acase@tulane.edu / Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT) whereby cells adopt a motile and invasive phenotype through loss of epithelial markers, namely Cadherin 1/E-Cadherin (CDH1), and acquisition of mesenchymal markers, such as vimentin (VIM) and Cadherin 2/N-Cadherin (CDH2). While MAPK/ERK1/2 kinase inhibitors (MEKi) have shown promise as antitumor agents in the preclinical setting, application has had limited success clinically. Activation of compensatory signaling, potentially contributing to the emergence of drug resistance, has shifted the therapeutic strategy to combine MEK1/2 inhibitors with agents targeting oncoproteins (RAF) or parallel growth pathways (PI3K). Conventional MAPK family members have been well-characterized in modulation of cellular processes involved in tumor initiation and progression, yet the role of MEK5-ERK5 in cancer biology is not completely understood. Recent studies have highlighted the importance of the MEK5 pathway in metastatic progression of various cancer types, including those of the prostate, colon, bone and breast. Furthermore, elevated levels of ERK5 expression and activity observed in breast carcinomas are linked to worse prognosis in TNBC patients. The purpose of this work is to explore MEK5 regulation of the EMT axis and to evaluate a novel pan-MEK inhibitor on clinically aggressive TNBC cells. Our results show a distinction between the MEK1/2 and MEK5 cascades in maintenance of the mesenchymal phenotype, suggesting that the MEK5 pathway may be necessary and sufficient in EMT regulation while MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Furthermore, additive effects on MET induction are evident through the inhibition of both MEK1/2 and MEK5. Taken together, these data demonstrate the need for a better understanding of the individual roles of MEK1/2 and MEK5 signaling in breast cancer and provide rationale for combined targeting of these pathways to circumvent compensatory signaling and subsequent therapeutic resistance. / 1 / Van Hoang

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

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

Poly-(ADP-ribsoyl) Polymerases (PARPs) are a superfamily of enzymes comprised of 17 known isoforms. PARP inhibitors (PARPi) have shown success in clinical trials for the treatment of homologous recombination-deficient cancers. Though proven effective initially, tumors treated with PARPi eventually develop resistance. Combinatorial therapeutics targeting PARP and other pathways that may re-sensitize tumors to PARP inhibition, including PI3K/AKT/mTor pathway, and cell-cycle checkpoints (such as CDKs, CHK, and Wee) are being tested. In this context, the synthetic lethality of cyclin-dependent kinase 1 (CDK1) and PARP1 is known. Evaluation of PARP1 and CDK1 pharmacophores led to the development of the tetrahydro-arylazepinone (TAAP) scaffold as a potential dual PARP1/CDK1 inhibitor. We screened a handful of TAAP analogs against PARP1 in a cell-free assay that identified the low micromolar PARP1 inhibitor 1,2,3,4-tetrahydro-5H-benzo[e][1,4]-diazepin-5-one (TBAP), which served as the lead compound. The analogous 1,2,3,4-tetrahydro-5H-pyrido[2,3-e][1,4]-diazepin-5-one (TPAP) series showed a similar bioactivity profile. Satisfyingly, the N1-benzyl TPAP analogue showed activity in the low nanomolar range. The TAAP series (i.e., 6/7-membered scaffold) unfortunately lacked CDK1 inhibitory activity. Finally, many PARPi's show poor isoform-selectivity. The development of isoform-selective PARPi can clarify the specific function of each PARP isoform and may reduce the adverse side effects shown by PARPi. A handful of TAAP analogs were screened against 13 PARP isoforms, where some compounds demonstrated exquisite PARP1/2 selectivity. Concurrently, we discovered an inhibitor for PARP11, an isoform that lacks any known synthetic ligand. Future directions are suggested towards fine-tuning the structure-activity relationship of TAAP-isoform selective PARPi as well as developing a dual PARP1/CDK1 inhibitor. / Master of Science / The aim of this work is to explore the therapeutic potential of poly-(ADP-ribosyl) polymerase inhibitors (PARPi) for the treatment of ovarian and breast cancer, specifically triple negative breast cancer. Poly-(ADP-ribsoyl) Polymerases (PARPs) are a superfamily of enzymes comprised of 17 known isoforms. Currently, there are three FDA approved PARPi - olaparib, isoforms. Further, tumors have been shown to develop resistance to PARPi. Herein, we explored the 1,2,3,4-tetrahydro-5H-aryl[1,4]diazepin-5-one scaffold as a potential PARP1/2-selective rucaparib, and niraparib; however, these PARPi demonstrate non-selectivity amongst the PARP inhibitor and its possibility for targeted pharmacology with other kinases.

