LOW REACTIVE OXYGEN SPECIES AND HIGH GLYCOLYSIS IN GLIOBLASTOMA STEM CELLS:MECHANISMS AND THERAPEUTIC IMPLICATIONS

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor with poor prognosis due in part to drug resistance and high incidence of tumor recurrence. The drug resistant and cancer recurrence phenotype may be ascribed to the presence of glioblastoma stem cells (GSCs), which seem to reside in special stem-cell niches in vivo and require special culture conditions including certain growth factors and serum-free medium to maintain their stemness in vitro. Exposure of GSCs to fetal bovine serum (FBS) can cause their differentiation, the underlying mechanism of which remains unknown. Reactive oxygen species (ROS) play an important role in normal stem cell differentiation, but their role in affecting cancer stem cell fate remains unclear. Whether the metabolic characteristics of GSCs are different from other glioblastoma cells and can be targeted are also unknown. In this study, we used several stem-like glioblastoma cell lines derived from clinical tissues by typical neurosphere culture system or orthotopic xenografts, and showed that addition of fetal bovine serum to the medium induced an increase of ROS, leading to aberrant differentiation and decreases of stem cell markers such as CD133. We found that exposure of GSCs to serum induced their differentiation through activation of mitochondrial respiration, leading to an increase in superoxide (O2-) generation and a profound ROS stress response manifested by upregulation of oxidative stress response pathway. This increase in mitochondrial ROS led to a down-regulation of molecules including SOX2, and Olig2, and Notch1 that are important for stem cell function and an upregulation of mitochondrial superoxide dismutase SOD2 that converts O2- to H2O2. Neutralization of ROS by antioxidant N-acetyl-cysteine in the serum-treated GSCs suppressed the increase of superoxide and partially rescued the expression of SOX2, Olig2, and Notch1, and prevented the serum-induced differentiation phenotype. Additionally, GSCs showed high dependence on glycolysis for energy production. The combination of a glycolytic inhibitor 3-BrOP and a chemotherapeutic agent BCNU depleted cellular ATP and inhibited the repair of BCNU-induced DNA damage, achieving strikingly synergistic killing effects in drug resistant GSCs. This study uncovers the metabolic properties of glioblastoma stem cells and suggests that mitochondrial function and cellular redox status may profoundly affect the fates of glioblastoma stem cells via a ROS-mediated mechanism, and that the active glycolytic metabolism in cancer stem cells may provide a biochemical basis for developing novel therapeutic strategies to effectively eliminate GSCs.

ROLE OF MIR-19A RELEASED BY INFLAMMATORY BREAST CANCER CELLS IN THE REGULATION OF DENDRITIC CELL FUNCTIONS: IN VITRO MODEL OF CROSSTALK IN THE TUMOR MICROENVIRONMENT OF INFLAMMATORY BREAST CANCER

Inflammatory breast cancer (IBC) is a rare but very aggressive form of locally advanced breast cancer (1-6% of total breast cancer patients in United States), with a 5-year overall survival rate of only 40.5%, compared with 85% of the non-IBC patients. So far, a unique molecular signature for IBC able to explain the dramatic differences in the tumor biology between IBC and non-IBC has not been identified. As immune cells in the tumor microenvironment plays an important role in regulating tumor progression, we hypothesized that tumor-associated dendritic cells (TADC) may be responsible for regulating the development of the aggressive characteristics of IBC. MiRNAs can be released into the extracellular space and mediate the intercellular communication by regulating target gene expression beyond their cells of origin. We hypothesized that miRNAs released by IBC cells can induce an increased activation status, secretion of pro-inflammatory cytokines and migration ability of TADC. In an in vitro model of IBC tumor microenvironment, we found that the co-cultured of the IBC cell line SUM-149 with immature dendritic cells (iDCSUM-149) induced a higher degree of activation and maturation of iDCSUM-149 upon stimulation with lipopolysaccharide (LPS) compared with iDCs co-cultured with the non-IBC cell line SUM-159 (iDCSUM-159), resulting in: increased expression of the costimulatory and activation markers; higher production of pro-inflammatory cytokines (TNF-a, IL-6); and 3) higher migratory ability. These differences were due to the exosome-mediated transfer of miR-19a and miR-146a from SUM-149 and SUM-159, respectively, to iDCs, causing the downregulation of the miR-19a target genes PTEN, SOCS-1 and the miR-146a target genes IRAK1, TRAF6. PTEN, SOCS-1 and IRAK1, TRAF6 are important negative and positive regulator of cytokine- and TLR-mediated activation/maturation signaling pathway in DCs. Increased levels of IL-6 induced the upregulation of miR-19a synthesis in SUM-149 cells that was associated with the induction of CD44+CD24-ALDH1+ cancer stem cells (CSCs) with epithelial-to-mesenchymal transition (EMT) characteristics. In conclusion, in IBC tumor microenvironment IL-6/miR-19a axis can represent a self-sustaining loop able to maintain a pro-inflammatory status of DCs, leading to the development of tumor cells with high metastatic potential (EMT CSCs) responsible of the poor prognosis in IBC patients.

