No description available.

No description available.

No description available.

One of the most consistent biochemical changes associated with colon cancer progression is the altered expression of cell-associated carbohydrates. For example, the elevated expression of β1-6 branched N-linked oligosaccharides correlates with the presence of metastatic disease in colon cancer patients. Thus, it has become desirable to identify glycoproteins that are modified by these cancer-associated carbohydrates. Previous work in our laboratory identified the tumor-associated antigen 90 kDa (TAA90K) as a carrier of these cancer-associated carbohydrates. Since TAA90K has been previously implicated in cancer progression and metastasis, we examined its expression and function in human colon tumors. Immunohistochemical analysis revealed elevated expression of TAA90K in all tumor samples analyzed compared to normal colon. To examine the function of TAA90K in colon cancer, we performed protein binding and cellular assays with TAA90K purified from HT-29 human colon cancer cells infected with recombinant vaccinia virus expressing TAA90K. Purified TAA90K bound to ECM proteins including fibronectin, collagen IV, laminins-1, -5 and -10 and galectin-3. Unlike TAA90K isolated from other cell types, TAA90K isolated from HT-29 cells failed to mediate adhesion of colon cancer and normal cell lines, due in part to cell-specific glycosylation differences. Although TAA90K did not directly mediate cellular adhesion, it did modulate galectin-3-dependent adhesion of HT-29 cells. In addition, TAA90K bound to and was a substrate for MMP-7, a matrix metalloproteinase previously implicated in colon cancer progression. MMP-7-cleavage of TAA90K had little effect on its binding to pro- and active MMP-7, laminin-1 and galectin-3, but reduced significantly its binding to fibronectin and laminin-10. In addition, treatment of cells with MMP-7-cleaved TAA90K resulted in lower levels of proMMP-7 in the conditioned medium than cells treated with intact TAA90K. This may be mediated by the reduced binding of MMP-7-cleaved TAA90K to IL-6 and IL-1β, cytokines previously implicated in enhanced proMMP-7 expression in prostate cancer cells. Thus, a possible mechanism by which TAA90K may contribute to colon cancer progression is by modulating tumor cell adhesion to extracellular proteins and extracellular matrix remodeling through interactions with MMP-7 and galectin-3.

One of the most consistent biochemical changes associated with colon cancer progression is the altered expression of cell-associated carbohydrates. For example, the elevated expression of β1-6 branched N-linked oligosaccharides correlates with the presence of metastatic disease in colon cancer patients. Thus, it has become desirable to identify glycoproteins that are modified by these cancer-associated carbohydrates. Previous work in our laboratory identified the tumor-associated antigen 90 kDa (TAA90K) as a carrier of these cancer-associated carbohydrates. Since TAA90K has been previously implicated in cancer progression and metastasis, we examined its expression and function in human colon tumors. Immunohistochemical analysis revealed elevated expression of TAA90K in all tumor samples analyzed compared to normal colon. To examine the function of TAA90K in colon cancer, we performed protein binding and cellular assays with TAA90K purified from HT-29 human colon cancer cells infected with recombinant vaccinia virus expressing TAA90K. Purified TAA90K bound to ECM proteins including fibronectin, collagen IV, laminins-1, -5 and -10 and galectin-3. Unlike TAA90K isolated from other cell types, TAA90K isolated from HT-29 cells failed to mediate adhesion of colon cancer and normal cell lines, due in part to cell-specific glycosylation differences. Although TAA90K did not directly mediate cellular adhesion, it did modulate galectin-3-dependent adhesion of HT-29 cells. In addition, TAA90K bound to and was a substrate for MMP-7, a matrix metalloproteinase previously implicated in colon cancer progression. MMP-7-cleavage of TAA90K had little effect on its binding to pro- and active MMP-7, laminin-1 and galectin-3, but reduced significantly its binding to fibronectin and laminin-10. In addition, treatment of cells with MMP-7-cleaved TAA90K resulted in lower levels of proMMP-7 in the conditioned medium than cells treated with intact TAA90K. This may be mediated by the reduced binding of MMP-7-cleaved TAA90K to IL-6 and IL-1β, cytokines previously implicated in enhanced proMMP-7 expression in prostate cancer cells. Thus, a possible mechanism by which TAA90K may contribute to colon cancer progression is by modulating tumor cell adhesion to extracellular proteins and extracellular matrix remodeling through interactions with MMP-7 and galectin-3.

