Pancreatic cancer cells express CD44 variant 9 and multidrug resistance protein 1 during mitosis

https://doi.org/10.1016/j.yexmp.2014.12.001Get rights and content

Highlights

  • PCC lines with an epithelial phenotype upregulate CD44v9 expression during mitosis.

  • Sorted CD44v9-negative cells resume CD44v9 expression during mitosis.

  • PCCs upregulate intracellular expression of MDR1 during mitosis.

  • Expression of CD44v9 and MDR1 may render dividing PCCs resistant to therapy.

Abstract

Pancreatic cancer is one of the most lethal cancers with high metastatic potential and strong chemoresistance. Its intractable natures are attributed to high robustness in tumor cells for their survival. We demonstrate here that pancreatic cancer cells (PCCs) with an epithelial phenotype upregulate cell surface expression of CD44 variant 9 (CD44v9), an important cancer stem cell marker, during the mitotic phases of the cell cycle. Of five human CD44+ PCC lines examined, three cell lines, PCI-24, PCI-43 and PCI-55, expressed E-cadherin and CD44 variants, suggesting that they have an epithelial phenotype. By contrast, PANC-1 and MIA PaCa-2 cells expressed vimentin and ZEB1, suggesting that they have a mesenchymal phenotype. PCCs with an epithelial phenotype upregulated cell surface expression of CD44v9 in prophase, metaphase, anaphase and telophase and downregulated CD44v9 expression in late-telophase, cytokinesis and interphase. Sorted CD44v9-negative PCI-55 cells resumed CD44v9 expression when they re-entered the mitotic stage. Interestingly, CD44v9bright mitotic cells expressed multidrug resistance protein 1 (MDR1) intracellularly. Upregulated expression of CD44v9 and MDR1 might contribute to the intractable nature of PCCs with high proliferative activity.

Introduction

Pancreatic cancer is one of the most lethal cancers with a 5-year survival rate less than 5%. This discouraging outcome is assumed to be due to the intrinsic biologic aggressiveness and robustness of pancreatic cancer cells (PCCs), characterized by extensive local invasion and strong resistance to conventional therapies (Li et al., 2004). Consequently, there have been no improvements of survival in patients with advanced pancreatic cancer even after the introduction of gemcitabine (Burris et al., 1997, Li et al., 2004).

Cancer stem cells (CSCs) are defined as a small fraction of tumor cells that display properties of self-renewal, high tumorigenicity and drug resistance, resulting in cancer metastasis and recurrence (Huntly and Gilliland, 2005, Mani et al., 2008). Accumulating evidence indicates the existence of pancreatic CSCs (Li et al., 2007). CD44, an important marker for CSCs, is a membrane glycoprotein involved in cell–cell and cell–extracellular matrix adhesion as well as cell migration, differentiation and survival (Zöller, 2011) and can directly reprogram stem cell properties in colon cancer cells (Su et al., 2011). The CD44 gene is encoded by 20 exons and generates a standard isoform (CD44s) and many variant isoforms (CD44v) by alternative splicing (Zöller, 2011). Importantly, expression of alternatively spliced variants reflects the grade of malignancy in gastric and breast cancers (Brown et al., 2011, Lau et al., 2014), with CD44s signaling the acquisition of a mesenchymal phenotype (Mima et al., 2012, Okabe et al., 2014). In breast cancer, splicing isoforms are switched from CD44v to CD44s as cancer cells undergo epithelial–mesenchymal transition (EMT) (Brown et al., 2011).

Although the precise functions of variant isoforms still remain to be elucidated, a recent study has shown that expression of the CD44 variant containing exons 8–10 (CD44v8-10 or CD44v9) contributes to the defense against reactive oxygen species by upregulating the synthesis of a major antioxidant, glutathione (Ishimoto et al., 2011), thereby making cancer cells resistant to chemotherapy and radiotherapy. CD44v9 also interacts with the death receptor FAS in lipid rafts, and interferes with death receptor signaling, leading to apoptosis resistance (Mielgo et al., 2006). Clinically, CD44v9 expression represents a potential predictive marker for recurrence in patients with colorectal (Yamaguchi et al., 1996) and multiple early gastric cancers (Hirata et al., 2013).

We demonstrate here that PCC lines with an epithelial phenotype upregulate the expression of CD44v9 and multidrug resistance protein 1 (MDR1) during the mitotic phases of the cell cycle. This observation suggests that the upregulated expression of CD44v9 and MDR1 might contribute to the intractable nature of PCCs with high proliferative activity.

Section snippets

Cells

Five human PCC lines, PCI-24, PCI-43, PCI-55, PANC-1 and MIA PaCa-2, were used. PCI-24, PCI-43 and PCI-55 were established in our laboratory from pancreatic ductal adenocarcinoma tissues of primary sites surgically resected at Hokkaido University Hospital (Kishimoto et al., 1996). MIA PaCa-2 cells were obtained from the RIKEN Bio-Resource Center (Tsukuba, Japan). PCC lines were maintained in Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum and penicillin/streptomycin.

PCI-24, PCI-43 and PCI-55 cells exhibit an epithelial phenotype, and PANC-1 and MIA PaCa-2 cells show a mesenchymal phenotype

We first analyzed the expression patterns of EMT markers in the following PCC lines: PCI-24, PCI-43, PCI-55, PANC-1 and MIA PaCa-2 (Fig. 1A). Transcriptional expression of E-cadherin, a prototypical epithelial cell marker (Zeisberg and Neilson, 2009), was observed in PCI-24, PCI-43 and PCI-55 cells. By contrast, PANC-1 and MIA PaCa-2 cells did not express E-cadherin, but expressed vimentin (Vuoriluoto et al., 2011) and ZEB1, a zinc finger transcription factor suppressing E-cadherin expression (

Discussion

In the present study, we have demonstrated that PCC lines with an epithelial phenotype strongly upregulate CD44v9 expression during mitosis (Fig. 2, Fig. 3, Fig. 4). Cell surface expression of CD44v8-10 plays an important role in the defense against reactive oxygen species, leading to ultimate survival of CSCs (Ishimoto et al., 2011). Therefore, our observation suggests that dividing PCCs can acquire strong resistance against oxidative stress. The malignant features of pancreatic cancer depend

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by Grants-in-Aid for Scientific Research Grant Number 26460464 from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT).

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