Vahur Valvere

Metabolic control analysis of cellular respiration in situ in intraoperational samples of human breast cancer

The aim of this study was to analyze quantitatively cellular respiration in intraoperational tissue samples taken from human breast cancer (BC) patients. We used oxygraphy and the permeabilized cell techniques in combination with Metabolic Control Analysis (MCA) to measure a corresponding flux control coefficient (FCC). The activity of all components of ATP synthasome, and respiratory chain complexes was found to be significantly increased in human BC cells in situ as compared to the adjacent control tissue. FCC(s) were determined upon direct activation of respiration with exogenously-added ADP and by titrating the complexes with their specific inhibitors to stepwise decrease their activity. MCA showed very high sensitivity of all complexes and carriers studied in human BC cells to inhibition as compared to mitochondria in normal oxidative tissues. The sum of FCC(s) for all ATP synthasome and respiratory chain components was found to be around 4, and the value exceeded significantly that for normal tissue (close to 1). In BC cells, the key sites of the regulation of respiration are Complex IV (FCCu2009=u20090.74), ATP synthase (FCCu2009=u20090.61), and phosphate carrier (FCCu2009=u20090.60); these FCC(s) exceed considerably (~10-fold) those for normal oxidative tissues. In human BC cells, the outer mitochondrial membrane is characterized by an increased permeability towards adenine nucleotides, the mean value of the apparent Km for ADP being equal to 114.8u2009±u200913.6xa0μM. Our data support the two-compartment hypothesis of tumor metabolism, the high sum of FCC(s) showing structural and functional organization of mitochondrial respiratory chain and ATP synthasome as supercomplexes in human BC.

Metabolic control analysis of respiration in human cancer tissue

Bioenergetic profiling of cancer cells is of great potential because it can bring forward new and effective therapeutic strategies along with early diagnosis. Metabolic Control Analysis (MCA) is a methodology that enables quantification of the flux control exerted by different enzymatic steps in a metabolic network thus assessing their contribution to the system‘s function. Our main goal is to demonstrate the applicability of MCA for in situ studies of energy metabolism in human breast and colorectal cancer cells as well as in normal tissues. We seek to determine the metabolic conditions leading to energy flux redirection in cancer cells. A main result obtained is that the adenine nucleotide translocator exhibits the highest control of respiration in human breast cancer thus becoming a prospective therapeutic target. Additionally, we present evidence suggesting the existence of mitochondrial respiratory supercomplexes that may represent a way by which cancer cells avoid apoptosis. The data obtained show that MCA applied in situ can be insightful in cancer cell energetic research.

Identifying women at risk for delayed presentation of breast cancer: a cross-sectional study in Estonia

BackgroundSurvival from breast cancer remains lower in Estonia than in most other European countries. More advanced stage and larger tumors that have impact on survival may be a result of delay in seeking help for breast cancer symptoms. The aim of this study was to identify determinants of delayed presentation among breast cancer patients in Estonia.MethodsThe study population included women with primary breast cancer diagnosed in Estonia in 2008–2010. All data were collected using structured personal interviews carried out by trained nurses in the hospital setting. Only patients with self-discovered symptoms were included in this analysis. Patient delay was measured as time elapsing from symptom self-discovery to first medical consultation. The effect of different factors on the likelihood of prolonged delay (>90xa0days) was evaluated using logistic regression.ResultsAmong 703 eligible patients, median patient delay was 16xa0days (interquartile range 5–54xa0days). Seventeen percent of the patients had their first medical consultation more than three months after self-detection of symptoms. In multivariate analysis, the risk of prolonged delay was significantly associated with age 65xa0years and over (OR 2.27, 95% CI 1.23–4.20), current smoking (OR 2.09, 95% CI 1.21–3.61), symptoms other than painless breast lump or breast pain (OR 1.84, 95% CI 1.08–3.16), no history of mammograms (OR 1.83, 95% CI 1.13–2.95), having received no information on breast cancer during past year (OR 1.77, 95% CI 1.05–2.99), and previous benign breast problems (OR 1.65, 95% CI 1.01–2.67). Non-significant risk increase was seen with lower education.ConclusionsThis study provides evidence that factors associated with delayed presentation of breast cancer in Eastern Europe are similar to those observed in Western countries. The results suggest that educational messages to general population should aim at increasing awareness of “non-lump” symptoms of breast cancer and encouraging women of all ages to present in a timely manner. Women at risk for delayed presentation such as smokers and women with no history of mammograms could be identified in the primary care setting.

