Gregory G. Oakley

Spontaneous Metastasis of Prostate Cancer Is Promoted by Excess Hyaluronan Synthesis and Processing

Accumulation of extracellular hyaluronan (HA) and its processing enzyme, the hyaluronidase Hyal1, predicts invasive, metastatic progression of human prostate cancer. To dissect the roles of hyaluronan synthases (HAS) and Hyal1 in tumorigenesis and metastasis, we selected nonmetastatic 22Rv1 prostate tumor cells that overexpress HAS2, HAS3, or Hyal1 individually, and compared these cells with co-transfectants expressing Hyal1 + HAS2 or Hyal1 + HAS3. Cells expressing only HAS were less tumorigenic than vector control transfectants on orthotopic injection into mice. In contrast, cells co-expressing Hyal1 + HAS2 or Hyal1 + HAS3 showed greater than sixfold and twofold increases in tumorigenesis, respectively. Fluorescence and histological quantification revealed spontaneous lymph node metastasis in all Hyal1 transfectant-implanted mice, and node burden increased an additional twofold when Hyal1 and HAS were co-expressed. Cells only expressing HAS were not metastatic. Thus, excess HA synthesis and processing in concert accelerate the acquisition of a metastatic phenotype by prostate tumor cells. Intratumoral vascularity did not correlate with either tumor size or metastatic potential. Analysis of cell cycle progression revealed shortened doubling times of Hyal1-expressing cells. Both adhesion and motility on extracellular matrix were diminished in HA-overproducing cells; however, motility was increased twofold by Hyal1 expression and fourfold to sixfold by Hyal1/HAS co-expression, in close agreement with observed metastatic potential. This is the first comprehensive examination of these enzymes in a relevant prostate cancer microenvironment.

NBS1 mediates ATR-dependent RPA hyperphosphorylation following replication-fork stall and collapse.

Post-translational phosphorylation of proteins provides a mechanism for cells to switch on or off many diverse processes, including responses to replication stress. Replication-stress-induced phosphorylation enables the rapid activation of numerous proteins involved in DNA replication, DNA repair and cell cycle checkpoints, including replication protein A (RPA). Here, we report that hydroxyurea (HU)-induced RPA phosphorylation requires both NBS1 (NBN) and NBS1 phosphorylation. Transfection of both phosphospecific and nonphosphospecific anti-NBS1 antibodies blocked hyperphosphorylation of RPA in HeLa cells. Nijmegen breakage syndrome (NBS) cells stably transfected with an empty vector or with S343A-NBS1 or S278A/S343A phospho-mutants were unable to hyperphosphorylate RPA in DNA-damage-associated foci following HU treatment. The stable transfection of fully functional NBS1 in NBS cells restored RPA hyperphosphorylation. Retention of ATR on chromatin in both NBS cells and in NBS cells expressing S278A/S343A NBS1 mutants decreased after DNA damage, suggesting that ATR is the kinase responsible for RPA phosphorylation. The importance of RPA hyperphosphorylation is demonstrated by the ability of cells expressing a phospho-mutant form of RPA32 (RPA2) to suppress and delay HU-induced apoptosis. Our findings suggest that RPA hyperphosphorylation requires NBS1 and is important for the cellular response to DNA damage.

Helicobacter hepaticus Cytolethal Distending Toxin Causes Cell Death in Intestinal Epithelial Cells via Mitochondrial Apoptotic Pathway

Background:u2002 Helicobacter hepaticus, the prototype for enterohepatic Helicobacter species, colonizes the lower intestinal and hepatobiliary tracts of mice and causes typhlocolitis, hepatitis, and hepatocellular carcinoma in susceptible mouse strains. Cytolethal distending toxin (CDT) is the only known virulence factor found in H. hepaticus. CDT of several Gram‐negative bacteria is associated with double‐stranded DNA breaks resulting in cell cycle arrest and death of a wide range of eukaryotic cells in vitro. We previously observed H. hepaticus CDT (HhCDT) mediated apoptosis in INT407 cells. However, the exact mechanism for the induction of the apoptotic pathway by HhCDT is unknown. The objective of this study was to identify the apoptotic signaling pathway induced by HhCDT in INT407 cells.

