Amanda E. Brinker

Reduced O-GlcNAcase expression promotes mitotic errors and spindle defects

ABSTRACT Alterations in O-GlcNAc cycling, the addition and removal of O-GlcNAc, lead to mitotic defects and increased aneuploidy. Herein, we generated stable O-GlcNAcase (OGA, the enzyme that removes O-GlcNAc) knockdown HeLa cell lines and characterized the effect of the reduction in OGA activity on cell cycle progression. After release from G1/S, the OGA knockdown cells progressed normally through S phase but demonstrated mitotic exit defects. Cyclin A was increased in the knockdown cells while Cyclin B and D expression was reduced. Retinoblastoma protein (RB) phosphorylation was also increased in the knockdown compared to control. At M phase, the knockdown cells showed more compact spindle chromatids than control cells and had a greater percentage of cells with multipolar spindles. Furthermore, the timing of the inhibitory tyrosine phosphorylation of Cyclin Dependent Kinase 1 (CDK1) was altered in the OGA knockdown cells. Although expression and localization of the chromosomal passenger protein complex (CPC) was unchanged, histone H3 threonine 3 phosphorylation was decreased in one of the OGA knockdown cell lines. The Ewing Sarcoma Breakpoint Region 1 Protein (EWS) participates in organizing the CPC at the spindle and is a known substrate for O-GlcNAc transferase (OGT, the enzyme that adds O-GlcNAc). EWS O-GlcNAcylation was significantly increased in the OGA knockdown cells promoting uneven localization of the mitotic midzone. Our data suggests that O-GlcNAc cycling is an essential mechanism for proper mitotic signaling and spindle formation, and alterations in the rate of O-GlcNAc cycling produces aberrant spindles and promotes aneuploidy.

Mitochondrial genomic backgrounds affect nuclear DNA methylation and gene expression

Mitochondrial DNA (mtDNA) mutations and polymorphisms contribute to many complex diseases, including cancer. Using a unique mouse model that contains nDNA from one mouse strain and homoplasmic mitochondrial haplotypes from different mouse strain(s)-designated Mitochondrial Nuclear Exchange (MNX)-we showed that mtDNA could alter mammary tumor metastasis. Because retrograde and anterograde communication exists between the nuclear and mitochondrial genomes, we hypothesized that there are differential mtDNA-driven changes in nuclear (n)DNA expression and DNA methylation. Genome-wide nDNA methylation and gene expression were measured in harvested brain tissue from paired wild-type and MNX mice. Selective differential DNA methylation and gene expression were observed between strains having identical nDNA, but different mtDNA. These observations provide insights into how mtDNA could be altering epigenetic regulation and thereby contribute to the pathogenesis of metastasis. Cancer Res; 77(22); 6202-14. ©2017 AACR.

Mitochondrial Haplotype Alters Mammary Cancer Tumorigenicity and Metastasis in an Oncogenic Driver–Dependent Manner

Using a novel mouse model, a mitochondrial-nuclear exchange model termed MNX, we tested the hypothesis that inherited mitochondrial haplotypes alter primary tumor latency and metastatic efficiency. Male FVB/N-Tg(MMTVneu)202Mul/J (Her2) transgenic mice were bred to female MNX mice having FVB/NJ nuclear DNA with either FVB/NJ, C57BL/6J, or BALB/cJ mtDNA. Pups receiving the C57BL/6J or BALB/cJ mitochondrial genome (i.e., females crossed with Her2 males) showed significantly (P < 0.001) longer tumor latency (262 vs. 293 vs. 225 days), fewer pulmonary metastases (5 vs. 7 vs. 15), and differences in size of lung metastases (1.2 vs. 1.4 vs. 1.0 mm diameter) compared with FVB/NJ mtDNA. Although polyoma virus middle T-driven tumors showed altered primary and metastatic profiles in previous studies, depending upon nuclear and mtDNA haplotype, the magnitude and direction of changes were not the same in the HER2-driven mammary carcinomas. Collectively, these results establish mitochondrial polymorphisms as quantitative trait loci in mammary carcinogenesis, and they implicate distinct interactions between tumor drivers and mitochondria as critical modifiers of tumorigenicity and metastasis. Cancer Res; 77(24); 6941-9. ©2017 AACR.