Helicase-like Transcription Factor (HLTF) is a DNA helicase protein which is homologous to SNF/SWI family. It has been demonstrated to be a functional homolog of yeast Rad5, required for the maintenance of genomic stability. Although the physiologic role of HLTF is largely unknown,inactivation of HLTF by promoter hypermethylation has been found in more than 40% human colon cancers. In this study, we have applied mouse transgenic approaches to determine whether loss of HLTF function could be important for colorectal carcinogenesis. HLTF knockout mice were generated by the deletion of first 5 exons of the HLTF gene. The complete loss of HLTF expression in HLTF -/- mice was confirmed by northern blot and real time RT-PCR assays. HLTF -/- mice did not show any developmental defects within a 2-year observation indicating that HLTF is dispensable for mouse development. Furthermore, HLTF -/- mice were free of intestinal or colorectal tumors or other types of tumors, suggesting that loss of HLTF function alone is not sufficient to drive oncogenic transformation in intestinal track and other tissues. To determine whether loss of HLTF function could cooperate with other tumor suppressors in the formation of colorectal cancers, we have bred HLTF knockout mice with the mutant mice for APC (adenomtous polyposis coli) and P53. In HLTF -/-APC Min/+ mice, a significantly increased formation of intestinal adenocarcinoma and colorectal cancers were observed. Although very few HLTF -/-P53 -/- mice developed colorectal cancers, these mice had increased incidence of the formation of metastatic lymphomas. Cytogenetic analysis of colorectal cancer cells derived from HLTF -/-APC Min/+ mice demonstrated a high incidence of gross chromosomal instabilities, including Robertsonian fusions, fragments and aneuploidy. All these genetic alterations were not observed in the intestinal tumor cells from APC Min/+, implicating that loss of HLTF function could induce genomic instability which contributes to intestinal carcinogenesis. To further investigate the role of HLTF in colorectal carcinogenesis, we have also applied a shRNA knockdown approach to down-regulate HLTF expression in human HCT-116 colon cancer cells. HCT-116 cells highly express HLTF and show less chromosomal instability, making these cells as a very useful model to investigate the loss of function of HLTF in human colorectal carcinogenesis. Using Western blot approach, we confirmed that HLTF knockdown HCT-116 cells had less than 5% of HLTF expression as compared to the scramble controls. By inoculating HLTF knockdown HCT-116 cells to Rag1 -/-IL2 -/- immunocompromised mice, we further demonstrated that HLTF knockdown promote tumor growth and invasion. Moreover, spectral karyotyping analysis revealed that HLTF knockdown human colon cancer cells had significantly increased chromosomal instability, including both aneuploidy and chromosomal translocation. Taken together, our work strongly indicates that loss of HLTF function can promote the malignant transformation of intestinal or colonic adenomas to carcinomas by inducing genomic instability. Given the high frequency of epigenetic inactivation by hypermethylation of HLTF in human colon cancers, our studies strongly suggest that this epigenetic alteration could be directly involved in the development of colorectal cancer rather than a consequence of this carcinogenesis.

Helicase-like Transcription Factor (HLTF) is a DNA helicase protein which is homologous to SNF/SWI family. It has been demonstrated to be a functional homolog of yeast Rad5, required for the maintenance of genomic stability. Although the physiologic role of HLTF is largely unknown,inactivation of HLTF by promoter hypermethylation has been found in more than 40% human colon cancers. In this study, we have applied mouse transgenic approaches to determine whether loss of HLTF function could be important for colorectal carcinogenesis. HLTF knockout mice were generated by the deletion of first 5 exons of the HLTF gene. The complete loss of HLTF expression in HLTF -/- mice was confirmed by northern blot and real time RT-PCR assays. HLTF -/- mice did not show any developmental defects within a 2-year observation indicating that HLTF is dispensable for mouse development. Furthermore, HLTF -/- mice were free of intestinal or colorectal tumors or other types of tumors, suggesting that loss of HLTF function alone is not sufficient to drive oncogenic transformation in intestinal track and other tissues. To determine whether loss of HLTF function could cooperate with other tumor suppressors in the formation of colorectal cancers, we have bred HLTF knockout mice with the mutant mice for APC (adenomtous polyposis coli) and P53. In HLTF -/-APC Min/+ mice, a significantly increased formation of intestinal adenocarcinoma and colorectal cancers were observed. Although very few HLTF -/-P53 -/- mice developed colorectal cancers, these mice had increased incidence of the formation of metastatic lymphomas. Cytogenetic analysis of colorectal cancer cells derived from HLTF -/-APC Min/+ mice demonstrated a high incidence of gross chromosomal instabilities, including Robertsonian fusions, fragments and aneuploidy. All these genetic alterations were not observed in the intestinal tumor cells from APC Min/+, implicating that loss of HLTF function could induce genomic instability which contributes to intestinal carcinogenesis. To further investigate the role of HLTF in colorectal carcinogenesis, we have also applied a shRNA knockdown approach to down-regulate HLTF expression in human HCT-116 colon cancer cells. HCT-116 cells highly express HLTF and show less chromosomal instability, making these cells as a very useful model to investigate the loss of function of HLTF in human colorectal carcinogenesis. Using Western blot approach, we confirmed that HLTF knockdown HCT-116 cells had less than 5% of HLTF expression as compared to the scramble controls. By inoculating HLTF knockdown HCT-116 cells to Rag1 -/-IL2 -/- immunocompromised mice, we further demonstrated that HLTF knockdown promote tumor growth and invasion. Moreover, spectral karyotyping analysis revealed that HLTF knockdown human colon cancer cells had significantly increased chromosomal instability, including both aneuploidy and chromosomal translocation. Taken together, our work strongly indicates that loss of HLTF function can promote the malignant transformation of intestinal or colonic adenomas to carcinomas by inducing genomic instability. Given the high frequency of epigenetic inactivation by hypermethylation of HLTF in human colon cancers, our studies strongly suggest that this epigenetic alteration could be directly involved in the development of colorectal cancer rather than a consequence of this carcinogenesis.

