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Epidemiologic and histopathologic findings and the laying hen model support the long-standing incessant ovulation hypothesis and cortical inclusion cyst involvement in sporadic ovarian cancer development. MicroRNA-200 (miR-200) family is highly expressed in ovarian cancer. Herewith, we show that ovarian surface epithelial (OSE) cells with ectopic miR-200 expression formed stabilized cysts in three-dimensional (3D) organotypic culture with E-cadherin fragment expression and steroid hormone pathway activation, whereas ovarian cancer 3D cultures with miR-200 knockdown showed elevated TGF-β expression, mitotic spindle disorientation, increased lumenization, disruption of ROCK-mediated myosin II phosphorylation, and SRC signaling, which led to histotype-dependent loss of collective movement in tumor spread. Gene expression profiling revealed that epithelial-mesenchymal transition and hypoxia were the top enriched gene sets regulated by miR-200 in both OSE and ovarian cancer cells. The molecular changes uncovered by the in vitro studies were verified in both human and laying hen ovarian cysts and tumor specimens. As miR-200 is also essential for ovulation, our results of estrogen pathway activation in miR-200-expressing OSE cells add another intriguing link between incessant ovulation and ovarian carcinogenesis.

Members of microRNA-200 (miRNA-200) family have a regulatory role in epithelial to mesenchymal transition (EMT) by suppressing Zeb1 and Zeb2 expression. Consistent with its role in suppressing EMT, Hsa-miR-200c-3p (miR-200c), a member of miR-200 family is poorly expressed in mesenchymal-like triple-negative breast cancer (TNBC) cells and ectopic miR-200c expression suppresses cell migration. In this study, we demonstrated that miR-200c potently inhibited TNBC cell growth and tumor development in a mechanism distinct from its ability to downregulate Zeb1 and Zeb2 expression, because silencing them only marginally affected TNBC cell growth. We identified phosphodiesterase 7B (PDE7B) as a bona fide miR-200c target. Importantly, miR-200c-led inhibition in cell growth and tumor development was prevented by forcing PDE7B transgene expression, while knockdown of PDE7B effectively inhibited cell growth. These results suggest that miR-200c inhibits cell growth by targeting PDE7B mRNA. To elucidate mechanism underlying miR-200c/PDE7B regulation of TNBC cell growth, we showed that cAMP concentration was lower in TNBC cells compared with estrogen receptor-positive (ER + ) cells, and that both miR-200c and PDE7B siRNAs were able to increase cAMP concentration in TNBC cells. High level of cellular cAMP has been shown to induce cell cycle arrest and apoptosis in TNBC cells. Our observation that ectopic expression of miR-200c triggered apoptosis indicates that it does so by elevating level of cellular cAMP. Analysis of breast tumor gene expression datasets revealed an inverse association between miR-200c and PDE7B expression. Especially, both low miR-200c and high PDE7B expression were correlated with poor survival of breast cancer patients. Our study supports a critical role of miR-200c/PDE7B relationship in TNBC tumorigenesis.

TNFα is a pleiotropic cytokine which fuels tumor cell growth, invasion, and metastasis in some malignancies, while in others it induces cytotoxic cell death. However, the molecular mechanism by which TNFα exerts its diverse effects on breast cancer subtypes remains elusive. Using in vitro assays and mouse xenografts, we show here that TNFα contributes to the aggressive properties of triple negative breast cancer (TNBC) cell lines via upregulation of TNFAIP3(A20). In a striking contrast, TNFα induces a potent cytotoxic cell death in luminal (ER+) breast cancer cell lines which fail to upregulate A20 expression. Overexpression of A20 not only protects luminal breast cancer cell lines from TNFα-induced cell death via inducing HSP70-mediated anti-apoptotic pathway but also promotes a robust EMT/CSC phenotype by activating the pStat3-mediated inflammatory signaling. Furthermore, A20 overexpression in luminal breast cancer cells induces aggressive metastatic properties in mouse xenografts via generating a permissive inflammatory microenvironment constituted by granulocytic-MDSCs. Collectively, our results reveal a mechanism by which A20 mediates pleiotropic effects of TNFα playing role in aggressive behaviors of TNBC subtype while its deficiency results in TNFα-induced apoptotic cell death in luminal breast cancer subtype.

Although it has been demonstrated that transformed progenitor cell population can contribute to tumor initiation, factors contributing to this malignant transformation are poorly known. Using in vitro and xenograft-based models, previous studies demonstrated that miR-489 acts as a tumor suppressor miRNA by targeting various oncogenic pathways. It has been demonstrated that miR-489 directly targets HER2 and inhibits the HER2 signaling pathway; however, its role in mammary gland development and HER2-induced tumor initiation hasn't been studied. To dissect the role of miR-489, we sorted different populations of mammary epithelial cells and determined that miR-489 was highly expressed in mammary stem cells. MMTV-miR-489 mice that overexpressed miR-489 in mammary epithelial cells were developed and these mice exhibited an inhibition of mammary gland development in early ages with a specific impact on highly proliferative cells. Double transgenic MMTV-Her2-miR489 mice were then generated to observe how miR-489 overexpression affects HER2-induced tumorigenesis. miR-489 overexpression delayed HER2-induced tumor initiation significantly. Moreover, miR-489 overexpression inhibited tumor growth and lung metastasis. miR-489 overexpression reduced mammary progenitor cell population significantly in preneoplastic mammary glands of MMTV-Her2 mice which showed a putative transformed population in HER2-induced tumorigenesis. The miR-489 overexpression reduced CD49f hi CD61 hi populations in tumors that have stem-like properties, and miR-489 overexpression altered the HER2 signaling pathway in mammary tumors. Altogether, these data indicate that the inhibition of HER2-induced tumorigenesis by miR-489 overexpression was due to altering progenitor cell populations while decreasing tumor growth and metastasis via influencing tumor promoting genes DEK and SHP2.

Aberrant expression of Forkhead box (FOX) transcription factors plays vital roles in carcinogenesis. However, the function of the FOX family member FOXC1 in maintenance of colorectal cancer (CRC) malignancy is unknown. Herein, FOXC1 expression in CRC specimens in The Cancer Genome Atlas (TCGA) cohort was analyzed and validated using immunohistochemistry with a tissue microarray. The effect of FOXC1 expression on proliferation of and glycolysis in CRC cells was assessed by altering its expression in vitro and in vivo. Mechanistic investigation was carried out using cell and molecular biological approaches. Our results showed that FOXC1 expression was higher in CRC specimens than in adjacent benign tissue specimens. Univariate survival analyses of the patients from whom the study specimens were obtained, and validated cohorts indicated that ectopic FOXC1 expression was significantly correlated with shortened survival. Silencing FOXC1 expression in CRC cells inhibited their proliferation and colony formation and decreased their glucose consumption and lactate production. In contrast, FOXC1 overexpression had the opposite effect. Furthermore, increased expression of FOXC1 downregulated that of a key glycolytic enzyme, fructose-1,6-bisphosphatase 1 (FBP1). Mechanistically, FOXC1 bound directly to the promoter regions of the FBP1 gene and negatively regulated its transcriptional activity. Collectively, aberrant FBP1 expression contributed to CRC tumorigenicity, and decreased FBP1 expression coupled with increased FOXC1 expression provided better prognostic information than did FOXC1 expression alone. Therefore, the FOXC1/FBP1 axis induces CRC cell proliferation, reprograms metabolism in CRCs, and constitutes potential prognostic predictors and therapeutic targets for CRC.

Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide. The poor survival may be due to a high proportions of tumor recurrence and metastasis. Kinesin family member C1 (KIFC1) is highly expressed in a variety of neoplasms and is a potential marker for non-small cell lung cancer or ovarian adenocarcinoma metastasis. Nevertheless, the role of KIFC1 in HCC metastasis remains obscure. We investigated this in the present study using HCC cell lines and clinical specimens. Our results indicated that increased levels of KIFC1 were associated with poor prognosis and metastasis in HCC. In addition, KIFC1 induced epithelial-to-mesenchymal transition (EMT) and HCC metastasis both in vitro and in vivo. This tumorigenic effect depended on gankyrin; inhibiting gankyrin activity reversed EMT via activation of protein kinase B (AKT)/Twist family BHLH transcription factor 1 (AKT/TWIST1). We also found that KIFC1 was directly regulated by the microRNA miR-532-3p, whose downregulation was associated with metastatic progression in HCC. These results denote that a decrease in miR-532-3p levels results in increased KIFC1 expression in HCC, leading to metastasis via activation of the gankyrin/AKT/TWIST1 signaling pathway.