Abstract 5882: Bench-to-bedside translation of ciclopirox prodrug for the treatment of non-muscle invasive and muscle-invasive bladder cancer

Ciclopirox (CPX) is contained in a number of FDA-approved topical antifungal drug products as the free acid and olamine salt. CPX possesses anticancer activity in a number of in vitro and in vivo preclinical models. Its clinical utility is limited as an oral anticancer agent, however. The oral bioavailability of CPX is quite low due to extensive first pass effect. The poor water solubility of CPX and its olamine salt prevent formulation as an injectable drug product. Thirdly, dose-limiting gastrointestinal toxicities were observed following four times daily oral dosing of CPX in patients with advanced hematologic malignancies. Ciclopirox Prodrug (CPX-POM), in contrast, has demonstrated excellent bioavailability via injectable routes of administration. Here we describe the preclinical characterization of CPX-POM, a novel anticancer agent being developed for the treatment of non-muscle invasive (NMIBC) and muscle invasive (MIBC) bladder cancer. Following IV, SQ and IP administration to mice, CPX-POM is rapidly and completely metabolized to CPX in blood via circulating phosphatases. CPX and its major, inactive glucuronide metabolite are extensively eliminated in urine. At well-tolerated doses, steady-state urine concentrations of CPX exceed in vitro IC50 values in mice by 15-30 fold. CPX inhibited cell proliferation, colony formation, and bladdosphere formation in vitro in T24 (NMIBC) and 253JBV (MIBC) human cell lines in both concentration- and time-dependent manners with IC50 values of 2-4 µM. CPX exposure increased the percentage of NMIBC and MIBC cells arrested at the S and G0/G1 phases, and induced cell death. CPX exposure significantly reduced expression of genes at the mRNA level involved in cancer stem cell signaling pathways including Notch, Wnt, and Hedgehog. CPX was shown to inhibit bladder cancer cell growth in vitro by inhibiting the Notch 1 signaling pathway. The validated N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) chemical carcinogen mouse model of bladder cancer was employed to establish in vivo preclinical proof of principle for CPX-POM. Over the once-daily IP dose range of 25-200 mg/kg, CPX-POM treatment resulted in significant decreases in bladder weight, a clear migration to lower stage tumors, dose-dependent reduction in Ki67 and PCNA staining, as well as a reduction in PCNA-expressing cells. All CPX-POM doses were well tolerated with no evidence of toxicity to the urinary tract based on blinded pathologic evaluation. There were also dose-dependent decreases in Notch 1, Presenilin 1, and Hey 1 in bladder cancer tissues obtained from CPX-POM treated animals. Tumor response was similar, in vivo, following once-daily and three-times weekly CPX-POM administration. CPX-POM has received FDA clearance to proceed to Phase I, and is currently being evaluated in a first-in-human trial in patients with advanced solid tumors. Citation Format: Scott J. Weir, Partha Ranjarajan, Robyn Wood, Karl Schorno, Prabhu Ramamoorthy, Lian Rajweski, Kathy Heppert, Michael J. McKenna, William McCulloch, Greg A. Reed, Amanda Brinker, Michael J. Baltezor, Roy A. Jensen, John A. Taylor, Shrikant Anant. Bench-to-bedside translation of ciclopirox prodrug for the treatment of non-muscle invasive and muscle-invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5882.

Abstract 1696: Mitochondrial haplotype alters metastasis in a non-cell autonomous manner

