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Cancer cells show characteristic gene expression profiles. Recent studies support the potential importance of micro-RNA (miRNA) expression signatures as biomarkers and therapeutic targets. The membrane-anchored protease regulator RECK is downregulated in many cancers, and forced expression of RECK in tumor cells results in decreased malignancy in animal models. RECK is also essential for mammalian development. In this study, we found that RECK is a target of at least three groups of miRNAs (miR-15b/16, miR-21 and miR-372/373); that RECK mutants lacking the target sites for these miRNA show augmented tumor/metastasis-suppressor activities; and that miR-372/373 are upregulated in response to hypoxia through HIF1a and TWIST1, whereas miR-21 is upregulated by RAS/ERK signaling. These data indicate that the hypoxia-and RAS-signaling pathways converge on RECK through miRNAs, cooperatively downregulating this tumor suppressor and thereby promoting malignant cell behavior.

The mammalian target of rapamycin (mTOR) is a Ser/Thr kinase that plays essential roles in the regulation of a wide array of growthrelated processes such as protein synthesis, cell sizing, and autophagy. mTOR forms two functionally distinct complexes, termed the mTOR complex 1 (mTORC1) and 2 (mTORC2); only the former of which is inhibited by rapamycin. Based on the similarity between the cellular responses caused by rapamycin treatment and by nutrient starvation, it has been widely accepted that modulation in the mTORC1 activity in response to nutrient status directs these cellular responses, although direct evidence has been scarce. Here we report isolation of hyperactive mutants of mTOR. The isolated mTOR mutants exhibited enhanced kinase activity in vitro and rendered cells refractory to the dephosphorylation of the mTORC1 substrates upon amino acid starvation. Cells expressing the hyperactive mTOR mutant displayed larger cell size in a normal growing condition and were resistant to cell size reduction and autophagy induction in an amino acid-starved condition. These results indicate that the activity of mTORC1 actually directs these cellular processes in response to nutrient status and confirm the biological functions of mTORC1, which had been proposed solely from lossof-function analyses using rapamycin and (molecular)genetic techniques. Additionally, the hyperactive mTOR mutant did not induce cellular transformation of NIH/3T3 cells, suggesting that concomitant activation of additional pathways is required for tumorigenesis. This hyperactive mTOR mutant will be a valuable tool for establishing physiological consequences of mTOR activation in cells as well as in organisms.

Kirsten-ras-revertant 1 (Krev-1) cDNA encodes a ras-related protein and exhibits an activity of inducing flat revertants at certain frequencies (2-5% of total transfectants) when introduced into a v-K-ras-transformed mouse NIH 3T3 cell line, DT. Toward understanding the mechanism of action of Krev-l protein, we constructed a series of point mutants of Krev-1 cDNA and tested their biological activities in DT cells and HT1080 human fibrosarcoma cells harboring the activated N-ras gene. Substitutions of the amino acid residues in the putative guanine nucleotide-binding regions (Asp'7 and Asn'I'), in the putative effector-binding domain (residue 38), at the putative acylation site (Cys"'5), and at the unique Thr" all decreased the transformation suppressor activity. On the other hand, substitutions such as Gly'2 to Val'2 and Gln63 to Glu63were found to significantly increase the transformation suppressor/tumor suppressor activity of Krev-1. These findings are consistent with the idea that Krev-1 protein is regulated like many other G proteins by the guanine triphosphate/ guanine diphosphate-exchange mechanism probably in response to certain negative growth-regulatory signals.Kirsten-ras-revertant 1 (Krev-1) cDNA was recovered from one of the flat revertants isolated from populations of a v-Kras-transformed NIH 3T3 cell derivative, DT, following transfection with a human fibroblast cDNA expression library (1). When transfected into DT cells, the plasmid expressing Krev-1 cDNA induces flat revertants at certain frequencies (2-5% of total transfectants), and relatively high levels of expression seem to be required to induce morphological reversion (2). Krev-1 (also known as rap 1A and smg p21) encodes a guanine-nucleotide binding protein with a molecular weight of 21,000 whose amino acid sequence shares strong similarity (around 50% amino acid identity) with the products of the classical ras protooncogenes-namely, H-ras, K-ras, and N-ras (2-4). Similarities are especially high in the essential regions known in H-ras protein as guanine nucleotide-binding regions, the effector-binding region, and the C-terminal acylation site (see Fig. 1 for locations), which have been determined by in vitro mutagenesis (5-10) and later by x-ray crystallography (11, 12). The classical ras oncoproteins are also known to be activated by specific point mutations, such as amino acid substitutions at residues 12, 59, 61, and 63, to become highly transforming (reviewed in refs. 13 and 14). These mutations are believed to somehow disturb the guanine triphosphate (GTP)-hydrolyzing (GTPase) activity associated with ras proteins, thereby arresting these proteins in their active, GTP-bound form. In Krev-1 protein, some of the critical target amino acids for mutational activation of ras are identical (Gly12 and Thr59), and others are distinct from the corresponding amino acids in normal ras proteins (Thr61/ Gln63 in Krev-1 vs. Gln61/Glu63 in ras) (2).In this study we took advantage of the strong structural similarity between Krev-1 protei...

