<p> Although castration-resistant prostate cancers no longer respond to anti-androgen therapies, the androgen receptor (AR) is still required to promote tumor survival. However, the signaling pathways downstream of AR that promote this survival are not well known. We recently identified an AR-dependent survival pathway whereby AR induction of integrin &alpha;6&beta;1 and adhesion to laminin activates NF-&kappa;B/RelA signaling and Bcl-xL. This pathway acts in parallel with the PI3K/Akt pathway in Pten-null tumor cells such that combined inhibition of both PI3K and integrin &alpha;6&beta;1 is required to effectively kill tumor cells adherent to laminin. However, PTEN-null castration-resistant tumors were not effectively killed by this combination. I discovered that BNIP3, a hypoxia-induced BH3-only, pro-mitophagic Bcl-2 family member, is induced by androgen in castration-resistant cells through integrin &alpha;6&beta;1 and HIF1&alpha;. Furthermore, castration-resistant cells adherent to laminin were much more efficient at inducing autophagy in response to androgen. Androgen blocked the ability of the PI3K inhibitor PX866 to kill castration-resistant tumors, but this was reversed by loss of BNIP3. Although BNIP3 was dispensable for androgen-induced autophagy, its mitophagy function was required for BNIP3 to promote resistance to PX866. Thus, enhanced hypoxia signaling in cooperation with AR/&alpha;6&beta;1/HIF1&alpha; signaling on laminin in castration-resistant cells drives the expression of BNIP3 and enhances autophagy, both of which contribute to PX866 resistance through induction of mitophagy.</p><p>

<p> Metastasis is the most lethal step in the progression of cancer, and the five-year survival rate for metastatic lung cancer patients is less than 5%. An essential step for metastasis is resistance to anoikis, a cell death program physiologically induced by detachment of cells from the extracellular matrix. Heat shock transcription factor 1 (HSF1) is the master regulator of heat shock proteins (HSP), and HSF1 and HSP promote cell survival and protein homeostasis during stress. In cancer, HSF1 dynamically controls a network of genes beyond HSP, is a mediator of malignant transformation, and promotes metastasis. HSF1 has been linked to anchorage-independent growth, but whether it exerts its effect by supporting anoikis resistance is largely unknown. Using NSCLC cells, we identified HSF1 as a novel supporter of anoikis resistance. Knockdown of HSF1 sensitizes NSCLC cells to anoikis, yet HSF1 expression or activation does not confer anoikis resistance to normal bronchial epithelial cells, suggesting parallel oncogenic pathways may be required to inhibit anoikis. Consistent with the ability of HSF1 to regulate HSP, HSF1 knockdown partially inhibited HSP72, HSP40, and HSP27. However, targeted inhibition of each HSP did not induce anoikis, suggesting the mechanism of HSF1 is unrelated to these HSP. Intriguingly, HSF1 activation markers were increased in response to cell detachment in H460 cells. Except for HSP60 in A549 cells, cell detachment did not induce HSP, further suggesting an alternative mechanism for HSF1. Interestingly, knockdown of HSP60 sensitized A549 cells to anoikis, despite HSF1 knockdown having no effect on HSP60. This work provides novel evidence that HSF1 and HSP60 can promote anchorage independence by supporting anoikis resistance, and may be valuable targets for future efforts to therapeutically suppress metastasis.</p><p>

<p> The majority of breast cancers express the estrogen receptor (ER+) and are treated with anti-estrogen therapies, particularly the inhibitor tamoxifen. However, many women treated with tamoxifen develop resistance, leading to metastatic disease, which is responsible for the majority of breast cancer deaths. Using small molecule inhibitors, phospho-mimetic proteins, tamoxifen sensitive and resistant breast cancer cells, a patient derived tamoxifen-resistant xenograft model, and genome-wide transcription and translation studies, we show that tamoxifen resistance is mediated by selective mRNA translational reprogramming. Tamoxifen resistant translation is mediated by increased expression of translation factor eIF4E, increased mTOR activity to promote eIF4E availability, and increased MNK activity to promote eIF4E Ser209 phosphorylation. Tamoxifen re-sensitization is restored only by reducing eIF4E expression or mTOR activity and blocking MNK1-directed eIF4E phosphorylation. Of the translationally upregulated mRNAs specific to tamoxifen resistant cells, we show that Runx2, which encodes a regulator of ER signaling that antagonizes estrogen responses and promotes breast cancer metastasis, significantly increases tamoxifen resistance and restores sensitivity when silenced. Moreover, tamoxifen resistant but not sensitive patient ER+ breast cancer specimens demonstrate strongly increased levels of mTOR and MNK activity and eIF4E protein. eIF4E levels, availability and phosphorylation therefore promote tamoxifen resistance in ER+ breast cancer through translatome reprogramming.</p><p>

