Treatment response assessment with (R)-[11CPAQ PET in the MMTV-PyMT mouse model of breast cancer.

BACKGROUND: The goal of the study was to assess the potential of the vascular endothelial growth factor receptor (VEGFR)-2-targeting carbon-11 labeled (R)-N-(4-bromo-2-fluorophenyl)-6-methoxy-7-((1-methyl-3-piperidinyl)methoxy)-4-quinazolineamine ((R)-[11C]PAQ) as a positron emission tomography (PET) imaging biomarker for evaluation of the efficacy of anticancer drugs in preclinical models. METHODS: MMTV-PyMT mice were treated with vehicle alone (VEH), murine anti-VEGFA antibody (B20-4.1.1), and paclitaxel (PTX) in combination or as single agents. The treatment response was measured with (R)-[11C]PAQ PET as standardized uptake value (SUV)mean, SUVmax relative changes at the baseline (day 0) and follow-up (day 4) time points, and magnetic resonance imaging (MRI)-derived PyMT mammary tumor volume (TV) changes. Expression of Ki67, VEGFR-2, and CD31 in tumor tissue was determined by immunohistochemistry (IHC). Non-parametric statistical tests were used to evaluate the relation between (R)-[11C]PAQ radiotracer uptake and therapy response biomarkers. RESULTS: The (R)-[11C]PAQ SUVmax in tumors was significantly reduced after 4 days in the B20-4.1.1/PTX combinational and B20-4.1.1 monotherapy groups (p < 0.0005 and p < 0.003, respectively). No significant change was observed in the PTX monotherapy group. There was a significant difference in the SUVmax change between the VEH group and B20-4.1.1/PTX combinational group, as well as between the VEH group and the B20-4.1.1 monotherapy group (p < 0.05). MRI revealed significant decreases in TV in the B20-4.1.1/PTX treatment group (p < 0.005) but not the other therapy groups. A positive trend was observed between the (R)-[11C]PAQ SUVmax change and TV reduction in the B20-4.1.1/PTX group. Statistical testing showed a significant difference in the blood vessel density between the B20-4.1.1/PTX combinational group and the VEH group (p < 0.05) but no significant difference in the Ki67 positive signal between treatment groups. CONCLUSIONS: The results of this study are promising. However, additional studies are necessary before (R)-[11C]PAQ can be approved as a predictive radiotracer for cancer therapy response.

Optitrain: a randomised controlled exercise trial for women with breast cancer undergoing chemotherapy.

BACKGROUND: Women with breast cancer undergoing chemotherapy suffer from a range of detrimental disease and treatment related side-effects. Exercise has shown to be able to counter some of these side-effects and improve physical function as well as quality of life. The primary aim of the study is to investigate and compare the effects of two different exercise regimens on the primary outcome cancer-related fatigue and the secondary outcomes muscle strength, function and structure, cardiovascular fitness, systemic inflammation, skeletal muscle gene activity, health related quality of life, pain, disease and treatment-related symptoms in women with breast cancer receiving chemotherapy. The second aim is to examine if any effects are sustained 1, 2, and 5 years following the completion of the intervention and to monitor return to work, recurrence and survival. The third aim of the study is to examine the effect of attendance and adherence rates on the effects of the exercise programme. METHODS: This study is a randomised controlled trial including 240 women with breast cancer receiving chemotherapy in Stockholm, Sweden. The participants are randomly allocated to either: group 1: Aerobic training, group 2: Combined resistance and aerobic training, or group 3: usual care (control group). During the 5-year follow-up period, participants in the exercise groups will receive a physical activity prescription. Measurements for endpoints will take place at baseline, after 16 weeks (end of intervention) as well as after 1, 2 and 5 years. DISCUSSION: This randomised controlled trial will generate substantial information regarding the effects of different types of exercise on the health of patients with breast cancer undergoing chemotherapy. We expect that dissemination of the knowledge gained from this study will contribute to developing effective long term strategies to improve the physical and psychosocial health of breast cancer survivors. TRIAL REGISTRATION: OptiTrain - Optimal Training Women with Breast Cancer (OptiTrain), NCT02522260 ; Registration: June 9, 2015, Last updated version Feb 29, 2016. Retrospectively registered.

