Molecular Cancer Research recent issues

Systems-Level Analysis of ErbB4 Signaling in Breast Cancer: A Laboratory to Clinical Perspective

Chuu, C.-P., Chen, R.-Y., Barkinge, J. L., Ciaccio, M. F., Jones, R. B. — 2008-06-20

Although expression of the ErbB4 receptor tyrosine kinase in breast cancer is generally regarded as a marker for favorable patient prognosis, controversial exceptions have been reported. Alternative splicing of ErbB4 pre-mRNAs results in the expression of distinct receptor isoforms with differential susceptibility to enzymatic cleavage and different downstream signaling protein recruitment potential that could affect tumor progression in different ways. ErbB4 protein expression from nontransfected cells is generally low compared with ErbB1 in most cell lines, and much of our knowledge of the role of ErbB4 in breast cancer is derived from the ectopic overexpression of the receptor in non–breast-derived cell lines. One of the primary functions of ErbB4 in vivo is in the maturation of mammary glands during pregnancy and lactation induction. Pregnancy and extended lactation durations have been correlated with reduced risk of breast cancer, and the role of ErbB4 in tumor suppression may therefore be linked with its role in lactation. Most reports are consistent with a role for ErbB4 in reversing growth stimuli triggered by other ErbB family members during puberty. In this report, we provide a systems-level examination of several reports highlighting the seemingly opposing roles of ErbB4 in breast cancer and potential explanations for the discrepancies and draw the conclusion that future studies examining the function of ErbB4 in breast cancer should also take into account the pregnancy history, lactation status, and hormone supplementation or ablation history of the patient from whom the tumor or tumor cells are derived. (Mol Cancer Res 2008;6(6):885–91)

Zinc {alpha}2-Glycoprotein: A Multidisciplinary Protein

Hassan, Md. I., Waheed, A., Yadav, S., Singh, T. P., Ahmad, F. — 2008-06-20

Zinc 2-glycoprotein (ZAG) is a protein of interest because of its ability to play many important functions in the human body, including fertilization and lipid mobilization. After the discovery of this molecule, during the last 5 decades, various studies have been documented on its structure and functions, but still, it is considered as a protein with an unknown function. Its expression is regulated by glucocorticoids. Due to its high sequence homology with lipid-mobilizing factor and high expression in cancer cachexia, it is considered as a novel adipokine. On the other hand, structural organization and fold is similar to MHC class I antigen-presenting molecule; hence, ZAG may have a role in the expression of the immune response. The function of ZAG under physiologic and cancerous conditions remains mysterious but is considered as a tumor biomarker for various carcinomas. There are several unrelated functions that are attributed to ZAG, such as RNase activity, regulation of melanin production, hindering tumor proliferation, and transport of nephritic by-products. This article deals with the discussion of the major aspects of ZAG from its gene structure to function and metabolism. (Mol Cancer Res 2008;6(6):892–906)

Tumor Environment Dictates Medulloblastoma Cancer Stem Cell Expression and Invasive Phenotype

2008-06-20

The neural precursor surface marker CD133 is thought to be enriched in brain cancer stem cells and in radioresistant DAOY medulloblastoma-derived tumor cells. Given that membrane type-1 matrix metalloproteinase (MT1-MMP) expression is a hallmark of highly invasive, radioresistant, and hypoxic brain tumor cells, we sought to determine whether MT1-MMP and other MMPs could regulate the invasive phenotype of CD133(+) DAOY cells. We found that when DAOY medulloblastoma or U87 glioblastoma cells were implanted in nude mice, only those cells specifically implanted in the brain environment generated CD133(+) brain tumors. Vascular endothelial growth factor and basic fibroblast growth factor gene expression increases in correlation with CD133 expression in those tumors. When DAOY cultures were induced to generate in vitro neurosphere-like cells, gene expression of CD133, MT1-MMP, MMP-9, and MDR-1 was induced and correlated with an increase in neurosphere invasiveness. Specific small interfering RNA gene silencing of either MT1-MMP or MMP-9 reduced the capacity of the DAOY monolayers to generate neurospheres and concomitantly abrogated their invasive capacity. On the other hand, overexpression of MT1-MMP in DAOY triggered neurosphere-like formation which was further amplified when cells were cultured in neurosphere medium. Collectively, we show that both MT1-MMP and MMP-9 contribute to the invasive phenotype during CD133(+) neurosphere-like formation in medulloblastoma cells. Increases in MMP-9 may contribute to the opening of the blood-brain barrier, whereas increased MT1-MMP would promote brain tumor infiltration. Our study suggests that MMP-9 or MT1-MMP targeting may reduce the formation of brain tumor stem cells. (Mol Cancer Res 2008;6(6):907–16)

Effects of Raf Kinase Inhibitor Protein Expression on Metastasis and Progression of Human Epithelial Ovarian Cancer

2008-06-20

Loss of function of metastasis suppressor genes is an important step in the progression to a malignant tumor type. Studies in cell culture and animal models have suggested a role of Raf kinase inhibitor protein (RKIP) in suppressing the metastatic spread of prostate cancer, breast cancer, and melanoma cells. However, the function of RKIP in ovarian cancer (OVCA) has not been reported. To explore the potential role of RKIP in epithelial OVCA metastasis, we detected the expression levels of RKIP protein in tissue samples from patients with epithelial OVCA. Consequently, the expression of RKIP is reduced in the poorly differentiated OVCA than in the well-differentiated and moderately differentiated OVCA. In addition, in vitro cell invasion assay indicated that the RKIP expression was inversely associated with the invasiveness of five OVCA cell lines. Consistent with this result, the cell proliferation, anchorage-independent growth, cell adhesion, and invasion were decreased in RKIP overexpressed cells but increased in RKIP down-regulated cells. Further investigation indicated that RKIP inhibited OVCA cell proliferation by altering cell cycle progression rather than promoting apoptosis. Furthermore, the overexpression of RKIP suppressed the ability of human OVCA cells to metastasize when the tumor cells were transplanted into nude mice. Our data show the effect of RKIP on the proliferation, migration, or adhesion of OVCA cells. These results indicate that RKIP is also a metastasis suppressor gene of human epithelial OVCA. (Mol Cancer Res 2008;6(6):917–28)

Bone Marrow Subsets Differentiate into Endothelial Cells and Pericytes Contributing to Ewing's Tumor Vessels

Reddy, K., Zhou, Z., Schadler, K., Jia, S.-F., Kleinerman, E. S. — 2008-06-20

Hematopoietic progenitor cells arising from bone marrow (BM) are known to contribute to the formation and expansion of tumor vasculature. However, whether different subsets of these cells have different roles in this process is unclear. To investigate the roles of BM-derived progenitor cell subpopulations in the formation of tumor vasculature in a Ewing's sarcoma model, we used a functional assay based on endothelial cell and pericyte differentiation in vivo. Fluorescence-activated cell sorting of human cord blood/BM or mouse BM from green fluorescent protein transgenic mice was used to isolate human CD34⁺/CD38^–, CD34⁺/CD45⁺, and CD34^–/CD45⁺ cells and mouse Sca1⁺/Gr1⁺, Sca1^–/Gr1⁺, VEGFR1⁺, and VEGFR2⁺ cells. Each of these progenitor subpopulations was separately injected intravenously into nude mice bearing Ewing's sarcoma tumors. Tumors were resected 1 week later and analyzed using immunohistochemistry and confocal microscopy for the presence of migrated progenitor cells expressing endothelial, pericyte, or inflammatory cell surface markers. We showed two distinct patterns of stem cell infiltration. Human CD34⁺/CD45⁺ and CD34⁺/CD38^– and murine VEGFR2⁺ and Sca1⁺/Gr1⁺ cells migrated to Ewing's tumors, colocalized with the tumor vascular network, and differentiated into cells expressing either endothelial markers (mouse CD31 or human vascular endothelial cadherin) or the pericyte markers desmin and -smooth muscle actin. By contrast, human CD34^–/CD45⁺ and mouse Sca1^–/Gr1⁺ cells migrated predominantly to sites outside of the tumor vasculature and differentiated into monocytes/macrophages expressing F4/80 or CD14. Our data indicate that only specific BM stem/progenitor subpopulations participate in Ewing's sarcoma tumor vasculogenesis. (Mol Cancer Res 2008;6(6):929–36)

Cell Cycle Regulator Gene CDC5L, a Potential Target for 6p12-p21 Amplicon in Osteosarcoma

2008-06-20

Osteosarcoma is a primary malignant tumor of bone arising from primitive bone-forming mesenchymal cells and accounts for ~60% of malignant bone tumors. Our comparative genomic hybridization (CGH) studies have identified frequent amplification at 6p12-p21, 12q13-q15, and 17p11.2 in osteosarcoma. Of these amplified regions, 6p12-p21 is particularly interesting because of its association with progression and poor prognosis in patients with osteosarcoma. In an attempt to identify aberrantly expressed gene(s) mapping to the 6p12-p21 amplicon, a region-specific array was generated using 108 overlapping BAC and P1 clones covering a 28.8-Mb region at 0.26-Mb intervals. Based on array CGH analysis, the 6p amplicon was refined to 7.9 Mb between the clones RP11-91E11 and RP1-244F2 and 10 amplified clones, with possible target genes, were identified. To study the expression pattern of the target genes from the hotspot amplicon and known candidate genes from 6p12-21, we did quantitative reverse transcription-PCR analysis of MAPK14, MAPK13, CDKN1A, PIM1, MDGA1, BTB9, DNAH8, CCND3, PTK7, CDC5L, and RUNX2 on osteosarcoma patient samples and seven cell lines. The combined array CGH and quantitative reverse transcription-PCR analysis identified amplification and overexpression of CDC5L, CCND3, and RUNX2. We screened these three genes for protein expression by Western blotting and immunohistochemistry and detected overexpression of CDC5L. Furthermore, we used an in vivo assay to show that CDC5L possesses potential oncogenic activity. These results indicate that CDC5L, a cell cycle regulator important for the G₂-M transition, is the most likely candidate oncogene for the 6p12-p21 amplicon found in osteosarcoma. (Mol Cancer Res 2008;6(6):937–46)