Ovarian carcinoma is the fifth common cause of cancer death in women, despite advanced therapeutic approaches. αvβ3 integrin, a plasma membrane receptor, binds thyroid hormones (L-thyroxine, T4; 3,5,3'-triiodo-L-thyronine, T3) and is overexpressed in ovarian cancer. We have demonstrated selective binding of fluorescently labeled hormones to αvβ3-positive ovarian cancer cells but not to integrin-negative cells. Physiologically relevant T3 (1 nM) and T4 (100 nM) concentrations in OVCAR-3 (high αvβ3) and A2780 (low αvβ3) cells promoted αv and β3 transcription in association with basal integrin levels. This transcription was effectively blocked by RGD (Arg-Gly-Asp) peptide and neutralizing αvβ3 antibodies, excluding T3-induced β3 messenger RNA, suggesting subspecialization of T3 and T4 binding to the integrin receptor pocket. We have provided support for extracellular regulated kinase (ERK)-mediated transcriptional regulation of the αv monomer by T3 and of β3 monomer by both hormones and documented a rapid (30-120 min) and dose-dependent (0.1-1000 nM) ERK activation. OVCAR-3 cells and αvβ3-deficient HEK293 cells treated with αvβ3 blockers confirmed the requirement for an intact thyroid hormone-integrin interaction in ERK activation. In addition, novel data indicated that T4, but not T3, controls integrin's outside-in signaling by phosphorylating tyrosine 759 in the β3 subunit. Both hormones induced cell proliferation (cell counts), survival (Annexin-PI), viability (WST-1) and significantly reduced the expression of genes that inhibit cell cycle (p21, p16), promote mitochondrial apoptosis (Nix, PUMA) and tumor suppression (GDF-15, IGFBP-6), particularly in cells with high integrin expression. At last, we have confirmed that hypothyroid environment attenuated ovarian cancer growth using a novel experimental platform that exploited paired euthyroid and severe hypothyroid serum samples from human subjects. To conclude, our data define a critical role for thyroid hormones as potent αvβ3-ligands, driving ovarian cancer cell proliferation and suggest that disruption of this axis may present a novel treatment strategy in this aggressive disease.

The process of epithelial-mesenchymal transition (EMT), in addition to being an initiating event for tumor metastasis, is implicated in conferring several clinically relevant properties to disseminating cancer cells. These include stem cell-like properties, resistance to targeted therapies and ability to evade immune surveillance. Enrichment analysis of gene expression changes during transforming growth factor-β (TGF-β)-induced EMT in lung cancer cells identified complement cascade as one of the significantly enriched pathway. Further analysis of the genes in the complement pathway revealed an increase in the expression of complement inhibitors and a decrease in the expression of proteins essential for complement activity. In this study, we tested whether EMT confers resistance to complement-dependent cytotoxicity (CDC) in lung cancer cells and promotes tumor progression. CD59 is a potent inhibitor of membrane attack complex that mediates complement-dependent cell lysis. We observed a significant increase in the CD59 expression on the surface of cells after TGF-β-induced EMT. Furthermore, CD59 knockdown restored susceptibility of cells undergoing EMT to cetuximab-mediated CDC. TGF-β-induced CD59 expression during EMT is dependent on Smad3 but not on Smad2. Chromatin immunoprecipitation analysis confirmed that Smad3 directly binds to the CD59 promoter. Stable knockdown of CD59 in A549 cells inhibited experimental metastasis. These results demonstrate that TGF-β-induced EMT and CD59 expression confers an immune-evasive mechanism to disseminating tumor cells facilitating tumor progression. Together, our data demonstrates that CD59 inhibition may serve as an adjuvant to enhance the efficacy of antibody-mediated therapies, as well as to inhibit metastasis in lung cancer.

The presence of hypoxic regions in solid tumors is an adverse prognostic factor for patient outcome. Here, we show that hypoxia induces the expression of Ephrin-A3 through a novel hypoxia-inducible factor (HIF)-mediated mechanism. In response to hypoxia, the coding EFNA3 mRNA levels remained relatively stable, but HIFs drove the expression of previously unknown long noncoding (lnc) RNAs from EFNA3 locus and these lncRNA caused Ephrin-A3 protein accumulation. Ephrins are cell surface proteins that regulate diverse biological processes by modulating cellular adhesion and repulsion. Mounting evidence implicates deregulated ephrin function in multiple aspects of tumor biology. We demonstrate that sustained expression of both Ephrin-A3 and novel EFNA3 lncRNAs increased the metastatic potential of human breast cancer cells, possibly by increasing the ability of tumor cells to extravasate from the blood vessels into surrounding tissue. In agreement, we found a strong correlation between high EFNA3 expression and shorter metastasis-free survival in breast cancer patients. Taken together, our results suggest that hypoxia could contribute to metastatic spread of breast cancer via HIF-mediated induction of EFNA3 lncRNAs and subsequent Ephrin-A3 protein accumulation.

Glioblastomas are aggressive adult brain tumors, characterized by inadequately organized vasculature and consequent nutrient and oxygen (O2)-depleted areas. Adaptation to low nutrients and hypoxia supports glioblastoma cell survival, progression and therapeutic resistance. However, specific mechanisms promoting cellular survival under nutrient and O2 deprivation remain incompletely understood. Here, we show that miR-124 expression is negatively correlated with a hypoxic gene signature in glioblastoma patient samples, suggesting that low miR-124 levels contribute to pro-survival adaptive pathways in this disease. As miR-124 expression is repressed in various cancer types (including glioblastoma), we quantified miR-124 abundance in normoxic and hypoxic regions in glioblastoma patient tissue, and investigated whether ectopic miR-124 expression compromises cell survival during tumor ischemia. Our results indicate that miR-124 levels are further diminished in hypoxic/ischemic regions within individual glioblastoma patient samples, compared with regions replete in O2 and nutrients. Importantly, we also show that increased miR-124 expression affects the ability of tumor cells to survive under O2 and/or nutrient deprivation. Moreover, miR-124 re-expression increases cell death in vivo and enhances the survival of mice bearing intracranial xenograft tumors. miR-124 exerts this phenotype in part by directly regulating TEAD1, MAPK14/p38α and SERP1, factors involved in cell proliferation and survival under stress. Simultaneous suppression of these miR-124 targets results in similar levels of cell death as caused by miR-124 restoration. Importantly, we further demonstrate that SERP1 reintroduction reverses the hypoxic cell death elicited by miR-124, indicating the importance of SERP1 in promoting tumor cell survival. In support of our experimental data, we observed a significant correlation between high SERP1 levels and poor patient outcome in glioblastoma patients. Collectively, among the many pro-tumorigeneic properties of miR-124 repression in glioblastoma, we delineated a novel role in promoting tumor cell survival under stressful microenvironments, thereby supporting tumor progression.

FOXM1 is implicated in genotoxic drug resistance but its role and mechanism of action remain unclear. Here, we establish that γH2AX foci, indicative of DNA double-strand breaks (DSBs), accumulate in a time-dependent manner in the drug-sensitive MCF-7 cells but not in the resistant counterparts in response to epirubicin. We find that FOXM1 expression is associated with epirubicin sensitivity and DSB repair. Ectopic expression of FOXM1 can increase cell viability and abrogate DSBs sustained by MCF-7 cells following epirubicin, owing to an enhancement in repair efficiency. Conversely, alkaline comet and γH2AX foci formation assays show that Foxm1-null cells are hypersensitive to DNA damage, epirubicin and γ-irradiation. Furthermore, we find that FOXM1 is required for DNA repair by homologous recombination (HR) but not non-homologous end joining (NHEJ), using HeLa cell lines harbouring an integrated direct repeat green fluorescent protein reporter for DSB repair. We also identify BRIP1 as a direct transcription target of FOXM1 by promoter analysis and chromatin-immunoprecipitation assay. In agreement, depletion of FOXM1 expression by small interfering RNA downregulates BRIP1 expression at the protein and mRNA levels in MCF-7 and the epirubicin-resistant MCF-7 Epi(R) cells. Remarkably, the requirement for FOXM1 for DSB repair can be circumvented by reintroduction of BRIP1, suggesting that BRIP1 is an important target of FOXM1 in DSB repair. Indeed, like FOXM1, BRIP1 is needed for HR. These data suggest that FOXM1 regulates BRIP1 expression to modulate epirubicin-induced DNA damage repair and drug resistance.

