There are no practical options to reduce breast cancer risk in American women that are without side-effects. Almost all preventive strategies are designed to diminish the role of estrogens in promoting breast cell proliferation. Brassica vegetables (e.g., broccoli) contain indole glucosinolates that can shift estrogen metabolism away from the highly estrogenic 16α-hydroxyestrone (16HE) and toward 2-hydroxyestrone (2HE), which has little estrogenic activity on breast cells. The relative strength of these two pathways is measured in urine as the ratio 2HE/16HE (2/16). In controlled trials that have enrolled premenopausal women or young men, large quantities of Brassica vegetables or indole glucosinolate derivatives decreased urinary 16HE levels relative to 2HE levels, possibly lowering breast cancer risk. However, in order to be an acceptable and effective preventative, Brassica vegetables must shift estrogen metabolism as Brassica consumption is practiced within free-living women. The goal of this research is to determine if Brassica should be further explored as a strategy to prevent breast cancer. In order to do so, it was determined that three issues must be addressed: (1) Brassica consumption must increase the 2/16 ratio among healthy free-living women, without serious side-affects, (2) the 2/16 ratio must be a valid indicator of breast cancer risk, and (3) a reliable and valid method to estimate Brassica consumption must be identified. As part of this research, thirty-seven healthy postmenopausal women participated in a dietary intervention designed to facilitate daily Brassica consumption. The diet, 2/16, and other information were measured before, during, and after the intervention. The 2/16 ratio significantly increased with greater Brassica intake. However, urinary 2/16 levels between participants were sensitive to dietary fat and fiber intake, and future studies evaluating the 2/16-breast cancer association should control for macronutrient intake. Dithiocarbamate excretion, a biomarker of Brassica consumption, inconsistently predicted self-reported Brassica intake and is sensitive to the types of vegetables consumed. However, DTC may be useful to rank-order participants in epidemiological studies. Overall, to the extent that lower 2/16 values are associated with increased breast cancer risk, Brassica vegetables may be an important component of any strategy to reduce breast cancer risk.

Leiomyomata uteri, also know as uterine fibroids, are the most common tumors of the female reproductive tract. Although the majority of leiomyomata are asymptomatic, more than 30% of affected women suffer from one or more symptoms including abnormal uterine bleeding, anemia, pelvic pain, pelvic pressure, urinary problems and infertility. Our objective was to identify patient-, physician- and hospital-related factors that are associated with patterns of care for this condition. Two approaches were used to identify determinants of treatment among women with a primary diagnosis of leiomyoma. First, we used administrative data from the Massachusetts Health Data Consortium to identify determinants of hysterectomy in Massachusetts women over a two year period. Second, we conducted a chart review study of leiomyoma patients at Baystate Medical Center in Springfield, Massachusetts to identify determinants of treatment pattern during the same time period. Both sets of analyses identified factors associated with treatment decision. In the analysis of the Massachusetts Health Data Consortium data, significant determinants of hysterectomy included older age, White or Hispanic race, hospital location outside of Boston and hospital lacking specialized OB/GYN services. In the analysis using hospital records, we found that younger age; Black race, insurance through an HMO, more recent physician graduation, and nulliparity were significantly associated with a conservative treatment approach. Presence of symptoms including heavy bleeding and urinary problems were significantly associated with receiving no conservative treatment prior to hysterectomy. This pair of studies has clearly shown that a variety of clinical, patient, hospital and physician characteristics are important determinants of treatment in women with a primary diagnosis of leiomyoma. Future research should focus on examining the interrelationships of these various factors in determining patterns of care for various sub-sets of patients diagnosed with leiomyomata. New research efforts should be centered in large physician practice groups where longitudinal studies of patients can be used to assess the extent to which physician and patient attitudes and behaviors influence the decision making process with regard to this condition.

