Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Anna Zolkiewska / Mitogen-activated protein kinase (MAPK) signaling plays an important role in the proliferation, survival, and therapy resistance of breast cancer cells. Two important protein kinases involved in the MAPK pathway are MEK and ERK. The MEK/ERK signaling cascade can be stimulated by activation of the epidermal growth factor receptor (EGFR) upon binding of EGF-like ligands, which are released from cells by ADAM proteases. EGFR is frequently overexpressed in triple-negative breast cancer (TNBC), a particularly aggressive form of breast cancer. Our analysis of clinical data revealed that high expression of ADAM12, but not other ADAMs, in TNBC is associated with poor patient survival. Thus, we hypothesized that ADAM12 plays a critical role in the progression of TNBC, possibly by stimulating MEK/ERK activity in an EGFR-dependent manner. To test this hypothesis, ADAM12 was knocked-down (KD) in SUM159PT TNBC cells, which express high levels of the endogenous ADAM12 protein. An antibody array assay indicated a significant decrease in the activation of the MAPK pathway in SUM159PT cells after ADAM12 KD. The decrease in MAPK activity was further confirmed by Western blotting using phospho-MEK and phospho-ERK specific antibodies. Additionally, conditioned media from ADAM12-deficient SUM159PT cells failed to support the survival of MCF10A cells, suggesting that ADAM12 KD reduced the release of pro-survival growth factors from SUM159PT cells. Based upon this data, we propose that ADAM12 is a novel regulator of the MAPK pathway and a potential therapeutic target in breast cancer.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Anna Zolkiewska / ADAM12 (A Disintegrin and Metalloprotease 12) is a cell surface protease, which is deregulated in many human diseases. High expression of ADAM12 in triple-negative breast cancers (lacking estrogen receptor, progesterone receptor, and HER2 expression) is associated with poor patient prognosis. My dissertation focused on the understanding of the biological functions of ADAM12 in triple-negative breast cancers. I found that ADAM12 is significantly upregulated in the claudin-low molecular subtype of breast cancer. Claudin-low tumors are typically triple-negative and are enriched in cancer stem cells. Here, I demonstrated that the loss of ADAM12 expression not only decreased the number of cancer stem-like cells in vitro but also significantly compromised the tumor-initiating capabilities of breast cancer cells in vivo. This is the first evidence showing that ADAM12 might regulate the cancer stem cell-like phenotype in triple-negative breast cancers. I also discovered a novel mechanism of ADAM12-regulated signaling by transforming growth factor β (TGFβ) through modulation of TGFBR1 mRNA expression in breast cancer cells. Lastly, I characterized the effects of six different somatic mutations in the ADAM12 gene found in human breast cancers on the intracellular trafficking, post-translational processing, and function of ADAM12 protein. Collectively, the findings of this study support the notion that ADAM12 with catalytically active metalloprotease domain is required for the progression of triple-negative breast cancers.

Les récepteurs métabotropes du glutamate (mGluR) sont des récepteurs couplés aux protéines G qui régulent la transmission synaptique. Ce sont des cibles de choix pour le traitement de maladies neurologiques et psychiatriques telles que la maladie de Parkinson et la schizophrénie.J'ai développé une stratégie d'étude de l'assemblage multimérique des protéines membranaires dans des cellules vivantes, à l'aide de techniques de marquage orthogonal et de FRET en temps-résolu. De façon inattendue, j'ai montré que certaines sous-unités de mGluR, en plus de former des récepteurs homodimériques, peuvent former des récepteurs hétérodimériques fonctionnels. D'autre part, j'ai appliqué ces techniques à l'étude du mécanisme d'activation des mGluR et de leur régulation allostérique. J'ai démontré qu'un mouvement relatif des domaines extracellulaires au sein du dimère était responsable de l'action du glutamate.Ce travail a permis de mieux comprendre le fonctionnement des mGluR, et permet la conception de nouveaux tests de criblage. / Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors that regulate synaptic transmission. They are relevant therapeutic targets for neurological and psychiatric disorders, such as Parkinson disease and schizophrenia.I developed a strategy to study the multimeric assembly of membrane proteins in living cells, through a combination of orthogonal labeling and time-resolved FRET. Unexpectedly, some subunits of mGluRs, in addition to forming homodimeric receptors, were found capable of forming functional heterodimeric receptors. Then, I applied these techniques to study the activation mechanism of mGluRs and their allosteric regulation. I demonstrated that a conformational change of the dimeric extracellular domain is responsible for the action of glutamate.In addition to increase our understandings of how mGluRs function, this work opens new avenues for the design of drug screening tests.