Seven morphologically nontransformed (flat) revertants with reduced tumorigenicity in vivo have been isolated from populations of Kirsten sarcoma virus-transformed NIH 3T3 cells transfected with a cDNA expression library ofnormal human fibroblasts. Each revertant harbors 1-10 recombinant plis per cell and retains a rescuable transforming virus as well as high level expression of v-Ki-ras-specific RNA and the viral oncogene product, p21v-i-. Transformed phenotypes are suppressed in cell hybrids generated by fusing each revertant to v-Ki-rastransformed NIH 3T3 cells. From two of the revertant lines, plasmids capable ofgiving rise to flat secondary transfectants have been recovered. Thus, in some, if not all, of the revertants, transfected cDNAs seem to be responsible for the suppression of specific transformed phenotypes.Flat revertants from rodent fibroblast cell lines transformed by various agents have been isolated and characterized by several groups of investigators as an approach to define and, ultimately, resolve the mechanism(s) of malignant cell transformation (reviewed in refs. 1 and 2). This approach has been most successfully applied in defining and characterizing those genes that are responsible for the transforming activities of various retroviruses (e.g., see refs. 3 and 4). There are also additional reports describing flat revertants, which appear to have resulted from mutations in specific cellular genes rather than viral-encoded transforming genes (1,(5)(6)(7)(8). Such revertants may be useful in revealing cellular macromolecules that might either directly or indirectly participate in the process of cellular transformation. Previously, we described two nontumorigenic flat revertants isolated from populations of the DT line of Kirsten murine sarcoma virus (Ki-MSV)-transformed cells (7). These revertants seem to harbor dominant mutations in cellular genes, which in some way suppress certain malignant characteristics usually associated with Ki-MSV-induced transformation (7, 9-12). The putative gene or genes responsible for suppression of the malignant characteristics have not yet been identified.In this report, we show that flat revertants with reduced tumorigenicity can be isolated from Ki-MSV-transformed DT cells after transfection of normal human fibroblast cDNA expression library. The properties of some, if not all, of the revertants are consistent with the idea that expression of the introduced cDNA clones contributes to a reversal of the ras-induced transformed phenotype. MATERIALS AND METHODSCell Lines and Viruses. The derivations and methods for maintenance of NIH 3T3, DT (Ki/HGPRT-NIH 3T3), and Ki/TK-NIH have been described (7). DTneoR was derived from DT cells by transfection of pL2 DNA (13) followed by *G418 selection. Rescue for replication-defective sarcoma viruses, focus formation, and soft agar assays were performed as described (7, 9). Growth medium consisted of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS) (KC Biological, Lenexa, KS) and 4 mM...

The membrane-anchored protease regulator RECK plays important roles in mammalian development and tumor suppression. The biochemical bases of these bioactivities, however, remain poorly understood. Here we report on the properties of a recombinant RECK protein expressed in mouse fibroblasts and purified to near homogeneity. Multiple lines of evidence indicate that RECK forms dimers. Single particle reconstruction using transmission electron microscopy revealed a unique cowbell-like shaped RECK dimer. RECK is cleaved by MMP-2 and MMP-7 and competitively inhibits MMP-7-catalyzed cleavage of fibronectin. Forced RECK expression in HT1080 cells, whose endogenous RECK expression is minimal, leads to an increase in the amount of fibronectin associated with the cell. Our data demonstrate the ability of RECK to protect fibronectin from MMP-mediated degradation.

Accumulating evidence indicates that Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a membrane-anchored matrix metalloproteinase regulator, plays crucial roles in mammalian development and tumor suppression. Its mechanisms of action at the single cell level, however, remain largely unknown. In mouse fibroblasts, RECK is abundant around the perinuclear region, membrane ruffles and cell surface. Cells lacking Reck show decreased spreading, ambiguous anteriorposterior (AP) polarity, and increased speed and decreased directional persistence in migration; these characteristics are also found in transformed fibroblasts and fibrosarcoma cells with low RECK expression. RECK-deficient cells fail to form discrete focal adhesions, have increased levels of GTP-bound Rac1 and Cdc42, and a marked decrease in the level of detyrosinated tubulin, a hallmark of stabilized microtubules. RECK-deficient cells also show elevated gelatinolytic activity and decreased fibronectin fibrils. The phenotype of RECK-deficient cells is largely suppressed when the cells are plated on fibronectin-coated substrates. These findings suggest that RECK regulates pericellular extracellular matrix degradation, thereby allowing the cells to form proper cell-substrate adhesions and to maintain AP polarity during migration; this mechanism is compromised in malignant cells.

BackgroundDevelopmental angiogenesis proceeds through multiple morphogenetic events including sprouting, intussusception, and pruning. Mice lacking the membrane-anchored metalloproteinase regulator Reck die in utero around embryonic day 10.5 with halted vascular development; however, the mechanisms by which this phenotype arises remain unclear.ResultsWe found that Reck is abundantly expressed in the cells associated with blood vessels undergoing angiogenesis or remodelling in the uteri of pregnant female mice. Some of the Reck-positive vessels show morphological features consistent with non-sprouting angiogenesis. Treatment with a vector expressing a small hairpin RNA against Reck severely disrupts the formation of blood vessels with a compact, round lumen. Similar defects were found in the vasculature of Reck-deficient or Reck conditional knockout embryos.ConclusionsOur findings implicate Reck in vascular remodeling, possibly through non-sprouting angiogenesis, in both maternal and embyornic tissues.