Since its original cloning by subtraction hybridization in 2002, it is now evident that Astrocyte elevated gene-1 (AEG-1) is a key contributor to the carcinogenic process in diverse organs. AEG-1 protein expression is elevated in advanced stages of many cancers, which correlates with poor survival. In specific cancers, such as breast and liver cancer, the AEG-1 gene itself is amplified, further supporting a seminal role in tumorigenesis. Overexpression and inhibition studies both in in vitro and in in vivo models reveal the importance of AEG-1 in regulating multiple physiologically and pathologically relevant processes including proliferation, invasion, metastasis, and gene expression. AEG-1 is a single-pass transmembrane protein with multiple nuclear localization signals and no known domains or motifs. Although pertinent roles of AEG-1 in the carcinogenic process are established, its potential function (promotion of metastasis only versus functioning as a bona fide oncogene) as well as localization (cell surface versus nucleus) remain areas requiring further clarification. The present review critically evaluates what is currently known about AEG-1 and provides new perspectives relative to this intriguing molecule that may provide a rational target for intervening in the cancer phenotype. [Cancer Res 2009;69(22):8529–35]

Cell fusion plays an essential role in fertilization, formation of placenta, bone and muscle tissues, immune response, tissue repair, and regeneration. Increasing recognition of cell fusion in somatic cell dynamics has revitalized the century-old hypothesis that cell fusion may contribute to the initiation and progression of cancer. In this review, we discuss findings from experimental and clinical studies that suggest a potentially multifaceted involvement of cell fusion in different stages of tumor progression, including aneuploidy and tumor initiation, origin of cancer stem cells, multidrug resistance, and the acquisition and diversification of metastatic abilities. [Cancer Res 2009;69(22):8536–9]

Aurora B is critically involved in ensuring proper cytokinesis and maintaining genomic stability. The tumor suppressor RASSF1A regulates cell cycle progression by regulating mitotic progression, G₁-S transition, and microtubule stability. We previously reported that both Aurora A and Aurora B phosphorylate RASSF1A, and showed that phosphorylation of RASSF1A by Aurora A blocks the inhibitory function of RASSF1A toward anaphase-promoting complex-Cdc20. However, the role of Aurora B–mediated RASSF1A phosphorylation remains unknown. Here, we show that phosphorylation of RASSF1A on Ser203 by Aurora B during late mitosis has a critical role in regulating cytokinesis. Notably, RASSF1A interacts with Syntaxin16, a member of the t-SNARE family, at the midzone and midbody during late mitosis. Aurora B is required for this interaction and for the subsequent recruitment of Syntaxin16 to the midzone and midbody, a prerequisite for the successful completion of cytokinesis. Furthermore, Aurora B depletion results in a failure of Syntaxin16 to properly localize to the midzone and midbody, a mislocalization that was prevented by overexpression of the phosphomimetic RASSF1A (S203D) mutant. Finally, either depletion of Syntaxin16 or expression of the nonphosphorylatable RASSF1A (S203A) mutant results in cytokinesis defects. Our findings implicate Aurora B–mediated phosphorylation of RASSF1A in the regulation of cytokinesis. [Cancer Res 2009;69(22):8540–4]

Glioblastoma multiforme (GBM) is the most common and lethal primary human brain tumor. GBMs are characterized by a variety of genetic alterations, among which oncogenic mutations of epidermal growth factor receptor (EGFRvIII) is most common. GBMs harboring EGFRvIII have increased proliferation and invasive characteristics versus those expressing wild-type (wt) EGFR. To identify the molecular basis of this increased tumorgenic phenotype, we used iTRAQ-labeling differential proteomic analysis. Among several differentially expressed proteins, we selected CRMP1, a protein implicated in cellular invasion that was markedly decreased in GBMs expressing EGFRvIII, for further study. The differential expression of CRMP1 was confirmed in a panel of human GBM cell lines and operative specimens that express wtEGFR or mutant EGFRvIII by quantitative real-time PCR, Western blot, and immunohistochemical analysis. In human GBM samples, decreased expression of CRMP1 correlated with EGFRvIII positivity. Knockdown of CRMP1 by siRNA resulted in increased invasion of wtEGFR expressing human GBM cells (U87 and U373) to those found in isogenic GBM cells. Exogenous expression of EGFRvIII in these wtEGFR-expressing GBM cells promoted their ability to invade and was accompanied by decreased expression of CRMP1. Rescuing CRMP1 expression decreased invasion of the EGFRvIII-expressing GBM cells by tilting the balance between Rac and Rho. Collectively, these results show that the loss of CRMP1 contribute to the increased invasive phenotype of human GBMs expressing mutant EGFRvIII. [Cancer Res 2009;69(22):8545–54]

We have shown previously that Pten deletion leads to the expansion of subset of prostate cancer cells positive for CK5 and p63. Although this subpopulation may be involved in tumor initiation or progression, studies to date have not functionally validated this hypothesis. Using in vitro sphere-forming assay and in vivo prostate reconstitution assay, we show here the presence of a tumor-initiating subpopulation in the Pten prostate cancer mouse model. Specifically, we show that the Lin^–Sca-1⁺CD49f^high (LSC) subpopulation overlaps with CK5⁺;p63⁺ cells and is significantly increased during prostate cancer initiation and progression and after castration. Mutant spheres mimic the structural organization of the epithelial compartment in the Pten-null primary tumor. Sorted LSC cells from either Pten-null spheres or primary tumors are able to regenerate prostate epithelial structure with cancerous morphology, closely mimicking that of primary cancers. Therefore, the LSC subpopulation is capable of initiating a cancerous phenotype that recapitulates the pathology seen in the primary lesions of the Pten mutant prostate model. [Cancer Res 2009;69(22):8555–62]

