Kazuya Sato

Hedgehog Promotes Neovascularization in Pancreatic Cancers by Regulating Ang-1 and IGF-1 Expression in Bone-Marrow Derived Pro-Angiogenic Cells

Background The hedgehog (Hh) pathway has been implicated in the pathogenesis of cancer including pancreatic ductal adenocarcinoma (PDAC). Recent studies have suggested that the oncogenic function of Hh in PDAC involves signaling in the stromal cells rather than cell autonomous effects on the tumor cells. However, the origin and nature of the stromal cell type(s) that are responsive to Hh signaling remained unknown. Since Hh signaling plays a crucial role during embryonic and postnatal vasculogenesis, we speculated that Hh ligand may act on tumor vasculature specifically focusing on bone marrow (BM)-derived cells. Methodology/Principal Findings Cyclopamine was utilized to inhibit the Hh pathway in human PDAC cell lines and their xenografts. BM transplants, co-culture systems of tumor cells and BM-derived pro-angiogenic cells (BMPCs) were employed to assess the role of tumor-derived Hh in regulating the BM compartment and the contribution of BM-derived cells to angiogenesis in PDAC. Cyclopamine administration attenuated Hh signaling in the stroma rather than in the cancer cells as reflected by decreased expression of full length Gli2 protein and Gli1 mRNA specifically in the compartment. Cyclopamine inhibited the growth of PDAC xenografts in association with regression of the tumor vasculature and reduced homing of BM-derived cells to the tumor. Host-derived Ang-1 and IGF-1 mRNA levels were downregulated by cyclopamine in the tumor xenografts. In vitro co-culture and matrigel plug assays demonstrated that PDAC cell-derived Shh induced Ang-1 and IGF-1 production in BMPCs, resulting in their enhanced migration and capillary morphogenesis activity. Conclusions/Significance We identified the BMPCs as alternative stromal targets of Hh-ligand in PDAC suggesting that the tumor vasculature is an attractive therapeutic target of Hh blockade. Our data is consistent with the emerging concept that BM-derived cells make important contributions to epithelial tumorigenesis.

Regulation of p38 phosphorylation and topoisomerase IIα expression in the B-cell lymphoma line Jiyoye by CD26/dipeptidyl peptidase IV is associated with enhanced in vitro and in vivo sensitivity to doxorubicin

CD26 is a Mr 110,000 surface-bound glycoprotein with diverse functional properties, including having a key role in normal T-cell physiology and the development of certain cancers. In this article, we show that surface expression of CD26, especially its intrinsic dipeptidyl peptidase IV (DPPIV) enzyme activity, results in enhanced topoisomerase IIalpha level in the B-cell line Jiyoye and subsequent in vitro sensitivity to doxorubicin-induced apoptosis. In addition, we show that expression of CD26/DPPIV is associated with increased phosphorylation of p38 and its upstream regulators mitogen-activated protein kinase kinase 3/6 and apoptosis signal-regulating kinase 1 and that p38 signaling pathway plays a role in the regulation of topoisomerase IIalpha expression. Besides demonstrating that CD26 effect on topoisomerase IIalpha and doxorubicin sensitivity is applicable to cell lines of both B-cell and T-cell lineages, the potential clinical implication of our work lies with the fact that we now show for the first time that our in vitro results can be extended to a severe combined immunodeficient mouse model. Our findings that CD26 expression can be an in vivo marker of tumor sensitivity to doxorubicin treatment may lead to future treatment strategies targeting CD26/DPPIV for selected human cancers in the clinical setting. Our article thus characterizes the biochemical linkage among CD26, p38, and topoisomerase IIalpha while providing evidence that CD26-associated topoisomerase IIalpha expression results in greater in vitro and in vivo tumor sensitivity to the antineoplastic agent doxorubicin.

Transplanting Normal Vascular Proangiogenic Cells to Tumor-Bearing Mice Triggers Vascular Remodeling and Reduces Hypoxia in Tumors

Blood vessels deliver oxygen and nutrients to tissues, and vascular networks are spatially organized to meet the metabolic needs for maintaining homeostasis. In contrast, the vasculature of tumors is immature and leaky, resulting in insufficient delivery of nutrients and oxygen. Vasculogenic processes occur normally in adult tissues to repair injured blood vessels, leading us to hypothesize that bone marrow mononuclear cells (BMMNC) may be able to restore appropriate vessel function in the tumor vasculature. Culturing BMMNCs in endothelial growth medium resulted in the early outgrowth of spindle-shaped attached cells expressing CD11b/Flt1/Tie2/c-Kit/CXCR4 with proangiogenic activity. Intravenous administration of these cultured vascular proangiogenic cells (VPC) into nude mice bearing pancreatic cancer xenografts and Pdx1-Cre;LSL-Kras(G12D);p53(lox/+) genetically engineered mice that develop pancreatic ductal adenocarcinoma significantly reduced areas of hypoxia without enhancing tumor growth. The resulting vasculature structurally mimicked normal vessels with intensive pericyte coverage. Increases in vascularized areas within VPC-injected xenografts were visualized with an ultrasound diagnostic system during injection of a microbubble-based contrast agent (Sonazoid), indicating a functional normalization of the tumor vasculature. In addition, gene expression profiles in the VPC-transplanted xenografts revealed a marked reduction in major factors involved in drug resistance and stemness of cancer cells. Together, our findings identify a novel alternate approach to regulate abnormal tumor vessels, offering the potential to improve the delivery and efficacy of anticancer drugs to hypoxic tumors.