An in situ study of bioenergetic properties of human colorectal cancer: The regulation of mitochondrial respiration and distribution of flux control among the components of ATP synthasome

The aim of this study is to characterize the function of mitochondria and main energy fluxes in human colorectal cancer (HCC) cells. We have performed quantitative analysis of cellular respiration in post-operative tissue samples collected from 42 cancer patients. Permeabilized tumor tissue in combination with high resolution respirometry was used. Our results indicate that HCC is not a pure glycolytic tumor and the oxidative phosphorylation (OXPHOS) system may be the main provider of ATP in these tumor cells. The apparent Michaelis-Menten constant (Km) for ADP and maximal respiratory rate (Vm) values were calculated for the characterization of the affinity of mitochondria for exogenous ADP: normal colon tissue displayed low affinity (Km = 260 ± 55 μM) whereas the affinity of tumor mitochondria was significantly higher (Km = 126 ± 17 μM). But concurrently the Vm value of the tumor samples was 60-80% higher than that in control tissue. The reason for this change is related to the increased number of mitochondria. Our data suggest that in both HCC and normal intestinal cells tubulin β-II isoform probably does not play a role in the regulation of permeability of the MOM for adenine nucleotides. The mitochondrial creatine kinase energy transfer system is not functional in HCC and our experiments showed that adenylate kinase reactions could play an important role in the maintenance of energy homeostasis in colorectal carcinomas instead of creatine kinase. Immunofluorescent studies showed that hexokinase 2 (HK-2) was associated with mitochondria in HCC cells, but during carcinogenesis the total activity of HK did not change. Furthermore, only minor alterations in the expression of HK-1 and HK-2 isoforms have been observed. Metabolic Control analysis showed that the distribution of the control over electron transport chain and ATP synthasome complexes seemed to be similar in both tumor and control tissues. High flux control coefficients point to the possibility that the mitochondrial respiratory chain is reorganized in some way or assembled into large supercomplexes in both tissues.

Metabolic remodeling in human colorectal cancer and surrounding tissues: alterations in regulation of mitochondrial respiration and metabolic fluxes

The aim of the work was to evaluate whether or not there is glycolytic reprogramming in the neighboring cells of colorectal cancer (CRC). Using postoperative material we have compared the functional capacity of oxidative phosphorylation (OXPHOS) in CRC cells, their glycolytic activity and their inclination to aerobic glycolysis, with those of the surrounding and healthy colon tissue cells. Experiments showed that human CRC cannot be considered a hypoxic tumor, since the malignancy itself and cells surrounding it exhibited even higher rates of OXPHOS than healthy large intestine. The absence of acute hypoxia in colorectal carcinomas was also confirmed by their practically equal glucose-phosphorylating capacity as compared with surrounding non-tumorous tissue and by upregulation of VEGF family and their ligands. Studies indicated that human CRC cells in vivo exert a strong distant effect on the energy metabolism of neighboring cells, so that they acquire the bioenergetic parameters specific to the tumor itself. The growth of colorectal carcinomas was associated with potent downregulation of the creatine kinase system. As compared with healthy colon tissue, the tumor surrounding cells display upregulation of OXPHOS and have high values of basal and ADP activated respiration rates. Strong differences between the normal and CRC cells in the affinity of their mitochondria for ADP were revealed; the corresponding Km values were measured as 93.6±7.7 µM for CRC cells and 84.9±9.9 µM for nearby tissue; both these apparent Km (ADP) values were considerably (by almost 3 times) lower in comparison with healthy colon tissue cells (256±34 µM).