Mdm2 and Mdm4 Loss Regulates Distinct p53 Activities

Barboza, J. A., Iwakuma, T., Terzian, T., El-Naggar, A. K., Lozano, G. — 2008-06-20

Mutational inactivation of p53 is a hallmark of most human tumors. Loss of p53 function also occurs by overexpression of negative regulators such as MDM2 and MDM4. Deletion of Mdm2 or Mdm4 in mice results in p53-dependent embryo lethality due to constitutive p53 activity. However, Mdm2^–/– and Mdm4^–/– embryos display divergent phenotypes, suggesting that Mdm2 and Mdm4 exert distinct control over p53. To explore the interaction between Mdm2 and Mdm4 in p53 regulation, we first generated mice and cells that are triple null for p53, Mdm2, and Mdm4. These mice had identical survival curves and tumor spectrum as p53^–/– mice, substantiating the principal role of Mdm2 and Mdm4 as negative p53 regulators. We next generated mouse embryo fibroblasts null for p53 with deletions of Mdm2, Mdm4, or both; introduced a retrovirus expressing a temperature-sensitive p53 mutant, p53A135V; and examined p53 stability and activity. In this system, p53 activated distinct target genes, leading to apoptosis in cells lacking Mdm2 and a cell cycle arrest in cells lacking Mdm4. Cells lacking both Mdm2 and Mdm4 had a stable p53 that initiated apoptosis similar to Mdm2-null cells. Additionally, stabilization of p53 in cells lacking Mdm4 with the Mdm2 antagonist nutlin-3 was sufficient to induce a cell death response. These data further differentiate the roles of Mdm2 and Mdm4 in the regulation of p53 activities. (Mol Cancer Res 2008;6(6):947–54)

G-Quadruplex Stabilizer 3,6-Bis(1-Methyl-4-Vinylpyridinium)Carbazole Diiodide Induces Accelerated Senescence and Inhibits Tumorigenic Properties in Cancer Cells

2008-06-20

Carbazole derivatives that stabilized G-quadruplex DNA structure formed by human telomeric sequence have been designed and synthesized. Among them, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) showed an increase in G-quadruplex melting temperature by 13°C and has a potent inhibitory effect on telomerase activity. Treatment of H1299 cancer cells with 0.5 µmol/L BMVC did not cause acute toxicity and affect DNA replication; however, the BMVC-treated cells ceased to divide after a lag period. Hallmarks of senescence, including morphologic changes, detection of senescence-associated β-galactosidase activity, and decreased bromodeoxyuridine incorporation, were detected in BMVC-treated cancer cells. The BMVC-induced senescence phenotype is accompanied by progressive telomere shortening and detection of the DNA damage foci, indicating that BMVC caused telomere uncapping after long-term treatments. Unlike other telomerase inhibitors, the BMVC-treated cancer cells showed a fast telomere shortening rate and a lag period of growth before entering senescence. Interestingly, BMVC also suppressed the tumor-related properties of cancer cells, including cell migration, colony-forming ability, and anchorage-independent growth, indicating that the cellular effects of BMVC were not limited to telomeres. Consistent with the observations from cellular experiments, the tumorigenic potential of cancer cells was also reduced in mouse xenografts after BMVC treatments. Thus, BMVC repressed tumor progression through both telomere-dependent and telomere-independent pathways. (Mol Cancer Res 2008;6(6):955–64)

Active Ras Triggers Death in Glioblastoma Cells through Hyperstimulation of Macropinocytosis

Overmeyer, J. H., Kaul, A., Johnson, E. E., Maltese, W. A. — 2008-06-20

Expression of activated Ras in glioblastoma cells induces accumulation of large phase-lucent cytoplasmic vacuoles, followed by cell death. This was previously described as autophagic cell death. However, unlike autophagosomes, the Ras-induced vacuoles are not bounded by a double membrane and do not sequester organelles or cytoplasm. Moreover, they are not acidic and do not contain the autophagosomal membrane protein LC3-II. Here we show that the vacuoles are enlarged macropinosomes. They rapidly incorporate extracellular fluid-phase tracers but do not sequester transferrin or the endosomal protein EEA1. Ultimately, the cells expressing activated Ras detach from the substratum and rupture, coincident with the displacement of cytoplasm with huge macropinosome-derived vacuoles. These changes are accompanied by caspase activation, but the broad-spectrum caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone does not prevent cell death. Moreover, the majority of degenerating cells do not exhibit chromatin condensation typical of apoptosis. These observations provide evidence for a necrosis-like form of cell death initiated by dysregulation of macropinocytosis, which we have dubbed "methuosis." An activated form of the Rac1 GTPase induces a similar form of cell death, suggesting that Ras acts through Rac-dependent signaling pathways to hyperstimulate macropinocytosis in glioblastoma. Further study of these signaling pathways may lead to the identification of other chemical and physiologic triggers for this unusual form of cell death. (Mol Cancer Res 2008;6(6):965–77)

Senescence-Dependent MutS{alpha} Dysfunction Attenuates Mismatch Repair

2008-06-20

DNA damage and mutations in the genome increase with age. To determine the potential mechanisms of senescence-dependent increases in genomic instability, we analyzed DNA mismatch repair (MMR) efficiency in young and senescent human colonic fibroblast and human embryonic lung fibroblast. It was found that MMR activity is significantly reduced in senescent cells. Western blot and immunohistochemistry analysis revealed that hMSH2 and MSH6 protein (MutS complex), which is a known key component in the MMR pathway, is markedly down-regulated in senescent cells. Moreover, the addition of purified MutS to extracts from senescent cells led to the restoration of MMR activity. Semiquantitative reverse transcription-PCR analysis exhibited that MSH2 mRNA level is reduced in senescent cells. In addition, a decrease in E2F transcriptional activity in senescent cells was found to be crucial for MSH2 suppression. E2F1 small interfering RNA expression reduced hMSH2 expression and MMR activity in young human primary fibroblast cells. Importantly, expression of E2F1 in quiescent cells restored the MSH2 expression as well as MMR activity, whereas E2F1-infected senescent cells exhibited no restoration of MSH2 expression and MMR activity. These results indicate that the suppression of E2F1 transcriptional activity in senescent cells lead to stable repression of MSH2, followed by a induction of MutS dysfunction, which results in a reduced cellular MMR capacity in senescent cells. (Mol Cancer Res 2008;6(6):978–89)

The RNA Exosome Component hRrp6 Is a Target for 5-Fluorouracil in Human Cells

Kammler, S., Lykke-Andersen, S., Jensen, T. H. — 2008-06-20

The drug 5-fluorouracil (5-FU) is a widely used chemotherapeutic in the treatment of solid tumors. Recently, the essential 3'-5' exonucleolytic multisubunit RNA exosome was implicated as a target for 5-FU in yeast. Here, we show that this is also the case in human cells. HeLa cells depleted of the inessential exosome component hRrp6, also called PM/Scl100, are significantly growth impaired relative to control cells after 5-FU administration. The selective stabilization of bona fide hRrp6 RNA substrates on 5-FU treatment suggests that this exosome component is specifically targeted. Consistently, levels of hRrp6 substrates are increased in two 5-FU–sensitive cell lines. Interestingly, whereas down-regulation of all tested core exosome components results in decreased hRrp6 levels, depletion of hRrp6 leaves levels of other exosome components unchanged. Taken together, our data position hRrp6 as a promising target for antiproliferative intervention. (Mol Cancer Res 2008;6(6):990–5)

Activation of Epidermal Growth Factor Receptor and Its Downstream Signaling Pathway by Nitric Oxide in Response to Ionizing Radiation

2008-06-20

Epidermal growth factor receptor (EGFR) is activated by ionizing radiation (IR), but the molecular mechanism for this effect is unknown. We have found that intracellular generation of nitric oxide (NO) by NO synthase (NOS) is required for the rapid activation of EGFR phosphorylation by IR. Treatment of A549 lung cancer cells with IR increased NOS activity within minutes, accompanied by an increase of NO. 2-Phenyl-4,4,5,5,-tetramethylimidazolline-1-oxyl-3-oxide, an NO scavenger, and N^G-monomethyl-l-arginine, an NOS inhibitor, abolished the increase in intracellular NO and activation of EGFR by IR. In addition, an NO donor alone induced EGFR phosphorylation. Transient transfection with small interfering RNA for endothelial NOS reduced IR-induced NO production and suppressed IR-induced EGFR activation. Overexpression of endothelial NOS increased IR-induced NO generation and EGFR activation. These results indicate a novel molecular mechanism for EGFR activation by IR-induced NO production via NOS. (Mol Cancer Res 2008;6(6):996–1002)

