Carnitine palmitoyltransferase 1C promotes cell survival and tumor growth under conditions of metabolic stress.

Tumor cells gain a survival/growth advantage by adapting their metabolism to respond to environmental stress, a process known as metabolic transformation. The best-known aspect of metabolic transformation is the Warburg effect, whereby cancer cells up-regulate glycolysis under aerobic conditions. However, other mechanisms mediating metabolic transformation remain undefined. Here we report that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific metabolic enzyme, may participate in metabolic transformation. CPT1C expression correlates inversely with mammalian target of rapamycin (mTOR) pathway activation, contributes to rapamycin resistance in murine primary tumors, and is frequently up-regulated in human lung tumors. Tumor cells constitutively expressing CPT1C show increased fatty acid (FA) oxidation, ATP production, and resistance to glucose deprivation or hypoxia. Conversely, cancer cells lacking CPT1C produce less ATP and are more sensitive to metabolic stress. CPT1C depletion via siRNA suppresses xenograft tumor growth and metformin responsiveness in vivo. CPT1C can be induced by hypoxia or glucose deprivation and is regulated by AMPKα. Cpt1c-deficient murine embryonic stem (ES) cells show sensitivity to hypoxia and glucose deprivation and altered FA homeostasis. Our results indicate that cells can use a novel mechanism involving CPT1C and FA metabolism to protect against metabolic stress. CPT1C may thus be a new therapeutic target for the treatment of hypoxic tumors.

Metabolic targeting of HIF-dependent glycolysis reduces lactate, increases oxygen consumption and enhances response to high-dose single-fraction radiotherapy in hypoxic solid tumors.

BACKGROUND: A high rate of glycolysis leading to elevated lactate content has been linked to poor clinical outcomes in patients with head and neck and cervical cancer treated with radiotherapy. Although the biological explanation for this relationship between lactate and treatment response remains unclear, there is a continued interest in evaluating strategies of targeting metabolism to enhance the effectiveness of radiotherapy. The goal of this study was to investigate the effect of metabolic-targeting through HIF-1α inhibition and the associated changes in glycolysis, oxygen consumption and response on the efficacy of high-dose single-fraction radiotherapy (HD-SFRT). METHODS: HIF-1α wild-type and HIF-1α knockdown FaDu and ME180 xenograft tumors were grown in the hind leg of mice that were placed in an environmental chamber and exposed to different oxygen conditions (air-breathing and hypoxia). Ex vivo bioluminescence microscopy was used to measure lactate and ATP levels and the hypoxic fraction was measured using EF5 immunohistochemical staining. The oxygen consumption rate (OCR) in each cell line in response to in vitro hypoxia was measured using an extracellular flux analyzer. Tumor growth delay in vivo was measured following HD-SFRT irradiation of 20 Gy. RESULTS: Targeting HIF-1α reduced lactate content, and increased both oxygen consumption and hypoxic fraction in these tumors after exposure to short-term continuous hypoxia. Tumors with intact HIF-1α subjected to HD-SFRT immediately following hypoxia exposure were less responsive to treatment than tumors without functional HIF-1α, and tumors irradiated under air breathing conditions regardless of HIF-1α status. CONCLUSIONS: Blocking the HIF1 response during transient hypoxic stress increased hypoxia, reduced lactate levels and enhanced response to HD-SFRT. This strategy of combining hypofractionated radiotherapy with metabolic reprogramming to inhibit anaerobic metabolism may increase the efficacy of HD-SFRT through increased oxygen consumption and complementary killing of radiosensitive and hypoxic, radioresistant cells.

Interstitial fluid pressure, vascularity and metastasis in ectopic, orthotopic and spontaneous tumours.