Severe Hepatic Injury Caused by Imatinib Mesylate Administered for the Treatment of Chronic Myeloid Leukemia and the Efficacy of Prednisolone for its Management

Imatinib mesylate is a specific inhibitor of BCR-ABL tyrosine kinase, which is now widely used for the treatment of chronic myeloid leukemia (CML) with a high efficacy. Although severe hepatic injury caused by imatinib mesylate is rare, such a side effect may force patients to discontinue taking imatinib mesylate. In the present paper, we report on the case of a 51-year-old woman with CML who experienced hepatic injury with severe hyperbilirubinemia caused by imatinib mesylate. The findings from a liver biopsy specimen and her clinical course suggested the hepatic injury to presumably have been caused by an allergic mechanism. The co-administration of prednisolone was thus tried, and she has been able to continue imatinib mesylate administration without any liver dysfunction and finally was able to obtain a complete cytogenetic response. We therefore recommend that prednisolone should be tried when severe hepatic injury caused by imatinib mesylate is observed, since it might enable such patients to continue imatinib mesylate treatment and thereby improve the prognosis in such cases.

Loss of ABCB7 gene: pathogenesis of mitochondrial iron accumulation in erythroblasts in refractory anemia with ringed sideroblast with isodicentric (X)(q13)

An isodicentric (X)(q13) (idicXq13) is a rare, acquired chromosomal abnormality originated by deletion of the long arm from Xq13 (Xq13-qter), and is found in female patients with hematological disorders involving increased ringed sideroblasts (RSs), which are characterized by mitochondrial iron accumulation around the erythroblast nucleus. The cause of increased RSs in idicXq13 patients is not fully understood. Here, we report the case of a 66-year-old female presenting with refractory anemia with ringed sideroblasts (RARS), and idicXq13 on G-banded analysis. We identify the loss of the ABCB7 (ATP-binding cassette subfamily B member-7) gene, which is located on Xq13 and is involved in mitochondrial iron transport to the cytosol, by fluorescent in situ hybridization (FISH) analysis and the decreased expression level of ABCB7 mRNA in the patient’s bone marrow cells. Further FISH analyses showed that the ABCB7 gene is lost only on the active X-chromosome, not on the inactive one. We suggest that loss of ABCB7 due to deletion of Xq13-qter at idicXq13 formation may have contributed to the increased RSs in this patient. These findings suggest that loss of the ABCB7 gene may be a pathogenetic factor underlying mitochondrial iron accumulation in RARS patients with idicXq13.

Induction of leukemia-specific antibodies by immunotherapy with leukemia-cell-derived heat shock protein 70

Cancer immunotherapy using heat shock protein (HSP) derived from autologous tumor requires cluster of differentiation (CD)4+ as well as CD8+ T‐cells for the prolongation of patient survival, suggesting that a humoral immune response through CD4+ T‐cells is important in addition to cellular immunity. However, the role of humoral responses in HSP‐based autologous tumor immunotherapy remains unclear. In the present study, we investigated whether leukemia‐specific antibodies and antibody‐mediated cytotoxicity against autologous leukemia cells have a crucial role in a mouse A20 leukemia model by immunizing A20‐derived HSP70. Immunization with A20‐derived HSP70 induced the production of anti‐A20‐antibodies and the antibodies recognized HSP70‐binding peptides derived from A20. One of those was a major histocompatibility complex (MHC) class‐I binding peptide, which has been clarified as the target peptide of CD8+ cytotoxic T‐cells (CTL) against A20. The anti‐A20‐antibodies produced by immunization with A20‐derived HSP70 induced complement‐dependent cytotoxicity (CDC) against A20 in vitro. In addition, immunization with A20‐derived HSP70 increased intracellular interleukin‐4 (IL4)‐production of CD4+ T‐cells, confirming the activation of type‐2 helper T‐cells. Taken together, immunization with leukemia‐cell‐derived HSP70 induces antibodies against leukemia‐cell‐specific peptides and might play a crucial role in the eradication of leukemia cells by CDC in mice. These findings will enable future establishment of a novel therapeutic strategy using antileukemia antibodies in HSP‐based autologous tumor immunotherapy. (Cancer Sci 2008; 99: 1427–1434)

Heterogeneous expressions of hepcidin isoforms in hepatoma‐derived cells detected using simultaneous LC‐MS/MS

