published_or_final_version / Pathology / Master / Master of Medical Sciences

Thesis (M. Med. Sc.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 67-76).

Renal cell carcinoma (RCC) accounts for 85% of kidney cancer incidence in the US. Since 1950 there has been a 126% increase in kidney cancer incidence in the US. Thirty percent of new patients present with a localized easily treatable carcinoma while 30% of patients present with a high-grade metastatic carcinoma. Five-year survival rates for metastatic RCC is 6-12 months (Lipworth et al, 2006). Current disease treatment options for metastasis include chemotherapy and radiation (8% response rate), immunotherapy (15-30% response rate) and newly developed angiogenesis inhibitors which are in phase III trials (Staehler et el, 2005). In RCC cells, it has been shown that PPARγ agonists inhibit cell proliferation, induce apoptosis, and induce anti-angiogenic effects in vitro. Unlike most tumor types, PPARγ is downregulated in tissue samples from 47 RCC patients. However, in cell culture studies PPARγ expression does not correlate with growth inhibitory or apoptotic effects of PPARγ agonists in renal cell lines indicating that PPARγ independent responses may play a large role in actions associated with the PPARγ agonists (Yuan et al, 2006). 1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes containing p-trifluoromethyl, p-t-butyl and p-phenyl substituents activate peroxisome proliferator-activated receptor (PPAR) and inhibit growth of ACHN and 786-0 renal cell carcinoma cell lines. PPAR is overexpressed in ACHN cells and barely detectable in 786-0 cells, and treatment with the t-butyl analog (DIM-C-pPhtBu) induces cell cyle inhibition. DIM-C-pPhtBu also induced several common PPAR-independent proapoptotic responses in ACHN and 786-0 cells, including increased expression of nonsteroidal antiimflammatory drug-activated gene-1 (NAG-1) and endoplasmic reticulum stress which activates death receptor 5 and the extrinsic pathway of apoptosis. In addition, DIM-C-pPhtbu (40 mg/kg/d) also inhibited tumor growth in an orthotopic mouse model for renal carcinogenesis, and this was accompanied by induction of apoptosis in renal tumors treated with DIM-C-pPhtBu but not in tumors treated with the corn oil vehicle (control). Thus, DIM-C-pPhtBu and related compounds represent a novel class of mechanism-based drugs that have potential for treatment of renal adenocarcinoma for which there are currently limited options for successful chemotherapy.

WWOX is a tumor suppressor and the down-regulation of WWOX has been demonstrated in prostate, lung, breast, gastric cancers. However, the role of WWOX in renal cell carcinoma (RCC) remains unknown. It has been demonstrated that WWOX addressed in mitochondria, golgi apparatus, rough ER, lysosome, plasma membrane and nuclear. The Subcellular localization of WWOX has been controversial. There are two parts in this study: (I) The expression of WWOX in RCC and the probability of WWOX to be a diagnostic and a prognostic marker. (II) The regulation of WWOX by phosphorylation. For the study of WWOX expression in RCC, we prepared polyclonal WWOX antibody and characterized the specificity of the antibody. We applied this specific antibody to 33 NT paring RCC tissue specimen for immunoblotting study and 138 cases of paraffin-embedded specimens for IHC, respectively. Our results demonstrated that hWOX1 was specifically down-regulated in clear cell type RCC (p=0.018). The percentage of down-regulation in patient specimen is 60.7 % and 90.7 % in immunoblotting and IHC study, respectively. And in clear cell and clear-granular combined type RCC, down-regulation of WWOX was significantly correlated with the survival rate of patients (p=0.0482). Therefore, WWOX could be used as a diagnostic and a prognostic marker in clear cell type RCC. Besides, we performed bioinformatics to predict the phosphorylation site of WWOX and investigated the effect of phosphorylation on WWOX subcellular localization. Our results demonstrated that hWOX1 was phosphorylated by PKC at Thr49 and Thr102 and the phosphorylation regulated the subcellular localization of WWOX.

