Boryana K. Popivanova

Blocking TNF-α in mice reduces colorectal carcinogenesis associated with chronic colitis

The inflammatory bowel disease ulcerative colitis (UC) frequently progresses to colon cancer. To understand the mechanisms by which UC patients develop colon carcinomas, we used a mouse model of the disease whereby administration of azoxymethane (AOM) followed by repeated dextran sulfate sodium (DSS) ingestion causes severe colonic inflammation and the subsequent development of multiple tumors. We found that treating WT mice with AOM and DSS increased TNF-alpha expression and the number of infiltrating leukocytes expressing its major receptor, p55 (TNF-Rp55), in the lamina propria and submucosal regions of the colon. This was followed by the development of multiple colonic tumors. Mice lacking TNF-Rp55 and treated with AOM and DSS showed reduced mucosal damage, reduced infiltration of macrophages and neutrophils, and attenuated subsequent tumor formation. WT mice transplanted with TNF-Rp55-deficient bone marrow also developed significantly fewer tumors after AOM and DSS treatment than either WT mice or TNF-Rp55-deficient mice transplanted with WT bone marrow. Furthermore, administration of etanercept, a specific antagonist of TNF-alpha, to WT mice after treatment with AOM and DSS markedly reduced the number and size of tumors and reduced colonic infiltration by neutrophils and macrophages. These observations identify TNF-alpha as a crucial mediator of the initiation and progression of colitis-associated colon carcinogenesis and suggest that targeting TNF-alpha may be useful in treating colon cancer in individuals with UC.

Pim-3, a Proto-Oncogene with Serine/Threonine Kinase Activity, Is Aberrantly Expressed in Human Pancreatic Cancer and Phosphorylates Bad to Block Bad-Mediated Apoptosis in Human Pancreatic Cancer Cell Lines

Pancreatic cancer still remains a serious health problem with <5% 5-year survival rate for all stages. To develop an effective treatment, it is necessary to identify a target molecule that is crucially involved in pancreatic tumor growth. We previously observed that Pim-3, a member of the proto-oncogene Pim family that expresses serine/threonine kinase activity, was aberrantly expressed in human and mouse hepatomas but not in normal liver. Here, we show that Pim-3 is also expressed in malignant lesions of the pancreas but not in normal pancreatic tissue. Moreover, Pim-3 mRNA and protein were constitutively expressed in all human pancreatic cancer cell lines that we examined and colocalized with the proapoptotic protein Bad. The ablation of endogenous Pim-3 by small hairpin RNA transfection promoted apoptosis, as evidenced by increases in a proportion of cells in the sub-G(1) fraction of the cell cycle and in phosphatidyl serine externalization. A proapoptotic molecule, Bad, was phosphorylated constitutively at Ser(112) but not Ser(136) in human pancreatic cancer cell lines and this phosphorylation is presumed to represent its inactive form. Phosphorylation of Bad and the expression of an antiapoptotic molecule, Bcl-X(L), were reduced by the ablation of endogenous Pim-3. Thus, we provide the first evidence that Pim-3 can inactivate Bad and maintain the expression of Bcl-X(L) and thus prevent apoptosis of human pancreatic cancer cells. This may contribute to the net increase in tumor volume or tumor growth in pancreatic cancer.

Blockade of a Chemokine, CCL2, Reduces Chronic Colitis-Associated Carcinogenesis in Mice

