abstract: In the United States, 12% of women are typically diagnosed with breast cancer, where 20-30% of these cases are identified as Triple Negative Breast Cancer (TNBC). In the state of Arizona, 810 deaths occur due to breast cancer and more than 4,600 cases are diagnosed every year (American Cancer Society). The lack of estrogen, progesterone, and HER2 receptors in TNBC makes discovery of targeted therapies further challenging. To tackle this issue, a novel multi-component drug vehicle is presented. Previously, we have shown that mitoxantrone, a DNA damaging drug, can sensitize TNBC cells to TRAIL, which is a protein that can selectively kill cancer cells. In this current study, we have formulated aminoglycoside-derived nanoparticles (liposomes) loaded with mitoxantrone, PARP inhibitors, for delivery to cancer cells. PARP inhibitors are helpful in preventing cancer cells from repairing their DNA following damage with other drugs (e.g. mitoxantrone). Various treatment liposome groups, consisting of lipid-containing polymers (lipopolymers) synthesized in our laboratory, were formulated and characterized for their size, surface charge, and stability. PARP inhibitors and treatment of cells for in-vitro and in-vivo experiments with these liposomes resulted in synergistic death of cancer cells. Finally, studies to evaluate the pre-clinical efficacy of these approaches using immuno-deficient mouse models of TNBC disease have been initiated. / Dissertation/Thesis / Masters Thesis Chemical Engineering 2018

Tumor heterogeneity observed between patients has made it challenging to develop universal or broadly effective cancer therapies. Therefore, an ever-growing movement within cancer research aims to tailor cancer therapies to individual patients or specific tumor subtypes. Tumor stratification is generally dictated by the genomic mutation status of the tumor cells themselves. Importantly, non-genetic influences – such as interactions between tumor cells and other components of the tumor microenvironment – have largely been ignored. Therefore, in an effort to increase treatment predictability and efficacy, we investigated how tumor-stroma interactions contribute to drug sensitivity and drug resistance. I designed a high throughput co-culture screening platform to measure how tumor-stroma interactions alter drug mediated cell death. I identified tumor-stroma interactions that strongly desensitize or sensitize cancer cells to various drug treatments. The directionality of these observed phenotypes was dependent on the stromal cell tissue of origin. Further study revealed that interactions between tumor cells and fibroblasts modulate apoptotic priming in tumor cells to mediate sensitivity to chemotherapeutics. The principles uncovered in this study have important implications on the use of drugs that are designed to enhance apoptosis. For example, based on our screening data, I hypothesized and experimentally validated that the effectiveness of BH3 mimetic compounds would be strongly dependent on the fibroblast growth environment. Taken together, our study highlights the importance of understanding how environmental interactions alter the drug responses of cancer cells and reveals a mechanism by which stromal cells drive broad spectrum changes in tumor cell sensitivities to common chemotherapeutics.

No description available.