Multiple Signaling Pathways Are Responsible for Prostaglandin E2-Induced Murine Keratinocyte Proliferation

Ansari, K. M., Rundhaug, J. E., Fischer, S. M. — 2008-06-20

Although prostaglandin E₂ (PGE₂) has been shown by pharmacologic and genetic studies to be important in skin cancer, the molecular mechanism(s) by which it contributes to tumor growth is not well understood. In this study, we investigated the mechanisms by which PGE₂ stimulates murine keratinocyte proliferation using in vitro and in vivo models. In primary mouse keratinocyte cultures, PGE₂ activated the epidermal growth factor receptor (EGFR) and its downstream signaling pathways as well as increased cyclic AMP (cAMP) production and activated the cAMP response element binding protein (CREB). EGFR activation was not significantly inhibited by pretreatment with a c-src inhibitor (PP2), nor by a protein kinase A inhibitor (H-89). However, PGE₂-stimulated extracellularly regulated kinase 1/2 (ERK1/2) activation was completely blocked by EGFR, ERK1/2, and phosphatidylinositol 3-kinase (PI3K) pathway inhibitors. In addition, these inhibitors attenuated the PGE₂-induced proliferation, nuclear factor-B, activator protein-1 (AP-1), and CREB binding to the promoter regions of the cyclin D1 and vascular endothelial growth factor (VEGF) genes and expression of cyclin D1 and VEGF in primary mouse keratinocytes. Similarly, in vivo, we found that WT mice treated with PGE₂ and untreated cyclooxygenase-2–overexpressing transgenic mice had higher levels of cell proliferation and expression of cyclin D1 and VEGF, as well as higher levels of activated EGFR, nuclear factor-B, AP-1, and CREB, than vehicle-treated WT mice. Our findings provide evidence for a link between cyclooxygenase-2 overexpression and EGFR-, ERK-, PI3K-, cAMP-mediated cell proliferation, and the tumor-promoting activity of PGE₂ in mouse skin. (Mol Cancer Res 2008;6(6):1003–16)

Secreted Frizzled-Related Protein 4 Regulates Two Wnt7a Signaling Pathways and Inhibits Proliferation in Endometrial Cancer Cells

Carmon, K. S., Loose, D. S. — 2008-06-20

In the endometrium, hormonal effects on epithelial cells are often elicited through stromal hormone receptors via unknown paracrine mechanisms. Several lines of evidence support the hypothesis that Wnts participate in stromal-epithelial cell communication. Wnt7a is expressed in the luminal epithelium, whereas the extracellular modulator of Wnt signaling, secreted frizzled-related protein 4 (SFRP4), is localized to the stroma. Studies have reported that SFRP4 expression is significantly decreased in endometrial carcinoma and that both SFRP4 and Wnt7a genes are differentially regulated in response to estrogenic stimuli. Aberrant Wnt7a signaling irrevocably causes organ defects and infertility and contributes to the onset of disease. However, specific frizzled receptors (Fzd) that bind Wnt7a and the particular signal transduction pathway each Wnt7a-Fzd pair activates have not been identified. Additionally, the function of SFRP4 in the endometrium has not been addressed. We show here that Wnt7a coimmunoprecipitates with Fzd5, Fzd10, and SFRP4 in Ishikawa cells. Wnt7a binding to Fzd5 was shown to activate β-catenin/canonical Wnt signaling and increase cellular proliferation. Conversely, Wnt7a signaling mediated by Fzd10 induced a noncanonical c-Jun NH₂-terminal kinase–responsive pathway. SFRP4 suppresses activation of Wnt7a signaling in both an autocrine and paracrine manner. Stable overexpression of SFRP4 and treatment with recombinant SFRP4 protein inhibited endometrial cancer cell growth in vitro. These findings support a mechanism by which the nature of the Wnt7a signal in the endometrium is dependent on the Fzd repertoire of the cell and can be regulated by SFRP4. (Mol Cancer Res 2008;6(6):1017–28)

The Effect of Transforming Growth Factor {beta} on Human Neuroendocrine Tumor BON Cell Proliferation and Differentiation Is Mediated through Somatostatin Signaling

Leu, F. P., Nandi, M., Niu, C. — 2008-06-20

The dual effect of the ubiquitous inflammatory cytokine transforming growth factor β1 (TGFβ) on cellular proliferation and tumor metastasis is intriguing but complex. In epithelial cell– and neural cell–derived tumors, TGFβ serves as a growth inhibitor at the beginning of tumor development but later becomes a growth accelerator for transformed tumors. The somatostatin (SST) signaling pathway is a well-established antiproliferation signal, and in this report, we explore the interplay between the SST and TGFβ signaling pathways in the human neuroendocrine tumor cell line BON. We defined the SST signaling pathway as a determinant for neuroendocrine tumor BON cells in responding to TGFβ as a growth inhibitor. We also determined that TGFβ induces the production of SST and potentially activates the negative growth autocrine loop of SST, which leads to the downstream induction of multiple growth inhibitory effectors: protein tyrosine phosphatases (i.e., SHPTP1 and SHPTP2), p21^Waf1/Cip1, and p27^Kip1. Concurrently, TGFβ down-regulates the growth accelerator c-Myc protein and, collectively, they establish a firm antiproliferation effect on BON cells. Additionally, any disruption in the activation of either the TGFβ or SST signaling pathway in BON leads to "reversible" neuroendocrine-mesenchymal transition, which is characterized by the loss of neuroendocrine markers (i.e., chromogranin A and PGP 9.5), as well as the altered expression of mesenchymal proteins (i.e., elevated vimentin and Twist and decreased E-cadherin), which has previously been associated with elevated metastatic potential. In summary, TGFβ-dependent growth inhibition and differentiation is mediated by the SST signaling pathway. Therefore, any disruption of this TGFβ-SST connection allows BON cells to respond to TGFβ as a growth accelerator instead of a growth suppressor. This model can potentially apply to other cell types that exhibit a similar interaction of these pathways. (Mol Cancer Res 2008;6(6):1029–42)

Prostate-Specific Kallikreins-2 and -4 Enhance the Proliferation of DU-145 Prostate Cancer Cells through Protease-Activated Receptors-1 and -2

Mize, G. J., Wang, W., Takayama, T. K. — 2008-06-20

A major characteristic of prostate cancer is the elevation of serum levels of prostate-specific antigen (hK3) and hK2, which are tumor markers that correlate with advancing stages of disease. Including hK4, these three kallikrein serine proteases are almost exclusively produced by the prostate. Prostate cancer cells have been recently shown to overexpress protease-activated receptors (PAR), which can be potentially activated by kallikreins and can regulate tumor growth. Here, we show that recombinant hK2 and hK4 activate ERK1/2 signaling of DU-145, PC-3, and LNCaP prostate cancer cells, which express both PAR1 and PAR2. These kallikreins also stimulate the proliferation of DU-145 cells. Pretreatment of hK2 and hK4 with the serine protease inhibitor, aprotinin, blocks the responses in DU-145 cells, and small interfering RNA against PAR1 and PAR2 also inhibits ERK1/2 signaling. To determine which PAR is activated by hK2 and hK4, a cell line that expresses a single PAR, a PAR1 knockout mouse lung fibroblast cell line transfected with PAR1 (KOLF-PAR1) or PAR2 (KOLF-PAR2) was used. hK4 activates both PAR1 and PAR2, whereas hK2 activates PAR2. hK4 generates more phosphorylated ERK1/2 than hK2. These data indicate that prostatic kallikreins (hK2 and hK4) directly stimulate prostate cancer cell proliferation through PAR1 and/or PAR2 and may be potentially important targets for future drug therapy for prostate cancer. (Mol Cancer Res 2008;6(6):1043–51)

A Novel Unidirectional Cross-Talk from the Insulin-Like Growth Factor-I Receptor to Leptin Receptor in Human Breast Cancer Cells

Ozbay, T., Nahta, R. — 2008-06-20

Obesity is a major risk factor for the development and progression of breast cancer. Increased circulating levels of the obesity-associated hormones leptin and insulin-like growth factor-I (IGF-I) and overexpression of the leptin receptor (Ob-R) and IGF-I receptor (IGF-IR) have been detected in a majority of breast cancer cases and during obesity. Due to correlations between increased leptin, Ob-R, IGF-I, and IGF-IR in breast cancer, we hypothesized that molecular interactions may exist between these two signaling pathways. Coimmunoprecipitation and immunoblotting showed that IGF-IR and Ob-R interact in the breast cancer cell lines MDA-MB-231, MCF7, BT474, and SKBR3. Stimulation of cells with IGF-I promoted Ob-R phosphorylation, which was blocked by IGF-IR kinase inhibition. In addition, IGF-I activated downstream signaling molecules in the leptin receptor and IGF-IR pathways. In contrast to IGF-I, leptin did not induce phosphorylation of IGF-IR, indicating that receptor cross-signaling is unidirectional, occurring from IGF-IR to Ob-R. Our results show, for the first time, a novel interaction and cross-talk between the IGF-I and leptin receptors in human breast cancer cells. (Mol Cancer Res 2008;6(6):1052–8)

Targeting Nuclear Factor-{kappa}B Activation Pathway by Thymoquinone: Role in Suppression of Antiapoptotic Gene Products and Enhancement of Apoptosis

