Enhanced ALOX12 Gene Expression Predicts Therapeutic Susceptibility to 5-Azacytidine in Patients with Myelodysplastic Syndromes.

5-azacytidine (AZA), a representative DNA-demethylating drug, has been widely used to treat myelodysplastic syndromes (MDS). However, it remains unclear whether AZA's DNA demethylation of any specific gene is correlated with clinical responses to AZA. In this study, we investigated genes that could contribute to the development of evidence-based epigenetic therapeutics with AZA. A DNA microarray identified that AZA specifically upregulated the expression of 438 genes in AZA-sensitive MDS-L cells but not in AZA-resistant counterpart MDS-L/CDA cells. Of these 438 genes, the ALOX12 gene was hypermethylated in MDS-L cells but not in MDS-L/CDA cells. In addition, we further found that (1) the ALOX12 gene was hypermethylated in patients with MDS compared to healthy controls; (2) MDS classes with excess blasts showed a relatively lower expression of ALOX12 than other classes; (3) a lower expression of ALOX12 correlated with higher bone marrow blasts and a shorter survival in patients with MDS; and (4) an increased ALOX12 expression after AZA treatment was associated with a favorable response to AZA treatment. Taking these factors together, an enhanced expression of the ALOX12 gene may predict favorable therapeutic responses to AZA therapy in MDS.

Oncogenic Y-box binding protein-1 as an effective therapeutic target in drug-resistant cancer.

Y-box binding protein-1 (YBX1), a multifunctional oncoprotein containing an evolutionarily conserved cold shock domain, dysregulates a wide range of genes involved in cell proliferation and survival, drug resistance, and chromatin destabilization by cancer. Expression of a multidrug resistance-associated ATP binding cassette transporter gene, ABCB1, as well as growth factor receptor genes, EGFR and HER2/ErbB2, was initially discovered to be transcriptionally activated by YBX1 in cancer cells. Expression of other drug resistance-related genes, MVP/LRP, TOP2A, CD44, CD49f, BCL2, MYC, and androgen receptor (AR), is also transcriptionally activated by YBX1, consistently indicating that YBX1 is involved in tumor drug resistance. Furthermore, there is strong evidence to support that nuclear localization and/or overexpression of YBX1 can predict poor outcomes in patients with more than 20 different tumor types. YBX1 is phosphorylated by kinases, including AKT, p70S6K, and p90RSK, and translocated into the nucleus to promote the transcription of resistance- and malignancy-related genes. Phosphorylated YBX1, therefore, plays a crucial role as a potent transcription factor in cancer. Herein, a novel anticancer therapeutic strategy is presented by targeting activated YBX1 to overcome drug resistance and malignant progression.

N-myc downstream-regulated gene 1 promotes tumor inflammatory angiogenesis through JNK activation and autocrine loop of interleukin-1α by human gastric cancer cells.

The expression of N-myc downstream-regulated gene 1 (NDRG1) was significantly correlated with tumor angiogenesis and malignant progression together with poor prognosis in gastric cancer. However, the underlying mechanism for the role of NDRG1 in the malignant progression of gastric cancer remains unknown. Here we examined whether and how NDRG1 could modulate tumor angiogenesis by human gastric cancer cells. We established NU/Cap12 and NU/Cap32 cells overexpressing NDRG1 in NUGC-3 cells, which show lower tumor angiogenesis in vivo. Compared with parental NU/Mock3, NU/Cap12, and NU/Cap32 cells: 1) induced higher tumor angiogenesis than NU/Mock3 cells accompanied by infiltration of tumor-associated macrophages in mouse dorsal air sac assay and Matrigel plug assay; 2) showed much higher expression of CXC chemokines, MMP-1, and the potent angiogenic factor VEGF-A; 3) increased the expression of the representative inflammatory cytokine, IL-1α; 4) augmented JNK phosphorylation and nuclear expression of activator protein 1 (AP-1). Further analysis demonstrated that knockdown of AP-1 (Jun and/or Fos) resulted in down-regulation of the expression of VEGF-A, CXC chemokines, and MMP-1, and also suppressed expression of IL-1α in NDRG1-overexpressing cell lines. Treatment with IL-1 receptor antagonist (IL-1ra) resulted in down-regulation of JNK and c-Jun phosphorylation, and the expression of VEGF-A, CXC chemokines, and MMP-1 in NU/Cap12 and NU/Cap32 cells. Finally, administration of IL-1ra suppressed both tumor angiogenesis and infiltration of macrophages by NU/Cap12 in vivo. Together, activation of JNK/AP-1 thus seems to promote tumor angiogenesis in relationship to NDRG1-induced inflammatory stimuli by gastric cancer cells.