Cutaneous exposure to hypoxia does not affect skin perfusion in humans.

AIM: Experiments have indicated that skin perfusion in mice is sensitive to reductions in environmental O2 availability. Specifically, a reduction in skin-surface PO2 attenuates transcutaneous O2 diffusion, and hence epidermal O2 supply. In response, epidermal HIF-1α expression increases and facilitates initial cutaneous vasoconstriction and subsequent nitric oxide-dependent vasodilation. Here, we investigated whether the same mechanism exists in humans. METHODS: In a first experiment, eight males rested twice for 8 h in a hypobaric chamber. Once, barometric pressure was reduced by 50%, while systemic oxygenation was preserved by O2 -enriched (42%) breathing gas (HypoxiaSkin ), and once barometric pressure and inspired O2 fraction were normal (Control1 ). In a second experiment, nine males rested for 8 h with both forearms wrapped in plastic bags. O2 was expelled from one bag by nitrogen flushing (AnoxiaSkin ), whereas the other bag was flushed with air (Control2 ). In both experiments, skin blood flux was assessed by laser Doppler on the dorsal forearm, and HIF-1α expression was determined by immunohistochemical staining in forearm skin biopsies. RESULTS: Skin blood flux during HypoxiaSkin and AnoxiaSkin remained similar to the corresponding Control trial (P = 0.67 and P = 0.81). Immunohistochemically stained epidermal HIF-1α was detected on 8.2 ± 6.1 and 5.3 ± 5.7% of the analysed area during HypoxiaSkin and Control1 (P = 0.30) and on 2.3 ± 1.8 and 2.4 ± 1.8% during AnoxiaSkin and Control2 (P = 0.90) respectively. CONCLUSION: Reductions in skin-surface PO2 do not affect skin perfusion in humans. The unchanged epidermal HIF-1α expression suggests that epidermal O2 homoeostasis was not disturbed by HypoxiaSkin /AnoxiaSkin , potentially due to compensatory increases in arterial O2 extraction.

Negative regulation of HIF in skeletal muscle of elite endurance athletes: a tentative mechanism promoting oxidative metabolism.

The transcription factor hypoxia-inducible factor (HIF) has been suggested as a candidate for mediating training adaptation in skeletal muscle. However, recent evidence rather associates HIF attenuation with a trained phenotype. For example, a muscle-specific HIF deletion increases endurance performance, partly through decreased levels of pyruvate dehydrogenase kinase 1 (PDK-1). HIF activity is regulated on multiple levels: modulation of protein stability, transactivation capacity, and target gene availability. Prolyl hydroxylases (PHD1-3) induces HIF degradation, whereas factor-inhibiting HIF (FIH) and the histone deacetylase sirtuin-6 (SIRT6) repress its transcriptional activity. Together, these negative regulators introduce a mechanism for moderating HIF activity in vivo. We hypothesized that long-term training induces their expression. Negative regulators of HIF were explored by comparing skeletal muscle tissue from moderately active individuals (MA) with elite athletes (EA). In elite athletes, expression of the negative regulators PHD2 (MA 73.54 ± 9.54, EA 98.03 ± 6.58), FIH (MA 4.31 ± 0.25, EA 30.96 ± 7.99) and SIRT6 (MA 0.24 ± 0.07, EA 11.42 ± 2.22) were all significantly higher, whereas the response gene, PDK-1 was lower (MA 0.12 ± 0.03, EA 0.04 ± 0.01). Similar results were observed in a separate 6-wk training study. In vitro, activation of HIF in human primary muscle cell culture by PHD inactivation strongly induced PDK-1 (0.84 ± 0.12 vs 4.70 ± 0.63), providing evidence of a regulatory link between PHD activity and PDK-1 levels in a relevant model system. Citrate synthase activity, closely associated with aerobic exercise adaptation, increased upon PDK-1 silencing. We suggest that training-induced negative regulation of HIF mediates the attenuation of PDK-1 and contributes to skeletal muscle adaptation to exercise.

