Christina R. Ross

Alternatively expressed domains of AU-rich element RNA- binding protein 1 (AUF1) regulate RNA-binding affinity, RNA-induced protein oligomerization, and the local conformation of bound RNA ligands

AU-rich element RNA-binding protein 1 (AUF1) binding to AU-rich elements (AREs) in the 3′-untranslated regions of mRNAs encoding many cytokines and other regulatory proteins modulates mRNA stability, thereby influencing protein expression. AUF1-mRNA association is a dynamic paradigm directed by various cellular signals, but many features of its function remain poorly described. There are four isoforms of AUF1 that result from alternative splicing of exons 2 and 7 from a common pre-mRNA. Preliminary evidence suggests that the different isoforms have varied functional characteristics, but no detailed quantitative analysis of the properties of each isoform has been reported despite their differential expression and regulation. Using purified recombinant forms of each AUF1 protein variant, we used chemical cross-linking and gel filtration chromatography to show that each exists as a dimer in solution. We then defined the association mechanisms of each AUF1 isoform for ARE-containing RNA substrates and quantified relevant binding affinities using electrophoretic mobility shift and fluorescence anisotropy assays. Although all AUF1 isoforms generated oligomeric complexes on ARE substrates by sequential dimer association, sequences encoded by exon 2 inhibited RNA-binding affinity. By contrast, the exon 7-encoded domain enhanced RNA-dependent protein oligomerization, even permitting cooperative RNA-binding activity in some contexts. Finally, fluorescence resonance energy transfer-based assays showed that the different AUF1 isoforms remodel bound RNA substrates into divergent structures as a function of protein:RNA stoichiometry. Together, these data describe isoform-specific characteristics among AUF1 ribonucleoprotein complexes, which likely constitute a mechanistic basis for differential functions and regulation among members of this protein family.

A dimer interface mutation in glyceraldehyde 3-phosphate dehydrogenase regulates its binding to AU-rich RNA.

DOI 10.1074/jbc.A114.618165 A dimer interface mutation in glyceraldehyde 3-phosphate dehydrogenase regulates its binding to AU-rich RNA. Michael R. White, Mohd M. Khan, Daniel Deredge, Christina R. Ross, Royston Quintyn, Beth E. Zucconi, Vicki H. Wysocki, Patrick L. Wintrode, Gerald M. Wilson, and Elsa D. Garcin PAGE 1780: The charge state distributions shown for the mass spectra peaks in Fig. 7B were not correct. The correct charge state distributions are shown in the revised Fig. 7B. This correction does not affect the results or conclusions of this work. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 7, p. 4129, February 13, 2015

Antiproliferative activities of halogenated thieno[3,2-d]pyrimidines

The in vitro evaluation of thieno[3,2-d]pyrimidines identified halogenated compounds 1 and 2 with antiproliferative activity against three different cancer cell lines. A structure activity relationship study indicated the necessity of the chlorine at the C4-position for biological activity. The two most active compounds 1 and 2 were found to induce apoptosis in the leukemia L1210 cell line. Additionally, the compounds were screened against a variety of other microbial targets and as a result, selective activity against several fungi was also observed. The synthesis and preliminary biological results are reported herein.

Coordinated expression of tristetraprolin post-transcriptionally attenuates mitogenic induction of the oncogenic Ser/Thr kinase Pim-1.

The serine/threonine kinase Pim-1 directs selected signaling events that promote cell growth and survival and is overexpressed in diverse human cancers. Pim-1 expression is tightly controlled through multiple mechanisms, including regulation of mRNA turnover. In several cultured cell models, mitogenic stimulation rapidly induced and stabilized PIM1 mRNA, however, vigorous destabilization 4–6 hours later helped restore basal expression levels. Acceleration of PIM1 mRNA turnover coincided with accumulation of tristetraprolin (TTP), an mRNA-destabilizing protein that targets transcripts containing AU-rich elements. TTP binds PIM1 mRNA in cells, and suppresses its expression by accelerating mRNA decay. Reporter mRNA decay assays localized the TTP-regulated mRNA decay element to a discrete AU-rich sequence in the distal 3′-untranslated region that binds TTP. These data suggest that coordinated stimulation of TTP and PIM1 expression limits the magnitude and duration of PIM1 mRNA accumulation by accelerating its degradation as TTP protein levels increase. Consistent with this model, PIM1 and TTP mRNA levels were well correlated across selected human tissue panels, and PIM1 mRNA was induced to significantly higher levels in mitogen-stimulated fibroblasts from TTP-deficient mice. Together, these data support a model whereby induction of TTP mediates a negative feedback circuit to limit expression of selected mitogen-activated genes.

