Christine M. Coticchia

Hypercholesterolemia induces angiogenesis and accelerates growth of breast tumors in vivo.

Obesity and metabolic syndrome are linked to an increased prevalence of breast cancer among postmenopausal women. A common feature of obesity, metabolic syndrome, and a Western diet rich in saturated fat is a high level of circulating cholesterol. Epidemiological reports investigating the relationship between high circulating cholesterol levels, cholesterol-lowering drugs, and breast cancer are conflicting. Here, we modeled this complex condition in a well-controlled, preclinical animal model using innovative isocaloric diets. Female severe combined immunodeficient mice were fed a low-fat/no-cholesterol diet and then randomized to four isocaloric diet groups: low-fat/no-cholesterol diet, with or without ezetimibe (cholesterol-lowering drug), and high-fat/high-cholesterol diet, with or without ezetimibe. Mice were implanted orthotopically with MDA-MB-231 cells. Breast tumors from animals fed the high-fat/high-cholesterol diet exhibited the fastest progression. Significant differences in serum cholesterol level between groups were achieved and maintained throughout the study; however, no differences were observed in intratumoral cholesterol levels. To determine the mechanism of cholesterol-induced tumor progression, we analyzed tumor proliferation, apoptosis, and angiogenesis and found a significantly greater percentage of proliferating cells from mice fed the high-fat/high-cholesterol diet. Tumors from hypercholesterolemic animals displayed significantly less apoptosis compared with the other groups. Tumors from high-fat/high-cholesterol mice had significantly higher microvessel density compared with tumors from the other groups. These results demonstrate that hypercholesterolemia induces angiogenesis and accelerates breast tumor growth in vivo.

Urinary MMP-2 and MMP-9 predict the presence of ovarian cancer in women with normal CA125 levels

OBJECTIVE To determine whether urinary matrix metalloproteinases (MMPs) predict the presence of ovarian cancer in patients with CA125 levels below the normal threshold of 35U/mL, a critical group of patients for whom no ovarian cancer biomarker is currently available. To determine whether these noninvasive biomarkers provide clinically useful information in the general ovarian cancer patient population as well. METHODS ELISA analyses and substrate gel electrophoresis detected the levels and activity of urinary MMP-2, MMP-9, MMP-9/neutrophil gelatinase-associated lipocalin (NGAL) complex, and MMP-9 dimer in all ovarian cancer patients (n=97), those with CA125 <35U/mL (n=26) and controls (n=81). RESULTS In patients with CA125 <35U/mL, receiver-operating characteristic (ROC) area under curve (AUC) analysis demonstrated that either urinary MMP-2 or MMP-9 or NGAL significantly discriminated between controls and ovarian cancer patients with normal CA125. Multivariate logistic regression revealed that the combination of urinary MMP-2 and MMP-9 provided the best diagnostic accuracy when multiplexed. When further multiplexed with age, the diagnostic accuracy of these biomarkers increased to a significant AUC of 0.820. These findings were consistent among the general ovarian cancer population studied as well, where the combination of urinary MMP-2 and MMP-9 multiplexed with age resulted in a highly significant AUC of 0.881. Pearson chi-square analysis revealed that higher urinary levels of either MMP-2 or MMP-9 were strongly associated with the increasing percentage of women with ovarian cancer independent of CA125 levels. CONCLUSION This study demonstrates the potential utility of urinary MMP-2 and MMP-9 to differentiate between ovarian cancer patients with normal CA125 levels and controls and suggests that urinary MMP-2 and MMP-9 may be a clinically useful aid in the diagnosis of advanced or recurrent ovarian cancer.

Abstract 173: Hypercholesterolemia induces angiogenesis and causes accelerated growth of breast tumors in vivo.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: While many risk factors for breast cancer (BC) such as family history and age have been firmly established, evidence suggests that dietary factors may also play a role in BC. Studies have linked intake of saturated fat as well as metabolic syndrome to an increased prevalence of BC. A common feature shared by metabolic syndrome and a typical American diet is a high level of circulating cholesterol, however a number of epidemiological reports investigating the relationship between high cholesterol levels, cholesterol lowering drugs and BC are conflicting. To determine the relationship between dietary cholesterol and BC in vivo, we modeled this complex condition in a well controlled animal model that utilized an innovative isocaloric diet. We hypothesized that hypercholesterolemia promotes BC progression while hypocholesterolemia has the opposite effect and retards BC progression. Methods: Female SCID mice were fed a low fat/no cholesterol (LFNC) diet for 2 weeks and then randomized to 4 diet groups with calorie set at 21.2 Kcal/day. These isocaloric diets consisted of a LFNC diet with or without ezetimibe (a drug that specifically blocks cholesterol uptake in the gut, thereby lowering serum cholesterol) and a high fat/high cholesterol (HFHC) diet with and without ezetimibe. Significant differences in serum cholesterol level between groups were achieved and maintained throughout the study. Mice were then implanted orthotopically with MDA-MB-231 BC cells into the 4th mammary fat pad and tumor growth was monitored every 3 days until sacrifice when tumors were harvested. Results: No differences in tumor take were observed (>95% for all groups). Tumors from animals fed the HFHC diet exhibited the fastest progression. While significant differences in serum cholesterol were observed between the diet groups, no differences were observed in the intratumoral cholesterol levels. To determine the underlying mechanism of cholesterol-induced tumor progression, we assessed tumor proliferation, apoptosis and angiogenesis. Ki67 analyses of tumor proliferation demonstrated a significantly greater percentage of proliferating cells in tumor from mice fed the HFHC diet (48%) compared to animals on the HFHC diet with ezetimibe or the LFNC diet with and without ezetimibe (32%, 33% and 30% respectively). Tumors from hypercholesterolemic animals displayed significantly less apoptosis, as determined by percentage of TUNEL positive cells (39%), compared to the other diet groups (49%, 52% and 58%). Finally, when tumor angiogenesis was evaluated by CD31 staining and microvessel density was quantified, tumors from HFHC mice had vessel areas that were 70% greater, on average, compared to tumors from the other groups. Conclusion: These results demonstrate that hypercholesterolemia induces angiogenesis and accelerates breast tumor growth in vivo. Supported by the Breast Cancer Research Foundation and NIH P01 [CA045548][1] Citation Format: Christine M. Coticchia, Kristine Pelton, Adam S. Curatolo, David Zurakowski, Carl P. Schaffner, Keith R. Solomon, Marsha A. Moses. Hypercholesterolemia induces angiogenesis and causes accelerated growth of breast tumors in vivo . [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 173. doi:10.1158/1538-7445.AM2013-173 [1]: /lookup/external-ref?link_type=GEN&access_num=CA045548&atom=%2Fcanres%2F73%2F8_Supplement%2F173.atom

