Efficient delivery of small interfering RNAs targeting particular mRNAs into pancreatic cancer cells inhibits invasiveness and metastasis of pancreatic tumors.

We report the use of small interfering RNAs (siRNAs) against ARHGEF4, CCDC88A, LAMTOR2, mTOR, NUP85, and WASF2 and folic acid (FA)-modified polyethylene glycol (PEG)-chitosan oligosaccharide lactate (COL) nanoparticles for targeting, imaging, delivery, gene silencing, and inhibition of invasiveness and metastasis in an orthotopic xenograft model. In vitro assays revealed that these siRNA-FA-PEG-COL nanoparticles were specifically inserted into pancreatic cancer cells compared to immortalized normal pancreatic epithelial cells and knocked down expression of the corresponding targets in pancreatic cancer cells. Cell motility and invasion were significantly inhibited by adding target siRNA-FA-PEG-COL nanoparticles into the culture medium. In vivo mouse experiments confirmed that when intravenously delivered, these siRNA-FA-PEG-COL nanoparticles became incorporated into human pancreatic cancer cells in mouse pancreatic tumors. Little accumulation was seen in the normal pancreas and vital organs. All target siRNA-FA-PEG-COL nanoparticles significantly inhibited retroperitoneal invasion. The siRNA-FA-PEG-COL nanoparticles against LAMTOR2, mTOR, and NUP85, which strongly inhibited retroperitoneal invasion and significantly inhibited peritoneal dissemination compared to the other nanoparticles, improved prognosis of the mice. Our results imply that siRNA-FA-PEG-COL nanoparticles against these six targets could have great potential as biodegradable drug carriers. In particular, siRNA nanoparticles against LAMTOR2, mTOR, and NUP85 may hold significant clinical promise.

Circulating pancreatic cancer exosomal RNAs for detection of pancreatic cancer.

Diagnostic biomarkers for the early diagnosis of pancreatic cancer are needed to improve prognosis for this disease. The aim of this study was to investigate differences in the expression of four messenger RNAs (mRNAs: CCDC88A, ARF6, Vav3, and WASF2) and five small nucleolar RNAs (snoRNAs: SNORA14B, SNORA18, SNORA25, SNORA74A, and SNORD22) in serum of patients with pancreatic cancer and control participants for use in the diagnosis of pancreatic cancer. Results were compared with the expression of sialylated Lewis (a) blood group antigen CA19-9, the standard clinical tumor biomarker. Reverse transcription quantitative real-time PCR showed that all of the mRNAs and snoRNAs, except CCDC88A, were encapsulated in exosomes and secreted from cultured pancreatic cancer cells, and present in cell culture medium. In a discovery-stage clinical study involving 27 pancreatic cancer patients and 13 controls, the area under the receiver operating characteristic curve (AUC) of two mRNAs (WASF2 and ARF6) and two snoRNAs (SNORA74A and SNORA25) was > 0.9 for distinguishing pancreatic cancer patients from controls; the AUC of CA19-9 was 0.897. Comparing serum levels of WASF2, ARF6, SNORA74A, SNORA25, and CA19-9 revealed that levels of WASF2 were the most highly correlated with the risk of pancreatic cancer. The AUCs of WASF2, ARF6, SNORA74A, and SNORA25 in serum from patients in the early stages of pancreatic cancer (stages 0, I, and IIA) were > 0.9, compared with an AUC of 0.93 for the level of CA19-9. The results of this study suggest that WASF2, ARF6, SNORA74A, and SNORA25 may be useful tools for the early detection of pancreatic cancer. Monitoring serum levels of WASF2 mRNA may be particularly useful, as it was the most highly correlated with pancreatic cancer risk.

Measurement of serum PODXL concentration for detection of pancreatic cancer.

BACKGROUND: The aim of this study was to investigate the use of podocalyxin (PODXL) and secretoglobin family 1D, member 2 (SCGB1D2) expressions in whole blood as diagnostic biomarkers to distinguish between patients with pancreatic cancer and control participants, in comparison with serum cancer antigen 19-9 (CA19-9), which is the current clinical standard. PATIENTS AND METHODS: Flow cytometric analysis was performed to determine the expressions of PODXL and SCGB1D2 on the surface of cultured pancreatic cancer cells. Immunoblotting was performed to determine whether PODXL and SCGB1D2 were detectable in the media of cultured pancreatic cancer cells. A discovery-stage clinical study was performed in a cohort of 23 patients with pancreatic cancer and 51 control individuals without pancreatic disease who had been treated in the Department of Gastroenterology and Hepatology at Kochi Medical School Hospital from April 2014 to January 2016. Serum PODXL and SCGB1D2 levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: PODXL and SCGB1D2 accumulated in the protrusions of cultured pancreatic cancer cells, and they were detectable both on the cell surface and in the cultured media from these cells. The discovery-stage clinical study showed that the area under the receiver-operating characteristic curve (AUC) was 0.96 (95% confidence interval [CI] 0.91-1.000) for PODXL, 0.80 (95% CI 0.67-0.94) for SCGB1D2, and 0.78 (95% CI 0.66-0.90) for CA19-9. The AUC for PODXL was thus significantly higher than that for CA19-9 (P = 0.006). The combination of SCGB1D2 with CA19-9 did not significantly increase the AUC (0.83; 95% CI 0.70-0.96) compared with the AUC for either SCGB1D2 or CA19-9 alone (P = 0.563). CONCLUSION: PODXL may be a novel, non-invasive diagnostic biomarker for the detection of pancreatic cancer.

