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Neutralizing Anti-Matrix Metalloproteinase 7 Antibody Inhibits Growth of Human Colon Cancer Xenografts
Figure 1. Tumor volume after tumor implantation (1x 10^6 cells) in syngeneic Balb/C mice (n=5 per group). Tumor volume = b^2 x a/2 (a= longer diameter and b= shorter diameter of the tumor).
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Tu1765
Neutralizing Anti-Matrix Metalloproteinase 7 Antibody Inhibits Growth of Human Colon Cancer Xenografts Zhongsheng Peng, Jean-Pierre Raufman, Guofeng Xie
Mutual Regulation of Expression Between MUC4 and CDx2 in Colon Cancer Cells Ashish K. Tiwari, Dhananjay Kunte, Jeffrey T. Brasky, Tina P. Gibson, Mart DeLaCruz, Preethi Subramanian, Ramesh K. Wali, Hemant K. Roy
Marix metalloproteinase (MMP) 7 is over-expressed in the majority of human colon tumors and is thought to play a key role in colon cancer cell growth, invasion and metastasis. In the ApcMin/+ mouse model of intestinal neoplasia, genetic ablation of MMP7 attenuates tumor formation. In clinical trials during the last decade, because of the complexity of the MMP family (> 30 members - some pro-proliferative and others anti-proliferative), synthetic broad-spectrum MMP inhibitors were mostly ineffective for the treatment of human cancer. However, the efficacy of specific therapies against individual MMPs has not been tested. Our previous In Vitro study showed that neutralizing anti-MMP7 antibody robustly attenuated cell proliferation in H508 human colon cancer cells. The objective of the present study was to determine whether MMP7 antibody inhibits human tumor growth In Vivo in a xenograft mouse model. To generate tumor implants, five million H508 cells were injected subcutaneously into the flanks of 6-week-old male nude mice. Tumor size was measured weekly using a caliper and tumor volumes were calculated using the formula: tumor volume = (length ×width2)/2. When tumors grew to a suitable size (~1000 mm3), tumors were excised and cut into small fragments. Ten-mg fragments were then injected subcutaneously into the flanks of 6-8 week-old nude mice. Three weeks after injection of tumor fragments, neutralizing polyclonal anti-MMP7 antibodies (50 μg; R&D systems) in 100 μL PBS or an equal volume of PBS alone was injected intraperitoneally twice weekly for 4 consecutive weeks. Mice were euthanized at week 7, and subcutaneous tumors, livers and lungs were examined with necropsy. Tumor volumes of control (N=14) and MMP7 antibody-treated (N=14) mice remained similar through the first 4 weeks (mean ± SE in mm3: 2nd wk: 113.8 ± 16.80 vs. 125.0 ± 17.8; 3rd wk: 522.5 ± 108.8 vs. 495.4 ± 104.2; 4th wk: 1031.6 ± 163.5 vs. 1078.9 ± 183.7; all p > 0.05). One week after starting treatment, tumor volumes in MMP7 antibody-treated mice were less than those in PBS-treated mice; this persisted through the end of treatment (5th wk: 2760.0 ± 403.2 vs. 3795.5 ± 715.6; 6th wk: 3849.6 ± 614.1 vs. 5447.9 ± 1242.1; 7th wk: 4993.6 ± 725.8 vs. 6795.8 ± 1028.1; all p < 0.01). At the end of treatment, tumor weights in MMP7 antibody-treated mice were less than those in PBStreated mice (mean ± SE, 3.2 ± 0.8 g vs. 3.8 ± 0.7 g, p=0.05). In both groups, no metastatic tumors were found in the lungs and livers. These data indicate that neutralizing anti-MMP7 antibody inhibits growth of human colon cancer xenografts in nude mice. This is the first report demonstrating the effectiveness of a MMP biological agent in reducing human tumor growth In Vivo. The results raise the possibility that such antibodies may be useful therapeutic agents for human colon neoplasia.