Breast cancer is the most prevalent cancer in women in the UK with over 50,000 new cases diagnosed each year. Almost all breast cancer deaths are due to metastatic disease. The RUNX1-CBFbeta transcription factor complex has been implicated in the development of human breast cancer and recent evidence from our laboratory indicated that it might have a role in metastasis. The aim of this project was therefore to determine the role of the RUNX1 transcription factor in breast cancer metastasis. Initial experiments to knockdown RUNX1 by shRNA also decreased the expression of RUNX2. Therefore, due to the off-target effect of shRUNX1, CRISPR-Cas9n was used to establish a RUNX1-negative cell line by targeting the first exon of the RUNX1 gene. Migration and invasion capacity of the cells decreased in the absence of RUNX1 and it was comparable to the absence of RUNX2 and CBFbeta respectively, which are known to play roles in migration and invasion of MDA-MB-231 cells. The cells also formed spherical clusters in 3D culture which was associated with the changes in cell morphology from stellate to round shape in the absence of RUNX1. The expression of the metastasis-related genes MMP13, MMP9, OPN and SLUG also decreased in parallel with the loss of the mesenchymal-like phenotype whilst the expression of the epithelial markers cytokeratin, desmoplakin and E-cadherin increased concomitantly. Importantly, re-expression of RUNX1 in the RUNX1-negative cell lines using an inducible expression system rescued migration and invasion. Therefore, RUNX1 is required to maintain the mesenchymal-like phenotype of MDA-MB-231 cells and hence is important for the epithelial to mesenchyme transition (EMT), a key characteristic of metastatic cells. The transcription factor SLUG is a known regulator of EMT. Data obtained shows that RUNX1 down-regulates the expression of SLUG. ChIP analysis demonstrated that RUNX1 was bound to the SLUG promoter and RUNX1 was subsequently shown to activate the promoter activity. Finally, experiments to inhibit the activity of the RUNX transcription factors pharmacologically showed changes in cell differentiation and also affected cell viability, possibly by off-target effects. Taken together, data presented in this work demonstrates that RUNX1 is required for EMT in the metastatic breast cancer cells and it is therefore a potential therapeutic target to prevent breast cancer metastasis.

Bioimpedance presents a versatile, label-free means of monitoring biological cells and their responses to physical, chemical and biological stimuli. Breast cancer is the second most common type of cancer among women in the United States. Although significant progress has been made in diagnosis and treatment of this disease, there is a need for robust, easy-to-use technologies that can be used for the identification and discrimination of critical subtypes of breast cancer in biopsies obtained from patients. This dissertation makes contributions in three major areas towards addressing the goal. First, we developed miniaturized bioimpedance sensors using MEMS and microfluidics technology that have the requisite traits for clinical use including reliability, ease-of-use, low-cost and disposability. Here, we designed and fabricated two types of bioimpedance sensors. One was based on electric cell-substrate impedance sensing (ECIS) to monitor cell adhesion based events and the other was a microfluidic device with integrated microelectrodes to examine the biophysical properties of single cells. Second, we examined a panel of triple negative breast cancer (TNBC) cell lines and a hormone therapy resistant model of breast cancer in order to improve our understanding of the bioimpedance spectra of breast cancer subtypes. Third, we explored strategies to improve the sensitivity of the microelectrodes to bioimpedance measurements from breast cancer cells. We investigated nano-scale coatings on the surface of the electrode and geometrical variations in a branched electrode design to accomplish this. This work demonstrates the promise of bioimpedance technologies in monitoring diseased cells and their responses to pharmaceutical agents, and motivates further research in customization of this technique for use in personalized medicine. / Ph. D.

