No description available.

Gene expression profiles have identified five major molecular breast cancer subtypes (Luminal A, Luminal B, Basal-like, HER2+/estrogen receptor− , and Normal Breast-like) that show significant differences in survival. The cost and complexity of gene expression technology has impeded its clinical implementation. By comparison, immunohistochemistry is an economical technique applicable to the standard formalin-fixed, paraffin-embedded material commonly used in hospital labs, and has the advantage of simultaneously interpretation with histomorphology. In this thesis, I hypothesize that a surrogate panel of immunohistochemical biomarkers can be developed to discriminate the breast cancer biological subtypes. The main study cohort consists of over 4000 primary invasive breast tumors, assembled into tissue microarrays. These patients were referred to the British Columbia Cancer Agency between 1986-1992 and have staging, pathology, treatment and follow-up information. In summary, our results demonstrate that (1) the rabbit monoclonal antibody, SP1, is an improved standard for immunohistochemiscal estrogen receptor assessment in breast cancer; (2) the transcription factor, GATA-3, is almost exclusively expressed among estrogen receptor positive tumors but does not seem to predict for tamoxifen response among estrogen receptor positive patients; (3) the proliferation marker, Ki-67, together with HER2 can segregate Luminal A from Luminal B subtypes, which carry distinct risks for breast cancer relapse and death; and (4) the inclusion of the basal markers EGFR and ck5/6 to “triple negative” breast cancers provides a more specific definition of basal-like breast cancer that better predicts patient survival. These results consistently demonstrate that an immunopanel of six biomarkers (estrogen receptor, progesterone receptor, HER2, Ki-67, epidermal growth factor receptor and cytokeratin 5/6) can be readily applied to standard pathology specimens to subtype breast cancer samples based on their underlying molecular biology. These findings have been considered sufficient to justify application of this panel onto NCIC (MA5, MA12) and CALGB (9341 and 9741) clinical trials specimens. This followup work which is underway and will determine if the six marker immunopanel can guide decisions about which patients need aggressive systemic drug treatment, and thereby ensure patients get the most effective, individualized adjuvant systemic therapy for their breast tumor. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Cancer survivors are at greater risk of cardiovascular diseases in comparison to the general population. Cardiovascular disorders are among the most prominent comorbidities in breast cancer patients. In order to gain a better understanding of the prescribing practices and cardiovascular risks associated with oncology drugs, this thesis encompasses a detailed review of the molecular and physiological mechanisms leading to drug‐induced cardiotoxicity and an evaluation of the cardiovascular risks associated with cancer drug therapies. Using a nested case‐control design, we evaluated whether these cancer drugs were associated with adverse cardiovascular outcomes under real‐world conditions of use. Although only few oncology drugs are indicated for breast cancer treatment, we were interested in prescribing practices and whether breast cancer treatments are restricted to labelled indications. The characterization of prescribing practices provides insights on the range of antineoplastic agents that should be considered in the evaluation of treatmentrelated adverse reactions such as cardiotoxicity. We found that selective estrogen receptor modulators demonstrated a better safety profile than aromatase inhibitors based on their mechanism of action on the cardiovascular system. These observations were corroborated by our findings from logistic regression analyses where aromatase inhibitors were associated with a higher risk of heart failure in a heterogeneous population of breast cancer patients. We reported that off‐label prescribing is common strategy in breast cancer treatment. While this practice tends to be associated with specific socio‐demographic and disease characteristics, the majority of off-label encounters are evidence‐based decisions. Because these off‐label treatments have their own inherent safety profiles, a comprehensive approach, covering all antineoplastic agents administered should be adopted in the evaluation of breast cancer treatment-induced cardiotoxicity. Careful monitoring of patients is crucial for the early detection of warning signs of cardiotoxicity to prevent long‐term deleterious effects. The information contained in this thesis provides useful considerations for the prospective surveillance of cancer drug‐induced cardiac events. These findings point to the need for a multi‐disciplinary approach to facilitate the rapid diagnosis and treatment of cardiac complications secondary to cancer therapy and to ensure that treatment decisions will maximize tumor response while minimizing adverse outcomes.