Intravenous administration of ovokinin(2-7), a cleavage peptide derived from ovalbumin, dose-dependently (0.1-5 mg/kg) lowered the mean arterial pressure (MAP) that was not accompanied by a significant change in the heart rate (HR) of urethane-anesthetized rats. The hypotensive effects of ovokinin(2-7) were five orders of magnitude lower compared to that of bradykinin and were largely prevented by pretreatment with the bradykinin B2 receptor antagonist HOE140 (81.6±18.4%) and moderately affected by the B1 receptor antagonist [des-Arg10]-HOE140 (26.3±15.5%). Intracellular Ca2+ levels, as measured by Fur 2-AM, were significantly elevated in cultured aorta smooth muscle cells by ovokinin(2-7). The increases were abolished by HOE140 and unaffected by [des-Arg10]-HOE140. The elevation of intracellular Ca2+ by ovokinin(2-7) was dependent on Ca2+ entry from extracellular space as it was reduced in a Ca2+-free solution. Pretreatment of the cells with the phospholipase C inhibitor U73122 (2 μM) eliminated the Ca 2+ increase by the peptide. PA phosphohydrolase and phospholipase A2 inhibitors significantly reduced the responses as well. Our results show that ovokinin(2-7) modulates cardiovascular activity by interacting with B2 bradykinin receptors.

<p> This thesis focuses on fundamental and applied aspects of sol-gel based affinity assays for screening membrane receptor targets. Fundamental studies investigate the role of non-specific interactions between polycationic polymers and sol-gel derived monoliths prepared from sodium silicate precursors. Previous studies from our group using time-resolved fluorescence anisotropy (TRFA) have shown that both the sidechain motion (Φ1) and backbone motion (Φ2) of polycationic polymers, such as poly-D-lysine, can be measured by fitting anisotropy decays of fluorescein labeled polymers to a two component hindered rotor model. These studies demonstrated that polycationic polymers remain fairly mobile in sol-gel derived materials made from sodium silicate precursors, despite the strong electrostatic interactions between the cationic polymers and the anionic silica materials. The first objective of my work was to assess the nature of the electrostatic interactions between several polycationic polymers and a silica material made from sodium silicate using a novel two-point labeling technique with a pyranine dye to determine if previous studies were indeed correct. Our results show that the two-point labeling technique with pyranine provides a more rigid interaction between the polymer and the dye compared to the previous labeling method using fluorescein, allowing for more accurate monitoring of dynamic motions of cationic polymers in sol-gel derived materials. The dynamics of poly-D-lysine entrapped in sol-gel derived materials was indeed seen to be highly restricted, contrary to results obtained in previous studies.</p> <p> While the first project provided a framework for understanding the effect of electrostatic interactions on the dynamics of biomolecules, it also provide valuable insight into the effect of non-specific interactions between cationic species and sol-gel derived materials in general. These considerations are important when using sol-gel based affinity columns for small molecule screening. The second study outlines a competitive affinity-based screening method using mass spectrometry (MS) detection that can help reduce the effect of non-specific interactions between test compounds and the column matrix in small molecule screening applications. This technique relies on the use of a high-affinity indicator compound pre-equilibrated on column to identify weak affinity ligands in mixtures through transient spikes in the indicator signal that result from the competition between the indicator and test compounds. The results of this study demonstrate the ability to identify weak affinity ligands for the nicotinic acetylcholine receptor (nAChR) using low to sub-picomole amount of active receptor on column. The technique results in a reproducible signal output that can potentially be used to obtain quantitative data on the binding affinities of target-ligand interactions. The assay is amenable to automation and can be performed at high speeds, thereby demonstrating the potential of this technique as a high-throughput screening tool for screening membrane receptors.</p> / Thesis / Master of Science (MSc)