Despite extensive studies on the role of tumor suppressor p53 protein and its homologues, p73 and p63, following their overexpression or cellular stress, very little is known about the regulation of the three proteins in cells during physiologic cell cycle progression. We report a role for p73 and p63 in supporting cellular proliferation through the transcriptional activation of the genes involved in G₁-S and G₂-M progression. We found that in MCF-7 cells, p73 and p63, but not p53, are modulated during the cell cycle with a peak in S phase, and their silencing determines a significant suppression of proliferation compared with the control. Chromatin immunoprecipitation analysis shows that in cycling cells, p73 and p63 are bound to the p53-responsive elements (RE) present in the regulatory region of cell cycle progression genes. On the contrary, when the cells are arrested in G₀-G₁, p73 detaches from the REs and it is replaced by p53, which represses the expression of these genes. When the cells move in S phase, p73 is recruited again and p53 is displaced or is weakly bound to the REs. These data open new possibilities for understanding the involvement of p73 and p63 in cancer. The elevated concentrations of p73 and p63 found in many cancers could cause the aberrant activation of cell growth progression genes and therefore contribute to cancer initiation or progression under certain conditions. [Cancer Res 2009;69(22):8563–71]

Wnt and Hedgehog signaling pathways play central roles in embryogenesis, stem cell maintenance, and tumorigenesis. However, the mechanisms by which these two pathways interact are not well understood. Here, we identified a novel mechanism by which Wnt signaling pathway stimulates the transcriptional output of Hedgehog signaling. Wnt/β-catenin signaling induces expression of an RNA-binding protein, CRD-BP, which in turn binds and stabilizes GLI1 mRNA, causing an elevation of GLI1 expression and transcriptional activity. The newly described mode of regulation of GLI1 seems to be important to several functions of Wnt, including survival and proliferation of colorectal cancer cells. [Cancer Res 2009;69(22):8572–8]

Lipocalin 2 (LCN2; also known as NGAL) is a secreted glycoprotein and its elevated expression has been observed in breast cancers. However, the importance of LCN2 in breast tumorigenesis is unclear. Here, we employed a spontaneous mammary tumor mouse model showing that MMTV-ErbB2(V664E) mice lacking mouse LCN2 had significantly delayed mammary tumor formation and metastasis with reduced matrix metalloproteinase-9 activity in the blood. LCN2 expression is upregulated by HER2/phosphoinositide 3-kinase/AKT/NF-B pathway. Decreasing LCN2 expression significantly reduced the invasion and migration ability of HER2⁺ breast cancer cells. Furthermore, injecting an anti-mouse LCN2 antibody into mice bearing established murine breast tumors resulted in significant blockage of lung metastasis. Our findings indicate that LCN2 is a critical factor in enhancing breast tumor formation and progression possibly in part by stabilizing matrix metalloproteinase-9. Our results suggest that inhibition of LCN2 function by an inhibitory monoclonal antibody has potential for breast cancer therapy, particularly by interfering with metastasis in aggressive types of breast cancer. [Cancer Res 2009;69(22):8579–84]

The KCl cotransporter (KCC) is a major determinant of osmotic homeostasis and plays an emerging role in tumor biology. This study stresses the important role of KCC4 in tumor malignant behavior. Real-time reverse transcription-PCR on samples collected by laser microdissection and immunofluorescent stainings with different KCC isoform antibodies indicate that KCC4 is abundant in metastatic cervical and ovarian cancer tissues. Insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) stimulate KCC4 recruitment from a presumably inactive cytoplasmic pool of endoplasmic reticulum and Golgi to plasma membrane along actin cytoskeleton that is significantly inhibited by LY294002 and wortmannin. Throughout the trafficking process, KCC4 is incorporated into lipid rafts that function as a platform for the association between KCC4 and myosin Va, an actin-dependent motor protein. KCC4 and ezrin, a membrane cytoskeleton linker, colocalize at lamellipodia of migratory cancer cells. Interference with KCC activity by either an inhibitor or a dominant-negative loss-of-function mutant profoundly suppressed the IGF-I–induced membrane trafficking of KCC4 and the structural interaction between KCC4 and ezrin near the cell surface. Endogenous cancer cell invasiveness was significantly attenuated by small interfering RNA targeting KCC4, and the residual invasiveness was much less sensitive to IGF-I or EGF stimulation. In the metastatic cancer tissues, KCC4 colocalizes with IGF-I or EGF, indicating a likely in vivo stimulation of KCC4 function by growth factors. Thus, blockade of KCC4 trafficking and surface expression may provide a potential target for the prevention of IGF-I– or EGF-dependent cancer spread. [Cancer Res 2009;69(22):8585–93]

Invadopodia are ventral membrane protrusions through which invasive cancer cells degrade the extracellular matrix. They are thought to function in the migration of cancer cells through tissue barriers, which is necessary for cancer invasion and metastasis. Although many protein components of invadopodia have been identified, the organization and the role of membrane lipids in invadopodia are not well understood. In this study, the role of lipid rafts, which are cholesterol-enriched membrane microdomains, in the assembly and function of invadopodia in human breast cancer cells was investigated. Lipid rafts are enriched, internalized, and dynamically trafficked at invadopodia sites. Perturbation of lipid raft formation due to depleting or sequestering membrane cholesterol blocked the invadopodia-mediated degradation of the gelatin matrix. Caveolin-1 (Cav-1), a resident protein of lipid rafts and caveolae, accumulates at invadopodia and colocalizes with the internalized lipid raft membranes. Membrane type 1 matrix metalloproteinase (MT1-MMP), a matrix proteinase associated with invadopodia, is localized at lipid raft-enriched membrane fractions and cotrafficked and colocalized with Cav-1 at invadopodia. The small interfering RNA–mediated silencing of Cav-1 inhibited the invadopodia-mediated and MT1-MMP–dependent degradation of the gelatin matrix. Furthermore, Cav-1 and MT1-MMP are coexpressed in invasive human breast cancer cell lines that have an ability to form invadopodia. These results indicate that invadopodia are the sites where enrichment and trafficking of lipid rafts occur and that Cav-1 is an essential regulator of MT1-MMP function and invadopodia-mediated breast cancer cell invasion. [Cancer Res 2009;69(22):8594–602]

