Kyle P. Feeley

Mitochondrial Genetics Regulate Breast Cancer Tumorigenicity and Metastatic Potential.

Current paradigms of carcinogenic risk suggest that genetic, hormonal, and environmental factors influence an individuals predilection for developing metastatic breast cancer. Investigations of tumor latency and metastasis in mice have illustrated differences between inbred strains, but the possibility that mitochondrial genetic inheritance may contribute to such differences in vivo has not been directly tested. In this study, we tested this hypothesis in mitochondrial-nuclear exchange mice we generated, where cohorts shared identical nuclear backgrounds but different mtDNA genomes on the background of the PyMT transgenic mouse model of spontaneous mammary carcinoma. In this setting, we found that primary tumor latency and metastasis segregated with mtDNA, suggesting that mtDNA influences disease progression to a far greater extent than previously appreciated. Our findings prompt further investigation into metabolic differences controlled by mitochondrial process as a basis for understanding tumor development and metastasis in individual subjects. Importantly, differences in mitochondrial DNA are sufficient to fundamentally alter disease course in the PyMT mouse mammary tumor model, suggesting that functional metabolic differences direct early tumor growth and metastatic efficiency.

Endothelial Cell Bioenergetics and Mitochondrial DNA Damage Differ in Humans Having African or West Eurasian Maternal Ancestry.

Background—We hypothesized that endothelial cells having distinct mitochondrial genetic backgrounds would show variation in mitochondrial function and oxidative stress markers concordant with known differential cardiovascular disease susceptibilities. To test this hypothesis, mitochondrial bioenergetics were determined in endothelial cells from healthy individuals with African versus European maternal ancestries. Methods and Results—Bioenergetics and mitochondrial DNA (mtDNA) damage were assessed in single-donor human umbilical vein endothelial cells belonging to mtDNA haplogroups H and L, representing West Eurasian and African maternal ancestries, respectively. Human umbilical vein endothelial cells from haplogroup L used less oxygen for ATP production and had increased levels of mtDNA damage compared with those in haplogroup H. Differences in bioenergetic capacity were also observed in that human umbilical vein endothelial cells belonging to haplogroup L had decreased maximal bioenergetic capacities compared with haplogroup H. Analysis of peripheral blood mononuclear cells from age-matched healthy controls with West Eurasian or African maternal ancestries showed that haplogroups sharing an A to G mtDNA mutation at nucleotide pair 10398 had increased mtDNA damage compared with those lacking this mutation. Further study of angiographically proven patients with coronary artery disease and age-matched healthy controls revealed that mtDNA damage was associated with vascular function and remodeling and that age of disease onset was later in individuals from haplogroups lacking the A to G mutation at nucleotide pair 10398. Conclusions—Differences in mitochondrial bioenergetics and mtDNA damage associated with maternal ancestry may contribute to endothelial dysfunction and vascular disease.

An ex-vivo model for evaluating bioenergetics in aortic rings.

Cardiovascular disease (CVD) is the leading cause of death worldwide and it exhibits a greatly increasing incidence proportional to aging. Atherosclerosis is a chronic condition of arterial hardening resulting in restriction of oxygen delivery and blood flow to the heart. Relationships between mitochondrial DNA damage, oxidant production, and early atherogenesis have been recently established and it is likely that aspects of atherosclerotic risk are metabolic in nature. Here we present a novel method through which mitochondrial bioenergetics can be assessed from whole aorta tissue. This method does not require mitochondrial isolation or cell culture and it allows for multiple technical replicates and expedient measurement. This procedure facilitates quantitative bioenergetic analysis and can provide great utility in better understanding the link between mitochondria, metabolism, and atherogenesis.

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

Abstract 4326: Mitochondrial genetics and cellular metabolism regulate tumorigenicity and metastatic potential

