Randolph C. Elble

p53 represses c-Myc through induction of the tumor suppressor miR-145

The tumor suppressor p53 negatively regulates a number of genes, including the proto-oncogene c-Myc, in addition to activating many other genes. One mechanism of the p53-mediated c-Myc repression may involve transcriptional regulation. However, it is not clear whether microRNAs (miRNAs) play a role in the p53-mediated posttranscriptional regulation of c-Myc. In this study, we show that a putative tumor suppressor, miR-145, is expressed through the phosphoinositide-3 kinase (PI-3K)/Akt and p53 pathways. Importantly, p53 transcriptionally induces the expression of miR-145 by interacting with a potential p53 response element (p53RE) in the miR-145 promoter. We further show that c-Myc is a direct target for miR-145. Although miR-145 silences the expression of c-Myc, anti-miR-145 enhances its expression. This specific silencing of c-Myc by miR-145 accounts at least in part for the miR-145-mediated inhibition of tumor cell growth both in vitro and in vivo. Finally, the blockade of miR-145 by anti-miR-145 is able to reverse the p53-mediated c-Myc repression. Together, these results define the role of miR-145 in the posttranscriptional regulation of c-Myc by p53 and suggest that, as a new member of the p53 regulatory network, miR-145 provides a direct link between p53 and c-Myc in this gene regulatory network.

Saccharomyces cerevisiae protein involved in plasmid maintenance is necessary for mating of MATα cells

We previously reported the isolation of yeast mutants that seem to affect the function of certain autonomously replicating sequences (ARSs). These mutants are known as mcm for their defect in the maintenance of minichromosomes. We have now characterized in more detail one ARS-specific mutation, mcm1-1. This Mcm1 mutant has a second phenotype; MAT alpha mcm1-1 strains are sterile. MCM1 is non-allelic to other known alpha-specific sterile mutations and, unlike most genes required for mating, it is essential for growth. The alpha-specific sterile phenotype of the mcm1-1 mutant is manifested by its failure to produce a normal amount of the mating pheromone, alpha-factor. In addition, transcripts of the MF alpha 1 and STE3 genes, which encode the alpha-factor precursor and the alpha-factor receptor, respectively, are greatly reduced in this mutant. These and other properties of the mcm1-1 mutant suggest that the MCM1 protein may act as a transcriptional activator of alpha-specific genes. We have cloned, mapped and sequenced the wild-type and mutant alleles of MCM1, which is located on the right arm of chromosome XIII near LYS7. The MCM1 gene product is a protein of 286 amino acid residues and contains an unusual region in which 19 out of 20 residues are either aspartic or glutamic acid, followed by a series of glutamine tracts. MCM1 has striking homology to ARG80, a regulatory gene of the arginine metabolic pathway located about 700 base-pairs upstream from MCM1. A substitution of leucine for proline at amino acid position 97, immediately preceding the polyanionic region, was shown to be responsible for both the alpha-specific sterile and minichromosome-maintenance defective phenotypes of the mcm1-1 mutant.

Lung Endothelial Dipeptidyl Peptidase IV Promotes Adhesion and Metastasis of Rat Breast Cancer Cells via Tumor Cell Surface-associated Fibronectin

Endothelial cell adhesion molecules are partly responsible for the distinct organ distribution of cancer metastases. Dipeptidyl peptidase IV (DPP IV) expressed on rat lung capillary endothelia is shown here to be an adhesion receptor for rat breast cancer cells and to mediate lung colonization by these tumor cells. Fibronectin (FN) assembled on breast cancer cell surfaces into multiple, randomly dispersed globules from cellular and plasma FN is identified as the principal ligand for DPP IV. Ligand expression correlates quantitatively with the tumor cells’ capabilities to bind to DPP IV and to metastasize to the lungs. DPP IV/FN-mediated adhesion and metastasis are blocked when tumor cells are incubated with soluble DPP IV prior to conducting adhesion and lung colony assays. Adhesion is also blocked by anti-DPP IV monoclonal antibody 6A3 and anti-FN antiserum. However, adhesion to immobilized FN is unaffected by soluble plasma FN and, thus, can happen during hematogenous spread of cancer cells at high plasma FN concentrations. The ability of many cancer cells to capture FN molecules on their surface and to augment such deposits by FN self-association during passage in the blood suggests that DPP IV/FN binding may be a relatively common mechanism for lung metastasis.