Wnt inhibitory factor-1 (WIF-1) has been identified as one of the secreted antagonists that bind Wnt protein. WIF-1 has been described as a tumor suppressor in various types of cancer. However, the molecular function of WIF-1 gene has never been examined in human renal cell carcinoma (RCC). Therefore, we hypothesized that WIF-1 functions as a tumor suppressor gene and overexpression of this gene may induce apoptosis and inhibit tumor growth in RCC cells. Immunohistochemistry and real-time reverse transcription-PCR revealed that WIF-1 was significantly downregulated in RCC samples and RCC cell lines, respectively. Bisulfite sequencing of the WIF-1 promoter region in RCC cell lines showed it to be densely methylated, whereas there was no methylation of WIF-1 promoter in normal kidney. Significant inhibition of cell growth and colony formation in WIF-1–transfected cells compared with controls were observed. WIF-1 transfection significantly induced apoptosis and suppressed in vivo tumor growth. Also, Wnt signaling activity and β-catenin expression were reduced by WIF-1 transfection. In conclusion, this is the first report documenting that the WIF-1 is downregulated by promoter methylation and functions as a tumor suppressor gene by inducing apoptosis in RCC cells. [Cancer Res 2009;69(22):8603–10]

Transformed cells in lymphomas usually maintain the phenotype of the postulated normal lymphocyte from which they arise. By contrast, anaplastic large cell lymphoma (ALCL) is a T-cell lymphoma with aberrant phenotype because of the defective expression of the T-cell receptor and other T-cell–specific molecules for still undetermined mechanisms. The majority of ALCL carries the translocation t(2;5) that encodes for the oncogenic tyrosine kinase NPM-ALK, fundamental for survival, proliferation, and migration of transformed T cells. Here, we show that loss of T-cell–specific molecules in ALCL cases is broader than reported previously and involves most T-cell receptor–related signaling molecules, including CD3, ZAP70, LAT, and SLP76. We further show that NPM-ALK, but not the kinase-dead NPM-ALK^K210R, downregulated the expression of these molecules by a STAT3-mediated gene transcription regulation and/or epigenetic silencing because this downregulation was reverted by treating ALCL cells with 5-aza-2-deoxycytidine or by knocking down STAT3 through short hairpin RNA. Finally, NPM-ALK increased the methylation of ZAP70 intron 1-exon 2 boundary region, and both NPM-ALK and STAT3 regulated the expression levels of DNA methyltransferase 1 in transformed T cells. Thus, our data reveal that oncogene-deregulated tyrosine kinase activity controls the expression of molecules that determine T-cell identity and signaling. [Cancer Res 2009;69(22):8611–9]

Resistance to trastuzumab, the monoclonal antibody targeting human epidermal growth factor receptor 2 (HER-2), is a major concern for HER-2–positive metastatic breast cancer (MBC) patients. To date, HER-2 status is the only available biomarker for selecting patients for trastuzumab-based therapy. β₁-Integrin, an adhesion molecule involved in cell survival and drug resistance, shares common downstream signaling elements with HER-2, such as the phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase-1/2 (ERK1/2) pathways. The significance of β₁-integrin expression in HER-2–positive breast cancer and its involvement in a patient's response to trastuzumab-based therapy are unknown. We show here that overexpression of β₁-integrin is an independent negative prognostic factor for tumor progression of HER-2–positive MBC patients treated with trastuzumab-based chemotherapy. Enforced overexpression of β₁-integrin, its small interfering RNA–induced knockdown or treatment with a β₁-integrin–blocking antibody in HER-2–positive breast cancer cells, identified a strong inverse relationship between expression level of β₁-integrin and in vitro sensitivity to trastuzumab. Notably, β₁-integrin overexpression increased the phosphorylation of Akt-Ser473 and ERK1/2, thereby promoting survival and mitogenic signals to bypass the antiproliferative effects of trastuzumab. Our findings show that β₁-integrin provides a novel independent prognostic biomarker of trastuzumab response in HER-2–positive MBC patients and suggest a new target to augment the antiproliferative effects of trastuzumab. [Cancer Res 2009;69(22):8620–8]

In previous studies, we reported that key antioxidant and DNA repair genes are regulated differently in normal bronchial epithelial cells of lung cancer cases compared with non–lung cancer controls. In an effort to develop a biomarker for lung cancer risk, we evaluated the transcript expressions of 14 antioxidant, DNA repair, and transcription factor genes in normal bronchial epithelial cells (HUGO names CAT, CEBPG, E2F1, ERCC4, ERCC5, GPX1, GPX3, GSTM3, GSTP1, GSTT1, GSTZ1, MGST1, SOD1, and XRCC1). A test comprising these 14 genes accurately identified the lung cancer cases in two case-control studies. The receiver operating characteristic–area under the curve was 0.82 (95% confidence intervals, 0.68–0.91) for the first case-control set (25 lung cancer cases and 24 controls), and 0.87 (95% confidence intervals, 0.73–0.96) for the second set (18 cases and 22 controls). For each gene included in the test, the key difference between cases and controls was altered distribution of transcript expression among cancer cases compared with controls, with more lung cancer cases expressing at both extremes among all genes (Kolmorogov-Smirnov test, D = 0.0795; P = 0.041). A novel statistical approach was used to identify the lower and upper boundaries of transcript expression that optimally classifies cases and controls for each gene. Based on the data presented here, there is an increased prevalence of lung cancer diagnosis among individuals that express a threshold number of key antioxidant, DNA repair, and transcription factor genes at either very high or very low levels in the normal airway epithelium. [Cancer Res 2009;69(22):8629–35]