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Current paradigms of carcinogenic risk suggest that genetic, hormonal, and environmental factors combine to influence an individuals predilection for breast cancer and related metastatic tumor formation. The genetic component, in particular, has become the focus of many emergent studies. A renewed focus on cancer metabolism and the Warburg Effect has similarly cast a spotlight on the role, if any, of the mitochondrion in directing disease progression. Analysis of the direct contribution of mitochondrial DNA on tumorigenicity is made possible through the use of mitochondrial-nuclear exchange (MNX) mice in which nuclei from normal FVB mice (the background strain of the tg: MMTV-PyMT) were transferred onto cytoplasms containing C57BL/6 or BALB/c mitochondria. Crossing male FVB:tg:MMTV:PyMT mice with FVB(nDNA)C57BL/6(mtDNA) or FVB(nDNA)BALB/c(mtDNA) females maintained nuclear FVB nDNA and takes advantage of maternal inheritance of mtDNA. These PyMT transgene positive female progeny are then scored for primary tumor onset and pulmonary metastatic density. Present data indicate primary tumor latency segregating by mitochondrial DNA as PyMT-FVB wild-type animals develop primary tumors in 57 days compared to PyMT-FVB(n)C57BL/6(mt) which develop primary tumors in 65 days and PyMT-FVB(n)BALB/c(mt) animals having detectable tumors in 52 days. One group of animals were aged 40 days following primary tumor detection and a second group were sacrificed when aged to 70 days, allowing for evaluation of metastatic severity and confirmation of differential primary tumor growth, respectively. This work hypothesizes that the pre-existent “normal” mitochondrial haplotype harbored by an individual conveys risk in determining tumor latency and metastatic susceptibility. Furthermore, these changes in susceptibility will be accompanied by altered mitochondrial functional characteristics that can be attributed to differences in mitochondrial haplotype. To address those mitochondrial differences, primary mammary epithelial cells were isolated from resected tumors which were then assessed for Complex I and Complex IV activity. In addition, isolated mammary epithelial cells from tumor and healthy animals had bioenergetic profiles generated using the Seahorse XF24 analyzer. Markers of ROS production will also be assessed as they too have been implicated increasingly frequently in cancer aggressiveness. Citation Format: Kyle P. Feeley, Alexander W. Bray, Jessica L. Fetterman, David G. Westbrook, Larry W. Johnson, Robert A. Kesterson, Danny R. Welch, Scott W. Ballinger. Mitochondrial genetics and cellular metabolism regulate tumorigenicity and metastatic potential. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4326. doi:10.1158/1538-7445.AM2014-4326

Abstract SY20-03: Nuclear-mitochondrial cross-talk: A key determinant of cancer metastasis

Despite the well known energy requirements and stresses of metastasis, the relationships between metabolism, mitochondrial genetics and metastasis are still underdeveloped. Two lines of investigation point to more intimate involvement of mtDNA than is widely appreciated. First, recent data demonstrate that the metastasis suppressor KISS1 essentially reverses the so-called Warburg Effect by regulating mitochondrial biogenesis. KISS1 re-expression results in higher pH[Ex] due to reduced lactate secretion concomitant with reduced glycolysis and a shift toward oxidative phosphorylation. KISS1-expressing cells have 30-50% more mitochondrial mass, which appears to be due to higher expression of PPARγ co-activator 1α (PGC1α), a master regulator of mitochondrial biogenesis. shRNA-mediated knockdown of KISS1 and PGC1α establish a pathway between these molecules, mitochondrial biogenesis and metastatic potential. Second, genetic crosses with a newly described MNX (mitochondrial-nuclear exchange) mice suggest that mitochondrial polymorphisms (haplotypes) may control susceptibility to metastasis. Transgenic FVB/N-tg:MMTV-PyMT which spontaneously develop mammary tumors and lung metastasis with high penetrance were crossed with female MNX mice having the same nuclear background (FVB - wild-type) but with C57BL/6 and BALB/c mitochondrial backgrounds. Using this strategy, the mtDNA contributions to metastasis can be discriminated. Results demonstrate that tumor and metastasis incidence do not appear to be significantly different. However, metastasis size is greatly affected. Taken together, these data strongly support the concept that mitochondrial-nuclear cross-talk is a more significant determinant of metastasis than generally appreciated. SUPPORT: NCI-CA134981; Natl Fndn Cancer Res, Susan G. Komen SAC11037, Hall Family Fndn, KS Bioscience Auth. Citation Format: Danny R. Welch, Wen Liu, Kyle P. Feeley, Scott W. Ballinger. Nuclear-mitochondrial cross-talk: A key determinant of cancer metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY20-03. doi:10.1158/1538-7445.AM2014-SY20-03

Abstract A090: Mitochondrial genetics in the regulation of tumorigenicity and metastatic potential