The regulation of genes involved in proliferation and cell cycle control plays a critical role in normal development and differentiation. Aberrant expression of genes promoting proliferation (oncogenes) or loss of genes involved in restraining cell growth (tumor suppressors) can result in cancer. The p53 tumor suppressor protein has been demonstrated to play a critical role in both tumorigenesis and normal developmental processes. The mdm2 proto-oncogene can regulate the activity and stability of p53 protein. This suggested that mdm2 functions in development and tumorigenesis through p53-dependent mechanisms. However, mdm2 has also been shown to interact with factors involved in the regulation of cell cycle control, transcription and ribosome biosynthesis. The aim of this dissertation was to examine whether mdm2 expression during breast tumorigenesis and during normal mammary gland development in the mouse was regulated by p53-dependent or -independent mechanisms. The first segment of this work involved the analysis of alternative splicing of mdm2 mRNA during breast tumorigenesis in mice and humans. The second element of this dissertation examined the role of p53 in regulating the expression of mdm2 mRNA in adult tissues from the mouse and during normal mammary gland development in the mouse. Results from these experiments demonstrated that truncated mdm2 mRNA are expressed in mouse and human breast tissues. These truncated transcripts are predicted to code for mdm2 proteins that have lost C-terminal sequences involved in regulating proteolytic cleavage of mdm2 itself and in targeting p53 for ubiquitin mediated proteosomal degradation.

Human papillomavirus-associated head and neck squamous cell carcinoma (HPV[+] HNSCC) is rapidly increasing in incidence and, in this regard, the effects of preventative HPV vaccines have yet to be realized. While HPV(+) HNSCC carries a more favorable prognosis than many other tumor types, a significant number of patients still experience treatment failure. Disease recurrence and metastasis are often fatal. Thus, factors that promote disease progression and underlie aggressive tumor phenotypes must be identified, and, most importantly, novel therapeutic approaches must be developed. Interestingly, β-adrenergic signaling plays a tumorigenic role in several cancer types and β-adrenergic antagonists (β-blockers) have recently garnered interest as anti-cancer agents. Herein, we identify increased β2-adrenergic receptor (β2AR) expression in an aggressive, recurrent/metastatic model of HPV(+) HNSCC as compared to the primary tumor model from which it was derived. This led us to hypothesize that β2AR contributes to an aggressive disease phenotype in HPV(+) HNSCC and that β-blockers would serve as an effective therapeutic tool in this setting. Here, we show that propranolol, a non-selective β-blocker, significantly delays primary tumor growth and decreases metastatic burden in mice implanted with this aggressive tumor model. Additionally, genetic deletion studies indicate that the model’s aggressive phenotype is critically dependent on expression of β2AR. Importantly, we identify that these effects are associated with inhibition of tumor cell mitochondrial metabolism and show that propranolol-mediated perturbations in mitochondrial respiration render tumor cells more sensitive to glucose starvation. Thus, we further hypothesized that concurrent glycolytic inhibition would enhance the anti-cancer activity of propranolol. To this end, we have developed a novel, metabolism-targeting treatment combination in propranolol and dichloroacetate (DCA), a glycolytic inhibitor. This combination dramatically attenuates tumor cell metabolism, displays significant anti-cancer effects in vitro, and delays tumor growth in vivo. Together, these data provide support for further investigation into the therapeutic potential of propranolol in the setting of HPV(+) HNSCC, and suggest that its effectiveness may be improved through combination with other metabolism-targeting drugs. Importantly, as a clinically-available agent, propranolol has the potential for swift translation from the bench to the bedside.

