Cancer Letters

Cancer Letters

Volume 402, 28 August 2017, Pages 1-8
Cancer Letters

Original Article
Chemosensitization and inhibition of pancreatic cancer stem cell proliferation by overexpression of microRNA-205

https://doi.org/10.1016/j.canlet.2017.05.007Get rights and content

Highlights

  • Differential downregulation of miR-205 (miR-205-5p) in pancreatic cancer tissues and cells.

  • Compared to gemcitabine (GEM)-sensitive cells, miR-205 is highly downregulated in GEM-resistant pancreatic cancer cells.

  • miR-205 regulates pancreatic cancer stem cells (CSCs), which in turn result in chemoresistance.

  • miR-205 overexpression in GEM-resistant cells reduces CSCs proliferation and inhibits tumor growth by resensitizing to GEM.

Abstract

Treatment of pancreatic cancer with gemcitabine (GEM) is limited due to its rapid plasma metabolism and development of chemoresistance. MicroRNA (miRNA) regulates cancer stem cell (CSC) maintenance and induces chemoresistance in cancer cells. In this study, we observed differential downregulation of miR-205 (miR-205-5p) in human pancreatic cancer tissues and cells. Compared to GEM-sensitive MIA PaCa-2 cells, miR-205 was highly downregulated in GEM-resistant MIA PaCa-2R cells. Lentivirus-mediated overexpression of miR-205 inhibits MIA PaCa-2R cell proliferation after GEM-treatment. Further investigation confirmed that miR-205 alone significantly reduces the proliferation of CSCs and tumor growth in mouse models. However, miR-205 in combination with GEM was more efficient in reducing the proliferation of CSCs and 3D spheroids. Moreover, miR-205 overexpressing MIA PaCa-2R cells induced orthotopic tumor growth was significantly inhibited after intravenous administration of GEM-conjugated methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate)-graft-gemcitabine-graft-dodecanol (mPEG-b-PCC-g-GEM-g-DC) (mPEG-b-PCC-g-GEM-g-DC) polymeric micelles. Also, a reduction in CSCs, EMT and chemoresistance markers was observed in miR-205 overexpressing MIA PaCa-2R cells. Immunohistochemical analysis of orthotopic tumors showed a decrease in drug resistance protein caveolin-1 and cell proliferation marker Ki-67 in combination treatment. Overall, our findings suggest that miR-205 resensitizes GEM-resistant pancreatic cancer cells to GEM and acts as a tumor suppressor miRNA.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is aggressive cancer to treat, with the limited success in the therapeutic management of patients. As per American Cancer Society, PDAC accounts for 3% of all cancers in the United States and about 7% of cancer-related deaths in 2016. Mortality rates for PDAC have increased by 0.4% per year since 2000 with estimated 41,780 deaths in 2016. Due to its late-stage clinical prognosis, 85% of patients have metastatic disease making surgical and therapeutic interventions ineffective [1], [2]. Gemcitabine (GEM) is a first-line FDA-approved drug used alone or in combination to improve the lifespan of PDAC patients. Although the mechanisms by which GEM induces cell death are well known, their effectiveness is incomplete due to the acquisition of drug resistance after prolonged treatment and desmoplastic microenvironment [3]. Several therapeutic interventions have been developed for GEM-resistant PDAC. For instance, zidovudine sensitizes GEM-resistant pancreatic cancer cells through Akt/GSK3β-Snail pathway [4]. Similar effects have also been shown by eribulin mesylate and the combination of cotylenin A and phenethyl isothiocyanate in killing GEM-resistant pancreatic cancer cells [5], [6]. Although the combination of GEM with nab-paclitaxel [7] and the chemotherapy regimen FOLFIRINOX has shown the overall survival of metastatic PDAC patients; a significant rate of grade 3/4 toxicity was observed [8].

Pancreatic cancer stem cell (CSC) displays enhanced chemoresistance and metastatic activity, which in turn is regulated by miRNAs. Aberrantly expressed miRNAs contribute to many biological processes including tumor initiation and progression, apoptosis, maintenance of CSCs and EMT phenotype [9], [10], [11]. Studies including ours have shown the importance of miRNAs and CSCs in cancer progression and metastasis, making their significance as biomarkers and potential targets for reversing drug resistance [12], [13]. Although miR-15a [14], miR-21 [15], miR-34 [16], miR-181b [17], miR-200b/c and let-7 family [18], [19] have shown their association with GEM-resistant PDAC, only few have been examined for their roles in drug resistance.

The development of miRNA-based therapeutics in combination with small molecule provides an unprecedented opportunity to counteract chemoresistance. Therefore, efficient delivery of miRNAs to the target organ and cells could improve treatment outcome in a broad range of human cancers. However, in vivo delivery of miRNAs remains a challenge due to their poor biostability, rapid excretion, immunogenicity, poor cellular uptake, endosomal escape and improper intracellular release. Our earlier study has shown miR-205 (miR-205-5p) to associate with CSC proliferation, resulting in chemosensitization of GEM-resistant pancreatic cancer cells after its transient expression [12]. Therefore, in this study, instead of transiently overexpressing miR-205, we used lentivirus particles to overexpress this miRNA in GEM-resistant pancreatic cancer cells to determine its long-term effect alone or in combination with GEM.

Section snippets

Human pancreatic cancer tissue samples and cells

Human frozen specimens from Rapid Autopsy Program (RAP) were obtained from surgical resections following well-known protocols and the informed consent waiver provided by the Institutional Review Board, University of Nebraska Medical Center (UNMC). Tissues were classified based on the diagnosis made by a pathologist. All cells were purchased from ATCC. GEM-resistant MIA PaCa-2R cells were maintained in DMEM containing GEM supplemented with 1% penicillin/streptomycin and 10% FBS throughout the

miR-205 suppressed GEM-resistant pancreatic cancer

miRNA profiling from primary pancreatic cancer lesions showed differential expression of various miRNAs, where miR-205 was significantly downregulated in cancer lesions compared to unaffected pancreatic tissue from matched patients (Fig. 1A). However, it was highly downregulated in a patient with the highest metastatic site (10 sites) and less in the least metastatic sites (2 sites). Similar expression pattern of miR-205 was also evident in GEM-sensitive MIA PaCa-2, HPAF-II and BXPC-3

Discussion

In this study, we investigated the expression of miRNAs in clinically defined pancreatic cancer lesions. Like others, we also observed downregulation of miR-205 in cancer lesions where it was highly downregulated in a patient with highest metastasis sites and low with least metastasis sites (Fig. 1A) [12]. Since most cancer-related mortality is metastasis-dependent, our observation suggests metastasis in PDAC might relate to the level of miR-205 expression. Although GEM is the first-line

Acknowledgments

This work was supported by the National Institutes of Health (1R01EB017853, P50CA127297), Fred and Pamela Buffet Cancer Center and the Faculty Start-up fund to RIM.

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