Molecular and Functional Characterization of a Calcium-sensitive Chloride Channel from Mouse Lung

A protein (mCLCA1) has been cloned from a mouse lung cDNA library that bears strong sequence homology with the recently described bovine tracheal, Ca2+-sensitive chloride channel protein (bCLCA1), bovine lung endothelial cell adhesion molecule-1 (Lu-ECAM-1), and the human intestinal Ca2+-sensitive chloride channel protein (hCLCA1). In vitro, its 3.1-kilobase message translates into a 100-kDa protein that can be glycosylated to an approximately 125-kDa product. SDS-polyacrylamide gel electrophoresis from lysates of mCLCA1 cDNA-transfected transformed human embryonic kidney cells (HEK293) reveals proteins of 130, 125, and 90 kDa as well as a protein triplet in the 32–38 kDa size range. Western analyses with antisera raised against Lu-ECAM-1 peptides show that the N-terminal region of the predicted open reading frame is present only in the larger size proteins (i.e. 130, 125, and 90 kDa), whereas the C-terminal region of the open reading frame is observed in the 32–38 kDa size proteins, suggesting a posttranslational, proteolytic processing of a precursor protein (125/130 kDa) into 90 kDa and 32–38 kDa components similar to that reported for Lu-ECAM-1. Hydrophobicity analyses predict four transmembrane domains for the 90-kDa protein. The mCLCA1 mRNA is readily detected by Northern analysis and byin situ hybridization in the respiratory epithelia of trachea and bronchi. Transient expression of mCLCA1 in HEK293 cells was associated with an increase in whole cell Cl−current that could be activated by Ca2+ and ionomycin and inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, dithiothreitol, and niflumic acid. The discovery of mCLCA1 opens the door for further investigating the possible contribution of a Ca2+-sensitive chloride conductance to the pathogenesis of cystic fibrosis.

The Breast Cancer β4 Integrin and Endothelial Human CLCA2 Mediate Lung Metastasis

Adhesion of blood-borne cancer cells to the endothelium is a critical determinant of organ-specific metastasis. Here we show that colonization of the lungs by human breast cancer cells is correlated with cell surface expression of the α6β4 integrin and adhesion to human CLCA2 (hCLCA2), a Ca2+-sensitive chloride channel protein that is expressed on the endothelial cell luminal surface of pulmonary arteries, arterioles, and venules. Tumor cell adhesion to endothelial hCLCA2 is mediated by the β4 integrin, establishing for the first time a cell-cell adhesion property for this integrin that involves an entirely new adhesion partner. This adhesion is augmented by an increased surface expression of the α6β4 integrin in breast cancer cells selected in vivo for enhanced lung colonization but abolished by the specific cleavage of the β4 integrin with matrilysin. β4 integrin/hCLCA2 adhesion-blocking antibodies directed against either of the two interacting adhesion molecules inhibit lung colonization, while overexpression of the β4 integrin in a model murine tumor cell line of modest lung colonization potential significantly increases the lung metastatic performance. Our data clearly show that the β4/hCLCA2 adhesion is critical for lung metastasis, yet expression of the β4 integrin in many benign breast tumors shows that this integrin is insufficient to bestow metastatic competence on cells that lack invasiveness and other established properties of metastatic cells.

Ca2+-Activated Cl– Channels: A Newly Emerging Anion Transport Family

Abstract. A new family of chloride transport proteins has recently emerged. These proteins have extensive homology to a protein previously isolated from bovine tracheal epithelium that acts as a Ca2+-sensitive Cl– channel (CaCC) when heterologously expressed or when reconstituted into planar lipid bilayers. Several new members of this family have been identified in human, murine, and bovine epithelia, in addition to some other tissues, and are associated with Ca2+-sensitive conductive chloride transport when heterologously expressed in Xenopus oocytes or HEK 293 cells. The expressed current is also sensitive to inhibitors such as DIDS and niflumic acid. In addition, at least one family member acts as an endothelial cell adhesion molecule. This emerging family may underlie the Ca2+-mediated Cl– conductance responsible for rescue of the cystic fibrosis (CF) knockout mouse from significant airway disease.