Oncolytic viroimmunotherapy combines the inflammatory nature of viral vectors with the delivery of virally-encoded tumor associated antigens (TAAs) to induce robust anti-tumor T cell responses. We have previously reported that vesicular stomatitis virus (VSV) vectors expressing truncated forms of the TAAs NRAS, Cytochrome-C1, and TYRP1 provide superior therapeutic benefit to mice bearing B16 melanoma tumors compared to treatment with VSV expressing the full-length form of these proteins. Based on structural modeling, we determined that each carboxyl-(C)-terminal truncation disrupted domains important for antigen stability. Others have demonstrated a direct association between the stability of an antigen, its degradation rate, and the generation of immunogenic epitopes that can be utilized to activate cognate T cell responses. Thus, we hypothesized that modulation of tumor antigen stability and degradation would increase the availability of immunogenic epitopes for presentation on MHC complexes, thereby improving cognate T cell responses in the context of VSV immunotherapy. In this work, we first demonstrated that the 19 amino acid C-terminal truncation of the melanoma antigen, TYRP1, identified in the cDNA library significantly reduced the protein’s half-life. We found that truncated TYRP1 underwent autophagy-mediated degradation, consistent with the CD4+ T cell-dependent responses observed in vivo. To extend these findings, we used the crystal-structure of the model antigen ovalbumin (OVA) to identify putative mutations which would reduce protein stability and promote antigen degradation. We found that one mutant exhibited a significantly reduced half-life compared to the wild-type antigen and was degraded in a 26S-proteasomal dependent manner. Infection of murine antigen presenting cells with VSV vectors expressing the destabilized OVA antigen resulted in enhanced presentation of the immunogenic OVA epitope, SIINFEKL, and activation of cognate T cells in vitro and in vivo compared to expression of the wild-type antigen. Correspondingly, SIINFEKL-specific T cell activation by the virally delivered destabilized OVA resulted in improved long term survival in a murine B16OVA melanoma model. In a final approach, we aimed to utilize pharmacological activation of the unfolded protein response (UPR), a cellular mechanism to promote degradation of misfolded proteins, to promote antigen presentation. We found that UPR activation using the small molecule thapsigarigin (Tg) induced proteasomal-mediated degradation of endogenously encoded OVA in our B16OVA murine melanoma cell line. This was associated with enhanced presentation of the SIINFEKL epitope and improved recognition and activation of cognate CD8+ T cells in vitro. Intratumoral administration of Tg in combination with systemic VSV-OVA immunotherapy significantly reduced early tumor growth and had modest improvements in overall survival. Overall, the mechanisms investigated here provide a framework for improving therapeutic outcomes with viral immunotherapeutic agents by manipulating the stability and degradation of virally-encoded TAAs to promote anti-tumor T cell responses.

abstract: The analysis focuses on a two-population, three-dimensional model that attempts to accurately model the growth and diffusion of glioblastoma multiforme (GBM), a highly invasive brain cancer, throughout the brain. Analysis into the sensitivity of the model to changes in the diffusion, growth, and death parameters was performed, in order to find a set of parameter values that accurately model observed tumor growth for a given patient. Additional changes were made to the diffusion parameters to account for the arrangement of nerve tracts in the brain, resulting in varying rates of diffusion. In general, small changes in the growth rates had a large impact on the outcome of the simulations, and for each patient there exists a set of parameters that allow the model to simulate a tumor that matches observed tumor growth in the patient over a period of two or three months. Furthermore, these results are more accurate with anisotropic diffusion, rather than isotropic diffusion. However, these parameters lead to inaccurate results for patients with tumors that undergo no observable growth over the given time interval. While it is possible to simulate long-term tumor growth, the simulation requires multiple comparisons to available MRI scans in order to find a set of parameters that provide an accurate prognosis. / Dissertation/Thesis / Masters Thesis Mathematics 2020

Loss of p53 function can lead to a variety of cancers, including breast cancer. Mice heterozygous for the p53 gene (designated Trp53 +/−) develop spontaneous mammary tumors, but this depends on the strain background and has been linked to a locus on chromosome 7 (designated SuprMam1). Mammary tumors are common in BALB/c-Trp53 +/−females, but are rare in C57BL/6-Trp53 +/− mice. Prevalence of genomic instability appears to contribute to the phenotype as loss of heterozygosity (LOH) is significantly more common among tumors arising in BALB/c-Trp53+/− mice compared to C57BL/6J-Trp53+/− mice. This increased LOH in BALB/c-Trp53+/− tumors was shown to be due to recombination events. The BALB/c strain has been shown to have a deficiency in non-homologous end joining (NHEJ) of DNA double strand breaks (dsb), however, this does not account for the increase of LOH events in tumors. Our hypothesis was that BALB/c-Trp53 +/− mice are more susceptible to mammary tumors due to impaired Homologous Recombination Repair (HRR) leading to LOH. Using the COMET assay, we demonstrate that dsbs persist longer in BALB/c-Trp53 +/− mouse embryonic fibroblasts (MEFs) compared to C57BL/6J- Trp53+/− MEFs. Similarly, co-localization of H2AX and the homologous recombination protein RAD51 remain at dsbs longer in BALB/c-Trp53+/− MEFs compared to C57BL/6-Trp53+/− MEFs. Palb2 , a gene that lies within the SuprMam1 interval and has been shown to contribute to heritable breast cancer, was chosen as an initial candidate gene. No coding SNPs or expression differences of Palb2 were found in the mammary glands between the two strains. Additional fine mapping and use of a filtering criteria in the SuprMam1 region yielded an additional 34 candidate genes. We demonstrate no significant differences in any of these genes in whole mammary glands and primary mammary epithelial cells between the two strains. Finally, using a congenic mouse strain, we demonstrate the lack of irradiation (IR) sensitivity alleles within the SuprMam1 region. These results suggest a possible defect in HRR in the BALB/c strain that is unlikely related to Palb2. The gene or genes responsible for increased mammary tumor incidence in the BALB/c-Trp53+/− remain to be identified.

