Indiana University-Purdue University Indianapolis (IUPUI) / 2021-11-30

Triple negative breast cancer (TNBC) is an aggressive subtype of the disease with dismal clinical outcome. Immune checkpoint blockade (ICB), which blocks inhibitory pathways on T cells, has surged to the forefront of cancer therapy with clinical success in a variety of cancer types. However, ICB for TNBC only benefits 10-20% of patients. Thus, a deeper understanding of the immune landscape in TNBC is required to develop efficacious therapies and delineate prognostic biomarkers of disease. We have developed combination therapy platforms that sensitize TNBC tumors to ICB. Using a clinical chemotherapy (FEC) combined with oncolytic virotherapy (oHSV-1) we show enhanced tumor-infiltrating lymphocytes (TILs), upregulation of B cell receptor signaling pathways, suppression of myeloid-derived suppressor cells (MDSCs) and improved survival. In vivo depletion studies revealed that B cells were required to achieve cures with treatment. Furthermore, the absence of B cells resulted in the expansion of MDSCs. This crucial finding of the importance of B cells for mediation and downregulation of MDSCs is a novel and significant contribution to the field. RNA sequencing revealed that two of the top upregulated genes in mice treated with FEC + oHSV-1 were S100A8 and S100A9, calcium binding proteins highly expressed in myeloid cells. These genes have controversial findings in the literature with both pro- and antitumorigenic functions being reported. Investigation of data from the Cancer Genome Atlas revealed that high levels of S100A8 and S100A9 correlate with improved prognostic outcomes in breast cancer patients. In line with the clinical data, our data suggests that increased levels of S100A8 and S100A9 results in improved responses to immunotherapy treatments and that this increased expression is involved in macrophage-mediated epigenetic reprogramming of the tumor microenvironment. Our second therapeutic platform used a radiolabeled biomolecule containing the beta-emitting radioisotope, lutetium-177. We found that two doses of radiotherapy, combined with ICB improved overall survival in murine TNBC tumors, increased TILs and suppressed circulating MDSCs. These findings offer insight into the newly explored field of combination radioimmunotherapy and again highlight the importance of suppressing MDSCs to alleviate tumor immunosuppression. / Thesis / Doctor of Philosophy (PhD) / Triple-negative breast cancer (TNBC) has poor prognostic outcomes due to lack of expression of targets for therapy. As such, patients routinely undergo aggressive treatment regimens with many harsh side effects, including high levels of toxicity. Immunotherapy, a form of therapy that boosts the immune system to fight cancer cells, has gained increasing prominence largely due to its safety and low toxicity to the patient. In the work within this dissertation, we have developed therapeutic platforms and studied them in a murine model of TNBC. The completed studies show the use of clinical therapies, in combination with immunotherapy and investigate the fundamental biology associated with therapeutic outcomes. These findings contribute knowledge to progress clinical regimens for TNBC patients as well as to better identify patients that will respond to therapy. Although this proposal is specific to breast cancer, the underlying concepts can be applied to many other forms of cancer.

Oncolytic viruses (OV) are a promising alternative cancer therapy due to their specificity and lack of debilitating side effects, such as those which typically accompany conventional therapeutics such as chemotherapy and radiation. Bovine herpesvirus type 1 (BHV-1) is an alphaherpesvirus with the ability to infect and kill multiple human tumor cell types. In comparison to other species-specific viruses, for which deficiencies in type I interferon signalling pathways dictates cellular sensitivity to infection, mutations in KRAS were found to correlate with high levels of BHV-1 replication. Interestingly, BHV-1 is able to induce cellular cytotoxicity in the absence of a productive infection. In contrast to current breast cancer (BC) treatments, which are largely based on receptor expression status, BHV-1 is able to infect and kill BC cells and breast cancer initiating cells (BCICs) from luminal and basal subtypes. Furthermore, BHV-1-infected BC cells are significantly diminished in their capacity to form tumors in vivo, suggesting that BHV-1 reduces the tumor forming capacity of BCICs. Combination therapy involving OVs has been used to exploit differences in the mechanism of tumor cell death elicited by individual treatments. Treatment with epigenetic modifiers such as 5-Azacytidine (5-Aza), a DNA methyltransferase inhibitor, has been shown to increase the antitumor activity of OVs. Our data indicates that 5-Aza strongly synergises with BHV-1, increasing virus replication and cytotoxicity in vitro. In vivo, BHV-1 monotherapy did not significantly impact tumor growth or survival of CR bearing subcutaneous breast tumors; however, combination therapy with 5-Aza significantly decreased the number of secondary lesions compared to BHV-1 monotherapy. Overall, the data presented in this dissertation indicate that BHV-1 is a promising broad spectrum OV with a unique mechanism of tumor cell targeting, and the ability to infect and kill tumor cells independent of a productive infection. / Thesis / Doctor of Philosophy (Medical Science)