Ligand-induced activation of peroxisome proliferator-activated receptor (PPAR) inhibits proliferation in cancer cells in vitro and in vivo; however, the downstream targets remain undefined. We report the identification of a peroxisome proliferator response element in the promoter region of the Na⁺/H⁺ transporter gene NHE1, the overexpression of which has been associated with carcinogenesis. Exposure of breast cancer cells expressing high levels of PPAR to its natural and synthetic agonists resulted in downregulation of NHE1 transcription as well as protein expression. Furthermore, the inhibitory effect of activated PPAR on tumor colony-forming ability was abrogated on overexpression of NHE1, whereas small interfering RNA–mediated gene silencing of NHE1 significantly increased the sensitivity of cancer cells to growth-inhibitory stimuli. Finally, histopathologic analysis of breast cancer biopsies obtained from patients with type II diabetes treated with the synthetic agonist rosiglitazone showed significant repression of NHE1 in the tumor tissue. These data provide evidence for tumor-selective downregulation of NHE1 by activated PPAR in vitro and in pathologic specimens from breast cancer patients and could have potential implications for the judicious use of low doses of PPAR ligands in combination chemotherapy regimens for an effective therapeutic response. [Cancer Res 2009;69(22):8636–44]

Lung cancer is the commonest cancer killer. Small cell lung cancer (SCLC) is initially chemosensitive, but rapidly relapses in a chemoresistant form with an overall survival of <5%. Consequently, novel therapies are urgently required and will likely arise from an improved understanding of the disease biology. Our previous work showed that fibroblast growth factor-2 induces proliferation and chemoresistance in SCLC cells. Here, we show that the selective fibroblast growth factor receptor (FGFR) inhibitor PD173074 blocks H-510 and H-69 SCLC proliferation and clonogenic growth in a dose-dependent fashion and prevents FGF-2–induced chemoresistance. These effects correlate with the inhibition of both FGFR1 and FGFR2 transphosphorylation. We then determined the efficacy of daily oral administration of PD173074 for 28 days in two human SCLC models. In the H-510 xenograft, tumor growth was impaired similar to that seen with single-agent cisplatin administration, increasing median survival compared with control sham–treated animals. Crucially, the effect of cisplatin was significantly potentiated by coadministration of PD173074. More dramatically, in H-69 xenografts, PD173074 induced complete responses lasting >6 months in 50% of mice. These effects were not a consequence of disrupted tumor vasculature but instead correlated with increased apoptosis (caspase 3 and cytokeratin 18 cleavage) in excised tumors. Moreover, in vivo imaging with 3'-deoxy-3'-[¹⁸F]fluorothymidine–positron emission tomography ([¹⁸F]FLT-PET) showed decreased intratumoral proliferation in live animals treated with the compound at 7 to 14 days. Our results suggest that clinical trials of FGFR inhibitors should be undertaken in patients with SCLC and that [¹⁸F]FLT-PET imaging could provide early in vivo evidence of response. [Cancer Res 2009;69(22):8645–51]

Genomic instability in solid tumors participates in the oncogenetic process and is associated with the activation of the DNA damage response pathway. Here, we report the activation of the constitutive DNA damage and checkpoint pathway associated with complex karyotypes in samples from patients with acute myeloid leukemia (AML). We show that antagonizing CHK1 kinase with a small inhibitory compound or by RNA interference strongly reduces the clonogenic properties of high–DNA damage level AML samples, particularly those with complex karyotypes. Moreover, we observe a beneficial effect of CHK1 inhibition in high–DNA damage level AML samples treated with 1-β-d-arabinofuranosylcytosine. In contrast, CHK1 inhibition has no effect on the clonogenic properties of normal hematopoietic progenitors. All together, our results indicate that CHK1 inhibition may represent an attractive therapeutic opportunity in AML with complex karyotype. [Cancer Res 2009;69(22):8652–61]

We have recently discovered that de-N-acetyl GM3 [NeuNH₂LacCer, d-GM3], a derivative of ganglioside GM3, is specifically expressed in metastatic tumor cells and that its expression correlates with an enhanced metastatic phenotype. Although the classic N-acetylated form of GM3 (NeuAcLacCer, c-GM3) is found in both normal and tumor cells, metastatic tumor cells (but not other cells) predominantly express d-GM3 (82–95% of total GM3). d-GM3 expression is mainly found in metastatic melanomas, but not in benign nevi or the majority of primary melanomas. Using metastatic (d-GM3–positive) and poorly invasive (d-GM3–negative) human melanoma cell lines, we found that d-GM3 stimulates cell migration and invasion by increasing the expression and activation of urokinase-like plasminogen activator (uPA). Further studies showed that d-GM3 activates matrix metalloproteinase-2 (MMP-2), but not MMP-9, when uPA receptor signaling is activated. These results implicate d-GM3 as a specific marker for metastatic melanoma and a novel therapeutic target for neoplastic diseases. [Cancer Res 2009;69(22):8662–9]

Aromatase inhibitors are important drugs to treat estrogen receptor (ER)–positive postmenopausal breast cancer patients. However, development of resistance to aromatase inhibitors has been observed. We examined whether the heat shock protein 90 (HSP90) inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) can inhibit the growth of aromatase inhibitor–resistant breast cancers and the mechanisms by which 17-DMAG affects proliferation. Aromatase inhibitor–responsive MCF-7aro and aromatase inhibitor–resistant LTEDaro breast epithelial cells were used in this study. We observed that 17-DMAG inhibited proliferation in both MCF-7aro and LTEDaro cells in a dose-dependent manner. 17-DMAG induced apoptosis and G₂ cell cycle arrest in both cell lines. Although inhibition of HSP90 decreased the levels of ER, the ER transcriptional activity was not affected when cells were treated with 17-DMAG together with estradiol. Moreover, detailed mechanistic studies suggested that 17-DMAG inhibits cell growth via degradation of HSP90 client proteins AKT and HER2. Collectively, results from this study provide data to support that HSP90 inhibitors may be an effective therapy to treat aromatase inhibitor–resistant breast cancers and that improved efficacy can be achieved by combined use of a HSP90 inhibitor and an AKT inhibitor. [Cancer Res 2009;69(22):8670–7]