Many studies show that estradiol (E2) and consumption of soy and its primary phytoestrogen component genistein (GEN) can inhibit the formation of colon tumors. However, the effects of E2 and GEN at physiologically relevant levels in non-diseased colonocytes have yet to be investigated. We hypothesized that E2 and GEN could prove to be chemo-protective agents in the colon by moderately increasing apoptosis and decreasing proliferation in a healthy system. Thus, the presented studies focused on evaluating the effects of E2 and GEN in non-malignant colonocytes in vitro and in vivo to determine how the compounds influence the physiology of these cells. E2 (1 nM/L) and GEN treatments (1 and 10 microM/L) decreased cell growth, increased apoptosis, and increased p53 transcriptional activity in young adult mouse colonocytes, a non-malignant cell line. To study further the effects of E2 and GEN in healthy colonic epithelia, we evaluated physiologic changes in colonic crypts in ovariectomized mice given an E2 pellet, 1,000 ppm GEN diet, or a phytoestrogen free diet. As seen in vitro, E2 treated animals had significantly higher rates of apoptosis with GEN trending in the same fashion. These data demonstrate that E2 and GEN alter the physiology of non-malignant colonocytes. Collectively, with our previous data, this suggests that E2 and GEN influence colonocyte physiology and this state may partially explain how these compounds decrease risk of colon cancer.

Hepatic metastases are a frequent cause of mortality in colon cancer patients. Many patients with hepatic metastasis have large tumour burden, signaling the need for therapies capable of down-staging metastatic disease. Research evidence indicates that immune cells promote the metastasis of various primary cancers. We wish to determine whether immune cells play a role in the promotion of hepatic colon cancer metastasis. Using the well-characterised method of intrasplenic tumour cell injection, we developed hepatic metastases in both immunocompetent and immunoincompetent mice using a range of murine and human cancer cell lines. We analysed the immune cell infiltrates associated with hepatic metastases using flow cytometry and identified chemokines responsible for their recruitment using targeted protein arrays. The effect of immune cell depletion or inhibition of immune cell recruitment was determined using various in-vivo imaging techniques. Hepatic metastases developed using the murine colon cancer cell line MC38 were associated with CD11b⁺/Gr1^mid/CCR2⁺ monocytes, the recruitment of which was delayed by inhibition of tumour-derived CCL2. In contrast, human HT29, HCT-116 and LoVo hepatic metastases in SCID mice were associated with infiltrates of CD45⁺/CXCR2⁺ neutrophils recruited in response to tumour-derived Macrophage Inhibitory Factor (MIF). Depletion of Gr1^mid cells in CD11b-DTR transgenic mice delayed MC38 metastasis development, whilst neutrophil depletion using anti-Ly6G antibodies significantly inhibited the growth of HT29, HCT-116 and LoVo hepatic metastases. The neutrophils recruited to HT29, HCT-116 and LoVo hepatic metastases promoted angiogenesis, potentially through the expression of fibtroblast growth factor-2. This work demonstrates a role for myeloid cells in the development of hepatic metastasis from colon cancer and in doing so identifies various potential therapeutic targets.

No description available.