Meningiomas are frequent, mostly benign intracranial or spinal tumors. A small subset of meningiomas is characterized by histological features of atypia or anaplasia that are associated with more aggressive biological behavior resulting in increased morbidity and mortality. Infiltration into the adjacent brain tissue is a major factor linked to higher recurrence rates. The molecular mechanisms of progression, including brain invasion are still poorly understood. We have studied the role of micro-RNA 145 (miR-145) in meningiomas and detected significantly reduced miR-145 expression in atypical and anaplastic tumors as compared with benign meningiomas. Overexpression of miR-145 in IOMM-Lee meningioma cells resulted in reduced proliferation, increased sensitivity to apoptosis, reduced anchorage-independent growth and reduction of orthotopic tumor growth in nude mice as compared with control cells. Moreover, meningioma cells with high miR-145 levels had impaired migratory and invasive potential in vitro and in vivo. PCR-array studies of miR145-overexpressing cells suggested that collagen type V alpha (COL5A1) expression is downregulated by miR-145 overexpression. Accordingly, COL5A1 expression was significantly upregulated in atypical and anaplastic meningiomas. Collectively, our data indicate an important anti-migratory and anti-proliferative function of miR-145 in meningiomas.

Different classes of non-coding RNAs, including microRNAs, have recently been implicated in the process of tumourigenesis. In this study, we examined the expression and putative functions of a novel class of non-coding RNAs known as transcribed ultraconserved regions (T-UCRs) in neuroblastoma. Genome-wide expression profiling revealed correlations between specific T-UCR expression levels and important clinicogenetic parameters such as MYCN amplification status. A functional genomics approach based on the integration of multi-level transcriptome data was adapted to gain insights into T-UCR functions. Assignments of T-UCRs to cellular processes such as TP53 response, differentiation and proliferation were verified using various cellular model systems. For the first time, our results define a T-UCR expression landscape in neuroblastoma and suggest widespread T-UCR involvement in diverse cellular processes that are deregulated in the process of tumourigenesis.

Cancer cells have complex, unique characteristics that distinguish them from normal cells, such as increased growth rates and evasion of anti-proliferative signals. Global inhibition of class I and II histone deacetylases (HDACs) stops cancer cell proliferation in vitro and has proven effective against cancer in clinical trials, at least in part, through transcriptional reactivation of the p21(WAF1/Cip1)gene. The HDACs that regulate p21(WAF1/Cip1) are not fully identified. Using small interfering RNAs, we found that HDAC4 participates in the repression of p21(WAF1/Cip1) through Sp1/Sp3-, but not p53-binding sites. HDAC4 interacts with Sp1, binds and reduces histone H3 acetylation at the Sp1/Sp3 binding site-rich p21(WAF1/Cip1) proximal promoter, suggesting a key role for Sp1 in HDAC4-mediated repression of p21(WAF1/Cip1). Induction of p21(WAF1/Cip1) mediated by silencing of HDAC4 arrested cancer cell growth in vitro and inhibited tumor growth in an in vivo human glioblastoma model. Thus, HDAC4 could be a useful target for new anti-cancer therapies based on selective inhibition of specific HDACs.

Effective control of both cell survival and cell proliferation is critical to the prevention of oncogenesis and to successful cancer therapy. Using functional expression cloning, we have identified GAS5 (growth arrest-specific transcript 5) as critical to the control of mammalian apoptosis and cell population growth. GAS5 transcripts are subject to complex post-transcriptional processing and some, but not all, GAS5 transcripts sensitize mammalian cells to apoptosis inducers. We have found that, in some cell lines, GAS5 expression induces growth arrest and apoptosis independently of other stimuli. GAS5 transcript levels were significantly reduced in breast cancer samples relative to adjacent unaffected normal breast epithelial tissues. The GAS5 gene has no significant protein-coding potential but expression encodes small nucleolar RNAs (snoRNAs) in its introns. Taken together with the recent demonstration of tumor suppressor characteristics in the related snoRNA U50, our observations suggest that such snoRNAs form a novel family of genes controlling oncogenesis and sensitivity to therapy in cancer.

Most human cancers are characterized by genetic aberrations accompanied by altered expression and function of numerous genes. Applying genome-wide, microarray gene expression analysis to identify deregulated genes in different tumour types can provide potential gene candidates as diagnostic and prognostic tools and promising targets for drug development. However, the detection of deregulated genes with low levels of expression remains a major challenge. In this study, we have designed a strategy, termed modified suppression subtractive hybridization (mSSH), to identify genes encoding rare transcripts. The strategy entails incorporating the T(7)-promoter sequence at the 5' end of the noncoding cDNA strand during first strand cDNA synthesis to generate unidirectional antisense RNA from the resultant cDNA following conventional SSH. These transcripts are subsequently analysed by Affymetrix oligonucleotide gene arrays. Here, we have used five hepatocellular carcinoma (HCC), five breast carcinoma and four nasopharyngeal carcinoma (NPC) biopsies separately as testers and their corresponding normal biopsies as drivers to enrich for low abundance tumour type-specific transcripts. The total detectable number of probe sets following mSSH was reduced almost 10-fold in comparison to those detected for the same resected tumour tissues without undergoing subtraction, thus yielding a subtraction efficacy of over 90%. Using this experimental approach, we have identified 48 HCC-specific, 45 breast carcinoma-specific, and 83 NPC-specific genes. In addition, 115 genes were upregulated in all the three cancer types. When compared to gene-profiling data obtained without mSSH, the majority of these identified transcripts were of low abundance in the original cancer tissues. mSSH can therefore serve as a comprehensive molecular strategy for pursuing functional genomic studies of human cancers.

Directed by microarray analyses, we report that autocrine human growth hormone (hGH) increased the mRNA and protein expression of DNA methyltransferase 1 (DNMT1), DNMT3A and DNMT3B in mammary carcinoma cells. Autocrine hGH stimulation of DNMT3A and DNMT3B expression was mediated by JAK2 and Src kinases, and treatment of mammary carcinoma cells with the DNMT inhibitor, 5'-aza-2'-deoxycytidine (AZA), abrogated autocrine hGH-stimulated cellular proliferation, apoptosis and anchorage-independent growth. AZA reversed the epitheliomesenchymal transition of mammary carcinoma cells induced by autocrine hGH, to an epithelioid morphology and abrogated cell migration stimulated by autocrine hGH. Autocrine hGH-stimulated hypermethylation of the first exon of the PLAKOGLOBIN gene and AZA abrogated the ability of autocrine hGH to repress plakoglobin gene transcription. Small interfering RNA (siRNA)-mediated depletion of the individual DNMT molecules did not release autocrine hGH repression of PLAKOGLOBIN promoter activity nor did individual DNMT depletion affect autocrine hGH-stimulated migration. However, concomitant siRNA-mediated depletion of both DNMT3A and DNMT3B abrogated hypermethylation of the PLAKOGLOBIN gene stimulated by autocrine hGH and subsequent repression of plakoglobin gene transcription and increased cell migration. Thus, the autocrine hGH-stimulated increases in DNMT3A and DNMT3B expression mediate repression of plakoglobin gene transcription by direct hypermethylation of its promoter and consequent phenotypic conversion of mammary carcinoma cells. Autocrine hGH, therefore, utilizes DNA methylation as a mechanism to exert its oncogenic effects in mammary carcinoma cells.

Human papillomaviruses (HPVs) are involved in the pathogenesis of cancer of the cervix (CaCx). MicroRNA (miRNA) expression analysis using Ambion (Austin, TX, USA) arrays showed that three miRNAs were overexpressed and 24 underexpressed in cervical cell lines containing integrated HPV-16 DNA compared to the normal cervix. Furthermore, nine miRNAs were overexpressed and one underexpressed in integrated HPV-16 cell lines compared to the HPV-negative CaCx cell line C-33A. Based on microarray and/or quantitative real-time PCR and northern blot analyses, microRNA-218 (miR-218) was specifically underexpressed in HPV-positive cell lines, cervical lesions and cancer tissues containing HPV-16 DNA compared to both C-33A and the normal cervix. Expression of the E6 oncogene of high-risk HPV-16, but not that of low-risk HPV-6, reduced miR-218 expression, and conversely, RNA interference of E6/E7 oncogenes in an HPV-16-positive cell line increased miR-218 expression. We also demonstrate that the epithelial cell-specific marker LAMB3 is a target of miR-218. We also show that LAMB3 expression is increased in the presence of the HPV-16 E6 oncogene and this effect is mediated through miR-218. These findings may contribute to a better understanding of the molecular mechanisms involved in cervical carcinogenesis.

The transmembrane protein-tyrosine phosphatase (PTP) DEP-1 (density-enhanced phosphatase) is a candidate tumor suppressor in the colon epithelium. We have explored the function of DEP-1 in colon epithelial cells by inducible re-expression in a DEP-1-deficient human colon cancer cell line. Density-enhanced phosphatase-1 re-expression led to profound inhibition of cell proliferation and cell migration, and was associated with cytoskeletal rearrangements. These effects were dependent on the PTP activity of DEP-1 as they were not observed with cells expressing the catalytically inactive DEP-1 C1239S variant. shRNA-mediated suppression of DEP-1 in a colon epithelial cell line with high endogenous DEP-1 levels enhanced proliferation, further supporting the antiproliferative function of DEP-1. Nutrients, which are considered to be chemoprotective with respect to colon cancer development, including butyrate, green tea and apple polyphenols, had the capacity to elevate transcription of endogenous DEP-1 mRNA and expression of DEP-1 protein. Upregulation of DEP-1 expression, and in turn inhibition of cell growth and migration may present a previously unrecognized mechanism of chemoprevention by nutrients.