Screening, surveillance and preventative medical interventions are being identified as best practices for women at high-risk of developing hereditary breast cancer. Genetic screening for the BRCA1/2 mutations associated with hereditary breast cancer is currently recommended as an appropriate health intervention for younger women who have been affected by breast cancer, as well as for those who have been identified as high-risk due to their family histories of breast cancer. At present, however, little is known about the psychosocial implications of genetic screening for BRCA1/2 mutations on young families. Using the common sense model of self-regulation (Leventhal et al., 1997), adapted for genetics (Cameron, 2003; Marteau & Weinman, 2006) as a guiding framework, the goals of this dissertation were to: (a) examine the relationships between threat representation and psychosocial functioning in BRCA1/2 counselees, (b) explore the impact of fear representation on women’s psychosocial functioning, and (c) assess how BRCA1/2 counselees’ threat representations and fear representations impact family functioning and the psychosocial adaptation of their adolescent daughters. Results indicated that total threat representation, including risk representation, illness representation and fear representation, was found to add to the prediction of mothers’ self-reported levels of anxiety, depressive symptoms and intrusive ideation regarding genetic counseling. Additionally, when the cognitive processes of the threat representation were controlled (i.e., risk representation and illness representation), the subjective-emotional processes (i.e., the fear representation) continued to emerge as a significant predictor of mothers’ self-reported anxiety, depressive and intrusive ideation symptoms. Additionally, support for the association between mothers’ threat representations and adolescent daughters’ reports of depressive symptoms and self-concept were noted. Cumulatively, these results provide support for the role of fear representation within the CSM framework and suggest that fear representation plays an important role in BRCA1/2 counselees’ psychosocial adaptation following genetic counseling.

The epidermal growth factor receptor (EGFR) is a well-recognized proto-oncogene and mediator of cancer cell growth and proliferation. Emerging evidence suggests the paradoxical role of EGFR activation, unique to metastatic breast cancer cells. Previously studies elucidated the role of EGFR mediated activation of Signal Transducer and Activator of Transcription 1, STAT1, is required to induce apoptosis in cells with increased metastatic potential. In this current study, we evaluate the effects of cells with mutations leading to aberrations in phosphatidylinositol 3-kinase, PI3K/AKT/protein kinase B signaling. Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA) is a key regulator of the PI3K/Akt pathway and is one of the most commonly mutated genes in breast cancer patients. Utilizing human breast cancer cells, MDA-MB-468 and BT-20s, characterized by amplified protein levels of EGFR and mutations in the PI3K signaling, we demonstrate activation of EGFR with EGF leads to increased pSTAT1 expression. We further demonstrate biased EGFR signaling toward STAT1 activation by pharmacological inhibition of downstream kinase activity with MEK1/2 inhibitor, trametinib, and the AKT inhibitor, Uprosertib or in combination with the PI3Kα specific inhibitor Alpelisib, trade name, PIQRAY. Combination MEK and Akt signaling inhibition followed by EGF stimulation show marked increase in apoptotic activity and decreased cell viability. Moreover, we demonstrate changes in EGFR mediated apoptosis in murine breast cancer cell line derived from metastatic lung nodules, delineate from cells derived from the primary tumor. These finding support the notion of differential evolution of cancer cells as they metastasize to secondary organs. Furthermore, EGFR expression was observed to be vital in EGF mediated pSTAT1 in human breast cancer cell lines. To this end we explored alternative stimulators of pSTAT1 using interferon activation. Addition of INFγ led to robust pSTAT1 in cells that did not respond to EGF and levels of pSTAT1 were not attenuated with our combination treatment. Together, our findings demonstrate the differential role of EGFR expression and signaling in metastatic cells and tolerance for novel combination therapies for patients of late stage breast cancer.

In 2016, there were 250,000 new cases of invasive cancer and 60,000 of ductal carcinoma in situ. Mammograms are used to screen for cases of disease, but the literature shows that mammograms are highly dependent on patient characteristics and do not majorly impact mortality rates from invasive cancer. Additionally, they are prone to false-positives, false-negatives, and overdiagnosis in cases of in situ cancer, with overdiagnosis exposing patients to the side effects of treatment. Better screening tests are needed, and a potential solution can be to extend molecular screening methods often used in advanced stage 1 and higher cancers to stage 0 ductal carcinoma in situ cases. This new test would prevent overdiagnosis, be more accurate, and prevent unnecessary screening as well as be in line with the future of cancer care in the US.

