Kira G. Hartman

Modeling inflammation and oxidative stress in gastrointestinal disease development using novel organotypic culture systems

Gastroesophageal reflux disease (GERD), Barretts esophagus (BE), graft-versus-host disease (GVHD), and inflammatory bowel diseases such as ulcerative colitis and Crohns disease are common human gastrointestinal diseases that share inflammation as a key driver for their development. A general outcome resulting from these chronic inflammatory conditions is increased oxidative stress. Oxidative stress is caused by the generation of reactive oxygen and nitrogen species that are part of the normal inflammatory response, but are also capable of damaging cellular DNA, protein, and organelles. Damage to DNA can include DNA strand breaks, point mutations due to DNA adducts, as well as alterations in methylation patterns leading to activation of oncogenes or inactivation of tumor suppressors. There are a number of significant long-term consequences associated with chronic oxidative stress, most notably cancer. Infiltrating immune cells and stromal components of tissue including fibroblasts contribute to dynamic changes occurring in tissue related to disease development. Immune cells can potentiate oxidative stress, and fibroblasts have the capacity to contribute to advanced growth and proliferation of the epithelium and any resultant cancers. Disease models for GERD, BE, GVHD, and ulcerative colitis based on three-dimensional human cell and tissue culture systems that recapitulate in vivo growth and differentiation in inflammatory-associated microphysiological environments would enhance our understanding of disease progression and improve our ability to test for disease-prevention strategies. The development of physiologically relevant, human cell-based culture systems is therefore a major focus of our research. These novel models will be of enormous value, allowing us to test hypotheses and advance our understanding of these disorders, and will have a translational impact allowing us to more rapidly develop therapeutic and chemopreventive agents. In summary, this work to develop advanced human cell-based models of inflammatory conditions will greatly improve our ability to study, prevent, and treat GERD, BE, GVHD, and inflammatory bowel disease. The work will also foster the development of novel therapeutic and preventive strategies that will improve patient care for these important clinical conditions.

The evolution of S100B inhibitors for the treatment of malignant melanoma

Malignant melanoma continues to be an extremely fatal cancer due to a lack of viable treatment options for patients. The calcium-binding protein S100B has long been used as a clinical biomarker, aiding in malignant melanoma staging and patient prognosis. However, the discovery of p53 as a S100B target and the consequent impact on cell apoptosis redirected research efforts towards the development of inhibitors of this S100B-p53 interaction. Several approaches, including computer-aided drug design, fluorescence polarization competition assays, NMR, x-ray crystallography and cell-based screens have been performed to identify compounds that block the S100B-p53 association, reactivate p53 transcriptional activities and induce cancer cell death. Eight promising compounds, including pentamidine, are presented in this review and the potential for future modifications is discussed. Synthesis of compound derivatives will likely exhibit increased S100B affinity and mimic important S100B-target dynamic properties that will result in high specificity.

Covalent Small Molecule Inhibitors of Ca(2+)-Bound S100B.

Elevated levels of the tumor marker S100B are observed in malignant melanoma, and this EF-hand-containing protein was shown to directly bind wild-type (wt) p53 in a Ca2+-dependent manner, dissociate the p53 tetramer, and inhibit its tumor suppression functions. Likewise, inhibiting S100B with small interfering RNA (siRNAS100B) is sufficient to restore wild-type p53 levels and its downstream gene products and induce the arrest of cell growth and UV-dependent apoptosis in malignant melanoma. Therefore, it is a goal to develop S100B inhibitors (SBiXs) that inhibit the S100B–p53 complex and restore active p53 in this deadly cancer. Using a structure–activity relationship by nuclear magnetic resonance approach (SAR by NMR), three persistent binding pockets are found on S100B, termed sites 1–3. While inhibitors that simultaneously bind sites 2 and 3 are in place, no molecules that simultaneously bind all three persistent sites are available. For this purpose, Cys84 was used in this study as a potential means to bridge sites 1 and 2 because it is located in a small crevice between these two deeper pockets on the protein. Using a fluorescence polarization competition assay, several Cys84-modified S100B complexes were identified and examined further. For five such SBiX–S100B complexes, crystallographic structures confirmed their covalent binding to Cys84 near site 2 and thus present straightforward chemical biology strategies for bridging sites 1 and 3. Importantly, one such compound, SC1982, showed an S100B-dependent death response in assays with WM115 malignant melanoma cells, so it will be particularly useful for the design of SBiX molecules with improved affinity and specificity.