Accumulating evidence indicates the crucial contribution of chronic inflammation to various types of carcinogenesis, including colon carcinoma associated with ulcerative colitis and asbestosis-induced malignant mesothelioma. Ulcerative colitis-associated colon carcinogenesis can be recapitulated in mice by azoxymethane administration followed by repetitive dextran sulfate sodium ingestion. In the course of this carcinogenesis process, the expression of a macrophage-tropic chemokine, CCL2, was enhanced together with intracolonic massive infiltration of macrophages, which were a major source of cyclooxygenase (COX)-2, a crucial mediator of colon carcinogenesis. Mice deficient in CCL2-specific receptor, CCR2, exhibited less macrophage infiltration and lower tumor numbers with attenuated COX-2 expression. Moreover, CCL2 antagonists decreased intracolonic macrophage infiltration and COX-2 expression, attenuated neovascularization, and eventually reduced the numbers and size of colon tumors, even when given after multiple colon tumors have developed. These observations identify CCL2 as a crucial mediator of the initiation and progression of chronic colitis-associated colon carcinogenesis and suggest that targeting CCL2 may be useful in treating colon cancers, particularly those associated with chronic inflammation.

Aberrant expression of serine/threonine kinase Pim-3 in hepatocellular carcinoma development and its role in the proliferation of human hepatoma cell lines.

Most cases of human hepatocellular carcinoma develop after persistent chronic infection with human hepatitis B virus or hepatitis C virus, and host responses are presumed to have major roles in this process. To recapitulate this process, we have developed the mouse model of hepatocellular carcinoma using hepatitis B virus surface antigen transgenic mice. To identify the genes associated with hepatocarcinogenesis in this model, we compared the gene expression patterns between pre‐malignant lesions surrounded by hepatocellular carcinoma tissues and control liver tissues by using a fluorescent differential display analysis. Among the genes that were expressed differentially in the pre‐malignant lesions, we focused on Pim‐3, a member of a proto‐oncogene Pim family, because its contribution to hepatocarcinogenesis remains unknown. Moreover, the unavailability of the nucleotide sequence of full‐length human Pim‐3 cDNA prompted us to clone it from the cDNA library constructed from a human hepatoma cell line, HepG2. The obtained 2,392 bp human Pim‐3 cDNA encodes a predicted open reading frame consisting of 326 amino acids. Pim‐3 mRNA was selectively expressed in human hepatoma cell lines, but not in normal liver tissues. Moreover, Pim‐3 protein was detected in human hepatocellular carcinoma tissues and cell lines but not in normal hepatocytes. Furthermore, cell proliferation was attenuated and apoptosis was enhanced in human hepatoma cell lines by the ablation of Pim‐3 gene with RNA interference. These observations suggest that aberrantly expressed Pim‐3 can cause autonomous cell proliferation or prevent apoptosis in hepatoma cell lines.

Prostaglandin E2 Signaling and Bacterial Infection Recruit Tumor-Promoting Macrophages to Mouse Gastric Tumors

BACKGROUND & AIMS Helicobacter pylori infection induces an inflammatory response, which can contribute to gastric tumorigenesis. Induction of cyclooxygenase-2 (COX-2) results in production of prostaglandin E(2) (PGE(2)), which mediates inflammation. We investigated the roles of bacterial infection and PGE(2) signaling in gastric tumorigenesis in mice. METHODS We generated a germfree (GF) colony of K19-Wnt1/C2mE mice (Gan mice); these mice develop gastric cancer. We examined tumor phenotypes, expression of cytokines and chemokines, and recruitment of macrophages. We also investigated PGE(2) signaling through the PGE(2) receptor subtype 4 (EP4) in Gan mice given specific inhibitors. RESULTS Gan mice raised in a specific pathogen-free facility developed large gastric tumors, whereas gastric tumorigenesis was significantly suppressed in GF-Gan mice; reconstitution of commensal flora or infection with Helicobacter felis induced gastric tumor development in these mice. Macrophage infiltration was significantly suppressed in the stomachs of GF-Gan mice. Gan mice given an EP4 inhibitor had decreased expression of cytokines and chemokines. PGE(2) signaling and bacterial infection or stimulation with lipopolysaccharide induced expression of the chemokine C-C motif ligand 2 (CCL2) (which attracts macrophage) in tumor stromal cells or cultured macrophages, respectively. CCL2 inhibition suppressed macrophage infiltration in tumors, and depletion of macrophages from the tumors of Gan mice led to signs of tumor regression. Wnt signaling was suppressed in the tumors of GF-Gan and Gan mice given injections of tumor necrosis factor-α neutralizing antibody. CONCLUSIONS Bacterial infection and PGE(2) signaling are required for gastric tumorigenesis in mice; they cooperate to up-regulate CCL2, which recruits macrophage to gastric tumors. Macrophage-derived tumor necrosis factor-α promotes Wnt signaling in epithelial cells, which contributes to gastric tumorigenesis.