BACKGROUND: High tumour interstitial fluid pressure (IFP) has been adversely linked to poor drug uptake in patients, and to treatment response following radiotherapy in cervix cancer patients. In this study we measured IFP values in a selection of murine and xenograft models, spontaneously arising or transplanted either intramuscularly (i/m) or orthotopically and analysed their relationship to tumour vascularity and metastatic spread. METHODS: KHT-C murine fibrosarcoma, ME180 and SiHa human cervix carcinoma were grown either intramuscularly (i/m), sub-cutaneously (s/c) or orthotopically. Polyoma middle-T (MMTV-PyMT) transgenic spontaneous mammary tumours were studied either as spontaneous tumours or following orthotopic or i/m transplantation. IFP was measured in all tumours using the wick-in-needle method. Spontaneous metastasis formation in the lungs or lymph nodes was assessed in all models. An immunohistochemical analysis of tumour hypoxia, vascular density, lymphatic vascular density and proliferation was carried out in ME180 tumours grown both i/m and orthotopically. Blood flow was also assessed in the ME180 model using high-frequency micro-ultrasound functional imaging. RESULTS: Tumour IFP was heterogeneous in all the models irrespective of growth site: KHT-C i/m: 2-42 mmHg, s/c: 1-14 mmHg, ME180: i/m 5-68 mmHg, cervix 4-21 mmHg, SiHa: i/m 20-56 mmHg, cervix 2-26 mmHg, MMTV-PyMT: i/m: 13-45 mmHg, spontaneous 2-20 mmHg and transplanted 2-22 mmHg. Additionally, there was significant variation between individual tumours growing in the same mouse, and there was no correlation between donor and recipient tumour IFP values. Metastatic dissemination to the lungs or lymph nodes demonstrated no correlation with tumour IFP. Tumour hypoxia, proliferation, and lymphatic or blood vessel density also showed no relationship with tumour IFP. Speckle variance analysis of ultrasound images showed no differences in vascular perfusion between ME180 tumours grown i/m versus orthotopically despite differences in IFP. CONCLUSION: Our studies across a range of different tumour models showed substantial heterogeneity in tumour IFP, suggesting differences in the vascular development and interstitial fluid dynamics in the individual tumours. The results demonstrate a strong stochastic aspect to tumour IFP development, notably the variation apparent between different tumours within the same animal and the lack of correlation between donor and recipient tumours.

Analysis of the effects of exposure to acute hypoxia on oxidative lesions and tumour progression in a transgenic mouse breast cancer model.

BACKGROUND: Tumour hypoxia is known to be a poor prognostic indicator, predictive of increased risk of metastatic disease and reduced survival. Genomic instability has been proposed as one of the potential mechanisms for hypoxic tumour progression. Both of these features are commonly found in many cancer types, but their relationship and association with tumour progression has not been examined in the same model. METHODS: To address this issue, we determined the effects of 6 week in vivo acute hypoxic exposure on the levels of mutagenic lipid peroxidation product, malondialdehyde, and 8-oxo-7,8-dihydro-2'-deoxyguanosine DNA (8-oxo-dG) lesions in the transgenic polyomavirus middle T (PyMT) breast cancer mouse model. RESULTS: We observed significantly increased plasma lipid peroxidation and 8-oxo-dG lesion levels in the hypoxia-exposed mice. Consumption of malondialdehyde also induced a significant increase in the PyMT tumour DNA lesion levels, however, these increases did not translate into enhanced tumour progression. We further showed that the in vivo exposure to acute hypoxia induced accumulation of F4/80 positive tumour-associated macrophages (TAMs), demonstrating a relationship between hypoxia and macrophages in an experimental model. CONCLUSION: These data suggest that although exposure to acute hypoxia causes an increase in 8-oxo-dG lesions and TAMs in the PyMT tumours, these increases do not translate into significant changes in tumour progression at the primary or metastatic levels in this strong viral oncogene-driven breast cancer model.

"I'm Just Guessing These Answers!" An Evaluation of the (In)Accuracy of Patient-Reported Medical History Collected as Part of a Breast Imaging Program.