Sethi, G., Ahn, K. S., Aggarwal, B. B. — 2008-06-20

Thymoquinone (TQ), derived from the medicinal plant Nigella sativa, exhibits antiinflammatory and anticancer activities through mechanism(s) that is not fully understood. Because numerous effects modulated by TQ can be linked to interference with the nuclear factor-B (NF-B) signaling, we investigated in detail the effect of this quinone on NF-B pathway. As examined by DNA binding, we found that TQ suppressed tumor necrosis factor–induced NF-B activation in a dose- and time-dependent manner and inhibited NF-B activation induced by various carcinogens and inflammatory stimuli. The suppression of NF-B activation correlated with sequential inhibition of the activation of IB kinase, IB phosphorylation, IB degradation, p65 phosphorylation, p65 nuclear translocation, and the NF-B–dependent reporter gene expression. TQ specifically suppressed the direct binding of nuclear p65 and recombinant p65 to the DNA, and this binding was reversed by DTT. However, TQ did not inhibit p65 binding to DNA when cells were transfected with the p65 plasmid containing cysteine residue 38 mutated to serine. TQ also down-regulated the expression of NF-B–regulated antiapoptotic (IAP1, IAP2, XIAP Bcl-2, Bcl-xL, and survivin), proliferative (cyclin D1, cyclooxygenase-2, and c-Myc), and angiogenic (matrix metalloproteinase-9 and vascular endothelial growth factor) gene products. This led to potentiation of apoptosis induced by tumor necrosis factor and chemotherapeutic agents. Overall, our results indicate that the anticancer and antiinflammatory activities previously assigned to TQ may be mediated in part through the suppression of the NF-B activation pathway, as shown here, and thus may have potential in treatment of myeloid leukemia and other cancers. (Mol Cancer Res 2008;6(6):1059–70)

TATA Box-Binding Protein-Associated Factor 12 Is Important for RAS-Induced Transformation Properties of Colorectal Cancer Cells

2008-06-20

Activating mutations in the RAS proto-oncogene result in constant stimulation of its downstream pathways, further leading to tumorigenesis. Transcription factor IID (TFIID) can be regulated by cellular signals to specifically alter transcription of particular subsets of genes. To investigate potential links between the regulation of TFIID function and the RAS-induced carcinogenesis, we monitored the expression of the TATA box-binding protein and its associated factors (TAF) in human colon carcinoma cells. We primarily identified TAF12 levels as being up-regulated in cell lines bearing natural RAS mutations or stably overexpressing a mutated RAS isoform via a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase–dependent pathway. We further showed by electrophoretic mobility shift assays and chromatin immunoprecipitation that the ETS1 protein was interacting with an ETS-binding site on the TAF12 promoter and was regulating TAF12 expression. The binding was enhanced in extracts from oncogenic RAS-transformed cells, pointing to a role in the RAS-mediated regulation of TAF12 expression. Reduction of TAF12 levels by small interfering RNA treatment induced a destabilization of the TFIID complex, enhanced E-cadherin mRNA and protein levels, and reduced migration and adhesion properties of RAS-transformed cells with epithelial to mesenchymal transition. Overall, our study indicates the importance of TAF12 in the process of RAS-induced transformation properties of human colon cells and epithelial to mesenchymal transition, most notably those related to increased motility, by regulating specifically expression of genes such as E-cadherin. (Mol Cancer Res 2008;6(6):1071–83)

Signal Transducer and Activator of Transcription-3: A Molecular Hub for Signaling Pathways in Gliomas

Brantley, E. C., Benveniste, E. N. — 2008-05-27

Glioblastoma is the most common and severe primary brain tumor in adults. Its aggressive and infiltrative nature renders the current therapeutics of surgical resection, radiation, and chemotherapy relatively ineffective. Accordingly, recent research has focused on the elucidation of various signal transduction pathways in glioblastoma, particularly aberrant activation. This review focuses on the signal transducer and activator of transcription-3 (STAT-3) signal transduction pathway in the context of this devastating tumor. STAT-3 is aberrantly activated in human glioblastoma tissues, and this activation is implicated in controlling critical cellular events thought to be involved in gliomagenesis, such as cell cycle progression, apoptosis, angiogenesis, and immune evasion. There are no reports of gain-of-function mutations in glioblastoma; rather, the activation of STAT-3 is thought to be a consequence of either dysregulation of upstream kinases or loss of endogenous inhibitors. This review provides detailed insight into the multiple mechanisms of STAT-3 activation in glioblastoma, as well as describing endogenous and chemical inhibitors of this pathway and their clinical significance. In glioblastoma, STAT-3 acts a molecular hub to link extracellular signals to transcriptional control of proliferation, cell cycle progression, and immune evasion. Because STAT-3 plays this central role in glioblastoma signal transduction, it has significant potential as a therapeutic target. (Mol Cancer Res 2008;6(5):675–84)

Zebrafish as a Cancer Model

Feitsma, H., Cuppen, E. — 2008-05-27

The zebrafish has developed into an important model organism for biomedical research over the last decades. Although the main focus of zebrafish research has traditionally been on developmental biology, keeping and observing zebrafish in the lab led to the identification of diseases similar to humans, such as cancer, which subsequently became a subject for study. As a result, about 50 articles have been published since 2000 in which zebrafish were used as a cancer model. Strategies used include carcinogenic treatments, transplantation of mammalian cancer cells, forward genetic screens for proliferation or genomic instability, reverse genetic target-selected mutagenesis to inactivate known tumor suppressor genes, and the generation of transgenics to express human oncogenes. Zebrafish have been found to develop almost any tumor type known from human, with similar morphology and, according to gene expression array studies, comparable signaling pathways. However, tumor incidences are relatively low, albeit highly comparable between different mutants, and tumors develop late in life. In addition, tumor spectra are sometimes different when compared with mice and humans. Nevertheless, the zebrafish model has created its own niche in cancer research, complementing existing models with its specific experimental advantages and characteristics. Examples of these are imaging of tumor progression in living fish by fluorescence, treatment with chemical compounds, and screening possibilities not only for chemical modifiers but also for genetic enhancers and suppressors. This review aims to provide a comprehensive overview of the state of the art of zebrafish as a model in cancer research. (Mol Cancer Res 2008;6(5):685–94)

Transforming Growth Factor-{beta}1, Transforming Growth Factor-{beta}2, and Transforming Growth Factor-{beta}3 Enhance Ovarian Cancer Metastatic Potential by Inducing a Smad3-Dependent Epithelial-to-Mesenchymal Transition

Do, T.-V., Kubba, L. A., Du, H., Sturgis, C. D., Woodruff, T. K. — 2008-05-27

Transforming growth factor-β (TGF-β) is thought to play a role in the pathobiological progression of ovarian cancer because this peptide hormone is overexpressed in cancer tissue, plasma, and peritoneal fluid. In the current study, we investigated the role of the TGF-β/Smad3 pathway in ovarian cancer metastasis by regulation of an epithelial-to-mesenchymal transition. When cancer cells were cultured on plastic, TGF-β1, TGF-β2, and TGF-β3 induced pro–matrix metalloproteinase (MMP) secretion, loss of cell-cell junctions, down-regulation of E-cadherin, up-regulation of N-cadherin, and acquisition of a fibroblastoid phenotype, consistent with an epithelial-to-mesenchymal transition. Furthermore, Smad3 small interfering RNA transfection inhibited TGF-β–mediated changes to a fibroblastic morphology, but not MMP secretion. When cancer cells were cultured on a three-dimensional collagen matrix, TGF-β1, TGF-β2, and TGF-β3 stimulated both pro-MMP and active MMP secretion and invasion. Smad3 small interfering RNA transfection of cells cultured on a collagen matrix abrogated TGF-β–stimulated invasion and MMP secretion. Analysis of Smad3 nuclear expression in microarrays of serous benign tumors, borderline tumors, and cystadenocarcinoma revealed that Smad3 expression could be used to distinguish benign and borderline tumors from carcinoma (P = 0.006). Higher Smad3 expression also correlated with poor survival (P = 0.031). Furthermore, a direct relationship exists between Smad3 nuclear expression and expression of the mesenchymal marker N-cadherin in cancer patients (P = 0.0057). Collectively, these results implicate an important role for the TGF-β/Smad3 pathway in mediating ovarian oncogenesis by enhancing metastatic potential. (Mol Cancer Res 2008;6(5):695–705)

Imatinib Mesylate Inhibits Proliferation and Exerts an Antifibrotic Effect in Human Breast Stroma Fibroblasts