Background: Mucins are a family of large glycoproteins that have been implicated in colorectal carcinogenesis. Our group has explored the role of MUC4, a transmembrane mucin, which is generally upregulated in most cancers (pancreas, lung etc), during colorectal carcinogenesis and noted that MUC4 is paradoxically downregulated in colorectal cancers. The progressive downregulation occurs since early stages of carcinogenesis (premalignant mucosa). However, the mechanism remains unclear. CDX2 is a caudal-related homeobox gene that has been known to be dysregulated in colon carcinogenesis. Intriguingly, a recent report implicated CDX2 in MUC4 regulation in Barrett's esophagus. We, therefore, wanted to elucidate the role of CDX2 in the MUC4 loss in colon carcinogenesis Materials and Methods:HT29 cells were incubated in standard conditions and transiently transfected with CDX2 siRNA using Lipofectamine. Separately, HT29 cells were stably transfected with MUC4 shRNA and suitable control vector (CV). MUC4 shRNA transfected HT29 cells and CV transfected HT29 cells were treated with PI3K inhibitor LY294002. Protein lysates and RNA were obtained from the transfected cells and western blotting and qRT-PCR performed to assess the expression of MUC4, CDX2 and pAKT (S473). Results:In HT29 cells, a modest decrease in CDX2 expression by SiRNA (34% decreased CDX2 expression) concomitantly reduced MUC4 expression by 20%. Conversely, MUC4 shRNA transfection of HT29 cells (75% reduced MUC4 expression; p=0.05) resulted in 80% reduction in CDX2 mRNA expression and 60% reduction (p<0.02) in CDX2 protein levels. Since PI3K-AKT signaling cascade is known to modulate CDX2 levels, we assessed this pathway in MUC4 shRNA transfected HT29 cells, and demonstrated 50% increase (p<0.02) in Serine 473 phosphyrated AKT, which was mitigated by PI3K inhibitor, LY294002. However, despite the fact that MUC4 knockdown activated AKT, there was no effect of LY294002 treatment on the expression of CDX2 in MUC4 shRNA transfected HT29 cells, indicating that MUC4 modulates CDX2 expression via PI3K/pAKT independent manner. Conclusions: We report, herein, for the first time, that the dysregulation of two putative tumor suppressor genes, MUC4 and CDX2, is intimately related in colon cancer. Not only do CDX2 and MUC4 mirror each other's expression but also mutually regulate each other. Further studies are ongoing to dissect this important novel pathway in colon carcinogenesis.
Tu1768 Lineage Tracing of Cytokeratin 19 Labels Gastric, Intestinal and Colonic Stem Cells Samuel Asfaha, Russell Ericksen, Johannes von Burstin, Michael Quante, Wataru Shibata, Anil K. Rustgi, Timothy C. Wang Introduction: Tissue stem cells, characterized by multipotentiality and self renewing ability, are not readily distinguishable from other resident cells. Tissue stem cells are believed to be present in small numbers that may remain quiescent, or divide frequently as previously shown with Lgr-5+ cells. In the gastric antrum, intestine and colon, epithelial cells derived from the stem cells at the crypt base rapidly migrate up towards to the mucosal surface, whereas in the gastric corpus, stem cells located in the isthmus are believed to give rise to epithelial progenitors that undergo bipolar migration and differentiation into pit and oxyntic cell lineages. Cytokeratin 19 (K19) has been shown to be expressed in cells in the gastric isthmus and colonic and intestinal crypts. Therefore, we aimed to use the K19 gene promoter to drive Cre recombinase expression and to track the lineage of K19+ cells in an inducible fashion. Methods: In order to recapitulate the endogenous expression pattern of K19, we used a BAC strategy and generated a transgenic mouse with a Tamoxifen inducible Cre under the control of the K19 promoter (K19-BAC-Cre-ERTM). K19CreERT2 mice were crossed to ROSA26rLacZ (or GFP) reporter mice, treated the mice with tamoxifen (4 mg/ day p.o. for 3 days) and sacrificed at various time points (3 days, 7 days, 26 weeks and 52 weeks) following tamoxifen induction. Results: Treatment of K19CreERT2/Rosa26r mice with tamoxifen resulted in the selective marking of about 30% of gastric and intestinal glands. Following tamoxifen induction, in the corpus a few gastric pit cells near the isthmus were labeled at 3 days, whereas, all of pit cells within the labeled glands stained positively by 7 days. Consistent with the labeling of progenitor or stem cells, all pit cells within marked glands stained positively even up to 52 weeks following tamoxifen induction. In contrast, in the antrum, intestine and colon nearly all epithelial cells were labeled within 3 days