INTRODUCTION: Hundreds of thousands of Ontario women receive breast screening imaging each year. The patient's medical history is a vital tool used to personalize breast screening approaches. This study evaluated the accuracy of self-reported medical and imaging history in patients about to receive breast imaging procedures in a large urban Canadian hospital. The patient experience with using a blank screening form vs. a novel prefilled screening form was also evaluated. METHODS AND MATERIALS: The study was conducted in two phases. Phase 1 compared patient-reported information (via the blank screening form) to information previously captured in the Radiology Information and Picture Archiving Communication Systems to assess data accuracy. In phase 2, study questionnaires were used to collect data on the patients' experience with the screening form in two cohorts (between the blank and a novel prefilled screening form). RESULTS: Data accuracy: for mammography (n = 60), 40% of the patients could accurately recall when and where their last mammogram was performed. For breast ultrasound (n = 43) and breast magnetic resonance imaging (n = 20), significantly fewer patients could accurately recall the date their last test was performed (14% and 10%, respectively). Of those who had previous breast surgery (n = 18), 100% were able to recall whether the surgery resulted in a benign or malignant diagnosis, and 61% were able to accurately recall the year they had the breast surgery. PATIENT EXPERIENCE: Of the returned questionnaires, 65 provided feedback on the blank screening form while 55 provided feedback on the prefilled form. Ninety percent preferred to fill out the new prefilled screening form. The themes acknowledged a general improvement in the screening form, a decrease in frustration associated with having to recall their medical history, and the patients felt that the institution better understood their medical history. CONCLUSIONS: The findings of this research indicate that asking patients to complete a blank medical screening form is a highly inaccurate method of gathering that important information. When that information was prefilled for the patient to review and update, patient frustration and stress were decreased, while satisfaction and confidence in the organization were increased.

Cancer initiating-cells are enriched in the CA9 positive fraction of primary cervix cancer xenografts.

Numerous studies have suggested that Cancer Initiating Cells (CIC) can be identified/enriched in cell populations obtained from solid tumors based on the expression of cell surface marker proteins. We used early passage primary cervix cancer xenografts to sort cells based on the expression of the intrinsic hypoxia marker Carbonic Anhydrase 9 (CA9) and tested their cancer initiation potential by limiting dilution assay. We demonstrated that CICs are significantly enriched in the CA9+ fraction in 5/6 models studied. Analyses of the expression of the stem cell markers Oct4, Notch1, Sca-1 & Bmi1 showed a trend toward an increase in the CA9+ populations, albeit not significant. We present evidence that enhanced autophagy does not play a role in the enhanced growth of the CA9+ cells. Our study suggests a direct in vivo functional link between hypoxic cells and CICs in primary cervix cancer xenografts.

Progression and metastasis in a transgenic mouse breast cancer model: effects of exposure to in vivo hypoxia.

Hypoxia is a predictor of poor patient survival in several cancers, including breast carcinomas. One possible mechanism is genomic instability induced by oxic stress. In this study we examined this possible mechanism by exposing an in vivo breast cancer model to hypoxia/reoxygenation. MMTV-Neu transgenic mice were exposed to cycling acute (AH) or chronic hypoxia (CH) before (early) or after (late) tumour detection to study effects of hypoxia on tumour initiation and progression, respectively. We observed no effect of the hypoxic exposures on times to first tumour detection, but we saw a trend of early AH-exposed mice to develop more tumours and macrometastases than CH-exposed mice. Unexpectedly, but consistent with these findings, we observed significantly reduced 8-oxo-dG lesions levels in the mammary tissue with the greatest difference observed between the air control (AC) and AH-exposed groups. In the late gassing group, there was a similar trend for reduced 8-oxo-dG lesion levels, but interestingly mice that developed macroscopic lung metastases demonstrated significantly increased levels of 8-oxo-dG lesions in their tumours relative to those that did not, irrespective of the gassing exposure. A trend for increased macrophage content was observed in tumours from mice exposed to acute hypoxia. Our results indicate that oxic stress induced by hypoxia/reoxygenation is unlikely to be a major factor driving tumour progression of established MMTV-Neu tumours but suggest that acute and chronic hypoxia may affect tumour incidence and metastasis when applied prior to tumour development.