Le cancer du sein est une maladie complexe et difficile à caractériser. Parmi les différents sous-types moléculaires, les tumeurs du sein Triple-Négatives (TN) sont particulièrement agressives et de mauvais pronostic. Elles sont caractérisées par une absence d’expression des récepteurs aux œstrogènes (ER), à la progestérone (PR), l’absence de surexpression du récepteur Human Epidermal growth factor 2 (HER2) et de fréquentes mutations sur les gènes BRCA1/2 (profil « BRCAness »). En absence de thérapies ciblées efficaces, de nombreux traitements ciblés notamment les inhibiteurs de poly-ADP-ribose polymérases (anti-PARPs) sont actuellement en cours de développement, en recherche préclinique et clinique. Basés sur le principe de létalité synthétique, les anti-PARPs ciblent les propriétés BRCAness des tumeurs TN. Dans ce contexte, ces travaux de recherche ont été orientés sur le développement d’outils diagnostics afin d’optimiser l’efficacité des anti-PARPs sur des tumeurs TN. Pour ce faire, dans un premier temps, des cultures cellulaires en 3D via la technique Liquid Overlay ainsi que des tests de cytotoxicités associés ont été développés, à partir des lignées cellulaires MDA-MB-231 et SUM1315 de phénotype TN. Ces deux modèles de sphéroïdes ont ensuite été optimisés/normalisés dans un milieu de culture synthétique intitulé OPTIPASS (BIOPASS). Dans un deuxième temps, l’efficacité d’un co-traitement combinant l’anti-PARP1 Olaparib à faibles et à fortes doses et la radiothérapie fractionnée (5x2 Gy) a été modélisée sur les deux lignées MDA-MB-231 et SUM1315, en conditions 2D et 3D. Ces expériences ont clairement mis en évidence un effet potentialisateur de l’Olaparib sur la radiothérapie (i) en présence de faibles doses de cet anti-PARP (5 µM ou inférieur) (ii) à long terme et (iii) en présence d’un fractionnement maximum (5x2 Gy). De plus, les lignées tumorales TN étudiées présentaient des différences de sensibilité vis-à-vis du co-traitement. Ainsi, une analyse transcriptomique in silico a mis en évidence des profils très différents de ces lignées hautement métastatiques et très agressives. Notamment, la lignée SUM1315 semblait présenter un engagement neuronal, suggérant son origine métastatique cérébrale. Ces résultats encourageants pourraient ouvrir de nouvelles perspectives pour le traitement des métastases cérébrales de tumeurs mammaires TN, très fréquentes chez ce sous-type. Dans un troisième temps, afin de mieux caractériser le mode d’action de l’Olaparib sur ces modèles de sphéroïdes, un dérivé fluorescent de l’Olaparib, l’Ola-FL, a été synthétisé et caractérisé. L’analyse de la pénétration et de la distribution de l’Ola-FL au sein des sphéroïdes MDA-MB-231 et SUM1315 a mis en évidence une distribution rapide et homogène du composé ainsi que sa persistance après 3h d’incubation, dans toute la profondeur des sphéroïdes et notamment dans les zones hypoxiques centrales. Enfin, l’analyse de la co-expression de deux pompes Multidrug Resistance (MDR) majeures, la MRP7 et la P-gp après le traitement des deux lignées TN avec l’Olaparib, a mis en évidence sur les cultures 2D, une expression de type relai de la MRP7 et la P-gp. Sur les sphéroïdes traités avec une faible dose d’Olaparib à long terme, une expression basale de la MRP7 et une surexpression de la P-gp ont été détectées, au sein des cellules résiduelles périphériques des sphéroïdes. Ces résultats mettent clairement en évidence l’implication des pompes d’efflux dans les mécanismes de résistances à l’Olaparib, dans ces tumeurs agressives. L’ensemble des résultats issus de la modélisation de l’action de l’Olaparib sur des sphéroïdes MDA-MB-231 et SUM1315 laissent supposer sa plus grande efficacité à faible dose et à long-terme, notamment dans les zones hypoxiques des sphéroïdes, probablement aussi à l’origine de son effet potentialisateur avec la radiothérapie. / Breast cancer is a very complex and heterogeneous disease. Among the different molecular subtypes, Triple-Negative (TN) breast cancers are particularly aggressive and of poor prognosis. TN tumours are characterized by a lack of estrogen receptors expression (ER), progesterone receptors expression (PR), the absence of Human Epidermal growth factor receptor 2 overexpression (HER2) of the frequent mutations on BRCA1 / 2 genes ("BRCAness" phenotype). In the absence of effective targeted therapies, many targeted therapies including poly-ADP-ribose polymerase inhibitors (anti-PARPs) are currently under development in preclinical and clinical studies. Based on the synthetic lethality concept, the anti-PARPs specifically target the BRCAness properties of TN tumors. In this context, these works were focused on the development of diagnostic tools for the optimization of TN tumours treatment with anti-PARPs. For this, firstly, 3D cell cultures formed with the Liquid Overlay technique as well as associated cytotoxicity tests were developed, from the TN breast cancer cell lines MDA-MB-231 and SUM1315. These two spheroid models were then optimized and standardized in a synthetic culture medium called OPTIPASS (BIOPASS). Secondly, the efficacy of a co-treatment combining anti-PARP1 Olaparib at low and high doses and fractioned radiotherapy (5x2 Gy) was analyzed on the two cell lines MDA-MB-231 and SUM1315 cultured in 2D and 3D conditions. These experiments clearly demonstrated a potentiating effect of Olaparib on radiotherapy (i) in presence of low doses of this anti-PARP (5 μM or inferior) (ii) at long term and (iii) in presence of the maximum fractionation (5x2 Gy). In addition, these two TN cell lines showed a heterogeneous sensitivity to the co-treatment. Thus, an in silico transcriptomic analysis revealed very different profiles of these highly metastatic and highly aggressive cell lines. Notably, the SUM1315 cell line presented a neuronal commitment, suggesting its cerebral metastatic origin. These promising results could open up new perspectives for the treatment of TN tumours brain metastases, which are very common in this subtype. Thirdly, in order to better characterize the mode of action of Olaparib on these spheroid models, a fluorescent derivative of Olaparib, Ola-FL, was synthesized and characterized. The analysis of Ola-FL penetration and distribution in MDA-MB-231 and SUM1315 spheroids showed a rapid and homogeneous distribution of the compound as well as its persistence after 3h of incubation, in all the depth of the spheroids and especially in the central hypoxic zones. Finally, the analysis of the co-expression of two major Multidrug Resistance (MDR) pumps, MRP7 and P-gp after the treatment of the two TN lines with Olaparib, revealed on 2D cultures, a relay type expression of the MRP7 and the P-gp. On spheroids treated with a low dose of Olaparib art long term (10 days), a basal expression of MRP7 and an overexpression of P-gp were detected in the peripheral residual cells of the spheroids. These results clearly highlighted the involvement of these efflux pumps in Olaparib resistance mechanisms, in these aggressive tumors. All the results resulting from the modeling of the action of Olaparib on MDA-MB-231 and SUM1315 spheroids suggest its greater efficacy at low dose and at long-term, especially in the hypoxic zones of the spheroids. This parameter might be probably at the origin of its potentiating effect with radiotherapy.

