Serk In Park

Development and Characterization of Gemcitabine-Resistant Pancreatic Tumor Cells

BackgroundPancreatic cancer is an exceptionally lethal disease with an annual mortality nearly equivalent to its annual incidence. This dismal rate of survival is due to several factors including late presentation with locally advanced, unresectable tumors, early metastatic disease, and rapidly arising chemoresistance. To study the mechanisms of chemoresistance in pancreatic cancer we developed two gemcitabine-resistant pancreatic cancer cell lines.MethodsResistant cells were obtained by culturing L3.6pl and AsPC-1 cells in serially increasing concentrations of gemcitabine. Stable cultures were obtained that were 40- to 50-fold increased in resistance relative to parental cells. Immunofluorescent staining was performed to examine changes in β-catenin and E-cadherin localization. Protein expression was determined by immunoblotting. Migration and invasion were determined by modified Boyden chamber assays. Fluorescence-activated cell sorting (FACS) analyses were performed to examine stem cell markers.ResultsGemcitabine-resistant cells underwent distinct morphological changes, including spindle-shaped morphology, appearance of pseudopodia, and reduced adhesion characteristic of transformed fibroblasts. Gemcitabine-resistant cells were more invasive and migratory. Gemcitabine-resistant cells were increased in vimentin and decreased in E-cadherin expression. Immunofluorescence and immunoblotting revealed increased nuclear localization of total β-catenin. These alterations are hallmarks of epithelial-to-mesenchymal transition (EMT). Resistant cells were activated in the receptor protein tyrosine kinase, c-Met and increased in expression of the stem cell markers CD (cluster of differentiation)24, CD44, and epithelial-specific antigen (ESA).ConclusionsGemcitabine-resistant pancreatic tumor cells are associated with EMT, a more-aggressive and invasive phenotype in numerous solid tumors. The increased phosphorylation of c-Met may also be related to chemoresistance and EMT and presents as an attractive adjunctive chemotherapeutic target in pancreatic cancer.

Signaling between Transforming Growth Factor β (TGF-β) and Transcription Factor SNAI2 Represses Expression of MicroRNA miR-203 to Promote Epithelial-Mesenchymal Transition and Tumor Metastasis

Background: TGF-β promotes tumor metastasis by inducing SNAI2. Results: TGF-β induces SNAI2 and promotes EMT by repressing miR-203. Conclusion: The SNAI2 and miR-203 feedback loop plays integral roles in EMT and tumor metastasis. Significance: Our findings provide new insights into molecular mechanisms of EMT and tumor metastasis and identify a therapeutic target for tumor invasion and metastasis. TGF-β promotes tumor invasion and metastasis by inducing an epithelial-mesenchymal transition (EMT). Understanding the molecular and epigenetic mechanisms by which TGF-β induces EMT may facilitate the development of new therapeutic strategies for metastasis. Here, we report that TGF-β induced SNAI2 to promote EMT by repressing miR-203. Although miR-203 targeted SNAI2, SNAI2 induced by TGF-β could directly bind to the miR-203 promoter to inhibit its transcription. SNAI2 and miR-203 formed a double negative feedback loop to inhibit each others expression, thereby controlling EMT. Moreover, we found that miR-203 was significantly down-regulated in highly metastatic breast cancer cells. The restoration of miR-203 in highly metastatic breast cancer cells inhibited tumor cell invasion in vitro and lung metastatic colonization in vivo by repressing SNAI2. Taken together, our results suggest that the SNAI2 and miR-203 regulatory loop plays important roles in EMT and tumor metastasis.

Osteal macrophages support physiologic skeletal remodeling and anabolic actions of parathyroid hormone in bone

Significance Cellular subpopulations in the bone marrow play distinct and unexplored functions in the regulation of the skeleton. A type of blood cell that resides in the bone marrow termed “osteal macrophage” was found to play a role in bone homeostasis by supporting bone formation and mediating parathyroid hormone-dependent bone regeneration. Furthermore, induction of cell death in mature macrophages activated the specialized process of efferocytosis (clearance of dead and dying cells), leading to a marrow microenvironment that supported bone formation. Cellular subpopulations in the bone marrow play distinct and unexplored functions in skeletal homeostasis. This study delineated a unique role of osteal macrophages in bone and parathyroid hormone (PTH)-dependent bone anabolism using murine models of targeted myeloid-lineage cell ablation. Depletion of c-fms+ myeloid lineage cells [via administration of AP20187 in the macrophage Fas-induced apoptosis (MAFIA) mouse model] reduced cortical and trabecular bone mass and attenuated PTH-induced trabecular bone anabolism, supporting the positive function of macrophages in bone homeostasis. Interestingly, using a clodronate liposome model with targeted depletion of mature phagocytic macrophages an opposite effect was found with increased trabecular bone mass and increased PTH-induced anabolism. Apoptotic cells were more numerous in MAFIA versus clodronate-treated mice and flow cytometric analyses of myeloid lineage cells in the bone marrow showed that MAFIA mice had reduced CD68+ cells, whereas clodronate liposome-treated mice had increased CD68+ and CD163+ cells. Clodronate liposomes increased efferocytosis (clearance of apoptotic cells) and gene expression associated with alternatively activated M2 macrophages as well as expression of genes associated with bone formation including Wnt3a, Wnt10b, and Tgfb1. Taken together, depletion of early lineage macrophages resulted in osteopenia with blunted effects of PTH anabolic actions, whereas depletion of differentiated macrophages promoted apoptotic cell clearance and transformed the bone marrow to an osteogenic environment with enhanced PTH anabolism. These data highlight a unique function for osteal macrophages in skeletal homeostasis.