Nuclear Y-box-binding protein-1 is a poor prognostic marker and related to epidermal growth factor receptor in uterine cervical cancer.

OBJECTIVE: Y-box binding protein-1 (YB-1) is a member of the cold shock protein family and functions in transcription and translation. Many studies indicate that YB-1 is strongly expressed in tumor cells and is considered a marker of tumor aggressiveness and clinical prognosis. Overexpression of epidermal growth factor receptor (EGFR) has been associated with poor outcomes in cervical cancer. Clinical trials of EGFR family-base therapy are currently being initiated in cervical cancer. Nuclear YB-1 expression correlates with EGFR expression in various types of cancer. However, the clinical significance of nuclear YB-1 expression in different settings, the correlation with EGFR, and the prognostic implications of YB-1 expression in cervical cancer remain elusive. PATIENTS AND METHODS: Nuclear YB-1 expression was immunohistochemically analyzed in tissue specimens obtained from 204 patients with cervical cancer who underwent surgery. Associations of nuclear YB-1 expression with clinicopathological factors such as survival, EGFR expression, and human epidermal growth factor receptor 2 (HER2) expression were investigated. RESULTS: Nuclear YB-1 expression was found in 41 (20.2%) of 204 cases of cervical cancer and correlated with disease stage, tumor diameter, stromal invasion, and lymph-node metastasis. Nuclear YB-1 expression also correlated with EGFR expression (P=0.0114) as well as HER2 expression (P=0.0053). Kaplan-Meier survival analysis showed that nuclear YB-1 expression was significantly associated with poor progression-free survival (P=0.0033) and overall survival (P=0.0003), respectively. CONCLUSION: Nuclear YB-1 expression is a prognostic marker and correlates with EGFR expression in cervical cancer.

Sorting nexin 2-mediated membrane trafficking of c-Met contributes to sensitivity of molecular-targeted drugs.

The sorting nexin (SNX) family is a diverse group of cytoplasmic and membrane-associated proteins that are involved in membrane-trafficking steps within the endocytotic network. SNX1 and SNX2 are components of the mammalian retromer complex and they also play critical roles in the membrane trafficking of growth factor receptors including epidermal growth factor receptor (EGFR) and c-Met. The human lung cancer cell lines, which harbor activating mutations in the kinase domain of EGFR gene, are sensitive to EGFR-targeted drugs gefitinib or erlotinib. However, a lung cancer cell line harboring gene amplification of c-Met is sensitive to the c-Met-targeted drug SU11274 but not to EGFR-targeted drugs. C-Met overexpression is identified as one of the bypass mechanisms for acquired resistance to EGFR-targeted drugs. Here we show that the siRNA-mediated knockdown of SNX2 decreases the cell-surface localization of c-Met, but not that of EGFR, resulting in lysosomal degradation of the c-Met protein. SNX2 specifically interacts with c-Met and treatment with lysosomal inhibitors almost completely annihilates downregulation of c-Met protein by SNX2 knockdown. Therefore, silencing of SNX2 markedly alters sensitivity to anticancer drugs targeted to c-Met (SU11274) and EGFR (gefitinib and erlotinib) through promotion of compensatory activation of the EGFR pathway in lung cancer cells. These findings suggest that development of drugs targeting SNX2 could be useful in overcoming drug resistance to EGFR-targeted drugs in lung cancer cells harboring c-Met gene amplification.