Proto-oncogene, Pim-3 with serine/threonine kinase activity, is aberrantly expressed in human colon cancer cells and can prevent Bad-mediated apoptosis

We previously observed that Pim‐3 with serine/threonine kinase activity, was aberrantly expressed in malignant lesions of endoderm‐derived organs, liver and pancreas. Because Pim‐3 protein was not detected in normal colon mucosal tissues, we evaluated Pim‐3 expression in malignant lesions of human colon, another endoderm‐derived organ. Pim‐3 was detected immunohistochemically in well‐differentiated (43/68 cases) and moderately differentiated (23/41 cases) but not poorly differentiated colon adenocarcinomas (0/5 cases). Moreover, Pim‐3 proteins were detected in adenoma (35/40 cases) and normal mucosa (26/111 cases), which are adjacent to adenocarcinoma. Pim‐3 was constitutively expressed in SW480 cells and the transfection with Pim‐3 short hairpin RNA promoted apoptosis. In the same cell line, a pro‐apoptotic molecule, Bad, was phosphorylated at Ser112 and Ser136 sites of phosphorylation that are representative of its inactive form. Ser112 but not Ser136 phosphorylation in this cell line was abrogated by Pim‐3 knockdown. Furthermore, in human colon cancer tissues, Pim‐3 co‐localized with Bad in all cases (9/9) and with phospho‐Ser112Bad in most cases (6/9). These observations suggest that Pim‐3 can inactivate Bad by phosphorylating its Ser112 in human colon cancer cells and thus may prevent apoptosis and promote progression of human colon cancer. (Cancer Sci 2007; 98: 321–328)

Crucial involvement of the CX3CR1-CX3CL1 axis in dextran sulfate sodium-mediated acute colitis in mice.

Ingestion of DSS solution can induce in rodents acute colitis with a massive infiltration of neutrophils and macropahges, mimicking pathological changes observed in the acute phase of UC patients. Concomitantly, DSS ingestion enhanced the expression of a potent macrophage‐tropic chemokine, CX3CL1/fractalkine, and its receptor, CX3CR1, in the colon. WT but not CX3CR1‐deficient mice exhibited marked body weight loss and shortening of the colon after DSS ingestion. Moreover, inflammatory cell infiltration was attenuated in CX3CR1‐deficient mice together with reduced destruction of glandular architecture compared with WT mice. DSS ingestion enhanced intracolonic iNOS expression by macrophages and nitrotyrosine generation in WT mice, but iNOS expression and nitrotyrosine generation were attenuated in CX3CR1‐deficient mice. The analysis on bone marrow chimeric mice revealed that bone marrow‐derived but not non‐bone marrow‐derived CX3CR1‐expressing cells were a major source of iNOS. These observations would indicate that the CX3CL1‐CX3CR1 axis can regulate the expression of iNOS, a crucial mediator of DSS‐induced colitis. Thus, targeting the CX3CL1‐CX3CR1 axis may be effective for the treatment of IBDs such as UC.

Accumulation of microglial cells expressing ELR motif-positive CXC chemokines and their receptor CXCR2 in monkey hippocampus after ischemia-reperfusion.