Background Preclinical data suggests that metformin may have anti-cancer effects to reduce breast cancer incidence and improve cancer prognosis. However, the current evidence in observational studies is inconclusive. A systematic review and meta-analysis was conducted to assess the effect of metformin on the incidence and mortality of breast cancer in diabetic patients. Methods A comprehensive literature search was performed on Medline (Pubmed), EMBASE, and the Cochrane library from inception to November 2016 with no language restrictions. Outcomes were incidence of breast cancer and all-cause mortality. Risk of bias and overall quality of evidence was assessed using the Newcastle Ottawa Scale and GRADE respectively. A meta-analysis was performed using the most adjusted odds ratios (ORs) or hazard ratios (HRs) and 95% confidence intervals (95% CI) as effect measures. Results A total of 12 observational studies were included for breast cancer incidence and 11 studies for all-cause mortality. No significant association was found between metformin exposure and incidence of breast cancer (OR: 0.93, 95% CI: 0.85-1.03, I2 = 35%). A 45% risk reduction was observed for all-cause mortality (HR: 0.55, 95%CI: 0.44-0.70, I2=81%). Presence of publication bias is strongly suspected for both outcomes. Conclusion The use of metformin in standard cancer therapy may improve overall survival of diabetic patients with breast cancer. No effect of metformin on the incidence of breast cancer was observed. Interpretation of results is limited by the observational nature of the studies and methodological biases. Clinical trials are warranted to determine the role of metformin in breast cancer risk reduction and prognosis. / Thesis / Master of Health Sciences (MSc)

Breast Cancer (BC) is the leading cause of cancer related deaths among women worldwide. Its etiology includes inactivation of tumor suppressors. In line with this notion, our laboratory has recently reported that CYB5D2 possesses tumor suppressing activities in cervical cancer; evidence also suggests CYB5D2 as a novel tumor suppressor of BC. I thus hypothesize that CYB5D2 mitigates cell propagation in vitro. To examine this possibility, I transiently expressed CYB5D2 in two typical BC subtype cell lines, HCC 1954 (HER2+) and MCF7 (Luminal A). Remarkably, cell population was diminished over a period of at least five days post-transfection when compared to empty vector (EV). To characterize CYB5D2-derived inhibition of BC cell proliferation, I generated a Tet-On inducible system in both cell lines in which CYB5D2 expression is induced upon addition of doxycycline. As expected, induction of CYB5D2 led to a decline of cell propagation and colony formation based on cell proliferation and colony formation assays respectively. Moreover, knockdown of CYB5D2 via lentivirus-based shCYB5D2 transfection in HCC 1954 and MCF7 cells resulted in an incline of cell propagation and colony formation when compared to the shCTRL (control) line. To examine possible apoptotic mechanism of tumor suppressor, we performed TUNEL assay analysis in cell lines expressing CYB5D2. Both HCC 1954 and MCF7 obtained similar results exhibiting evidence of apoptotic cells when compared to their respective controls. Interestingly, upon CYB5D2 expression HCC 1954 also displayed evidence of halting G1 phase progression when performing cell cycle analysis, suggesting a promising alternate tumor suppressing mechanism. Lastly, to corroborate with previous observations of an existing molecular interaction between CYB5D2 and tumor suppressor Phosphatase and tensin homolog (PTEN) we performed a Co-Immunoprecipitation (Co-Ip) assay. As expected, endogenous CYB5D2 and PTEN lysate from HCC 1954 and MCF7 were demonstrated to interact on a molecular level. These observations support the notion that CYB5D2 elicits tumor-suppressing activities in both Her2+ and Luminal A breast cancer cell lines. It is important to note that we detected a greater tumor suppressing impact of CYB5D2 in HCC 1954 when compared to MCF7 suggesting a potential mechanism to favour HER2+ status. Additional research in determining potential tumor-suppressing pathway of CYB5D2 in BC is encouraged as we established promising insight of novel tumor suppressor. / Thesis / Master of Health Sciences (MSc)