Parmi les sous-types moléculaires de cancers du sein, le cancer du sein triple-négatif (TNBC) est caractérisé par un très mauvais pronostic et ne bénéficie actuellement d’aucune thérapie ciblée efficace. Dans ce projet, nous avons analysé le profil de certaines altérations oncogéniques dans les tumeurs TNBC et évalué le potentiel thérapeutique de leur ciblage à l’aide des modèles de xénogreffes (PDX).Nous avons d'abord démontré que le récepteur à activité tyrosine kinase RET est surexprimé dans une sous-population de tumeurs du sein TN et HER2+. Le ciblage de RET par son inhibiteur Vandetanib a été testé in vivo dans trois modèles de PDX TNBC et un modèle de PDX HER2+ caractérisés par des niveaux différents d’expression de RET et d’EGFR (les cibles principales du Vandetanib). Le Vandetanib a induit une régression tumorale dans les trois modèles de PDX surexprimant RET ou EGFR. L’effet du Vandetanib a été associé à une inhibition de la voie MAPK, une inhibition de l'angiogenèse et une induction de la nécrose.Nous avons également étudié les altérations de la voie PI3K/AKT/mTOR dans une large série de PDX de cancers du sein incluant des PDX TNBC. La voie PI3K/AKT/mTOR a été trouvée activée dans le cancer du sein triple-négatif. L’altération principalement retrouvée dans cette voie est la perte des deux suppresseurs de la voie, PTEN et/ou INPP4B. Sept des quinze modèles de PDX triple-négatifs testés ont montré une réponse à l’Everolimus. L'analyse des tumeurs traitées a montré que la phosphorylation post-traitement d’AKT est significativement plus fréquente dans les modèles répondeurs par rapport aux non-répondeurs. En conclusion, mon travail de thèse a permis de montrer que le Vandetanib et l'Everolimus pourraient être efficaces pour traiter le cancer du sein triple-négatif. Des études complémentaires sont nécessaires pour valider les biomarqueurs prédictifs de réponse à ces deux thérapies ciblées. / Among breast cancer subtypes, Triple-negative breast cancer (TNBC) has a very poor prognosis. There are currently no known targeted therapies for this subgroup of patients. In this project, we analyzed the profile of certain oncogenic alterations in the TN tumors and evaluated in vivo the therapeutic potential of targeting these alterations in TNBC.We first demonstrated that the tyrosine kinase receptor RET is overexpressed in a subset of TN and HER2+ tumors. Targeting RET by his inhibitor Vandetanib was tested in vivo in three PDX models of TNBC and one model of HER2+ BC with different expression levels of RET and EGFR. Vandetanib induced tumor regression in the three PDX models with high expression of RET or EGFR. Vandetanib effect was associated with inhibition of MAPK pathway, inhibition of angiogenesis and induction of necrosis. PI3K pathway alterations were investigated in an important number of BC PDX including TNBC PDX. PI3K pathway was shown to be activated in TNBC PDX possibly by the loss of the two pathway suppressors, PTEN and/or INPP4B. Treatment by Everolimus induced response in seven among the fifteen TNBC PDX tested. Analysis of treated tumors showed that post-treatment phosphorylation of AKT was more pronounced in responder PDX. The combination of Everolimus with chemotherapy was tested in one PDX and resulted in increased efficacy.In conclusion, in this work we showed that Vandetanib and Everolimus could be effective in treating TNBC. Further investigations are still needed to validate response related biomarkers.

No description available.

Breast cancer is a multifaceted disease that affects 1 in every 8 women. Triple negative breast cancer (TNBC) accounts for ~15-20% of all diagnosed breast cancers and is characterized by the absence of ER, PR, and HER2 on the tumor cell surface. As most cancer therapies to date target these cell surface receptors, TNBC is the only subtype of breast cancer without a targeted therapy and thus prognosis for it remains poor. The heterogeneity of TNBC also makes finding a targeted therapy particularly difficult. This work focuses on different methods of targeting distinct subpopulations of TNBC in order to identify potential novel therapeutic nodes to exploit as targeted therapies. The first chapter describes the use of a directed siRNA synthetic lethality screen to target vulnerabilities associated with basal TNBC, the most common TNBC subtype. The screen identified multiple dependency genes associated with RNA splicing, particularly those in the U4/U6.U5 tri-snRNP complex (PRPF8, PRPF38A). Depletion of these genes or the upstream splicing inhibitor E7107 in basal TNBC cell lines resulted in intronic retention and altered splicing of transcripts in pathways necessary for TNBC survival, including mitosis and apoptosis. In vivo, E7107 hindered the growth of both basal cell line and patient derived xenographs, a phenotype that was enhanced with the addition of the proteasome inhibitor bortezomib. This suggests that splicing and proteasome inhibition could be an effective basal TNBC treatment. The second chapter investigates the role of G-Protein Pathway Suppressor 2 (GPS2) as a tumor suppressor in the PI3K/AKT pathway in TNBC. Previous work has shown that GPS2 acts as a negative regulator of this pathway through inhibition of Ubc13-mediated activation of AKT in the insulin signaling pathway. In this study, MDA-MB231-GPS2KO cells were found to have increased proliferative, migratory, and invasive properties, which were rescued upon treatment with the allosteric AKT inhibitor MK2206. In vivo, GPS2 depleted cells conferred greater tumor burden in an orthotopic mouse model that was also responsive to AKT inhibition. Transcriptomic analysis showed significant overlap between MB231-GPS2KO and MB231 cells modified to have constitutively active AKT, indicating that the phenotypes observed in MB231-GPS2KO were at least in part due to loss of GPS2-mediated regulation of AKT activation. These studies point to GPS2 as a potential biomarker for a subclass of breast cancers that would be responsive to PI3K-class inhibitor drugs. In sum, these studies elucidate interactions and processes that seem to specifically adversely affect TNBC cells, which broaden our knowledge of TNBC biology and its potential weaknesses. / 2022-03-03T00:00:00Z