Cell Cycle Regulatory Roles of Estrogen Receptor alpha (ERα) in Breast Cancer Cells

Previous studies have shown that Estrogen Receptor alpha (ERα) is an important indicator for diagnosis, prognosis and treatment of breast cancers. However, the question remains as to the role of ERα in the cell in the presence versus absence of 17-β estradiol In this dissertation the role of ERα in both its unliganded and liganded state, with respect to the cell cycle will be explored. The cell line models used in this project are ER-positive MCF-7 cells with and without siRNA to ERα and ER-positive MDA-MB-231 cells that have been engineered to express ERα. Cells were synchronized and the cell cycle progression was monitored by flow cytometric analysis. Using these methods, two specific questions were addressed: Does ERα modulate the cell cycle differently under liganded versus unliganded conditions? And, does the presence of ERα regulate cell cycle phase transitions? The results show for the first time that ERα is cell cycle regulated and modulates the progression of cells through S and G2/M phases of the cell cycle. Ligand bound ERα increases progression through S and G2/M phases, whereas unliganded ERα acts as an inhibitor of cell cycle progression. To further investigate the cell cycle regulated effects of liganded ERα, a luciferase assay was performed and showed that the transcription of target genes such as Progestrone Receptor (PgR) and Trefoil protein (pS2) increased duing S and G2/M phases when ERα is bound to ligand. Additionally, complex formation between cyclin B and ER α was shown by immunoprecipitation and led to the discovery that anaphase promoting complex (APC) is the E3 ligase for both cyclin B and ERα at the termination of M phase. Our findings suggest that unliganded ERα has an inhibitory effect on the progression of the cell cycle. Therefore, it is reasonable to speculate that the combination of drugs that lower estrogen level (such as aromatase inhibitors) and preserves ERα from degradation would provide better outcome for breast cancer treatment. We have shown that APC functions as the E3 ligase for ERα and thus might provide a target to design a specific inhibitor of ERα degradation.

DOWNREGULATION OF PAX2 SUPPRESSES OVARIAN CANCER CELL GROWTH

PAX2 is one of nine PAX genes regulating tissue development and cellular differentiation in embryos. PAX2 promotes cell proliferation, oncogenic transformation, cell-lineage specification, migration, and survival. Unattenuated PAX2 has been found in several cancer types. We therefore sought to elucidate the role of PAX2 in ovarian carcinomas. We found that PAX2 was expressed in low-grade serous, clear cell, endometrioid and mucinous cell ovarian carcinomas, which are relatively chemoresistant compared to high grade serous ovarian carcinomas. Four ovarian cancer cell lines, RMUGL (mucinous), TOV21G (clear cell), MDAH-2774 (endometrioid) and IGROV1 (endometrioid), which express high-levels of PAX2, were used to study the function of PAX2. Lentiviral shRNAs targeting PAX2 were used to knock down PAX2 expression in these cell lines. Cellular proliferation and motility assays subsequently showed that PAX2 stable knockdown had slower growth and migration rates. Microarray gene expression profile analysis further identified genes that were affected by PAX2 including the tumor suppressor gene G0S2. Reverse phase protein array (RPPA) data showed that PAX2 knockdown affected several genes that are involved in apoptosis, which supports the fact that downregulation of PAX2 in PAX2-expressing ovarian cancer cells inhibits cell growth. We hypothesize that this growth inhibition is due to upregulation of the tumor suppressor gene G0S2 via induction of apoptosis. PAX2 represents a potential therapeutic target for chemoresistant PAX2-expressing ovarian carcinomas.