<p>With 2.1 million new cases, breast cancer is the most common cancer in women. Triple negative breast cancer (TNBC), which is 15-20% of these breast cancer cases is clinically negative for expression of estrogen and progesterone receptors (ER/PR) and human epidermal growth factor receptor 2 (HER2) receptors<a>.</a> It is characterized by its unique molecular profile, aggressive behavior, distinct patterns of metastasis, and lack of targeted therapies. TNBCs utilize glycolysis for growth, proliferation, invasiveness, chemotherapeutic resistance and hence has poor therapeutic response. There is an urgent need for novel/alternate therapeutic strategies beyond current standard of treatment for this subset of high-risk patients. Electrical pulse-based chemotherapy, known as electrochemotherapy (ECT) could be a viable option for TNBC therapy. ECT involves the local application of precisely controlled electrical pulses to reversibly permeabilize the cell membrane for enhanced uptake. ECT can increase the cytotoxicity of the chemotherapeutics up-to 1000 times, facilitating a potent local cytotoxic effect. </p> <p>The high cost and severe side-effects of conventional chemotherapeutics motivate the application of effective natural compounds. Combining electrical pulses with natural compounds will enhance the treatment efficacy. This dissertation focuses on curcumin, the yellow pigment of natural herb turmeric, that has been used for over 5000 years for its excellent anticancer properties. Previous studies have demonstrated the effectiveness of curcumin for treating multiple cancers, including TNBC, with limited side effects. The potency of curcumin can be enhanced further by combining it with ECT to provide an attractive and cost-effective alternative for TNBC treatment. </p> <p>Towards this we studied the effect of ECT with curcumin on MDA-MB-231 cell line, a human adenocarcinoma epithelial TNBC cell line. We performed various assays, including cell viability, colony forming, cell cycle, apoptosis, H₂O₂ reactive oxygen species (ROS), immunoblotting, real time quantitative PCR (qPCR), and cellular metabolites detection to study the impact of ECT with curcumin on MDA-MB-231 cells. In addition, to better understand the underlying mechanisms, we used high throughput, label-free quantitative proteomics. While several studies have attempted to define the mechanism of action of curcumin on cancer cells, little is known on the action mechanism of the curcumin delivered with electrical pulses. This work unravels the molecular mechanism behind the enhanced effects observed under the ECT-based curcumin therapy in TNBC cells, employing a high-throughput, quantitative, label-free mass spectroscopy-based proteomics approach. The proteomics approach provides information on the thousands of cellular proteins involved in the cellular process, allowing a comprehensive understanding of the electro-curcumin-therapy mechanism. Similar studies were also performed for ECT with cisplatin to compare the efficacy of the electro-curcumin-therapy to the standard stand-alone cisplatin-based therapy.</p> <p>Our results revealed a switch in the metabolism from glycolysis to mitochondrial metabolic pathways. This metabolic switch caused an excessive production of H₂O₂ ROS to inflict apoptotic cell death in MDA-MB-231 cells, demonstrating the potency of this ECT based curcumin therapy. These results encourage further studies to extend the application of ECT for clinical practice.</p>