Notch Signaling Activated by Replication Stress–Induced Expression of Midkine Drives Epithelial–Mesenchymal Transition and Chemoresistance in Pancreatic Cancer

Abstract

The incidence of pancreatic ductal adenocarcinoma (PDAC) nearly equals its mortality rate, partly because most PDACs are intrinsically chemoresistant and thus largely untreatable. It was found recently that chemoresistant PDAC cells overexpress the Notch-2 receptor and have undergone epithelial-mesenchymal transition (EMT). In this study, we show that these two phenotypes are interrelated by expression of Midkine (MK), a heparin-binding growth factor that is widely overexpressed in chemoresistant PDAC. Gemcitabine, the front-line chemotherapy used in PDAC treatment, induced MK expression in a dose-dependent manner, and its RNAi-mediated depletion was associated with sensitization to gemcitabine treatment. We identified an interaction between the Notch-2 receptor and MK in PDAC cells. MK-Notch-2 interaction activated Notch signaling, induced EMT, upregulated NF-kB, and increased chemoresistance. Taken together, our findings define an important pathway of chemoresistance in PDAC and suggest novel strategies for its clinical attack. Cancer Res; 71(14);

The Multifunctional Growth Factor Midkine Promotes Proliferation and Migration in Pancreatic Cancer

“…Moreover, we have identified that gemcitabine dose-dependently induced expression and secretion of MK followed by activation of Notch signaling to promote epithelial-mesenchymal transition (EMT) and intrinsic as well as acquired chemotherapy resistance through upregulation of NF-kB. In addition, our results explained for the first time how MK may trigger cancer cells to become more resistant against cell death and how it promotes cell viability (9). Recently, we and others have shown that the secretion of MK in tumor cells may serve as biomarker for gastrointestinal tumors and glioblastomas (10,11).…”

“…The list of MK interacting receptors on cell surface is still growing, since we were recently able to identify the Notch-2 receptor as a new MK interactor in PDAC cells whose activation by extracellular MK ended in a highly chemoresistant phenotype accompanied by EMT (9).…”

“…Interestingly, several studies have already discussed the possibility of successfully neutralizing MK by using antibodies, antisense RNA, or aptamers and thereby blocking tumor growth or reversing chemotherapeutic resistance (9). Presumably, in case of PDAC, the appliance of TNF-a antagonists such as infliximab might be conceivable to counteract elevated MK expression in general or even during chemotherapeutical treatment modalities.…”

“…We observed a statistically significant mRNA overexpression (P < 0.05), whereas no expression was detected in HPDE cells. In a previously published report, we already showed that MK expression in patients with PDAC is significantly upregulated in more than 50% of the investigated samples (9). To verify that the MK mRNA expression resulted in robust protein expression, we investigated the MK protein expression in cancer cell lines and HPDE cells using specific antibodies (Fig.…”

“…For analyses of S-MK, cell culture media were collected and filtered (0.22 mM). Supernatants were resolved by SDS-PAGE and followed by immunoblotting as previously described (9). Western blot analyses and immunostainings were performed using MK antibodies (rab.…”