N-Myc Downstream Regulated Gene-1 May Play an Important Role in the Prognosis of Ovarian Cancer, in Its Association with Beta-Catenin.

NDRG1 is a nickel- and calcium-inducible gene that plays important roles in the primary growth of malignant tumors, as well as in invasion and metastasis. This study investigated the associations of NDRG1 expression with cell adhesion and other clinicopathological factors in ovarian cancer. The clinical records of 123 women who underwent surgery for ovarian cancer in our institute were reviewed retrospectively. The expression of NDRG1, E-cadherin, and beta-catenin in surgical specimens were evaluated immunohistochemically. The NDRG1 expression level was significantly associated with beta-catenin expression, peritoneal metastasis outside the pelvic cavity, lymph node metastasis, and FIGO stages. The Kaplan-Meier analysis showed a significant association between the NDRG1 expression level and progression-free survival: high NDRG1 expression was related to poor survival. Our results suggest that the increased expression of NDRG1 is associated with cell adhesion and may be a poor prognostic indicator in women with ovarian cancer.

The antitumor effect of a novel angiogenesis inhibitor (an octahydronaphthalene derivative) targeting both VEGF receptor and NF-κB pathway.

Development of a novel type of angiogenesis inhibitor will be essential for further improvement of therapeutics against cancer patients. We examined whether an octahydronaphthalene derivative, AMF-26, which was screened as an inhibitor of intercellular adhesion molecule-1 (ICAM-1) production stimulated by inflammatory stimuli in vascular endothelial cells, could block angiogenesis in response to vascular endothelial growth factor (VEGF) and/or inflammatory cytokines. Low dose AMF-26 effectively inhibited the tumor necrosis factor-α (TNF-α)- or the interleukin-1ß (IL-1ß)-induced production of ICAM-1 in human umbilical vascular endothelial cells (HUVECs). We found that the TNF-α-induced phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) and nuclear translocation of p65 were impaired by AMF-26 in both endothelial cells and cancer cells. AMF-26 was found to inhibit the phosphorylation of VEGF receptor 1 (VEGFR1), VEGFR2 and the downstream signaling molecules Akt, extracellular signal-regulated kinase (ERK)1/2 stimulated by VEGF in HUVECs. Therefore, the VEGF-induced proliferation, migration and tube formation of vascular endothelial cells was highly susceptible to inhibition by AMF-26. Oral administration of AMF-26 significantly blocked VEGF- or IL-1ß-induced angiogenesis in the mouse cornea, and also tumor angiogenesis and growth. Together, our results indicate that AMF-26 inhibits angiogenesis through suppression of both VEGFR1/2 and nuclear factor-κB (NF-κB) signaling pathways when stimulated by VEGF or inflammatory cytokines. AMF-26 could be a promising novel candidate drug for cancer treatments.

Macrophage infiltration predicts a poor prognosis for human ewing sarcoma.

Ewing sarcoma-primitive neuroectodermal tumor (EWS) is associated with the most unfavorable prognosis of all primary musculoskeletal tumors. The objective of the present study was to investigate whether tumor-associated macrophages (TAMs) affect the development of EWS. TAMs were isolated from mouse xenografts using CD11b magnetic beads and examined for their cytokine expression and osteoclastic differentiation. To evaluate the role of TAMs in xenograft formation, liposome-encapsulated clodronate was used to deplete TAMs in mice. Macrophage infiltration and tumor microvascular density were histologically evaluated in 41 patients with EWS, and association with prognosis was examined using Kaplan-Meier survival analysis. In mouse EWS xenografts, TAMs expressed higher concentrations of cytokines including interleukin-6, keratinocyte-derived chemokine, and monocyte chemotactic protein-1. TAMs were more capable than normal monocytes of differentiating into tartrate-resistant acid phosphatase-positive giant cells. Depleting macrophages using liposome-encapsulated clodronate significantly inhibited development of EWS xenografts. In human EWS samples, higher levels of CD68-positive macrophages were associated with poorer overall survival. In addition, enhanced vascularity, increase in the amount of C-reactive protein, and higher white blood cell counts were also associated with poor prognosis and macrophage infiltration. TAMs seem to enhance the progression of EWS by stimulating both angiogenesis and osteoclastogenesis. Further investigation of the behavior of TAMs may lead to development of biologically targeted therapies for EWS.