The role of functional tumor suppressor 15-lipoxygenase 2 (15-LOX2) in normal human prostate epithelial cells and prostate cancer development

15-Lipoxygenase 2 (15-LOX2) is a recently cloned human lipoxygenase that shows tissue-restricted expression in prostate, lung, skin, and cornea. The protein level and enzymatic activity of 15-LOX2 have been shown to be down-regulated in prostate cancers compared with normal and benign prostate tissues. We report the cloning and functional characterization of 15-LOX2 and its three splice variants (termed 15-LOX2sv-a, 15-LOX2sv-b, and 15-LOX2sv-c) from primary prostate epithelial (NHP) cells. Western blotting with multiple NHP cell strains and prostate cancer (PCa) cell lines reveals that the expression of 15-LOX2 is lost in all PCa cell lines, accompanied by decreased enzymatic activity. 15-LOX2 is expressed at multiple subcellular locations, including cytoplasm, cytoskeleton, cell-cell border, and nucleus. Surprisingly, the three splice variants of 15-LOX2 are mostly excluded from the nucleus. To elucidate the relationship between nuclear localization, enzymatic activity, and tumor suppressive functions, we established PCa cell clones stably expressing 15-LOX2 or 15-LOX2sv-b. The 15-LOX2 clones express 15-LOX2 in the nuclei and possess robust enzymatic activity, whereas 15-LOX2sv-b clones show neither nuclear protein localization nor arachidonic acid-metabolizing activity. Interestingly, both 15-LOX2- and 15-LOX2sv-b-stable clones proliferate much slower in vitro when compared with control clones. When orthotopically implanted in nude mouse prostate, both 15-LOX2 and 15-LOX2sv-b suppress PC3 tumor growth in vivo. Finally, cultured NHP cells lose the expression of putative stem/progenitor cell markers, slow down in proliferation, and enter senescence. Several pieces of evidence implicate 15-LOX2 plays a role in replicative senescence of NHP cells: (1) promoter activity and the mRNA and protein levels of 15-LOX2 and its splice variants are upregulated in serially passaged NHP cells, which precede replicative senescence and occur in a cell-autonomous manner; (2) PCa cells stably expressing 15-LOX2 or 15-LOX2sv-b show a passage-related senescence-like phenotype; (3) enforced expression of 15-LOX2 or 15-LOX2sv-b in young NHP cells induce partial cell-cycle arrest and senescence-like phenotypes. Together, these results suggest that 15-LOX2 suppress prostate tumor development and do not necessarily depend on arachidonic acid-metabolizing activity and nuclear localization. Also, 15-LOX2 may serve as an endogenous prostate senescence gene and its tumor-suppressing functions might be associated with its ability to induce cell senescence. ^

ROLE OF STAT3 IN KERATINOCYTE STEM CELLS DURING SKIN TUMORIGENESIS

STATs play crucial roles in a wide variety of biological functions, including development, proliferation, differentiation, migration and in cancer development. In the present study, we examined the impact of Stat3 deletion or activation on behavior of keratinocytes, including keratinocyte stem cells (KSCs). Deletion of Stat3 specifically in the bulge region of the hair follicle using K15.CrePR1 X Stat3fl/fl mice led to decreased tumor development by altering survival of bulge region KSCs. To further understand the role of KSCs in skin tumorigenesis, K5.Stat3C transgenic (Tg) mice which express a constitutively active/dimerized form of Stat3 called Stat3C via the bovine keratin 5 (K5) promoter were studied. The number of CD34 and α6 integrin positive cells was significantly reduced in Tg mice as compared to non-transgenic (NTg) littermates. There was a concomitant increase in the progenitor populations (Lgr-6, Lrig-1 and Sca-1) in the Tg mice vs. the stem cell population (CD34 and Keratin15). To investigate the mechanism underlying the increase in the progenitor population at the expense of bulge region KSCs we examined if Stat3C expression was involved in inducing migration of the bulge region KSCs. There was altered β-catenin and α6-integrin expression in the hair follicles of Tg mice, which may have contributed to reduced adhesive interactions between the epithelial cells and the basement membrane facilitating migration out of the niche. To further study the effect of Stat3 on differentiation of keratinocytes we analyzed the epidermal keratinocytes in K5.Cre X Stat3fl/fl mice. There was an increase in the expression of epidermal differentiation markers in the Stat3 knockout mice. These data suggest that deletion of Stat3 in the epidermis and hair follicle induced differentiation in these cells. Preliminary studies done with the BK5.Stat3C mouse model suggests that multiple hair follicle stem/progenitor populations may be involved in skin tumor development and progression in this model of skin tumorigenesis. Overall, these data suggest that Stat3 plays an important role in differentiation as well as migration of keratinocytes and that these effects may play a role during epithelial carcinogenesis.

