Cancer Letters

Cancer Letters

Volume 330, Issue 1, 1 March 2013, Pages 11-21
Cancer Letters

Original Articles
Delta like ligand 4 induces impaired chemo-drug delivery and enhanced chemoresistance in pancreatic cancer

https://doi.org/10.1016/j.canlet.2012.11.015Get rights and content

Abstract

The stubborn chemoresistance of pancreatic ductal adenocarcinoma (PDA) is simultaneously influenced by tumor parenchymal and stromal factors, and the ctritical role of Notch ligand Delta-like 4 (DLL4) in the regulation of tumor malignancies has been observed. DLL4 positive expression ratio between duct cells from clinical tumor and adjacent tissues was statistically significant, and the overactivation of DLL4/Notch pathway enhanced the phenotype of EMT and cancer stem cell, even can induce multi-chemoresistance in vitro. Notably, the accompanied defective angiogenesis directly induced inefficient chemo-drug delivery in vivo. Collectively, overexpressed DLL4 on neoplastic cells can enhance chemoresistance through angiogenesis-dependent/independent mechanisms in PDA.

Highlights

► DLL4 positive immunoreactivity was observed in most cases of duct cells from tumor tissues. ► Chemo-drug distribution was broadly consistent with intratumoral blood perfusion. ► Defective angiogenesis caused by DLL4/Notch pathway induced the inefficient drug delivery. ► DLL4 enhanced chemoresistance through the angiogenesis dependent and independent mechanisms.

Introduction

Pancreatic ductal adenocarcinoma (PDA) is a hypovascular scirrhous carcinoma with highly chemoresistance, and carries a dismal 5-year survival rate of about 5% [1]. Since less than 20% of patients present with surgically resectable disease at initial diagnosis [2], chemotherapeutics remains the primary modality for advanced PDA [3]. Unfortunately, even with Gemcitabine, the current most effective chemo agent for advanced PDA, the tumor response rate remains poor (<10%) and the median survival is approximately only 6 months [3], [4]. Thus, there is an urgent need for the design of new therapeutic strategies for conquering such refractory resistance.

Previous research has demonstrated that factors like genetic mutations, cancer stem cell (CSC) phenotype and drug metabolism are deeply involved in intrinsic and acquired chemoresistance in PDA [5], [6]. In addition, a recently eminent argument indicated that inefficient drug delivery caused by poor vascularization is another important contributor to stubborn chemoresistance in PDA [7]. These research progressions point out the chemoresistance of PDA is simultaneously influenced by tumor parenchymal factors and tumor stromal factors, and further investigation in the mechanisms underlying the stubborn chemoresistance of PDA could lead to novel efficient treatment.

Cumulative research reveals that both aberrant expression and over activation of Notch pathway components are prerequisites for the angiogenesis, tumorigenesis, maintenance and progression of many solid tumors, including PDA [8], [9]. The inactivation of Notch components could be a potential approach for overcoming the intractable chemoresistance of PDA [10], [11]. In humans, the NOTCH signaling pathway is evolutionarily conserved, and consists of four receptors (Notch1–4) and five transmembrane ligands (Jagged1, 2, Delta like ligands 1, 3, 4). Previous studies have shown that among the five ligands of Notch, Delta like ligand 4 (DLL4), which mainly activates Notch1 and Notch4, is a key negative regulator of angiogenesis both in embryonic and postnatal development [12]. DLL4 was once found mainly expressed on endothelial cells lining the tumor vasculature [13], [14], and significant progress has been made in understanding the key role of DLL4 in negative regulation of tumor vascularization [15], [16], [17]. But a recent study has reported the widespread DLL4 protein expression is observed in the cytoplasms of neoplastic cells from majority of tumors, including PDA, and stimulus like hypoxic condition can even promoted DLL4 translocating to the nuclei [18]. Meanwhile, it has been reported that DLL4 induced Notch activation is intimately related to tumor invasion [19] and tumor-initiating cell frequency [20], [21]. Clinical study has also revealed that the high DLL4 expression is significantly associated with poor prognosis in PDA patients [13]. In combination with the commonly observed ligand-dependent Notch activation in PDA [22], these researches indicated the biological role of DLL4 is far beyond a regulator of angiogenesis.

In our present study, for the first time, we systematically evaluated the influence of DLL4/Notch pathway on drug delivery and chemosensitivity in PDA. We first investigated the expression level of DLL4 and its main receptors Notch1/4 in clinical PDA specimens by using IHC or qPCR, and then we assessed the pathological function of overexpressed DLL4 in the efficacy of chemotherapeutics in PDA. Collectively, our data showed the expressions of these Notch components are up-regulated within the tumor cells/tissues in different levels, and overactivation of DLL4/Notch pathway can simultaneously impair chemo drug delivery and enhance chemoresistance in PDA.

Section snippets

Pancreatic carcinoma specimens preparation

The study was approved by the ethics committees of Zhejiang University. 42 tumor specimens (30 paraffin-embedded tissue blocks and 12 freshly-frozen tissues were collected from January 2007 to January 2010) and their corresponding normal tissues (pancreas tissue which was 2 cm apart from tumor tissue in each case) were randomly obtained from the tissue bank belonging to the Department of Surgery, Second Affiliated Hospital of Zhejiang University. All patients underwent resection for human

The DLL4 is overexpressed in PDA specimens, and the expression profile of major associated pathway factors is aberrant

Consistent with previous reports, DLL4 staining was observed in endothelial cells from tumor vasculature [13] as well as in normal pancreas epithelium cells [18] (Fig. 1A,1B). Cytoplasmic and membrane DLL4 positive immunoreactivity of variable intensity was observed in most cases of neoplastic cells (24/30, 80%) (Fig. 1D), and less frequently staining was detected in adjacent tissues (14/30, 46.67%) (Fig. 1C). Although, there was no statistical correlation between the presence of DLL4 in tumor

Discussion

Prognosis in patients with pancreatic cancer is extremely grim, and stubborn chemoresistance is the major cause of treatment failure. Recently, studies argue that both tumor parenchymal factors (such as EMT and cancer stem cell) [5], [6] and tumor stromal factors (such as vascular perfusion) [7] have profound impacts on drug resistance in PDA. Therefore, the combination of tumor angiogenesis inhibition and conventional chemotherapeutics is expected to be a new promising therapeutic approach for

Acknowledgements

This work was supported by the Grants from National Natural Science Foundation of China (Grant Numbers: 81172158, 81071960, 30901445, 81001094, and 81100549).

We thank XP Xu and YY Pan for help in laboratory animal feeding.

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