Ying-Yi Li

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.

CCL3-CCR5 Axis Regulates Intratumoral Accumulation of Leukocytes and Fibroblasts and Promotes Angiogenesis in Murine Lung Metastasis Process

Metastasis proceeds through interaction between cancer cells and resident cells such as leukocytes and fibroblasts. An i.v. injection of a mouse renal cell carcinoma, Renca, into wild-type mice resulted in multiple metastasis foci in lungs and was associated with intratumoral accumulation of macrophages, granulocytes, and fibroblasts. A chemokine, CCL3, was detected in infiltrating cells and, to a lesser degree, tumor cells, together with an infiltration of leukocytes expressing CCR5, a specific receptor for CCL3. A deficiency of the CCL3 or CCR5 gene markedly reduced the number of metastasis foci in the lung, and the analysis using bone marrow chimeric mice revealed that both bone marrow- and non-bone marrow-derived cells contributed to metastasis formation. CCL3- and CCR5-deficient mice exhibited a reduction in intratumoral accumulation of macrophages, granulocytes, and fibroblasts. Moreover, intratumoral neovascularization, an indispensable process for metastasis, was attenuated in these gene-deficient mice. Intrapulmonary expression of matrix metalloproteinase (MMP)-9 and hepatocyte growth factor (HGF) was enhanced in wild-type mice, and the increases were markedly diminished in CCL3- and CCR5-deficient mice. Furthermore, MMP-9 protein was detected in macrophages and granulocytes, the cells that also express CCR5 and in vitro stimulation by CCL3-induced macrophages to express MMP-9. Intratumoral fibroblasts expressed CCR5 and HGF protein. In vitro CCL3 stimulated fibroblasts to express HGF. Collectively, the CCL3-CCR5 axis appears to regulate intratumoral trafficking of leukocytes and fibroblasts, as well as MMP-9 and HGF expression, and as a consequence to accelerate neovascularization and subsequent metastasis formation.

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)

Accelerated hepatocellular carcinoma development in mice expressing the Pim-3 transgene selectively in the liver

Pim-3, a proto-oncogene with serine/threonine kinase activity, was enhanced in hepatocellular carcinoma (HCC) tissues. To address the roles of Pim-3 in HCC development, we prepared transgenic mice that express human Pim-3 selectively in liver. The mice were born at a Mendelian ratio, were fertile and did not exhibit any apparent pathological changes in the liver until 1 year after birth. Pim-3-transgenic mouse-derived hepatocytes exhibited accelerated cell cycle progression. The administration of a potent hepatocarcinogen, diethylnitrosamine (DEN), induced accelerated proliferation of liver cells in Pim-3 transgenic mice in the early phase, compared with that observed for wild-type mice. Treatment with DEN induced lipid droplet accumulation with increased proliferating cell numbers 6 months after the treatment. Eventually, wild-type mice developed HCC with a frequency of 40% until 10 month after the treatment. Lipid accumulation was accelerated in Pim-3 transgenic mice with higher proliferating cell numbers, compared with that observed for wild-type mice. Pim-3 transgenic mice developed HCC with a higher incidence (80%) and a heavier burden, together with enhanced intratumoral CD31-positive vascular areas, compared with that observed for wild-type mice. These observations indicate that Pim-3 alone cannot cause, but can accelerate HCC development when induced by a hepatocarcinogen, such as DEN.

Roles of Pim‐3, a novel survival kinase, in tumorigenesis

Pim‐3 is a member of the Provirus integrating site Moloney murine leukemia virus (Pim) family, which belongs to the Ca2+/calmodulin‐dependent protein kinase (CaMK) group and exhibits serine/threonine kinase activity. Similar to other members of the Pim family (i.e. Pim‐1 and Pim‐2), Pim‐3 can prevent apoptosis and promote cell survival and protein translation, thereby enhancing cell proliferation of normal and malignant cells. Pim‐3 is expressed in vital organs, such as the heart, lung, and brain. However, minimal phenotypic changes in Pim‐3‐deficient mice suggest that Pim‐3 may be physiologically dispensable. Pim‐3 expression is enhanced in several cancer tissues, particularly those of endoderm‐derived organs, including the liver, pancreas, colon, and stomach. The development of hepatocellular carcinoma is accelerated in mice expressing the Pim‐3 gene selectively in the liver only when these mice are treated with a hepatocarcinogen, indicating that Pim‐3 can act as a promoter but not as an initiator. Moreover, inhibition of Pim‐3 expression can retard in vitro cell proliferation of hepatocellular, pancreatic, and colon carcinoma cell lines by promoting cell apoptosis. Furthermore, a Pim‐3 kinase inhibitor has been reported to inhibit cell proliferation in an in vivo xenograft model using a human pancreatic cancer cell line without inducing any major adverse effects. Thus, Pim‐3 kinase may be a candidate molecule for the development of molecular targeting drugs against cancer. (Cancer Sci 2011; 102: 1437–1442)

Tumor cell apoptosis induces tumor-specific immunity in a CC chemokine receptor 1- and 5-dependent manner in mice.

