Melissa M. Alegre

Mechanics behind Breast Cancer Prevention - Focus on Obesity, Exercise and Dietary Fat

Cancer prevention is rapidly emerging as a major strategy to reduce cancer mortality. In the field of breast cancer, significant strides have recently been made in the understanding of underlying preventive mechanisms. Currently, three major strategies have been linked to an increase in breast cancer risk: obesity, lack of physical exercise, and high levels of saturated dietary fat. As a result, prevention strategies for breast cancer are usually centered on these lifestyle factors. Unfortunately, there remains controversy regarding epidemiological studies that seek to determine the benefit of these lifestyle changes. We have identified crucial mechanisms that may help clarify these conflicting studies. For example, recent reports with olive oil have demonstrated that it may influence crucial transcription factors and reduce breast tumor aggressiveness by targeting HER2. Similarly, physical exercise reduces sex hormone levels, which may help protect against breast cancer. Obesity promotes tumor cell growth and cell survival through upregulation of leptin and insulin-like growth factors. This review seeks to discuss these underlying mechanisms, and more behind the three major prevention strategies, as a means of understanding how breast cancer can be prevented.

Thymidine Kinase 1 Upregulation Is an Early Event in Breast Tumor Formation

Prognostic markers play an important role in our understanding of tumors and how to treat them. Thymidine kinase 1 (TK1), a proliferation marker involved in DNA repair, has been shown to have independent prognostic potential. This prognostic potential includes the novel concept that upregulation of serum TK1 levels is an early event in cancer development. This same effect may also be seen in tumor tissue. In order to demonstrate that TK1 upregulation is an early event in tumor tissue formation, tissue arrays were obtained and stained for TK1 by immunohistochemistry. Using a progressive breast tissue array, precancerous tissue including breast adenosis, simple hyperplasia, and atypical hyperplasia stained positive for TK1 expression. Different stages of breast carcinoma tissue also stained positive for TK1 including nonspecific infiltrating duct, infiltrating lobular, and infiltrating duct with lymph node metastasis carcinomas. This indicates that TK1 upregulation is an early event in breast carcinoma development, and may be useful in identifying precancerous tissue. Further work is needed to better understand the differences seen between TK1 positive and negative tissues.

DNA damage caused by inorganic particulate matter on Raji and HepG2 cell lines exposed to ultraviolet radiation

Epidemiological studies have correlated exposure to ultraviolet-irradiated particulate matter with cardiovascular, respiratory, and lung diseases. This study investigated the DNA damage induced by two major inorganic particulate matter compounds found in diesel exhaust, ammonium nitrate and ammonium sulfate, on Burkitts lymphoma (Raji) and hepatocellular carcinoma (HepG2) cell lines. We found a dose-dependent positive correlation of accumulated DNA damage at concentrations of ammonium nitrate (25 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml, 400 μg/ml) with ultraviolet exposure (250 J/m(2), 400 J/m(2), 600 J/m(2), 850 J/m(2)), as measured by the comet assay in both cell lines. There was a significant difference between the treated ammonium nitrate samples and negative control samples in Raji and HepG2 cells (p<0.001). Apoptosis was shown in Raji and HepG2 cells when exposed to high concentrations of ammonium nitrate (200 μg/ml and 400 μg/ml) for 1h in samples without ultraviolet exposure, as assessed by the comet assay. However, the level of apoptosis greatly diminished after ultraviolet exposure at these concentrations. Over a 24h period, at intervals of 1, 4, 8, 12, 18, and 24h, we also observed that ammonium nitrate decreased viability in Raji and HepG2 cell lines and inhibited cell growth. Ammonium sulfate-induced DNA damage was minimal in both cell lines, but there remained a significant difference (p<0.05) between the ultraviolet radiation treated and negative control samples. These results indicate that the inorganic particulate compound, ammonium nitrate, induced DNA strand breaks at all concentrations, and indications of apoptosis at high concentrations in Raji and HepG2 cells, with ultraviolet radiation preventing apoptosis at high concentrations. We hypothesize that ultraviolet radiation may inhibit an essential cellular mechanism, possibly involving p53, thereby explaining this phenomenon. Further studies are necessary to characterize the roles of apoptosis inhibition induced by DNA damage caused by inorganic particulate matter.

