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-

Autophagy is an evolutionally conserved "self-eating" process. Although the genes essential for autophagy (termed Atg) have been identified in yeast, the molecular mechanism of how these Atg proteins control autophagosome formation in mammalian cells remains to be elucidated. Here, we demonstrate that Bif-1 (also known as Endophilin B1) interacts with Beclin 1 through UVRAG and acts as a positive mediator of the class III PI3-kinase (PI3KC3). In response to nutrition deprivation, Bif-1 localizes to autophagosomes where it colocalizes with Atg5, as well as LC3. Furthermore, loss of Bif-1 suppresses autophagosome formation. While the SH3 domain of Bif-1 is sufficient for binding to UVRAG, both the BAR and SH3 domains are required for Bif-1 to activate PI3KC3 and induce autophagosome formation. We also found that Bif-1 ablation prolongs cell survival under nutrient starvation. Moreover, knockout of Bif-1 significantly enhances the development of spontaneous tumors in mice. These findings suggest that Bif-1 joins the UVRAG-Beclin 1 complex as a potential activator of autophagy and tumor suppressor.Autophagy is a tightly orchestrated intracellular process for bulk degradation of cytoplasmic proteins or organelles that appears to be essential for many physiological processes such as cellular homeostasis, development, differentiation, tissue remodeling, cell survival and death, innate immunity, and pathogenesis in various organisms 1-4 . The process of autophagic degradation is initiated when a portion of the cytosolic components are sequestered in cupshaped membrane structures called isolation membranes 1, 2, 5, 6 . The isolation membranes are elongated and eventually sealed to become double-membrane vesicles called autophagosomes, which are then fused with lysosomes resulting in degradation of the enclosed components. Eighteen autophagy-related (Atg) genes have been characterized in S. cerevisiae and can be categorized into four functional groups: (1) the Atg1 protein kinase complex regulating the induction of autophagy, (2) the class III PI3-kinase (PI3KC3) lipid kinase complex controlling vesicle nucleation, (3) the Atg12-Atg5 and Atg8-phosphatidylethanolamine conjugation pathways for vesicle expansion and completion, and (4) the Atg protein retrieval system 2, 7 . Beclin 1, the mammalian homologue of yeast Atg6, is a key component of the PI3KC3 complex, which plays an essential role in autophagosome formation 8-11 . Although the phosphatidylinositol 3-phosphate (PtdIns-3-P) generated by PI3KC3 has been proposed to control membrane dynamics during autophagosome formation 3 , the molecular mechanism underlying this process remains unknown. Results Loss of Bif-1 suppresses caspase-independent cell deathWe have previously reported that Bif-1 localizes to mitochondria and regulates the activation of Bax and Bak during apoptosis induced by intrinsic death stimuli 21 . To examine Bif-1 localization in mouse embryonic fibroblast (MEF) cells during serum deprivation, we added a pancaspase inhibitor, z-VAD-fmk, to ...

Malignant melanoma is the skin cancer with the most significant impact on man, carrying the highest risk of death from metastasis. Both incidence and mortality rates continue to rise each year, with no effective long-term treatment on the horizon. In part, this reflects lack of identification of critical genes involved and specific therapies targeted to correct these defects. We report that selective activation of the Akt3

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...

The AKT2 gene is one of the human homologues of v-akt, the transduced oncogene of the AKT8 virus, which induces lymphomas in mice. In previous studies, AKT2, which codes for a serine-threonine protein kinase, was shown to be amplified and overexpressed in some human ovarian carcinoma cell lines and amplified in primary tumors of the ovary. To confirm and extend these findings, we conducted a large-scale, multicenter study of AKT2 alterations in ovarian and breast cancer. Southern-blot analysis demonstrated AKT2 amplification in 16 of 132 (12.1%) ovarian carcinomas and in 3 of 106 (2.8%) breast carcinomas. No AKT2 alteration was detected in 24 benign or borderline tumors. Northern-blot analysis revealed overexpression of AKT2 in 3 of 25 fresh ovarian carcinomas which were negative for AKT2 amplification. The difference in the incidence of AKT2 alterations in ovarian and breast cancer suggests a specific role for this gene in ovarian oncogenesis. No significant association was found between AKT2 amplification and amplification of the proto-oncogenes MYC and ERBB2, suggesting that amplification of AKT2 defines an independent subset of breast and ovarian cancers. Ovarian cancer patients with AKT2 alterations appear to have a poor prognosis. Amplification of AKT2 was especially frequent in undifferentiated tumors (4 of 8, p = 0.019), suggesting that AKT2 alterations may be associated with tumor aggressiveness.