Current paradigms of carcinogenic risk suggest that genetic, hormonal, and environmental factors combine to influence an individual9s predilection for breast cancer and related metastatic tumor formation. The genetic component, in particular, has become the focus of emergent studies which have determined a role for nuclear genetic differences directing breast cancer susceptibility. Studies examining tumor latency and metastatic formation in mice have demonstrated clear differences between inbred strains. However, these studies fail to exclude the possibility that mitochondrial genetic inheritance is responsible for the observed changes in tumor onset and metastatic spread due to maternal inheritance of the mitochondrial genome. Although mitochondrial mutations within the tumor cell have recently been implicated as contributing to metastatic potential, studies have not directly addressed the effects of the mitochondrial DNA (mtDNA) background of the host on disease susceptibility. This work hypothesizes that the pre-existent “normal” mitochondrial haplotype harbored by an individual conveys risk in determining tumor latency and metastatic susceptibility. Furthermore, these changes in susceptibility will be accompanied by altered mitochondrial functional characteristics that can be attributed to differences in mitochondrial haplotype. Analysis of the direct contribution of mitochondrial DNA on tumorigenicity is made possible through the use of mitochondrial-nuclear exchange (MNX) mice in which nuclei from normal FVB mice (the background strain of the tg: MMTV-PyMT) were transferred onto cytoplasms containing C57BL/6 or BALB/c mitochondria. Crossing male FVB:tg:MMTV:PyMT mice with FVB(nDNA)C57BL/6(mtDNA) or FBV(nDNA)BALB/c(mtDNA) females maintained nuclear FVB nDNA and takes advantage of maternal inheritance of mtDNA. Present data indicate primary tumor latency segregating by mitochondrial DNA as PyMT-FVB wild-type animals develop primary tumors in 57 days compared to PyMT-FVB(n)C57BL/6(mt) which develop primary tumors in 65 days. Bioenergetic analyses using the Seahorse XF-24 as well as electron transport complex enzymatic assays will be conducted to more precisely delineate the functional metabolic differences contributing to altered tumorigenicity. MNX crosses suggest that cross-talk between mtDNA and nDNA has a greater influence on metastasis than previously appreciated and that mtDNA may be used clinically to improve patient prognosis. Citation Format: Kyle P. Feeley, Alexander W. Bray, Jessica L. Fetterman, David G. Westbrook, Larry W. Johnson, Robert A. Kesterson, Danny R. Welch, Scott W. Ballinger. Mitochondrial genetics in the regulation of tumorigenicity and metastatic potential. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A090.

Abstract 3866: The KISS1 metastasis suppressor appears to reverse the Warburg effect by enhancing mitochondria biogenesis.

Cancer cells tend to utilize aerobic glycolysis even under normoxic conditions, which is commonly called the “Warburg Effect.” Aerobic glycolysis often directly correlates with malignant potential. Though its purpose remains unclear, the “Warburg Effect” is thought to confer advantages to proliferation, survival and dissemination to cancer cells by increasing uptake of nutrients into biomass. KISS1 protein is secreted and proteolytically cleaved into kisspeptins (KP) that block the colonization of disseminated metastatic C8161.9 human melanoma cells at secondary sites. In this study, we hypothesized that KISS1 metastasis suppression occurs via regulation of aerobic glycolysis. Comparison of bioenergetic and metabolic aspects of glucose metabolism showed that all KISS1-secreting clones were less invasive, took up less glucose, produced less lactate which corresponds to higher pH[Ex], effects which were reversed when cells were transduced with shRNA to KISS1. The metabolism, invasion, and metastasis changes did not occur when KISS1 was missing the signal peptide (ΔSS). Utilizing a Seahorse bioanalyzer, KISS1, but not ΔSS cells showed significantly decreased extracellular acidification rates, increased O2 consumption and elevated mitochondria reserve capacity, an indicator of mitochondrial condition and a parameter thought to improve the cells’ ability to cope with oxidative stress. KISS1-expressing cells have 30-50% more mitochondria compared to vector or ΔSS-expressing cells. Increased mitochondrial mass was accompanied by significantly increased expression of mitochondrial genes involved in apoptosis and mitophagy, protein processing and trafficking. Increased mitochondrial mass correlated with higher PGC1α considered to be a master co-activator that regulates mitochondrial mass and metabolism. Interestingly, KISS1 differentially affects PGC1α-mediated downstream pathways, i.e. fatty acid synthesis and β-oxidation. KISS1-mediated up-regulation of mitochondria biogenesis appears to rely on KISS1 interaction with NRF1, a major transcription factor of mitochondria biogenesis. KP10 (which can activate the KISS1 receptor) does not alter pH[Ex] since the metastatic tumor cells do not express KISS1R. This paradox - metastasis and metabolic changes require secretion, but responding cells do not have the receptor - raises questions regarding the mechanism. Nonetheless, these data appear to directly connect changes in mitochondria mass, cellular glucose metabolism and metastasis. [Support: CA134581, Natl. Fndn. Cancer Res., Komen SAC110037]. Citation Format: Wen Liu, Benjamin H. Beck, Kedar S. Vaidya, Kevin T. Nash, Anne R. Diers, Kyle P. Feeley, Aimee Landar, Scott W. Ballinger, Danny R. Welch. The KISS1 metastasis suppressor appears to reverse the Warburg effect by enhancing mitochondria biogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3866. doi:10.1158/1538-7445.AM2013-3866