<p> Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo <i>de novo</i> vessel formation.&nbsp;Quantitative assessment of vasculogenesis is a central readout of endothelial progenitor cell functionality. However, current assays lack kinetic measurements. To address this issue, new approaches were developed to quantitatively assess <i> in vitro</i> endothelial colony forming cell (ECFC) network formation in real time. Eight parameters of network structure were quantified using novel Kinetic Analysis of Vasculogenesis (KAV) software. KAV assessment of structure complexity identified two phases of network formation. This observation guided the development of additional vasculogenic readouts, including a tissue cytometry approach to quantify the frequency and localization of dividing ECFCs within cell networks. Additionally, FIJI TrackMate was used to quantify ECFC displacement and speed at the single cell level during network formation. These novel approaches were then applied to determine how intrauterine exposure to maternal type 2 diabetes mellitus (T2DM) impairs fetal ECFC vasculogenesis, and whether increased Transgelin 1 (TAGLN) expression in ECFCs from pregnancies complicated by gestational diabetes (GDM) was sufficient to impair vasculogenesis. Fetal ECFCs exposed to maternal T2DM formed fewer initial network structures, which were not stable over time. Correlation analyses identified that ECFC samples with greater division in branches formed fewer closed network structures and that reductions in ECFC movement decreased structural connectivity. To identify specific cellular mechanisms and signaling pathways altered in ECFCs following intrauterine GDM exposure, these new techniques were also applied in TAGLN expression studies. Similarly, ECFCs from GDM pregnancies and ECFCs overexpressing TAGLN exhibited impaired vasculogenesis and decreased migration. Both ECFCs from GDM pregnancies as well as ECFCs over-expressing TAGLN exhibited increased phosphorylation of myosin light chain. Reduction of myosin light chain phosphorylation via Rho kinase inhibition increased ECFC migration; therefore, increased TAGLN was sufficient to impair ECFC vasculogenic function. Overall, identification of these novel phenotypes provides evidence for the molecular mechanisms contributing to aberrant ECFC vasculogenesis. Determining how intrauterine exposure to maternal T2DM and GDM alters fetal ECFC function will enable greater understanding of the chronic vascular pathologies observed in children from pregnancies complicated by diabetes mellitus.</p><p>

<p> Enhancers are regulatory elements that orchestrate cell type specific gene expression patterns. They can be separated from their target genes by large distances and activate transcription by coming into physical proximity with promoters in three-dimensional nuclear space. Complex regulatory networks with multiple enhancers often cooperate to control the same target gene. Antigen receptor loci have proved to be a rich ground for understanding enhancer-mediated gene regulation. The loci undergo somatic recombination of their V, (D), J segments to create a diverse repertoire of antigen receptors that can counteract a wide range of foreign antigens. V(D)J recombination relies on the presence of proximal enhancers that activate the antigen receptor loci in a lineage and cell stage specific manner. Unexpectedly we find that both active and inactive antigen receptor loci enhancers cooperate to disseminate their effects in a localized and long-range mode. We demonstrate the importance of short-range contacts between active enhancers that constitute an <i>Igk</i> super-enhancer in B cells. Deletion of one element reduces the interaction frequency between other enhancers in the hub, which compromises the transcriptional output of each enhancer. We further establish that in T cells, the <i> Igk</i> enhancer MiE&kappa; exerts a long-range effect on another antigen receptor locus <i>Tcrb</i> located 29MB away on chromosome 6. MiE&kappa; deletion leads to inefficient <i>Tcrb</i> recombination resulting in a block in T cell development, an effect that is associated with a long-range contact and cooperation between the MiE&kappa; and the <i> Tcrb</i> enhancer, E&beta;. MiE&kappa; deletion alters enrichment of the transcription factor CBF&beta; on E&beta; in a manner that impacts <i> Tcrb</i> recombination. These findings underline the complexities of enhancer regulation and point to a role for localized and long-range enhancer sharing between active and inactive elements in lineage and stage specific control. Finally, we expand our assessment of enhancer cooperation to the entire chromosome 6. We detect nearly nine hundred putative regulatory elements that are active in either DP or pre-B cells with less than 20% common to the two cell types. We also demonstrate how long-range contacts between enhancers and promoters coincide with target gene expression status, and provide a resource for identifying the regulatory elements that control T and B cell specific gene expression patterns.</p><p>