Regulation of angiogenesis and vascular permeability by Src family kinases: opportunities for therapeutic treatment of solid tumors.

Aberrant expression or activation of protein tyrosine kinases, including Src and related Src family kinases, is a common occurrence in many human cancers, resulting in deregulation of expression of numerous mediators of cellular functions, including pro-angiogenic molecules. In addition, Src activation regulates vascular permeability of endothelial cells. How these processes contribute to tumor progression and metastasis are the subjects of this review. As Src-selective inhibitors have entered clinical trials for a number of solid tumors, further understanding of the roles of Src kinases in mediating tumor angiogenesis as well as modulating tumor/microenvironment interactions will provide insights into the best use of these inhibitors in treating patients afflicted with tumors in which Src is activated.

Preclinical Mouse Models of Human Prostate Cancer and Their Utility in Drug Discovery

In vivo animal experiments are essential to current prostate cancer research, and are particularly critical to studying interactions between tumor cells and their microenvironment. Numerous pre-clinical animal models of prostate cancer are currently available, including transgenic mouse models and human prostate cancer xenograft mouse models. In contrast to transgenic mouse models producing more heterogeneous cohorts of tumors, xenograft mouse models provide more controlled approaches. This unit describes the detailed procedures necessary to establish several distinct pre-clinical mouse models of human prostate cancer, including an orthotopic prostate xenograft model, an orthotopic bone metastasis model, an experimental metastasis model of intra-cardiac injection, and a vossicle model of tumor-bone interaction.

AFAP-110 is overexpressed in prostate cancer and contributes to tumorigenic growth by regulating focal contacts

The actin filament-associated protein AFAP-110 is an actin cross-linking protein first identified as a substrate of the viral oncogene v-Src. AFAP-110 regulates actin cytoskeleton integrity but also functions as an adaptor protein that affects crosstalk between Src and PKC. Here we investigated the roles of AFAP-110 in the tumorigenic process of prostate carcinoma. Using immunohistochemistry of human tissue arrays, we found that AFAP-110 was absent or expressed at very low levels in normal prostatic epithelium and benign prostatic hyperplasia but significantly increased in prostate carcinomas. The level of AFAP-110 in carcinomas correlated with the Gleason scores. Downregulation of AFAP-110 in PC3 prostate cancer cells inhibited cell proliferation in vitro and tumorigenicity and growth in orthotopic nude mouse models. Furthermore, downmodulation of AFAP-110 resulted in decreased cell-matrix adhesion and cell migration, defective focal adhesions, and reduced integrin beta1 expression. Reintroduction of avian AFAP-110 or a mutant disabling its interaction with Src restored these properties. However, expression of an AFAP-110 lacking the PKC-interacting domain failed to restore properties of parental cells. Thus, increased expression of AFAP-110 is associated with progressive stages of prostate cancer and is critical for tumorigenic growth, in part by regulating focal contacts in a PKC-dependent mechanism.

Activation of NF-kappa B signaling promotes growth of prostate cancer cells in bone.

Patients with advanced prostate cancer almost invariably develop osseous metastasis. Although many studies indicate that the activation of NF-κB signaling appears to be correlated with advanced cancer and promotes tumor metastasis by influencing tumor cell migration and angiogenesis, the influence of altered NF-κB signaling in prostate cancer cells within boney metastatic lesions is not clearly understood. While C4-2B and PC3 prostate cancer cells grow well in the bone, LNCaP cells are difficult to grow in murine bone following intraskeletal injection. Our studies show that when compared to LNCaP, NF-κB activity is significantly higher in C4-2B and PC3, and that the activation of NF-κB signaling in prostate cancer cells resulted in the increased expression of the osteoclast inducing genes PTHrP and RANKL. Further, conditioned medium derived from NF-κB activated LNCaP cells induce osteoclast differentiation. In addition, inactivation of NF-κB signaling in prostate cancer cells inhibited tumor formation in the bone, both in the osteolytic PC3 and osteoblastic/osteoclastic mixed C4-2B cells; while the activation of NF-κB signaling in LNCaP cells promoted tumor establishment and proliferation in the bone. The activation of NF-κB in LNCaP cells resulted in the formation of an osteoblastic/osteoclastic mixed tumor with increased osteoclasts surrounding the new formed bone, similar to metastases commonly seen in patients with prostate cancer. These results indicate that osteoclastic reaction is required even in the osteoblastic cancer cells and the activation of NF-κB signaling in prostate cancer cells increases osteoclastogenesis by up-regulating osteoclastogenic genes, thereby contributing to bone metastatic formation.