Renal cell carcinoma (RCC) accounts for 85% of kidney cancer incidence in the US. Since 1950 there has been a 126% increase in kidney cancer incidence in the US. Thirty percent of new patients present with a localized easily treatable carcinoma while 30% of patients present with a high-grade metastatic carcinoma. Five-year survival rates for metastatic RCC is 6-12 months (Lipworth et al, 2006). Current disease treatment options for metastasis include chemotherapy and radiation (8% response rate), immunotherapy (15-30% response rate) and newly developed angiogenesis inhibitors which are in phase III trials (Staehler et el, 2005). In RCC cells, it has been shown that PPARγ agonists inhibit cell proliferation, induce apoptosis, and induce anti-angiogenic effects in vitro. Unlike most tumor types, PPARγ is downregulated in tissue samples from 47 RCC patients. However, in cell culture studies PPARγ expression does not correlate with growth inhibitory or apoptotic effects of PPARγ agonists in renal cell lines indicating that PPARγ independent responses may play a large role in actions associated with the PPARγ agonists (Yuan et al, 2006). 1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes containing p-trifluoromethyl, p-t-butyl and p-phenyl substituents activate peroxisome proliferator-activated receptor (PPAR) and inhibit growth of ACHN and 786-0 renal cell carcinoma cell lines. PPAR is overexpressed in ACHN cells and barely detectable in 786-0 cells, and treatment with the t-butyl analog (DIM-C-pPhtBu) induces cell cyle inhibition. DIM-C-pPhtBu also induced several common PPAR-independent proapoptotic responses in ACHN and 786-0 cells, including increased expression of nonsteroidal antiimflammatory drug-activated gene-1 (NAG-1) and endoplasmic reticulum stress which activates death receptor 5 and the extrinsic pathway of apoptosis. In addition, DIM-C-pPhtbu (40 mg/kg/d) also inhibited tumor growth in an orthotopic mouse model for renal carcinogenesis, and this was accompanied by induction of apoptosis in renal tumors treated with DIM-C-pPhtBu but not in tumors treated with the corn oil vehicle (control). Thus, DIM-C-pPhtBu and related compounds represent a novel class of mechanism-based drugs that have potential for treatment of renal adenocarcinoma for which there are currently limited options for successful chemotherapy.

published_or_final_version / Medicine / Master / Master of Medical Sciences

While extirpative surgery is curative for localized clear cell renal cell carcinoma (ccRCC), many patients develop recurrences or present with metastatic disease. Several aspects of ccRCC biology have been investigated, but these have been done in cell lines, which are known to poorly represent the tumour. Since cell lines are amenable to a wide array of experimental testing, the studies presented here demonstrate a novel method to generate ccRCC cell lines from primary tumours, which increases the rate of primary tumour cell line generation four fold. Additionally, ccRCC cells do not grow in serum-free media, which has been shown to be beneficial in other cancers. Therefore, we interrogated the effect of exogenous growth factors to optimize our serum-free media growth conditions, among which TGFb1 appeared to elicit the largest mitogenic effect. Once optimized, these findings will provide a valuable tool for understanding ccRCC tumour cell biology and identifying therapeutic targets.

While extirpative surgery is curative for localized clear cell renal cell carcinoma (ccRCC), many patients develop recurrences or present with metastatic disease. Several aspects of ccRCC biology have been investigated, but these have been done in cell lines, which are known to poorly represent the tumour. Since cell lines are amenable to a wide array of experimental testing, the studies presented here demonstrate a novel method to generate ccRCC cell lines from primary tumours, which increases the rate of primary tumour cell line generation four fold. Additionally, ccRCC cells do not grow in serum-free media, which has been shown to be beneficial in other cancers. Therefore, we interrogated the effect of exogenous growth factors to optimize our serum-free media growth conditions, among which TGFb1 appeared to elicit the largest mitogenic effect. Once optimized, these findings will provide a valuable tool for understanding ccRCC tumour cell biology and identifying therapeutic targets.

T cells are a crucial component of the immune system and play an important role in responses to pathogens, tumours, and transplanted tissues. In many human cancers, elevated numbers of tumour-infiltrating CD8+ killer T cells are associated with favourable outcomes, suggesting that enhancing T-cell responses could provide major therapeutic benefit for cancer patients. Thus, identifying factors that can promote protective T-cell responses is of great clinical importance. The cytokine interleukin-2 (IL-2) is a major inducer of T-cell proliferation and differentiation, and is used clinically to treat melanoma and renal cell carcinoma. The first two chapters of this thesis focus on the biochemical mechanisms by which IL-2 induces T-cell proliferation. By using mutant and chimeric cytokine receptors expressed in lymphocyte cell lines, the interplay between Shc and STAT5, two major mitogenic signaling pathways activated by the IL-2 receptor, are investigated, revealing an essential synergy between the two pathways for optimal lymphocyte proliferation. The third chapter of this thesis describes work done to identify cytokines that promote T-cell responses within the ovarian cancer microenvironment. In human diseases such as HIV/AIDS and cancer, high numbers of “polyfunctional” T cells (i.e., T cells capable of multiple effector functions) are associated with favourable outcomes. Using clinical ovarian cancer samples in a novel ex vivo assay, it was found that the ovarian tumour environment inhibits polyfunctional T-cell responses to varying extents among patients. After surveying a large panel of cytokines, the cytokine combination of IL-2, IL-12, and IL-18 was found to overcome the immunosuppressive environment to potently enhance CD8+ T-cell proliferation and polyfunctionality in all patient samples. The polyfunctional profiles induced by these cytokines are associated with protective immunity in various human conditions. Thus, these findings suggest that given the right signals, T cells can become highly polyfunctional effectors in the ovarian cancer microenvironment, which offers promise for the development of effective T-cell based therapies for this clinically challenging disease. / Graduate / 0982

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisors: Cheryl L. Walker and John H. Richburg. Vita. Includes bibliographical references. Also available from UMI.