<p> Autophagy is a cellular recycling process in which cytoplasmic proteins and organelles are sequestered in a double membrane vesicle, delivered to the lysosome, and degraded following fusion of the two vesicles. A key part of the initiation signaling for autophagy is the generation of phosphoinositol 3-phosphate (P13P) by class III phosphoinositol 3-kinase also knows as Vps 34. In humans there are eight P13K isoforms divided into three classes, four class I enzymes, three class II enzymes, and a single class III enzyme. Of these eight enzymes, only the class III isoform is thought to participate directly in autophagic signaling. A quantitative microscopy based, loss-of-function survey of all eight P13K isoforms was used to determine their relative contribution to autophagic signaling, as measured by LC3 positive autophagic vesicles. As predicted, knockdown of P13K-class III reduced the number of autophagic vesicles in cells. Interestingly, knockdown of the P13K-class II&alpha; isoform had an even more potent effect on reducing the number of autophagic vesicles than knockdown of P13K-class III. In follow up studies, knockdown of P13K-class II&alpha; reduced endogenous LC3 conversion, caused the accumulation of p62 and lipid droplets, and colocalized with endosomal markers. These results suggest P13K-class II&alpha; may act to promote autophagy through the shuttling of endosomal vesicles into the autophagic pathway and approaches to test this hypothesis will be discussed. The requirement of P13K-class II&alpha; for autophagy is an important finding as it indicates a role for class II P13Ks in autophagy.</p>

<p> Chemoresistance is a highly significant problem affecting a diverse array of cancers at all clinical stages. In an attempt to identify molecular mechanisms leading to chemoresistance, we performed a RNAi screen against all known and putative kinases and phosphatases in the human genome. The knockdown of one of these genes, MK-STYX, resulted in potent chemoresistance in response to a diverse array of chemotherapeutic agents. As many of these drugs function through the induction of the apoptotic program, we hypothesized that the RNAi-mediated knockdown of MK-STYX blocks the cellular response to chemotherapeutic-induced apoptosis. </p><p> To investigate this hypothesis, we determined the ability of both control and MK-STYX knockdown cells to undergo apoptosis after exposure to an array of cell death inducing agents with different mechanisms of action. The results of these experiments demonstrated that MK-STYX knockdown protects against intrinsic, but not extrinsic apoptotic stimuli. These data were recapitulated with knockdown of the pro-apoptotic genes caspase-9 and Bax/Bak, suggesting that MK-STYX may modulate the regulation of one of these key apoptotic regulatory nodes. We demonstrated that the loss of MK-STYX blocks cytochrome c release, placing the apoptotic deficiency at the level of Bax/Bak-mediated mitochondrial outer membrane permeabilization, or MOMP. MK-STYX was found to localize to the mitochondria, but is neither released from the mitochondria upon apoptotic stress nor localized proximal to the machinery currently known to control MOMP. These results are summarized in Chapter 2. </p><p> In an effort to more fully define molecular mechanism of MK-STYX, we performed an unbiased TAP-tagging experiment to identify its interaction partners. The most significant and unique protein identified was the mitochondrial phosphatase PTPMT1. Interestingly, MK-STYX is a catalytically inactive dual specificity phosphatase, and catalytically inactive phosphatases have a precedent for regulating the activity and/or localization of active phosphatases. Because of this potential phosphatase regulatory mechanism, as well as similar localization patterns of both genes, we chose to further explore the interaction between PTPMT1 and MK-STYX. </p><p> Due to the robust survival phenotype seen in MK-STYX knockdown cells when treated with chemotherapeutic, we predicted that the knockdown of PTPMT1 may have a similar phenotype. Surprisingly, we found that PTPMT1 knockdown causes a Bax/Bak dependent cell death, suggesting that MK-STYX and PTPMT1 may functionally oppose one another in the mitochondria. Experiments in which both enzymes are downregulated show that PTPMT1 is epistatic to MK-STYX, as cells are resensitized to chemotherapeutic agents and cytochrome c release under these conditions. Interestingly, PTPMT1 was recently shown to be an important enzyme in the cardiolipin biosynthetic pathway, positively regulating the synthesis of this mitochondrial lipid. The genetic interaction provided by the robust changes in viability seen when these enzymes are downregulated suggests that MK-STYX may function to dampen PTPMT1 enzymatic activity. This allows us to hypothesize that the loss of MK-STYX results in increased cardiolipin biosynthesis, leading to altered mitochondrial membrane composition and subsequently, an altered apoptotic response. These results are summarized in Chapters 3 and 4. </p><p> We further hypothesize that the upregulation of cardiolipin levels directly inhibits the ability of Bax/Bak to permeabilize the outer mitochondrial membrane, effectively blocking the induction of mitochondrial apoptosis. These data suggest a novel mechanism by which dysregulated cardiolipin can facilitate chemoresistance, and suggest that this pathway could be exploited by recurrent cancers to evade therapies.</p><p>

