Molecular mechanisms of drug resistance, invasion and metastasis in pancreatic carcinoma

The research outlined in this thesis aims to further our knowledge of the biological characteristics underlying the aggressive behaviour by which pancreatic cancer invades and metastasises at an early stage and is refractory to most chemotherapeutic drugs. Investigation into drug resistance in pancreatic cancer involved pulse-selection of three cell lines with epirubicin, taxotere and gemcitabine. The drug-selected cell lines, in general, displayed low changes in sensitivity to their respective selecting drugs and no obvious cross-resistance profile. However, drug treatment modulated the invasive potential of the pulse-selected cell lines. Expression of multi-drug resistant proteins (P-gp and MRP-1) was also determined by IHC in 45 pancreatic tumour specimens, results suggested a contributory role for P-gp expression in pancreatic cancer. An in vitro pancreatic invasion model was established by single cell cloning of MiaPaCa-2, yielding sub-clones displaying diverse invasive properties. The malignant phenotypes of MiaPaCa-2, Clone #3 (highly invasive) and Clone #8 (poorly invasive) were characterised, comparative proteomic profiling by 2-D DIGE, MALDI-TOF MS and RNAi revealed three targets, ALDH1A1, VIM and STIP1 with functional involvement in the invasion process. ALDH1A1 appeared to play a role in the aggressive invasive phenotype; expression was associated with increased invasion, drug resistance and decreased adhesion. VIM and STIP expression was also linked to increased invasion and decreased adhesion, with STIP1 displaying a role in proliferation, which has not previously been described. Factors secreted into conditioned media of Clone #3 and Clone #8 was also examined by proteomics. Targets, GSN and ALDH1A1 were chosen for functional analysis and their potential involvement in cancer cell invasion assessed. EGFR/HER2 expression was analysed in 5 pancreatic cancer cell lines, and two cell lines which co-expressed EGFR and HER2 were tested with lapatinib. Lapatinib, a dual EGFR/HER2 inhibitor in combination with chemotherapeutic drugs showed additive interactions, suggesting the possibility of a therapeutic role for lapatinib in combination with chemotherapy in pancreatic cancer patients co-expressing EGFR/HER2

Digital Assets and Fiduciaries

This chapter addresses the appropriate treatment of a person\u27s digital life when the account holder can no longer manage it. As the Internet becomes an increasingly important presence in our daily lives, the law has a significant role to play in determining the management of digital assets upon the account holder\u27s incapacity or death. In the past, people put hard copies of photos in albums, listened to record albums, and paid bills with a stamped envelope. Today, most people use the Internet to store photos, listen to music, and pay bills. Yet few people have considered how to dispose of their digital assets. This chapter explores the legal issues for trusts, estates, conservatorships, and powers of attorney. It addresses the importance of fiduciaries being able to manage an account holder\u27s digital assets, and the obstacles under federal and state law to a fiduciary assuming that role. Finally, it shows how the Uniform Fiduciary Access to Digital Assets Act provides a solution to ensure effectuation of the account holder\u27s intent

Digital Assets and Fiduciaries

This chapter addresses the appropriate treatment of a person\u27s digital life when the account holder can no longer manage it. As the Internet becomes an increasingly important presence in our daily lives, the law has a significant role to play in determining the management of digital assets upon the account holder\u27s incapacity or death. In the past, people put hard copies of photos in albums, listened to record albums, and paid bills with a stamped envelope. Today, most people use the Internet to store photos, listen to music, and pay bills. Yet few people have considered how to dispose of their digital assets. This chapter explores the legal issues for trusts, estates, conservatorships, and powers of attorney. It addresses the importance of fiduciaries being able to manage an account holder\u27s digital assets, and the obstacles under federal and state law to a fiduciary assuming that role. Finally, it shows how the Uniform Fiduciary Access to Digital Assets Act provides a solution to ensure effectuation of the account holder\u27s intent

Identification of pancreatic cancer invasion-related proteins by proteomic analysis

Background – Markers of pancreatic cancer invasion were investigated in two clonal populations of the cell line, MiaPaCa-2, Clone #3 (high invasion) and Clone #8 (low invasion) using proteomic profiling of an in vitro model of pancreatic cancer. Materials and methods – Using 2D-DIGE followed by MALDI-TOF MS, two clonal sub-populations of the pancreatic cancer cell line, MiaPaCa-2 with high and low invasive capacities were incubated on matrigel 24 hours prior to analysis to stimulate cell-ECM contact and mimic in vivo interaction with the basement membrane. Results - Sixty proteins were identified as being differentially expressed (>1.2 fold change and p ≤ 0.05) between Clone #3 and Clone #8. Proteins found to have higher abundance levels in the highly invasive Clone #3 compared to the low invasive Clone #8 include members of the chaperone activity proteins and cytoskeleton constituents whereas metabolism-associated and catalytic proteins had lower abundance levels. Differential protein expression levels of ALDH1A1, VIM, STIP1 and KRT18 and GAPDH were confirmed by immunoblot. Using RNAi technology, STIP1 knockdown significantly reduced invasion and proliferation of the highly invasive Clone #3. Knockdown of another target, VIM by siRNA in Clone #3 cells also resulted in decreased invasion abilities of Clone #3. Elevated expression of STIP1 was observed in pancreatic tumour tissue compared to normal pancreas, whereas ALDH1A1 stained at lower levels in pancreatic tumours, as detected by immunohistochemistry. Conclusion - Identification of targets which play a role in the highly invasive phenotype of pancreatic cancer may help to understand the biological behaviour, the rapid progression of this cancer and may be of importance in the development of new therapeutic strategies for pancreatic cancer