Breast cancer (BC) is the second most common cause of cancer-related deaths for women in the United States, estimated to affect 1 in 8 women. Difficulties arise in BC treatment due to the hormone sensitivity and heterogeneity of the malignancies, and the poor prognosis after metastases. Due to the immense physical and psychological effects of conventional surgical methods, minimally invasive, non-thermal, focal electroporation-based ablation therapies are being investigated for the treatment of BC. Irreversible Electroporation (IRE) delivers a series of long, monopolar electrical pulses via electrodes inserted directly into the targeted tissue which disrupt cellular membranes by creating nano-scale pores, killing the cells via loss of homeostasis while promoting an immune response. However, IRE requires cardiac synchronization and a full-body paralytic to mitigate unwanted muscle contractions, which motivated the creation of second generation High-Frequency IRE or H-FIRE. H-FIRE delivers short, bipolar pulses to destroy cancer cells without muscle contractions and nerve excitation, and allows for more tunable treatment parameters. Throughout my thesis, I discuss investigations of H-FIRE for the treatment of triple-negative and hormone-sensitive BC cell lines and compare efficacy to IRE outcomes. To further establish the translation and understanding of H-FIRE for BC applications, my master's thesis focuses on: (1) determining the lethal electric field threshold of both cell lines in a 3D hydrogel matrix after H-FIRE and IRE; and (2) employ those values in a single bipolar probe numerical model to simulate in vivo treatments. The culmination of this thesis advances the use of H-FIRE in breast tissues, as well as demonstrates how in vitro data can be used to develop clinically relevant numerical models to better predict in vivo treatment outcome. / Master of Science / Breast cancer (BC) is one of the most deadly forms of cancer for women in the United States, affecting every 1 in 8 women. Difficulties arising in the treatment of BC include the hormone sensitivity of malignancies, metastatic tendencies, and the diversity of the tissue that characterizes the breast. Surgical options like mastectomy or lumpectomy are most often used when treating BC; however, these are incredibly taxing on the patient. This reason has sparked investigations of focused ablation modalities for the treatment of BC, specifically non-thermal mechanisms like electroporation-based therapies. Electroporation explains the phenomenon that cells subjected to a high enough electric field will result in increased membrane permeability, allowing for the entrance of therapeutic agents in reversible mechanisms, or cell death beyond an irreversible point. Irreversible Electroporation (IRE) has shown success for the treatment of prostate, liver, kidney, and pancreas. However, due to some drawbacks, second generation High-Frequency IRE (H-FIRE) is increasingly being investigated for certain cancer types and is the main focus of this thesis project. Within this thesis, I discuss investigations of H-FIRE with applications to treat malignant breast cell lines. Specifically, my thesis focuses on: (1) determining the point at which cancer cells damage is irreversible; and (2) incorporate those values into a numerical model used to simulate electroporation treatment if a tumor were embedded in a layer of fatty connective breast tissue. The culmination of this thesis enhances our understanding of H-FIRE in the breast, with the hopes of future transition of application into animal studies and ultimately the clinic.