Mitochondrial Nuclear Exchange (MNX) mice, created by transferring the nucleus from an oocyte from strain x into an enucleated oocyte of strain y, showed that mammary tumor formation and metastasis can be regulated by inherited mitochondrial polymorphisms (PMID26471915). Besides genetic (cell autonomous) changes observed, we asked whether mitochondrial polymorphisms in non-cancer compartments could exert effects on tumor formation or metastasis. Tumor cells were injected into syngeneic wild type mice and tumor growth and metastasis were compared to similarly injected cells into MNX mice sharing the same nuclear but different mtDNA backgrounds. Orthotopic tumor growth rates were equal for all of the cell lines tested. However, the ability to form experimental lung metastases following i.v. injection were dramatically altered. Compared to injections into wild-type, syngeneic mice, E0771 mammary carcinoma and B16-F10 melanoma cells (both syngeneic to C57BL/6J), formed significantly (P MNX(C3H/HeN) and K1735-M2 melanoma cells (syngeneic to C3H/HeN) formed significantly fewer lung metastases in C3H/HeN mtMNXC57BL/6J . These results have been replicated at least three times using >10 mice per experiment. Interestingly, C57BL/6J mitochondria confer resistance to metastasis in both cell autonomous and non-cell autonomous experiments. Basal metabolic differences comparing mouse embryonic fibroblasts isolated from wild-type and MNX mice are among mechanisms being explored. Together, our findings highlight the striking influences that mitochondrial haplotypes can exert on tumorigenicity and metastasis via both intrinsic and extrinsic mechanisms. Support: Susan G. Komen for the Cure (SAC11037), Natl Fndn Cancer Res, Steiner Family Fund for Metastasis Research, Kansas Bioscience Authority, CA134981, P30-CA168524 Citation Format: Amanda E. Brinker, Carolyn J. Vivian, Danny R. Welch. Mitochondrial haplotype alters metastasis in a non-cell autonomous manner. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1696.

Abstract 2784: Mitochondrial genomic backgrounds affect nuclear DNA methylation and gene expression

Increasing evidence shows mitochondrial DNA (mtDNA) genetics contribute to complex diseases like cancer, but the mechanisms responsible are mostly unknown. A new genetically engineered mouse model was created that contains nuclear DNA from one mouse strain and mtDNA from different mouse strain(s), designated Mitochondrial Nuclear Exchange (MNX). We have demonstrated that specific mtDNA-nDNA combinations could modify metastasis efficiency. The underlying mechanism is hypothesized to be due to mtDNA-driven changes in nDNA expression, but how mtDNA regulates the coordinated gene expression is not yet known. Since DNA methylation is an important epigenetic modification that occurs in all vertebrate genomes and retrograde and anterograde cross-talk exists between the nuclear and mitochondrial genomes, we hypothesized that the nuclear epigenome, specifically methylation, changes as a result of mitochondrial haplotype. To test this hypothesis, we performed Meth-Seq using the Agilent Mouse SureSelectXT, RNA-Seq using Illumina HiSeq 2500®, and Affymetrix GeneChip® Mouse Transcriptome Array. Initial studies were performed using four male mouse brains (8 wk) that were pooled from different litters and cages of wild-type and MNX mice. Significant and selective differential DNA methylation and gene expression patterns were observed and indicate that there are some global, some subtle, but mostly reproducible, differences between these strains. Pathway analysis show clustering of changes involving cell adhesion, ion channels, cell surface receptors, metabolism and molecules involved in transport. Together these observations provide insights into how mtDNA could be altering epigenetic regulation and thereby contribute to cancer pathogenesis. Support: Susan G. Komen for the Cure (SAC11037), Natl Fndn Cancer Res, Steiner Family Fund for Metastasis Research, Kansas Bioscience Authority CA134981, P30-CA168524 Citation Format: Carolyn J. Vivian, Amanda E. Brinker, Gerald C. Gooden, Christophe Legendre, Samuel Turpin, Devin C. Koestler, Bodour Salhia, Danny R. Welch. Mitochondrial genomic backgrounds affect nuclear DNA methylation and gene expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2784.

