MicroRNAs (miRNA) represent a novel class of genes that function as negative regulators of gene expression. Recently, miRNAs have been implicated in several cancers. However, aberrant miRNA expression and its clinicopathologic significance in human ovarian cancer have not been well documented. Here, we show that several miRNAs are altered in human ovarian cancer, with the most significantly deregulated miRNAs being miR-214, miR-199a*, miR-200a, miR-100, miR125b, and let-7 cluster. Further, we show the frequent deregulation of miR-

Transforming growth factor ␤ (TGF-␤) signaling facilitates metastasis in advanced malignancy. While a number of protein-encoding genes are known to be involved in this process, information on the role of microRNAs (miRNAs) in TGF-␤-induced cell migration and invasion is still limited. By hybridizing a 515-miRNA oligonucleotide-based microarray library, a total of 28 miRNAs were found to be significantly deregulated in TGF-␤-treated normal murine mammary gland (NMuMG) epithelial cells but not Smad4 knockdown NMuMG cells. Among upregulated miRNAs, miR-155 was the most significantly elevated miRNA. TGF-␤ induces miR-155 expression and promoter activity through Smad4. The knockdown of miR-155 suppressed TGF-␤-induced epithelial-mesenchymal transition (EMT) and tight junction dissolution, as well as cell migration and invasion. Further, the ectopic expression of miR-155 reduced RhoA protein and disrupted tight junction formation. Reintroducing RhoA cDNA without the 3 untranslated region largely reversed the phenotype induced by miR-155 and TGF-␤. In addition, elevated levels of miR-155 were frequently detected in invasive breast cancer tissues. These data suggest that miR-155 may play an important role in TGF-␤-induced EMT and cell migration and invasion by targeting RhoA and indicate that it is a potential therapeutic target for breast cancer intervention.Metastasis accounts for the majority of deaths of cancer patients, and thus, it is crucial to understand the molecular and cellular mechanisms that cause primary tumors to metastasize. The most critical step in the conversion of primary tumors to metastases is attributed to the process known as epithelialmesenchymal transition (EMT). EMT is a remarkable example of cellular plasticity that involves the dissolution of epithelial tight junctions, the intonation of adherens junctions, the remodeling of the cytoskeleton, and the loss of apical-basal polarity (49, 55). In cells undergoing EMT, the loss of epithelial cell adhesion and cytoskeletal components is coordinated with a gain of mesenchymal components and the initiation of a migratory phenotype.Transforming growth factor ␤ (TGF-␤) has emerged as a key regulator of EMT in late-stage carcinomas, where it promotes invasion and metastasis (54). TGF-␤ binds to a heteromeric complex of transmembrane serine/threonine kinases, the type I and II TGF-␤ receptors (T␤RI and T␤RII). Following ligand binding to T␤RII, the type I receptor is recruited to the ligand-receptor complex, where the constitutively active T␤RII transactivates T␤RI. Activated T␤RI phosphorylates the receptor-specific Smad2 and Smad3. Phosphorylated Smad2/ Smad3 associates with Smad4 as a heteromeric complex and translocates to the nucleus. This complex binds directly to Smad-binding elements and associates with a plethora of transcription factors, coactivators or corepressors, thus leading to the transcriptional induction or repression of a diverse array of genes (54). A number of genes that are associated with tumor growth and metastasis have been show...

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MicroRNA-155 (miR-155) is frequently up-regulated in various types of human cancer; however, its role in cancer angiogenesis remains unknown. Here, we demonstrate the role of miR-155 in angiogenesis through targeting von Hippel-Lindau tumour suppressor (VHL) in breast cancer. Ectopic expression of miR-155 induced whereas knockdown of miR-155 inhibited HUVEC network formation, proliferation, invasion, and migration. Furthermore, mammary fat pad xenotransplantation of ectopically expressed miR-155 resulted in extensive angiogenesis, proliferation, tumour necrosis, and recruitment of pro-inflammatory cells such as tumour associated macrophages. Expression of VHL abrogated these miR-155 effects. Moreover, miR-155 expression inversely correlates with VHL expression level and is associated with late stage, lymph node metastasis, and poor prognosis as well as triple-negative tumour in breast cancer. These findings indicate that miR-155 plays a pivotal role in tumour angiogenesis by downregulation of VHL, and provide a basis for miR-155-expressing tumours to embody an aggressive malignant phenotype, and therefore, miR-155 is an important therapeutic target in breast cancer.