Genetic variants within the Wnt/ B-catenin signaling pathway as indicators of bladder cancer clinical outcomes

The genetic factors that influence bladder cancer clinical outcomes are largely unknown. In this clinical outcomes study, I assessed genetic variations in the Wnt/β-catenin stem-cell pathway genes for association with recurrence and progression. A total of 230 SNPS in 40 genes from the Wnt/β-catenin pathway were genotyped in 419 histologically confirmed non-muscle invasive bladder cancer cases. Several significant associations were observed in the clinical outcomes analysis. Under the dominant model WNT8B: rs4919464 (HR: 1.55, 95% CI: 1.17-2.06, P=2.2x10-3) and WNT8B: rs3793771 (HR: 1.54, 95% CI: 1.09-1.62, P=4.6x10-3 ) were statistically significantly associated with an increase risk of recurrence while two other variants, APC2: rs11668593 (HR: 2.50, 95% CI: 1.43-4.35, P=1.2x10-3) and LRP5 : rs312778 (HR: 1.81, 95% CI: 1.23-2.65, P=2.7x10-3), were significantly associated with recurrence risk under the recessive model of inheritance. Four SNPs in the recessive model were associated with an increased risk of progression (AXIN2: rs1544427, LRP5: rs312778, AXIN1: rs370681, AXIN1: rs2301522). LRP5: rs312778 had the most significant increased risk of progression with a 2.68 (95% CI: 1.52-4.72, P=6.4x10-4)-fold increased risk. Stratification analysis based on treatment regimen (transurethral resection (TUR) and Bacillus Calmette-Guérin (BCG)) was also performed. Individuals with at least one variant in AXIN2: rs2007085 were found to have a 2.09 (95% CI: 1.24-3.52, P=5.4x10-3) -fold increased risk of recurrence in those that received TUR only, and no statistically significant effect was seen in those that received BCG. Individuals who received TUR with at least one variant in LEF1: rs10516550 were found to have a 2.26 (95% CI: 1.22-4.18, P=9.7x10-3)-fold increase risk of recurrence and no statistically significant effect was found in individuals who received BCG. Also, the recessive model of LRP6: rs2302684 in TUR only treatment was shown to have a 1.95 (95%CI: 1.18-3.21, P=8.8x10 -3)-fold increased risk of recurrence, and a suggested protective effect associated with a (HR: 0.83, 95% CI: 0.51-1.37, P=0.468) decreased risk of recurrence. Together, these findings implicate the Wnt/β-catenin stem-cell pathway as playing a role in bladder cancer clinical outcomes and have important implications for personalization of future treatment regimens. ^