Internal fertilization usually implies that a spermatozoon, with intact attributes for zygote formation, passes all hurdles during its transport through the female genitalia and reaches the oocyte. During this journey, millions to billions of other spermatozoa perish. Spermatozoa are highly differentiated motile cells without synthetic capabilities. They generate energy via glycolysis and oxidative phosphorylation to sustain motility and to maintain the stability and functionality of their plasma membrane. In vivo, they spend their short lifespan bathing in female genital tract fluids of different origins, or are in vitro exposed to defined media during diverse sperm handling i.e. extension, cryopreservation, in vitro fertilization, etc. Being excitable cells, spermatozoa respond in vivo to various stimuli during pre-fertilization (capacitation, hyperactivation, oocyte location) and fertilization (acrosome reaction, interaction with the oocyte) events, mediated via diverse membrane ion-conducting channels and ligand-gated receptors. The present Thesis has mapped the presence and reactivity (sperm intactness and kinematics) of selected receptors, water and ion channels in ejaculated boar spermatozoa. The final aim was to find a relevant alternative cell type for in vitro bioassays that could ease the early scrutiny of candidate drugs as well as decreasing our needs for experimental animals according to the 3R principles. Spermatozoa are often extended, cooled and thawed to warrant their availability as fertile gametes for breeding or in vitro testing. Such manipulations stress the cells via osmotic variations and hence spermatozoa need to maintain membrane intactness by controlling the exchange of water and the common cryoprotectant glycerol, via aquaporins (AQPs). Both AQPs-7 and -9 were studied for membrane domain changes in cauda- and ejaculated spermatozoa (un-processed, extended, chilled or frozen-thawed). While AQP-9 maintained location through source and handling, thawing of ejaculated spermatozoa clearly relocated the labelling of AQP-7, thus appearing as a relevant marker for non-empirical studies of sperm cryopreservation. Alongside water, spermatozoa interact with calcium (Ca2+) via the main Ca2+ sperm channel CatSper. Increments in intracellular Ca2+ initiate motility hyperactivation and the acrosome reaction. The four subunits of the CatSper channel were present in boar spermatozoa, mediating changes in sperm motility under in vitro capacitation-inducing conditions (increased extracellular Ca2+ availability and bicarbonate) or challenge by the CatSper antagonists mibefradil and NNC 55-0396. Uterine and oviduct fluids are richest in endogenous opioids as β-endorphins during mating and ovulation. Both μ- and δ- opioid receptors were present in boar spermatozoa modulating sperm motility, as in vitro challenge with known agonists (μ: morphine; δ: DPDPE and κ: U 50488) and antagonists (μ: naloxone; δ: naltrindole and κ: nor-binaltrorphimine) showed that the μ-opioid receptor maintained or increased motility while the δ-opioid receptor mediated decreased motility over time. Finally, boar spermatozoa depicted dose-response effects on sperm kinematics and mitochondrial potential following in vitro challenge with 130 pharmacological drugs and toxic compounds as well as with eight known mito-toxic compounds. In conclusion, boar spermatozoa expressing functional water (AQPs-7 and -9) and ion (CatSper 1-4) channels as well as μ- and δ-opioid receptors are able to adapt to stressful environmental variations, capacitation and pharmacological compounds and drug components. Ejaculated sperm suspensions are easily and painlessly obtained from breeding boars, and are suitable biosensors for in vitro drug-induced testing, complying with the 3R principles of reduction and replacement of experimental animals, during early toxicology screening.