Retroviral vector-mediated overexpression of c-myc in embryonic bursal precursors induces multi-staged tumorigenesis beginning with preneoplastic-transformed follicles (TF) and progressing to clonal metastatic B-cell lymphomas. Using a 13K chicken cDNA microarray, specifically enriched for chicken immune system expressed sequence tagged (ESTs), we carried out array-based comparative genomic hybridization (array-CGH) and detected significant DNA copy number change at many loci on most or all chromosomes in both early TF and end-stage lymphomas. Formation of long palindromes, through breakage-fusion-bridge cycles, is thought to play an early role in gene amplification. Employing genome-wide analysis of palindrome formation (GAPF), we detected extensive palindrome formation in early TF and end-stage lymphomas. The population of loci showing amplification by array-CGH was enriched for palindromes detected by GAPF providing strong evidence for genetic instability early in Myc-induced tumorigenesis and further support for the role of palindromes in gene amplification. Comparing gene copy number change and RNA expression changes profiled on the same cDNA array, we detected very little consistent contribution of gene copy number change to RNA expression changes. Palindromic loci in TF and tumors, however, were expressed, many at high levels, suggesting an abundance of RNA species with long double-stranded segments generated during tumorigenesis.

Aberrant activation of the Wnt signaling pathway has been reported during neoplastic progression in Barrett's esophagus (BE). However, mutations in APC and CTNNB1 genes were rarely observed. In this study, expression pattern of Wnt ligands, Frizzled receptors and APC, as well as the methylation status of the APC, SFRP1 and SFRP2 promoter genes were investigated in normal esophageal mucosa and in preneoplastic and neoplastic lesions of BE patients. Promoter methylation of APC was found in all BE samples and in 95% of esophageal adenocarcinomas (EAC). Full methylation of APC correlated with lack of expression. In EAC, nuclear translocation of beta-catenin was observed regardless of the expression of APC. WNT2 expression was higher in dysplasia and EAC than in BE, with 20/26 (77%) of the EAC showing high expression of WNT2. SFRP1 methylation occurred in all BE samples and in 96% of EAC, while SFRP2 was methylated in 73% of the normal squamous esophageal mucosa samples. In conclusion, (1) alterations of key regulators of the Wnt signaling are frequent in the pathogenesis of BE; (2) the APC and SFRP1 genes are inactivated by promoter methylation in BE; (3) the WNT2 gene is upregulated along the progression from low-grade dysplasia to EAC.

The pulmonary adenoma susceptibility 1 (Pas1) locus affects inherited predisposition and resistance to chemically induced lung tumorigenesis in mice. The A/J and C57BL/6J mouse strains carry the susceptibility and resistance allele, respectively. We identified and genotyped 65 polymorphisms in the Pas1 locus region in 29 mouse inbred strains, and delimited the Pas1 locus to a minimal region of 468 kb containing six genes. That region defined a core Pas1 haplotype with 42 tightly linked markers, including intragenic polymorphisms in five genes (Bcat1, Lrmp, Las1, Ghiso, and Kras2) and amino-acid changes in three genes (Lrmp, Las1, Lmna-rs1). In (A/J x C57BL/6J)F1 mouse lung tumors, the Lmna-rs1 gene was completely downregulated, whereas allele-specific downregulation of the C57BL/6J-derived allele was observed at the Las1 gene, suggesting the potential role of these genes in tumor suppression. These results indicate a complex multigenic nature of the Pas1 locus, and point to a functional role for both intronic and exonic polymorphisms of the six genes of the Pas1 haplotype in lung tumor susceptibility.

Retinoids, the natural and synthetic derivatives of vitamin A, have a role in cancer treatment and prevention. There is a need to reveal mechanisms that account for retinoid response or resistance. This study identified candidate all-trans-retinoic acid (RA) target genes linked to growth suppression in BEAS-2B human bronchial epithelial cells. Microarray analyses were performed using Affymetrix arrays. A total of 11 RA-induced species were validated by reverse transcription polymerase chain reaction (RT-PCR), Western or Northern analyses. Three of these species were novel candidate RA-target genes in human bronchial epithelial cells. These included: placental bone morphogenetic protein (PLAB), polyamine oxidase isoform 1 (PAOh1) and E74-like factor 3 (ELF3). Expression patterns were studied in RA-resistant BEAS-2B-R1 cells. In BEAS-2B-R1 cells, RA dysregulated the expression of the putative lymphocyte G0/G1 switch gene (G0S2), heme oxygenase 1 (HMOX1), tumor necrosis factor-alpha-induced protein 2 (TNFAIP2), inhibitor of DNA binding 1(Id1), fos-like antigen 1 (FOSL1), transglutaminase 2 (TGM2), asparagine synthetase (ASNS), PLAB, PAOh1 and ELF3, while prominent induction of insulin-like growth-factor-binding protein 6 (IGFBP6) still occurred. In summary, this study identified 11 candidate RA-target genes in human bronchial epithelial cells including three novel species. Expression studies in BEAS-2B-R1 cells indicated that several were directly implicated in RA signaling, since their aberrant expression was linked to RA resistance of human bronchial epithelial cells.

Identification of distinct molecular subtypes is a critical challenge for cancer biology. In this study, we used Affymetrix high-density oligonucleotide arrays to identify the global gene expression signatures associated with gliomas of different types and grades. Here, we show that the global transcriptional profiles of gliomas of different types and grades are distinct from each other and from the normal brain. To determine whether our data could be used to uncover molecular subtypes without prior knowledge of pathologic type and grade, we performed K-means clustering analysis and found evidence for three clusters with the aid of multidimensional scaling plots. These clusters corresponded to glioblastomas, lower grade astrocytomas and oligodendrogliomas (P<0.00001). A predictor constructed from the 170 genes that are most differentially expressed between the subsets correctly identified the type and grade of all samples, indicating that a relatively small number of genes can be used to distinguish between these molecular subtypes. These results further define molecular subsets of gliomas which may potentially be used for patient stratification, and suggest potential targets for treatment.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear receptor for eicosanoids that promotes differentiation of human epithelial and mesenchymal cells in vitro and in vivo. PPARgamma was proposed as a target for drug-induced differentiation therapy of cancer. Caveolin-1 is a constituent of plasma membrane caveolae in epithelial cells that is often downregulated upon oncogenic transformation. Caveolin-1 has growth-inhibitory activities and its disruption is sufficient to induce transformation in fibroblasts. Herein we have tested the hypothesis that caveolins are transcriptional target genes for PPARgamma. In human HT-29 colon carcinoma cells, thiazolidinedione PPARgamma ligands increased the levels of caveolin-1 and caveolin-2 proteins two to fivefold in a concentration-dependent manner within 24 h. In human MCF-7 breast adenocarcinoma cells, nonthiazolidinedione PPARgamma ligands elevated caveolin-2 protein three to fourfold, while the thiazoli-dinediones were less effective. Caveolin-1 mRNA levels were found to be upregulated by PPARgamma ligands already after 3 h in both the cell lines. Ectopic expression of a dominant-negative PPARgamma construct attenuated ligand-induced upregulation of caveolins in both HT-29 and HEK-293T cells, indicating that ligand action is mediated by PPARgamma. Ligand-treated MCF-7 cells exhibited a differentiated phenotype, as evinced by analysis of cell-specific differentiation markers: protein levels of maspin were elevated and perinuclear lipid droplets accumulated. In contrast, in HT-29 cells, caveolin expression was not correlated with differentiation. Interestingly, PPARgamma partially cofractionated in lipid rafts and could be coimmunoprecipitated from cell lysates with caveolin-1, indicating that PPARgamma and caveolin-1 may coexist in a complex. Our data indicate that PPARgamma participates in the regulation of caveolin gene expression in human carcinoma cells and suggest that caveolin-1 may mediate some of the phenotypic changes induced by this nuclear receptor in cancer cells. These findings may have potentially important functional implications in the context of cancer differentiation therapy and multidrug resistance.