DNA damage repair deficiency in pancreatic ductal adenocarcinoma: preclinical models and clinical perspectives

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide, and survival rates have barely improved in decades. In the era of precision medicine, treatment strategies tailored to disease mutations have revolutionized cancer therapy. Next generation sequencing has found that up to a third of all PDAC tumors contain deleterious mutations in DNA damage repair (DDR) genes, highlighting the importance of these genes in PDAC. The mechanisms by which DDR gene mutations promote tumorigenesis, therapeutic response, and subsequent resistance are still not fully understood. Therefore, an opportunity exists to elucidate these processes and to uncover relevant therapeutic drug combinations and strategies to target DDR deficiency in PDAC. However, a constraint to preclinical research is due to limitations in appropriate laboratory experimental models. Models that effectively recapitulate their original cancer tend to provide high levels of predictivity and effective translation of preclinical findings to the clinic. In this review, we outline the occurrence and role of DDR deficiency in PDAC and provide an overview of clinical trials that target these pathways and the preclinical models such as 2D cell lines, 3D organoids and mouse models [genetically engineered mouse model (GEMM), and patient-derived xenograft (PDX)] used in PDAC DDR deficiency research

WNT5A regulates adipose tissue angiogenesis via antiangiogenic VEGF-A165b in obese humans

Experimental studies have suggested that Wingless-related integration site 5A (WNT5A) is a proinflammatory secreted protein that is associated with metabolic dysfunction in obesity. Impaired angiogenesis in fat depots has been implicated in the development of adipose tissue capillary rarefaction, hypoxia, inflammation, and metabolic dysfunction. We have recently demonstrated that impaired adipose tissue angiogenesis is associated with overexpression of antiangiogenic factor VEGF-A165b in human fat and the systemic circulation. In the present study, we postulated that upregulation of WNT5A is associated with angiogenic dysfunction and examined its role in regulating VEGF-A165b expression in human obesity. We biopsied subcutaneous and visceral adipose tissue from 38 obese individuals (body mass index: 44 ± 7 kg/m2, age: 37 ± 11 yr) during planned bariatric surgery and characterized depot-specific protein expression of VEGF-A165b and WNT5A using Western blot analysis. In both subcutaneous and visceral fat, VEGF-A165b expression correlated strongly with WNT5A protein (r = 0.9, P \u3c 0.001). In subcutaneous adipose tissue where angiogenic capacity is greater than in the visceral depot, exogenous human recombinant WNT5A increased VEGF-A165b expression in both whole adipose tissue and isolated vascular endothelial cell fractions (P \u3c 0.01 and P \u3c 0.05, respectively). This was associated with markedly blunted angiogenic capillary sprout formation in human fat pad explants. Moreover, recombinant WNT5A increased secretion of soluble fms-like tyrosine kinase-1, a negative regulator of angiogenesis, in the sprout media (P \u3c 0.01). Both VEGF-A165b-neutralizing antibody and secreted frizzled-related protein 5, which acts as a decoy receptor for WNT5A, significantly improved capillary sprout formation and reduced soluble fms-like tyrosine kinase-1 production (P \u3c 0.05). We demonstrated a significant regulatory nexus between WNT5A and antiangiogenic VEGF-A165b in the adipose tissue of obese subjects that was linked to angiogenic dysfunction. Elevated WNT5A expression in obesity may function as a negative regulator of angiogenesis

Aldehyde dehydrogenase 1A1 and gelsolin identified as novel invasion-modulating factors in conditioned medium of pancreatic cancer cells

Conditioned medium (CM) from clonal sub-populations of the pancreatic cancer cell line, MiaPaCa-2 with differing invasive abilities, were examined for their effect on in vitro invasion. Conditioned medium from Clone #3 (CM#3) strongly promoted invasion, while CM from Clone #8 (CM#8) inhibited invasion in vitro. 2D DIGE followed by MALDI-TOF MS analysis of CM#3 and CM#8 identified 41 proteins which were differentially regulated; 27 proteins were down-regulated and 14 proteins up-regulated in the invasion-promoting CM#3 when compared to CM#8. Western blotting analysis confirmed the down-regulated expression of gelsolin and the up-regulation of aldehyde dehydrogenase 1A1 in CM#3. Down-regulation of aldehyde dehydrogenase 1A1 in Clone #3 CM and gelsolin levels in Clone #8 CM by siRNA transfection revealed an important involvement of these proteins in promoting and inhibiting invasion in these pancreatic cancer cell lines