2008-05-27

Tumor stroma plays an important role in cancer development. In a variety of tumors, such as breast carcinomas, a desmoplastic response, characterized by stromal fibroblast and collagen accumulation, is observed having synergistic effects on tumor progression. However, the effect of known anticancer drugs on stromal cells has not been thoroughly investigated. Imatinib mesylate is a selective inhibitor of several protein tyrosine kinases, including the receptor of platelet-derived growth factor, an important mediator of desmoplasia. Recently, we have shown that imatinib inhibits the growth and invasiveness of human epithelial breast cancer cells. Here, we studied the effect of imatinib on the proliferation and collagen accumulation in breast stromal fibroblasts. We have shown that it blocks the activation of the extracellular signal-regulated kinase and Akt signaling pathways and up-regulates cyclin-dependent kinase inhibitor p21^WAF1, leading to the inhibition of fibroblast proliferation, by arresting them at the G₀/G₁ phase of the cell cycle. Imatinib inhibits more potently the platelet-derived growth factor–mediated stimulation of breast fibroblast proliferation. By using specific inhibitors, we have found that this is due to the inhibition of the Akt pathway. In addition, imatinib inhibits fibroblast-mediated collagen accumulation. Conventional and quantitative PCR analysis, as well as gelatin zymography, indicates that this is due to the down-regulation of mRNA synthesis of collagen I and collagen III—the main collagen types in breast stroma—and not to the up-regulation or activation of collagenases matrix metalloproteinase 2 and matrix metalloproteinase 9. These data indicate that imatinib has an antifibrotic effect on human breast stromal fibroblasts that may inhibit desmoplastic reaction and thus tumor progression. (Mol Cancer Res 2008;6(5):706–14)

A Suppressive Role of Mitogen Inducible Gene-2 in Mesenchymal Cancer Cell Invasion

Shi, X., Wu, C. — 2008-05-27

Cancer cell invasion of extracellular matrix (ECM) is essential for dissemination of cancer cells and metastasis. In this study, we have investigated the role of mitogen inducible gene-2 (Mig-2, also known as kindlin-2), a focal adhesion protein whose expression is altered in several types of human cancers, in mesenchymal cancer cell invasion. Mig-2 is abundantly expressed in SK-LMS-1 leiomyosarcoma cells. The level of Mig-2, however, is considerably lower in more invasive HT-1080 fibrosarcoma cells. Overexpression of Mig-2 in HT-1080 and SK-LMS-1 cells substantially reduced their ability to invade ECM in an in vitro Matrigel invasion assay. Conversely, knockdown of Mig-2 markedly increased the invasiveness of these cells. Consistent with a suppressive role in mesenchymal cancer cell invasion, Mig-2 inhibits urokinase-type plasminogen activator (uPA) secretion and pericellular proteolysis. Overexpression of Mig-2 increased uPA accumulation at the intracellular face of cell-ECM adhesions and reduced the level of secreted uPA. Conversely, knockdown of Mig-2 reduced uPA accumulation at the intracellular face of cell-ECM adhesions and increased uPA secretion. Our results reveal an important role of Mig-2 in suppression of mesenchymal cancer cell invasion and shed new light on how altered Mig-2 expression could influence cancer cell invasion. (Mol Cancer Res 2008;6(5):715–24)

The Fibroblast Growth Factor-Inducible 14 Receptor Is Highly Expressed in HER2-Positive Breast Tumors and Regulates Breast Cancer Cell Invasive Capacity

2008-05-27

Genomic characterization is beginning to define a molecular taxonomy for breast cancer; however, the molecular basis of invasion and metastasis remains poorly understood. We report a pivotal role for the fibroblast growth factor–inducible 14 (Fn14) receptor in this process. We examined whether Fn14 and its ligand tumor necrosis factor–like weak inducer of apoptosis (TWEAK) were expressed in breast tumors and whether deregulation of Fn14 levels affected malignant behavior of breast cancer cell lines. Analysis of TWEAK and Fn14 in publicly available gene expression data indicated that high Fn14 expression levels significantly correlated with several poor prognostic indicators (P < 0.05). Fn14 expression was highest in the HER2-positive/estrogen receptor–negative (HER2⁺/ER^–) intrinsic subtype (P = 0.0008). An association between Fn14 and HER2 expression in breast tumors was confirmed by immunohistochemistry. Fn14 levels were elevated in invasive, ER^– breast cancer cell lines. Overexpression of Fn14 in weakly invasive MCF7 and T47D cells resulted in a marked induction of invasion and activation of nuclear factor-B (NF-B) signaling. Ectopic expression of Fn14tCT, a Fn14 deletion mutant that cannot activate NF-B signaling, was not able to induce invasion. Moreover, ectopic expression of Fn14tCT in highly invasive MDA-MB-231 cells reduced their invasive capability. RNA interference–mediated inhibition of Fn14 expression in both MDA-MB-231 and MDA-MB-436 cells reduced invasion. Expression profiling of the Fn14-depleted cells revealed deregulation of NF-B activity. Our findings support a role for Fn14-mediated NF-B pathway activation in breast tumor invasion and metastasis. (Mol Cancer Res 2008;6(5):725–34)

A Functional Screen Identifies miR-34a as a Candidate Neuroblastoma Tumor Suppressor Gene

2008-05-27

MicroRNAs are small noncoding RNAs that have critical roles in regulating a number of cellular functions through transcriptional silencing. They have been implicated as oncogenes and tumor suppressor genes (oncomirs) in several human neoplasms. We used an integrated genomics and functional screening strategy to identify potential oncomirs in the pediatric neoplasm neuroblastoma. We first identified microRNAs that map within chromosomal regions that we and others have defined as frequently deleted (1p36, 3p22, and 11q23-24) or gained (17q23) in high-risk neuroblastoma. We then transiently transfected microRNA precursor mimics or inhibitors into a panel of six neuroblastoma cell lines that we characterized for these genomic aberrations. The majority of transfections showed no phenotypic effect, but the miR-34a (1p36) and miR-34c (11q23) mimics showed dramatic growth inhibition in cell lines with 1p36 hemizygous deletion. In contrast, there was no growth inhibition by these mimics in cell lines without 1p36 deletions. Quantitative reverse transcription-PCR showed a perfect correlation of absent miR-34a expression in cell lines with a 1p36 aberration and phenotypic effect after mimetic add-back. Expression of miR-34a was also decreased in primary tumors (n = 54) with 1p36 deletion (P = 0.009), but no mutations were discovered in resequencing of the miR-34a locus in 30 neuroblastoma cell lines. Flow cytometric time series analyses showed that the likely mechanism of miR-34a growth inhibition is through cell cycle arrest followed by apoptosis. BCL2 and MYCN were identified as miR-34a targets and likely mediators of the tumor suppressor phenotypic effect. These data support miR-34a as a tumor suppressor gene in human neuroblastoma. (Mol Cancer Res 2008;6(5):735–42)

HMGA2 Participates in Transformation in Human Lung Cancer

2008-05-27

Although previous studies have established a prominent role for HMGA1 (formerly HMG-I/Y) in aggressive human cancers, the role of HMGA2 (formerly HMGI-C) in malignant transformation has not been clearly defined. The HMGA gene family includes HMGA1, which encodes the HMGA1a and HMGA1b protein isoforms, and HMGA2, which encodes HMGA2. These chromatin-binding proteins function in transcriptional regulation and recent studies also suggest a role in cellular senescence. HMGA1 proteins also appear to participate in cell cycle regulation and malignant transformation, whereas HMGA2 has been implicated primarily in the pathogenesis of benign, mesenchymal tumors. Here, we show that overexpression of HMGA2 leads to a transformed phenotype in cultured lung cells derived from normal tissue. Conversely, inhibiting HMGA2 expression blocks the transformed phenotype in metastatic human non–small cell lung cancer cells. Moreover, we show that HMGA2 mRNA and protein are overexpressed in primary human lung cancers compared with normal tissue or indolent tumors. In addition, there is a statistically significant correlation between HMGA2 protein staining by immunohistochemical analysis and tumor grade (P < 0.001). Our results indicate that HMGA2 is an oncogene important in the pathogenesis of human lung cancer. Although additional studies with animal models are needed, these findings suggest that targeting HMGA2 could be therapeutically beneficial in lung cancer and other cancers characterized by increased HMGA2 expression. (Mol Cancer Res 2008;6(5):743–50)

BRAF Silencing by Short Hairpin RNA or Chemical Blockade by PLX4032 Leads to Different Responses in Melanoma and Thyroid Carcinoma Cells

2008-05-27

BRAF-activating mutations have been reported in several types of cancer, including melanoma (~70% of cases), thyroid (30-70%), ovarian (15-30%), and colorectal cancer (5-20%). Mutant BRAF has constitutive kinase activity and causes hyperactivation of the mitogen-activated protein kinase pathway. BRAF silencing induces regression of melanoma xenografts, indicating the essential role of BRAF for cell survival. We set up an inducible short hairpin RNA system to compare the role of oncogenic BRAF in thyroid carcinoma versus melanoma cells. Although BRAF knockdown led to apoptosis in the melanoma cell line A375, the anaplastic thyroid carcinoma cell ARO underwent growth arrest upon silencing, with little or no cell death. Reexpression of the thyroid differentiation marker, sodium iodide symporter, was induced after long-term silencing. The different outcome of BRAF down-regulation in the two cell lines was associated with an opposite regulation of p21^CIP1/WAF1 expression levels in response to the block of the BRAF mitogenic signal. These results were confirmed using a specific BRAF small-molecule inhibitor, PLX4032. Restoration of p21^CIP1/WAF1 expression rescued melanoma cells from death. Altogether, our data indicate that oncogenic BRAF inhibition can have a different effect on cell fate depending on the cellular type. Furthermore, we suggest that a BRAF-independent mechanism of cell survival exists in anaplastic thyroid cancer cells. (Mol Cancer Res 2008;6(5):751–9)

Gene Expression Changes in an Animal Melanoma Model Correlate with Aggressiveness of Human Melanoma Metastases