Tumour oxygenation: implications for breast cancer prognosis.

There are areas of limited oxygen availability in most solid tumours, including breast cancer. Hypoxia in solid tumours is mainly a consequence of poor perfusion. Structural and functional abnormalities of newly formed tumour vessels cause spatial and temporal heterogeneity of tissue perfusion. The two principal mediators of hypoxia response, HIF-1 and HIF-2, are known to be stabilized at different oxygen levels and to have different temporal responses to hypoxia. Recently, stromal HIF-1 and HIF-2 have been suggested to have opposing roles in breast cancer progression. There is an established link between intralesional, severe hypoxia near areas of necrosis with high levels of HIF-1 and poor prognosis in breast cancer. However, the biological effects of moderate hypoxia and the hypoxic response of stromal cells are currently topics of intense investigation.

Reduction in plasma leucine after sprint exercise is greater in males than in females.

There is a pronounced gender difference in the accumulation of plasma ammonia after sprint exercise. Ammonia is a key intermediate in amino acid metabolism, which implies that gender-related differences in plasma and muscle amino acid concentrations after sprint exercise exist. To study this, three bouts of 30-s sprint exercise were performed by healthy females (n=8) and males (n=6). Blood leucine and muscle leucine were collected over the exercise period. Basal arterial plasma and skeletal muscle leucine were 40% higher in males than females (P<0.010 and P<0.020). Plasma, but not muscle, leucine decreased by sprint exercise and more so in males than females (g × t: P=0.025). Increase in ammonia was higher in males than females in both plasma and muscle (g × t: P<0.001 and P=0.003). An opposite pattern was shown for plasma glutamine, where an increase was found in females (P<0.001), but not in males. In conclusion, the lower plasma ammonia after sprint exercise in females seems to be explained by a lower accumulation of ammonia in skeletal muscle and by a buffering of ammonia in the form of glutamine in females. The greater reduction in plasma leucine in males seems to be related to their greater increase in muscle ammonia after sprint exercise.

Targeting of distinct signaling cascades and cancer-associated fibroblasts define the efficacy of Sorafenib against prostate cancer cells.

Sorafenib, a multi-tyrosine kinase inhibitor, kills more effectively the non-metastatic prostate cancer cell line 22Rv1 than the highly metastatic prostate cancer cell line PC3. In 22Rv1 cells, constitutively active STAT3 and ERK are targeted by sorafenib, contrasting with PC3 cells, in which these kinases are not active. Notably, overexpression of a constitutively active MEK construct in 22Rv1 cells stimulates the sustained phosphorylation of Bad and protects from sorafenib-induced cell death. In PC3 cells, Src and AKT are constitutively activated and targeted by sorafenib, leading to an increase in Bim protein levels. Overexpression of constitutively active AKT or knockdown of Bim protects PC3 cells from sorafenib-induced killing. In both PC3 and 22Rv1 cells, Mcl-1 depletion is required for the induction of cell death by sorafenib as transient overexpression of Mcl-1 is protective. Interestingly, co-culturing of primary cancer-associated fibroblasts (CAFs) with 22Rv1 or PC3 cells protected the cancer cells from sorafenib-induced cell death, and this protection was largely overcome by co-administration of the Bcl-2 antagonist, ABT737. In summary, the differential tyrosine kinase profile of prostate cancer cells defines the cytotoxic efficacy of sorafenib and this profile is modulated by CAFs to promote resistance. The combination of sorafenib with Bcl-2 antagonists, such as ABT737, may constitute a promising therapeutic strategy against prostate cancer.

Sprint exercise enhances skeletal muscle p70S6k phosphorylation and more so in women than in men.