AXL/CDCP1/SRC axis confers acquired resistance to osimertinib in lung cancer.

Osimertinib, a third-generation EGFR-TKI, has nowadays been applied to non-small cell lung cancer harboring activated EGFR mutation with or without T790M, but ultimately develop resistance to this drug. Here we report a novel mechanism of acquired resistance to osimertinib and the reversal of which could improve the clinical outcomes. In osimertinib-resistant lung cancer cell lines harboring T790M mutation that we established, expression of multiple EGFR family proteins and MET was markedly reduced, whereas expression of AXL, CDCP1 and SRC was augmented along with activation of AKT. Surprisingly, AXL or CDCP1 expression was induced by osimertinib in a time-dependent manner up to 3 months. Silencing of CDCP1 or AXL restored the sensitivity to osimertinib with reduced activation of SRC and AKT. Furthermore, silencing of both CDCP1 and AXL increased the sensitivity to osimertinib. Either silencing of SRC or dasatinib, a SRC family kinase (SFK) inhibitor, suppressed AKT phosphorylation and cell growth. Increased expression of AXL and CDCP1 was observed in refractory tumor samples from patients with lung cancer treated with osimertinib. Together, this study suggests that AXL/SFK/AKT and CDCP1/SFK/AKT signaling pathways play some roles in acquired osimertinib resistance of non-small cell lung cancer.

Identification of sites subjected to serine/threonine phosphorylation by SGK1 affecting N-myc downstream-regulated gene 1 (NDRG1)/Cap43-dependent suppression of angiogenic CXC chemokine expression in human pancreatic cancer cells.

We have recently reported that N-myc downstream-regulated gene 1 (NDRG1)/Ca(2+)-associated protein with a molecular mass of 43kDa (Cap43) suppresses angiogenesis and tumor growth of pancreatic cancer through marked decreases in both the expression of CXC chemokines and phosphorylation of a NF-kappaB signaling molecule, inhibitor of kappaB kinase (IkappaBalpha). NDRG1/Cap43 is phosphorylated at serine/threonine sites in its C-terminal domain by serum- and glucocorticoid-regulated kinase 1 (SGK1). In this study, we attempted to clarify the domain or site of NDRG1/Cap43 responsible for its suppression of CXC chemokine expression in pancreatic cancer cells. Expression of the deletion constructs CapDelta2 [deletion of amino acids (AA) 130-142] and CapDelta4 [deletion of AA 180-294] as well as the wild-type full sequence of NDRG1/Cap43 (F-Cap), suppressed the production of CXC chemokines such as Groalpha/CXCL1 and ENA-78/CXCL5, whereas no or low suppression was observed in cell expressing the CapDelta5 mutant [deletion of AA 326-350] and CapDelta6 mutant [deletion of AA 326-394]. We further introduced mutations at the serine and threonine sites at 328 [T328A], 330 [S330A] and 346 [T346A], which are susceptible to phosphorylation by SGK1, and also constructed double mutants [T328A, S330A], [T328A, T346A] and [S330A, T346A]. Expression of all these mutants, with the exception of [S330A, T346A], suppressed the production of CXC chemokine to similar levels as their wild-type counterpart. IkappaBalpha was found to be specifically phosphorylated by this double mutant [S330A, T346A] and the CapDelta5 mutant at levels comparable to that induced in their wild-type counterpart. Phosphorylation of NDRG1/Cap43 at both serine330 and threonine346 is required for its suppressive action on the NF-kappaB signaling pathway and CXC chemokine expression in pancreatic cancer cells.