Eribulin is a non-taxane microtubule inhibitor approved for the treatment of metastatic breast carcinoma after two prior chemotherapeutic regimens. We report a patient with extended progression-free survival (PFS) of more than 57 months with metastatic breast carcinoma treated with eribulin in the third-line setting. A 48-year-old lady was diagnosed with stage IIA (pT2N0M0), high grade, triple-negative, invasive ductal carcinoma (IDC) of the left breast on core needle biopsy. She underwent neoadjuvant chemotherapy with adriamycin, and cyclophosphamide followed by a negative sentinel lymph node (SLN) biopsy. Subsequent mastectomy and axillary lymph node dissection revealed a 2.5 cm, high grade, triple-negative IDC with three additional lymph nodes negative for metastatic carcinoma, consistent with the initial diagnosis. Eight months into the surveillance program, the patient developed a 2.8 cm right lower lobe (RLL) lung mass with standard uptake value (SUV) of 27 on positron emission tomography-computed tomography (PET/CT). Core needle biopsy of the lung lesion revealed sheets of poorly differentiated carcinoma, immunophenotypically compatible with the initial diagnosis of breast pathology. She then commenced single-agent paclitaxel in the 1st line metastatic setting with a significant decrease in RLL lung mass to less than 1 cm with an SUV of 1.7 noted. The patient developed progression after seven months and started 2nd line gemcitabine noting initial improvement and subsequent stable disease for a period of 12 months. Eventual progression of RLL lung nodule measuring 2.1 cm with SUV of 10 noted. Initiated 3rd line eribulin with a notable response on imaging studies within three months and with no evidence of disease (NED) on scans over the subsequent 57 months. Eribulin related mild neuropathy superimposed on previous paclitaxel associated grade 2 neuropathy required a 20% eribulin dose reduction. The patient is currently clinically and radiographically stable with plateaued serum tumor markers. Our patient has shown excellent response and tolerance to eribulin with PFS of over 57 months (nineteen times the norm) which is rare.