DLC-1 (deleted in liver cancer) gene is frequently deleted in hepatocellular carcinoma. However, little is known about the genetic status and the expression of this gene in gastric cancer. In this study, Northern and Southern analysis showed that seven of nine human gastric cancer cell lines did not express DLC-1 mRNA, but contained the DLC-1 gene. To identify the mechanism of the loss of DLC-1 mRNA expression in these cell lines, we investigated the methylation status of DLC-1 gene by using methylation-specific PCR (MSP) and Southern blot, and found that five of seven DLC-1 nonexpressing gastric cancer cell lines were methylated in the DLC-1 CpG island. Treatment with 5-aza-2'-deoxycytidine (5-Aza-dC) induced DLC-1 mRNA expression in the gastric cancer cell lines that have the methylated alleles. Studies using SNU-601 cell line with methylated DLC-1 alleles revealed that nearly all CpG sites within DLC-1 CpG island were methylated, and that the in vitro methylation of the DLC-1 promoter region is enough to repress DLC-1 mRNA expression, regardless of the presence of transcription factors capable of inducing this gene. In all, 29 of 97 (30%) primary gastric cancers were also shown to be methylated, demonstrating that methylation of the DLC-1 CpG island is not uncommon in gastric cancer. In addition, we demonstrated that DLC-1 mRNA expression was induced, and an increase in the level of acetylated H3 and H4 was detected by the treatment with trichostatin A (TSA) in two DLC-1 nonexpressing cell lines that have the unmethylated alleles. Taken together, the results of our study suggest that the transcriptional silencing of DLC-1, by epigenetic mechanism, may be involved in gastric carcinogenesis.

p62 is a multifunctional cytoplasmic protein able to noncovalently bind ubiquitin and several signaling proteins, suggesting a regulatory role connected to the ubiquitin-proteasome pathway. No studies to date have linked p62 protein expression with pathological states. Here we demonstrate the overabundance of p62 protein in malignant breast tissue relative to normal breast tissue. The proteasome inhibitor PSI increased p62 mRNA and protein; however, PSI treatment of breast epithelial cells transfected with the p62 promoter did not affect promoter activity. High levels of prostate-derived Ets factor (PDEF) mRNA have been identified in breast cancer compared to normal breast. Only the PSA and maspin promoters have been identified as targets of this transcription factor. Here we show that PDEF stimulates the p62 promoter through at least two sites, and likely acts as a coactivator. PSI treatment abrogates the PDEF-stimulated increase of p62 promoter activity by 50%. Thus, multiple mechanisms for the induction of p62 exist. We conclude that (1) p62 protein is overexpressed in breast cancer; (2) p62 mRNA and protein increase in response to PSI, with no change of basal promoter activity; (3) PDEF upregulates p62 promoter activity through at least two sites; and (4) PSI downregulates PDEF-induced p62 promoter activation through one of these sites.

DNA amplifications are important mechanisms for proto-oncogene activation. Comparative genomic hybridization (CGH) to metaphase chromosome preparations has revealed amplifications in 10-20% of B-cell lymphomas (B-NHL). We analysed a series of 16 aggressive non-Hodgkin lymphomas by the new approach termed Matrix-CGH (M-CGH) using genomic DNA microarrays as hybridization target. For M-CGH, a dedicated B-cell lymphoma chip was constructed containing 496 genomic targets covering oncogenes, tumor suppressor genes as well as chromosome regions frequently altered in B-NHL. In 10 of 16 samples a total of 15 DNA amplifications were identified. The amplicons included BCL2, REL, CCND1, CCND2, JAK2, FGF4 and MDM2. Four of the 15 amplifications remained undetected by chromosomal CGH. The respective amplicons mapped to bands 2p13, 9p13-p21 and 12q24 and, were confirmed by fluorescence in situ hybridization. Furthermore, for four genomically amplified genes real-time quantitative reverse transcription polymerase chain reaction revealed elevated mRNA expression levels. These data show the superior diagnostic sensitivity of the newly developed diagnostic tool. As only a small portion of the genome (approximately 1.5%) has been analysed by the present DNA array, it is likely that gene amplifications are much more common in aggressive lymphomas than previously assumed.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a compound found in cooked meat, is a mammary gland carcinogen in rats. Comparative genomic hybridization of PhIP-induced rat mammary gland carcinomas revealed loss in the centromeric region of 2q, a region known to carry the mammary carcinoma susceptibility 1 (Mcs1) gene and several other genes relevant to carcinogenesis. Allelic imbalance, specifically microsatellite instability and loss of heterozygosity, was examined in mammary gland carcinomas induced by PhIP in Sprague-Dawley (SD)xWistar Furth F1 hybrid rats. In a polymerase chain reaction (PCR)-based assay with 34 microsatellite markers coinciding to 2q11-2q16, nine markers revealed allelic imbalance. The frequency of imbalance in the tumors varied from 10 to 100% depending on the specific marker. However, none of the markers coinciding with the Mcs1 gene locus showed allelic imbalance, suggesting that alterations at this locus were not associated with PhIP-induced rat mammary gland cancer. The expression of several genes physically mapped to 2q11-2q16 and potentially involved in carcinogenesis including Ccnb (cyclin B1), Ccnh (cyclin H), Rasa (Ras GAP), Rasgrf2, Pi3kr1 (p85alpha), and Il6st (gp130) was also examined by quantitative real-time PCR and immunohistochemistry (IHC) across a large bank of PhIP-induced SD rat mammary gland carcinomas. By quantitative real-time PCR, the mRNA expression of Rasa, Pi3kr1, Ccnh, and Il6st in carcinomas was, respectively, 22-, 20-, three- and threefold higher in carcinomas than in control mammary gland tissues (P<0.05, Student's t-test). A statistically sixfold lower expression of Rasgrf2 was detected in carcinomas whereas no significant change in Ccnb1 expression was observed. The findings from quantitative real-time PCR were confirmed by IHC for each gene. In addition, the proliferation index in mammary gland carcinomas as assessed by PCNA was found to correlate with the overexpression of Cyclin H by IHC analysis (P<0.05, Spearman Rank Order Correlation). The findings from the current study implicate molecular alterations in the proximal region of 2q in PhIP-induced rat mammary gland carcinomas.

Primary effusion lymphoma (PEL) is a lymphoproliferative disease of B-cell origin that is associated with HHV-8 infection. PEL cells harbor a non-B, non-T phenotype and lack significant surface immunoglobulin (Ig) expression, a characteristic that has not been fully explained. In the present study, we demonstrate that PEL cells constitutively express interferon regulatory factor (IRF)-4, a transcription factor that regulates the activity of the immunoglobulin light-chain enhancer elements lambdaB and kappaE3' through binding to a composite Ets-IRF site. IRF-4 activity requires its physical interaction with PU.1, an Ets family member involved in the activation of genes essential for B-cell development. However, in PEL-derived B-cell lines, PU.1 expression was completely abrogated; expression of the B cell specific transcription factor Oct-2, which is known to regulate PU.1 expression, was also abolished. Moreover, the B-cell-specific coactivator of octamer factors, BOB-1/OcaB, was expressed at very decreased levels in PEL cells. Ectopic expression of Oct-2 was able to fully restore PU.1 promoter activity in the PEL cell line BCBL-1, while PU.1 expression also reconstituted the activity of the lambdaB Ets-IRF site. In addition, protein levels of BSAP/Pax-5 and IRF-8/ICSBP were undetectable in PEL cells. The pattern of transcription factor ablation observed in PEL was found to be comparable to that observed in classical Hodgkin's disease-derived cell lines, which also lack B-cell-specific surface markers. These observations indicate that disruption of the B-cell-specific transcriptional program is likely to contribute to the incomplete B-cell phenotype characteristic of PEL cells.

The membrane type 1 matrix metalloproteinase (MT1-MMP) has been identified as a major activator of MMP-2 - a process involving the formation of a trimolecular complex with TIMP-2. We previously identified the IGF-I receptor as a positive regulator of MMP-2 synthesis. Here, we investigated the role of IGF-IR in the regulation of MT1-MMP. Highly invasive Lewis lung carcinoma subline H-59 cells express MT1-MMP and utilize it to activate their major extracellular matrix degrading proteinase-MMP-2. These cells were transiently transfected with a plasmid vector expressing a luciferase reporter gene downstream of the mouse MT1-MMP promoter. IGF-I treatment increased luciferase activity in the transfected cells by up to 10-fold and augmented endogenous MT1-MMP mRNA and protein synthesis by up to 2-3-fold, relative to controls. MT1-MMP induction and invasion were blocked by the PI 3-kinase inhibitors LY294002 and wortmannin and by rapamycin, but not by the MEK inhibitor PD98059. Overexpression of a dominant negative Akt mutant or of the tumor suppressor phosphatase and tensin homologue, PTEN, in these cells also caused a significant reduction in MT1-MMP expression and invasion. The results demonstrate that IGF-IR controls tumor cell invasion by coordinately regulating MMP-2 expression and its MT1-MMP-mediated activation and identify PI 3-kinase/Akt/mTOR signaling as critical to this regulation.