A simple method to verify the total treatment time generated by the treatment planning system (TPS) when the CONTURA MLB or the SAVI applicator are used for APBI treatments has been developed. The method compares the time generated by the TPS to a predicted time, calculated by inserting parameters obtained from the TPS in equations generated in this Thesis. The equations were generated by linearly fitting data from clinical cases that had been treated using the Contura MLB or the SAVI applicator at the Lynn Cancer Institute of the Boca Raton Regional Hospital. The parameters used were the PTV coverage, Air Kerma Strength, Balloon Volume (Contura data fit) and Evaluation PTV (SAVI data fit). As an outcome of this research, it is recommended that the plan should be reevaluated when the percent difference between the generated and the predicted times exceeds 5% for the Contura MLB, or 10% for the SAVI. / by Andreas Kyriacou. / Thesis (M.S.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

Introduction: Basal-like (BL) breast cancer is an aggressive sub-type of breast cancer for which there is no targeted systemic therapy. C-Met is a receptor tyrosine kinase implicated in breast cancer. Clinical trials assessing the efficacy of anti-c-Met therapy are underway, yet few studies have analysed the clinical significance of c-Met expression and/or activation in breast cancer, in particular whether there is a correlation with molecular sub-type. The aims of this study are: 1) to establish the clinical significance of c-Met expression in invasive breast cancer, 2) evaluate the novel proximity ligation assay (PLA) as a method of measuring c-Met activation and 3) address the effect of hepatocyte growth factor (HGF)-mediated c-Met phosphorylation on migration and protein expression in cell lines representative of the BL sub-type. Methods: Immunohistochemistry for c-Met was performed on 1455 cases of breast cancer using tissue microarray (TMA) technology. The PLA was performed on TMAs constructed from 181 breast cancers. C-Met expression and the PLA product were correlated with clinico-pathological parameters and survival. The effects of HGF on cell migration and protein expression were assessed using migration assays, western blots and immunofluorescent studies. Results: C-Met expression was independently associated with BL breast cancer (odds ratio = 6.44, 95% confidence interval (CI) = 1.74-23.78, p = 0.005) and reduced overall survival (hazard ratio = 1.81, 95% CI = 1.07-3.06), p = 0.026). The PLA signal was not associated with molecular sub-type or survival. HGF stimulation was associated with a significant increase in BL cell migration (p < 0.01) but no evidence of epithelial-mesenchymal transition was observed. Conclusion: My findings suggest BL breast cancer patients should be included in future trials of anti-c-Met therapy. Further work is necessary to establish the prognostic utility of the PLA as a measure of c-Met activation and the mechanisms driving HGF-mediated cell migration.

Breast cancer is the most common cancer in the UK and Basal-like breast cancer (a highly aggressive subtype) accounts for approximately 8-22% of all cases depending on ethnicity. Unlike most human malignancies and indeed other PAM50 breast cancer subtypes, the vast majority of Basal-like tumours are positive for wild type p16. This p16 signature is associated with a particularly poor prognosis and p16-positive Basal-like breast cancer remains the most clinically challenging subtype and is the focus of this project. Pro-senescence therapies are gaining momentum as attractive strategies for the treatment of those breast cancers with current unmet clinical need. To identify targets for pro-senescence therapy in p16-positive Basal-like breast cancer, a genome‐wide siRNA screen and two subsequent validation screens using two p16-positive cancer cell lines were performed. Screening revealed 20 siRNAs that induced senescence within both cancer cell lines. Strikingly, 11 of these 20 siRNAs targeted ribosomal proteins, implicating disrupted ribosomal biosynthesis in senescence activation in p16-positive Basal-like breast cancer. Importantly, subsequent experiments in normal human mammary epithelial cells established that specific ribosomal protein knockdown is well tolerated by normal cells. Analysis of the METABRIC data set showed a high degree of ribosomal dysregulation in Basal-like tumours and revealed that all 11 ribosomal hits identified were frequently overexpressed in p16-positive Basal-like breast cancers. Kaplan Meier analysis confirmed that elevated expression of six of the 11 ribosomal proteins correlates with a reduced overall survival in these women, further supporting a role for these proteins as drivers of disease. These six ribosomal hits, associated with the poorest patient survival, were prioritised for further validation. Senescence induction was found to be highly stable, and associated with dramatic changes to nucleolar morphology, reminiscent of the nucleolar signature observed upon premature senescence induction in normal human mammary epithelial cells. In addition, siRNA rescue experiments indicated that senescence initiation is dependent on p16 and p21 expression and is accompanied by p16 nuclear translocation and p21 degradation. Further, ribosomal protein silencing in MDA-MB-231 cells (p16-null Basal-like breast cancer cell line) resulted in a 'death-like' phenotype, partially dependent on p21 expression suggesting that, within a cancer context, ribosomal protein silencing may induce a differential response depending on the status of p16. In conclusion, it is proposed that these six ribosomal candidates may form the basis of a novel pro-senescence therapy for p16-positive Basal-like breast cancer. They may also represent novel prognostic biomarkers for this disease subset and may help to improve disease stratification and future directed personalised therapies.