Overexpression of class III beta-tubulin predicts good response to taxane-based chemotherapy in ovarian clear cell adenocarcinoma.

PURPOSE: Of the various microtubule-associated molecules, beta-tubulin III has been reported to be closely associated with the therapeutic efficacy of taxane-based chemotherapy against ovarian cancer. Stathmin and microtubule-associated protein 4 (MAP4) have been reported to play an important role in microtubule stabilization. In this study, we investigated whether expression of these microtubule-associated factors affects the therapeutic efficacy of taxane-based chemotherapy in ovarian clear cell adenocarcinoma. EXPERIMENTAL DESIGN: Drug sensitivity of paclitaxel or cisplatin was assessed in ovarian cancer cell lines treated with small interfering RNA of tubulin isoforms, MAP4, and stathmin. We examined 94 surgically resected ovarian clear cell adenocarcinoma specimens from patients treated with taxane-containing regimens (n=44) and with taxane-free regimens (n=50), using immunohistochemistry to detect expression of beta-tubulin III, stathmin, and MAP4. RESULTS: Knockdown of beta-tubulin III and IV specifically conferred drug resistance to paclitaxel in one ovarian cancer cell line, but not to other molecules. Estimated overall survival revealed a significant synergistic effect between taxane and beta-tubulin III in patients with ovarian clear cell adenocarcinoma. Of three microtubule-related molecules, among the taxane-based chemotherapy group, cases with higher beta-tubulin III expression were associated with a significantly more favorable prognosis compared with those having lower beta-tubulin III expression. By contrast, there was no statistical significance in the synergistic relationships between stathmin and taxane or between MAP4 and taxane. CONCLUSIONS: Taxane-based chemotherapy was effective for patients with ovarian clear cell adenocarcinomas who were positive for beta-tubulin III but not for those who were negative for these proteins.

NDRG1 activates VEGF-A-induced angiogenesis through PLCγ1/ERK signaling in mouse vascular endothelial cells.

Many diseases, including cancer, have been associated with impaired regulation of angiogenesis, of which vascular endothelial growth factor (VEGF)-A is a key regulator. Here, we test the contribution of N-myc downstream regulated gene 1 (NDRG1) to VEGF-A-induced angiogenesis in vascular endothelial cells (ECs). Ndrg1-/- mice exhibit impaired VEGF-A-induced angiogenesis in corneas. Tumor angiogenesis induced by cancer cells that express high levels of VEGF-A was also reduced in a mouse dorsal air sac assay. Furthermore, NDRG1 deficiency in ECs prevented angiogenic sprouting from the aorta and the activation of phospholipase Cγ1 (PLCγ1) and ERK1/2 by VEGF-A without affecting the expression and function of VEGFR2. Finally, we show that NDRG1 formed a complex with PLCγ1 through its phosphorylation sites, and the inhibition of PLCγ1 dramatically suppressed VEGF-A-induced angiogenesis in the mouse cornea, suggesting an essential role of NDRG1 in VEGF-A-induced angiogenesis through PLCγ1 signaling.

Bidirectional Regulation between NDRG1 and GSK3ß Controls Tumor Growth and Is Targeted by Differentiation Inducing Factor-1 in Glioblastoma.