AIM: Sprint exercise is characterized by repeated sessions of brief intermittent exercise at a high relative workload. However, little is known about the effect on mTOR pathway, an important link in the regulation of muscle protein synthesis. An earlier training study showed a greater increase in muscle fibre cross-sectional area in women than men. Therefore, we tested the hypothesis that the activation of mTOR signalling is more pronounced in women than in men. Healthy men (n=9) and women (n=8) performed three bouts of 30-s sprint exercise with 20-min rest in between. METHODS: Multiple blood samples were collected over time, and muscle biopsy specimens were obtained at rest and 140 min after the last sprint. RESULTS: Serum insulin increased by sprint exercise and more so in women than in men [gender (g) × time (t)]: P=0.04. In skeletal muscle, phosphorylation of Akt increased by 50% (t, P=0.001) and mTOR by 120% (t, P=0.002) independent of gender. The elevation in p70S6k phosphorylation was larger in women (g × t, P=0.03) and averaged 230% (P=0.006) as compared to 60% in men (P=0.04). Phosphorylation rpS6 increased by 660% over time independent of gender (t, P=0.003). Increase in the phosphorylation of p70S6k was directly related to increase in serum insulin (r=0.68, P=0.004). CONCLUSION: It is concluded that repeated 30-s all-out bouts of sprint exercise separated by 20 min of rest increases Akt/mTOR signalling in skeletal muscle. Secondly, signalling downstream of mTOR was stronger in women than in men after sprint exercise indicated by the increased phosphorylation of p70S6k.

Alternative splice variant PGC-1α-b is strongly induced by exercise in human skeletal muscle.

The present study investigated whether exercise induces the expression of PGC-1α splice variants in human skeletal muscle and the possible influence of metabolic perturbation on this response. The subjects exercised one leg for 45 min with restricted blood flow (R-leg), followed by 45 min of exercise using the other leg at the same absolute workload but with normal blood flow (NR-leg). This ischemic model (R-leg) has been shown previously to induce a greater metabolic perturbation and enhance the expression of PGC-1α beyond that observed in the NR-leg. Cultured human myotubes were used to test suggested exercise-induced regulatory stimuli of PGC-1α. We showed, for the first time, that transcripts from both the canonical promoter (PGC-1α-a) and the proposed upstream-located promoter (PGC-1α-b) are present in human skeletal muscle. Both transcripts were upregulated after exercise in the R-leg, but the fold change increase of PGC-1α-b was much greater than that of PGC-1α-a. No differences were observed between the two conditions regarding the marker for calcineurin activation, MCIP1, or p38 phosphorylation. AMPK phosphorylation increased to a greater extent in the R-leg, and AICAR stimulation of cultured human myotubes induced the expression of PGC-1α-a and PGC-1α-b. AICAR combined with norepinephrine yielded an additive effect on the PGC-1α-b expression only. Our results indicate clearly that exercise can activate an upstream promoter in humans and support AMPK as a major regulator of transcripts from the canonical PGC-1α promoter and the involvement of ß-adrenergic stimulation in combination with AMPK in the regulation of PGC-1α-b.

Training response of mitochondrial transcription factors in human skeletal muscle.