Inflammatory Breast Cancer: The Immune Perspective

Inflammatory breast cancer (IBC) is the most insidious form of locally advanced disease. Although rare and less than 2% of all breast cancer, IBC is responsible for up to 10% of all breast cancer deaths. Despite the name, very little is known about the role of inflammation or immune mediators in IBC. Therefore, we analyzed blood samples from IBC patients and non-IBC patients, as well as healthy donor controls to establish an IBC-specific profile of peripheral blood leukocyte phenotype and function of T cells and dendritic cells and serum inflammatory cytokines. Emerging evidence suggests that host factors in the microenviromement may interact with underlying IBC genetics to promote the aggressive nature of the tumor. An integral part of the metastatic process involves epithelial to mesenchymal transition (EMT) where primary breast cancer cells gain motility and stem cell-like features that allow distant seeding. Interestingly, the IBC consortium microarray data found no clear evidence for EMT in IBC tumor tissues. It is becoming increasingly evident that inflammatory factors can induce EMT. However, it is unknown if EMT-inducing soluble factors secreted by activated immune cells in the IBC microenvironment canπ account for the absence of EMT in studies of the tumor cells themselves. We hypothesized that soluble factors from immune cells are capable of inducing EMT in IBC. We tested the ability of immune conditioned media to induce EMT in IBC cells. We found that soluble factors from activated immune cells are able to induce the expression of EMT-related factors in IBC cells along with increased migration and invasion. Specifically, the pro-inflammatory cytokines TNF-α, IL-6 and TGF-β were able to induce EMT and blocking these factors in conditioned media abated the induction of EMT. Surprisingly, unique to IBC cells, this process was related to increased levels of E-cadherin expression and adhesion, reminiscent of the characteristic tightly packed tumor emboli seen in IBC samples. This data offers insight into the unique pathology of IBC by suggesting that tumor immune interactions in the tumor microenvironment contribute to the aggressive nature of IBC implying that immune induced inflammation can be a novel therapeutic target. Specifically, we showed that soluble factors secreted by activated immune cells are capable of inducing EMT in IBC cells and may mediate the persistent E-cadherin expression observed in IBC. This data suggests that immune mediated inflammation may contribute to the highly aggressive nature of IBC and represents a potential therapeutic target that warrants further investigation.

Inhibition of Deubiquitinase Activity and Ubiquitination of Jak2 Blocks Cytokine Signaling and Induces Tumor Cell Apoptosis

The Jak-stat pathway is critical for cellular proliferation and is commonly found to be deregulated in many solid tumors as well as hematological malignancies. Such findings have spurred the development of novel therapeutic agents that specifically inhibit Jak2 kinase, thereby suppressing tumor cell growth. Tyrphostin AG490, the first described Jak2 inhibitor, displays poor pharmacology and requires high concentrations for anti-tumor activities. Our research group screened a small library of AG490 structural analogues and identified WP1130 as a potent inhibitor of Jak2 signaling. However, unlike AG490, WP1130 did not directly inhibit Jak2 kinase activity. Our results show that WP1130 induces rapid ubiquitination and subsequent re-localization of Jak2 into signaling incompetent aggresomes. In addition to Jak2, WP1130 also induces accumulation of other ubiquitinated proteins without inhibiting 20S proteasome activity. Further analysis of the mechanism of action of WP1130 revealed that WP1130 acts as a partly selective DUB inhibitor. It specifically inhibits the deubiquitinase activity of USP9x, USP5, USP14 and UCH37. WP1130 mediated inhibition of tumor-associated DUBs resulted in down-regulation of anti-apoptotic and up-regulation of pro-apoptotic proteins, such as MCL-1 and p53 respectively. Our results demonstrate that chemical modification of a previously described Jak2 inhibitor results in the unexpected discovery of a novel compound which acts as a DUB inhibitor, suppressing Jak-Stat signaling by a novel mechanism.