The development of potent and selective therapeutic approaches to glioblastoma (GBM), one of the most aggressive primary brain tumors, requires identification of molecular pathways that critically regulate the survival and proliferation of GBM. Previous studies have reported that deregulated expression of N-myc downstream regulated gene 1 (NDRG1) affects tumor growth and clinical outcomes of patients with various types of cancer including glioma. Here, we show that high level expression of NDRG1 in tumors significantly correlated with better prognosis of patients with GBM. Loss of NDRG1 in GBM cells upregulated GSK3ß levels and promoted cell proliferation, which was reversed by selective inhibitors of GSK3ß. In contrast, NDRG1 overexpression suppressed growth of GBM cells by decreasing GSK3ß levels via proteasomal degradation and by suppressing AKT and S6 cell growth signaling, as well as cell-cycle signaling pathways. Conversely, GSK3ß phosphorylated serine and threonine sites in the C-terminal domain of NDRG1 and limited the protein stability of NDRG1. Furthermore, treatment with differentiation inducing factor-1, a small molecule derived from Dictyostelium discoideum, enhanced NDRG1 expression, decreased GSK3ß expression, and exerted marked NDRG1-dependent antitumor effects in vitro and in vivo. Taken together, this study revealed a novel molecular mechanism by which NDRG1 inhibits GBM proliferation and progression. Our study thus identifies the NDRG1/GSK3ß signaling pathway as a key growth regulatory program in GBM, and suggests enhancing NDRG1 expression in GBM as a potent strategy toward the development of anti-GBM therapeutics. SIGNIFICANCE: This study identifies NDRG1 as a potent and endogenous suppressor of glioblastoma cell growth, suggesting the clinical benefits of NDRG1-targeted therapeutics against glioblastoma.

Targeting Phosphorylation of Y-Box-Binding Protein YBX1 by TAS0612 and Everolimus in Overcoming Antiestrogen Resistance.

Nuclear expression of Y-box-binding protein (YBX1) is closely correlated with clinical poor outcomes and drug resistance in breast cancer. Nuclear translocation of YBX1 is facilitated by YBX1 phosphorylation at serine 102 by AKT, p70S6K, and p90RSK, and the phosphorylated YBX1 (pYBX1) promotes expression of genes related to drug resistance and cell growth. A forthcoming problem to be addressed is whether targeting the phosphorylation of YBX1 overcomes antiestrogen resistance by progressive breast cancer. Here, we found that increased expression of pYBX1 was accompanied by acquired resistance to antiestrogens, fulvestrant and tamoxifen. Forced expression of YBX1/S102E, a constitutive phosphorylated form, resulted in acquired resistance to fulvestrant. Inversely, YBX1 silencing specifically overcame antiestrogen resistance. Furthermore, treatment with everolimus, an mTORC1 inhibitor, or TAS0612, a novel multikinase inhibitor of AKT, p70S6K, and p90RSK, suppressed YBX1 phosphorylation and overcame antiestrogen resistance in vitro and in vivo IHC analysis revealed that expression of pYBX1 and YBX1 was augmented in patients who experienced recurrence during treatment with adjuvant endocrine therapies. Furthermore, pYBX1 was highly expressed in patients with triple-negative breast cancer compared with other subtypes. TAS0612 also demonstrated antitumor effect against triple-negative breast cancer in vivo Taken together, our findings suggest that pYBX1 represents a potential therapeutic target for treatment of antiestrogen-resistant and progressive breast cancer.

Overexpression of Cap43 is associated with malignant status of esophageal cancer.

Cap43 protein has been proven to be upregulated by nickel compounds or hypoxic stress, often during cell differentiation or cell growth arrest. However, the function of this gene remains unknown. Although, several studies have been performed, none of these have evaluated the expression of Cap43 in esophageal cancer. To clarify its function and role in esophageal cancer, a clinical archive of cancer specimens was examined for the expression of Cap43 by immunohistochemistry. The expression level of Cap43 protein was also investigated by Western blotting and mRNA by realtime RT-PCR using esophageal cancer cell lines. Immunohistochemistry results showed that overexpression of Cap43 was correlated with malignant status of esophageal cancer and that was considered as an independent prognostic marker. Interestingly, adenocarcinoma of the esophagus did not express Cap43. In esophageal cancer cell lines, Western blotting and real-time RT-PCR, showed a variation in the expression level of Cap43 and there was no obvious correlation between protein and mRNA levels. The present report shows for the first time that the expression of the Cap43 gene has a function in tumor progression and that its expression correlates independently with patient survival. Cap43 gene could be considered as a new and important cancer marker.