2008-05-27

Metastasis is the deadliest phase of cancer progression. Experimental models using immunodeficient mice have been used to gain insights into the mechanisms of metastasis. We report here the identification of a "metastasis aggressiveness gene expression signature" derived using human melanoma cells selected based on their metastatic potentials in a xenotransplant metastasis model. Comparison with expression data from human melanoma patients shows that this metastasis gene signature correlates with the aggressiveness of melanoma metastases in human patients. Many genes encoding secreted and membrane proteins are included in the signature, suggesting the importance of tumor-microenvironment interactions during metastasis. (Mol Cancer Res 2008;6(5):760–9)

Interferon Regulatory Factors IRF5 and IRF7 Inhibit Growth and Induce Senescence in Immortal Li-Fraumeni Fibroblasts

2008-05-27

Cellular immortalization is one of the prerequisite steps in carcinogenesis. By gene expression profiling, we have found that genes in the interferon (IFN) pathway were dysregulated during the spontaneous cellular immortalization of fibroblasts from Li-Fraumeni syndrome (LFS) patients with germ-line mutations in p53. IFN signaling pathway genes were down-regulated by epigenetic silencing during immortalization, and some of these same IFN-regulated genes were activated during replicative senescence. Bisulfite sequencing of the promoter regions of two IFN regulatory transcription factors (IRF5 and IRF7) revealed that IRF7, but not IRF5, was epigenetically silenced by methylation of CpG islands in immortal LFS cells. The induction of IRF7 gene by IFN in immortal LFS cells was potentiated by pretreatment with the demethylation agent 5-aza-2'-deoxycytidine. Overexpression of IRF5 and IRF7 revealed that they can act either alone or in tandem to activate other IFN-regulated genes. In addition, they serve to inhibit the proliferation rate and induce a senescence-related phenotype in immortal LFS cells. Furthermore, polyinosinic:polycytidylic acid treatment of the IRF-overexpressing cells showed a more rapid induction of several IFN-regulated genes. We conclude that the epigenetic inactivation of the IFN pathway plays a critical role in cellular immortalization, and the reactivation of IFN-regulated genes by transcription factors IRF5 and/or IRF7 is sufficient to induce cellular senescence. The IFN pathway may provide valuable molecular targets for therapeutic interventions at early stages of cancer development. (Mol Cancer Res 2008;6(5):770–84)

Decorin-Induced Growth Inhibition Is Overcome through Protracted Expression and Activation of Epidermal Growth Factor Receptors in Osteosarcoma Cells

2008-05-27

Decorin is an established natural oncosuppressive factor whose action is being studied in detail. Recently, decorin gene therapy formulations using adenoviral vectors have been shown in several animal models with very promising results. The present study describes the first exception to the established oncosuppression model using human osteosarcoma cells. MG-63 osteosarcoma cells were found to constitutively produce decorin, and furthermore, to be resistant to decorin-induced growth arrest. On the contrary, decorin seemed to be beneficial to osteosarcoma cells because it was necessary for MG-63 cell migration and acted as a mediator, counteracting the transforming growth factor-β2–induced cytostatic function. Efforts to determine how MG-63 cells could overcome the decorin-induced cytostatic effect established that decorin in MG-63 cells does not induce p21 expression nor does it cause protracted retraction and inactivation of the epidermal growth factor receptor. Conversely, epidermal growth factor receptor seemed to be overexpressed and continuously phosphorylated. In view of the proposed design of decorin-based anticancer therapeutic strategies, our study provides new data on pathways that cancer cells might employ to overcome the established decorin-induced growth suppression. (Mol Cancer Res 2008;6(5):785–94)

Neutral Sphingomyelinase-3 Is a DNA Damage and Nongenotoxic Stress-Regulated Gene That Is Deregulated in Human Malignancies

Corcoran, C. A., He, Q., Ponnusamy, S., Ogretmen, B., Huang, Y., Sheikh, M. S. — 2008-05-27

In this study, we report the characterization of a novel genotoxic and nongenotoxic stress-regulated gene that we had previously named as SKNY. Our results indicate that SKNY encodes the recently identified neutral sphingomyelinase-3 (nSMase3; hereafter SKNY is referred to as nSMase3). Examination of nSMase3 subcellular distribution reveals nSMase3 to localize to the endoplasmic reticulum (ER), and deletion of a COOH-terminal region containing its putative transmembrane domain and ER targeting signal partly alters its compartmentalization to the ER. Treatment with genotoxic Adriamycin and nongenotoxic tumor necrosis factor- up-regulates endogenous nSMase3 expression, albeit with different kinetics. Tumor necrosis factor- up-regulates nSMase3 expression within 2 h that lasts beyond 24 h and declines to control levels by 36 h. Adriamycin up-regulation of nSMase3 is transient, occurs within 30 min, and declines to control levels by 120 min. Prolonged treatment with Adriamycin by 24 h and beyond, however, causes a down-regulation in nSMase3 expression. Activation of wild-type p53 also down-regulates nSMase3 expression, suggesting that DNA damage-mediated nSMase3 down-regulation seems to occur partly through the tumor suppressor p53. Overexpression of exogenous nSMase3 sensitizes cells to Adriamycin-induced cell killing, a finding consistent with the proposed proapoptotic role of nSMase enzymes and nSMase-generated ceramide. We further investigated nSMase3 expression in various human malignancies and found its expression to be deregulated in several types of primary tumors when compared with their matching normal tissues. Collectively, our results have identified nSMase3 to be an important molecule that is linked to tumorigenesis and cellular stress response. (Mol Cancer Res 2008;6(5):795–807)

Impairment of the DNA Repair and Growth Arrest Pathways by p53R2 Silencing Enhances DNA Damage-Induced Apoptosis in a p53-Dependent Manner in Prostate Cancer Cells

2008-05-27

p53R2 is a p53-inducible ribonucleotide reductase that contributes to DNA repair by supplying deoxynucleotide triphosphate pools in response to DNA damage. In this study, we found that p53R2 was overexpressed in prostate tumor cell lines compared with immortalized prostatic epithelial cells and that the protein was induced upon DNA damage. We investigated the effects of p53R2 silencing on DNA damage in LNCaP cells (wild-type p53). Silencing p53R2 potentiated the apoptotic effects of ionizing radiation and doxorubicin treatment as shown by increased sub-G₁ content and decreased colony formation. This sensitizing effect was specific to DNA-damaging agents. Comet assay and -H2AX phosphorylation status showed that the decreased p53R2 levels inhibited DNA repair. Silencing p53R2 also reduced the levels of p21^WAF1/CIP1 at the posttranscriptional level, suggesting links between the p53-dependent DNA repair and cell cycle arrest pathways. Using LNCaP sublines stably expressing dominant-negative mutant p53, we found that the sensitizing effect of p53R2 silencing is mediated by p53-dependent apoptosis pathways. In the LNCaP sublines (R273H, R248W, and G245S) that have defects in inducing p53-dependent apoptosis, p53R2 silencing did not potentiate DNA damage–induced apoptosis, whereas p53R2 silencing was effective in a LNCaP subline (P151S) which retains the ability to induce p53-dependent apoptosis. This study shows that p53R2 is a potential therapeutic target that could be used to enhance the effectiveness of ionizing radiation or DNA-damaging chemotherapy in a subset of patients with prostate cancer. (Mol Cancer Res 2008;6(5):808–18)

Characterization of a Novel Primary Mammary Tumor Cell Line Reveals that Cyclin D1 Is Regulated by the Type I Insulin-Like Growth Factor Receptor

Jones, R. A., Campbell, C. I., Petrik, J. J., Moorehead, R. A. — 2008-05-27

The importance of type I insulin-like growth factor receptor (IGF-IR) overexpression in mammary tumorigenesis was recently shown in two separate transgenic models. One of these models, the MTB-IGFIR transgenics, was generated in our lab to overexpress IGF-IR in mammary epithelial cells in a doxycycline (Dox)-inducible manner. To complement this transgenic model, primary cells that retained Dox-inducible expression of IGF-IR were isolated from a transgenic mammary tumor. This cell line, RM11A, expressed high levels of IGF-IR, phosphorylated Akt, and phosphorylated extracellular signal–regulated kinase 1/2 in the presence of Dox. IGF-IR overexpression provided the primary tumor cells with a survival advantage in serum-free media and seemed to induce ligand-independent activation of the IGF-IR because RM11A cells cultured in the presence of Dox were largely nonresponsive to exogenous IGFs. IGF-IR overexpression also augmented the growth of RM11A cells in vivo because injection of these cells into mammary glands of wild-type mice produced palpable tumors in 15.8 ± 3.4 days when the mice were administered Dox, compared with 57.8 ± 6.3 days in the absence of Dox. DNA microarray analysis revealed a number of genes regulated by IGF-IR, one of which was cyclin D1. Suppression of IGF-IR expression in vitro or in vivo was associated with a decrease in cyclin D1 protein, suggesting that at least some of the proliferative actions of IGF-IR are mediated through cyclin D1. Therefore, this article characterizes the first primary murine mammary tumor cell line with inducible IGF-IR expression. These cells provide a powerful in vitro/in vivo model to examine the function of IGF-IR in mammary tumorigenesis. (Mol Cancer Res 2008;6(5):819–28)

The Biphasic Role of the Hypoxia-Inducible Factor Prolyl-4-Hydroxylase, PHD2, in Modulating Tumor-Forming Potential