Whereas interleukin-13 receptor 2 chain (IL-13R2) is overexpressed in a variety of human solid cancers including pancreatic cancer, we investigated its significance in cancer invasion and metastasis. We used two pancreatic cancer cell lines, IL-13R2–negative HPAF-II and IL-13R2–positive HS766T, and generated IL-13R2 stably transfected HPAF-II as well as IL-13R2 RNA interference knocked-down HS766T cells. Ability of invasion and signal transduction was compared between IL-13R2–negative and IL-13R2–positive cells and tumor metastasis was assessed in murine model for human pancreatic cancer with orthotopic implantation of tumors. IL-13 treatment enhanced cell invasion in IL-13R2–positive cancer cell lines but not in IL-13R2–negative cell lines. Furthermore, gene transfer of IL-13R2 in negative cell lines enhanced invasion, whereas its silencing downmodulated invasion of pancreatic cell lines in a Matrigel invasion assay. In vivo study revealed that IL-13R2–positive cancer metastasized to lymph nodes, liver, and peritoneum at a significantly higher rate compared with IL-13R2–negative tumors. The expression of IL-13R2 in metastatic lesions was found to be increased compared with primary tumors, and mice with IL-13R2–positive cancer displayed cachexia and poor prognosis. Invasion and metastasis also correlated with increased matrix metalloproteinase protease activity in these cells. Mechanistically, IL-13 activated extracellular signal-regulated kinase 1/2 and activator protein-1 nuclear factors in IL-13R2–positive pancreatic cancer cell lines but not in IL-13R2–negative cell lines. Taken together, our results show for the first time that IL-13 can signal through IL-13R2 in pancreatic cancer cells and IL-13R2 may serve as a prognostic biomarker of invasion and metastasis in pancreatic cancer. [Cancer Res 2009;69(22):8678–85]

S1P₂ sphingosine 1-phosphate receptor signaling can regulate proliferation, survival, morphology, and migration in many cell types in vitro. Here, we report that S1P₂^–/– mice develop clonal B-cell lymphomas with age, such that approximately half of the animals display this neoplasm by 1.5 to 2 years of age. Histologic, immunophenotypic, and molecular analyses revealed a uniform tumor phenotype with features of germinal center (GC)–derived diffuse large B-cell lymphoma (DLBCL). Tumor formation was preceded by increases in GC B cells and CD69⁺ T cells, as well as an increased formation of spontaneous GCs, suggesting that S1P₂ loss may promote lymphomagenesis in part by disrupting GC B-cells homeostasis. With the sole exception of rare lung tumors, the effect of S1P₂ gene disruption is remarkably restricted to DLBCL. In humans, 28 of 106 (26%) DLBCL samples were found to harbor multiple somatic mutations in the 5' sequences of the S1P₂ gene. Mutations displayed features resembling those generated by the IgV-associated somatic hypermutation mechanism, but were not detected at significant levels in normal GC B cells, indicating a tumor-associated aberrant function. Collectively, our data suggest that S1P₂ signaling may play a critical role in suppressing DLBCL formation in vivo. The high incidence of DLBCL in S1P₂^–/– mice, its onset at old age, and the relative lack of other neoplasms identify these mice as a novel, and potentially valuable, model for this highly prevalent and aggressive human malignancy. [Cancer Res 2009;69(22):8686–92]

Tumors grow in the presence of antigen-specific T cells, suggesting the existence of intrinsic cancer cell escape mechanisms. We hypothesized that a histone deacetylase (HDAC) inhibitor could sensitize tumor cells to immunotherapy because this class of agents has been reported to increase tumor antigen expression and shift gene expression to a proapoptotic milieu in cancer cells. To test this question, we treated B16 murine melanoma with the combination of the HDAC inhibitor LAQ824 and the adoptive transfer of gp100 melanoma antigen-specific pmel-1 T cells. The combined therapy significantly improved antitumor activity through several mechanisms: (a) increase in MHC and tumor-associated antigen expression by tumor cells; (b) decrease in competing endogenous lymphocytes in recipient mice, resulting in a proliferative advantage for the adoptively transferred cells; and (c) improvement in the functional activity of the adoptively transferred lymphocytes. We confirmed the beneficial effects of this HDAC inhibitor as a sensitizer to immunotherapy in a different model of prophylactic prime-boost vaccination with the melanoma antigen tyrosinase-related protein 2, which also showed a significant improvement in antitumor activity against B16 melanoma. In conclusion, the HDAC inhibitor LAQ824 significantly enhances tumor immunotherapy through effects on target tumor cells as well as improving the antitumor activity of tumor antigen-specific lymphocytes. [Cancer Res 2009;69(22):8693–9]

To define the factors that modulate regulatory T (Treg) cells in the tumor setting, we cocultured various tumor cells with either purified Treg cells, or with unfractionated splenocytes. We found that Treg expansion occurred only with unfractionated splenocytes, suggesting that accessory cells and/or factors produced by them play an essential role in tumor-induced Treg expansion. We performed gene expression profiling on tumor-associated Treg cells to identify candidate signaling molecules and studied their effects on tumor-induced Treg expansion. We inadvertently discovered that interleukin (IL)-12 treatment blocked Treg expansion in an IL-12 receptor–dependent fashion. Additional studies showed that IL-12 acts by stimulating IFN- mediated inhibition of Treg cell proliferation, which may partially account for the antitumor effects of IL-12. Furthermore, IL-12 treatment was found to decrease IL-2 production, which may lead to IFN-–independent inhibition of Treg cells, as IL-2 is required for their survival and expansion. Mechanistic studies revealed that IFN- signaling directly causes cell cycle arrest in Treg cells. This study shows that an IL-12–IFN- axis can suppress tumor-induced Treg proliferation. This mechanism may counteract the ability of Treg cells to promote tumor growth in vivo. [Cancer Res 2009;69(22):8700–9]

Toll-like receptor (TLR) agonists are considered adjuvants in clinical trials of cancer immunotherapy. Here, we investigated the modulation of T cell–mediated tumor cell lysis by TLR ligands. T-cell cytotoxicity and granzyme A/B production were enhanced after pretreatment of tumor cells with TLR3 [poly(I:C)] or TLR7 ligand (imiquimod). We examined TLR3- and TLR7-expressing pancreatic adenocarcinomas, squamous cell carcinomas of head and neck and lung carcinomas. Poly(I:C) treatment of pancreatic adenocarcinomas followed by coculture with T cells resulted in an upregulation of CD54 on the tumor cells. The interaction of CD54 and the corresponding ligand CD11a/CD18 expressed on T cells is responsible for triggering effector function in T cells. Moreover, treatment with imiquimod downregulated MHC class I molecules on tumor cells possibly resulting in a reduced binding affinity for inhibitory receptor NKG2A expressed on T cells. These results indicate that TLR3 or TLR7 ligand stimulation of tumor cells enhances the cytotoxic activity of expanded T cells of cancer patients in vitro. [Cancer Res 2009;69(22):8710–7]

