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Targeting MRP3 in Pancreatic Cancer In Vitro

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Thesis(3.33 MB)

Date

2020

Authors

Supervisor

Li, Yan
Merien, Fabrice
Liu, Dong-Xu

Item type

Thesis

Degree name

Doctor of Philosophy

Journal Title

Journal ISSN

Volume Title

Publisher

Auckland University of Technology

Abstract

Pancreatic cancer is currently one of the most aggressive and resistant cancers. The incidences rates of pancreatic cancer are comparatively low. However, the death rates of pancreatic cancer are within the top 10 of all cancers worldwide. The five-year survival rate is currently 5%, meaning that 95% of patients diagnosed with pancreatic cancer will not live more than five years. Most pancreatic cancer patients will succumb to the disease within one year of diagnosis. The current treatments for pancreatic cancer have not been significantly improved over the past three decades. The mechanism of action for gemcitabine in cancer killing is that the nucleoside analogue prevents cell cycle progression and proliferation. However, the major limiting factor with the use of gemcitabine and many other drugs is multidrug resistance. Multidrug resistance is the resistance of cancer towards multiple drugs that are distinct both in structure and pharmacological target. ABC transporters have modulated multidrug resistance across a wide range of cancers. Each ABC transporter has displayed unique substrate specificity, tissue distribution and unique molecules that inhibited its activity. P-gp, BCRP2 and MRP1 were among the first ABC transporters discovered and are the best characterized of the ABC transporters. However, recent studies have shown that other ABC transporters such as MRP2, MRP3 and MRP5 are also overexpressed in a variety of cancer types, which have been suggested to confer multidrug resistance. MRP3 has for instance, shown overexpression in pancreatic cancer cells, has a unique affinity for glucuronide metabolites and has modulated anticancer drug resistance (MTX, teniposide and etoposide). This study initially investigated the expression of MRP3 across three bioinformatic platforms (ONCOMINE, Kmplot and STRING). The results confirmed the wide-reaching overexpression of ABCC3/MRP3 in kidney, lung and pancreatic cancer. The well-established genomic editing tool CRISPR-Cas9 was used to knockout the expression of MRP3 in PANC1 cells by liposome-delivered three guide RNAs and Cas9 protein. Silencing MRP3 in knockout cell lines increased cellular accumulation of a model MRP3 substrate, 5-chloromethylfluorescein (CMF) by 47%-fold, 76% and 38%. The same mixed knockout populations also showed decreased resistance towards methotrexate (MTX, a well-characterised MRP3 drug substrate) and gemcitabine (GEM). In addition to CRISPR-Cas9 modulation, the activity of MRP3 was also targeted using the potential inhibitors, suramin, curcumin and EF24. Suramin, curcumin and EF24 all successfully increased CMF accumulation by 2.11-fold, 2.43-fold and 2.4-fold. Whether this would translate into reduced resistance was investigated. Within PANC1 cells, the combination of MTX and EF24 proved to be the most promising, with synergistic values ranging from 0.137 to 0.412. While, MIAPACA2 cells showed highly synergistic results across all combinations tested, especially the combination of MTX and suramin. This study was able to identify suramin, curcumin and EF24, three novel MRP3 inhibitors. Furthermore, for the first-time gemcitabine resistance was modulated by MRP3 expression in PANC1 cells. Taken together, our results suggest that MRP3 confers resistance to gemcitabine. Modulation of ABCC3 increased the sensitivity of PANC-1 and MiaPaCa-2 cells to gemcitabine and modulation of ABCC3 activity may represent a novel strategy to reverse gemcitabine resistance in pancreatic cancer cells. Screening tumour MRP3 expression levels to select patients for treatment with gemcitabine-based regimen alone or in combination with MRP3 modulation, could improve outcomes of pancreatic cancer treatment.  

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Keywords

Cancer, Pancreatic cancer, MRP3, ABCC3, In vitro, Molecular biology, ABC Transporters, Bioinformatics, CRISPR Cas9, Drug resistance, Multidrug resistance, Suramin, Curcumin, EF24, Gemcitabine, Methotrexate, Benzbromarone, CMFDA, Flow cytometry, Transport protein, Inhibition, Drug study

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Permanent link

https://hdl.handle.net/10292/13397

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Doctoral Theses