Abstract Although the critical role of the small GTPases Ras and Ral in oncogenesis has been well documented, much remains to be investigated about the molecular mechanism by which these GTPases regulate malignant transformation. The work under this thesis made two major contributions to this field. The first is the discovery that K-Ras, RalA and/or RalB are required for the maintenance of the high levels of the anti-apoptotic protein survivin in some human cancer cells, and the second is the demonstration that down regulation of K-Ras, RalA and/or RalB, but not Raf-1 or Akt1/2, stabilizes the tumor suppressor p53 and reactivates it to inhibit malignant transformation in human cancer cells with mutant K-Ras and wild type p53. Here we found that depletion of K-Ras leads to decreased survivin levels in human cancer cells that harbor mutant K-Ras but not those with wild type Ras. The mechanism by which this occurs involves ubiquitination and subsequent proteasome-mediated degradation of survivin. The presence of mutant K-Ras alone was not sufficient to predict the effects of RalA/B depletion on survivin levels. Indeed, depletion of RalA and/or RalB reduces survivin levels in human cancer cells with wild type p53 and mutant K-Ras, but not in those with mutant p53 and/or wild type K-Ras. The functional relevance of these findings to malignant transformation was further supported by the demonstration that compromising the expression of survivin by siRNA leads to reduction of mutant K-Ras-driven invasion and anchorage-independent growth. Furthermore, in this thesis, we have discovered that down regulation of K-Ras, RalA and/or RalB using siRNA leads to increased levels of functional p53 that is capable of regulating its target genes. The mechanism by which depleting K-Ras, RalA and RalB increases the levels of p53 involves an increase in the half-life of the p53 protein concurrent with an increase in the phosphorylation of serine-15 of p53, a marker of p53 stability. Finally, we demonstrated that depletion of K-Ras, RalA and/or RalB interferes with cell cycle progression, anchorage-independent growth and invasion in a p53-dependent manner. In summary, the studies suggest that mutant K-Ras contributes to the maintenance of the aberrantly-high survivin levels by regulating its stability, and that the ability of mutant K-Ras to induce malignant transformation is, at least in part, dependent of these high levels of survivin. The work of this thesis also suggests that the expression of K-Ras, RalA and/or RalB proteins is critical to maintain low levels of p53, and that down regulation of these GTPases reactivates p53 by significantly enhancing its stability, and this contributes to suppression of malignant transformation.

Pomegranate extract (PE) is a standardized whole fruit extract of pomegranate (Punica granatum L.), a fruit with known anti inflammatory and anti cancer properties. In the present study, the effects of PE on two different cell lines of human pancreatic cancer cells, AsPC-1 and PANC-1 were examined. Both cell lines are epithelial cancers but differ morphologically and in their response to treatment with PE. PE inhibited proliferation of both cancer cell lines in a dose dependent and time dependent manner. The reason for this was the potency of PE to cause significant cell cycle arrest in the pancreatic cancer cells, which was noted to be cell specific. AsPC-1 was arrested in G₂ phase while PANC-1 was arrested in G₀/G₁phase of the cell cycle. PE also checked the viability of confluent cancer cells in a concentration dependent manner. In case of AsPC-1 this was due to the action of PE to induce apoptosis, as demonstrated by the Annexin-V assay. In case of PANC-1, a highly invading cancer greatly affected by the presence of surface stem cell markers CD44⁺/CD24⁺, PE was capable of modifying the stem cell markers and induce an increase in the non tumorigenic sub population of cells. A study was also conducted to look at the action of PE in causing inhibition of pancreatic cancer cell proliferation, in comparison to a clinically used chemotherapeutic agent, Paclitaxel. The results indicated that the effect of PE, at clinically feasible doses was superior to that of clinically used doses of Paclitaxel. This data suggests that PE, which is proven to be a safe dietary supplement, looks promising in the prevention and treatment of human pancreatic cancer cells without the side effects of standard drug therapy and could possibly have a role in controlling stem cancer cell population / text