We have investigated interferon- (IFN-) regulation in the context of human papillomavirus (HPV)–induced carcinogenesis using primary human foreskin keratinocytes (HFK), immortalized HFKs encoding individual oncoproteins of HPV16 (E6, E7, and E6/E7), and cervical carcinoma cells. Here, IFN- was suppressed in the presence of E6, whereas its expression was not affected in HFKs or E7-immortalized HFKs. Transcription could be reactivated after DNA demethylation but was decreased again upon drug removal. Partial reactivation could also be accomplished when E6 was knocked down, suggesting a contribution of E6 in IFN- de novo methylation. We identified a single CpG island near the transcriptional start site as being involved in selective IFN- expression. To prove the functional relevance of IFN- in building up an antiviral response, IFN- was ectopically expressed in cervical carcinoma cells where protection against vesicular stomatitis virus–mediated cytolysis could be achieved. Reconstitution of IFN- was accompanied by an increase of p53, MxA, and IFN-regulatory factors, which was reversed by knocking down either IFN- or p53 by small interfering RNA. This suggests the existence of a positive feedback loop between IFN-, p53, and components of IFN signaling pathway to maintain an antiviral state. Our in vitro findings were further corroborated in biopsy samples of cervical cancer patients, in which IFN- was also downregulated when compared with normal donor tissue. This is the first report showing an epigenetic silencing of type I IFN after HPV16 oncogene expression and revealing a novel strategy on how high-risk HPVs can abolish the innate immune response in their genuine host cells. [Cancer Res 2009;69(22):8718–25]

One of the current models of cancer proposes that oncogenes activate a DNA damage response (DDR), which would limit the growth of the tumor in its earliest stages. In this context, and in contrast to studies focused on the acute responses to a one-time genotoxic insult, understanding how cells respond to a persistent source of DNA damage might become critical for future studies in the field. We here report the discovery of a novel damage-responsive pathway, which involves p27^Kip1 and retinoblastoma tumor suppressors and is only implemented after a persistent exposure to clastogens. In agreement with its late activation, we show that this pathway is critical for the maintenance, but not the initiation, of the cell cycle arrest triggered by DNA damage. Interestingly, this late response is independent of the canonical ataxia telangiectasia mutated–dependent and ataxia telangiectasia mutated and Rad3-related–dependent DDR but downstream of p38 mitogen-activated protein kinase. Our results might help to reconcile the oncogene-induced DNA damage model with the clinical evidence that points to non-DDR members as the most important tumor suppressors in human cancer. [Cancer Res 2009;69(22):8726–32]

Lung cancer is the leading cause of cancer deaths, accounting for more deaths than breast, colon, and prostate cancer combined. The retinoblastoma (Rb)/p16 tumor suppressive pathway is deregulated in most cancers. Loss of p16 occurs more frequently than Rb loss, suggesting that p16 suppresses cancer by regulating Rb as well as the related proteins p107 and p130. However, direct evidence demonstrating that p130 or p107 cooperate with Rb to suppress epithelial cancers associated with p16 loss is currently lacking. Moreover, the roles of p130 and p107 in lung cancer are not clear. In the present studies, Rb ablation was targeted to the lung epithelium in wild-type, p107, or p130 null mice to determine unique and overlapping Rb family functions critical in tumor suppression. Rb ablation during development resulted in marked epithelial abnormalities despite p107 upregulation. In contrast, p130 and p107 were not required during development but had distinct functions in the Rb-deficient epithelium: p107 was required to suppress proliferation, whereas a novel proapoptotic function was identified for p130. Adult Rb-ablated lungs lacked the epithelial phenotype seen at birth and showed compensatory p107 upregulation and p16 induction in epithelial cell lineages that share phenotypic characteristics with human non–small cell lung cancers (NSCLC) that frequently show p16 loss. Importantly, Rb/p107-deficient, but not Rb/p130-deficient, lungs developed tumors resembling NSCLC. Taken together, these studies identify distinct Rb family functions critical in controlling epithelial cell growth, and provide direct evidence that p107 cooperates with Rb to protect against a common adult cancer. [Cancer Res 2009;69(22):8733–41]

Rho-associated kinase (ROCK) signaling plays a fundamental role in regulating cell morphology, adhesion, and motility. Aberrant expression of ROCK is related to tumor metastases and poor clinical outcome. Here, we show that ROCK expression is increased in metastatic human mammary tumors and breast cancer cell lines compared with nonmetastatic tumors and cell lines. Overexpression of ROCK confers a metastatic phenotype on the nonmetastatic MCF-7 cell line. Inhibition of ROCK activity, by either a specific ROCK inhibitor (Y27632) or ROCK-targeted small interfering RNAs, reduces cell migration and proliferation in vitro and metastasis to bone in vivo using a novel "human breast cancer metastasis to human bone" mouse model. Expression of the c-Myc–regulated miR-17-92 cluster is shown to be elevated in metastatic breast cancer cells compared with nonmetastatic cells and diminished by Y27632 treatment. Furthermore, blockade of miR-17 is shown to decrease breast cancer cell invasion/migration in vitro and metastasis in vivo. Together, these findings suggest that augmented ROCK signaling contributes to breast cancer metastasis. The effects of ROCK on tumor cell invasion/motility and growth may derive from regulating cytoskeletal actin-myosin contraction and modulating the c-Myc pathway, including c-Myc–dependent microRNAs. Inhibition of ROCK or the pathway it stimulates, therefore, may represent a novel approach for treatment of breast cancer metastases. [Cancer Res 2009;69(22):8742–51]