Hepcidin, a key regulator of iron homeostasis, is known to have three isoforms: hepcidin‐20, ‐22, and ‐25. Hepcidin‐25 is thought to be the major isoform and the only one known to be involved in iron metabolism; the physiological roles of other isoforms are poorly understood. Because of its involvement in the pathophysiology of hereditary hemochromatosis and the anemia of chronic disease, the regulatory mechanisms of hepcidin expression have been extensively investigated, but most studies have been performed only at the transcriptional level. Difficulty in detecting hepcidin has impeded in vitro research. In the present study, we developed a novel method for simultaneous quantification of hepcidin‐20, ‐22, and ‐25 in the media from hepatoma‐derived cell lines. Using this method, we determined the expression patterns of hepcidin isoforms and the patterns of responses to various stimuli in human hepatoma‐derived cultured cells. We found substantial differences among cell lines. In conclusion, a novel method for simultaneous quantification of hepcidin isoforms is presented. Heterogeneous expressions of hepcidin isoforms in human hepatoma‐derived cells were revealed by this method. We believe our method will facilitate quantitative investigation of the role hepcidin plays in iron homeostasis.

Two cases of mycosis fungoides treated by reduced‐intensity cord blood transplantation

Mycosis fungoides is a cutaneous T‐cell lymphoma, which is clinically divided into three stages: patch, plaque and tumor. Despite a variety of treatments the prognosis is poor in advanced mycosis fungoides. Recently, allogeneic hematopoietic stem cell transplantation has been successfully applied for such cases. We performed reduced‐intensity umbilical cord blood transplantation for two advanced mycosis fungoides patients. Case 1 was a 56‐year‐old man and case 2 was a 30‐year‐old woman. Tumors of each case were refractory to conventional chemotherapy. Although radiation therapy was considerably effective, tumors relapsed after several months. Reduced‐intensity umbilical cord blood transplantation was performed because case 1 had no human leukocyte antigen‐identical siblings and the sibling of case 2 did not agree to be the donor. The male patient died of pulmonary failure 23u2003days after reduced‐intensity umbilical cord blood transplantation. The case 2 patient succeeded in reduced‐intensity umbilical cord blood transplantation and remained in complete/partial remission for 13u2003months. However, chemotherapy‐resistant tumors relapsed, and allogeneic hematopoietic stem cell transplantation was performed at 17u2003months. She died of cerebral hemorrhage 23u2003days after the procedure. Reduced‐intensity umbilical cord blood transplantation may be included in the treatments for advanced mycosis fungoides, where graft‐versus‐lymphoma effect seems to be a significant factor for the success of the treatment.

Donor-type myelodysplastic syndrome with t(2;3) and monosomy 7 after allogeneic peripheral blood stem cell transplantation and liver transplantation in a patient with severe-type aplastic anemia.

Secondary clonal hematological disease in donor cells has rarely been reported as a complication of allogeneic stem cell transplantation in hematological disease. We report a case of myelodysplastic syndrome that showed cytogenetic abnormalities of t(2;3) and monosomy 7, which developed 2 years after peripheral blood stem cell transplantation for aplastic anemia and 1 year after liver transplantation for drug-induced hepatic failure. This secondary malignancy of donor origin is most frequently seen in patients with leukemia. We suspect that the chromosomal abnormalities are related to hepatitis-associated aplastic anemia, administration of granulocyte colony-stimulating factor and erythropoietin for posttransplantion pancytopenia, and repeated infections after liver transplantation.

Development of POEMS syndrome after an initial manifestation of solitary plasmacytoma

A 44-year-old male was admitted for numbness in the left arm. CT showed a tumor impacting on the spinal cord with an adjacent thoracic vertebral osteosclerotic lesion. The histopathology of the tumor showed diffuse proliferation of atypical plasma cells with expressed vascular endothelial growth factor (VEGF), which is a known etiological factor in POEMS syndrome. Though serum VEGF (sVEGF) level was elevated, a diagnosis of solitary plasmacytoma with an osteosclerotic lesion was made as the patient presented no polyneuropathy, organomegaly, endocrinopathy, or skin changes. The patient experienced muscle weakness of the lower limbs and skin pigmentation/hemangioma one year after irradiation of the osteosclerotic lesion. Laboratory tests revealed hypothyroidism, hyperglycemia, serum monoclonal gammopathy, further elevation of sVEGF, and increased atypical bone marrow plasma cells. CT imaging showed splenomegaly, and a nerve conduction test revealed demyelinating motor peripheral neuropathy. The patient was therefore diagnosed with POEMS syndrome. Plasmacytoma is very rare as an initial manifestation of POEMS syndrome. Patients presenting with plasmacytoma with an osteosclerotic lesion should be carefully observed and evaluated for the expression of sVEGF and development of POEMS syndrome, as most bone plasmacytomas in POEMS syndrome patients are reported to be osteosclerotic. This is to our knowledge the first case of osteosclerotic plasmacytoma that progressed to POEMS syndrome, with an increase of sVEGF.