Inflammatory mechanisms influence tumor development and metastatic progression1. Of interest is the role of such mechanisms in metastatic spread of tumors whose etiology does not involve pre-existing inflammation or infection, such as breast and prostate cancers. We found that prostate cancer metastasis is associated with lymphocyte infiltration into advanced tumors and elevated expression of the tumor necrosis factor (TNF) family members receptor activator of NF-κB (RANK) ligand (RANKL) and lymphotoxin (LT)2. But the source of RANKL and its role in metastasis were not established. RANKL and its receptor RANK control proliferation of mammary lobuloalveolar cells during pregnancy3 through activation of IκB kinase α (IKKα)4, a protein kinase that is required for self-renewal of mammary cancer progenitors5 and prostate cancer metastasis2. We therefore examined whether RANKL, RANK and IKKα are also involved in mammary/breast cancer metastasis. Indeed, RANK signaling in mammary carcinoma cells that overexpress the ErbB2 (c-Neu) proto-oncogene6, which is frequently amplified in metastatic human breast cancers7,8, was important for pulmonary metastasis. Metastatic spread of ErbB2-transformed carcinoma cells was also dependent on CD4+CD25+ T cells, whose major pro-metastatic function appeared to be RANKL production. RANKL-producing T cells were mainly FoxP3+ and found in close proximity to smooth muscle actin (SMA)-positive stromal cells in mouse and human breast cancers. The T cell-dependence of pulmonary metastasis was replaced by administration of exogenous RANKL, a procedure that also stimulated pulmonary metastasis of RANK-positive human breast carcinoma cells. These results are consistent with the adverse prognostic impact of tumor-infiltrating CD4+ or FoxP3+ T cells on human breast cancer9,10 and suggest that targeting of RANKL-RANK signaling can be used in conjunction with other therapies to prevent subsequent metastatic disease.

We isolated cDNA clones containing the entire coding region of the putative oncogene AKT2. Sequence analysis and in vitro translation demonstrated that AKT2 encodes a 56-kDa protein with homology to serine/threonine kinases; moreover, this protein contains a Src homology 2-like domain. AKT2 was shown to be amplified and overexpressed in 2 of 8 ovarian carcinoma cell lines and 2 of 15 primary ovarian tumors. AKT2 was mapped to chromosome region 19q13.1-q13.2 by fluorescence in situ hybridization. In the two ovarian carcinoma cell lines exhibiting amplification of AKT2, the amplified sequences were localized within homogeneously staining regions. We conclude that AKT2 belongs to a distinct subfamily of protein-serine/threonine kinases containing Src homology 2-like domains and that alterations of AKT2 may contribute to the pathogenesis of ovarian carcinomas. akt, the protooncogene transduced by the acute transforming retrovirus AKT8 (1, 2), encodes a protein-serine/threonine kinase containing a Src homology 2-like (SH2-like) domain (3). Two putative human cellular homologs, AKTI and AK12, were cloned by screening a human genomic DNA library with a v-akt probe under conditions of reduced stringency (4). Recently, we obtained AKTI clones from a normal human thymus cDNA library by using an AKTI genomic probe. Sequence analysis of portions of AKTI cDNA clones revealed that AKTI is the true human homolog of v-akt and is identical to the recently cloned RAC gene, which has been shown to encode a kinase related to members of the protein kinase C (PKC) family and the cyclic adenosine monophosphate-dependent protein kinase (cAMP-PK) (5). AKTI has been mapped to human chromosome band 14q32 (6), proximal to the immunoglobulin heavy-chain locus, and has been shown to be amplified in a gastric adenocarcinoma (4).In this communication, we report the cDNA cloning, sequence analysis,t and chromosomal mapping ofAKT2 and demonstrate that this putative oncogene encodes a protein belonging to a subfamily of serine/threonine kinases containing SH2-like domains. Moreover, we show that AKT2 is amplified and overexpressed in some human ovarian carcinoma cell lines and primary tumors, suggesting that it contributes to the development of common epithelial tumors of the ovary.

We previously demonstrated that the putative oncogene AKT2 is amplified and overexpressed in some human ovarian carcinomas. We have now identified amplification ofAKT2 in -10% of pancreatic carcinomas (2 of 18 cell lines and 1 of 10 primary tumor specimens). The (18). The following conditions were used for amplification of AKT2: 94°C for 1 min, 52°C for 1 min, and 72°C for 2 min for 35 cycles; after the final cycle, the reaction was held at 72°C for 10 min. As a control, a portion of the glyceraldehyde-3-phosphate dehydrogenase gene was also amplified using the same PCR conditions described above. The resulting PCR products were electrophoresed on a 1.5% agarose gel. The primers used for a portion of the open reading frame ofAKT2Abbreviations: AS, antisense; S, sense.§To whom reprint requests should be addressed.3636