THE ROLE OF PLATELETS IN OVARIAN CARCINOMA

Platelets represent one of the largest storage pools of angiogenic and oncogenic growth factors in the human body. The observation that thrombocytosis (platelet count >450,000/uL) occurs in patients with solid malignancies was made over 100 years ago. However, the clinical and biological implications as well as the underlying mechanism of paraneoplastic thrombocytosis associated with ovarian carcinoma remains unknown and were the focus of the current study. Following IRB approval, patient data were collected on 619 patients from 4 U.S. centers and used to test associations between platelet count at initial diagnosis, clinicopathologic factors, and outcome. In vitro effects of plasma-purified platelets on ovarian cancer cell proliferation, docetaxel-induced apoptosis, and migration were evaluated using BrdU-PI flow cytometric and two-chamber chemotaxis assays. In vivo effects of platelet depletion on tumor growth, proliferation, apoptosis, and angiogenesis were examined using an anti-platelet antibody (anti-mouse glycoprotein 1ba, Emfret) to reduce platelets by 50%. Complete blood counts and number of mature megakaryocytes in the spleen and bone marrow were compared between control mice and ovarian cancer-bearing mice. Plasma levels of key megakaryo- and thrombopoietic factors including thrombopoietin (TPO), IL-1a, IL-3, IL-4, IL-6, IL-11, G-CSF, GM-CSF, stem cell factor, and FLT-3 ligand were assayed in a subset of 150 patients at the time of initial diagnosis with advanced stage, high grade epithelial ovarian cancer using immunobead-based cytokine profiling coupled with the Luminex® xMAP platform. Plasma cytokines significantly associated with thrombocytosis in ovarian cancer patients were subsequently evaluated in mouse models of ovarian cancer using ELISA immunoassays. The results of human and mouse plasma cytokine profiling were used to inform subsequent in vivo studies evaluating the effect of siRNA-induced silencing of select megakaryo- and thrombopoietic cytokines on paraneoplastic thrombocytosis. Thirty-one percent of patients had thrombocytosis at initial diagnosis. Compared to patients with normal platelet counts, women with thrombocytosis were significantly more likely to have advanced stage disease (p<0.001) and poor median progression-free (0.94 vs 1.35 years, p<0.001) and overall survival (2.62 vs 4.65 years, p<0.001). On multivariate analysis, thrombocytosis remained an independent predictor of decreased overall survival. Our analysis revealed that thrombocytosis significantly increases the risk of VTE in ovarian cancer patients and that thrombocytosis is an independent predictor of increased mortality in women who do develop a blood clot. Platelets increased ovarian cancer cell proliferation and migration by 4.1- and 2.8-fold (p<0.01), respectively. Platelets reduced docetaxel-induced apoptosis in ovarian cancer cells by 2-fold (p<0.001). In vivo, platelet depletion reduced tumor growth by 50%. Staining of in vivo specimens revealed decreased tumor cell proliferation (p<0.001) and increased tumor and endothelial cell apoptosis (p<0.01). Platelet depletion also significantly decreased microvessel density and pericyte coverage (p<0.001). Platelet counts increase by 31-130% in mice with invasive ovarian cancer compared to controls (p<0.01) and strongly correlate with mean megakaryocyte counts in the spleen and bone marrow (r=0.95, p<0.05). Plasma levels of TPO, IL-6, and G-CSF were significantly increased in ovarian cancer patients with thrombocytosis. Plasma levels of the same cytokines were found to be significantly elevated in orthotopic mouse models of ovarian cancer, which consistently develop paraneoplastic thromocytosis. Silencing TPO, IL-6, and G-CSF significantly abrogated paraneoplastic thrombocytosis in vivo. This study provides new understanding of the clinical and biological significance of paraneoplastic thrombocytosis in ovarian cancer and uncovers key humoral factors driving this process. Blocking the development of paraneoplastic thrombocytosis and interfering with platelet-cancer cell interactions could represent novel therapeutic strategies.

THE CONSEQUENCES OF DISRUPTING THE MDM2-P53 BALANCE IN HEMATOPOIESIS

The bone marrow accommodates hematopoietic stem cells and progenitors. These cells provide an indispensible resource for replenishing the blood constituents throughout an organism’s life. A tissue with such a high turn-over rate mandates intact cycling checkpoint and apoptotic pathways to avoid inappropriate cell proliferation and ultimately the development of leukemias. p53, a major tumor suppressor, is a transcription factor that regulates cell cycle, and induces apoptosis and senescence. Mice inheriting a hypomorphic p53 allele in the absence of Mdm2, a p53 inhibitor, have elevated p53 cell cycle activity and die by postnatal day 13 due to hematopoietic failure. Hematopoiesis progresses normally during embryogenesis until it moves to the bone marrow in late development. Increased oxidative stress in the bone marrow compartment postnatally is the impediment for normal hematopoiesis via activation of p53. p53 in turn stimulates the generation of more reactive oxygen species and depletes bone marrow cellularity. Also, p53 exerts various defects on the hematopoietic niche by increasing mesenchymal lineage populations and their differentiation. Hematopoietic defects are rescued with antioxidants or when cells are cultured at low oxygen levels. Deletion of p16 partially rescues bone marrow cellularity and progenitors via a p53-independent pathway. Thus, although p53 is required to inhibit tumorigenesis, Mdm2 is required to control ROS-induced p53 levels for sustainable hematopoiesis and survival during homeostasis.