Breast cancer is generated through a multistep genetic and epigenetic process including activations of oncogenes and inactivations of tumor suppressor genes. Here, we report a critical role of ubiquitin-conjugating enzyme E2T (UBE2T), an E2 ubiquitin-conjugating enzyme, in mammary carcinogenesis. Immunocytochemical staining and in vitro binding assay revealed that UBE2T interacted and colocalized with the BRCA1/BRCA1-associated RING domain protein (BARD1) complex. Knocking down of UBE2T expression with small interfering RNA drastically suppressed the growth of breast cancer cells. Interestingly, in vivo ubiquitination assay indicated BRCA1 to be polyubiquitinated by incubation with wild-type UBE2T protein, but not with C86A-UBE2T protein, an E2 activity–dead mutant, in which the 86th residue of cysteine was replaced with alanine. Furthermore, knocking down of UBE2T protein induced upregulation of BRCA1 protein in breast cancer cells, whereas its overexpression caused the decrease of the BRCA1 protein. Our data imply a critical role of UBE2T in development and/or progression of breast cancer through the interaction with and the regulation of the BRCA1/BARD1 complex. [Cancer Res 2009;69(22):8752–60]

Upon a wide range of cellular stresses, p53 is activated and inhibits malignant transformation through the transcriptional regulation of its target genes related to apoptosis, cell cycle arrest, and DNA repair. However, its involvement in posttranslational modifications of proteins has not yet been well characterized. Here, we report the novel role of p53 in the regulation of protein citrullination. p53 transactivated peptidylarginine deiminase type 4 (PADI4) through an intronic p53-binding site. The PADI4 gene encodes an enzyme catalyzing the citrullination of arginine residues in proteins, and ectopic expression of p53 or PADI4 induced protein citrullination. In addition, various proteins were citrullinated in response to DNA damage, but knockdown of PADI4 or p53 remarkably inhibited their citrullination, indicating the regulation of protein citrullination in a p53/PADI4-dependent manner. We found that PADI4 citrullinated the histone chaperone protein, nucleophosmin (NPM1), at the arginine 197 residue in vivo under physiologic conditions. Citrullination of NPM1 by PADI4 resulted in its translocation from the nucleoli to the nucleoplasm, whereas PADI4 did not alter the localization of mutant NPM1 (R197K). Furthermore, ectopic expression of PADI4 inhibited tumor cell growth, and concordantly, the knockdown of PADI4 attenuated p53-mediated growth-inhibitory activity, demonstrating the significance of PADI4-mediated protein citrullination in the p53 signaling pathway.[Cancer Res 2009;69(22):8761–9]

Breast cancer is a major contributor to overall morbidity and mortality in women. Several genes predisposing to breast cancer have been identified, but the majority of risk factors remain unknown. Even less is known about the inherited risk factors underlying canine mammary tumors (CMT). Clear breed predispositions exist, with 36% of English springer spaniels (ESS) in Sweden being affected. Here, we evaluate 10 human breast cancer genes (BRCA1, BRCA2, CHEK2, ERBB2, FGFR2, LSP1, MAP3K1, RCAS1, TOX3, and TP53) for association with CMTs. Sixty-three single-nucleotide polymorphisms (SNPs; four to nine SNPs per gene) were genotyped by iPLEX in female ESS dogs, 212 CMT cases and 143 controls. Two genes, BRCA1 and BRCA2, were significantly associated with CMT (Bonferroni corrected P = 0.005 and P = 0.0001, respectively). Borderline association was seen for FGFR2. Benign and malignant cases were also analyzed separately. Those findings supported the association to BRCA1 and BRCA2 but with a stronger association to BRCA1 in malignant cases. Both BRCA1 and BRCA2 showed odds ratios of ~4. In conclusion, this study indicates that BRCA1 and BRCA2 contribute to the risk of CMT in ESS, suggesting that dogs may serve as a good model for human breast cancer. [Cancer Res 2009;69(22):8770–4]

Fanconi anemia is a cancer-prone inherited bone marrow failure and cancer susceptibility syndrome with at least 13 complementation groups (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, and FANCN). Our laboratory has previously described several regulatory phosphorylation events for core complex member proteins FANCG and FANCA by phosphorylation. In this study, we report a novel phosphorylation site serine 331 (S331) of FANCD2, the pivotal downstream player of the Fanconi anemia pathway. Phosphorylation of S331 is important for its DNA damage–inducible monoubiquitylation, resistance to DNA cross-linkers, and in vivo interaction with FANCD1/BRCA2. A phosphomimetic mutation at S331 restores all of these phenotypes to wild-type. In vitro and in vivo experiments show that phosphorylation of S331 is mediated by CHK1, the S-phase checkpoint kinase implicated in the Fanconi anemia DNA repair pathway. [Cancer Res 2009;69(22):8775–83]

Titanium dioxide (TiO₂) nanoparticles are manufactured worldwide in large quantities for use in a wide range of applications including pigment and cosmetic manufacturing. Although TiO₂ is chemically inert, TiO₂ nanoparticles can cause negative health effects, such as respiratory tract cancer in rats. However, the mechanisms involved in TiO₂-induced genotoxicity and carcinogenicity have not been clearly defined and are poorly studied in vivo. The present study investigates TiO₂ nanoparticles–induced genotoxicity, oxidative DNA damage, and inflammation in a mice model. We treated wild-type mice with TiO₂ nanoparticles in drinking water and determined the extent of DNA damage using the comet assay, the micronuclei assay, and the -H2AX immunostaining assay and by measuring 8-hydroxy-2'-deoxyguanosine levels and, as a genetic instability endpoint, DNA deletions. We also determined mRNA levels of inflammatory cytokines in the peripheral blood. Our results show that TiO₂ nanoparticles induced 8-hydroxy-2'-deoxyguanosine, -H2AX foci, micronuclei, and DNA deletions. The formation of -H2AX foci, indicative of DNA double-strand breaks, was the most sensitive parameter. Inflammation was also present as characterized by a moderate inflammatory response. Together, these results describe the first comprehensive study of TiO₂ nanoparticles–induced genotoxicity in vivo in mice possibly caused by a secondary genotoxic mechanism associated with inflammation and/or oxidative stress. Given the growing use of TiO₂ nanoparticles, these findings raise concern about potential health hazards associated with TiO₂ nanoparticles exposure. [Cancer Res 2009;69(22):8784–9]

