BICC1 Drives Pancreatic Cancer Progression by Inducing VEGF-Independent Angiogenesis

VEGF inhibitors are one of the most successful antiangiogenic drugs in the treatment of many solid tumors. Nevertheless, pancreatic adenocarcinoma (PAAD) cells can reinstate tumor angiogenesis via activation of VEGF-independent pathways, thereby conferring resistance to VEGF inhibitors. Bioinformatic analysis showed that BICC1 was one of the top genes involved in the specific angiogenesis process of PAAD. The analysis of our own cohort confirmed that BICC1 was overexpressed in human PAAD tissues and was correlated to increased microvessel density and tumor growth, and worse prognosis. In cells and mice with xenograft tumors, BICC1 facilitated angiogenesis in pancreatic cancer in a VEGF-independent manner. Mechanistically, as an RNA binding protein, BICC1 bounds to the 3\u27UTR of Lipocalin-2 (LCN2) mRNA and post-transcriptionally up-regulated LCN2 expression in PAAD cells. When its level is elevated, LCN2 binds to its receptor 24p3R, which directly phosphorylates JAK2 and activates JAK2/STAT3 signal, leading to increased production of an angiogenic factor CXCL1. Blocking of the BICC1/LCN2 signalling reduced the microvessel density and tumor volume of PAAD cell grafts in mice, and increased the tumor suppressive effect of gemcitabine. In conclusion, BICC1 plays a pivotal role in the process of VEGF-independent angiogenesis in pancreatic cancer, leading to resistance to VEGF inhibitors. BICC1/LCN2 signaling may serve as a promising anti-angiogenic therapeutic target for pancreatic cancer patients

Biological risk based on preoperative serum CA19‐9 and histological grade predicts prognosis and improves accuracy of classification in patients with pancreatic ductal adenocarcinoma

Abstract Background Carbohydrate antigen (CA) 19‐9 and histological grade can serve as indicators of the biological characteristics of pancreatic ductal adenocarcinoma (PDAC). Aims The aim of this study was to investigate the combined impact of preoperative CA19‐9 levels and histological grade on prognosis and classification accuracy in PDAC patients. Methods and results A retrospective cohort study was conducted on 612 patients with PDAC who underwent curative pancreatectomy, and a biological risk model based on preoperative CA19‐9 levels and histology grade was established. The prognostic importance of the biological risk model was evaluated, and its validity was confirmed through a validation cohort of 218 patients. The survival of patients with PDAC was independently associated with preoperative CA19‐9 levels and histology grade, indicating a biological risk. This biological risk was incorporated into the eighth edition of the TNM staging system, leading to the development of a modified TNM (mTNM) staging system. Receiver operating characteristic (ROC) curves demonstrated that the mTNM staging system had a significantly larger area under the curve (AUC) than the TNM staging system. The discriminatory capacity of the mTNM staging system was further validated in an independent cohort. Conclusion Biological risk based on preoperative CA19‐9 and histological grade could predict the survival of patients with PDAC. The incorporation of biological risk into the TNM staging system has the potential to enhance the accuracy of patient classification in PDAC, predicting patient survival and enabling the development of individualized treatment plans

CypA, a gene downstream of HIF-1α, promotes the development of PDAC.

Hypoxia-inducible factor-1α (HIF-1α) is a highly important transcription factor involved in cell metabolism. HIF-1α promotes glycolysis and inhibits of mitochondrial respiration in pancreatic ductal adenocarcinoma (PDAC). In response to tumor hypoxia, cyclophilin A (CypA) is over-expressed in various cancer types, and is associated with cell apoptosis, tumor invasion, metastasis, and chemoresistance in PDAC. In this study, we showed that both HIF-1α and CypA expression were significantly associated with lymph node metastasis and tumor stage. The expression of CypA was correlated with HIF-1α. Moreover, the mRNA and protein expression of CypA markedly decreased or increased following the suppression or over-expression of HIF-1α in vitro. Chromatin immunoprecipitation analysis showed that HIF-1α could directly bind to the hypoxia response element (HRE) in the CypA promoter regions and regulated CypA expression. Consistent with other studies, HIF-1α and CypA promoted PDAC cell proliferation and invasion, and suppressed apoptosis in vitro. Furthermore, we proved the combination effect of 2-methoxyestradiol and cyclosporin A both in vitro and in vivo. These results suggested that,CypA, a gene downstream of HIF-1α, could promote the development of PDAC. Thus, CypA might serve as a potential therapeutic target for PDAC