Abstract 1866: Detection of early-stage lung cancer using a novel ELISA for thymidine kinase 1

Lung cancer is the number one cause of cancer mortality and early detection will increase the survival of this disease. Thymidine kinase 1 (TK1) is a biomarker indicative of cell proliferation that is elevated in numerous malignancies including lung cancer. TK1 has been found to be elevated early in the development of lung cancer and therefore is an ideal target as an early detection biomarker. We have developed an enzyme-linked immunosorbent assay (ELISA) to detect TK1 in serum. Forty patients with pulmonary nodules and 18 healthy control individuals had serum collected under standard conditions, prior to operative removal of any lesions. All serum samples were analyzed using TK1 radioassay to measure TK1 activity, and TK1 ELISA to measure TK1 concentration. TK1 was significantly elevated in all patients with confirmed lung cancer. This was true even in stage IA, where patients (n=16) had significantly higher TK1 levels than controls. The clearest cut-off was 4.9 nM with an area under the curve of 0.792 for early stage lung cancer. The corresponding sensitivity and specificity was 82.4 and 83.3, respectively. TK1 concentration was a more sensitive and accurate indicator of lung cancer than TK1 activity. Overall, TK1 is significantly elevated in serum of lung cancer patients. This TK1 ELISA is sensitive and specific for detection of lung cancer and further trials with a larger patient base can confirm the potential of this method of detection. Citation Format: Madison K. Ramsden, Melissa M. Alegre, Michael J. Weyant, Daine T. Bennett, Jessica A. Yu, Atif Elnaggar, Richard A. Robison, Kim L. O9Neill. Detection of early-stage lung cancer using a novel ELISA for thymidine kinase 1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1866. doi:10.1158/1538-7445.AM2014-1866

Abstract 1790: Mutagenic effects of inorganic particulate matter on Raji and HepG2 cell lines exposed to ultraviolet radiation.

Epidemiological studies have correlated exposure to ultraviolet-irradiated particulate matter with cardiovascular, respiratory, and malignant lung diseases. This study investigated the DNA damage induced by two major inorganic particulate matter compounds found in diesel exhaust, ammonium nitrate and ammonium sulfate on Burkitt9s lymphoma (Raji) and hepatocellular carcinoma (HepG2) cell lines. We found a dose-dependent positive correlation of accumulated DNA damage in concentrations of ammonium nitrate and ammonium sulfate (25 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml, 400 μg/ml) with ultraviolet exposure (250 J/m 2 , 400 J/m 2 , 600 J/m 2 , 850 J/m 2 ), as measured by the Comet Assay in both cell lines. There was a significant difference between the treated ammonium nitrate samples and negative control samples in Raji and HepG2 (p Citation Format: Michael Xiao, Atif El-Naggar, Albert V. Helsing, Philip M. Lynch, Melissa M. Alegre, Richard A. Robison, Kim L. O9Neill. Mutagenic effects of inorganic particulate matter on Raji and HepG2 cell lines exposed to ultraviolet radiation. [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 1790. doi:10.1158/1538-7445.AM2013-1790

Abstract 5528: Setting the clock: Tumor TK1 is a prognostic marker for disease recurrence

Cancer is a devastating disease which affects millions of people every year. There is a need for reliable markers which indicate more aggressive tumors. Thymidine kinase 1 (TK1) has been shown to have prognostic potential in the serum of cancer patients, correlating with disease stage and recurrence. Serum TK1 has been studied extensively, but relatively few studies have investigated the prognostic potential of TK1 in tumor tissue. We created a breast array of samples from 300 breast cancer patients with various disease stages and treatment methods. We compared several potential prognostic markers to clinically relevant markers, including TK1, fatty acid synthase (FAS), annexin I, and Ki67. These markers were also compared to tumor progesterone and estrogen receptor status (PR and ER), HER2neu status, type of chemotherapy, and type of hormone therapy. Whether or not the patients received radiation therapy was also considered. Tumors were scored for TK1 staining on a scale from 0 to 2. A TK1 score of 0 indicated that 0-5% of tumor cells stained positive for TK1. A score of 1 or 2 indicated 10%-20% TK1 staining and 20% or more TK1 staining respectively. The relationships between various markers and therapies were analyzed using logistic regression and chi square analysis. Interestingly, tumor TK1 staining did not correlate to tumor stage, and was only moderately correlated to tumor grade. This is different from the results of serum TK1 (sTK1) studies, which have shown correlations between sTK1 and disease stage. Tumor TK1 levels did correlate with Ki67 (p=6.333e-11), but there were no correlations between TK1 and FAS (p=.7927), annexin I (p=.1169), or diagnosis (p=.4303). Markers were also analyzed for their predictive value. Distant metastasis, PR status, and the presence of positive nodes were strong predictors of disease outcome and time to recurrence. If the disease had not metastasized at the time of evaluation, patients were 82.85% more likely to survive. No metastasis also predicted 1.4 years to disease recurrence. Positive PR status also indicated that patients were 4.6 times more likely to die of their disease. Additionally, for every positive node present, patients were 9.8% more likely to die of their disease. The presence of positive nodes also predicted shorter time to disease recurrence by 0.04 years per node. Higher TK1 scores were also found to predict shorter times to recurrence. On the other hand, low TK1 scores were found to be predictive of longer time to recurrence. A score of 2 indicated on average 1.8375 years to disease recurrence. Scores of 0 and 1 indicated 2.8 and 2.4 years on average to recurrence, respectively. Further studies are needed to elucidate the differences between tumor TK1 and sTK1. Additional studies involving larger patient groups may be needed to confirm the relationship between these predictors and recurrence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5528. doi:1538-7445.AM2012-5528