Lee, K., Lynd, J. D., O'Reilly, S., Kiupel, M., McCormick, J. J., LaPres, J. J. — 2008-05-27

Hypoxia is a common feature of solid tumors. The cellular response to hypoxic stress is controlled by a family of prolyl hydroxylases (PHD) and the transcription factor hypoxia-inducible factor 1 (HIF1). To investigate the relationship between PHD and HIF1 activity and cellular transformation, we characterized the expression levels of PHD isoforms across a lineage of cell strains with varying transformed characteristics. We found that PHD2 is the primary functional isoform in these cells and its levels are inversely correlated to tumor-forming potential. When PHD2 levels were altered with RNA interference in nontumorigenic fibroblasts, we found that small decreases can lead to malignant transformation, whereas severe decreases do not. Consistent with these results, direct inhibition of PHD2 was also shown to influence tumor-forming potential. Furthermore, we found that overexpression of PHD2 in malignant fibroblasts leads to loss of the tumorigenic phenotype. These changes correlated with HIF1 activity, glycolytic rates, vascular endothelial growth factor expression, and the ability to grow under hypoxic stress. These findings support a biphasic model for the relationship between PHD2 activity and malignant transformation. (Mol Cancer Res 2008;6(5):829–42)

Identification and Functional Characterization of the Human Glutathione S-Transferase P1 Gene as a Novel Transcriptional Target of the p53 Tumor Suppressor Gene

Lo, H.-W., Stephenson, L., Cao, X., Milas, M., Pollock, R., Ali-Osman, F. — 2008-05-27

The glutathione S-transferase P1 (GSTP1) is involved in multiple cellular functions, including phase II metabolism, stress response, signaling, and apoptosis. The mechanisms underlying the significantly high GSTP1 expression in many human tumors are, however, currently not well understood. We report here that the GSTP1 gene is a heretofore unrecognized downstream transcriptional target of the tumor suppressor p53. We identified a p53-binding motif comprising two consecutive half-sites located in intron 4 of the GSTP1 gene and is highly homologous to consensus p53-binding motifs in other p53-responsive genes. Using a combination of electrophoretic mobility shift assay and DNase I footprinting analyses, we showed that wild-type p53 protein binds to the GSTP1 p53 motif and luciferase reporter assays showed the motif to be transcriptionally functional in human tumor cells. In a temperature-sensitive p53-mutant cells, levels of both p21/WAF1 and GSTP1 gene transcripts increased time dependently when cells were switched from the inactive mutant state to the wild-type p53 state. Small interfering RNA–mediated reduction of p53 expression resulted in a specific decrease in GSTP1 expression and in tumor cells with mutated p53; adenovirally mediated expression of wild-type p53 increased GSTP1 expression significantly. In a panel of early-passage brain tumor cultures from patients, high levels of GSTP1 transcripts and protein were associated with wild-type p53 and, conversely, low GSTP1 levels with mutant p53. p53 expression knockdown by small interfering RNA increased cisplatin sensitivity. The ability of wild-type p53 to transcriptionally activate the human GSTP1 gene defines a novel mechanism of protecting the genome and, potentially, of tumor drug resistance. (Mol Cancer Res 2008;6(5):843–50)

Growth Factor Regulation of Estrogen Receptor Coregulator PELP1 Functions via Protein Kinase A Pathway

2008-05-27

PELP1 (proline-rich, glutamic acid–rich, and leucine-rich protein-1) is a potential proto-oncogene that functions as a coregulator of estrogen receptor (ER), and its expression is deregulated during breast cancer progression. Emerging evidence suggests growth factor signaling crosstalk with ER as one possible mechanism by which breast tumors acquire resistance to therapy. In this study, we examined mechanisms by which growth factors modulate PELP1 functions, leading to activation of ER. Using in vivo labeling assays, we have found that growth factors promote phosphorylation of PELP1. Utilizing a panel of substrate-specific phosphorylated antibodies, we discovered that growth factor stimulation promotes phosphorylation of PELP1 that is recognized by a protein kinase A (PKA) substrate–specific antibody. Accordingly, growth factor–mediated PELP1 phosphorylation was effectively blocked by PKA-specific inhibitor H89. Utilizing purified PKA enzyme and in vitro kinase assays, we obtained evidence of direct PELP1 phosphorylation by PKA. Using deletion and mutational analysis, we identified PELP1 domains that are phosphorylated by PKA. Interestingly, site-directed mutagenesis of the putative PKA site in PELP1 compromised growth factor–induced activation and subnuclear localization of PELP1 and also affected PELP1-mediated transactivation function. Utilizing MCF-7 cells expressing a PELP1 mutant that cannot be phosphorylated by PKA, we provide mechanistic insights by which growth factor signaling regulates ER transactivation in a PELP1-dependent manner. Collectively, these findings suggest that growth factor signals promote phosphorylation of ER coactivator PELP1 via PKA pathway, and such modification may have functional implications in breast tumors with deregulated growth factor signaling. (Mol Cancer Res 2008;6(5):851–61)

TLS-ERG Leukemia Fusion Protein Deregulates Cyclin-Dependent Kinase 1 and Blocks Terminal Differentiation of Myeloid Progenitor Cells

2008-05-27

TLS-ERG fusion protein is derived from the t(16;21) translocation found in human myeloid leukemia. Here, we show that retroviral transduction of TLS-ERG confers a growth advantage to L-G myeloid progenitor cells and blocks terminal differentiation. We found that the level of cyclin-dependent kinase 1 (Cdk1) protein was significantly decreased in controls but unchanged in TLS-ERG–expressing cells after granulocyte colony-stimulating factor treatment or interleukin-3 withdrawal. Injection of TLS-ERG–expressing L-G cells induced rapid development of a leukemia-like disease in syngeneic mice. Through site-directed mutagenesis, we showed that transformation and deregulation of Cdk1 by TLS-ERG require an intact ets DNA-binding domain within the fusion protein. Interestingly, treatment of TLS-ERG–expressing L-G cells with 5-aza-2'-deoxycytidine (Decitabine) or trichostatin A resulted in down-regulation of Cdk1 and induction of terminal differentiation. To investigate whether Cdk1 deregulation is indeed responsible for transformation by TLS-ERG, we constructed lentiviral vectors for delivery of Cdk1 mutants and small interfering RNA (siRNA). Both dominant-negative inhibition and siRNA knockdown of Cdk1 were able to restore the ability of TLS-ERG–expressing L-G cells to undergo terminal differentiation. In addition, siRNA knockdown of Cdk1 in YNH-1 cells derived from a t(16;21) acute myelogenous leukemia patient also resulted in terminal differentiation. As restoration of terminal myeloid differentiation to TLS-ERG cells is dependent on cell cycle arrest, our findings suggest an important role for Cdk1 in cellular transformation and may be useful in the search for new treatments of TLS-ERG–associated myeloid leukemia. (Mol Cancer Res 2008;6(5):862–72)

Inhibition of Histone Deacetylases Promotes Ubiquitin-Dependent Proteasomal Degradation of DNA Methyltransferase 1 in Human Breast Cancer Cells

Zhou, Q., Agoston, A. T., Atadja, P., Nelson, W. G., Davidson, N. E. — 2008-05-27

Histone deacetylases (HDAC) play a critical role in chromatin modification and gene expression. Recent evidence indicates that HDACs can also regulate functions of nonhistone proteins by catalyzing the removal of acetylated lysine residues. Here, we show that the HDAC inhibitor LBH589 down-regulates DNA methyltransferase 1 (DNMT1) protein expression in the nucleus of human breast cancer cells. Cotreatment with the proteasomal inhibitor MG-132 abolishes the ability of LBH589 to reduce DNMT1, suggesting that the proteasomal pathway mediates DNMT1 degradation on HDAC inhibition. Deletion of the NH₂-terminal 120 amino acids of DNMT1 diminishes LBH589-induced ubiquitination, indicating that this domain is essential for its proteasomal degradation. DNMT1 recruits the molecular chaperone heat shock protein 90 (Hsp90) to form a chaperone complex. Treatment with LBH589 induces hyperacetylation of Hsp90, thereby inhibiting the association of DNMT1 with Hsp90 and promoting ubiquitination of DNMT1. In addition, inactivation of HDAC1 activity by small interfering RNA and MS-275 is associated with Hsp90 acetylation in conjunction with reduction of DNMT1 protein expression. We conclude that the stability of DNMT1 is maintained in part through its association with Hsp90. Disruption of Hsp90 function by HDAC inhibition is a unique mechanism that mediates the ubiquitin-proteasome pathway for DNMT1 degradation. Our studies suggest a new role for HDAC1 and identify a novel mechanism of action for the HDAC inhibitors as down-regulators of DNMT1. (Mol Cancer Res 2008;6(5):873–83)

DNA Damage Responses: Mechanisms and Roles in Human Disease: 2007 G.H.A. Clowes Memorial Award Lecture