Abstract 5128: Changes in mitochondrial background affect nuclear DNA methylation

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA There is emerging evidence demonstrating that mitochondrial DNA (mtDNA) contributes to complex genetic diseases such as cancer. To test directly the role of mtDNA in tumorigenicity and metastasis, a new genetically engineered mouse model was created that contains nuclear DNA from one mouse strain and mtDNA contribution from different mouse strain(s). Taking advantage of maternal inheritance, Mitochondrial Nuclear Exchange (MNX) mice can be bred to mice sharing the same nuclear DNA background. As a result, MNX serves as an important tool to determine if and how mitochondrial genetic background contributes to cancer, negating the concern for mixing maternal and paternal nuclear DNA polymorphisms. MNX mice are healthy, breed normally, show no overt differences in cell cycle or apoptosis and are genetically stable for multiple (>10) generations. Nonetheless, they are distinct strains based upon behaviors and gene expression patterns. The mechanisms controlling gene expression in different MNX strains are not yet known; however, retrograde and anterograde cross-talk exists between the nuclear and mitochondrial genomes. Our hypothesis is that important epigenetic changes occur in the nuclear genome depending upon mitochondrial genetics. To test this hypothesis, we performed Meth-Seq using the Agilent Mouse SureSelect platform and Affymetrix gene expression arrays on four mouse brains (8 wk) that were pooled from different litters and cages of wild-type and MNX to minimize age, developmental, inter-individual and generational heterogeneity. Both differential DNA methylation and gene expression patterns were observed but analyses are ongoing to better define the observed differences. Together these observations provide novel insights into how mtDNA could be altering epigenetic regulation and thereby contribute to cancer pathogenesis. Support: Susan G. Komen for the Cure (SAC11037), Natl Fndn Cancer Res., Kansas Bioscience Authority, Steiner Family Fund. Citation Format: Carolyn J. Vivian, Amanda E. Brinker, Gerald C. Gooden, Christophe Legendre, Bodour Salhia, Danny R. Welch. Changes in mitochondrial background affect nuclear DNA methylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5128. doi:10.1158/1538-7445.AM2015-5128

Abstract 3262: Mitochondrial haplotype effects on tumor formation and metastasis are both cell autonomous and non-cell autonomous

Increasing data support roles for mitochondrial genomes in complex diseases, including cancer. We hypothesize that primary tumor formation and metastasis can arise from inherited mitochondrial differences. To test directly the role of mitochondrial DNA (mtDNA) in mammary cancer tumorigenicity and metastasis, we generated Mitochondrial Nuclear Exchange (MNX) mice. This unique animal model is created by moving the nucleus from an oocyte of one strain into an enucleated oocyte of a different strain. By exchanging the nucleus of mouse strains promoting or inhibiting metastatic efficiency, mtDNA effects can be distinguished from phenotypes which would occur due to nuclear admixing. To determine if a change in the mtDNA background impacts metastasis in a cell autonomous manner, two FVB transgenic mouse strains encoding either Her2 or PyMT oncogenes were crossed with MNX mice with FVB nuclear DNA and mtDNA from either BALB/cJ or C57BL6J strains. The mtDNA were chosen because of higher or lower metastatic efficiency, respectively (PMID9679770, PMID16491073). Latency of mammary tumor formation in MNX mice with C57BL/6 mtDNA is longer for both Her2 and PyMT. Lung metastases are smaller in C57BL6 but larger in BALB/c MNX crosses with the PyMT. Studies measuring metastasis efficiency in the MNX crosses with Her2 are still in progress. To determine whether the mitochondrial haplotype alters tumorigenicity or metastasis in a non-cell autonomous manner, syngeneic tumor cells were injected orthotopically and ectopically (i.v.). Tumor formation and metastasis differ in a tumor and mtDNA-dependent manner. For example, E0771 forms significantly more lung metastases in C57BL/6n:C3H/HeNmt mice compared to controls. To explore underlying mechanisms, metabolic differences were observed in MNX and matched wild type mouse embryonic fibroblasts using the Seahorse bioanalyzer. Conclusion: mtDNA affects mammary cancer development and progression via both genetic and non-cell autonomous mechanisms in the tumor microenvironment. Support: Susan G. Komen for the Cure (SAC11037), Natl Fndn Cancer Res, Steiner Family Fund for Metastasis Research, Kansas Bioscience Authority, CA134981, P30-CA168524 Citation Format: Amanda E. Brinker, Carolyn J. Vivian, Kyle P. Feeley, Scott W. Ballinger, Danny R. Welch. Mitochondrial haplotype effects on tumor formation and metastasis are both cell autonomous and non-cell autonomous. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3262. doi:10.1158/1538-7445.AM2015-3262