Contributions of the Histidine Side Chain and the N-Terminal α-Amino Group to the Binding Thermodynamics of Oligopeptides to Nucleic Acids as a Function of pH

Interactions of histidine with nucleic acid phosphates and histidine pK(a) shifts make important contributions to many protein-nucleic acid binding processes. To characterize these phenomena in simplified systems, we quantified binding of a histidine-containing model peptide HWKK ((+)NH(3)-His-Trp-Lys-Lys-NH(2)) and its lysine analogue KWKK ((+)NH(3)-Lys-Trp-Lys-Lys-NH(2)) to a single-stranded RNA model, polyuridylate (polyU), by changes in tryptophan fluorescence as a function of salt concentration and pH. For both HWKK and KWKK, equilibrium binding constants, K(obs), and magnitudes of log-log salt derivatives, SK(obs) identical with (partial differential logK(obs)/partial differential log[Na(+)]), decreased with increasing pH in the manner expected for a titration curve model in which deprotonation of the histidine and alpha-amino groups weakens binding and reduces its salt-dependence. Fully protonated HWKK and KWKK exhibit the same K(obs) and SK(obs) within uncertainty, and these SK(obs) values are consistent with limiting-law polyelectrolyte theory for +4 cationic oligopeptides binding to single-stranded nucleic acids. The pH-dependence of HWKK binding to polyU provides no evidence for pK(a) shifts nor any requirement for histidine protonation, in stark contrast to the thermodynamics of coupled protonation often seen for these cationic residues in the context of native protein structure where histidine protonation satisfies specific interactions (e.g., salt-bridge formation) within highly complementary binding interfaces. The absence of pK(a) shifts in our studies indicates that additional Coulombic interactions across the nonspecific-binding interface between RNA and protonated histidine or the alpha-amino group are not sufficient to promote proton uptake for these oligopeptides. We present our findings in the context of hydration models for specific vs nonspecific nucleic acid binding.

Tristetraprolin induces cell cycle arrest in breast tumor cells through targeting AP-1/c-Jun and NF-κB pathway

The main characteristic of cancers, including breast cancer, is the ability of cancer cells to proliferate uncontrollably. However, the underlying mechanisms of cancer cell proliferation, especially those regulated by the RNA binding protein tristetraprolin (TTP), are not completely understood. In this study, we found that TTP inhibits cell proliferation in vitro and suppresses tumor growth in vivo through inducing cell cycle arrest at the S phase. Our studies demonstrate that TTP inhibits c-Jun expression through the C-terminal Zn finger and therefore increases Wee1 expression, a regulatory molecule which controls cell cycle transition from the S to the G2 phase. In contrast to the well-known function of TTP in regulating mRNA stability, TTP inhibits c-Jun expression at the level of transcription by selectively blocking NF-κB p65 nuclear translocation. Reconstitution of NF-κB p65 completely abolishes the inhibition of c-Jun transcription by TTP. Moreover, reconstitution of c-Jun in TTP-expressing breast tumor cells diminishes Wee1 overexpression and promotes cell proliferation. Our results indicate that TTP suppresses c-Jun expression that results in Wee1 induction which causes cell cycle arrest at the S phase and inhibition of cell proliferation. Our study provides a new pathway for TTP function as a tumor suppressor which could be targeted in tumor treatment.

Mitotic arrest of breast cancer MDA-MB-231 cells by a halogenated thieno[3,2-d]pyrimidine

Halogenated thieno[3,2-d]pyrimidines exhibit antiproliferative activity against a variety of cancer cell models, such as the mouse lymphocytic leukemia cell line L1210 in which they induce apoptosis independent of cell cycle arrest. Here we assessed these activities on MDA-MB-231 cells, a well-established model of aggressive, metastatic breast cancer. While 2,4-dichloro[3,2-d]pyrimidine was less toxic to MDA-MB-231 cells than previously observed in the L1210 model, flow cytometry analysis showed that MDA-MB-231 cell death involved arrest at the G2/M stage of the cell cycle. Conversely, the introduction of bromine at C7 of the 2,4-dichloro[3,2-d]pyrimidine eliminated cell type-dependent differences in cytotoxicity or cell cycle status. Together, these data indicate that a substituent at C7 can profoundly modify the cytotoxic mechanism of halogenated thieno[3,2-d]pyrimidines in a cell type-specific manner.

Abstract 1970: The mRNA-destabilizing protein tristetraprolin suppresses tumorigenic phenotypes in a triple-negative breast cancer cell model via a non-canonical mechanism