The augmented expression of the cytidine deaminase gene by 5-azacytidine predicts therapeutic efficacy in myelodysplastic syndromes.

5-Azacytidine (5AC), a hypomethylating agent, is clinically used for the treatment of patients with myelodysplastic syndromes (MDS). Cytidine deaminase (CDA) is a key enzyme in the detoxification of 5AC. We investigated whether the CDA expression could predict response to 5AC in MDS. Among leukemia-derived cell lines, MDS-L, an MDS-derived cell line with a relatively low CDA expression level, was found to be the most sensitive to 5AC. Combination with tetrahydrouridine, an inhibitor of CDA, synergistically potentiated the cytotoxic effect of 5AC. Treatment with 5AC markedly enhanced the expression level of CDA mRNA and showed demethylation at CpG sites in the 5'-flanking region of the CDA gene. We further compared the protein expression levels of CDA in matched clinical samples before and after treatment with 5AC in bone marrow cells from 8 MDS patients by an immunohistochemical analysis. The CDA expression level showed an approximately 2- to 3-fold increase after 5AC treatment in 3 of these cases, and these three patients with relatively higher CDA expression levels after 5AC treatment all showed better clinical responses to 5AC. In contrast, the 5 remaining patients, whose CDA expression showed no augmentation, observed no clinical benefit. Taken together, the optimized determination of the CDA expression levels before and after 5AC treatment, and the methylation status at CpG sites of 5'-flanking region of the CDA gene, may contribute to the development of precise 5AC therapy for MDS.

Different expression profiles of Y-box-binding protein-1 and multidrug resistance-associated proteins between alveolar and embryonal rhabdomyosarcoma.

Nuclear expression of the Y-box-binding protein-1 (YB-1) has been reported to regulate the expression of both P-glycoprotein (P-gp) and major vault protein (MVP), and to regulate proliferative activities in human malignancies. Based on morphology and molecular biology, rhabdomyosarcoma (RMS) can be divided into two major types: embryonal type and the more aggressive alveolar type. Thirty-five cases of embryonal RMS (ERMS) and 28 cases of alveolar RMS (ARMS) were examined immunohistochemically for the nuclear expression of YB-1 and the intrinsic expression of P-gp, multidrug resistance (MDR)-associated protein (MRP) 1, 2, and 3, breast-cancer resistant protein (BCRP) and MVP, and the findings were compared with proliferative activities as evaluated by the MIB-1-labeling index (LI). Moreover, mRNA levels of these MDR-related molecules were assessed using a quantitative reverse transcriptase-PCR method in 18 concordant frozen materials. P-gp expression was more frequently observed ARMS, compared with ERMS (P = 0.0332), whereas immunoreactivity for BCRP was more frequently recognized in ERMS (P = 0.0184). Nuclear expression of YB-1 protein was correlated with P-gp (P = 0.0359) and MVP (P = 0.0044) expression, and a higher MIB-1-labeling index (P = 0.0244) in ERMS, however, in ARMS no such relationships were observed. These immunohistochemical results indicate that different expression profiles of MDR-related molecules and their correlation with YB-1 nuclear expression support the concept that ERMS and ARMS are molecular biologically distinct neoplasms. Apart from ERMS, frequent P-gp expression in ARMS may be independent from YB-1 regulation. However, YB-1 may be a candidate for a molecular target in rhabdomyosarcoma therapy, especially in ERMS.

Ets regulates peroxiredoxin1 and 5 expressions through their interaction with the high-mobility group protein B1.