Tu1766 Increased ECM Density Increases Rock's Ability to Modulate Colon Cancer Proliferation but Does Not Change Its Impact on Invasion Sarah C. Glover, Ramana V. Vishnubhotla, Michael Cho, Shruthi Bharadwaj Both the density of the cells and the density of the extracellular matrix (ECM) proteins contribute to overall tissue density in both malignant and non-malignant tissues. To date, our work has focused on how the cellular aspects of a colon cancer impact ROCK's behavior. Specifically, we have shown that ROCK-II localizes to invadopodia in colon cancer where it appears to regulate the activities of MMP-2 and MMP-13 (Lab Invest, 87:1149) and, have
S-829
AGA Abstracts
AGA Abstracts
also demonstrated that ROCK-II is more active in colon cancer and hat its activity is greatest in the most cellularly dense portions of a tumor (Gastro. 138 (5 Supp 1): S-736). In this study, we sought to understand the contribution of the ECM density to the behavior both ROCK-I and ROCK-II to colon cancer cell proliferation and invasion. To accomplish this, SW620 cells, an aggressive colon cancer cell line, were seeded onto type I collagen-based hydrogels with collagen concentrations ranging from 1.5 to 4.0 mg/ml. ROCK-I or ROCKII expression was then knocked down using siRNA and after 72 hours, cell-containing scaffolds were evaluated for proliferation and invasion. We found that colon cancer cell proliferation increased by 50% in scaffolds that were 2.5 mg/ml and 250% in the 4 mg/ml scaffolds. Furthermore, knockdown of both ROCK-I and II led to a reduction in proliferation by half in cells grown in 2.5 mg/ml and 4 mg/ml scaffolds. Colon cancer cell invasion, however, was not significantly impacted by a change in collagen density but intriguingly, knockdown of ROCK I led to a more than 50% increase in cell invasion in cells grown in 1.5 mg/ml and 4 mg/ml scaffolds. ROCK-II knockdown led to decreased invasion in cells grown in all three ECM densities. Taken together, this data suggests that changes in the ECM density have a greater impact on colon cancer cell proliferation and that this is in part, ROCK mediated. Invasion, however, does not appear to be impacted by ECM density and as previously shown, ROCK-II knockdown led to decreased colon cancer invasion regardless of ECM density.
Tu1767
Tu1765
Neutralizing Anti-Matrix Metalloproteinase 7 Antibody Inhibits Growth of Human Colon Cancer Xenografts Zhongsheng Peng, Jean-Pierre Raufman, Guofeng Xie
Mutual Regulation of Expression Between MUC4 and CDx2 in Colon Cancer Cells Ashish K. Tiwari, Dhananjay Kunte, Jeffrey T. Brasky, Tina P. Gibson, Mart DeLaCruz, Preethi Subramanian, Ramesh K. Wali, Hemant K. Roy
Marix metalloproteinase (MMP) 7 is over-expressed in the majority of human colon tumors and is thought to play a key role in colon cancer cell growth, invasion and metastasis. In the ApcMin/+ mouse model of intestinal neoplasia, genetic ablation of MMP7 attenuates tumor formation. In clinical trials during the last decade, because of the complexity of the MMP family (> 30 members - some pro-proliferative and others anti-proliferative), synthetic broad-spectrum MMP inhibitors were mostly ineffective for the treatment of human cancer. However, the efficacy of specific therapies against individual MMPs has not been tested. Our previous In Vitro study showed that neutralizing anti-MMP7 antibody robustly attenuated cell proliferation in H508 human colon cancer cells. The objective of the present study was to determine whether MMP7 antibody inhibits human tumor growth In Vivo in a xenograft mouse model. To generate tumor implants, five million H508 cells were injected subcutaneously into the flanks of 6-week-old male nude mice. Tumor size was measured weekly using a caliper and tumor volumes were