Cancer metastasis is a multi-stage process initiated by the cancer cell acquiring the ability to migrate. The protein profile of such a cell undergoes dramatic changes including changes in integrin expression. Integrins play a major role in cell adhesion, motility, differentiation, blood clotting, tissue organization and cell growth as well as cancer cell migration, invasion and metastasis. Integrin a6, which can pair with integrin b4 or b1 is a laminin receptor and is detected in epithelial cells. Earlier studies have reported uPA mediated integrin a6 cleavage in prostate cancer resulting in loss of the ligand binding domain. Site-directed mutagenesis studies have identified the cleavage site to be at R594R595 located in the "stalk" region of the integrin a6. Prostate cancer cells PC3N-a6-RR cells, bearing a R594R595 to A594A595 mutation, engineered to express the uncleavable form of integrin a6 were found to migrate 6.4 folds lesser on Laminin-1 as compared to the PC3N-a6-WT cells which expressed the wild-type integrin a6. This result suggests that integrin a6 cleavage enhances migration. Prostate cancer is known to metastasize to the bone. Injection of the PC3N-a6-WT cells in mouse femurs resulted in increased bone destruction and pain behavior when compared to the femurs injected with PC3N-a6-RR cells indicating that the integrin a6 cleavage could affect and modify the bone microenvironment. An observation that complete conversion of integrin a6 to a6p was not observed in cell lines even in presence of excess uPA suggested a regulatory mechanism. Integrins are known to associate with many proteins including tetraspanins, which are transmembrane proteins, that function as protein adapters. Integrin a6 was found to be refractory to uPA mediated cleavage when complexed with tetraspanin CD151. The amount of integrin a6 available for cleavage increased when CD151 levels were decreased by CD151 siRNA treatment. These results suggest that the integrin a6 available and unavailable for cleavage can be modulated by interaction with CD151 and hence affect the migratory potential of the cell. Collectively these data suggest that integrin a6 cleavage can enhance cell migration, initiate signals to modify the tumor microenvironment and can be regulated by interaction with tetraspanin CD151.

Expression of A6 integrin, a laminin receptor, on tumor cell surfaces is associated with reduced patient survival and increased metastasis in a variety of tumors. In prostate cancer, tumor extra capsular escape occurs in part via laminin coated nerves and vascular dissemination, resulting in clinically significant bone metastases. Our group previously identified a novel form of A6 integrin, called A6p, generated by urokinase (uPA) dependent cleavage of the laminin binding domain from the tumor cell surface. Although functional consequences of cleavage have been characterized, little is known about how this process is regulated.Regulation of uPA mediated cleavage was identified by a laterally interacting protein expressed on the cellular surface. A direct interaction between the urokinase receptor (uPAR) and A6 integrin was characterized. This direct interaction was responsible for the extracellular cleavage of A6. Transient knockout of A3 integrin, a known interacting partner of uPAR, increased uPAR association with A6 integrin and enhanced production of A6p. Analysis of tissue obtained from human prostate tumors confirmed uPAR and A6 integrin expression in invasive disease. Taken together the results demonstrate a novel and dynamic role for uPAR regulation of integrin dependent adhesion through lateral interaction.Using the known conformation sensitivity of integrin function to I determined if engagement of the extracellular domain by antibodies inhibited integrin cleavage and the extravasation step of metastasis. Both endogenous and inducible levels of A6p were inhibited by engaging the extracellular domain of A6 with monoclonal antibody J8H. J8H inhibited tumor cell invasion through Matrigel. A SCID mouse model of extravasation and bone metastasis produced detectable, progressive osteolytic lesions within three weeks of intracardiac injections. Injection of tumor cells, pre-treated with J8H, delayed the appearance of metastases. Validation of the A6 cleavage effect on extravasation was confirmed through a genetic approach using tumor cells transfected with uncleavable A6 integrin. Uncleavable A6 integrin significantly delayed the onset and progression of osseous metastases out to 6 weeks post injection. The results suggest that A6 integrin cleavage permits extravasation of human prostate cancer cells from circulation to bone and can be manipulated to prevent metastasis.