Peroxiredoxins (Prdxs) are thiol-specific antioxidant proteins that are highly expressed in human cancer cells. Prdxs have been shown to be involved in tumor cell proliferation under conditions of microenvironmental stress such as hypoxia. We hypothesized that Prdxs could be categorized into two groups, stress-inducible and non-inducible ones. In this study, we analyzed the promoter activity and expression levels of five Prdx family members in human cancer cells. We found that both Prdx1 and Prdx5 are inducible after treatment with hydrogen peroxide or hypoxia, but that Prdx2, Prdx3, and Prdx4 are not or are only marginally inducible. We also found that Ets transcription factors are the key activators for stress-inducible expression. High-mobility group protein HMGB1 was shown to function as a coactivator through direct interactions with Ets transcription factors. The DNA binding of Ets transcription factors was significantly enhanced by HMGB1. Silencing of Ets1, Ets2, Prdx1, and Prdx5 expression sensitized cells to oxidative stress. These data indicate that transcription of Prdx genes mediated by Ets/HMG proteins might protect cells from oxidative stress.

Inhibition of bone and muscle metastases of lung cancer cells by a decrease in the number of monocytes/macrophages.

Attention has recently focused on the critical role of inflammatory responses in the tumor stroma that provide favorable conditions for cancer-cell growth and invasion/metastasis. In particular, macrophages recruited into the tumor stroma and activated, known as tumor-associated macrophages, are suggested to promote tumorigenesis. In this study, we examined the effect of a decrease in the number of monocytes/macrophages in peripheral blood and the tumor stroma on the development of bone and muscle metastases by lung cancer cells. Treatment with clodronate encapsulated by liposomes (Cl(2)MDP-LIP) has been developed for the depletion of monocytes/macrophages in an animal model. Subcutaneous administration of Cl(2)MDP-LIP markedly reduced the number of monocytes in peripheral blood, resulting in efficient suppression of both bone metastasis and muscle metastasis when lung cancer HARA-B cells were injected into the left cardiac ventricle of mice. Treatment with Cl(2)MDP-LIP significantly reduced the number of macrophages in tumors and the number of osteoclasts in bone marrow, as well as peripheral monocytes in mice harboring lung cancer cells. In contrast, treatment with an osteoclast-targeting antibiotic, reveromycin A, inhibited bone metastasis by lung cancer cells, but not muscle metastasis. The survival of human macrophages in culture was found to be specifically blocked by Cl(2)MDP-LIP, but not by reveromycin A. Cl(2)MDP-LIP thus exerted antimetastatic effects in both bone and muscle whereas reveromycin A did so only in bone. Liposome-encapsulated bisphosphonate may modulate metastasis through decreasing the number of monocytes/macrophages in both peripheral blood and the tumor stroma, suggesting that tumor-associated macrophages might be suitable targets for antimetastatic therapy.

Infiltration of COX-2-expressing macrophages is a prerequisite for IL-1 beta-induced neovascularization and tumor growth.

Inflammatory angiogenesis is a critical process in tumor progression and other diseases. The inflammatory cytokine IL-1beta promotes angiogenesis, tumor growth, and metastasis, but its mechanisms remain unclear. We examined the association between IL-1beta-induced angiogenesis and cell inflammation. IL-1beta induced neovascularization in the mouse cornea at rates comparable to those of VEGF. Neutrophil infiltration occurred on day 2. Macrophage infiltration occurred on days 4 and 6. The anti-Gr-1 Ab-induced depletion of infiltrating neutrophils did not affect IL-1beta- or VEGF-induced angiogenesis. The former was reduced in monocyte chemoattractant protein-1-deficient (MCP-1(-/-)) mice compared with wild-type mice. After day 4, clodronate liposomes, which kill macrophages, reduced IL-1beta-induced angiogenesis and partially inhibited VEGF-induced angiogenesis. Infiltrating macrophages near the IL-1beta-induced neovasculature were COX-2 positive. Lewis lung carcinoma cells expressing IL-1beta (LLC/IL-1beta) developed neovasculature with macrophage infiltration and enhanced tumor growth in wild-type but not MCP-1(-/-) mice. A COX-2 inhibitor reduced tumor growth, angiogenesis, and macrophage infiltration in LLC/IL-1beta. Thus, macrophage involvement might be a prerequisite for IL-1beta-induced neovascularization and tumor progression.