As breast cancer screening rates increase, smaller and more numerous lesions are being identified earlier, leading to more breast-conserving surgical procedures. Achieving a clean surgical margin represents a technical challenge with important clinical implications. Optical coherence tomography (OCT) is introduced as an intraoperative high-resolution imaging technique that assesses surgical breast tumor margins by providing real-time microscopic images up to 2 mm beneath the tissue surface. In a study of 37 patients split between training and study groups, OCT images covering 1 cm² regions were acquired from surgical margins of lumpectomy specimens, registered with ink, and correlated with corresponding histologic sections. A 17-patient training set used to establish standard imaging protocols and OCT evaluation criteria showed that areas of higher scattering tissue with a heterogeneous pattern were indicative of tumor cells and tumor tissue in contrast to lower scattering adipocytes found in normal breast tissue. The remaining 20 patients were enrolled into the feasibility study. Of these lumpectomy specimens, 11 were identified with a positive or close surgical margin and 9 were identified with a negative margin under OCT. Based on histologic findings, 9 true positives, 9 true negatives, 2 false positives, and 0 false negatives were found, yielding a sensitivity of 100% and specificity of 82%. These results show the potential of OCT as a real-time method for intraoperative margin assessment in breast-conserving surgeries. [Cancer Res 2009;69(22):8790–6]

Tumor-microenvironment interactions are increasingly recognized to influence tumor progression. To understand the competitive dynamics of tumor cells in diverse microenvironments, we experimentally parameterized a hybrid discrete-continuum mathematical model with phenotypic trait data from a set of related mammary cell lines with normal, transformed, or tumorigenic properties. Surprisingly, in a resource-rich microenvironment, with few limitations on proliferation or migration, transformed (but not tumorigenic) cells were most successful and outcompeted other cell types in heterogeneous tumor simulations. Conversely, constrained microenvironments with limitations on space and/or growth factors gave a selective advantage to phenotypes derived from tumorigenic cell lines. Analysis of the relative performance of each phenotype in constrained versus unconstrained microenvironments revealed that, although all cell types grew more slowly in resource-constrained microenvironments, the most aggressive cells were least affected by microenvironmental constraints. A game theory model testing the relationship between microenvironment resource availability and competitive cellular dynamics supports the concept that microenvironmental independence is an advantageous cellular trait in resource-limited microenvironments. [Cancer Res 2009;69(22):8797–806]

The tumor suppressor gene TP53 is known to be a key regulator in cancer, and more than half of human cancers exhibit mutations in this gene. Recent evidence shows that point mutations in TP53 not only disrupt its function but also possess gain-of-function and dominant-negative effects on wild-type copies, thus making the mutated gene an oncogene. Hence, this brings about the possibility that TP53 mutations may be under selection for increasing the overall translation efficiency (TE) of defected TP53 in cancerous cells. Here, we perform the first large-scale analysis of TE in human cancer mutated TP53 variants, identifying a significant increase in TE that is correlated with the frequency of TP53 mutations. Furthermore, mutations with a known oncogenic effect significantly increase their TE compared with the other TP53 mutations. Further analysis shows that TE may have influence both on selecting the location of the mutation and on its outcome: codons with lower TE show stronger selection toward nonsynonymous mutations and, for each codon, frequent mutations show stronger increase in TE compared with less frequent mutations. Additionally, we find that TP53 mutations have significantly higher TE increase in progressive versus primary tumors. Finally, an analysis of TP53 NCI-60 cell lines points to a coadaptation between the mutations and the tRNA pool, increasing the overall TP53 TE. Taken together, these results show that TE plays an important role in the selection of TP53 cancerous mutations. [Cancer Res 2009;69(22):8807–13]

Even after a tumor is established, it can early on enter a state of dormancy marked by balanced cell proliferation and cell death. Disturbances to this equilibrium may affect cancer risk, as they may cause the eventual lifetime clinical presentation of a tumor that might otherwise have remained asymptomatic. Previously, we showed that cell death, proliferation, and migration can play a role in shifting this dynamic, making the understanding of their combined influence on tumor development essential. We developed an individual cell-based computer model of the interaction of cancer stem cells and their nonstem progeny to study early tumor dynamics. Simulations of tumor growth show that three basic components of tumor growth—cell proliferation, migration, and death—combine in unexpected ways to control tumor progression and, thus, clinical cancer risk. We show that increased proliferation capacity in nonstem tumor cells and limited cell migration overall lead to space constraints that inhibit proliferation and tumor growth. By contrast, increasing the rate of cell death produces the expected tumor size reduction in the short term, but results ultimately in paradoxical accelerated long-term growth owing to the liberation of cancer stem cells and formation of self-metastases.[Cancer Res 2009;69(22):8814–21]

Metastasis continues to be one of the major causes of mortality from prostate cancer. Because human malignant cell lines metastasize more readily from orthotopic sites than from heterotopic sites, to identify metastasis-permissive tumor microenvironments, we used noninvasive imaging to compare the in vivo vascular, metabolic, and physiologic characteristics of a human prostate cancer xenograft implanted orthotopically in the prostate or s.c. in the flank. Hypoxia was detected in these xenografts by placing an enhanced green fluorescence protein optical reporter under the control of a hypoxia response element. A multiparametric analysis of hypoxia, extracellular pH, vascularization, and metabolism provided a characterization of environments that are permissive for metastasis to occur. We found that orthotopic tumors, which metastasized more easily, were characterized by higher vascular volume, permeability, and total choline and a more acidic extracellular pH. Interestingly, metastatic deposits in the lymph nodes as well as cancer cells in ascites fluid were found to be hypoxic, explaining, in part, the refractory nature of metastatic disease. These results also provide the basis for clinically translatable noninvasive imaging markers for predicting metastatic risk in prostate cancer. [Cancer Res 2009;69(22):8822–9]