The first step in the generation of tumor immunity is the migration of dendritic cells (DCs) to the apoptotic tumor, which is presumed to be mediated by various chemokines. To clarify the roles of chemokines, we induced apoptosis using suicide gene therapy and investigated the immune responses following tumor apoptosis. We injected mice with a murine hepatoma cell line, BNL 1ME A.7R.1 (BNL), transfected with HSV‐thymidine kinase (tk) gene and then treated the animals with ganciclovir (GCV). GCV treatment induced massive tumor cell apoptosis accompanied with intratumoral DC infiltration. Tumor‐infiltrating DCs expressed chemokine receptors CCR1 and CCR5, and T cells and macrophages expressed CCL3, a ligand for CCR1 and CCR5. Moreover, tumor apoptosis increased the numbers of DCs migrating into the draining lymph nodes and eventually generated a specific cytotoxic cell population against BNL cells. Although GCV completely eradicated HSV‐tk‐transfected BNL cells in CCR1‐, CCR5‐, or CCL3‐deficient mice, intratumoral and intranodal DC infiltration and the subsequent cytotoxicity generation were attenuated in these mice. When parental cells were injected again after complete eradication of primary tumors by GCV treatment, the wild‐type mice completely rejected the rechallenged cells, but the deficient mice exhibited impairment in rejection. Thus, we provide definitive evidence indicating that CCR1 and CCR5 and their ligand CCL3 play a crucial role in the regulation of intratumoral DC accumulation and the subsequent establishment of tumor immunity following induction of tumor apoptosis by suicide genes.

Essential contribution of Ets-1 to constitutive Pim-3 expression in human pancreatic cancer cells.

We previously demonstrated that the proto‐oncogene Pim‐3 with serine/threonine kinase activity was aberrantly expressed in cancer cells but not in the normal cells of the pancreas. In order to elucidate the molecular mechanism underlying aberrant Pim‐3 expression in pancreatic cancer cells, we constructed luciferase expression vectors linked to 5′‐flanking deletion mutants of the human Pim‐3 gene and transfected human pancreatic cancer cells with the resultant vectors. The region up to –264 bp was essential for constitutive Pim‐3 gene expression, and the mutation in the Ets‐1 binding site (between –216 and –211 bp) reduced luciferase activities. Moreover, Ets‐1 mRNA and protein were constitutively expressed together with Pim‐3 in human pancreatic cancer cell lines. Chromatin immunoprecipitation assay demonstrated constitutive binding of Ets‐1 to the 5′‐flanking region of human Pim‐3 gene between –249 and –183 bp. Pim‐3 promoter activity and its protein expression were induced by transfection with wild type‐Ets‐1 and were reduced by transfection with dominant negative‐Ets‐1 or Ets‐1 small‐interfering RNA (siRNA). Furthermore, dominant negative‐Ets‐1 and Ets‐1 siRNA reduced the amount of Bad phosphorylated at its Ser112 and induced apoptosis, when they were transfected into human pancreatic cancer cells. Finally, Pim‐3 cDNA transfection reversed Ets‐1 siRNA‐induced increase in apoptosis and decrease in Bad phosphorylation at its Ser112. These observations would indicate that the transcription factor Ets‐1 can induce aberrant Pim‐3 expression and subsequently prevent apoptosis in human pancreatic cancer cells. (Cancer Sci 2009; 100: 396–404)

Protective Roles of the Fractalkine/CX3CL1-CX3CR1 Interactions in Alkali-Induced Corneal Neovascularization through Enhanced Antiangiogenic Factor Expression