AIM: Mitochondrial function is essential for physical performance and health. Aerobic fitness is positively associated with mitochondrial (mt) biogenesis in muscle cells through partly unknown regulatory mechanisms. The present study aimed to investigate the influence of exercise and training status on key mt transcription factors in relation to oxidative capacity in human skeletal muscle. METHODS: The basal mRNA and protein levels of mitochondrial transcription factor A (TFAM), mitochondrial transcription factors B1 (TFB1M) or B2 (TFB2M), and mRNA levels of mitochondrial transcription termination factor (mTERF), were measured in a cross-sectional study with elite athletes (EA) and moderately active (MA) and the basal mRNA levels of these factors were measured during a 10-day endurance training programme with (R-leg) and without (NR-leg) restricted blood flow to the working leg. RESULTS: TFAM protein expression was significantly higher in the EA than in the MA, while protein levels of TFB1M and TFB2M were not different between the groups. There was no difference between EA and MA, or any effect with training on TFAM mRNA levels. However, the mRNA levels of TFB1M, TFB2M and mTERF were higher in EA compared with MA. For TFB1M and TFB2M, the mRNA expression was increased in the R-leg after 10 days of training, but not in the NR-leg. mTERF mRNA levels were higher in EA compared with MA. CONCLUSION: This study further establishes that TFAM protein levels are higher in conditions with enhanced oxidative capacity. The mRNA levels of TFB1M and TFB2M are influenced by endurance training, possibly suggesting a role for these factors in the regulation of exercise-induced mitochondrial biogenesis.

The influence of physical training on the angiopoietin and VEGF-A systems in human skeletal muscle.

Eleven subjects performed one-legged exercise four times per week for 5 wk. The subjects exercised one leg for 45 min with restricted blood flow (R leg), followed by exercise with the other leg at the same absolute workload with unrestricted blood flow (UR leg). mRNA and protein expression were measured in biopsies from the vastus lateralis muscle obtained at rest before the training period, after 10 days, and after 5 wk of training, as well as 120 min after the first and last exercise bouts. Basal Ang-2 and Tie-1 mRNA levels increased in both legs with training. The Ang-2-to-Ang-1 ratio increased to a greater extent in the R leg. The changes in Ang-2 mRNA were followed by similar changes at the protein level. In the R leg, VEGF-A mRNA expression responded transiently after acute exercise both before and after the 5-wk training program. Over the course of the exercise program, there was a concurrent increase in basal VEGF-A protein and VEGFR-2 mRNA in the R leg. Ki-67 mRNA showed a greater increase in the R leg and the protein was localized to the endothelial cells. In summary, the increased translation of VEGF-A is suggested to be caused by the short mRNA burst induced by each exercise bout. The concurrent increase in the Ang-2-to-Ang-1 ratio and the VEGF-expression combined with the higher level of Ki-67 mRNA in the R leg indicate that changes in these systems are of importance also in nonpathological angiogenic condition such as voluntary exercise in humans. It further establish that hypoxia/ischemia-related metabolic perturbation is likely to be involved as stimuli in this process in human skeletal muscle.

A single bout of exercise activates matrix metalloproteinase in human skeletal muscle.

The aims of this study were 1) to characterize changes in matrix metalloproteinase (MMP), endostatin, and vascular endothelial growth factor (VEGF)-A expression in skeletal muscle in response to a single bout of exercise in humans; and 2) to determine if any exchange of endostatin and VEGF-A between circulation and the exercising leg is associated with a change in the tissue expression or plasma concentration of these factors. Ten healthy males performed 65 min of cycle exercise, and muscle biopsies were obtained from the vastus lateralis muscle at rest and immediately and 120 min after exercise. In the muscle biopsies, measurements of mRNA expression levels of MMP-2, MMP-9, MMP-14, and tissue inhibitor of metalloproteinase; VEGF and endostatin protein levels; and MMP activities were performed. Femoral arterial and venous concentrations of VEGF-A and endostatin were determined before, during, and 120 min after exercise. A single bout of exercise increased MMP-9 mRNA and activated MMP-9 protein in skeletal muscle. No measurable increase of endostatin was observed in the skeletal muscle or in plasma following exercise. A concurrent increase in skeletal muscle VEGF-A mRNA and protein levels was induced by exercise, with no signs of peripheral uptake from the circulation. However, a decrease in plasma VEGF-A concentration occurred following exercise. Thus 1) a single bout of exercise activated the MMP system without any resulting change in tissue endostatin protein levels, and 2) the increased VEGF-A protein levels are due to changes in the skeletal muscle tissue itself. Other mechanisms are responsible for the observed exercise-induced decrease in VEGF-A in plasma.