Complex Formation between S100B Protein and the p90 Ribosomal S6 Kinase (RSK) in Malignant Melanoma Is Calcium-dependent and Inhibits Extracellular Signal-regulated Kinase (ERK)-mediated Phosphorylation of RSK

Background: S100B is overexpressed in malignant melanoma and contributes to cancer progression. Results: The S100B-RSK complex was found to be Ca2+-dependent, block phosphorylation of RSK at Thr-573, and sequester RSK to the cytosol. Conclusion: The Ca2+-dependent S100B-RSK complex provides a new link between the MAPK and Ca2+ signaling pathways. Significance: S100B inhibitors may restore normal MAPK and Ca2+ signaling in malignant melanoma. S100B is a prognostic marker for malignant melanoma. Increasing S100B levels are predictive of advancing disease stage, increased recurrence, and low overall survival in malignant melanoma patients. Using S100B overexpression and shRNAS100B knockdown studies in melanoma cell lines, elevated S100B was found to enhance cell viability and modulate MAPK signaling by binding directly to the p90 ribosomal S6 kinase (RSK). S100B-RSK complex formation was shown to be Ca2+-dependent and to block ERK-dependent phosphorylation of RSK, at Thr-573, in its C-terminal kinase domain. Additionally, the overexpression of S100B sequesters RSK into the cytosol and prevents it from acting on nuclear targets. Thus, elevated S100B contributes to abnormal ERK/RSK signaling and increased cell survival in malignant melanoma.

Modeling human gastrointestinal inflammatory diseases using microphysiological culture systems

Gastrointestinal illnesses are a significant health burden for the US population, with 40 million office visits each year for gastrointestinal complaints and nearly 250,000 deaths. Acute and chronic inflammations are a common element of many gastrointestinal diseases. Inflammatory processes may be initiated by a chemical injury (acid reflux in the esophagus), an infectious agent (Helicobacter pylori infection in the stomach), autoimmune processes (graft versus host disease after bone marrow transplantation), or idiopathic (as in the case of inflammatory bowel diseases). Inflammation in these settings can contribute to acute complaints (pain, bleeding, obstruction, and diarrhea) as well as chronic sequelae including strictures and cancer. Research into the pathophysiology of these conditions has been limited by the availability of primary human tissues or appropriate animal models that attempt to physiologically model the human disease. With the many recent advances in tissue engineering and primary human cell culture systems, it is conceivable that these approaches can be adapted to develop novel human ex vivo systems that incorporate many human cell types to recapitulate in vivo growth and differentiation in inflammatory microphysiological environments. Such an advance in technology would improve our understanding of human disease progression and enhance our ability to test for disease prevention strategies and novel therapeutics. We will review current models for the inflammatory and immunological aspects of Barretts esophagus, acute graft versus host disease, and inflammatory bowel disease and explore recent advances in culture methodologies that make these novel microphysiological research systems possible.