Macrophages accumulate during the course of corneal neovascularization, but its mechanisms and roles still remain elusive. To address these points, we herein examined corneal neovascularization after alkali injury in mice deficient in fractalkine receptor/CX3CR1, which is normally expressed by macrophages. After alkali injury, the mRNA expression of CX3CR1 was augmented along with accumulation of F4/80-positive macrophages and Gr-1-positive neutrophils in the corneas. Compared with wild-type mice, CX3CR1-deficient mice exhibited enhanced corneal neovascularization 2 wk after injury, as evidenced by enlarged CD31-positive areas. Concomitantly, the accumulation of F4/80-positive macrophages, but not Gr-1-positive neutrophils, was markedly attenuated in CX3CR1-deficient mice compared with wild-type mice. The intraocular mRNA expression of vascular endothelial growth factor (VEGF) was enhanced to similar extents in wild-type and CX3CR1-deifient mice after the injury. However, the mRNA expression of antiangiogenic factors, thrombospondin (TSP) 1, TSP-2, and a disintegrin and metalloprotease with thrombospondin (ADAMTS) 1, was enhanced to a greater extent in wild-type than CX3CR1-deificient mice. A double-color immunofluorescence analysis demonstrated that F4/80-positive cells also expressed CX3CR1 and ADAMTS-1 and that TSP-1 and ADAMTS-1 were detected in CX3CR1-positive cells. CX3CL1 enhanced TSP-1 and ADAMTS-1, but not VEGF, expression by peritoneal macrophages. Moreover, topical application of CX3CL1 inhibited corneal neovascularization at 2 wk, along with enhanced intraocular expression of TSP-1 and ADAMTS-1 but not VEGF. Thus, these observations indicate that accumulation of CX3CR1-positive macrophages intraocularly can dampen alkali-induced corneal neovascularization by producing antiangiogenic factors such as TSP-1 and ADAMTS-1 and suggest the potential therapeutic efficacy of using CX3CL1 against alkali-induced corneal neovascularization.

Identification of stemonamide synthetic intermediates as a novel potent anticancer drug with an apoptosis-inducing ability.

We previously demonstrated that Pim‐3, a protooncogene with serine/threonine kinase activity, was aberrantly expressed in malignant lesions but not in normal tissues of endoderm‐derived organs, including pancreas, liver, colon and stomach. Moreover, aberrantly expressed Pim‐3 can prevent tumor cell apoptosis by inactivating a proapoptotic molecule, Bad, and enhancing the expression of an antiapoptotic molecule, Bcl‐XL. These observations prompted us to speculate that a chemical targeting Pim‐3 kinase may be a good candidate for a novel type of anticancer drug. Hence, we screened various low‐molecule compounds by examining their capacity to inhibit Pim‐3 kinase activity in vitro. We observed that some synthetic intermediates of stemonamide can inhibit in vitro activities of Pim‐3 kinase and its related kinases, such as Pim‐1 and Pim‐2. Moreover, these compounds inhibit in vitro cell proliferation of various human pancreatic, hepatocellular and colon cancer cell lines. Furthermore, the compounds can induce apoptosis of human pancreatic cancer cell lines in vitro by reducing the amount of phospho‐Ser112‐Bad, but not total amounts of Bad and Pim‐3. Finally, when the compound was administered to nude mice injected with a human pancreatic cancer cell line, it retarded tumor growth by increasing apoptotic cell numbers and decreasing proliferating cell numbers without causing serious adverse effects on blood counts. These observations indicate that the chemicals and its related compounds may be effective for the treatment of tumors of endoderm‐derived organs, particularly the pancreas.

Identification of a phenanthrene derivative as a potent anticancer drug with Pim kinase inhibitory activity

Pim‐3, a proto‐oncogene with serine/threonine kinase activity, is aberrantly expressed in malignant lesions, but not in normal tissues, of endoderm‐derived organs, including the pancreas, liver, colon, and stomach. Furthermore, the development of hepatocellular carcinoma is accelerated in mice expressing Pim‐3 transgene selectively in the liver when these mice are treated with a hepatocarcinogen. These observations suggest that a chemical targeting Pim‐3 kinase may be a novel type of anticancer drug. In the present study, we screened low molecular weight chemicals and observed that the phenanthrene derivative T26 potently inhibited Pim‐3 and Pim‐1, but only weakly inhibited Pim‐2. Moreover, T26 markedly inhibited the in vitro growth of human pancreatic cancer cell lines by inducing apoptosis and G2/M arrest. The growth inhibitory effects of T26 were reversed by overexpression of Pim‐3 cDNA in human pancreatic cancer cells, indicating that T26 acts primarily on Pim‐3. Furthermore, T26 inhibited the growth of a human pancreatic cancer cell line in nude mice without causing apparent adverse effects when it was administered after tumor formation was evident. These observations imply that the chemical and its related compounds may be effective for the treatment of cancers in which there is aberrant Pim‐3 expression. (Cancer Sci 2012; 103: 107–115)