Role and Regulation of EphA2 in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cancer cause of death in the US. Gemcitabine is the first-line therapy for this disease, but unfortunately it shows only very modest benefit. The focus of the current study was to investigate the role and regulation of EphA2, a receptor tyrosine kinase expressed in PDAC, to further understand this disease and identify new therapeutic targets. The role of EphA2 was determined in PDAC by siRNA mediated silencing. In combination with gemcitabine, silencing of EphA2 caused a dramatic increase in apoptosis even in highly resistant cells in vitro. Furthermore, EphA2 silencing was found to be useful in 2 orthotopic models in vivo: 1) shRNA-pretreated Miapaca-2 cells, and 2) in vivo delivery of siRNA to established MPanc96 tumors. Silencing of EphA2 alone reduced tumor growth in Miapaca-2 cells. In MPanc96, only the combination treatment of gemcitabine plus siEphA2 significantly reduced tumor growth, as well as the number of lung and liver metastases. Taken together, these observations support EphA2 as a target for combination therapies for PDAC. The regulation of EphA2 was further explored with a focus on the role of Ras. K-Ras activating mutations are the most important initiating event in PDAC. We demonstrated that Ras regulates EphA2 expression through activation of MEK2 and phosphorylation of ERK. Downstream of ERK, silencing of the transcription factor AP-1 subunit c-Jun or inhibition of the ERK effector RSK caused a decrease in EphA2 expression, supporting their roles in this process. Further examination of Ras/MEK/ERK pathway modulators revealed that PEA-15, a protein that sequesters ERK to the cytoplasm, inhibited expression of EphA2. A significant inverse correlation between EphA2 and PEA-15 levels was observed in mouse models of PDAC. In cells where an EGFR inhibitor reduced phospho-Erk, expression of EphA2 was also reduced, indicating that changes in EphA2 levels may allow monitoring the effectiveness of anti-Ras/MEK/ERK therapies. In conclusion, EphA2 levels may be a good prognostic factor for anti-EGFR/anti-Ras therapies, and EphA2 itself is a relevant target for the development of new therapies.

Altered Responses to Endoplasmic Reticulum Stress in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) represents the fourth most common cause of cancer-associated death in the United States. Little progress has been made in understanding how proteotoxic stress affects rapidly proliferating pancreatic tumor cells. Endoplasmic reticulum (ER) stress occurs when protein homeostasis in the ER lumen is perturbed. ER stress activates the unfolded protein response (UPR) to reduce the protein load in the ER. Under conditions of moderate ER stress, the UPR promotes cell cycle arrest which allows time for successful protein load reduction and enables cell survival. However, under conditions of high levels of ER stress the UPR induces cellular apoptosis. In this dissertation, I investigated the role of endoplasmic reticulum (ER) stress and its effects on the cell cycle in pancreatic cancer cells. Activation of the unfolded protein response after ER stress induction was determined by comparing expression of key UPR mediators in non-tumorigenic pancreatic ductal cells to pancreatic cancer cells. Two arms of the UPR were assessed: eIF2α/ATF4/CHOP and IRE1α/XBP1s. Pancreatic cancer cells exhibited altered UPR activation characterized by a delay in both phosphorylation of eIF2α and induction of spliced XBP1. Further evaluation of the UPR-mediated effects on cell cycle progression revealed that pancreatic cancer cells showed a compromised ability to inhibit G1 to S phase progression after ER stress. This reduced ability to arrest proliferation was found to be due to an impaired ability to downregulate cyclin D1, a key gatekeeper of the G1/S checkpoint. Abrogation of cyclin D1 repression was mediated through a slow induction of phosphorylation of eIF2α, a critical mediator of translational attenuation in response to ER stress. In conclusion, pancreatic cancer cells demonstrate a globally compromised ability to regulate the unfolded protein response. This deficiency results in reduced cyclin D1 repression through an eIF2α-mediated mechanism. These findings indicate that pancreatic cancer cells have increased tolerance for elevated ER stress compared to normal cells, and this tolerance results in continued tumor cell proliferation under proteotoxic conditions.