Kastan, M. B. — 2008-04-10

Significant progress has been made in recent years in elucidating the molecular controls of cellular responses to DNA damage in mammalian cells. Much of our understanding of the mechanisms involved in cellular DNA damage response pathways has come from studies of human cancer susceptibility syndromes that are altered in DNA damage responses. Ataxia-telangiectasia mutated (ATM), the gene mutated in the disorder ataxia-telangiectasia, codes for a protein kinase that is a central mediator of responses to DNA double-strand breaks (DSB) in cells. Once activated, ATM phosphorylates numerous substrates in the cell that modulate the response of the cell to the DNA damage. We recently developed a novel system to create DNA DSBs at defined endogenous sites in the human genome and used this system to detect protein recruitment and loss at and around these breaks by chromatin immunoprecipitation. Results from this system showed the functional importance of ATM kinase activity and phosphorylation in the response to DSBs and supported a model in which ordered chromatin structure changes that occur after DNA breakage and that depend on functional NBS1 and ATM facilitate DNA DSB repair. Insights about these pathways provide us with opportunities to develop new approaches to benefit patients. Examples and opportunities for developing inhibitors that act as sensitizers to chemotherapy or radiation therapy or activators that could improve responses to cellular stresses, such as oxidative damage, are discussed. Relevant to the latter, we have shown benefits of an ATM activator in disease settings ranging from metabolic syndrome to cancer prevention. (Mol Cancer Res 2008;6(4):517–24)

Vascular Endothelial Growth Factor Stimulates Organ-Specific Host Matrix Metalloproteinase-9 Expression and Ovarian Cancer Invasion

2008-04-10

Vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP) regulate each other, contributing to tumor progression. We have previously reported that MMP9 induces the release of tumor VEGF, promoting ascites formation in human ovarian carcinoma xenografts. The aim of this study was to investigate whether tumor-derived VEGF regulated the expression of gelatinase by the stroma, influencing the invasive properties of ovarian tumors. Tumor variants derived from 1A9 human ovarian carcinoma, stably expressing VEGF₁₂₁ in the sense (1A9-VS-1) and antisense orientations (1A9-VAS-3), were used. In vivo, zymographic analysis of tumors from 1A9-VS-1 implanted in the peritoneal cavity of nude mice showed higher levels of gelatinases, particularly murine MMP9, indicating that VEGF stimulates host expression of the matrix-degrading enzyme. Murine MMP9 expression was also high in the ovaries of mice bearing 1A9-VS-1 tumors. The effect on host MMP9 activity was organ-specific. The levels of host pro-MMP9 in ovaries correlated with the plasma levels of tumor VEGF and with the selective invasion of the ovaries. Induction of host MMP9 expression in tumors and ovaries was independent of the site of tumor growth as it was seen in mice carrying both intraperitoneal and subcutaneous tumors. The anti-VEGF antibody bevacizumab (Avastin) inhibited MMP9 expression and tumor invasion in the ovaries of mice bearing 1A9-VS-1 tumors. These findings point to a complex cross-talk between VEGF and MMPs in the progression of ovarian tumor and suggest the possibility of using VEGF inhibitors to affect MMP-dependent tumor invasion. (Mol Cancer Res 2008;6(4):525–34)

Arachidonic Acid-Induced Ca2+ Entry Is Involved in Early Steps of Tumor Angiogenesis

2008-04-10

Growth factor–induced intracellular calcium signals in endothelial cells regulate cytosolic and nuclear events involved in the angiogenic process. Among the intracellular messengers released after proangiogenic stimulation, arachidonic acid (AA) plays a key role and its effects are strictly related to calcium homeostasis and cell proliferation. Here, we studied AA-induced intracellular calcium signals in endothelial cells derived from human breast carcinomas (B-TEC). AA promotes B-TEC proliferation and organization of vessel-like structures in vitro. The effect is directly mediated by the fatty acid without a significant contribution of its metabolites. AA induces Ca²⁺_i signals in the entire capillary-like structure during the early phases of tubulogenesis in vitro. No such responses are detectable in B-TECs organized in more structured tubules. In B-TECs growing in monolayer, AA induces two different signals: a Ca²⁺_i increase due to Ca²⁺ entry and an inhibition of store-dependent Ca²⁺ entry induced by thapsigargin or ATP. An inhibitor of Ca²⁺ entry and angiogenesis, carboxyamidotriazole, significantly and specifically decreases AA-induced B-TEC tubulogenesis, as well as AA-induced Ca²⁺ signals in B-TECs. We conclude that (a) AA-activated Ca²⁺ entry is associated with the progression through the early phases of angiogenesis, mainly involving proliferation and tubulogenesis, and it is down-regulated during the reorganization of tumor-derived endothelial cells in capillary-like structures; and (b) inhibition of AA-induced Ca²⁺ entry may contribute to the antiangiogenic action of carboxyamidotriazole. (Mol Cancer Res 2008;6(4):535–45)

CXCL16 Functions as a Novel Chemotactic Factor for Prostate Cancer Cells In vitro

2008-04-10

A variety of tumor cells produce chemokines that promote tumor cell proliferation and chemotaxis. We previously reported that CXCL16 production is increased in aggressive prostate cancer cells compared with the less aggressive tumor cells and benign cells as identified in a cytokine antibody array. The functional contribution of CXCL16 in prostate cancer development has not yet been evaluated. Accordingly, mRNA expression of CXCL16 and its receptor, CXCR6, were determined by real-time reverse transcription-PCR in various cancer cell lines, including prostate cancer and tissues obtained from localized and metastatic prostate cancer. Consistent with our finding on CXCL16 protein production by prostate cancer cells, aggressive prostate cancer C4-2B and PC3 cells, as well as bone and liver metastatic tissues, expressed higher levels of both CXCL16 and CXCR6 mRNA compared with the less aggressive prostate cancer LNCaP cells, nonneoplastic PrEC and RWPE-1 cells, and benign prostate tissues, respectively. Furthermore, CXCR6 and CXCL16 protein expressions were examined in tissue specimens by immunohistochemistry. Immunohistochemical examination of CXCR6 expression showed strong epithelial staining that correlated with Gleason score, whereas CXCL16 staining was not. Finally, we found that both interleukin-1β and tumor necrosis factor significantly induced CXCL16 production by prostate epithelial cells, thereby indicating that inflammatory cytokines may play a role in the CXCL16 induction. CXCL16 was found to promote prostate cancer cell migration and invasion in vitro. Therefore, we concluded that CXCL16 functions, through CXCR6, as a novel chemotactic factor for prostate cancer cells. (Mol Cancer Res 2008;6(4):546–54)

MUC1 Initiates Src-CrkL-Rac1/Cdc42-Mediated Actin Cytoskeletal Protrusive Motility after Ligating Intercellular Adhesion Molecule-1

2008-04-10

MUC1, a transmembrane glycoprotein of the mucin family, when aberrantly expressed on breast cancer cells is correlated with increased lymph node metastases. We have previously shown that MUC1 binds intercellular adhesion molecule-1 (ICAM-1) on surrounding accessory cells and facilitates transendothelial migration of MUC1-bearing cells. Nevertheless, the underlying molecular mechanism is still obscure. In the present study, we used a novel assay of actin cytoskeletal reorganization to show that by ligating ICAM-1, MUC1 triggers Rac1- and Cdc42-dependent actin cytoskeletal protrusive activity preferentially at the heterotypic cell-cell contact sites. Further, we show that these MUC1/ICAM-1 interaction–initiated lamellipodial and filopodial protrusions require Src family kinase and CT10 regulator of kinase like (CrkL) accompanied by the rapid formation of a Src-CrkL signaling complex at the MUC1 cytoplasmic domain. Through inhibition of Src kinase activity, we further revealed that Src is required for recruiting CrkL to the MUC1 cytoplasmic domain as well as mediating the observed actin cytoskeleton dynamics. These findings suggest a novel MUC1-Src-CrkL-Rac1/Cdc42 signaling cascade following ICAM-1 ligation, through which MUC1 regulates cytoskeletal reorganization and directed cell motility during cell migration. (Mol Cancer Res 2008;6(4):555–67)

Gene-Viral Cancer Therapy Using Dual-Regulated Oncolytic Adenovirus with Antiangiogenesis Gene for Increased Efficacy

2008-04-10

Conditionally replicative adenovirus (CRAD) represents a promising approach for cancer therapy. Several CRADs controlled by the human telomerase reverse transcriptase promoter have been developed. However, because of their replicative capacity, the importance of cancer specificity for CRADs needs to be further emphasized. In this study, we have developed a novel dual-regulated CRAD, CNHK500-mE, which has its E1a and E1b gene controlled by the human telomerase reverse transcriptase promoter and the hypoxia response element, respectively. It also carries a mouse endostatin expression cassette controlled by the cytomegalovirus promoter. These properties allow for increased cancer cell targeting specificity and decreased adverse side effects. We showed that CNHK500-mE preferentially replicated in cancer cells. Compared with a replication-defective vector carrying the same endostatin expression cassette, CNHK500-mE–mediated transgene expression level was markedly increased via viral replication within cancer cells. In the nasopharyngeal tumor xenograft model, CNHK500-mE injection resulted in antitumor efficacy at day 7 after therapy. Three weeks later, it led to significant inhibition of xenograft tumor growth due to the combined effects of viral oncolytic therapy and antiangiogenesis gene therapy. Pathologic examination showed that most cancer cells were positive for adenoviral capsid protein and for apoptotic terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling in the CNHK500-mE–treated tumor tissues, and the microvessels in these tumor tissues were diminished in quantity and abnormal in morphology. These results suggest that, as a potential cancer therapeutic agent, the CNHK500-mE is endowed with higher specificity to cancer cells and low cytotoxicity to normal cells. (Mol Cancer Res 2008;6(4):568–75)