Abstract 5243: The novel calcium-dependent direct binding of S100B to p90 ribosomal S6 kinase (RSK) decreases MAP kinase-mediated RSK activation in malignant melanoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC S100B is an effective and extensively used prognostic marker for melanoma, with increasing serum S100B being predictive of disease stage, increased recurrence, and low overall patient survival. Establishing the mechanism by which S100B alters cell signaling provides insight into how it may facilitate the progression of melanoma and aid in developing new pharmacological drugs to inhibit cancer advancement. To evaluate specific roles for S100B in malignant melanoma, our S100B knock-down and over-expression studies established a correlation between S100B expression and cell viability. ERK phosphorylation was also found to correlate with S100B levels. We have discovered RSK, a downstream ERK target, as a novel binding partner of S100B. RSK is known to regulate MAPK signaling in melanoma but the mechanism is unclear. We observed an inverse relationship between activated and phosphorylated RSK and S100B levels. We used a Ca2+-binding mutant of S100B (E31A + E72A) to demonstrate that the S100B-RSK interaction was calcium-dependent. Pull-down experiments revealed the C-terminal domain of RSK to be necessary for S100B binding. In vitro, S100B was found to reduce ERK-dependent phosphorylation of RSK at Thr573. Our cellular fractionation and immuno-fluorescence studies showed that S100B protein prevented nuclear localization of phosphorylated RSK. These data are consistent with a mechanism in which elevated S100B binds directly to RSK in a calcium-dependent manner, preventing ERK-mediated phosphorylation of RSK and inhibiting RSK localization to the nucleus. We have identified the S100B-RSK interaction as a novel drug target in melanoma cells. We are currently screening compounds for inhibitors of the S100B-RSK interaction. Citation Format: Adam D. Pierce, Kira G. Hartman, David Weber. The novel calcium-dependent direct binding of S100B to p90 ribosomal S6 kinase (RSK) decreases MAP kinase-mediated RSK activation in malignant melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5243. doi:10.1158/1538-7445.AM2013-5243

Abstract 2164: S100B affects MAPK signaling in malignant melanoma via a direct interaction with RSK

Establishing the mechanism by which S100B affects ERK and its downstream signaling provided insight into how S100B affects the progression of malignant melanoma and could aid in developing new pharmacological drugs. S100B is a 21.5 kDa symmetric homodimer that is highly conserved and expressed in a number of tissues and cell lines, including melanocytes. Generally, low levels of S100B have trophic effects, while higher levels can have dire consequences, as is the case in human malignant melanoma. S100B is an effective and widely used prognostic marker for malignant melanoma, with its increased level in serum being predictive of disease stage, increased recurrence, and low overall survival. More recently, S100B has been investigated as a potential contributor to cancer progression, which may be related to how it impacts cell signaling, including the MAPK pathway (BRAF-MEK-ERK). To further evaluate its significance, S100B knock-down clones were created from the WM115 melanoma cell line, and a positive correlation was found between S100B expression and cell viability, as measured by MTT assays. It was also discovered that cells with suppressed S100B expression showed significantly lower levels of ERK phosphorylation. Likewise, over-expression of S100B in the human melanoma cell line, 501-MEL, showed the reciprocal effect, with the introduction of high levels of S100B leading to increased cell viability and ERK phosphorylation. However, the phosphorylation status of ERK does not translate to all of its downstream targets. For example, increased RSK phosphorylation was observed in the S100B knock-down clones, and correspondingly, RSK phosphorylation was decreased with over-expression of S100B. Additionally, over-expression of a mutant S100B construct (E31A + E72A) that was incapable of binding calcium yielded neither effect, indicating that the effect of S100B on RSK phosphorylation was calcium-dependent. To determine if S100B interacted directly with RSK, pull-down assays were performed next. Consistent with the calcium-mutant data, RSK was detected in S100B pull-downs in the presence of calcium, but not in the presence of the calcium chelator EDTA. Changes in RSK localization was also observed, where RSK was enriched in the nucleus of WM115 cells when S100B was knocked down and diffuse in control cells. Together these data are consistent with a mechanism in which elevated S100B binds directly to RSK, preventing its phosphorylation by ERK and its subsequent translocation to the nucleus. Thus, the calcium-binding protein S100B affects MAPK signaling by increasing levels of phosphorylated ERK while simultaneously decreasing phosphorylated RSK. Together, these two effects of S100B on MAPK signaling could impact cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2164. doi:1538-7445.AM2012-2164