ELR(+) CXC chemokines including IL-8 are known to be involved in the ischemia-reperfusion injuries in various organs including rodent brain. However, the roles of these chemokines during the ischemia-reperfusion injuries of the primate brain still remain unknown. Here, we studied expressions of CXC chemokines and their receptor CXCR2 in monkey hippocampus known to develop total CA1 neuronal loss on day 5 after 20-min ischemia and reperfusion. ELR(+) chemokines and their receptor CXCR2 were not detected in the hippocampus of non-ischemic monkeys. On the contrary, at 30-60 min after the start of reperfusion, CD68-positive microglial cells increased significantly in the hippocampal CA1 sector, but there was negligible infiltration of neutrophils. These microglial cells expressed simultaneously growth regulated oncogene (Gro)-alpha and other ELR(+) CXC chemokines. Moreover, CD68-positive microglial cells also expressed the receptor for ELR(+) CXC chemokines. On day 4, capillary endothelial cells were significantly increased in the CA1 sector. Considering that ELR(+) CXC chemokines have potent angiogenic activities, the coordinate expression of ELR(+) CXC chemokines and their receptor CXCR2 in microglial cells may be related not only to the ischemic brain injuries but also to the microglial and capillary proliferation in the monkey hippocampus.

Activation of epidermal growth factor receptor signaling by the prostaglandin E2 receptor EP4 pathway during gastric tumorigenesis

Cyclooxygenase‐2 (COX‐2) plays an important role in tumorigenesis through prostaglandin E2 (PGE2) biosynthesis. It has been shown by in vitro studies that PGE2 signaling transactivates epidermal growth factor receptor (EGFR) through an intracellular mechanism. However, the mechanisms underlying PGE2‐induced EGFR activation in in vivo tumors are still not fully understood. We previously constructed transgenic mice that develop gastric tumors caused by oncogenic activation and PGE2 pathway induction. Importantly, expression of EGFR ligands, epiregulin, amphiregulin, heparin‐binding EGF‐like growth factor, and betacellulin, as well as a disintegrin and metalloproteinases (ADAMs), ADAM8, ADAM9, ADAM10, and ADAM17 were significantly increased in the mouse gastric tumors in a PGE2 pathway‐dependent manner. These ADAMs can activate EGFR by ectodomain shedding of EGFR ligands. Notably, the extensive induction of EGFR ligands and ADAMs was suppressed by inhibition of the PGE2 receptor EP4. Moreover, EP4 signaling induced expression of amphiregulin and epiregulin in activated macrophages, whereas EP4 pathway was required for basal expression of epiregulin in gastric epithelial cells. In contrast, ADAMs were not induced directly by PGE2 in these cells, suggesting indirect mechanism possibly through PGE2‐associated inflammatory responses. These results suggest that PGE2 signaling through EP4 activates EGFR in gastric tumors through global induction of EGFR ligands and ADAMs in several cell types either by direct or indirect mechanism. Importantly, gastric tumorigenesis of the transgenic mice was significantly suppressed by combination treatment with EGFR and COX‐2 inhibitors. Therefore, it is possible that inhibition of both COX‐2/PGE2 and EGFR pathways represents an effective strategy for preventing gastric cancer. (Cancer Sci 2011; 102: 713–719)

Tumor Necrosis Factor (TNF) and Chemokines in Colitis-Associated Cancer

The connection between inflammation and tumorigenesis has been well established, based on a great deal of supporting evidence obtained from epidemiological, pharmacological, and genetic studies. One representative example is inflammatory bowel disease, because it is an important risk factor for the development of colon cancer. Moreover, intratumoral infiltration of inflammatory cells suggests the involvement of inflammatory responses also in other forms of sporadic as well as heritable colon cancer. Inflammatory responses and tumorigenesis activate similar sets of transcription factors such as NF-κB, Stat3, and hypoxia inducible factor and eventually enhances the expression of inflammatory cytokines including tumor necrosis factor (TNF) and chemokines. The expression of TNF and chemokines is aberrantly expressed in a mouse model of colitis-associated carcinogenesis as well as in inflammatory bowel disease and colon cancer in humans. Here, after summarizing the presumed actions of TNF and chemokines in tumor biology, we will discuss the potential roles of TNF and chemokines in chronic inflammation-associated colon cancer in mice.