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

The breast tumor suppressor hCLCA2 is a putative chloride regulator that is expressed in normal breast epithelial cells and frequently down-regulated in breast cancers. The first CLCA protein was described as a calcium-activated, plasma-membrane chloride channel having four or five transmembrane pass structure that could form a channel pore. However, CLCA topology is inconsistent with chloride channel function. We showed that hCLCA2 itself is unlikely to form a channel as it has only a single transmembrane segment with a short cytoplasmic tail and is mostly extracellular. Moreover, the N-terminal 109-kDa ectodomain is cleaved at the cell surface and shed into the medium while the 35-kDa C-terminal product is retained by the cell membrane. The general goal of my project was to study the function of this novel protein and its role in breast cancer. In addition to its role in chloride regulation, hCLCA2 behaves as a tumor suppressor gene that is frequently down-regulated in breast cancer. We previously demonstrated that murine homologs of hCLCA2 are transcriptionally induced during mammary involution, when the gland shuts down and 80% of the mammary epithelial cells die by apoptosis. In cell culture, conditions that cause G1 arrest such as contact inhibition and depriving cells of growth factors and anchorage induced these genes. Therefore, one of the goals of this project was to find if this is true of hCLCA2 in human breast epithelial cells. We found that hCLCA2 was induced by the above mentioned stresses and by pharmacological blockage of cell survival signaling. In addition, we found that DNA-damaging agents doxorubicin and aphidicolin potently induced hCLCA2 in p53-positive cell lines such as MCF-7 but not in p53-deficient cells such as MDA-MB231. An adenovirus encoding p53 induced hCLCA2 expression in a broad spectrum of breast cancer cell lines while a control virus did not, suggesting that hCLCA2 is a p53-inducible gene. To further test the hypothesis, we performed chromatin immunoprecipitation (ChIP) to determine whether p53 bound to the hCLCA2 promoter. This analysis showed that p53 binds directly to the hCLCA2 promoter between -157 and -359bp upstream of the translation initiation site. This segment was required for the p53-dependent expression of an hCLCA2-luciferase fusion gene. Point mutation of the p53 consensus binding motif abolished this induction. Induction of hCLCA2 in MCF-7 cells by doxorubicin was inhibited by p53 knockdown and by p53 inhibitor pifithrin, indicating that p53 activates the endogenous hCLCA2 promoter in response to DNA damage. An adenovirus encoding hCLCA2 induced a cell cycle lag in G0/G1 phase, decreased intracellular pH from 7.49 to 6.7, caused Bax and Bad translocation to the mitochondria, activated caspases, induced PARP cleavage, and promoted apoptosis. Conversely, hCLCA2 knockdown enhanced proliferation of epithelial MCF10A cells and reduced sensitivity to doxorubicin. These results reveal the molecular mechanism of hCLCA2 induction and downstream events that may provide protection from tumorigenesis. Epithelial cells acquire mesenchymal characteristics by undergoing phenotypic and genotypic changes during cancer progression. An early step in the epithelial to mesenchymal transition (EMT) is the disruption of intercellular connections due to loss of epithelial cadherins. We find that expression of tumor suppressor hCLCA2 is strongly associated with epithelial differentiation and that induction of EMT by mesenchymal transcription factors represses its expression. Moreover, we found that knockdown of hCLCA2 by RNA interference results in disruption of cell-cell junctions by downregulating E-cadherin. This also imparts invasiveness and anoikis-resistance to epithelial cells but is insufficient to induce full EMT. However, activation of Ras oncogene in combination with hCLCA2 knockdown is sufficient to induce full EMT in vitro. These findings indicate that, like E-cadherin, hCLCA2 is required for epithelial differentiation and that its loss during tumor progression may contribute to metastasis.

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.

Breast cancer continues to be the most common diagnosed cancer among women, and radiation or chemotherapy generally fails to provide durable cure, especially in the context of advanced or metastatic disease. Tumours recurrence is believed to be driven by cancer stem cells, which resist anti-cancer therapy and survive to seed relapse after remission in breast cancer patients. Small molecules inhibitors of nicotinic acetylcholine receptors (nAChR) target cancer stem cells, however, the precise nAChR required for breast cancer stem cell activity is unknown. Hence, we propose to test the capacity of shRNAs that target each individual nAChR to inhibit breast cancer stem cell activity. Briefly, we performed a cancer stem cell based pooled lenti-vector shRNA screen, to identify receptors required for the propagation of breast cancer stem cell enriched cultures. Our results demonstrate that the suppression of multiple receptors can be detected and corresponding genes are essential for TIC viability and survival. We anticipate our approach will identify the relevant nAChR receptor required for breast cancer stem cell activity. Such receptors may represent useful drug targets for the development of anti-breast cancer stem cell therapeutics. / Thesis / Master of Science (MSc)