Deficient DNA Damage Signaling Leads to Chemoresistance to Cisplatin in Oral Cancer

Activation of the cellular DNA damage response (DDR) is an important determinant of cell sensitivity to cisplatin and other chemotherapeutic drugs that eliminate tumor cells through induction of DNA damage. It is therefore important to investigate whether alterations of the DNA damage-signaling pathway confer chemoresistance in cancer cells and whether pharmacologic manipulation of the DDR pathway can resensitize these cells to cancer therapy. In a panel of oral/laryngeal squamous cell carcinoma (SCC) cell lines, we observed deficiencies in DNA damage signaling in correlation with cisplatin resistance, but not with DNA repair. These deficiencies are consistent with reduced expression of components of the ataxia telangiectasia mutated (ATM)-dependent signaling pathway and, in particular, strong upregulation of Wip1, a negative regulator of the ATM pathway. Wip1 knockdown or inhibition enhanced DNA damage signaling and resensitized oral SCC cells to cisplatin. In contrast to the previously reported involvement of Wip1 in cancer, Wip1 upregulation and function in these SCC cells is independent of p53. Finally, using xenograft tumor models, we showed that Wip1 upregulation promotes tumorigenesis and its inhibition improves the tumor response to cisplatin. Thus, this study reveals that chemoresistance in oral SCCs is partially attributed to deficiencies in DNA damage signaling, and Wip1 is an effective drug target for enhanced cancer therapy. Mol Cancer Ther; 11(11); 2401–9. ©2012 AACR.

Small molecule inhibitor of the RPA70 N-terminal protein interaction domain discovered using in silico and in vitro methods

The pharmacological suppression of the DNA damage response and DNA repair can increase the therapeutic indices of conventional chemotherapeutics. Replication Protein A (RPA), the major single-stranded DNA binding protein in eukaryotes, is required for DNA replication, DNA repair, DNA recombination, and DNA damage response signaling. Through the use of high-throughput screening of 1500 compounds, we have identified a small molecule inhibitor, 15-carboxy-13-isopropylatis-13-ene-17,18-dioic acid (NSC15520), that inhibited both the binding of Rad9-GST and p53-GST fusion proteins to the RPA N-terminal DNA binding domain (DBD), interactions that are essential for robust DNA damage signaling. NSC15520 competitively inhibited the binding of p53-GST peptide with an IC(50) of 10 μM. NSC15520 also inhibited helix destabilization of a duplex DNA (dsDNA) oligonucleotide, an activity dependent on the N-terminal domain of RPA70. NSC15520 did not inhibit RPA from binding single-stranded oligonucleotides, suggesting that the action of this inhibitor is specific for the N-terminal DBD of RPA, and does not bind to DBDs essential for single-strand DNA binding. Computer modeling implicates direct competition between NSC15520 and Rad9 for the same binding surface on RPA. Inhibitors of protein-protein interactions within the N-terminus of RPA are predicted to act synergistically with DNA damaging agents and inhibitors of DNA repair. Novel compounds such as NSC15520 have the potential to serve as chemosensitizing agents.

A small molecule directly inhibits the p53 transactivation domain from binding to replication protein A

Replication protein A (RPA), essential for DNA replication, repair and DNA damage signalling, possesses six ssDNA-binding domains (DBDs), including DBD-F on the N-terminus of the largest subunit, RPA70. This domain functions as a binding site for p53 and other DNA damage and repair proteins that contain amphipathic alpha helical domains. Here, we demonstrate direct binding of both ssDNA and the transactivation domain 2 of p53 (p53TAD2) to DBD-F, as well as DBD-F-directed dsDNA strand separation by RPA, all of which are inhibited by fumaropimaric acid (FPA). FPA binds directly to RPA, resulting in a conformational shift as determined through quenching of intrinsic tryptophan fluorescence in full length RPA. Structural analogues of FPA provide insight on chemical properties that are required for inhibition. Finally, we confirm the inability of RPA possessing R41E and R43E mutations to bind to p53, destabilize dsDNA and quench tryptophan fluorescence by FPA, suggesting that protein binding, DNA modulation and inhibitor binding all occur within the same site on DBD-F. The disruption of p53–RPA interactions by FPA may disturb the regulatory functions of p53 and RPA, thereby inhibiting cellular pathways that control the cell cycle and maintain the integrity of the human genome.

Transforming growth factor‐β activates c‐Myc to promote palatal growth

During palatogenesis, the palatal mesenchyme undergoes increased cell proliferation resulting in palatal growth, elevation and fusion of the two palatal shelves. Interestingly, the palatal mesenchyme expresses all three transforming growth factor (TGF) β isoforms (1, 2, and 3) throughout these steps of palatogenesis. However, the role of TGFβ in promoting proliferation of palatal mesenchymal cells has never been explored. The purpose of this study was to identify the effect of TGFβ on human embryonic palatal mesenchymal (HEPM) cell proliferation. Our results showed that all isoforms of TGFβ, especially TGFβ3, increased HEPM cell proliferation by up‐regulating the expression of cyclins and cyclin‐dependent kinases as well as c‐Myc oncogene. TGFβ activated both Smad‐dependent and Smad‐independent pathways to induce c‐Myc gene expression. Furthermore, TBE1 is the only functional Smad binding element (SBE) in the c‐Myc promoter and Smad4, activated by TGFβ, binds to the TBE1 to induce c‐Myc gene activity. We conclude that HEPM proliferation is manifested by the induction of c‐Myc in response to TGFβ signaling, which is essential for complete palatal confluency. Our data highlights the potential role of TGFβ as a therapeutic molecule to correct cleft palate by promoting growth. J. Cell. Biochem. 113: 3069–3085, 2012.

