Date of Award

2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Drug Discovery and Biomedical Sciences

College

College of Graduate Studies

First Advisor

Christina Voelkel-Johnson

Second Advisor

Anna-Liisa Nieminen

Third Advisor

Douglas Pittman

Fourth Advisor

Patrick Woster

Fifth Advisor

Zhi Zhong

Abstract

Pancreatic cancer is extraordinarily resistant to chemotherapy. With current treatment options on average extending life by less than a year, advancements in sensitizing tumors to chemotherapy are a must. The combination of gemcitabine and nab-paclitaxel is the current standard of care for treating pancreatic cancer. Resistance to both of these drugs can occur through deregulation of the NFMκB pathway, MAPK pathway, Bcl-2 family members, as well as the balance of sphingolipids. More importantly, resistance to gemcitabine occurs through aberrations in its metabolism – mainly overexpression of the M2 subunit of ribonucleotide reductase. Additionally, overexpression of the transcription factor c-Myc has been shown to contribute to gemcitabine resistance. The specific sphingosine kinase 2 inhibitor ABC294640 has previously been shown to diminish the activation of ERK1/2 and NFMκB as well as alter the balance of Bcl-2 family members, thereby promoting cell death. ABC294640 can also diminish c-Myc protein levels. Preliminary studies revealed that ABC294640 synergized with both gemcitabine and paclitaxel in vitro in three pancreatic cancer cell lines. This led me to propose the central hypothesis that inhibition of nuclear SphK2 by ABC294640 synergizes with gemcitabine in different cell types by alteration of one or more common pathways that can be traceable to a common S1P mediated signaling event. Specific Aim 1 was to determine whether ABC294640 synergizes with gemcitabine through inhibition of the growth pathways known to promote gemcitabine resistance or alteration of the expression ratios of Bcl-2 family. Specific Aim 2 was to determine whether ABC294640 synergizes with gemcitabine by altering gemcitabine metabolism. In tracing the mechanism of the synergistic cell killing produced by the combination of ABC294640 and gemcitabine it was discovered that ABC294640 had no consistent impact on the NF-κB and MAPK pathways nor did it alter the ratio of Bcl-2 family members. However, treatment with ABC294640 decreased RRM2 and MYC transcript as well as their protein expression in three pancreatic cancer cell lines (BxPC-3, MiaPaCa-2, and Panc-1). Inhibition of RRM2 and c-Myc was shown to occur as a result of increased histone acetylation, which lead to increased expression of the cyclin-dependent kinase inhibitor p21. p21 prevents cyclinD/cdk4/6 mediated phosphorylation of Rb preventing release of the E2F transcription factors, thereby diminishing the transcription of its target genes two of which are RRM2 and MYC. ABC294640 caused a reduction in transcription of these genes. Due to the requirement of E2F in cell cycle progression, ABC294640 also caused G1 arrest in three pancreatic cancer cell lines. These data led to a model in which inhibition of SphK2 by ABC294640 in the nucleus leads to inhibition of histone deacetylase activity because of loss of nuclear S1P and/or elevation of nuclear sphingosine levels. The rise in histone acetylation leads to an increase in p21, which binds CDK/Cyclin-D1 complexes preventing the phosphorylation of Rb. Hypophosphorylated Rb sequesters the transcription factor E2F suppressing the expression of E2F target genes such as MYC and RRM2. This results in a decrease in proliferative signaling and an inhibition of cancer growth.

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