As a barrier to metastases, cells normally undergo apoptosis after they lose contact with their extra cellular matrix or their neighbouring cells. This cell death process has been termed “anoikis”. Tumour cells that acquire malignant potential have developed mechanisms to resist anoikis and thereby survive after detachment from their primary site and while travelling through the lymphatic and circulatory systems. The understanding of the molecular regulators of anoikis resistance will allow for a better understanding of the metastatic process and the development of novel anti-metastatic therapeutics. To better determine the molecular underpinnings of anoikis resistance, we have used both chemical biology and genetic approaches. Using chemical biological approaches such as small molecule screens, we determined that both FLIP and Na+/K+ ATPase could modulate a cell’s response to anoikis. Through the use of a shRNA genome wide lentiviral screen we determined that ABHD4 was able to inhibit a cell’s response to anoikis. We also showed the importance of anoikis resistance in the ability of malignant cancer cells to survive in circulation. By decreasing a cell’s ability to resist anoikis, one is able to decrease the ability of a cancer cell to survive in circulation and form tumours in distant organs. Taken together, we have identified novel regulators of anoikis resistance and demonstrated the importance of anoikis in metastatic progression, which may lead to the development of novel treatments for metastatic cancers.

Metastasis is the leading cause of mortality in human breast cancer. However, there are few predictive, prognostic, or therapeutic targets of breast cancer metastasis. Ezrin, a membrane cytoskeletal cross-linker, is frequently over-expressed in human breast cancer and is required for motility and invasion by cultured epithelial cells. Our group has recently shown that ezrin acts co-operatively with the non-receptor tyrosine kinase, Src, in the transformation of epithelial cells, in which ezrin is phosphorylated on specific tyrosines, such as Y477, by Src (91, 93). We therefore examined whether Src/ezrin interaction also regulates invasion and metastasis of breast cancer. This thesis presents the following results: 1) In a murine system, ezrin and Src are differentially localized in nulliparous, lactating mammary glands and PyMT-induced tumours, with pronounced apical expression in nulliparous mammary glands but non-polarized strong cytoplasmic expression in PyMT-induced tumours. 2) Increased expression and activation of ezrin, Src and Met in PyMT-induced tumours compared to normal breast tissues was observed. A concomitant increased expression of activated Stat3 and HGF was also observed in PyMT-induced tumours, consistent with the establishment of an HGF/Met autocrine loop. 3) In invasive human breast tumours, from a premenopausal patient cohort, ezrin showed significantly greater cytoplasmic localization compared to non-neoplastic epithelial ducts in normal mammoplasties. 4) In a mouse breast carcinoma xenograft model, a Y477F ezrin mutant (not phosphorylatable by Src), significantly reduced local invasion of primary tumours and spreading into visceral organs, yet, it did not significantly affect primary tumour growth rate. 5) Y477F ezrin-expressing tumours exhibited focal areas of incomplete membranous ezrin staining which was absent in control tumours. Moderate/strong cytoplasmic ezrin staining was evident in both tumour groups. Thus, ezrin is differentially localized in non-invasive versus invasive mammary tumours. Our study implicates a role of the Src/ezrin pathway in regulating local invasion and metastasis of breast carcinoma cells and provides a clinically relevant model for assessing the Src/ezrin pathway as a potential prognostic marker and treatment target for invasive breast cancer. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2010-04-28 12:24:25.286

No description available.

Metastasis is the spread of cancer cells from the primary tumor to distant sites. It is the most dangerous attribute of cancer, and also the principle cause of cancerrelated morbidity and mortality. Metastasis suppressor genes are a group of genes that suppress tumor metastasis without significant effect on tumorigenicity. NM23 was the first identified metastasis suppressor gene, and loss of its expression is a frequent hallmark of metastatic growth in multiple cancers (e.g. melanoma, carcinomas of breast, stomach and liver). NM23-H1 possesses at least three enzymatic activities, including nucleoside diphosphate kinase (NDPK), histidine kinase (hisK), and a more recently described 3f-5f exonuclease (EXO). While the hisK has been shown to be linked to the suppression of cell motility, the NDPK has been reported to be unrelated to the suppression of metastatic potential indirectly. Relevance of EXO has not been addressed. Other known 3f-5f exonuclease are closely associated with DNA repair functions, suggesting NM23-H1 may suppress mutations required for metastasis. As a transcription factor, NM23 has been shown to modestly downregulate the transcription on PDGF-A chain, a growth factor oncogene, either alone or in association with another transcriptional factor, Pur@. At the same time, identification of NM23-H1 as a 3f-5fexonuclease suggests the role of NM23-H1 in DNA repair. Etoposide and cisplatin elicited nuclear translocation of H1 within 4 h in HeLa and HepG2 cells, seen as accumulation of H1 in small intranuclear foci, strongly suggesting the DNA repair function of H1. To investigate the enzymatic function contributing to metastasis suppressor activity of H1, complementation system was used by transfecting NM23-H1 with individually disrupted enzymatic function into 2 melanoma cell lines, 1205LU and WM793. Overexpression of H1 in 1205LU suppressed lung metastasis in vivo without effect on indices of transformation (e.g. proliferation, soft agar colonization). EXO- deficient H1 and NDPK-deficient H1 lost suppression of lung metastasis, while hisK-deficient H1 maintained suppressor activity. Consistent with the results in 1205LU cells, EXO-deficient H1 and NDPKdeficient H1 lost suppression of the progression of WM793 cells in protein-free medium, while WT and hisK-deficient H1 prevented the progression. Taken together, these data suggest that the NDPK and/or 3f-5fEXO activity of H1 inhibits the progression of premetastatic cells to the metastatic phenotype, possibly via a DNA repair function or other structural transactions with DNA.

