Randolph Elble, PhD

1. Discovery of a novel epithelial differentiation mechanism. The disruption of epithelial homeostasis by EMT is a critical step in the evolution of the metastatic cell. EMT is prevented by inhibitory signaling from cell-cell junctions. My lab has identified CLCA2 as a critical mediator of this signaling. We found that CLCA2 expression is tightly coupled to that of epithelial markers such as E-cadherin; that it is induced by epithelial differentiation and repressed during EMT, that it is downregulated in basal and mesenchymal cell types; and that knocking down CLCA2 is sufficient to evoke EMT. This was published in Oncogene. Then, to determine how CLCA2 promotes epithelial differentiation, we identified interactors by two-hybrid screening and found that CLCA2 interacts with a cell-cell adhesion molecule called EVA1 and junctional signaling molecules. This work has been submitted to Oncogene. We subsequently found that another CLCA family member, CLCA4, also plays an important role in epithelial differentiation, published in PLOS One. These studies have revealed new, potentially targetable tumor-suppressive signaling pathways in breast cancer and point to the need for more in-depth studies of the biochemical and physiological functions of this ancient protein family. During these studies, we also discovered a way of fractionating epithelial cells into epithelial and mesenchymal/stem-like populations by differential trypsinization and showed that CLCA2 was characteristic of the most epithelioid, trypsin-resistant fraction and absent from the stem-like fraction. I conceived of this work, attracted funding and personnel, and supervised its performance.

Citations

    Walia V, Elble RC. Enrichment for breast cancer cells with stem/progenitor properties by differential adhesion. Stem Cells Dev. 2010 Aug;19(8):1175-82. PubMed PMID: 20222827.

    Yu Y, Ramena G, Elble RC. The role of cancer stem cells in relapse of solid tumors. Front Biosci (Elite Ed). 2012 Jan 1;4:1528-41. PubMed PMID: 22201973.

    Walia V, Yu Y, Cao D, Sun M, McLean JR, Hollier BG, Cheng J, Mani SA, Rao K, Premkumar L, Elble RC. Loss of breast epithelial marker hCLCA2 promotes epithelial-to-mesenchymal transition and indicates higher risk of metastasis. Oncogene. 2012 Apr 26;31(17):2237-46. PubMed PMID: 21909135; NIHMSID: NIHMS316494; PubMed Central PMCID: PMC4154589.

    Yu Y, Walia V, Elble RC. Loss of CLCA4 promotes epithelial-to-mesenchymal transition in breast cancer cells. PLoS One. 2013 Dec 26;8(12):e83943. PubMed PMID: 24386311; PubMed Central PMCID: PMC3873418.

2. Discovery of a novel stress-sensitive growth-arrest pathway. In 2009, we showed in Cancer Research that human CLCA2 is highly induced by p53 in response to DNA damage and other stresses, and that ectopic expression caused G1 arrest in normal cells but apoptosis or senescence-like phenotype in cancer cells. Knockdown rendered cells more sensitive to doxorubicin. Thus, it is a p53-inducible inhibitor of proliferation. These results have since been replicated by others, who have extended the observations to prostate cancer, osteosarcoma, melanoma, etc. This line of inquiry was inspired by my earlier discovery that murine Clca2 is induced during the involutionary, apoptotic phase of lactation. I found that the gene was induced by serum starvation and detachment and that tumor cells downregulated its expression by disrupting its splicing, while ectopic expression caused senescence. We later discovered that the true ortholog of hCLCA2, mClca5, was induced by G1 arrest, starvation, and detachment in normal cells but not metastatic cell lines. Ectopic expression restored a cobblestone growth pattern to metastatic 4T1 cells. These studies laid the foundation for investigating human CLCA2 role in breast cancer as a possible tumor suppressor and suggest re-establishing or mimicking CLCA function as a novel therapeutic avenue.

Citations

    Elble RC, Pauli BU. Tumor suppression by a proapoptotic calcium-activated chloride channel in mammary epithelium. J Biol Chem. 2001 Nov 2;276(44):40510-7. PubMed PMID: 11483609.

    Beckley JR, Pauli BU, Elble RC. Re-expression of detachment-inducible chloride channel mCLCA5 suppresses growth of metastatic breast cancer cells. J Biol Chem. 2004 Oct 1;279(40):41634-41. PubMed PMID: 15292178.

    Walia V, Ding M, Kumar S, Nie D, Premkumar LS, Elble RC. hCLCA2 Is a p53-Inducible Inhibitor of Breast Cancer Cell Proliferation. Cancer Res. 2009 Aug 15;69(16):6624-32. PubMed PMID: 19654313; NIHMSID: NIHMS125917; PubMed Central PMCID: PMC2745301.

