Jorming Goh

Mitochondrial targeted catalase suppresses invasive breast cancer in mice

BackgroundTreatment of invasive breast cancer has an alarmingly high rate of failure because effective targets have not been identified. One potential target is mitochondrial generated reactive oxygen species (ROS) because ROS production has been associated with changes in substrate metabolism and lower concentration of anti-oxidant enzymes in tumor and stromal cells and increased metastatic potential.MethodsTransgenic mice expressing a human catalase gene (mCAT) were crossed with MMTV-PyMT transgenic mice that develop metastatic breast cancer. All mice (33 mCAT positive and 23 mCAT negative) were terminated at 110 days of age, when tumors were well advanced. Tumors were histologically assessed for invasiveness, proliferation and metastatic foci in the lungs. ROS levels and activation status of p38 MAPK were determined.ResultsPyMT mice expressing mCAT had a 12.5 per cent incidence of high histological grade primary tumor invasiveness compared to a 62.5 per cent incidence in PyMT mice without mCAT. The histological grade correlated with incidence of metastasis with 56 per cent of PyMT mice positive for mCAT showing evidence of pulmonary metastasis compared to 85.4 per cent of PyMT mice negative for mCAT with pulmonary metastasis (p ≤ 0.05). PyMT tumor cells expressing mCAT had lower ROS levels and were more resistant to hydrogen peroxide-induced oxidative stress than wild type tumor cells, suggesting that mCAT has the potential of quenching intracellular ROS and subsequent invasive behavior. The metastatic tumor burden in PyMT mice expressing mCAT was 0.1 mm2/cm2 of lung tissue compared with 1.3 mm2/cm2 of lung tissue in PyMT mice expressing the wild type allele (p ≤ 0.01), indicating that mCAT could play a role in mitigating metastatic tumor progression at a distant organ site. Expression of mCAT in the lungs increased resistance to hydrogen peroxide-induced oxidative stress that was associated with decreased activation of p38MAPK suggesting ROS signaling is dependent on p38MAPK for at least some of its downstream effects.ConclusionTargeting catalase within mitochondria of tumor cells and tumor stromal cells suppresses ROS-driven tumor progression and metastasis. Therefore, increasing the antioxidant capacity of the mitochondrial compartment could be a rational therapeutic approach for invasive breast cancer.Please see related commentary article: http://www.biomedcentral.com/1741-7015/9/62

Mitochondrial‐targeted peptide rapidly improves mitochondrial energetics and skeletal muscle performance in aged mice

Mitochondrial dysfunction plays a key pathogenic role in aging skeletal muscle resulting in significant healthcare costs in the developed world. However, there is no pharmacologic treatment to rapidly reverse mitochondrial deficits in the elderly. Here, we demonstrate that a single treatment with the mitochondrial‐targeted peptide SS‐31 restores in vivo mitochondrial energetics to young levels in aged mice after only one hour. Young (5 month old) and old (27 month old) mice were injected intraperitoneally with either saline or 3 mg kg−1 of SS‐31. Skeletal muscle mitochondrial energetics were measured in vivo one hour after injection using a unique combination of optical and 31P magnetic resonance spectroscopy. Age‐related declines in resting and maximal mitochondrial ATP production, coupling of oxidative phosphorylation (P/O), and cell energy state (PCr/ATP) were rapidly reversed after SS‐31 treatment, while SS‐31 had no observable effect on young muscle. These effects of SS‐31 on mitochondrial energetics in aged muscle were also associated with a more reduced glutathione redox status and lower mitochondrial H2O2 emission. Skeletal muscle of aged mice was more fatigue resistant in situ one hour after SS‐31 treatment, and eight days of SS‐31 treatment led to increased whole‐animal endurance capacity. These data demonstrate that SS‐31 represents a new strategy for reversing age‐related deficits in skeletal muscle with potential for translation into human use.

Exercise Training in Transgenic Mice Is Associated with Attenuation of Early Breast Cancer Growth in a Dose-Dependent Manner