Abstract 1678: Visualization of thymidine kinase 1 on the surface of cancer cell lines: A potential diagnostic marker and therapeutic target

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Thymidine Kinase 1(TK1) is a salvage pathway enzyme that has been shown to be elevated in many different cancer types, including breast, prostate, and colon. Previous research in our lab using plasma membrane separation has shown an increase in TK1 levels associated with the plasma membrane of cancer cells when compared to normal lymphocytes. After membrane separation the plasma membrane of normal lymphocytes showed a TK1 activity of 1,012 CPM/mg protein/hr(N=20) whereas those of cancer cells showed: Jurkat 34,096(N=28), Raji 70,195(N=20), HL60 46,217(N=34), and MDA-MB-435 50,359(N=24). In this study we sought to further investigate this association by visualizing the surface staining of fluorescently tagged antibodies to TK1. Using a primary rabbit anti-TK1 antibody and a secondary goat anti-rabbit antibody conjugated with a FITC fluorochrome we were able to stain the cells and identify TK1 bound to the cell surface. Using a fluorescent microscope we were able to visualize the fluorescent staining of TK1 in various cancer cell lines, including: Raji, Jurkat, HL60, MDA-MB-435, H358, and MCF-7. We compared these results to our control of stained normal lymphocytes, which showed significantly greater levels of staining in cancer cells. Our results further confirm the presence of membrane associated TK1, and provide further evidence of TK1s usefulness as a diagnostic marker and potential therapeutic target. 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 1678. doi:10.1158/1538-7445.AM2011-1678

Abstract 897: Thymidine kinase 1, a novel biomarker specific to the plasma membrane of cancerous cell lines

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Thymidine Kinase 1 (TK1), an important nucleotide salvage pathway enzyme, has been shown to be upregulated in most malignant cancers. Recent studies have shown that TK1 not only has diagnostic and prognostic potential, but could also be used as a molecular target for therapeutic treatments. Previous research from our lab has shown the expression of TK1 on the plasma membrane of lymphoma. This study provides further insight on the role of TK1 as a diagnostic target for several different tumor cell lines. This study analyzed the localization of TK1 in ten different cancer cell lines using flow cytometry (FCM). The lines studied were A375, a malignant melanoma, CAL27, a squamous cell carcinoma derived from tongue, MDA-MB-231, a breast adenocarcinoma, MDA-MB-435S, previously described as a ductal carcinoma, HT29, a colorectal adenocarcinoma, SW620, a colorectal adenocarcinoma, A549, a lung carcinoma, NCI-H460, a large cell lung carcinoma, DU 145, a prostrate carcinoma, and PC3, a prostate adenocarcinoma. FCM was carried out using a polyclonal antibody against human TK1 as the primary antibody and a FITC conjugated secondary antibody. A sample stained with only secondary antibody was used as a negative control for the experiment. Five replicates were analyzed for each cell line. TK1 was found to bind between 48% and 96%, depending on the cell line. Non-cancerous tonsil tissue and normal lymphocytes were used as controls, where TK1 surface binding was less than 20%. These data indicate the feasibility of TK1 as a possible molecular target for immunotherapy in many types of cancer. Further research is ongoing to determine specific immunotherapy treatment options, and to verify the expression of TK1 in other cancer cell lines. 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 897. doi:10.1158/1538-7445.AM2011-897

