Despite the importance of stromal cells in tumor progression, our overall understanding of the molecular signals that regulate the complex cellular interactions within tumor stroma is limited. Here, we provide multiple lines of evidence that tumor-associated macrophages (TAM) preferentially traffic to stromal areas formed within tumors in a manner dependent on a hyaluronan (HA)-rich tumor microenvironment. To address the role of stroma-derived HA in macrophage recruitment, we disrupted the HA synthase 2 (Has2) gene in stromal fibroblasts using conditional gene targeting. The Has2 null fibroblasts showed severe impairment in recruiting macrophages when inoculated with tumor cells into nude mice, which shows the contribution of stroma-derived HA in intratumoral macrophage mobilization. Furthermore, a deficiency in stromal HA attenuated tumor angiogenesis and lymphangiogenesis concomitantly with impaired macrophage recruitment. Taken together, our results suggest that stromal HA serves as a microenvironmental signal for the recruitment of TAMs, which are key regulatory cells involved in tumor neovascularization. Cancer Res; 70(18); 7073-83. ©2010 AACR.

The aryl hydrocarbon receptor (AhR) plays a suppressive role in cecal carcinogenesis by CUL4B/AhR-mediated ubiquitylation and degradation of β-catenin, which is activated by xenobiotics and natural ligands. AhR-deficient (AhR(-)(/-)) mice develop cecal tumors with severe inflammation. To elucidate whether the tumors develop autonomously in AhR(-/-) mice due to impaired β-catenin degradation or in association with accelerated inflammation, we performed two kinds of experiments using germ-free (GF) AhR(-/-) mice and compound mutant mice lacking genes for AhR and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which plays an essential role in caspase-1 activation in inflammasomes. Both GF AhR(-/-) and AhR(-/-)•ASC(-/-) mice showed considerably reduced tumor development compared with that in AhR(-/-) mice albeit in a 'cancer-prone' state with aberrant β-catenin accumulation. Blocking of the interleukin (IL)-1β signaling pathway by treatment with a caspase-1 inhibitor, YVAD, reduced cecal tumorigenesis in AhR(-/-) mice. Signal transducers and activators of transcription 3 (STAT3) activation was detected in the cecal epithelium of the AhR(-/-) mice due to enhanced IL-6 production. An inhibitor of the STAT3 signaling pathway, AG490 suppressed the tumor formation. ASC-mediated inflammation was also found to play a critical role in tumor development in Apc(Min/+) mice, a mouse model of familial adenomatous polyposis. Collectively, these results revealed an important role of the bacteria-triggered or ASC-mediated inflammation signaling pathway in the intestinal tumorigenesis of mice and suggest a possible chemical therapeutic intervention, including AhR ligands and inhibitors of the inflammation pathway.

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Cholangiocarcinoma (CCC) is a strongly aggressive malignancy for which surgical resection is the only potential curative therapy. Sorafenib, a multikinase inhibitor of the RAF/MEK/ERK pathway, is a molecular-targeted drug that is approved for hepatocellular carcinoma (HCC) but not for CCC. The differences in signaling pathway characteristics under sorafenib treatment between HCC (HLF, Huh7, PLC/PRF/5) and CCC (RBE, YSCCC, Huh28) cell lines were therefore investigated using cell proliferation, western blotting, and apoptosis analyses. Sorafenib inhibited cell growth significantly less in CCC cells than in HCC cells, with lower suppression of ERK phosphorylation. Significantly decreased AKT Ser473 phosphorylation in HCC cells, and conversely enhanced phosphorylation of AKT Ser473 and mTORC2 in CCC cells, were observed with sorafenib treatment. Disassembly of the mTORC2 complex in RBE cells with siRNA targeting Rictor resulted in the downregulation of AKT Ser473 phosphorylation and enhanced apoptosis presumably via increased FOXO1, which consequently suppressed RBE cell proliferation. Phosphorylation of mTORC1 and autophagy were not influenced by sorafenib in CCC cells. Simultaneous administration of everolimus to suppress activated mTORC1 in RBE cells revealed that combined everolimus and sorafenib treatment under mTORC2 disassembly could enhance growth inhibition through the suppression of both sorafenib- and everolimus-dependent AKT Ser473 phosphorylation in addition to the inhibition of mTORC1 phosphorylation. Prevention of escape by AKT/mTOR signaling from the RAF/MEK/ERK pathway in sorafenib treatment by suppressing mTORC2 activity may lead to promising new approaches in CCC therapy.