calculated using the formula: tumor volume = (length ×width2)/2. When tumors grew to a suitable size (~1000 mm3), tumors were excised and cut into small fragments. Ten-mg fragments were then injected subcutaneously into the flanks of 6-8 week-old nude mice. Three weeks after injection of tumor fragments, neutralizing polyclonal anti-MMP7 antibodies (50 μg; R&D systems) in 100 μL PBS or an equal volume of PBS alone was injected intraperitoneally twice weekly for 4 consecutive weeks. Mice were euthanized at week 7, and subcutaneous tumors, livers and lungs were examined with necropsy. Tumor volumes of control (N=14) and MMP7 antibody-treated (N=14) mice remained similar through the first 4 weeks (mean ± SE in mm3: 2nd wk: 113.8 ± 16.80 vs. 125.0 ± 17.8; 3rd wk: 522.5 ± 108.8 vs. 495.4 ± 104.2; 4th wk: 1031.6 ± 163.5 vs. 1078.9 ± 183.7; all p > 0.05). One week after starting treatment, tumor volumes in MMP7 antibody-treated mice were less than those in PBS-treated mice; this persisted through the end of treatment (5th wk: 2760.0 ± 403.2 vs. 3795.5 ± 715.6; 6th wk: 3849.6 ± 614.1 vs. 5447.9 ± 1242.1; 7th wk: 4993.6 ± 725.8 vs. 6795.8 ± 1028.1; all p < 0.01). At the end of treatment, tumor weights in MMP7 antibody-treated mice were less than those in PBStreated mice (mean ± SE, 3.2 ± 0.8 g vs. 3.8 ± 0.7 g, p=0.05). In both groups, no metastatic tumors were found in the lungs and livers. These data indicate that neutralizing anti-MMP7 antibody inhibits growth of human colon cancer xenografts in nude mice. This is the first report demonstrating the effectiveness of a MMP biological agent in reducing human tumor growth In Vivo. The results raise the possibility that such antibodies may be useful therapeutic agents for human colon neoplasia.
Background: Mucins are a family of large glycoproteins that have been implicated in colorectal carcinogenesis. Our group has explored the role of MUC4, a transmembrane mucin, which is generally upregulated in most cancers (pancreas, lung etc), during colorectal carcinogenesis and noted that MUC4 is paradoxically downregulated in colorectal cancers. The progressive downregulation occurs since early stages of carcinogenesis (premalignant mucosa). However, the mechanism remains unclear. CDX2 is a caudal-related homeobox gene that has been known to be dysregulated in colon carcinogenesis. Intriguingly, a recent report implicated CDX2 in MUC4 regulation in Barrett's esophagus. We, therefore, wanted to elucidate the role of CDX2 in the MUC4 loss in colon carcinogenesis Materials and Methods:HT29 cells were incubated in standard conditions and transiently transfected with CDX2 siRNA using Lipofectamine. Separately, HT29 cells were stably transfected with MUC4 shRNA and suitable control vector (CV). MUC4 shRNA transfected HT29 cells and CV transfected HT29 cells were treated with PI3K inhibitor LY294002. Protein lysates and RNA were obtained from the transfected cells and western blotting and qRT-PCR performed to assess the expression of MUC4, CDX2 and pAKT (S473). Results:In HT29 cells, a modest decrease in CDX2 expression by SiRNA (34% decreased CDX2 expression) concomitantly reduced MUC4 expression by 20%. Conversely, MUC4 shRNA transfection of HT29 cells (75% reduced MUC4 expression; p=0.05) resulted in 80% reduction in CDX2 mRNA expression and 60% reduction (p<0.02) in CDX2 protein levels. Since PI3K-AKT signaling cascade is known to modulate CDX2 levels, we assessed this pathway in MUC4 shRNA transfected HT29 cells, and demonstrated 50% increase (p<0.02) in Serine 473 phosphyrated AKT, which was mitigated by PI3K inhibitor, LY294002. However, despite the fact that MUC4 knockdown activated AKT, there was no effect of LY294002 treatment on the expression of CDX2 in MUC4 shRNA transfected HT29 cells, indicating that MUC4 modulates CDX2 expression via PI3K/pAKT independent manner. Conclusions: We report, herein, for the first time, that the dysregulation of two putative tumor suppressor genes, MUC4 and CDX2, is intimately related in colon cancer. Not only do CDX2 and MUC4 mirror each other's expression but also mutually regulate each other. Further studies are ongoing to dissect this important novel pathway in colon carcinogenesis.