The transcription factor HOXB7 regulates ERK kinase activity and thereby stimulates the motility and invasiveness of pancreatic cancer cells.

HOX genes encode transcription factors that function as sequence-specific transcription factors that are involved in cellular proliferation, differentiation, and death. The aim of this study was to investigate the role of a HOX family protein, HOXB7, in the motility and invasiveness of pancreatic cancer cells. We previously identified a HOXB7 transcript that is one of a number of transcripts that are preferentially translated in membrane protrusions in pancreatic cancer cells. Immunocytochemistry showed that HOXB7 was localized to the cell protrusions of migrating pancreatic cancer cells. Knockdown of HOXB7 by transfection with HOXB7-specific siRNA decreased these protrusions and inhibited the motility and invasiveness of the cells. Transfection of a HOXB7-rescue construct into the HOXB7-knockdown cells restored peripheral actin structures in cell protrusions and abrogated the HOXB7 knockdown-induced decrease in cell protrusions. It is generally accepted that the Rho family of GTPases regulate the organization of actin filaments and contribute to the formation of cell protrusions. The levels of the active Rho GTPases were not influenced by HOXB7 in the cells; however, HOXB7 knockdown decreased the level of phosphorylated ERK1/2. This inactivation of ERK1/2 decreased cell protrusions, thereby inhibiting the invasiveness of pancreatic cancer cells. Further investigation showed that HOXB7/ERK1/2 signaling selectively stimulated JNK and HSP27 phosphorylation and thereby increased the motility and invasiveness of pancreatic cancer cells. These results suggested that HOXB7 stimulates ERK1/2 phosphorylation and provided evidence that HOXB7, besides its role in transcriptional regulation, also promotes cell motility and invasiveness.

Vav3 is linked to poor prognosis of pancreatic cancers and promotes the motility and invasiveness of pancreatic cancer cells.

BACKGROUND/OBJECTIVES: The aim of this study was to investigate the role of the guanine nucleotide exchange factor Vav3 in the motility and invasiveness of pancreatic ductal adenocarcinoma (PDAC) cells. METHODS: Immunohistochemistry was used to determine whether high Vav3 expression in human PDAC tissues is correlated with poor prognosis. Immunocytochemistry was used to determine the association and intracellular distribution of Vav3, Rac1 and Akt in PDAC cells. Phosphoprotein array analysis was performed to determine the Vav3-associated intracellular signaling pathways. Immunocytochemistry and Matrigel invasion assays were used to examine the effects of Vav3 on the formation of cell protrusions and PDAC cell invasion. RESULTS: Expression of Vav3 in PDAC tissue was significantly correlated with overall survival. Vav3 was localized in cell protrusions of migrating PDAC cells. Knockdown of Vav3 inhibited the motility and invasiveness of PDAC cells through a decrease in cell protrusions. The levels of active Rac1 or active Akt were not associated with the concentration of Vav3 in cell protrusions. The Vav3-dependent promotion of motility and invasiveness was not modulated by Rac1 or Akt. Additionally, knockdown of Vav3 increased phosphorylated WNK1 in PDAC cells, and knockdown of WNK1 inhibited the motility and invasiveness. This study suggests that Vav3 can be a useful marker for predicting the outcome of patients with PDAC and that Vav3 can promote PDAC cell motility and invasion through association with dephosphorylation of WNK1. CONCLUSIONS: Vav3 was accumulated in cell protrusions, contributed to the formation of membrane protrusions, and thereby increased the motility and invasiveness of PDAC cells.

A successful case of deceased donor liver transplantation for a patient with intrahepatic arterioportal fistula.

Intrahepatic arterioportal fistula (IAPF) is a rare cause of portal hypertension that is often difficult to treat with interventional radiology or surgery. Liver transplantation for IAPF is extremely rare. We report a case of bilateral diffuse IAPF with severe portal hypertension requiring deceased donor liver transplantation (DDLT). A 51-year-old woman with no past medical history was admitted to another hospital complaining of abdominal distension and marasmus. A computed tomography scan and digital subtraction angiography indicated a massive pleural effusion, ascites, and a very large IAPF. Several attempts of interventional embolization of the feeding artery failed to ameliorate arterioportal shunt flow. As ruptures of the esophageal varices became more frequent, hepatic encephalopathy worsened. After repeated, uncontrollable attacks of hepatic coma, the patient was referred to our facility for further treatment. Surgical approaches to IAPF other than liver transplantation were challenging because of diffuse collateralization; therefore, we placed the patient on the national waiting list for DDLT. Although her Model for End-Stage Liver Disease score was relatively low, she received a DDLT 2 months after the waiting period. The postoperative course was uneventful, and the patient was discharged 44 days after her transplant. Liver transplantation may be a valid treatment option for uncontrollable IAPF with severe portal hypertension.