<p> The mitochondrion is a complex, double membrane organelle that serves several important cellular functions including ATP synthesis, Ca ²⁺ buffering, and ROS homeostasis. Although classic mitochondrial diagrams depict the mitochondrion as a simple oval or &ldquo;bean&rdquo; shaped organelle, the mitochondria can form extensive tubular networks or numerous small spheres in response to various cellular environments through two opposing processes, mitochondrial fission and fusion. Deregulation of mitochondrial dynamics has been implicated in a wide range of diseases, including Parkinson&rsquo;s disease, heart disease and cancer. While significant emphasis for the last 15 years has been placed on the identification of the protein machinery responsible regulating mitochondrial morphology, it remains less clear how mitochondrial morphology affects various cellular functions and cellular fate outcomes. This thesis summarizes our findings on how mitochondrial morphology regulates cellular fate in the context of mitotic cell division and apoptosis. Using live cell microscopy and image analysis software we characterized mitochondrial dynamics with single cell resolution. We found that loss of key components of the mitochondrial fission machinery promotes a defect in cell cycle progression, characterized by an inability for cells to exit G2/M. Prolonged periods of mitochondrial fusion induced potent cell death, suggesting a novel mechanism to target the replicative potential of cancer cells. We also found that mitochondrial fission and fusion can alter the kinetics of cell death following apoptotic stimuli by inducing mitochondrial fusion prior to the commitment step in apoptosis, mitochondrial membrane permeabilization. This thesis summarizes our work in trying to elucidate how the structure of the mitochondria influences both mitochondrial and cellular fate.</p><p>

<p> Osteosarcoma (OS) is the most common type of solid bone cancer, mainly arising in children and young adults, and remains the second leading cause of cancer-related death in this age group. Chemotherapy resistance underlying latent recurrence and metastasis represent major contributors to poor outcome for many cancer patients especially those with OS. Tumor hypoxia is an essential element intrinsic to most solid tumor microenvironments and is associated with resistance to therapy and a malignant phenotype, while metastatic dissemination is dependent on a cells ability to resist anoikis, i.e., programmed cell death in the absence of attachment to an extracellular matrix. We sought to better characterize hypoxia and anoikis resistance in human OS using established and novel patient-derived OS cells and OS animal models with the long-term goal of identifying and validating targetable signaling pathways. We show that hypoxia-inducible factors (HIFs), canonical proteins associated with the hypoxic response, are present and can be induced in human OS cells. We demonstrate that the Wnt/&beta;-catenin signaling pathway, a key pathway in OS pathogenesis, is down-regulated in response to hypoxia in OS cells, and that this appears to result from both HIF-dependent and HIF-independent mechanisms. Hypoxia promotes resistance of human OS cells to standard chemotherapy, which is mitigated by treatment with Wnt/&beta;-catenin signaling inhibitors. Using an anchorage-independent growth model, we show that anoikis-resistant OS subpopulations have altered growth rates, increased resistance to standard chemotherapies, and display distinct changes in gene expression and DNA methylation. Finally, we validate the use of two FDA-approved epigenetic therapies predicted by expression profiling in both inhibiting anchorage-independent growth and sensitizing anoikis-resistant OS cells to chemotherapy. In summary, despite the heterogeneity of human OS, our work suggests that unique and effectively targetable signaling pathways underlie the phenotypic consequences in response to hypoxia and anoikis resistance.</p><p>

The factors controlling desensitization to ATP stimulation were investigated in PC12 cells. Reducing the concentration of ATP to produce half maximal response reduced the degree to which cells desensitize to ATP. Increasing external Me concentration, which produced a comparable decrease in the secretory response of the cells, had no effect on the degree of desensitization. Neither did decreasing external Ca2+ concentration, which produced a similar decrease in secretory response. Desensitizing cells in 0.5 mM Ca2+ did not result in a corresponding decrease in response when cells were subsequently tested in 2.2 mM Ca2+. PC12 cells desensitized in 2.2 mM Ca2+ were found to show the same degree of desensitization when tested in 0.5 mM Ca2+. A similar pattern was found when desensitization to 30 and 300 uM ATP was examined. The role of the ATP receptor subtypes, P2x and P2y, was studied using the ATP agonists 2-MeS ATP and UTP, respectively. 60 uM 2-MeS ATP was found to cause desensitization to the same degree as 30 uM ATP. As with ATP, the initial response to 2-MeS ATP was found to be sensitive to changes in external Mg2+. Unlike ATP, the desensitization to 2-MeS ATP was sensitive to changes in external Mg2+. When cells were co-stimulated with 2-MeS ATP and UTP, the sensitivity of 2-MeS ATP desensitization to Mg2+ remained. UTP in the background solution, however, increased desensitization to ATP and 2-MeS ATP. The effect of voltage-operated Ca2+ channel (VOCC) blockers on the response and desensitization to ATP and 2-MeS ATP was examined. Cd2+ produced a significant increase in the secretory response to both stimulants. Both nicardipine and Cd2+ increased the rate of desensitization to ATP. Only nicardipine was able to increase the rate of desensitization to 2-MeS ATP. These results were integrated to provide insights into the relationship between ATP receptors and VOCCs.