Serum insulin‐like growth factor binding protein 2 levels as biomarker for pancreatic ductal adenocarcinoma‐associated malnutrition and muscle wasting

Abstract Background Malnutrition and muscle wasting are common features frequently observed in pancreatic ductal adenocarcinoma (PDAC) patients with cancer cachexia. They are associated with reduced survival and quality of life. Nutrition therapy is an important part of multimodal cancer care in PDAC. However, due to the complexity of nutrition assessment, only 30–60% of patients with nutritional risks receive nutritional treatment at present. It is important to identify biomarkers that may be used to improve management of PDAC‐associated malnutrition. Serum insulin‐like growth factor binding protein 2 (IGFBP2) has emerged as a potential serum biomarker in a variety of tumours. However, its association with malnutrition and muscle wasting in PDAC is unclear. Methods We evaluated the tumour IGFBP2 expression and serum IGFBP2 level in 98 PDAC patients using immunohistochemistry and enzyme‐linked immunosorbent assay and analysed the correlation between them. Furthermore, we explored the relationship between IGFBP2 of both tumour and serum and nutritional status (Patient‐Generated Subjective Global Assessment and skeletal muscle index). Pan02 IGFBP2 stable transfection cell lines, Pan02 PLV‐IGFBP2 cells, and PLKO‐IGFBP2 cells were injected subcutaneously into the flank of C57BL/6 mouse. Serum IGFBP2 levels, food intake, and body weight of these mice were measured. The degree of muscle atrophy is characterized by haematoxylin and eosin, Oil Red O, and Masson's trichrome staining. The mRNA and protein expression of several essential muscle‐related signal proteins such as atrogin‐1 and muscle RING finger 1 was measured. Results Among 98 patients, we found that tumour IGFBP2 expression is related to plasma IGFBP2 levels (rs = 0.562, P < 0.001), and they significantly increased among patients with Patient‐Generated Subjective Global Assessment ≥9 and correlated with overall survival. Moreover, serum IGFBP2 level is negatively correlated with skeletal muscle index (rs = −0.600, P < 0.001) and Hounsfield units (rs = −0.532, P < 0.001). In mice injected with Pan02 PLV‐IGFBP2 cell, circulating IGFBP2 was elevated while body weight and food intake were decreased when compared with Pan02 PLV‐Control group. These mice also exhibited significantly aggravated muscle fibre atrophy, lipid deposition, and increased collagen tissue, and the expression of mRNA and protein of atrogin‐1 and muscle RING finger 1 in the gastrocnemius muscle is increased. Conversely, these symptoms were alleviated in the PLKO‐IGFBP2 group. Conclusions In the current study, there is a significant correlation between serum IGFBP2 levels, malnutrition, and muscle atrophy in PDAC. Our results suggested that serum IGFBP2 level might be a promising biomarker and intervention targets for PDAC‐associated severe malnutrition and muscle wasting

CypA expression is regulated by HIF-lα.

<p><b>A.</b> HIF-lα and CypA relative mRNA expression levels in different groups. HIF-lα and CypA relative mRNA expression levels in pancreatic cancer cell lines Mia-paca-2 and BXPC3 were determined by real-time PCR analysis. β-actin cDNA served as the internal control. Expression levels in both untransfected cell lines were set as 1. Columns, mean of triplicate samples from two different experiments; bars, SE. *: P<0.05. <b>B.</b> HIF-lα and CypA protein expression levels in different groups. HIF-lα and CypA protein expression levels were determined by western blot analysis. β-actin served as the loading control. Expression levels in both untransfected cell lines were set as 1. Columns, mean of triplicate samples from two different experiments; bars, SE. *: P<0.05. <b>C.</b> Binding of HIF-lα to the CypA promoter in BXPC3.: screening of the 5-flanking region of the CypA gene revealed 3 potential HIF-1α binding sites (−32∼−36 −311∼−315 −1269∼−1273) relative to the start site of transcription. D. ChIP results in different groups. ChIP assay was performed and the promoter fragment binding sites (−311∼−315) was specifically co-immuno-precipitated by anti-HIF-lα but not the control IgG or non-antibody. In chromatin fraction pulled down by anti-HIF-1α antibody, the CypA promoter PCR fragments binding sites (−32∼−36 or −1269∼−127) hardly has been detected. E. Dual luciferase results in different groups. The results showed that the full-length CypA promoter activity (CypA-promotor) increased significantly after over expression of HIF-1α compared to that treated with plasmids alone (P<0.05). But the mutant CypA promoter (CypA-promotor-mutation) had not increased, which was used as negative control. VEGF luciferase reporter construct (VEGF-promotor) was used as positive control for the HIF-1α response. Over-expression of HIF-1α increased about 3-fold of the VEGF promoter activity compared to controls (P<0.05).</p

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Validation of expression of HIF-lα and CypA in PDAC tissues and analysis of the clinical significance.