Cyclophosphamide Creates a Receptive Microenvironment for Prostate Cancer Skeletal Metastasis

A number of cancers predominantly metastasize to bone, due to its complex microenvironment and multiple types of constitutive cells. Prostate cancer especially has been shown to localize preferentially to bones with higher marrow cellularity. Using an experimental prostate cancer metastasis model, we investigated the effects of cyclophosphamide, a bone marrow-suppressive chemotherapeutic drug, on the development and growth of metastatic tumors in bone. Priming the murine host with cyclophosphamide before intracardiac tumor cell inoculation was found to significantly promote tumor localization and subsequent growth in bone. Shortly after cyclophosphamide treatment, there was an abrupt expansion of myeloid lineage cells in the bone marrow and the peripheral blood, associated with increases in cytokines with myelogenic potential such as C-C chemokine ligand (CCL)2, interleukin (IL)-6, and VEGF-A. More importantly, neutralizing host-derived murine CCL2, but not IL-6, in the premetastatic murine host significantly reduced the prometastatic effects of cyclophosphamide. Together, our findings suggest that bone marrow perturbation by cytotoxic chemotherapy can contribute to bone metastasis via a transient increase in bone marrow myeloid cells and myelogenic cytokines. These changes can be reversed by inhibition of CCL2.

Parathyroid Hormone–Related Protein Drives a CD11b+Gr1+ Cell–Mediated Positive Feedback Loop to Support Prostate Cancer Growth

In the tumor microenvironment, CD11b(+)Gr1(+) bone marrow-derived cells are a predominant source of protumorigenic factors such as matrix metalloproteinases (MMP), but how distal tumors regulate these cells in the bone marrow is unclear. Here we addressed the hypothesis that the parathyroid hormone-related protein (PTHrP) potentiates CD11b(+)Gr1(+) cells in the bone marrow of prostate tumor hosts. In two xenograft models of prostate cancer, levels of tumor-derived PTHrP correlated with CD11b(+)Gr1(+) cell recruitment and microvessel density in the tumor tissue, with evidence for mediation of CD11b(+)Gr1(+) cell-derived MMP-9 but not tumor-derived VEGF-A. CD11b(+)Gr1(+) cells isolated from mice with PTHrP-overexpressing tumors exhibited relatively increased proangiogenic potential, suggesting that prostate tumor-derived PTHrP potentiates this activity of CD11b(+)Gr1(+) cells. Administration of neutralizing PTHrP monoclonal antibody reduced CD11b(+)Gr1(+) cells and MMP-9 in the tumors. Mechanistic investigations in vivo revealed that PTHrP elevated Y418 phosphorylation levels in Src family kinases in CD11b(+)Gr1(+) cells via osteoblast-derived interleukin-6 and VEGF-A, thereby upregulating MMP-9. Taken together, our results showed that prostate cancer-derived PTHrP acts in the bone marrow to potentiate CD11b(+)Gr1(+) cells, which are recruited to tumor tissue where they contribute to tumor angiogenesis and growth.

Nuclear localization of parathyroid hormone-related peptide confers resistance to anoikis in prostate cancer cells

Prostate cancer remains a leading cause of cancer-related death in men, largely attributable to distant metastases, most frequently to bones. Despite intensive investigations, molecular mechanisms underlying metastasis are not completely understood. Among prostate cancer-derived factors, parathyroid hormone-related peptide (PTHrP), first discovered as an etiologic factor for malignancy-induced hypercalcemia, regulates many cellular functions critical to tumor growth, angiogenesis, and metastasis. In this study, the role of PTHrP in tumor cell survival from detachment-induced apoptosis (i.e. anoikis) was investigated. Reduction of PTHLH (encoding PTHrP) gene expression in human prostate cancer cells (PC-3) increased the percentage of apoptotic cells when cultured in suspension. Conversely, overexpression of PTHrP protected prostate cancer cells (Ace-1 and LNCaP, both typically expressing low or undetectable basal PTHrP) from anoikis. Overexpression of nuclear localization signal (NLS)-defective PTHrP failed to protect cells from anoikis, suggesting that PTHrP-dependent protection from anoikis is an intracrine event. A PCR-based apoptosis-related gene array showed that detachment increased expression of the TNF gene (encoding the proapoptotic protein tumor necrosis factor-α) fourfold greater in PTHrP-knockdown PC-3 cells than in control PC-3 cells. In parallel, TNF gene expression was significantly reduced in PTHrP-overexpressing LNCaP cells, but not in NLS-defective PTHrP overexpressing LNCaP cells, when compared with control LNCaP cells. Subsequently, in a prostate cancer skeletal metastasis mouse model, PTHrP-knockdown PC-3 cells resulted in significantly fewer metastatic lesions compared to control PC-3 cells, suggesting that PTHrP mediated antianoikis events in the bloodstream. In conclusion, nuclear localization of PTHrP confers prostate cancer cell resistance to anoikis, potentially contributing to prostate cancer metastasis.