Mitochondrial Respiration in Human Colorectal and Breast Cancer Clinical Material Is Regulated Differently

We conducted quantitative cellular respiration analysis on samples taken from human breast cancer (HBC) and human colorectal cancer (HCC) patients. Respiratory capacity is not lost as a result of tumor formation and even though, functionally, complex I in HCC was found to be suppressed, it was not evident on the protein level. Additionally, metabolic control analysis was used to quantify the role of components of mitochondrial interactosome. The main rate-controlling steps in HBC are complex IV and adenine nucleotide transporter, but in HCC, complexes I and III. Our kinetic measurements confirmed previous studies that respiratory chain complexes I and III in HBC and HCC can be assembled into supercomplexes with a possible partial addition from the complex IV pool. Therefore, the kinetic method can be a useful addition in studying supercomplexes in cell lines or human samples. In addition, when results from culture cells were compared to those from clinical samples, clear differences were present, but we also detected two different types of mitochondria within clinical HBC samples, possibly linked to two-compartment metabolism. Taken together, our data show that mitochondrial respiration and regulation of mitochondrial membrane permeability have substantial differences between these two cancer types when compared to each other to their adjacent healthy tissue or to respective cell cultures.

Abstract LB-268: Rate of temporal citrate efflux from malignant mitochondria predicts clinical aggressiveness in breast tumors

Background: Mitochondrial citrate has been shown to serve as a central substrate for many cytosolic processes critical for tumor progression. Experiments with stable isotopes and mass spectrometry have been used for this conclusion, but fixed pools of metabolites yielding from those studies do not help us understand kinetics of subcellular metabolic fluxes. New methods are necessary for such measurements. In clinical setting, luminal-A subtype of breast cancer progresses slower than triple negative subtype and we hypothesize that mitochondrial citrate efflux in more aggressive tumors must be greater than that in slow-growing tumors to support the faster progression. Methods: New permeabilization-based method for quantifying time-dependent mitochondrial fluxes was developed. Luminal-A type MCF7 and triple negative MDA-MB-231 cell lines were used; excised primary tumors were used from human subjects (luminal-A n=21, triple negative n=10). Mitochondrial substrate-to-citrate efflux was measured using glutamate (GM), pyruvate (PV) or palmitoylcarnitine (PC) as substrates. Finally, samples were analyzed using HPLC or UV-spectroscopy. Summary: Mitochondrial GM-to-citrate efflux in less aggressive MCF7 cells was 91.5% lower than that in the faster growing MDA-MB-231 cells (4.1 vs. 48.4 ng/min*106cell, respectively). Difference in PV-to-citrate efflux was smaller, yielding 25.0% lower result for MCF7 compared to MDA-MB-231. An opposite result was observed for PC-to-citrate efflux as the conversion of PC was 20.1% higher in MCF7 cells compared to MDA-MB-231. Significant amount of cytosolic GM-to-citrate was measured only for MDA-MB-231, possibly a result of reverse IDH1 action. In primary tumor samples from breast cancer patients, mitochondrial GM-to-citrate efflux in luminal-A subtype was 27.4% lower than that in more aggressive triple-negative subtype (92.9 vs. 127.9 ng/h*mg, respectively). The respective differences were 24.7% for PV-to-citrate efflux and 8.2% for PC-to-citrate. Conclusions: Here we showed for the first time that temporal mitochondrial fluxes can be directly quantified in cell lines and clinical tumors. Results from the new method showed that less aggressive breast cancer samples have significantly lower mitochondrial citrate efflux and GM-to-citrate increases the most as aggressiveness increases. Clinical cancer samples showed heterogeneity in citrate profile and it can be an addition in predicting individual disease progression as standard subtype is often not sufficient for it. Further studies are warranted to expand the use of this method to other mitochondrial targets (e.g. fluxes of folate or glutamine metabolism) and to conduct studies in tumor types other than breast cancer where usable aggressiveness markers are missing. In addition, suitability of this method for predicting tumor response to mitochondrial cancer drugs will be evaluated. Citation Format: Andre Koit, Natalja Timohhina, Vladimir Chekulayev, Vahur Valvere, Tuuli Kaambre. Rate of temporal citrate efflux from malignant mitochondria predicts clinical aggressiveness in breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-268. doi:10.1158/1538-7445.AM2017-LB-268