Sequence-specific small interfering RNA (siRNA) duplexes can be used for gene silencing in mammalian cells and as mechanistic probes for determining gene function. Transfection of siRNA for specificity protein 1 (Sp1) in MCF-7 or ZR-75 cells decreased Sp1 protein in nuclear extracts, and immunohistochemical analysis showed that Sp1 protein in transfected MCF-7 cells was barely detectable. Decreased Sp1 protein in MCF-7 was accompanied by a decrease in basal and estrogen-induced transactivation and cell cycle progression. These results clearly demonstrate the key role of Sp1 protein in regulating growth and gene expression of breast cancer cells. The aryl hydrocarbon (AhR) is a ligand-activated nuclear transcription factor. siRNA for the AhR decreased TCDD-induced CYP1A1 protein, CYP1A1dependent activity, and luciferase activity in cells transfected with an Ah-responsive construct. 17β-Estradiol (E2) induces proliferation of MCF-7 cells, and this response is inhibited in cells cotreated with E2 plus TCDD. The effects of TCDD on E2-induced cell cycle progression were partially blocked in MCF-7 cells transfected with siRNA for AhR. The decrease in AhR protein in MCF-7 cells was also accompanied by increased G0/G1 → S phase progression. Surprisingly, TCDD alone induced G0/G1 → S phase progression and exhibited estrogenic activity in MCF-7 cells transfected with siRNA for the AhR. In contrast, degradation of the AhR in HepG2 liver cancer cells resulted in decreased G0/G1 → S phase progression, and this was accompanied by decreased expression of cyclin D1, cyclin E, cdk2 and cdk4. In the absence of ligand, the AhR exhibits growth inhibitory (MCF-7) and growth promoting (HepG2) activity that is cell context-dependent. Sp family proteins play a complex role in regulation of pancreatic cancer cells growth and expression of genes required for growth, angiogenesis and apoptosis. Sp1, Sp3 and Sp4 cooperatively activate VEGF promoter constructs in these cells; however, only Sp3 regulates cell proliferation. siRNA for Sp3 inhibits phosphorylation of retinoblastoma protein, blocks G0/G1 → S phase progression of Panc-1 cells, and upregulates p27 protein/promoter activity. Thus, Sp3 plays a critical role in angiogenesis (VEGF upregulation) and the proliferation of Panc-1 cells by a novel mechanism of Sp3-dependent suppression of the cyclin-dependent kinase inhibitor p27.

Treatment of pancreatic cancer is an important medical problem. Most pancreatic cancer patients are diagnosed with advanced disease and their prognosis is poor. In Lithuania there were 440 new cases in year 2004. More than fifty percent of patients are diagnosed with stage IV disease [Kurtinaitis J et al., 2005]. Median survival of patients diagnosed with pancreatic cancer, depending on a stage of a disease, spread of the tumor, treatment method, functional status of a patient is from 6 to 20 months. Five-year survival is only 0–5 percent [Bramhall S et al., 1998; Oya N, 2004]. Today, pancreatic cancer is treated by combined methods: surgery combined with RT and/or chemotherapy. It is questionable if a patient diagnosed with pancreatic cancer in early stages must be treated by adjuvant treatment methods, or should patients diagnosed with unresectable cancer be treated with chemotherapy or ChRT; and what is more important for the patient: increased survival or better quality of life. Kaunas University of Medicine Clinic (KMUC) is a suitable hospital to treat patients diagnosed with pancreatic cancer. At the Clinic of Surgery pancreatic cancer patients are treated by radical and palliative surgery; and at the Clinic of Oncology patients with pancreatic cancer are treated applying contemporary methods of RT, chemotherapy and combined treatment methods. In this thesis treatment results of patients treated for resectable and unresectable pancreatic cancer from year 1987 to year... [to full text]

High-throughput mass spectrometry has discovered a plethora of candidates in the biomarker field, however, subsequent verification and validation studies are urgently needed to assess the potential of novel biomarkers in the detection of pancreatic cancer. We have conducted extensive verification and validation studies on two of our most promising biomarkers CUZD1 and LAMC2 with a total of 715 blood samples. In our study, both markers demonstrated consistent diagnostic ability of early- and CA19.9 negative-PDAC cases. When used in combination with CA19.9, CUZD1 and LAMC2 were shown to significantly improve the performance of CA19.9 alone in the diagnosis of PDAC patients. We speculate that CUZD1 and LAMC2 may be good candidates to be used in a panel for monitoring PDAC patients who do not express CA19.9 levels as well as for an aid in screening high risk populations. Further validation of these two proteins is warranted.

High-throughput mass spectrometry has discovered a plethora of candidates in the biomarker field, however, subsequent verification and validation studies are urgently needed to assess the potential of novel biomarkers in the detection of pancreatic cancer. We have conducted extensive verification and validation studies on two of our most promising biomarkers CUZD1 and LAMC2 with a total of 715 blood samples. In our study, both markers demonstrated consistent diagnostic ability of early- and CA19.9 negative-PDAC cases. When used in combination with CA19.9, CUZD1 and LAMC2 were shown to significantly improve the performance of CA19.9 alone in the diagnosis of PDAC patients. We speculate that CUZD1 and LAMC2 may be good candidates to be used in a panel for monitoring PDAC patients who do not express CA19.9 levels as well as for an aid in screening high risk populations. Further validation of these two proteins is warranted.