Metastasis is the most fearsome aspect of breast cancer (BC) a common disease in women, because it drives mortality. Although BC can invade almost any organ, it is most often found to invade the bone (31-79%), the brain (3-12%), the liver (8-18%) and the lung (11-13%). The site of distant metastasis is often associated with cause of death and length of survival. This dissertation examines whether the presence of select lifestyle and clinical factors can predict metastatic spread to the lung and/or the liver for a particular woman with advanced breast cancer. A systematic review of the literature identified tobacco use as a risk factor for lung metastasis in women with BC and suggested that obesity, hormone replacement therapy prior to BC diagnosis, hormonal therapy post diagnosis, and post-mastectomy radiation therapy may have an impact on this association. The review also uncovered that liver disease (i.e. hepatic steatosis, chronic hepatitis B infection, cirrhosis) is associated with the occurrence of liver metastasis in patients with colorectal cancer and that hyperglycemic and oxidative stress conditions as well as alcohol consumption were found to be associated with liver metastasis in colorectal or BC patients. We conducted a retrospective hospital-based case-control study of the association of select lifestyle and clinical factors with metastases detected in the lung and the liver among women diagnosed with stages II-IV BC and seen at the Columbia University Medical Center from 2008 to 2013. Select relevant clinical variables were extracted from the hospital patient charts and lifestyle factors from patients’ responses to a questionnaire developed for the purposes of this research. We examined whether smoking and / or post-mastectomy radiation therapy to the breast and/or the chest area were associated with an increased risk of 1st site lung metastasis in our sample of women with metastatic BC. We found that lifestyle factors such as smoking history or BMI at diagnosis did not affect the likelihood of 1st site lung metastasis in our sample of women. We also investigated whether a history of alcohol intake or chronic liver disease was associated with risk of developing a 1st site liver metastasis. Our analyses suggested that lifestyle factors such as alcohol intake or obesity might not affect the likelihood of 1st site liver metastasis in women with metastatic BC. We also report that a history of chronic liver disease significantly increased the odds of 1st site liver metastasis. Given our findings around adjuvant post mastectomy radiation therapy and chronic liver disease, we suggest collecting adjuvant treatment or relevant comorbid information in larger cohort studies. A better understanding of the relationship between these factors and the sites of metastasis has the potential to increase our understanding of the metastatic process. If we can find ways to identify women at high risk of metastatic disease, or develop preventive or therapeutic measures against lung or liver metastasis, we can hope to reduce mortality from metastases.

Second only to lung cancer, breast is the leading type of cancer among women in the US. Despite all the medical advances over the past few decades, toxicity and increased resistance to standard drug therapy still remains a significant problem. The heterogeneic nature of all cancers has led to a shift in treatment approaches, in that more research is being carried out with combination treatments in the hope that a multidirectional targeting of cancer will be far more effective than the current single treatment options. Our goal was to gain a better understanding of the molecular mechanisms of a nonhydrolyzable ether analog of RRR-[alpha]-tocopherol, 2, 5, 7, 8-tetramethyl-2R-(4R, 8R, 12-trimethyltridecyl)chroman-6-yloxyacetic acid (abbreviated [alpha] -TEA), and to investigate its efficacy when used in combination with known chemotherapeutics 9-nitro-camptothecin (9NC), and Paclitaxel (Taxol). The data presented here looks encouraging as it shows a clinically relevant delivery method using [alpha]-TEA and 9NC has the unique ability to reduce primary tumor burden as well as macro and micrometastatic lung and lymph node lesions in an aggressive syngeneic mouse mammary model, while displaying no obvious toxic side effects. The effect of combination treatments on tumor volume appears in part to be moderated by an increase in tumor cell apoptosis and a decrease in tumor cellular proliferation. Next, the intricate molecular mechanism of how [alpha]-TEA alone and in combination with 9NC is able to induce apoptosis in 66cl-4-GFP murine mammary cancer was investigated. The data suggest that the signaling pathway that ultimately leads to apoptosis is caspase dependent, is able to upregulate pro-death players while at the same time downregulate pro-survival proteins such as c-Flip and survivin. Finally, we investigated the efficacy of [alpha]-TEA used in an allograft mouse model following treatment with Taxol. Combination treatments were able to significantly reduce primary tumor burden, decrease lung and lymph node micrometastases, tumor cell proliferation, tumor blood vessel density as well as increase tumor cell apoptosis. Based on the results presented, we propose that [alpha]-TEA when used alone and in combination is an effective, non-toxic option for cancer treatment which warrants further investigation. / text