Epidemiological research suggests that regular physical activity confers beneficial effects that mediate an anti-tumor response and may reduce cancer recurrence. It is unclear what amount of physical activity is necessary to exert such a protective effect and what mechanisms are involved. We investigated the effects of voluntary wheel running on tumor progression and cytokine gene expression in the transgenic polyoma middle T oncoprotein (PyMT) mouse model of invasive breast cancer. Runners showed significantly reduced tumor sizes compared with non-runners after 3 weeks of running (p≤0.01), and the greater the running distance the smaller the tumor size (Pearsons r = −0.61, p≤0.04, R2 = 0.38). Mice running greater than 150 km per week had a significantly attenuated tumor size compared with non-runners (p≤0.05). Adipose tissue mass was inversely correlated with tumor size in runners (Pearsons r = −0.77, p = 0.014) but not non-runners. Gene expression of CCL22, a cytokine associated with recruitment of immunosuppressive T regulatory cells, was decreased in tumors of runners compared to non-runners (p≤0.005). No differences in tumor burden or metastatic burden were observed between runners and non-runners after ten weeks of running when the study was completed. We conclude that voluntary wheel running in PyMT mice correlates with an attenuation in tumor progression early during the course of invasive breast cancer. This effect is absent in the later stages of overwhelming tumor burden even though cytokine signaling for immunosuppressive regulatory T cells was down regulated. These observations suggest that the initiation of moderate exercise training for adjunctive therapeutic benefit early in the course of invasive breast cancer should be considered for further investigation.

Exercise enhances wound healing and prevents cancer progression during aging by targeting macrophage polarity

Physical activity, which can include regular and repetitive exercise training, has been shown to decrease the incidence of age-related diseases. Aging is characterized by aberrant immune responses, including impaired wound healing and increased cancer risk. The behavior and polarized phenotype of tissue macrophages are distinct between young and old organisms. The balance of M1 and M2 macrophages is altered in the aged tissue microenvironment, with a tilt towards an M2-dominant macrophage population, as well as its associated signaling pathways. These M2-type responses may result in unresolved inflammation and create an environment that impairs wound healing and is favorable for cancer growth. We discuss the concept that exercise training can improve the regulation of macrophage polarization and normalize the inflammatory process, and thereby exert anticancer effects and enhance wound healing in older humans.

A novel long term short interval physical activity regime improves body composition in mice

BackgroundExercise training (ET) and physical activity (PA) offer obvious health benefits in regular participants. In pre-clinical animal models, treadmills and running wheels are the models of choice for intervention studies using ET and PA. However, the frequency and duration necessary for positive effects on health are not completely understood. We investigated the impact of short duration voluntary wheel running on body composition in FVB × C57BL/6 F1 hybrid mice over 22 weeks. Mice were randomized and given access to voluntary wheel running (N = 6) or locked wheels (N = 5) for 1 hour per night during the dark cycle, 5 days per week.FindingAverage weekly running distance was generally cyclic in nature over the 22 weeks but did not change significantly from week to week, except for a difference between week 3 and week 9 (P = 0.05). Daily running distances ranged from 0.78 km to 1.45 km. Compared with non-runners, runners demonstrated significantly lower relative fat mass (9.98 ± 0.56% vs. 14.91 ± 1.47%, P = 0.0067) and significantly higher relative lean mass (79.18 ± 0.65% vs. 75.41 ± 1.28%, P = 0.019). No differences were observed with respect to glucose metabolism.ConclusionVoluntary wheel running for one hour a day five days a week over a five month period improved body composition in young adult mice. This repetitive short interval exercise regime should be a useful model to investigate the effects of structured moderate intensity physical activity on physiological performance and chronic disease conditions in mice.

Breast tumors in PyMT transgenic mice expressing mitochondrial catalase have decreased labeling for macrophages and endothelial cells.

We show by immunohistochemical labeling that prominent cell types in the tumor microenvironment of PyMT transgenic mice are tumor-associated macrophages (TAMs) and endothelial cells, and that both populations are decreased in the presence of mitochondrial targeted catalase (mCAT). This observation suggests that mitochondrial ROS can drive tumor invasiveness in conjunction with the presence of TAMs and increased angiogenesis. Since primary PyMT tumor cells expressing mCAT undergo increased apoptosis, mitochondrial antioxidants might be attractive anti-tumor agents.

Are Exercise and Mitochondrial Antioxidants Compatible in the Treatment of Invasive Breast Cancer

Breast cancer is now the leading cause of cancer mortality among women worldwide, with more that 40,000 American women dying from the disease annually. While these statistics are grim, numerous epidemiological studies generally support a protective effect of physical activity for breast cancer. Animal data using voluntary wheel running and invasive cancer models are in line with human epidemiological data suggesting that physical exercise has anti tumor affects and may be associated with an increase in reactive oxygen species (ROS). Elevated production of mitochondrial ROS (mtROS) is also associated with the promotion of tumor progression, and attenuation of oxidative stress with a mitochondrial targeted antioxidant has been shown to reduce tumor burden and metastasis in an invasive breast cancer mouse model. However, if an anti-tumor effect of running is associated with an increase in ROS, then there is a potential paradox in that any anti-oxidant activity directed to mitochondria might mitigate ROS and prevent anti-tumor affects. There are several mechanistic scenarios involving the tumor microenvironment and tumor associated macrophages where exercise and antioxidants may be compatible, thereby suggesting that physical activity and mitochondrial antioxidants could be complimentary and/or synergistic in suppressing invasive breast cancer by preventing or reversing the pro-tumor cell microenvironment and enhancing an anti-tumor microenvironment. Investigation of oxidative stress in the tumor microenvironment is an area highly relevant to understanding not just the biology of cancer, but also the mechanisms through which regular physical activity mediates changes in normal tissue during tumorigenesis and metastasis. Several antioxidant compounds that target mitochondria, such as the Szeto- Schiller (SS) peptides and mitoQ compounds, are being developed which could easily be tested in preclinical studies for compatibility with exercise training in the treatment and possible prevention of invasive breast cancer.