OATP1B3 belongs to the OATP (organic anion transporting polypeptides) superfamily, responsible for mediating the transport of various endogenous and xenobiotic substrates. OATP1B3 was initially reported to be expressed exclusively in the hepatocytes where it mediates the uptake of numerous endogenous substrates (e.g. bile acids, steroid hormone conjugates) and several clinically relevant drugs including anticancer drugs. Later, a number of studies reported that OATP1B3 is also frequently expressed in multiple types of cancers and may be associated with differing clinical outcomes. However, a detailed investigation on the expression, localization and functions of OATP1B3 expressed in cancer has been lacking. In this thesis work, we confirmed that colon and pancreatic cancer cells express a cancer-specific OATP1B3 variant (csOATP1B3), different from OATP1B3 wild-type (WT) expressed in the normal liver. The csOATP1B3 utilizes an alternative transcription initiation site and the translated product of csOATP1B3 lacks the first 28 amino acids at the N-terminus of OATP1B3 WT. Our results show that csOATP1B3 has modest uptake transporter functions and reduced plasma membrane localization compared to OATP1B3 WT. In our efforts to investigate the regulatory mechanism underlying the expression of csOATP1B3, we found that hypoxia inducible factor-1α (HIF-1α) may play a key role in the regulation of csOATP1B3 in colon and pancreatic cancer cells. In a separate study, we tested whether the N-terminal sequence of OATP1B3 WT plays an important role in the membrane trafficking. This is based on the observation that csOATP1B3 lacking the first 28 amino acids at N-terminus of OATP1B3 WT displays a predominantly cytoplasmic localization pattern. Using the constructs with N-terminal truncations and point mutations, we verified that the N-terminus of OATP1B3 WT contains important motifs in its membrane trafficking. In particular, the amino acids within a putative β-turn-forming tetrapeptide appear to be important in regulating the membrane trafficking of OATP1B3 WT. The findings from this thesis work provide important insights into the functional and clinical significance of OATP1B3 in cancer and normal liver.

Novel serological biomarkers to aid in the detection and clinical management of pancreatic cancer patients are urgently needed. In the present study, we performed in-depth proteomic analysis of conditioned media from six pancreatic cancer cell lines (MIA-PaCa2, PANC1, BxPc3, CAPAN1, CFPAC1 and SU.86.86), the normal pancreatic ductal epithelial cell line HPDE, and pancreatic juice samples from cancer patients for identification of novel biomarker candidates. Using 2D-LC-MS/MS, a total of 3479 non-redundant proteins were identified with ≥2 peptides. Subsequent label-free protein quantification and integrative analysis of the biological fluids resulted in the generation of candidate biomarkers, of which five proteins were shown to be significantly elevated in plasma from pancreatic cancer patients in a preliminary assessment. Further verification of two of the proteins in ~200 serum samples demonstrated the ability of these proteins to significantly improve the area under the receiver operating characteristic curve of CA19.9 from 0.84 to 0.91.

Novel serological biomarkers to aid in the detection and clinical management of pancreatic cancer patients are urgently needed. In the present study, we performed in-depth proteomic analysis of conditioned media from six pancreatic cancer cell lines (MIA-PaCa2, PANC1, BxPc3, CAPAN1, CFPAC1 and SU.86.86), the normal pancreatic ductal epithelial cell line HPDE, and pancreatic juice samples from cancer patients for identification of novel biomarker candidates. Using 2D-LC-MS/MS, a total of 3479 non-redundant proteins were identified with ≥2 peptides. Subsequent label-free protein quantification and integrative analysis of the biological fluids resulted in the generation of candidate biomarkers, of which five proteins were shown to be significantly elevated in plasma from pancreatic cancer patients in a preliminary assessment. Further verification of two of the proteins in ~200 serum samples demonstrated the ability of these proteins to significantly improve the area under the receiver operating characteristic curve of CA19.9 from 0.84 to 0.91.

Pancreatic cancer kills over 1700 people each year in Australia. In 2000, there were 1908 new cases diagnosed and it remains one of the least treatable malignancies. In the USA, it was the fourth leading cause of cancer death in 2004, with 31,860 new cases and 31,270 recorded deaths. Photodynamic therapy (PDT) is a novel, potentially useful treatment for locally advanced pancreatic cancer with only limited research and clinical work addressing this until now. PDT induces non-thermal, cytotoxic and ischaemic injury to a targeted volume of tissue. During PDT, a photosensitizer is activated by non-thermal light in the presence of oxygen, generating cytotoxic oxygen species and inducing cellular injury and microvascular occlusion. The aim of this thesis was to conduct an animal safety study using two second generation photosensitizers, talaporfin sodium and verteporfin, to assess the risks of pancreatic PDT by looking at injury to organs adjacent to the pancreas and assessing recovery from PDT treatment of the pancreas. The Syrian Golden hamster animal model was used to compare the results of this research to previous work by other authors. The study design incorporated a number of additional experiments, including quantitative tissue fluorescence techniques, plasma level analysis and histopathology techniques. The methods for the animal safety study were similar to the approach used in the clinical setting and provided vital data on the likely risks and side effects of phototherapy in humans. The first study, looking at talaporfin sodium, found likely risks of duodenal injury, gastric injury and death with a limited effect on normal pancreas at photosensitizer doses likely to be employed for pancreatic cancer PDT. The second study, using verteporfin, found similar results with a more potent effect on the normal pancreas at studied drug doses. Both agents had short drug-light intervals, ranging from 15 minutes to 2 hours, reducing the need for pre-treatment hospitalization and short photosensitivity periods of about one to two weeks. Some animals suffered minor cutaneous photosensitivity injuries. A human pancreatic cancer PDT pilot study is feasible and the risks and complications should be acceptable.