The purpose of this study was to define mechanisms by which the mRNA-destabilizing factor tristetraprolin (TTP) impacts tumorigenic phenotypes in an aggressive model of metastatic breast cancer. Widespread repression of TTP levels in human tumors and cancer cell lines relative to non-transformed tissues suggests that TTP may function as a tumor suppressor in diverse neoplastic contexts, and low TTP expression is a negative prognostic indicator in breast cancer. TTP is a tandem zinc finger protein that binds to AU-rich elements (AREs) and targets their associated mRNAs for degradation. AREs are potent cis-acting determinants of cytoplasmic mRNA turnover in mammalian cells, and are essential for limiting cellular production of many clinically important gene products including regulators of inflammation, cell proliferation, and apoptosis. In this study, we show that restoration of TTP levels attenuates several tumorigenic phenotypes in the aggressively metastatic breast cancer line MDA-MB-231. Notably, TTP-expressing cell lines replicate approximately 70% slower than non-transfected controls. Inhibition of cell proliferation did not result from apoptosis but rather by a delay at the G1/S checkpoint. TTP expression also significantly reduced the formation of cultured mammospheres based on both sphere-forming efficiency frequency and extreme limiting dilution analyses, indicating that this protein reduces stemness and non-adherent growth potential. Finally, cell motility was suppressed by TTP, demonstrated using wound healing assays. These findings are consistent with a tumor suppressor role for TTP. To identify potential mechanisms linking TTP to diminution of tumorigenic phenotypes, we surveyed the expression of genes encoding select pro-tumorigenic factors, including several known to encode TTP-targeted mRNAs. Expression of cyclin D1, cyclin E and c-Myc proteins were significantly reduced in TTP-expressing MDA-MB-231 cells, however, this was not mediated by accelerated mRNA decay. Interestingly, we observed that the mRNA-destabilizing function of TTP is abrogated by the constitutively active ERK signaling pathway in MDA-MB-231 cells, suggesting that TTP suppresses tumorigenic properties in this cell model independently of its canonical mRNA-destabilizing function. Consistent with this model, expressing a non-RNA-binding mutant form of TTP (C147R) robustly attenuated the same tumorigenic properties suppressed by the wild type protein. Together, these findings show that the mRNA-destabilizing activity of TTP is dispensable for its tumor suppressive properties in MDA-MB-231 cells, and by extension that TTP must limit diverse tumorigenic properties in these cells via an as yet uncharacterized non-canonical mechanism. Citation Format: Christina R. Ross, Gerald M. Wilson. The mRNA-destabilizing protein tristetraprolin suppresses tumorigenic phenotypes in a triple-negative breast cancer cell model via a non-canonical mechanism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1970. doi:10.1158/1538-7445.AM2015-1970

Abstract 3296: Tumor suppressive effects of tristetraprolin expression in metastatic breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The purpose of this study is to define mechanisms by which the mRNA-destabilizing factor tristetraprolin (TTP) impacts tumorigenic phenotypes in an aggressive model of metastatic breast cancer. Widespread suppression of TTP levels in human tumors and cancer cell lines relative to non-transformed tissues suggests that TTP may function as a tumor suppressor in diverse neoplastic contexts, and low TTP expression is a negative prognostic indicator in breast cancer. TTP is a tandem zinc finger protein that binds to AU-rich elements (AREs) and targets their associated mRNAs for degradation. AREs are potent cis-acting determinants of cytoplasmic mRNA turnover in mammalian cells, and are essential for limiting cellular production of many clinically important gene products including regulators of inflammation, cell proliferation, and apoptosis. Surveys of patient-derived samples showed that TTP expression is diminished in many neoplastic tissues, particularly those from aggressive cancers. In this study, we show that restoration of TTP levels attenuates several tumorigenic phenotypes in the cultured metastatic breast cancer line MDA-MB-231, consistent with a tumor suppressor role for TTP. Notably, TTP-expressing cell lines replicate approximately 70% slower than non-transfected controls. Inhibition of cell proliferation did not result from apoptosis but rather by a delay at the G1/S checkpoint. TTP expression also significantly reduced the formation of cultured mammospheres based on both sphere-forming efficiency frequency and extreme limiting dilution analyses, indicating that this protein reduces stemness and non-adherent growth potential. Cell motility was also suppressed by TTP, demonstrated using wound healing assays. To identify potential mechanisms linking TTP to diminution of tumorigenic phenotypes, we surveyed the expression of genes encoding selected pro-tumorigenic factors, including several known to encode TTP-targeted mRNAs. Expression of cyclins D1 and E as well as the angiogenic factor VEGF were significantly reduced in TTP-expressing MDA-MB-231 cells, however, this was not mediated by accelerated mRNA decay. Interestingly, we observed that the mRNA-destabilizing function of TTP is abrogated by the constitutively active ERK signaling pathway in this cell model, indicating that the mRNA-destabilizing activity of TTP may be dispensable for at least a subset of its tumor suppressive properties. Based on subsequent analyses of TTP effects on the expression of selected transcription factors at both the mRNA and protein levels, we have developed a working model whereby TTP may suppress diverse tumorigenic phenotypes in this metastatic cancer cell model by repressing the function of a strongly pro-oncogenic transcriptional regulatory circuit, even in the absence of its canonical mRNA-destabilizing activity. Citation Format: Christina R. Ross, Gerald M. Wilson. Tumor suppressive effects of tristetraprolin expression in metastatic breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3296. doi:10.1158/1538-7445.AM2014-3296