Pituitary tumor-transforming gene (pttg) is a distinct proto-oncogene which is expressed in certain normal tissues with high proliferation rate and in a variety of tumors. PTTG is the vertebrate analog of yeast securins Pds1 and Cut2 with a key role in the regulation of sister chromatid separation during mitosis. Impairment of PTTG regulated functions is expected to lead to chromosomal instability and aneuploidy. Human pttg (hpttg) is abundantly expressed in Jurkat T lymphoblastic lymphoma cells but not in normal peripheral blood leukocytes. To obtain additional data on the potential role of hpttg in lymphomagenesis we selected 150 cases of lymphoid tumors for the assessment of hpttg expression in tumor tissues. Immunohistochemical studies on formalin-fixed, paraffin-embedded tissues revealed hPTTG in 38.8% of B-cell lymphomas, 70.2% of T-cell lymphomas, and 73.1% of Hodgkin's lymphomas. Among B-cell lymphomas, the most frequently immunostained tumors were plasma cell tumors, diffuse large cell lymphomas, and follicle center cell lymphomas. In Hodgkin's disease, immunoreactivity was mainly noted in Reed-Sternberg cells. In conclusion, the frequent overexpression of hpttg in many histological subtypes of lymphoma suggests the involvement of this proto-oncogene in lymphomagenesis.

Stat3 mediates cellular responses associated with proliferation, survival and differentiation, but the mechanisms underlying the diverse effects of this signaling molecule remain unknown. M1 mouse myeloid leukemia cells arrest growth and differentiate into macrophages following treatment with interleukin 6 (IL-6) or leukemia inhibitory factor (LIF), and recent studies have shown that Stat3 plays a central role in this process. Utilizing representational difference analysis, we demonstrate that expression of the mouse BATF gene is upregulated as an early response to IL-6/LIF stimulation and Stat3 activation in this cell system. Immunoblots using antibodies to BATF detected an increase in BATF protein in response to LIF/IL-6 stimulation. BATF is a member of the AP-1 family of basic leucine zipper transcription factors and functions to inhibit the transcriptional and biological functions of AP-1 activity in mammalian cells. BATF forms complexes with c-Jun in M1 cells and forced expression of BATF in the absence of Stat3 signaling results in a reduced rate of cellular growth. These results indicate that Stat3 mediates cellular growth by modulating AP-1 activity through the induction of BATF.

Functional inactivation of tumor susceptibility gene tsg101 leads to cellular transformation and tumorigenesis in mice. While human TSG101 is located in a region where frequent loss of heterozygosity can be detected in a variety of cancers, no genomic deletion in TSG101 gene has been reported, casting a doubt on the role of TSG101 as a classical tumor suppressor. Some studies have revealed that TSG101 is a frequent target of splicing defects, which correlate with cellular stress and p53 status. Furthermore, recent reports have identified TSG101 as a part of the MDM2/p53 regulatory circuitry, a well-recognized circuitry that upon deregulation results in tumorigenesis. Interestingly, overexpression of tsg101 from an adventitious promoter also leads to neoplastic transformation. On the basis of this information, we have analysed TSG101 gene expression in 20 human papillary thyroid carcinomas (PTCs) by immunohistochemistry and demonstrated that the overexpression of TSG101 protein is closely associated with human PTCs. Further sequence analysis reveals no mutation in cDNA region encoding steadiness box in these PTC specimens, indicating that the upregulation of TSG101 protein is not caused by the alteration of this region. In situ hybridization analysis confirms that overexpression of TSG101 also occurs at the transcriptional level. In addition, semi-quantitative RT-PCR and subsequent Southern hybridization verify that the amounts of TSG101 transcripts are indeed lower in three normal thyroid tissues than in PTC specimens. Here we report the upregulation of TSG101 expression in PTC cells, providing the first evidence of the association of TSG101 overexpression with human tumors and suggesting that upregulation of TSG101 steady-state level might play a role in mediating tumorigenesis of human PTC.

Promoter hypermethylation is an important means for the transcriptional repression of a number of cancer-associated genes. However, the underlying mechanism of this aberration in cancer remains unclear. Here, we examined 5' CpG island methylation status and expression of the p14(ARF), p16(INK4a) and RASSF1A tumor suppressor genes, and investigated the relationship of these factors with the mRNA expression of DNA methyltransferases (DNMTs) and/or methyl-CpG-binding proteins (MBPs) in 30 lung cancer cell lines including 12 small cell lung cancers (SCLCs) and 18 non-small cell lung cancers (NSCLCs). When beta-actin was used as an internal control, the mRNA expression of three DNMTs (DNMT1, DNMT3A, and DNMT3B) and five MBPs (MBD1, MBD2, MBD3, MBD4, and MeCP2) was upregulated in SCLC, while only that of DNMT1, DNMT3B and MBD3 was upregulated in NSCLC, compared with normal lung tissues. However, when normalized using proliferating cell nuclear antigen (PCNA) as an internal control, these differences disappeared or diminished; there was even a significant reduction in the expression ratios of DNMT1, MBD2 and MeCP2 in SCLC and DNMT1, MBD2 and MBD4 in NSCLC. Furthermore, although significant correlations between PCNA expression and mRNA expression levels of the three DNMTs and four of the MBPs (excluding MeCP2) were observed, there was no obvious correlation between promoter hypermethylation of these tumor suppressor genes and the expression level of any of the DNMTs or MBPs. Our results suggest that upregulation of DNMTs and MBPs probably reflects an increased cell proliferation in human lung cancers and that there are likely to exist gene-specific mechanisms for epigenetic gene silencing.

We have identified a gene that is highly expressed in the androgen-dependent prostate cancer cell line, LNCaP. Sequence analysis revealed that it was identical to a recently cloned gene designated TMEFF2, which encodes a transmembrane protein containing an epidermal growth factor (EGF)-like motif and two follistatin domains. This gene was highly expressed only in primary samples of normal prostate and prostate cancer as well as normal brain. Expression of the gene was controlled by androgen as shown by dihydrotestosterone markedly increasing TMEFF2 expression in LNCaP cells. Also, androgen-dependent human prostate cancer xenografts (CWR22) expressed high levels of TMEFF2 and these levels markedly decreased by day 10 after castration of the mice. Furthermore, a large number of androgen-dependent xenografts (CWR22, LuCaP-35, LAPC-4AD, LAPC-9AD) exhibited higher levels of TMEFF2 mRNA than androgen-independent xenografts (CWR22R, LAPC-3AI, LAPC-4AI, LAPC-9AI). Ectopic expression of TMEFF2 in DU145 and PC3 cells resulted in their prominent inhibition of growth. Taken together, the results demonstrate that TMEFF2 is a androgen-regulated gene, which can suppress growth of prostate cancer cells and our xenograft data show that escape of prostate cancer cells from androgen modulation causes them to decrease their expression of this gene, which may result in their more malignant behavior.

The relation between thyroid status and diseases and cancer is unclear. No detailed analysis of thyroid hormone receptor (TR) expression in human breast cancer has been reported. We have analysed the expression and mutational status of the TRalpha1, encoded by the c-erbA proto-oncogene, TRbeta1 and TRbeta2 isoforms in 70 sporadic breast cancers. Alterations in the RNA level of TRbeta1, TRalpha1, or both were found in a number of patients. No expression of TRbeta2 RNA was detected. Western blotting analysis confirmed the differences in expression at the protein level in those cases where sufficient tumor sample was available. Additionally, tumor-specific truncated TRbeta1 RNA was found in six patients. Strikingly, three transcripts shared the same breakpoint. Only one tumor carried the corresponding deletion at the genomic DNA level, suggesting that the remaining abnormal TRbeta1 transcripts are aberrant splicing products. Though no significant correlation was found between TRbeta1 alteration and any clinical parameter, it showed a tendency to associate with early age of onset (<50 years). Our results reveal specific alterations in the expression of TRbeta and TRalpha genes in a subset of breast cancer patients, suggesting that deregulation of thyroid hormone target genes may be involved in the generation of this neoplasia.

Angiogenesis is an indispensable prerequisite for the progression and metastasis of solid malignancies. Tumor angiogenesis appears to be governed by alterations of tumor suppressor or oncogenes operant in a broad range of tumors. We have addressed this issue in neuroblastoma, a malignancy characterized by the near-exclusive amplification and overexpression of the N-Myc oncogene. Here, we report that N-Myc overexpression results in down-regulation of interleukin-6 (IL-6) and that IL-6 is an inhibitor of endothelial cell proliferation and VEGF-induced rabbit corneal angiogenesis. STAT3 is instrumental for IL-6 activity as infection with adenoviruses expressing a phosphorylation deficient STAT3 mutant renders endothelial cells insensitive to the antiproliferative action of IL-6. Finally, though IL-6 does not influence neuroblastoma cell growth, IL-6-expressing xenograft tumors in mice exhibit reduced neovascularization and suppressed growth. Our data shed new light on the mechanisms by which N-myc oncogene amplification enhances the malignant phenotype in neuroblastomas.