In the present study, we characterized the ability of a novel oncolytic rhabdovirus - Maraba MG1 to boost Natural Killer (NK) cell activity. In tandem, we addressed the ability of this enhanced NK cell functionality to reduce the incidence of post-cancer surgery micrometastases. Due to the potential safety barriers associated with the use of a live virus immediately prior to surgery in cancer patients, we generated a single cycle replication virus (MG1-Gless) and UV-inactivated MG1 to stimulate NK cell function and reduce post-operative metastases. Our in vivo data demonstrate that significant NK cell activation and a similar level of reduction in postoperative tumor metastases was achieved with live MG1, MG1-Gless and UV-inactivated MG1, concluding that viral replication is important, but not necessary for NK cell activation. Mechanistically, we observed that dendritic cells (DCs) are necessary intermediates for MG1-induced NK cell activation. Finally, we characterized and compared a panel of UV-inactivated MG1 (2mins to 2hrs) to better understand the requirements for NK cell activation. Our results suggest that intact viral particle and cellular recognition and association are essential for NK cell mediated anti-tumor responses. These findings provide the preclinical rationale to develop safe and viable virotherapy-based interventional protocols that might reduce the risk of metastatic disease after cancer surgery.

As a barrier to metastases, cells normally undergo apoptosis after they lose contact with their extra cellular matrix or their neighbouring cells. This cell death process has been termed “anoikis”. Tumour cells that acquire malignant potential have developed mechanisms to resist anoikis and thereby survive after detachment from their primary site and while travelling through the lymphatic and circulatory systems. The understanding of the molecular regulators of anoikis resistance will allow for a better understanding of the metastatic process and the development of novel anti-metastatic therapeutics. To better determine the molecular underpinnings of anoikis resistance, we have used both chemical biology and genetic approaches. Using chemical biological approaches such as small molecule screens, we determined that both FLIP and Na+/K+ ATPase could modulate a cell’s response to anoikis. Through the use of a shRNA genome wide lentiviral screen we determined that ABHD4 was able to inhibit a cell’s response to anoikis. We also showed the importance of anoikis resistance in the ability of malignant cancer cells to survive in circulation. By decreasing a cell’s ability to resist anoikis, one is able to decrease the ability of a cancer cell to survive in circulation and form tumours in distant organs. Taken together, we have identified novel regulators of anoikis resistance and demonstrated the importance of anoikis in metastatic progression, which may lead to the development of novel treatments for metastatic cancers.

Prostate cancer (CaP) is the 2nd most common cancer in North American men. Tumour management strategies are appropriate for early stage disease, but advanced disease has a poor prognosis and requires prompt treatment. Therefore, research into delay of tumour progression and efficacious treatment of aggressive cancer are of interest. Ganglioside was assessed for its role in altering markers of metastatic potential and susceptibility of CaP to adenovirus-mediated gene therapy. Healthy (RWPE-1) and malignant (DU-145, PC-3) prostate cells were cultured with or without mixed ganglioside. Differences in growth, ganglioside and integrin densities, and adenoviral infectivity were assessed between treatment and control groups. Ganglioside decreased (p<0.01) growth of PC-3 cells relative to untreated control. Ganglioside decreased (p<0.01) GD1a and increased (p<0.04) integrin densities in malignant prostate cells, suggesting ganglioside may increase metastatic potential of CaP. Ganglioside significantly increased adenovirus entry in PC-3 cells, thereby improving susceptibility of CaP to adenovirus-mediated gene therapy. / Nutrition and Metabolism

Thesis (Ph. D.)--University of New South Wales, 1999. / Also available online.