Hyaluronan suppresses prostate tumor cell proliferation through diminished expression of N-cadherin and aberrant growth factor receptor signaling.

Hyaluronan (HA) production has been functionally implicated in prostate tumorigenesis and metastasis. We previously used prostate tumor cells overexpressing the HA synthesizing enzyme HAS3 or the clinically relevant hyaluronidase Hyal1 to show that excess HA production suppresses tumor growth, while HA turnover accelerates spontaneous metastasis from the prostate. Here, we examined pathways responsible for effects of HAS3 and Hyal1 on tumor cell phenotype. Detailed characterization of cell cycle progression revealed that expression of Hyal1 accelerated cell cycle re-entry following synchronization, whereas HAS3 alone delayed entry. Hyal1 expressing cells exhibited a significant reduction in their ability to sustain ERK phosphorylation upon stimulation by growth factors, and in their expression of the cyclin-dependent kinase inhibitor p21. In contrast, HAS3 expressing cells showed prolonged ERK phosphorylation and increased expression of both p21 and p27, in asynchronous and synchronized cultures. Changes in cell cycle regulatory proteins were accompanied by HA-induced suppression of N-cadherin, while E-cadherin expression and β-catenin expression and distribution remained unchanged. Our results are consistent with a model in which excess HA synthesis suppresses cell proliferation by promoting homotypic E-cadherin mediated cell-cell adhesion, consequently signaling to elevate cell cycle inhibitor expression and suppress G1- to S-phase transition.

Protein interactomes of protein phosphatase 2A B55 regulatory subunits reveal B55-mediated regulation of replication protein A under replication stress

The specific function of PP2A, a major serine/threonine phosphatase, is mediated by regulatory targeting subunits, such as members of the B55 family. Although implicated in cell division and other pathways, the specific substrates and functions of B55 targeting subunits are largely undefined. In this study we identified over 100 binding proteins of B55α and B55β in Xenopus egg extracts that are involved in metabolism, mitochondria function, molecular trafficking, cell division, cytoskeleton, DNA replication, DNA repair, and cell signaling. Among the B55α and B55β-associated proteins were numerous mitotic regulators, including many substrates of CDK1. Consistently, upregulation of B55α accelerated M-phase exit and inhibited M-phase entry. Moreover, specific substrates of CDK2, including factors of DNA replication and chromatin remodeling were identified within the interactomes of B55α and B55β, suggesting a role for these phosphatase subunits in DNA replication. In particular, we confirmed in human cells that B55α binds RPA and mediates the dephosphorylation of RPA2. The B55-RPA association is disrupted after replication stress, consistent with the induction of RPA2 phosphorylation. Thus, we report here a new mechanism that accounts for both how RPA phosphorylation is modulated by PP2A and how the phosphorylation of RPA2 is abruptly induced after replication stress.

Phosphorylation and cellular function of the human Rpa2 N-terminus in the budding yeast Saccharomyces cerevisiae.

Maintenance of genome integrity is critical for proper cell growth. This occurs through accurate DNA replication and repair of DNA lesions. A key factor involved in both DNA replication and the DNA damage response is the heterotrimeric single-stranded DNA (ssDNA) binding complex Replication Protein A (RPA). Although the RPA complex appears to be structurally conserved throughout eukaryotes, the primary amino acid sequence of each subunit can vary considerably. Examination of sequence differences along with the functional interchangeability of orthologous RPA subunits or regions could provide insight into important regions and their functions. This might also allow for study in simpler systems. We determined that substitution of yeast Replication Factor A (RFA) with human RPA does not support yeast cell viability. Exchange of a single yeast RFA subunit with the corresponding human RPA subunit does not function due to lack of inter-species subunit interactions. Substitution of yeast Rfa2 with domains/regions of human Rpa2 important for Rpa2 function (i.e., the N-terminus and the loop 3-4 region) supports viability in yeast cells, and hybrid proteins containing human Rpa2 N-terminal phospho-mutations result in similar DNA damage phenotypes to analogous yeast Rfa2 N-terminal phospho-mutants. Finally, the human Rpa2 N-terminus (NT) fused to yeast Rfa2 is phosphorylated in a manner similar to human Rpa2 in human cells, indicating that conserved kinases recognize the human domain in yeast. The implication is that budding yeast represents a potential model system for studying not only human Rpa2 N-terminal phosphorylation, but also phosphorylation of Rpa2 N-termini from other eukaryotic organisms.