3. Discovery of the CLCA gene family, cloning and characterization of bovine, murine, and human family members. Starting with a peptide sequence from an immunopurified protein from bovine endothelial cells, I designed degenerate PCR primers to amplify and clone the founding member of this gene family, identifying several splice forms and unraveling the structure of the mature transcripts. I then used protein sequencing and DNA sequence to determine that bCLCA2 was a cleaved protein and mapped the site. These studies provided the template for discovery and modeling of the new protein family. I subsequently used similar methods to identify 4 murine homologs, mClca1, 2, 4, and 5, of what turned out to be 8 total. I later designed the strategy for cloning human homologs, hCLCA1, 2, 3, performed by a graduate student. hCLCA4 was identified by another lab. I contributed the following sequences to GenBank: bCLCA2 splice forms (originally LuECAM-1) AF001261, AF001261, AF001263, AF001264; mClca1, NM009899; mClca2, NM030601; mCLCA4, AY008277; Clca5, AY161007; hClca1, AF039400. Later, I used biochemical methods to establish hCLCA2 as a Type 1 transmembrane protein with only a single pass, correcting the previously published multipass model. This work was foundational for the CLCA field. A current manuscript will resolve the confusion over the previous designation of these proteins as channels.

Citations

    Elble RC, Widom J, Gruber AD, Abdel-Ghany M, Levine R, Goodwin A, Cheng HC, Pauli BU. Cloning and characterization of lung-endothelial cell adhesion molecule-1 suggest it is an endothelial chloride channel. J Biol Chem. 1997 Oct 31;272(44):27853-61. PubMed PMID: 9346932.

    Gruber AD, Elble RC, Ji HL, Schreur KD, Fuller CM, Pauli BU. Genomic cloning, molecular characterization, and functional analysis of human CLCA1, the first human member of the family of Ca2+-activated Cl- channel proteins. Genomics. 1998 Dec 1;54(2):200-14. PubMed PMID: 9828122.

    Elble RC, Ji G, Nehrke K, DeBiasio J, Kingsley PD, Kotlikoff MI, Pauli BU. Molecular and functional characterization of a murine calcium-activated chloride channel expressed in smooth muscle. J Biol Chem. 2002 May 24;277(21):18586-91. PubMed PMID: 11896056.

    Elble RC, Walia V, Cheng HC, Connon CJ, Mundhenk L, Gruber AD, Pauli BU. The putative chloride channel hCLCA2 has a single C-terminal transmembrane segment. J Biol Chem. 2006 Oct 6;281(40):29448-54. PubMed PMID: 16873362.

4. Earlier career: My graduate work centered on E. coli RNA1, the first example of a short interfering RNA. I characterized mutants that destabilized interaction with the primer of plasmid DNA replication. During my postdoc with Bik Tye, I studied MCM1, a transcription factor and regulator of MCM helicases that act at eukaryotic DNA replication origins. I helped sequence the gene(see GenBank X14187)and constructed Mcm1 mutants that evidenced its role in regulating mating-type genes and DNA replication. These studies helped establish Mcm1 as a master regulator of mating and cell division. In the course of that work, I discovered that Mcm1 is the yeast homolog of the mitogenic Serum Response Factor, a regulator of mammalian cell division and was inspired to pursue a career in cancer research. I also developed a widely used method for transfection of yeasts during this period.

Citations

    Fitzwater T, Zhang XY, Elble R, Polisky B. Conditional high copy number ColE1 mutants: resistance to RNA1 inhibition in vivo and in vitro. EMBO J. 1988 Oct;7(10):3289-97. PubMed PMID: 2460340; PubMed Central PMCID: PMC454759.

    Passmore S, Elble R, Tye BK. A protein involved in minichromosome maintenance in yeast binds a transcriptional enhancer conserved in eukaryotes. Genes Dev. 1989 Jul;3(7):921-35. PubMed PMID: 2673922.

    Elble R, Tye BK. Both activation and repression of a-mating-type-specific genes in yeast require transcription factor Mcm1. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10966-70. PubMed PMID: 1961765; PubMed Central PMCID: PMC53053.

    Elble R, Tye BK. Chromosome loss, hyperrecombination, and cell cycle arrest in a yeast mcm1 mutant. Mol Biol Cell. 1992 Sep;3(9):971-80. PubMed PMID: 1421579; PubMed Central PMCID: PMC275658.