Abstract 965: The KISS1 metastasis suppressor appears to reverse the ‘Warburg Effect’

In 1924, Otto Warburg described the preference of cancer cells for glycolytic metabolism, even under normoxic conditions and that these metabolic changes directly correlate with malignant potential of several cancers. Although its purpose remains unclear, the “Warburg Effect” is thought to confer proliferative and survival advantages by increasing uptake of nutrients into biomass. The KISS1 metastasis suppressor protein is secreted and proteolytically cleaved into so-called kisspeptins (KP) that block the colonization of metastatic C8161.9 human melanoma cells at secondary sites. We asked whether secreted KISS1 mediates its inhibitory effects on metastatic growth through regulation of the “Warburg Effect.” Comparing multiple bioenergetic and metabolic aspects of glucose metabolism in C8161.9 ± KISS1 showed that all KISS1-secreting clones had significantly (P 2 consumption. Interestingly, mitochondrial reserve capacity, an indicator thought to reflect a cell9s ability to cope with oxidative stresses, was also elevated in KISS1-expressing cells. Using mitochondrial-selective fluorescent probes, C8161.9 KISS1 melanoma and MDA-MB-435 KISS1 breast carcinoma cells have ∼30% more mitochondria compared to empty vector or KISS1ΔSS-expressing cells. Increased mitochondrial number in KISS1-expressing cells was correlated with higher levels of PGC-1α, a major mitochondrial biogenesis regulatory molecule, which was confirmed using siRNA to KISS1. Expression of KISS1 also protected C8161.9 cells from dichloroacetate-induced cell death. Unexpectedly, addition of KP10 to C8161.9 cells did not alter pHe, raising questions regarding the mechanism by which KISS1/KP alter PGC-1α in the absence of KISS1 receptor expression in the tumor cells. Nonetheless, these data appear to directly connect changes in mitochondrial number, metabolic pathway regulation and the metastatic process. Future studies will determine whether the increase in mitochondrial number is directly responsible for the change in glycolytic metabolism and whether these changes are necessary for KISS19s effects on metastatic growth. Support: RO1-CA134981, the National Foundation for Cancer Research, METAvivor, and UAB Med-into-Grad Fellowship. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 965. doi:10.1158/1538-7445.AM2011-965

Abstract 1941: Cancer-driven exhaustion of tumor-reactive γδ-T cells: Relevance to immunosurveillance and immunotherapy

In contrast to antigen-specific αβ-T cells (adaptive immunity), γδ-T cells can recognize and lyse malignantly-transformed cells almost immediately upon encounter in a manner that does not require the recognition of tumor-specific antigens (innate immunity). Given the well-accepted capability of γδ-T cells to innately kill a variety of cancerous cells, efforts are now actively underway to develop and refine the means by which to exploit the antitumor properties of γδ-T cells for clinical purposes. Here, we investigated a critical issue surrounding γδ-T cell-based cancer immunotherapies, which relates to the findings that γδ-T cells are numerically rare in the peripheral blood of some cancer patients, and only in a proportion of patients is it possible to activate and/or expand γδ-T cells either in vivo or ex vivo regardless of the methodology employed. This is in stark contrast to what is observed in healthy individuals, as γδ-T cells reliably respond to proliferative stimuli. Indeed, it is evident that such numerical or functional deficits may greatly hinder attempts to employ γδ-T cells in clinical settings. Therefore, the objective of the studies performed here was to examine the biological underpinnings that may explain the γδ-T cell impairments observed in cancer patients. To this end, we employed murine models of different malignancies (breast cancer, myeloma, and melanoma) and systematically examined γδ-T cell numbers in peripheral circulation, γδ-T cell proliferative capacity, and overall γδ-T viability and apoptosis status in healthy mice and mice bearing different tumors. We found that tumor-bearing mice had fewer γδ-T cells in peripheral blood, and γδ-T cells derived from tumor-bearing mice expanded poorly ex vivo using published expansion regimens. Importantly, we show that tumor cells were responsible for this numerical and functional exhaustion of γδ-T cells. Using both in vitro and in vivo models of different cancers we demonstrate that γδ-T cells undergo apoptosis after encounter with tumor cells and we have identified several putative genes involved in this γδ-T cell exhaustion. We propose that this cancer-associated γδ-T cell impairment could serve as a critical early event leading to impaired γδ-T cell anti-tumor immunosurveillance, which may contribute to the accelerated development or progression of different malignancies. Collectively, these findings will help facilitate the development of the next generation of clinical trials designed to exploit the tumor-reactive properties of γδ-T cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1941.