Cloning and characterization of lung-endothelial cell adhesion molecule- 1 suggest it is an endothelial chloride channel

Lung-endothelial cell adhesion molecule-1 (Lu-ECAM-1) is an endothelial cell surface molecule that mediates adhesion of metastatic melanoma cells to lung endothelium. Here we analyze the organization of the Lu-ECAM-1 protein complex, report the sequence of Lu-ECAM-1 cDNAs, and reveal a novel function of the protein. Lu-ECAM-1 immunopurified from bovine aortic endothelial cells (BAEC) consists of tightly associated glycoproteins of 90, 38, and 32 kDa, with minor components of 130 and 120 kDa. We present evidence that all of these protein species are encoded by a single open reading frame whose initial translation product is proteolytically processed to yield the other products. Correct processing in vitro was demonstrated by transfection of the longest cDNA into human embryonic kidney 293 cells; immunoblot analysis showed that the ∼120-kDa precursor gave rise to 90- and 38-kDa products. RNA blots of BAEC mRNA detected messages in agreement with the sizes of the cDNA clones in addition to several of high molecular weight. DNA blot analysis showed that Lu-ECAM-1 is conserved throughout its length in all mammals tested, usually as a single or low copy gene. In the bovine, Lu-ECAM-1 protein is 88% identical to a calcium-dependent chloride channel described recently in tracheal epithelium, Ca-CC. Probes for Lu-ECAM-1 mRNA and protein confirmed the presence of a homolog in this tissue. We show that messages for both proteins are present in lung while only Ca-CC is present in trachea and only Lu-ECAM-1 is present in BAEC. These results suggest that endothelial cells express a chloride channel that is related to, but distinct from, that expressed in tracheal epithelium. They further suggest that an adhesion molecule can also be a chloride channel.

Molecular characteristics and functional diversity of CLCA family members.

1. In the present brief review, we describe some of the molecular and functional characteristics of a novel mammalian family of putative Ca2+‐activated chloride channels (CLCA).

The role of cancer stem cells in relapse of solid tumors.

Recurrence at secondary locations, often years after removal of the primary tumor, accounts for most of the mortality associated with solid tumors. Metastasis, resistance to chemo- and radiotherapy, and eventual relapse have been attributed to a distinct tumor subpopulation known as cancer stem cells (CSCs). In this review, we consider the properties of CSCs that lead to these outcomes, in particular the relation between epithelial-to-mesenchymal transition, stemness, and tumor initiation. We compare recent clinical and laboratory studies of breast cancer, glioblastoma, and melanoma that illustrate how most current anticancer regimens select for cells with mesenchymal and CSC properties and therefore sow the seeds of relapse. Finally, we discuss the emerging paradigm of combined therapy that targets both CSC and non-CSC tumor components.

Loss of breast epithelial marker hCLCA2 promotes epithelial-to-mesenchymal transition and indicates higher risk of metastasis.

Transition between epithelial and mesenchymal states is a feature of both normal development and tumor progression. We report that expression of chloride channel accessory protein hCLCA2 is a characteristic of epithelial differentiation in the immortalized MCF10A and HMLE models, while induction of epithelial-to-mesenchymal transition by cell dilution, TGFβ or mesenchymal transcription factors sharply reduces hCLCA2 levels. Attenuation of hCLCA2 expression by lentiviral small hairpin RNA caused cell overgrowth and focus formation, enhanced migration and invasion, and increased mammosphere formation in methylcellulose. These changes were accompanied by downregulation of E-cadherin and upregulation of mesenchymal markers such as vimentin and fibronectin. Moreover, hCLCA2 expression is greatly downregulated in breast cancer cells with a mesenchymal or claudin-low profile. These observations suggest that loss of hCLCA2 may promote metastasis. We find that higher-than-median expression of hCLCA2 is associated with a one-third lower rate of metastasis over an 18-year period among breast cancer patients compared with lower-than-median (n=344, unfiltered for subtype). Thus, hCLCA2 is required for epithelial differentiation, and its loss during tumor progression contributes to metastasis. Overexpression of hCLCA2 has been reported to inhibit cell proliferation and is accompanied by increases in chloride current at the plasma membrane and reduced intracellular pH (pHi). We found that knockdown cells have sharply reduced chloride current and higher pHi, both characteristics of tumor cells. These results suggest a mechanism for the effects on differentiation. Loss of hCLCA2 may allow escape from pHi homeostatic mechanisms, permitting the higher intracellular and lower extracellular pH that are characteristic of aggressive tumor cells.