Abstract 2818: Urinary MMP-2 and MMP-9 predict the presence of ovarian cancer in women with normal CA125 levels

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Currently, the only widely used biomarker for monitoring ovarian cancer status and response to therapy is the cancer antigen 125 (CA125). Interestingly, the majority of newly developed ovarian cancer multi-marker assays also rely heavily on serum CA125 levels as one of the biomarkers in their panels. However, 20% of women with advanced ovarian cancer have blood CA125 levels within the normal range of 35 U/mL and below. These women represent a cohort of patients for whom no clinically useful biomarkers are available for monitoring disease status, stage or response to therapy. To address this need, we asked whether a panel of urinary biomarkers that we have previously validated for use in other cancers, could predict the presence of disease in women with ovarian cancer possessing normal CA125 levels. Urine samples were collected from 81 healthy controls and 26 ovarian cancer patients with blood CA125 levels below 35 U/mL. Using monospecific ELISAs, we determined that urinary levels of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) and the neutrophil gelatinase lipocalin 2 (NGAL) are detected at significantly higher levels in the urine of ovarian cancer patients than in the urine of healthy controls. Additionally, receiver-operating characteristic (ROC) area under the curve (AUC) analysis of these groups revealed that, individually, MMP-2, MMP-9 and NGAL each could significantly distinguish ovarian cancer patients with CA125 < 35 U/mL from controls. The best combination of biomarkers for predicting the presence of disease was MMP-2 plus MMP-9 with an ROC AUC of 0.7332 (P<0.01). When these biomarkers were multiplexed with age, a know risk factor of ovarian cancer, the diagnostic accuracy of these biomarkers increased to an AUC of 0.820 (P<0.001). This combination of urinary MMP-2, MMP-9 and age demonstrated excellent diagnostic capability in differentiating ovarian cancer patients from healthy controls among the cohort of patients for whom CA125 values were uninformative. Acknowledgments: The authors gratefully acknowledge the support of Ovations for the Cure of Ovarian Cancer (MAM and UAM) and the Stuart Weitzman Foundation (MAM). 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 2818. doi:10.1158/1538-7445.AM2011-2818

Abstract 2719: Non-invasive biomarker discovery for ovarian cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The majority of women with ovarian cancer are diagnosed with advanced disease (FIGO stage III and IV) and have a five year survival rate of approximately 20%. In contrast, the survival rate of women diagnosed with stage I ovarian cancer is greater than 90%, highlighting an unmet and dire need for accurate and reliable diagnostic tests for the early detection of ovarian cancer. We have previously demonstrated that urinary matrix metalloproteinases (MMPs) are independent predictors of disease status and stage in a variety of human cancers as well as predictors of breast cancer risk. We have also reported that neutrophil gelatinase-associated lipocalin (NGAL) is present in the urine of women with metastatic breast cancer and that the levels of urinary NGAL are significantly higher in these patients compared to normal controls. In the current study, we asked whether urinary MMPs and NGAL, alone or in combination, could provide useful clinical information with respect to the presence of ovarian cancer. Urine samples were obtained from a total of 166 women: 96 women with known FIGO stage III and IV ovarian cancer (newly diagnosed and recurrent) and 70 normal controls. All urine samples were analyzed for NGAL levels by ELISA and for urinary MMP levels by quantitative gelatin zymography. We found that urinary NGAL, MMP9-dimer, MMP9/NGAL complex, MMP9, and MMP2 were each significantly elevated in the urine of women with ovarian cancer compared to normal controls (p 55 years of age with significant measurable levels of urinary MMP9-dimer and MMP2 had a 95% probability of ovarian cancer (95% CI= 85-98% probability), AUC = 0.860 (95% CI = 0.802 – 0.915 (p<0.001)). Taken together, these data suggest that the panel of urinary biomarkers composed of gelatinases, their complexes and NGAL, along with patient age, may provide clinically useful information with respect to distinguishing between women who have ovarian cancer and those who do not. (Supported by NIH P01 CA045548, the Weitzman Family Foundation and Ovations for the Cure) 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 2719.