| BackgroundThe incidence of pancreatic cancer has been growing in the recent years in China.As traditional chemotherapy or existing targeting regiments failed to bring much benefit to the pancreatic cancer patients,pancreatic stellate cells,one of the key elements in the pancreatic cancer stroma,has become the spotlight in the search of novel therapeutic targets for the disease.Hyaluronan is a polysacccharide that is abundant in the pancreatic cancer stroma.Recent studies have revealed its role in promoting tumor growth and cell migration of pancreatic cancer cell lines.However,the source and the mechanism of bioactivity of hyaluronan remain unknown.The present study consists of two parts.In the first part,we explored the role of pancreatic stellate cells in the biosynthesis of hyaluronan.Afterwards,we stimulated a pancreatic cell line using exogenous hyaluronan to test for its bioactivity.The mechanisms were also investigated.Finally,we analyzed the expression of major regulators of hyaluronan metabolism in pancreatic cancer tissues,with the aim to associate it with clinicopathological parameters of pancreatic cancer.In the second part,we investigated the expression of 3 adaptor proteins,GAB2,FRS2,and CRKL,which were potential connectors of CD44,the most important receptor of hyalyuronan,and its downstream signaling components,in pancreatic cancer tissue to explore its clinicopathological and prognostic significances.Materials and methodsPart one.We analyzed the expression of HAS2 and HYAL1,which was respectively the most important synthesizing and hydrolyzing enzymes of hyaluron,in pancreatic cancer cell lines and pancreatic stellate cells using Real-Time PCR and Western Blot.Meanwhile,hyaluronan in the supernant of pancreatic cancer cell lines and pancreatic stellate cell,and that within the pancreatic cancer tissues was quantitatively and qualitatively examined using ELISA and agarose gel electrophoresis.Subsequently,exogenous hyaluronan or shRNA directed against HYAL1 was applied to pancreatic cancer cell line PANC-1 was HYAL1 expression in PANC-1 cell and changes of the biological behavior was documented.CD44 and RHAMM expression changes in response to hyaluronan treatment were also analyzed using westernblot to have a glimpse of the underlying mechanisms.Finally,we collected a few human pancreatic cancer specimens and analyzed the HAS2 and HYAL1 expressions using immunohistochemistry to explore the clinicopathological significances.Part two.Pancreatic ductal adenocarcinoma cases were retrieved from the archives of the pathology department of Peking Union Medical College using the following criteria:1)Date of registration between January 2011 and January 2016;2)Patient underwent radical resection of the tumor;3)Patient had not undergone preoperative adjuvant therapy.Pathological review and follow-up were conducted to obtain clinicopathological and prognostic information for the patients.The expression of GAB2,CRKL and FRS2 were analyzed using immunohistochemistry and their clinicopatholigcal and prognostic significances were explored.ResultsPart one.Activated pancreatic stellate cells show higher HAS2 expression in comparison to quiescent pancreatic stellate cells,while HAS2 and HYAL1 expressions were even higher in the pancreatic cell lines.The concentration of hyaluronan was higher in the supernatant of pancreatic stellate cells than that of the pancreatic cell lines in the form of high molecular weight hyaluronan,resembling the situation in pancreatic cancer tissues.High molecular weight hyaluronan treatment of the PANC-1 cell line promoted cell motility but impaired cell growth.Knock down of HYAL1 diminished the induced migration of PANC-1 cell,along with a decrease in the endocytosis of hyaluronan.Cleavage of CD44 was also observed in PANC-1 cells after hyaluronan treatment.In 18 pancreatic cancer cases,HAS2 and HYAL1 were positive in 85.17%and 100%of the cases,respectively.HAS2 expression was associated with larger tumor size and neuroinvasion.Part two.77 cases were enrolled in the present study.GAB2,CRKL,and FRS2 were positive in 40%,53.95%,and 35.06%of the cases.Median follow-up time was 16 months(range 1 to 48 months).GAB2,CRKL,and FRS2 expression alone,as well as GAB/CRKL/FRS2 co-expression were all indicators for poor prognosis regarding disease-free survival and overall survival.Among them,GAB2 expression was an independent prognosticator for disease-free survival,while GAB2/CRKL/FRS2 co-expression was an independent prognosticator for overall survival.ConclusionUp-regulated HAS2 in the activated pancreatic stellate cells was one source of the high molecular weight hyaluronan in the pancreatic cancer stroma.High molecular weight hyaluronan promoted pancreatic cancer cell motility in an HYAL1 dependent manner.The induced cell motility was associated with CD44 cleavage.Besides,GAB2,CRKL,and FRS2,the 3 adaptor proteins potentially downstream of CD44 were frequently expressed in pancreatic cancer.They all showed significant prognostic value. |
PDF Full Text Request