Background: KMD-3213 is an a 1A -adrenoceptor-selective antagonist currently being developed for the treatment of urinary outlet obstruction in patients with benign prostatic hyperplasia. In the present study, the uroselectivity of KMD-3213 was evaluated and compared with that of prazosin and tamsulosin in a decerebrate dog model. Methods: Intercollicular decerebration was carried out in male mongrel dogs under anesthesia. The inhibitory effects of intravenously and intraduodenally administered compounds on the increase in intraurethral pressure (IUP) induced by electrical stimulation of the hypogastric nerve were estimated. Systemic blood pressure was measured simultaneously. Results:The a 1 -antagonists tested produced a dose-dependent inhibition of the induced IUP response and decreased mean blood pressure (MBP). The ID 50 of KMD-3213, tamsulosin and prazosin for IUP (dose required to inhibit the increase in IUP by 50%) was 3.15, 1.73 and 11.8 mg/kg i.v., respectively, and the ED 20 for the hypotensive effect (dose required to reduce MBP by 20%) was 8.03, 0.59 and 2.46 mg/kg i.v., respectively. The data indicate that uroselectivity (ED 20 /ID 50 ) of KMD-3213 is 12-and 7.5-fold higher than that of prazosin and tamsulosin, respectively. When the drugs were administered intraduodenally, KMD-3213 was sufficiently absorbed from the digestive tract and continued to demonstrate at least 3.8-fold higher uroselectivity than tamsulosin. Conclusion: Based on these findings, KMD-3213 appears to be an effective orally active compound for decreasing urethral resistance during micturition that does not induce any negative cardiovascular effects in patients with benign prostatic hyperplasia.

We report a case of a patient with seminal vesicle-rectal fistula, an extremely rare complication of low anterior resection of the rectum. A 53-year-old man with rectal adenocarcinoma underwent low anterior resection in our hospital. The patient experienced diarrhea, pneumaturia, and low-grade fever on postoperative day 13. A computed tomography scan showed emphysema in the right seminal vesicle. We concluded that anastomotic leakage induced a seminal vesicle-rectal fistula. The patient underwent conservative therapy with total parenteral nutrition and oral intake of metronidazole. Diarrhea and pneumaturia rapidly improved after metronidazole administration and the patient was successfully cured without invasive therapy such as colostomy or surgical drainage. A seminal vesicle-rectal fistula is a rare complication of low anterior resection, and therapeutic strategies for this condition remain elusive. Our report provides valuable information on the successful conservative treatment of a secondary seminal vesicle-rectal fistula that developed after low anterior resection of the rectum in a patient.

ASC (apoptosis-associated speck-like protein containing a CARD) is a key adaptor molecule of inflammasomes that mediates inflammatory and apoptotic signals. Aberrant methylation-induced silencing of ASC has been observed in a variety of cancer cells, thus implicating ASC in tumor suppression, although this role remains incompletely defined especially in the context of closely neighboring cell proliferation. As ASC has been confirmed to be silenced by abnormal methylation in HT1080 fibrosarcoma cells as well, this cell line was investigated to characterize the precise role and mechanism of ASC in tumor progression. The effects of ASC were examined using in vitro cell cultures based on comparisons between low and high cell density conditions as well as in a xenograft murine model. ASC overexpression was established by insertion of the ASC gene into pcDNA3 and pMX-IRES-GFP vectors, the latter being packed into a retrovirus and subjected to reproducible competitive assays using parental cells as an internal control, for evaluation of cell viability. p21 and p53 were silenced using shRNA. Cell viability was suppressed in ASC-expressing transfectants as compared with control cells at high cell density conditions in in vitro culture and colony formation assays and in in vivo ectopic tumor formation trials. This suppression was not detected in low cell density conditions. Furthermore, remarkable progression of apoptosis was observed in ASC-introduced cells at a high cell density, but not at a low one. ASC-dependent apoptosis was mediated not by p21, p53, or caspase-1, but rather by cleavage of caspase-9 as well as by suppression of the NF-κB-related X-linked inhibitor-of-apoptosis protein. Caspase-9 cleavage was observed to be dependent on gap junction formation. The remarkable effect of ASC on the induction of apoptosis through caspase-9 and gap junctions revealed in this study may lead to promising new approaches in anticancer therapy.

Edited by Charles E. Samuel Fascin1 is an actin-bundling protein involved in cancer cell migration and has recently been shown also to have roles in virus-mediated immune cell responses. Because viral infection has been shown to activate immune cells and to induce interferon-␤ expression in human cancer cells, we evaluated the effects of fascin1 on virus-dependent signaling via the membrane-and actin-associated protein RIG-I (retinoic acid-inducible gene I) in colon cancer cells. We knocked down fascin1 expression with shRNA retrovirally transduced into a DLD-1 colon cancer and L929 fibroblast-like cell lines and used luciferase reporter assays and co-immunoprecipitation to identify fascin1 targets. We found that intracellular poly(I⅐C) transfection to mimic viral infection enhances the RIG-I/MDA5 (melanoma differentiation-associated gene 5)-mediated dimerization of interferon regulatory factor 3 (IRF-3). The transfection also significantly increased the expression levels of IRF-7, interferon-␤, and interferon-inducible cytokine IP-10 in fascin1-deleted cells compared with controls while significantly suppressing cell growth, migration, and invasion. We also found that fascin1 constitutively interacts with IB kinase ⑀ (IKK⑀) in the RIG-I signaling pathway. In summary, we have identified fascin1 as a suppressor of the RIG-I signaling pathway associating with IB kinase ⑀ in DLD-1 colon cancer cells to suppress immune responses to viral infection.