Breast cancer tops the list of new cancer cases and is predicted to be the second leading cause of cancer deaths in women in 2012. The primary objective of the present study was to provide insights into the molecular mechanisms underlying the aggressive growth and metastasis of triple-negative and basal-like breast cancers. To study increased growth and invasive behavior in triple-negative and basal-like breast cancers we utilize both an interesting and relevant cell culture model and examination of human tissue. In this study, we use the Tamoxifen-selected, MCF-7 derivative, TMX2-28 breast cancer cell line. TMX2-28 cells are triple-negative in that they lack expression of the estrogen receptor alpha (ERα), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). They also have acquired a mixed basal/luminal cytokeratin profile, suggestive of a more basal-like phenotype. TMX2-28 cells are highly proliferative and invasive. In addition to our cell culture model, we also examine human tissue. Thirty frozen breast carcinoma samples were evaluated for mRNA expression. Additionally, I analyzed protein expression, using immunohistochemistry (IHC), of 50 benign reduction mammoplasty and 188 breast tumors (formalin-fixed paraffin embedded). Of the 188 breast tumors, 93 were ERα-positive and 95 were ERα-negative. Of the 95 ERα-negative samples, 24 were further classified as non-triple negative (either PR or HER2 positive), 49 were classified as triple-negative, and 22 were not further classified due to unavailability of HER2 status and were used only in analyses of ERα-negative tumors. Thirty-seven of the 188 tumor samples were ductal carcinoma in situ, 138 were invasive ductal carcinomas, and 13 were classified as other. Lastly, 23 of the 188 tumors were grade 1, 48 were grade 2, 105 were grade 3, and 12 did not have grade data available. S-phase kinase-associated protein 2 (SKP2) plays an important role in cell cycle regulation by targeting p27 for degradation. The cyclin-dependent kinase (CDK) inhibitor p27 regulates G1/S transition by binding cyclin/CDK complexes and abrogating its activity. By targeting p27 for degradation, SKP2 frees the complexes needed to progress into the S phase of the cell cycle. Evaluation of SKP2 expression in TMX2-28 revealed significantly higher levels than in other breast cancer cell lines. Despite the high levels of SKP2 expression, p27 protein was not reduced. However, levels of the Serine 10 phosphorylated form of p27 (pSer10p27), which has been associated with increased proliferation rates, was found to be increased. Furthermore, suppression of SKP2 completely eliminated the pSer10p27 and slowed cycle progression confirming the role of SKP2 in the aggressive growth of TMX2-28 cells. Assessment of mRNA from 30 frozen human breast cancers demonstrated that SKP2 is more highly expressed in ERα-negative and basal-like breast cancers. Immunohistochemical analysis of 188 breast cancers and 50 benign reduction mammoplasty tissues confirmed that SKP2 is more highly expressed in ERα-negative breast cancers and for the first time demonstrated that triple-negative breast cancers are more likely to overexpress SKP2 than are non-triple-negative, but still ERα-negative, tumors. In contrast to some previous reports, we did not observe an inverse relationship between SKP2 and p27 expression. Only 11% of tumors expressed high SKP2 and low p27, while 32% of tumors had high SKP2 and high p27. Although no significant relationship between SKP2 and p27 expression was observed in human breast cancers, a significant positive relationship was discovered between SKP2 and pSer10p27. Furthermore, high levels of SKP2 and pSer10p27 were observed significantly more often in ERα-negative and triple negative breast tumors than in ERα-positive breast cancers. Based on these results and those of the cell culture experiments showing complete elimination of pSer10p27 after suppression of SKP2 it appears that levels of pSer10p27 may be a better indicator of SKP2-dependent p27 degradation than are levels of p27. Therefore, that inhibiting SKP2 in triple-negative breast cancers expressing high levels of both SKP2 and pSer10p27 regardless of p27 levels may be a valid therapeutic approach. A foremost threat to patients is tumor invasion and metastasis, with the greatest risk to patients diagnosed with triple-negative and basal-like breast cancers. Two distinct morphological/functional mechanisms are known for single cell migration in tissues: mesenchymal and amoeboid invasion. Mesenchymal movement involves the use of proteases that cause cellular lysis in tissues, thereby creating a path through which cells can invade. Amoeboid movement is protease-independent; cells find paths through the ECM by pushing and squeezing through regions of adequate size. Despite their invasive phenotype, TMX2-28 retains morphology similar to non-aggressive MCF-7 cells, suggesting that their invasion may be proteolytic-independent. We determined that TMX2-28 lack MMP-1 mRNA, and MMP-2/MMP-9 protein expression; each of which is important in protease-dependent invasion. Furthermore, TMX2-28 cells have low expression of other genes key to protease-dependent invasion including Slug, Zeb 1, Zeb 2, Vimentin, Fibronectin and N-cadherin. RhoA is a member of the Rho superfamily of GTPases that acts as a molecular switch to control signal transduction and is critical to the amoeboid invasion mechanism. TMX2-28 cells have high expression of protease-independent invasion genes such as RhoA, ROCK 1, ROCK 2, and E-cadherin. Finally, treating TMX2-28 cells with a RhoA pathway inhibitor or an shRNA targeting RhoA significantly reduces their invasiveness. These data suggest that TMX2-28 cells use a RhoA-dependent, proteolytic-independent invasion mechanism. Collectively, the data presented here demonstrate the roles of SKP2 and RhoA in triple-negative and basal-like breast cancers, making both genes, as well as their pathways, desirable therapeutic targets.

No description available.