Family members with a heritable mutation in the BRCA1 or BRCA2 gene have up to an 85% life-long risk of contracting one or more of the cancers associated with a genetic alteration to these genes. The inheritance risks of a BRCA alteration to first and second-degree relatives of affected individuals are 50% and 25%, respectively. Currently, members from high-risk families can be tested for the presence of BRCA1 or BRCA2 mutations by gene sequencing of blood cell DNA. This test is expensive and generally is not reimbursed by insurance carriers. Over 85% of BRCA1 and BRCA2 mutations are reported to result in the production of a truncated protein, from which the carboxy terminal end sequences of their respective protein is missing. In preliminary studies, using antibodies specific for the amino acid terminal or the carboxy terminal of the BRCA1 protein, we demonstrated that immunohistochemical analysis of surgical specimens, consisting of matched ovarian tumor and normal uninvolved tissue, identified BRCA1 protein truncations in two matched samples. Each BRCA1 protein truncation was shown to be indicative of the presence of a BRCA1 germline mutation. Human buccal cells were then investigated as a minimally invasive cell source for this assay. Buccal cells were shown to express BRCA1 and BRCA2 protein immunohistochemically using distinct antibodies for the BRCA1 and BRCA2 protein terminals. Transcription of the BRCA1 and BRCA2 gene within buccal cells was also shown to occur by RT-PCR. A double blind study was then conducted using blood DNA and buccal cells collected from thirteen high-risk patients for carrying a germline BRCA1 or BRCA2 mutation. Mutation analysis (PCR, SSCP, and gene sequencing) was performed on DNA. Immunohistochemical analysis for the BRCA1 and BRCA2 protein was performed on cytospun deposited buccal cells. The buccal cell assay correctly predicted the presence of six germline BRCA1 mutations, three germline BRCA2 mutations, and three with no mutations. One predicted germline BRCA2 mutation did not correlate with DNA sequencing results. Based on these results, this assay exhibited a 100% negative predictive value and 90% positive predictive value.

Hair loss has been ranked as a source of considerable distress and may add to the losses associated with the experience of cancer. Chemotherapy-induced hair loss (alopecia) is a public consequence of the non-selective action of specific antineoplastic agents on healthy tissue. The literature demonstrates a lack of research on hair loss. Nursing studies have focused on efforts to prevent hair loss or measure the impact of hair loss using body-image instruments. The purpose of this study was the detailed examination of the meanings of hair loss over time in a sample of women receiving alopecia-inducing cancer chemotherapy. A qualitative descriptive design using a semi-structured multiple interview format examined the meaning of hair loss over time. A purposive sample of ten women receiving alopecia-inducing chemotherapeutic agents at a metropolitan teaching hospital was used. Each woman was interviewed prior to hair loss, at the time of hair loss, and two-three months after initial hair loss. The specific research questions described the meanings of hair loss in the lives of women receiving alopecia-inducing agents. Supporting questions explored the status of hair loss among sources of cancer-related distress, the role of past experiences and expectations, and the role of other people and social demands on the experiences. Data analysis was based upon the words, metaphors, and language patterns used by participants in describing their feelings and experiences. Findings reflect the meaning and real substantive losses associated with both the threat and actual hair loss. Symptom responses are shaped by personal history, experiences as well as meanings of cancer images and one's hair. Analysis of the findings reflect three processes: affective anticipation rehearsal, confrontation of the hair losses, and management of the hair loss experience. The coping outcomes may be positive, regaining one's stride, or negative, not regaining one's stride. The findings are congruent with a number of theoretical frameworks, such as Lazarus, Benner, Mishel and Wright. The findings support both the significance of hair loss and its amenability to nursing treatment approaches. Nurses have the opportunity to explore the meanings of hair loss with an individual and to lessen the distress associated with the threat and actual impact of that experience.

No description available.