM-resistant pancreatic cancer.

The NuRD complex-mediated p21 suppression facilitates chemoresistance in BRCA-proficient breast cancer.

The Mi-2/nucleosome remodeling and deacetylase (NuRD) complex play a role in silencing gene expression. CHD4, the core component of the NuRD complex, which cooperates with histone deacetylase in reducing tumor suppressor genes (TSGs). To dissect the mechanisms underlying cancer promotion, we clarify the role of CHD4 in cyclin-dependent kinase inhibitor protein p21. Here, our data indicates that CHD4 deficiency impairs the recruitments of HDAC1 to the p21 promoter. ~ 300bp proximal promoter region is responsible for CHD4-HDAC1 axis-mediated p21 transcriptional activity. For identifying the role of anti-cancer drug response, knockdown of p21 overcomes cisplatin and poly-(ADP-ribose) polymerase (PARP) inhibitor-mediated growth suppression in CHD4-depleted cells. Consistent with in vitro data, tissue of patients and bioinformatics approach also showed positive correlation between CHD4 and p21. Overall, our findings not only identify that CHD4 deficiency preferentially impairs cell survival via increasing the level of p21, but also establishes targeting CHD4 as a potential therapeutic implication in BRCA-proficient breast cancer treatment.

G9a governs colon cancer stem cell phenotype and chemoradioresistance through PP2A-RPA axis-mediated DNA damage response.

BACKGROUND AND PURPOSE: Neoadjuvant concurrent chemoradiotherapy (CCRT) is a standard treatment of locally advanced colon cancer cell (CRC). In order to maximize efficacy and minimize toxicity, new drugs have been developed and used in combination with CCRT. Recently, it has been shown that G9a plays a role in mediating phenotypes of cancer stem cells (CSCs). This study aimed to characterize G9a as a biomarker in predicting therapy response to prevent overtreatment and adverse effects in CRC patients. EXPERIMENTAL DESIGN: The primary tumors from 39 patients who received CCRT for rectal cancer were selected. In vivo tumor xenograft models for tumorigenic properties in immunodeficient mice were developed. In vitro stemness ability was performed by tumor-sphere assays, cell response to anti-cancer agents and stemness-related genes analysis. RESULTS: Cells survived from radiation treatment, and displayed high levels of G9a. A significantly positive correlation was shown between G9a and CSCs marker CD133 in locally advanced rectal cancer patients with CCRT. Knockdown of G9a increased the sensitivity of cells to radiation treatment and sensitized cells to DNA damage agents through PP2A-RPA axis. CONCLUSIONS: Our study theorized that G9a might serve as a novel target in colon cancer, which offers exciting potential in prediction of response to preoperative chemoradiotherapy in patients with advanced CRC.

Krüpple-like factor 10 regulates radio-sensitivity of pancreatic cancer via UV radiation resistance-associated gene.

BACKGROUND AND PURPOSE: Krüpple-like factor 10 (Klf10), an early response gene of TGFß, was reported to be a prognostic biomarker for pancreatic cancer survival. The role of Klf10 in predicting tumor response to cancer treatment is unknown. MATERIALS AND METHODS: Genetically manipulated MiaPaCa and Panc-1 cells were established to evaluate clonogenic survival, autophagy, apoptosis and DNA repair after radiation. The interaction between Klf10 and UV radiation resistance-associated gene (UVRAG) was demonstrated by ChiP-PCR and luciferase reporter assay. Orthotopic murine tumor model and clinical specimens were used to evaluate radio-sensitivity of pancreatic cancer. RESULTS: We found Klf10 silencing correlates with enhanced pancreatic cancer clonogenic survival and murine tumor growth after radiation. UVRAG was an essential down-stream mediator transcriptionally suppressed by Klf10. Silencing UVRAG mRNA in Klf10 depleted Panc-1 cells reversed the radio-resistant phenotypes including decreased apoptosis and enhanced DNA repair as well as autophagy. Metformin, an anti-diabetic agent, was found to increase Klf10 and suppress UVRAG expression to improve radiation cytotoxicity in pancreatic cancer. The predictive value of Klf10 in radiation response and the inverse correlation with UVRAG were confirmed in cohorts of pancreatic cancer patients. CONCLUSIONS: Klf10 is a potential biomarker in predicting and sensitizing radiation effect in pancreatic cancer.

Full-length recombinant human SCF1-165 is more thermostable than the truncated SCF1-141 form.

Human stem cell factor initiates a diverse array of cellular responses, including hematopoiesis, cell proliferation, differentiation, migration and survival. To explore the relationship between its structure and function, we produced recombinant soluble human stem cell factor1-165 (wild type) and human stem cell factor1-141 (C-terminal truncated) in a yeast expression system and compared their biological activities and thermal stabilities. The biological activity of the two proteins was measured as a function of TF-1 cell viability and effects on downstream signaling targets after incubation. We found that these proteins enhanced cell viability and downstream signaling to a similar extent, in a dose-dependent manner. The biological activity of recombinant human stem cell factor1-165 was significantly greater than that of recombinant human stem cell factor1-141 after heating the proteins (100 ng/mL) at 25-110°C for 10 minutes (P<0.05 for all temperatures). In addition, circular dichroism spectral analysis indicated that ß-sheet structures were altered in recombinant human stem cell factor1-141 but not recombinant human stem cell factor1-165 after heating at 90°C for 15 or 30 min. Molecular modeling and limited proteolytic digestion were also used to compare the thermo stability between human stem cell factor1-165 and human stem cell factor1-141. Together, these data indicate that stem cell factor1-165 is more thermostable than stem cell factor1-141.