<p><b>A.</b> The IHC results of HIF-lα and CypA. CypA protein expression was significantly correlated with HIF-lα, as detected by immunohistochemical staining on PDAC. Left panels: various expression levels of HIF-lα protein. Right panels: expression of CypA protein of the same samples in the adjacent section. <b>B.</b> Cross tabulation of HIF-lα and CypA expression levels in 158 cases of PDAC. Expression of CypA was correlated with HIF-1α (P = 0.002). <b>C.</b> The survival analysis of HIF-lα IHC results. PDAC patients (n = 158) with mild positive HIF-lα protein expression (−/+/++, n = 119) had significantly better total survival than those with strong positive expression (+++, n = 39). HIF-1α expression was prognostic for overall survival. The two- and three-year survival rates were 25.2% and 13.4%, respectively, in patients with mild HIF-1α, but decreased to 7.7% or 2.6%, respectively, in patients with strong expression of HIF-1α. (P<0.001) <b>D.</b> The survival analysis of CypA IHC results. The same PDAC patients (n = 158) with negative CypA protein expression (−, n = 65) had significantly better total survival than those with positive expression (+, n = 93). Patients with negative CypA had two- and three-year survival rates of 32.3% and 20%, respectively, which significantly decreased to 12.9% and 4.3% in patients with positive CypA (P<0.001). <b>E.</b> The survival analysis of HIF-lα and CypA IHC results. The same PDAC patients (n = 158) with mild positive HIF-1α and negative CypA protein expression (n = 57) had significantly better total survival than those with strong positive HIF-1α and positive CypA expression (n = 31), the two- or three-year survival rate between them was significantly different, respectively (P<0.001). Besides, the same PDAC patients (n = 158) with mild positive HIF-1α and positive CypA protein expression (n = 62) or with strong positive HIF-1α and negative CypA protein expression (n = 8) had significantly better total survival than those with strong positive HIF-1α and positive CypA expression (n = 31), and had worse total survival than those with mild positive HIF-1α and negative CypA expression (n = 57). The two- or three-year survival rate between the above every combination of two groups was significantly different, respectively (P<0.05).</p

Association between HIF-1α, CypA expression levels and the clinical factors.

<p>Association between HIF-1α, CypA expression levels and the clinical factors.</p

Nuclear PLD1 combined with NPM1 induces gemcitabine resistance through tumorigenic IL7R in pancreatic adenocarcinoma

Objective: Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant gastrointestinal cancer with a 5-year survival rate of only 9%. Of PDAC patients, 15%-20% are eligible for radical surgery. Gemcitabine is an important chemotherapeutic agent for patients with PDAC; however, the efficacy of gemcitabine is limited due to resistance. Therefore, reducing gemcitabine resistance is essential for improving survival of patients with PDAC. Identifying the key target that determines gemcitabine resistance in PDAC and reversing gemcitabine resistance using target inhibitors in combination with gemcitabine are crucial steps in the quest to improve survival prognosis in patients with PDAC. Methods: We constructed a human genome-wide CRISPRa/dCas 9 overexpression library in PDAC cell lines to screen key targets of drug resistance based on sgRNA abundance and enrichment. Then, co-IP, ChIP, ChIP-seq, transcriptome sequencing, and qPCR were used to determine the specific mechanism by which phospholipase D1 (PLD1) confers resistance to gemcitabine. Results: PLD1 combines with nucleophosmin 1 (NPM1) and triggers NPM1 nuclear translocation, where NPM1 acts as a transcription factor to upregulate interleukin 7 receptor (IL7R) expression. Upon interleukin 7 (IL-7) binding, IL7R activates the JAK1/STAT5 signaling pathway to increase the expression of the anti-apoptotic protein, BCL-2, and induce gemcitabine resistance. The PLD1 inhibitor, Vu0155069, targets PLD1 to induce apoptosis in gemcitabine-resistant PDAC cells. Conclusions: PLD1 is an enzyme that has a critical role in PDAC-associated gemcitabine resistance through a non-enzymatic interaction with NPM1, further promoting the downstream JAK1/STAT5/Bcl-2 pathway. Inhibiting any of the participants of this pathway can increase gemcitabine sensitivity