Tamoxifen is widely applied as an antiestrogenic agent for adjuvant therapy in the treatment of breast cancer, while its estrogen-agonistic activity occasionally causes proliferative disorders or carcinogenesis at other sites, such as the uterus. We reported that estrogen activates telomerase in breast and endometrial cancer cells. The present study examines the effects of tamoxifen on the gene expression of human telomerase reverse transcriptase (hTERT) in breast and endometrial cancer cells. Tamoxifen inhibited the cell growth of MCF-7 cells, as well as hTERT mRNA expression in the presence of estrogen (E2), antagonizing the E2 effects. In contrast, tamoxifen stimulated the growth of Ishikawa cells and activated hTERT mRNA expression in the absence or presence of E2, exhibiting estrogen-agonistic action. Transient expression assays revealed that these actions of tamoxifen are achieved by transcriptional regulation of the hTERT promoter. An estrogen responsive element (ERE) in the hTERT 5' regulatory region was partly responsible for both the E2-antagonistic and -agonistic actions of tamoxifen. Tamoxifen activated the MAP kinase cascade in Ishikawa cells, but not in MCF-7 cells, and the activation of hTERT mRNA expression was effectively blocked by MEK inhibitor, suggesting that the MAP kinase pathway is involved in the tamoxifen-induced activation of hTERT. These findings indicate that tamoxifen regulates hTERT expression in a cell-type specific manner. Tamoxifen-induced activation of hTERT may be one component of estrogen agonistic function of tamoxifen that is involved in endometrial carcinogenesis induced by this agent.

Apo2L/TRAIL exhibits enhanced apoptotic activity in tumor xenograft models when used in combination with the topoisomerase 1 inhibitor CPT-11. To investigate the cellular mechanisms involved in this increased tumor-killing activity, a series of in vitro experiments were conducted using the human colon carcinoma cell line (HCT116). Apo2L/TRAIL induced a transient upregulation of DR5 mRNA, while CPT-11 increased both death and decoy receptor expression. Upregulation of decoy receptors by CPT-11 was partially inhibited by co-administration of Apo2L/TRAIL. CPT-11 treatment resulted in accumulation of cells at G(2)M-phase and correlated with a substantial increase in the protein levels of the cyclin-dependent kinase inhibitor p21. However, cells co-treated with CPT-11 and Apo2L/TRAIL, or pretreated with CPT-11 for up to 24 h followed by 2 h Apo2L/TRAIL, resulted in a caspase-dependent degradation of p21, reversal of G(2)-M phase arrest with a concomitant increase in apoptosis. The sequential treatment produced the greatest induction of DR5 and DR4, caspase-3-like cleavage/activation and p21 degradation, as well as increased apoptosis. These data indicate that the up-regulation of Apo2L/TRAIL ligand and its death receptors as well as cleavage of p21 protein in the Apo2L/TRAIL plus CPT-11 treatment contributes to the positive cooperation between these agents in enhancing tumor cell apoptosis.

A central role for polo-like kinases (PLK) in regulating several stages of mitotic progression has been born out in several species. Overexpression of PLK1 is observed in the majority of hitherto analysed human tumors. PLK1 overexpression is a negative prognostic factor in patients suffering from non-small cell lung cancer, head and neck tumors, esophageal carcinomas and melanomas. In order to define the role of PLK1 for mitotic progression of human cells and for neoplastic cell growth, phosphorothioate antisense oligonucleotides (ASOs) were tested to selectively downregulate PLK1 expression in MDA-MB-435 (breast cancer), HeLa S3 (cervical carcinoma) and A549 (non-small cell lung cancer) cells. ASOs were identified which suppress PLK1 mRNA and protein in a dose-dependent and sequence-specific manner. This approach also led to reduced PLK1 serine/threonine kinase activity. Downregulation of cellular PLK1 levels in cancer cells altered cell cycle progression moderately with an elevated percentage (20-30%) of cells in G(2)/M. Furthermore, cells with reduced PLK1 protein gained a rounded phenotype with multiple centrosomes. Moreover, ASO treatment resulted in potent antiproliferative effects in cell culture. Considerable antitumor activity was observed in vivo against A549 cells. This study suggests that antisense inhibitors targeted against PLK1 at well tolerated doses may be considered as a cancer therapeutic agent.

Although the prognosis of Hodgkin's disease is relatively good, around 20% of patients do not benefit from current therapies and succumb to their disease. A large-scale molecular characterization of disease might help improve HD management. Using cDNA arrays, we studied the mRNA expression levels of approximately 1000 selected genes in 34 benign and malignant lymphoid samples including 21 classical Hodgkin's disease (HD) tissue samples. Hierarchical clustering identified three main molecular groups of HD tumours relevant with respect to histology and clinical outcome (response to therapy and survival). Samples from all bad outcome HD (BOHD) patients clustered in one group whereas the two other groups contained most good outcome HD (GOHD) cases. The nodular sclerosis GOHD samples overexpressed genes involved in apoptotic induction and cell signalling, including cytokines, while the BOHD samples were characterized by the upregulation of genes involved in fibroblast activation, angiogenesis, extracellular matrix remodelling, cell proliferation, and the downregulation of tumour suppressor genes. Our results establish a molecular taxonomy of HD correlating with response to therapy and clinical outcome, thereby suggesting the possibility of improving the current prognostic classification.

Interleukin-6 (IL-6) is a major survival and proliferation factor of human malignant plasma cells and IL-6 dependent myeloma cell lines can be obtained from patients with terminal disease. We show here that mutated diphtheria toxin, a specific inhibitor of heparin-binding epidermal growth factor-like growth factor (HB-EGF), blocked the IL-6-induced growth of two myeloma cell lines (XG-1 and XG-14) and did not significantly affect that of two other cell lines (XG-6 and XG-13). The IL-6 mediated growth of myeloma cells was also inhibited by antibodies to ErbB1, a receptor for HB-EGF. The XG-1 and XG-14 cell lines that are sensitive to HB-EGF inhibitors overexpressed HB-EGF and EGF receptor (ErbB1) genes. They also overexpressed CD9, a tetraspanin that binds to the heparin-binding domain of HB-EGF and is critical for promoting ErbB1 activation by HB-EGF. The XG-6 and XG-13 myeloma cells that were not significantly sensitive to HB-EGF antagonists, poorly expressed HB-EGF, ErbB1 and CD9 genes or proteins. We demonstrated that recombinant HB-EGF supported the long-term growth of myeloma cells, as did IL-6. The myeloma cell growth factor activity of HB-EGF was completely inhited by antibodies to ErbB1, but also by antibodies to gp130 IL-6 transducer or to IL-6. These data indicate that in the XG-1 and XG-14 IL-6-dependent myeloma cell lines, the CD9/HB-EGF/erbB1 and the IL-6/IL-6R/gp130 pathways cooperate synergistically to trigger myeloma cell growth. They suggest that inhibitors of the EGF receptor or HB-EGF may be useful for inducing myeloma cell apoptosis in patients with multiple myeloma.

The chromosomal region 12q13-q15 is recurrently amplified in bladder cancer. Putative target genes located in this region include MDM2, CDK4, and GLI. To evaluate the involvement of these genes in bladder cancer, we screened a tissue microarray (TMA) containing 2317 samples by fluorescence in situ hybridization (FISH). Amplification was found for MDM2 in 5.1%, for CDK4 in 1.1%, and for GLI in 0.4% of interpretable tumors. Among tumors having amplification of at least one of these 12q13-q15 genes, 76.6% had amplification of MDM2 alone and 6.4% had amplification of CDK4 alone. Coamplifications were seen of MDM2 and CDK4 in 10.6%, and of CDK4 and GLI in 6.4%. Neither coamplifications of all three genes nor isolated GLI amplifications were found. These data suggest a prominent role of MDM2 as a 12q13-q15 amplification target in bladder cancer. However, independent CDK4 amplifications do also occur suggesting either two non-overlapping amplification sites or else a minimal overlapping region between MDM2 and CDK4 perhaps containing another yet unknown oncogene. The frequency of amplification increased significantly from stage pTa to pT1-4 (P<0.04) and from low to high grade (P<0.005). These data are consistent with a high level of genetic instability in invasively growing and high-grade bladder tumors.