Tu1768 Lineage Tracing of Cytokeratin 19 Labels Gastric, Intestinal and Colonic Stem Cells Samuel Asfaha, Russell Ericksen, Johannes von Burstin, Michael Quante, Wataru Shibata, Anil K. Rustgi, Timothy C. Wang Introduction: Tissue stem cells, characterized by multipotentiality and self renewing ability, are not readily distinguishable from other resident cells. Tissue stem cells are believed to be present in small numbers that may remain quiescent, or divide frequently as previously shown with Lgr-5+ cells. In the gastric antrum, intestine and colon, epithelial cells derived from the stem cells at the crypt base rapidly migrate up towards to the mucosal surface, whereas in the gastric corpus, stem cells located in the isthmus are believed to give rise to epithelial progenitors that undergo bipolar migration and differentiation into pit and oxyntic cell lineages. Cytokeratin 19 (K19) has been shown to be expressed in cells in the gastric isthmus and colonic and intestinal crypts. Therefore, we aimed to use the K19 gene promoter to drive Cre recombinase expression and to track the lineage of K19+ cells in an inducible fashion. Methods: In order to recapitulate the endogenous expression pattern of K19, we used a BAC strategy and generated a transgenic mouse with a Tamoxifen inducible Cre under the control of the K19 promoter (K19-BAC-Cre-ERTM). K19CreERT2 mice were crossed to ROSA26rLacZ (or GFP) reporter mice, treated the mice with tamoxifen (4 mg/ day p.o. for 3 days) and sacrificed at various time points (3 days, 7 days, 26 weeks and 52 weeks) following tamoxifen induction. Results: Treatment of K19CreERT2/Rosa26r mice with tamoxifen resulted in the selective marking of about 30% of gastric and intestinal glands. Following tamoxifen induction, in the corpus a few gastric pit cells near the isthmus were labeled at 3 days, whereas, all of pit cells within the labeled glands stained positively by 7 days. Consistent with the labeling of progenitor or stem cells, all pit cells within marked glands stained positively even up to 52 weeks following tamoxifen induction. In contrast, in the antrum, intestine and colon nearly all epithelial cells were labeled within 3 days
Tu1766 Increased ECM Density Increases Rock's Ability to Modulate Colon Cancer Proliferation but Does Not Change Its Impact on Invasion Sarah C. Glover, Ramana V. Vishnubhotla, Michael Cho, Shruthi Bharadwaj Both the density of the cells and the density of the extracellular matrix (ECM) proteins contribute to overall tissue density in both malignant and non-malignant tissues. To date, our work has focused on how the cellular aspects of a colon cancer impact ROCK's behavior. Specifically, we have shown that ROCK-II localizes to invadopodia in colon cancer where it appears to regulate the activities of MMP-2 and MMP-13 (Lab Invest, 87:1149) and, have
S-829
AGA Abstracts
AGA Abstracts
also demonstrated that ROCK-II is more active in colon cancer and hat its activity is greatest in the most cellularly dense portions of a tumor (Gastro. 138 (5 Supp 1): S-736). In this study, we sought to understand the contribution of the ECM density to the behavior both ROCK-I and ROCK-II to colon cancer cell proliferation and invasion. To accomplish this, SW620 cells, an aggressive colon cancer cell line, were seeded onto type I collagen-based hydrogels with collagen concentrations ranging from 1.5 to 4.0 mg/ml. ROCK-I or ROCKII expression was then knocked down using siRNA and after 72 hours, cell-containing scaffolds were evaluated for proliferation and invasion. We found that colon cancer cell proliferation increased by 50% in scaffolds that were 2.5 mg/ml and 250% in the 4 mg/ml scaffolds. Furthermore, knockdown of both ROCK-I and II led to a reduction in proliferation by half in cells grown in 2.5 mg/ml and 4 mg/ml scaffolds. Colon cancer cell invasion, however, was not significantly impacted by a change in collagen density but intriguingly, knockdown of ROCK I led to a more than 50% increase in cell invasion in cells grown in 1.5 mg/ml and 4 mg/ml scaffolds. ROCK-II knockdown led to decreased invasion in cells grown in all three ECM densities. Taken together, this data suggests that changes in the ECM density have a greater impact on colon cancer cell proliferation and that this is in part, ROCK mediated. Invasion, however, does not appear to be impacted by ECM density and as previously shown, ROCK-II knockdown led to decreased colon cancer invasion regardless of ECM density.