Abstract 3194: Thymidine Kinase 1 staining aids in lung cancer prognosis

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Lung cancer kills more patients than breast, colon, prostate, melanoma, and kidney cancers combined. One way to improve the high lung cancer mortality rate is through early detection and better prognosis. There have been attempts to establish spiral CT scan imaging protocols or early detection assays to improve the diagnosis of lung cancer. Although these detection methods show merit, they are often costly, and are only useful for a subset of the potential lung cancer population such as high-risk smokers. One biomarker that shows promise is Thymidine Kinase 1 (TK1). Recently, it has been demonstrated that TK1 has diagnostic potential in both small cell and non-small cell lung cancer. Therefore, the present study was conducted to elucidate whether TK1 could be used in early detection and lung cancer prognosis. Five tissue microarrays (TMA) including a lung carcinoma screen, lung adenocarcinoma grade III, large cell carcinoma, small cell carcinoma, and one normal lung TMA consisting of 376 lung cancer tissue cores from 172 patients and 63 control lung tissue cores from 22 normal individuals, were stained with a TK1 monoclonal antibody produced by our lab. Mesothelioma and some of the lung cancers including squamous cell, small cell, adenosquamous cell, undifferentiated, adenocarcinoma, and bronchioloalveolar carcinoma stained positive for TK1. Papillary carcinoma, sarcoma, neuroendrocrine carcinoma, and undifferentiated large cell carcinoma showed considerably less TK1 staining. Normal and inflammatory lung tissue showed zero to minimal staining which was significantly different from positively stained lung cancer tissue. There are various increasing grades of squamous cell carcinoma patients from grade I, I-II, II, II-III and III. There was a definitive increase in TK1 staining as the grade of the lung tumor increase. A similar pattern was found with adenocarcinoma tissue grade II, II-III, and III; the more progressive the tumor, the more intense the TK1 staining. This preliminary data supports further investigation of TK1 as a marker for early detection and prognosis of numerous lung cancer subtypes. 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 3194. doi:10.1158/1538-7445.AM2011-3194

Abstract 905: Localization of several potential biomarkers in various types of cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL DNA salvage pathway enzymes are known to be upregulated both within cancer cells and in the serum of cancer patients. These enzymes include TK1, APRT, HGPRT, and dCK. TK1 has been extensively studied and is known to be a cytosolic enzyme. However, relatively few studies have investigated the localization of the other three salvage pathway enzymes in cancer cells. The purpose of this study was to determine the localization of these salvage pathway enzymes in various types of cancer cells. Cancer cells and normal lymphocytes were embedded and prepared for immunolabeling transmission electron microscopy (TEM). Raji cells, a Burkitts lymphoma cell line known to express high levels of TK1, were used to optimize the TEM protocol. Using rabbit polyclonal antibodies, we were able to determine where the salvage pathway enzymes are localized in cancer cells. Cancer cell lines studied included Raji, Jurkat (acute T cell leukemia), HL60 (acute promyelocytic leukemia), HT29 (colorectal adenocarcinoma), SW620 (colorectal adenocarcinoma from lymph node), and H460 (large cell lung carcinoma). Normal lymphocytes (both unstimulated and stimulated with PHA) were also embedded and prepared for labeling. An antibody control, and anti-Macrophage marker, was used as an isotype control to detect non-specific binding. We observed that DNA salvage pathway enzymes were localized in distinctive parts throughout the cytoplasm, the cell membrane, and the nucleus. The expression of APRT appeared very similar to the expression of TK1 and it was visualized in approximately the same patterns. The same appeared to be true for HGPRT and dCK, but with much lower level of expression than TK1 and ARPT. In several cases, these salvage pathway enzymes appeared to localize near the nuclear pores, as well as other parts of the nuclear membrane. These enzymes also appeared dispersed throughout the cytoplasm, and in some cases formed clusters, especially near the cellular membrane, possibly indicating exocytosis routes. There was no significant difference between the various types of cancer cell lines in the localization of these enzymes Our normal lymphocyte controls show markedly lower expression, while the secondary only and isotype controls showed no staining. Further research is needed to determine the mechanisms of this localization and the role of these enzymes in tumor cell growth. 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 905. doi:10.1158/1538-7445.AM2011-905