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Estrogen receptor ␣ (ER␣)3 is an important marker for prognosis and is predictive of response to endocrine therapy in patients with breast cancer. Although the majority of primary breast cancers are ER␣-positive and respond to antiestrogen therapy, up to one-third of patients with breast cancer lack ER␣ at the time of diagnosis, and a fraction of breast cancers that are initially ER␣-positive lose ER␣ expression during tumor progression (1). These patients fail to respond to antiestrogen therapy and have a poor prognosis. Previous studies have shown that ER␣ absence is a result of hypermethylation of CpG islands in the 5Ј-regulatory regions of ER␣ in a fraction of breast cancers (1). However, the molecular mechanism of the rest of the ER␣-negative cases and the molecule(s) involving ER␣ hypermethylation remain largely unknown (1).MicroRNAs (miRNAs) are a new class of small (ϳ22 nucleotide) noncoding RNAs and negatively regulate protein-coding gene expression by targeting mRNA degradation or translation inhibition (2-5). Frequent deregulation of miRNAs has been detected in breast cancer, and some are associated with breast cancer metastasis and poor prognosis, suggesting an important role of miRNAs in breast oncogenesis and cancer progression (6 -9). In this study, we performed miRNA profiling in ER␣-negative versus ER␣-positive human breast cancer cell lines and primary tumors and identified the deregulation of a panel of miRNAs in ER␣-negative breast cancer. Of the elevated miRNAs, miR-221 and miR-222 were found to directly regulate ER␣ expression by interaction with the 3Ј-untranslated region (3Ј-UTR) of ER␣. Ectopic expression of miR-221 and/or miR-222 reduced ER␣ levels in MCF-7 and T47D cells, whereas knockdown of miR-221 and/or miR-222 restored ER␣ expression and tamoxifen sensitivity in MDA-MB-468 cells. These results indicate that miR-221 and miR-222 could play a pivotal role in the regulation of ER␣ expression in a subset of breast cancers. EXPERIMENTAL PROCEDURESCell Lines, Transfection, and Human Tumor Tissues-Human breast cancer cell lines (T47D, BT474, MDA-MB-361, MCF-7, MDA-MB-453, MDA-MB-157, SKBr3, MDA-MB-468, Hs578T, MDA-MB-231, and MDA-MB-435s) and spontaneously immortalized human breast epithelial cells (MCF-10A) were obtained from American Type Culture Collection. Breast cancer cell lines were grown in either RPMI 1640 medium (Sigma) or Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum. MCF-10A cells were cultured in mammary epithelium basal medium plus mammary epithelium growth medium (Clonetics). Transfection of 2Ј-O-MeantamiR oligonucleotides or pcDNA6.2-GW/EmGFP-miR (BLOCK-iT) plasmids was performed using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Stable cell lines were obtained by blasticidin selection. The sequences of 2Ј-O-Me-anta-miR-221 and 2Ј-O-Me-anta-miR-222 are 5Ј-GAAACCCAGCAGACAAUGUAGCU-3Ј and 5Ј-ACCCAGUAGCCAGAUGUAGCU-3Ј. Scrambled 2Ј-O-Me-* This work was supported, in whole or in part, by National Institutes of H...

Breast cancer is the second leading cause of cancer death in women. Despite improvement in treatment over the past few decades, there is an urgent need for development of targeted therapies. miR-155 (microRNA-155) is frequently up-regulated in breast cancer. In this study, we demonstrate the critical role of miR-155 in regulation of cell survival and chemosensitivity through down-regulation of FOXO3a in breast cancer. Ectopic expression of miR-155 induces cell survival and chemoresistance to multiple agents, whereas knockdown of miR-155 renders cells to apoptosis and enhances chemosensitivity. Further, we identified FOXO3a as a direct target of miR-155. Sustained overexpression of miR-155 resulted in repression of FOXO3a protein without changing mRNA levels, and knockdown of miR-155 increases FOXO3a. Introduction of FOXO3a cDNA lacking the 3-untranslated region abrogates miR-155-induced cell survival and chemoresistance. Finally, inverse correlation between miR-155 and FOXO3a levels were observed in a panel of breast cancer cell lines and tumors. In conclusion, our study reveals a molecular link between miR-155 and FOXO3a and presents evidence that miR-155 is a critical therapeutic target in breast cancer. MicroRNAs (miRs)3 are short single-stranded RNAs that have become known as important regulators of various cellular processes by controlling gene expression at the post-transcriptional level (1-4). Deregulated microRNAs in cancer function as either tumor suppressors (5-7) or oncogenes (8 -11) and play a central role in carcinogenesis. Accumulating evidence shows that miR-155 is an oncogenic microRNA. First, microRNA profiling studies indicated frequent increase of miR-155 in various types of human malignancy, including different forms of B cell lymphoma and carcinoma of breast, lung, colon, head/neck, and kidney (9,(11)(12)(13)(14)(15)(16) FKHRL1 (FOXO3a) is a major member of the forkhead transcriptional factor family (24). Members of this family are characterized by a distinctive forkhead DNA binding domain, which is negatively regulated by protein kinases Akt, SGK, and IKK␤ (25-28). The phosphorylation of FOXO3a by these kinases leads to its translocation from the nucleus to the cytoplasm and loss of the proapoptotic function. In the unphosphorylated active form, FOXO3a resides in the nucleus and induces cell death by up-regulation of apoptotic proteins, such as BIM, p27, BNIP3, and 24p3 (29 -32) and repression of antiapoptotic molecule FLIP and BCL-XL (33, 34).To date, the majority of validated miR-155 functional biology and protein targets define the importance of miR-155 in immunology (23,(35)(36)(37)(38)(39) and various forms of lymphoma (9,11,40,41); however, the role of miR-155 in human breast cancer remains elusive. Here, we report that miR-155 induces cell survival and plays an important role in chemoresistance in breast cancer. Its anti-apoptotic function is mediated by direct inhibition of FOXO3a. Thus, our findings not only demonstrate regulation of FOXO3a at post-transcriptional levels but also i...

Lung squamous cell carcinomas (LSCC) pathogenesis remains incompletely understood and biomarkers predicting treatment response remain lacking. Here we describe novel murine LSCC models driven by loss of Trp53 and Keap1, both of which are frequently mutated in human LSCCs. Homozygous inactivation of Keap1 or Trp53 promoted airway basal stem cell (ABSC) self-renewal, suggesting that mutations in these genes lead to expansion of mutant stem cell clones. Deletion of Trp53 and Keap1 in ABSCs, but not more differentiated tracheal cells, produced tumors recapitulating histological and molecular features of human LSCCs, indicating that they represent the likely cell of origin in this model. Deletion of Keap1 promoted tumor aggressiveness, metastasis, and resistance to oxidative stress and radiotherapy (RT). KEAP1/NRF2 mutation status predicted risk of local recurrence after RT in non-small lung cancer (NSCLC) patients and could be non-invasively identified in circulating tumor DNA. Thus, KEAP1/NRF2 mutations could serve as predictive biomarkers for personalization of therapeutic strategies for NSCLCs.