An immunohistochemical approach for monitoring effects of exercise on tumor stromal cells in old mice.

Epidemiological evidence supports a protective effect of physical activity for breast cancer in older women, but the mechanisms are not well understood. We used 18-month-old BALB/c mice injected in the mammary fat pad with syngeneic 4T1 tumor cells as a model of invasive breast cancer. During the tumor progression phase, there was a significant decrease in labeling for F4/80, a marker for mouse macrophages, and CD34, a marker for vascular endothelial cells, in primary tumors from mice that ran higher average distances compared to mice that ran lower average distances (p≤0.05). These observations suggest that immunohistochemistry can be used to monitor stromal cell populations in tumors from old mice under exercise conditions.

Abstract 2377: Variation in the BRCT1 domain of the DNA repair gene XRCC1 delays invasive breast cancer in mice

The breast cancer susceptibility protein BRCA1 C-terminal (BRCT) domain coordinates protein interactions in DNA repair and cell cycle control, and comprises over 100 member proteins. One of these, XRCC1, is involved in base excision repair and single strand break repair. In order to investigate the role of the XRCC1-BRCT1 domain in breast cancer, we generated a mouse line with a variation of leucine (L) from arginine (R) at position 360, and crossed it with PyMT transgenic mice with a highly penetrant metastatic breast cancer phenotype. PyMT mice with advanced tumors expressing the L360R variant (N=20) had an average tumor volume of 360±57 mm 3 compared to an average tumor volume of 730±69 mm 3 for PyMT/wild type littermates (N=20), significant at p less than 0.01. In addition, the L360R variant was associated with a significant decrease in metastatic foci in the lungs of PyMT mice, an increase in tumor cell apoptosis as measured by caspase 3 immunohistochemistry, a decrease in tumor cell proliferation as measured by ki-67 immunohistochemistry, and a decrease in PARP binding as determined by NADH depletion. These preliminary observations suggest that the XRCC1/BRCT1 domain is involved in invasive breast cancer, and that genetic variation of this domain can suppress the invasive phenotype by decreasing tumor cell proliferation and increasing apoptosis of tumor cells possibly in association with altered PARP binding and activity. The XRCC1 L360R mouse is a promising animal model to further investigate the potential of targeting the XRCC1/BRCT1 domain for adjunctive treatment of invasive breast cancer. 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 2377. doi:10.1158/1538-7445.AM2011-2377

Abstract 4180: The L360R point mutation in the DNA repair gene XRCC1 suppresses tumor progression

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Variations in the DNA repair gene X-ray repair cross-complementing group 1 (XRCC1) have been increasingly reported in cancer epidemiology investigations, but have yielded conflicting results and are lacking biological data. XRCC1 is an essential scaffolding protein that interacts with many proteins associated with base excision repair and single-strand break repair. Several polymorphisms and point mutations have been shown to be in the BRCT1 binding domain, where critical binding with the DNA damage sensor gene Poly (ADP-ribose) polymerase (PARP) occurs. The point mutation L360R interferes with PARP binding in cell culture systems. In order to further investigate the biological effects of L360R, we constructed a gene-targeted mouse line and showed the homozygous genotype to be embryonic lethal. We therefore used the heterozygous genotype to determine tumor susceptibility and showed that subcutaneously implanted B16 melanoma tumors developed more slowly in L360R mutant mice compared to wild type littermates (tumor volume 116±11 mm3 vs 263±21 mm3, respectively, p≤0.05) suggesting host mediated affects. We conducted a second experiment using azoxymethane (10mg/kg once a week for 6 weeks) to induce colon tumors and showed that after six months the tumor burden was 14.2±3 mm3 in mutants compared to 22.5±5 mm3 in wild type littermates (p≤ 0.05). These findings suggest that the XRCC1 L360R point mutation acts as a tumor suppressor, possibly through host mediated events. The molecular mechanism for the suppression is not yet known, but the XRCC1 L360R mouse line is a promising animal model to further investigate and determine the clinical relevance of this novel observation. 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 4180.