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Access Type

WSU Access

Date of Award

January 2020

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Cancer Biology

First Advisor

Q. Ping Dou

Abstract

Modified metabolism is a hallmark of cancer cell biology known as “The Warburg Effect,” characterized by increased glucose-consumption and lactate-production, providing the metabolites and energy necessary for rapid proliferation. Breast-cancer-associated-gene-2 (BCA2) is an E3-Ub-ligase known to modulate AMPK, the “master-regulator of energy-homeostasis.” BCA2 is an oncogene associated with poor patient-survival and breast cancer invasiveness. Phospho-proteomic analysis of siBCA2-MDA-MB-231 knockdown-(KD) revealed enrichment of Nicotinamide-Nucleotide-Adenylyl-transferase-1 (NMNAT1), an essential enzyme in nuclear NAD-synthesis and a tumor-suppressor.

Initial experiments demonstrated that glucose concentration was positively correlated with BCA2 and inversely with NMNAT1 protein expression. shBCA2-KD in MDA-MB-468/-231 reproduced the inverse correlation between BCA2 and NMNAT1. shBCA2 -KD resulted in a decrease in BCA2 protein (MDA-MB-468: 54% and 49%; MDA-MB-231: 31% and 19%), qPCR-transcript (MDA-MB-468: 45%; MDA-MB-231: 31%) expression.

shBCA2-KD also resulted in an inverse increase In NMNAT1 qPCR-transcript (MDA-MB-468: 146%; MDA-MB-231: 150%) and promoter levels (MDA-MB-468: 409% and 459%; MDA-MB-231: 175% and 169%). shBCA2-KD was also associated with a decrease in the ratio of key enzyme associated with Warburg metabolism (LDHA/LDHB); high LDHA/LDHB is associated with Warburg-like metabolism, and low LDHA/LDHB is associated with decreased Warburg-like metabolism. Consistent with these findings, shBCA2 -KD resulted in decreased glucose consumption (MDA-MB-468: 70% and 76%; MDA-MB-231: 75% and 77%), decreased lactate production (MDA-MB-468: 69% and 72%; MDA-MB-231: 68% and 64%), and decrease cellular proliferation (MDA-MB-468: 54% and 49%; MDA-MB-231: 31% and 19%). All of these data are consistent with a role for BCA2 in maintaining Warburg-like metabolism in breast cancer cell lines, and that inhibition of BCA2 decreases Warburg-like metabolism and tumor cell proliferation.

In order to investigate the reciprocal role of NMNAT1 in triple negative breast cancer cells NMNAT1 was knocked down. The shNMNAT1-KD induced BCA2 protein, indicating a potential reciprocal feedback loop. shNMNAT1 -KD resulted in a decrease in NMNAT1 protein (MDA-MB-468: 7070%, 6286 and 8427%; MDA-MB-231: 212%, 202% and 19%), qPCR-transcript (MDA-MB-468: 45%; MDA-MB-231: 31%) expression.

shBCA2-KD also resulted in an inverse increase in NMNAT1 qPCR-transcript (MDA-MB-468: 146%; MDA-MB-231: 150%) and promoter levels (MDA-MB-468: 409% and 459%; MDA-MB-231: 175% and 169%). shBCA2-KD was also associated with a decrease in the ratio of key enzyme associated with Warburg metabolism (LDHA/LDHB); high LDHA/LDHB is associated with Warburg-like metabolism, and low LDHA/LDHB is associated with decreased Warburg-like metabolism. Consistent with these findings, shBCA2 -KD resulted in decreased glucose consumption (MDA-MB-468: 70% and 76%; MDA-MB-231: 75% and 77%), decreased lactate production (MDA-MB-468: 69% and 72%; MDA-MB-231: 68% and 64%), and decrease cellular proliferation (MDA-MB-468: 54% and 49%; MDA-MB-231: 31% and 19%). All of these data are consistent with the role of BCA2 in maintaining Warburg-like metabolism in breast cancer cell lines, and that inhibition of BCA2 decreases Warburg-like metabolism and tumor cell proliferation.

In addition, shBCA2/NMNAT1-KD differentially regulates the expression of LDHA/LDHB. The ratio of LDHA/LDHB modulates lactate/pyruvate equilibria and is a recognized marker of the relative level of Warburg metabolism. High LDHA/LDHB is positively correlated with enhanced Warburg activity.

NMNAT1, differentially regulates glucose-consumption and lactate-production. shBCA2-KD diminished LDHA/LDHB-ratio, lactate-production, and glucose-consumption. shNMNAT1-KD increased the LDHA/LDHB-ratio and lactate-production significantly, with and on glucose-consumption. shBCA2/NMNAT1-double-KD reversed the effects of shBCA2-KD, resulting in significant increases in lactate production with an increase of LDHA and LDHB relative to shNT and shBCA2, and increased LDHA/LDHB ratios relative to shBCA2-KD, as well as rescuing the effects of shBCA2 on glucose consumption relative to shNT and shBCA2.

Collectively, these data suggest that BCA2 and NMNAT1 modulate breast-cancer-cell-metabolism. The role of energy status indicated by the role of glucose in the regulation of BCA2/NMNAT1 could provide clues to the significance of these findings and thereby direct future investigations.

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