THE ROLE AND MECHANISM OF THE HOMEOBOX GENE DLX4 IN TRANSFORMING GROWTH FACTOR-B RESISTANCE IN CANCER

Transforming growth factor-b (TGF-b) is a cytokine that plays essential roles in regulating embryonic development and tissue homeostasis. In normal cells, TGF-b exerts an anti-proliferative effect. TGF-b inhibits cell growth by controlling a cytostatic program that includes activation of the cyclin-dependent kinase inhibitors p15Ink4B and p21WAF1/Cip1 and repression of c-myc. In contrast to normal cells, many tumors are resistant to the anti-proliferative effect of TGF-b. In several types of tumors, particularly those of gastrointestinal origin, resistance to the anti-proliferative effect of TGF-b has been attributed to TGF-b receptor or Smad mutations. However, these mutations are absent from many other types of tumors that are resistant to TGF-b-mediated growth inhibition. The transcription factor encoded by the homeobox patterning gene DLX4 is overexpressed in a wide range of malignancies. In this study, I demonstrated that DLX4 blocks the anti-proliferative effect of TGF-b by disabling key transcriptional control mechanisms of the TGF-b cytostatic program. Specifically, DLX4 blocked the ability of TGF-b to induce expression of p15Ink4B and p21WAF1/Cip1 by directly binding to Smad4 and to Sp1. Binding of DLX4 to Smad4 prevented Smad4 from forming transcriptional complexes with Smad2 and Smad3, whereas binding of DLX4 to Sp1 inhibited DNA-binding activity of Sp1. In addition, DLX4 induced expression of c-myc, a repressor of p15Ink4B and p21WAF1/Cip1 transcription, independently of TGF-b signaling. The ability of DLX4 to counteract key transcriptional control mechanisms of the TGF-b cytostatic program could explain in part the resistance of tumors to the anti-proliferative effect of TGF-b. This study provides a molecular explanation as to why tumors are resistant to the anti-proliferative effect of TGF-b in the absence of mutations in the TGF-b signaling pathway. Furthermore, this study also provides insights into how aberrant activation of a developmental patterning gene promotes tumor pathogenesis.

Prime-boost vaccination with plasmid and adenovirus gene vaccines control HER2/neu+ metastatic breast cancer in mice.

INTRODUCTION: Once metastasis has occurred, the possibility of completely curing breast cancer is unlikely, particularly for the 30 to 40% of cancers overexpressing the gene for HER2/neu. A vaccine targeting p185, the protein product of the HER2/neu gene, could have therapeutic application by controlling the growth and metastasis of highly aggressive HER2/neu+ cells. The purpose of this study was to determine the effectiveness of two gene vaccines targeting HER2/neu in preventive and therapeutic tumor models. METHODS: The mouse breast cancer cell line A2L2, which expresses the gene for rat HER2/neu and hence p185, was injected into the mammary fat pad of mice as a model of solid tumor growth or was injected intravenously as a model of lung metastasis. SINCP-neu, a plasmid containing Sindbis virus genes and the gene for rat HER2/neu, and Adeno-neu, an E1,E2a-deleted adenovirus also containing the gene for rat HER2/neu, were tested as preventive and therapeutic vaccines. RESULTS: Vaccination with SINCP-neu or Adeno-neu before tumor challenge with A2L2 cells significantly inhibited the growth of the cells injected into the mammary fat or intravenously. Vaccination 2 days after tumor challenge with either vaccine was ineffective in both tumor models. However, therapeutic vaccination in a prime-boost protocol with SINCP-neu followed by Adeno-neu significantly prolonged the overall survival rate of mice injected intravenously with the tumor cells. Naive mice vaccinated using the same prime-boost protocol demonstrated a strong serum immunoglobulin G response and p185-specific cellular immunity, as shown by the results of ELISPOT (enzyme-linked immunospot) analysis for IFNgamma. CONCLUSION: We report herein that vaccination of mice with a plasmid gene vaccine and an adenovirus gene vaccine, each containing the gene for HER2/neu, prevented growth of a HER2/neu-expressing breast cancer cell line injected into the mammary fat pad or intravenously. Sequential administration of the vaccines in a prime-boost protocol was therapeutically effective when tumor cells were injected intravenously before the vaccination. The vaccines induced high levels of both cellular and humoral immunity as determined by in vitro assessment. These findings indicate that clinical evaluation of these vaccines, particularly when used sequentially in a prime-boost protocol, is justified.