Resistance of tumors to cytotoxic therapy may be due to disrupted apoptosis programs and remains a major obstacle in cancer treatment. Here, we report that IFNgamma sensitizes resistant tumor cells with absent or low caspase-8 expression for apoptosis induced by death-inducing ligands or cytotoxic drugs by upregulating caspase-8 through a Stat1/IRF1 dependent pathway. Combined treatment using IFNgamma with TRAIL, APO1, TNFalpha or cytotoxic drugs cooperated to trigger apoptosis in various resistant tumor cell lines derived from Ewing tumor, neuroblastoma or medulloblastoma, while single agents exerted only a minimal effect. Importantly, IFNgamma induced caspase-8 expression also in cells with inactivation of the caspase-8 gene by hypermethylation, although no direct effect of IFNgamma on the methylation status of regulatory sequences of the caspase-8 gene was found. IFNgamma-mediated facilitation of apoptosis was inhibited by the caspase-8 specific inhibitor zIETD.fmk or in caspase-8 mutant Jurkat cells implying a prominent role of caspase-8 in mediating sensitization by IFNgamma. Upregulation of caspase-8 and sensitization for apoptosis by IFNgamma was blocked by overexpression of dominant-negative mutants of Stat1 or in Stat1-deficient U3A cells, while complementation of Stat1-deficient U3A cells with wild-type Stat1 restored the IFNgamma effect. Moreover, ectopic expression of IRF1 induced caspase-8 expression thereby sensitizing cells for TRAIL-, APO1- or doxorubicin-induced apoptosis. These findings provide evidence that the Stat1/IRF1 pathway is involved in induction of caspase-8 expression and apoptosis initiated by IFNgamma and indicate that IFNgamma might be an effective strategy to sensitize various resistant tumor cells with deficient caspase-8 expression for chemotherapy- or death receptor-induced apoptosis.

The CHFR gene, which was recently cloned by Scolnick and Halazonetis in search for a novel mitotic checkpoint gene with fork-head association motifs, has been suggested to play a key role in the mitotic prophase checkpoint. In this study, we demonstrated tumor-specific aberrant hypermethylation of the promoter region of the CHFR gene in a significant fraction of lung cancers in association with loss of detectable levels of CHFR transcripts. Aberrant hypermethylation was observed in seven of 37 primary lung cancer cases. Treatment with the demethylating agent 5-aza-2'-deoxycytidine restored expression of the CHFR gene in lung cancer cell lines exhibiting aberrant hypermethylation and loss of its expression. In contrast, genetic alterations were found to be infrequent in lung cancers. This is the first description of aberrant hypermethylation of the CHFR gene in any type of human cancer, and provides further evidence of the involvement of multiple checkpoint alterations in lung cancer.

One isoform of the 14-3-3 family, 14-3-3sigma, plays a crucial role in the G2 checkpoint by sequestering Cdc2-cyclinB1 in the cytoplasm, and the expression of 14-3-3sigma is frequently lost in breast cancers. This loss of expression is thought to cause a G2 checkpoint defect, resulting in chromosomal aberrations. Since lung cancers frequently carry numerous chromosomal aberrations, we examined the DNA methylation status and expression level of the 14-3-3sigma gene in 37 lung cancer cell lines and 30 primary lung tumor specimens. We found that small cell lung cancer (SCLC) cell lines frequently showed DNA hypermethylation (9 of 13 lines, 69%), and subsequent silencing of the 14-3-3sigma gene. Among non-small cell lung cancers (NSCLC), large cell lung cancer cell lines showed frequent hypermethylation and silencing of 14-3-3sigma (4 or 7 lines, 57%). In contrast, in other NSCLC cell lines, hypermethylation occurred very rarely (1 of 17 lines, 6%). All eight primary SCLC specimens examined also showed a loss or significant reduction in 14-3-3sigma expression in vivo, while a loss or reduction of 14-3-3sigma expression was very rare in primary NSCLC specimens (1 of 22 tissues, 5%). This is the first description that indicates lung cancers frequently show significant inactivation of the 14-3-3sigma gene mainly due to DNA hypermethylation in SCLC, but rarely in NSCLC, suggesting involvement of the 14-3-3sigma gene in lung tumorigenesis in a histological type-specific manner.

NF-kappaB transcription factor is activated upon ubiquitination and subsequent proteolysis of its inhibitor IkappaB. The phosphorylation-dependent ubiquitination is mediated by SCF E3 ubiquitin ligase. In this study, we identified a novel murine F-box/WD40 repeat-containing protein, mHOS (a homologue of HOS/betaTrCP2). mHOS efficiently binds Skp1 protein (a 'core' component of SCF ubiquitin ligase), and phosphorylated IkappaB(alpha). We found that mHOS associates with SCF-ROC1 E3 ubiquitin ligase activity. We have also observed that mHOS is overexpressed in chemically-induced mouse skin tumors, and its overexpression (but not accelerated IkappaB phosphorylation) coincides with the accelerated degradation of IkappaB in vivo. The role of mHOS in the constitutive activation of NF-kappaB in skin carcinogenesis is discussed.

This study examined the role of TGF-beta1 in human keratinocyte malignancy. Two carcinoma-derived human oral keratinocyte cell lines, BICR 31 and H314, were selected on the basis of their known resistance to TGF-beta1-induced G(1) arrest, the presence of wild type TGF-beta cell surface receptors and normal Ras. Smad 4 protein was undetectable in both cell lines, but Smad 2 and Smad 3 were expressed at levels comparable with a fully TGF-beta responsive cell line, and treatment of the cells with TGF-beta1 resulted in the phosphorylation of Smad 2. Treatment with exogenous TGF-beta1 resulted in a failure to induce transcription from an artificial Smad-dependent promoter and a failure to down-regulate c-myc, but resulted in an up-regulation of AP-1 associated genes (Fra-1, JunB and fibronectin). Transient transfection of Smad 4 into BICR 31 restored TGF-beta1-induced growth inhibition and Smad-dependent transcriptional activation. Protracted treatment of cells with exogenous TGF-beta1 resulted in the attenuation of cell growth in vitro. To over-express TGF-beta1, both cell lines were transfected with latent TGF-beta1 cDNA; neutralization studies of conditioned media demonstrated that whilst the majority of the peptide was in the latent form, a small proportion was present as the active peptide. Cells that over-expressed endogenous TGF-beta1 grew more slowly in vitro compared to both the vector-only controls and cells that did not over-express the peptide. Orthotopic transplantation of cells that over-expressed endogenous TGF-beta1 to the floor of the mouth in athymic mice resulted in marked inhibition of primary tumor formation compared to controls. Expression of a dominant-negative TGF-beta type II receptor in cells that over-expressed endogenous TGF-beta1 resulted in enhanced cell growth in vitro and diminished the tumor suppressor effect of the ligand in vivo, indicating that the endogenous TGF-beta1 was acting in an autocrine capacity. The results demonstrate that over-expression of endogenous TGF-beta1 in human malignant oral keratinocytes leads to growth inhibition in vivo and tumor suppression in vitro by mechanisms that are independent of Smad 4 expression and TGF-beta1-induced G(1) arrest.

Fibulin-1 is an extracellular matrix protein induced by estradiol in estrogen receptor (ER) positive ovarian cancer cell lines. Alternative splicing of fibulin-1 mRNA results in four different variants named A, B, C and D that may have distinct biological functions. We studied the relative expression of fibulin-1 mRNA variants and their estrogen regulation in human ovarian cancer cells. In ovarian tissues and cancer cell lines, fibulin-1C and -1D are the predominant forms, whereas fibulin-1A and -1B are weakly expressed. We developed a competitive PCR assay based on coamplification of fibulin-1C and -1D to study the relative expression of these fibulin-1 variants in human ovarian samples. In ovarian cancer cell lines and ovarian cancer samples, there was a marked increase in the fibulin-1C:1D and fibulin-1C:HPRT mRNA ratios as compared to normal ovaries. In the BG1 estrogen receptor positive ovarian cancer cell line, fibulin-1C mRNA was induced by estradiol in a dose- and time-dependent manner. Since others and we have previously shown an increased expression of ERalpha as compared to ERbeta in ovarian cancer cells, we investigated whether ERalpha or ERbeta is involved in this induction. For this aim, MDA-MB-231 breast cancer cell line, which expresses both low basal levels of ERs and fibulin-1, was infected with recombinant ERalpha or ERbeta encoding adenovirus and treated with estradiol. Fibulin-1C was induced by estradiol in ERalpha- but not ERbeta-infected cells, suggesting that fibulin-1C induction is mediated through ERalpha. In ovarian tumors, a trend towards a correlation between fibulin-1C and ERalpha expression levels was noted. In conclusion, this study showed an increased fibulin-1C:-1D mRNA ratio in ovarian cancer cells as compared to normal ovaries. This finding suggests that the C variant may be involved in ovarian carcinogenesis. Fibulin-1C overexpression may thus be a clue for the understanding of a putative role of estrogens in ERalpha promoted ovarian tumor progression.