• S100B regulates IL-6 signaling via the p90 ribosomal S6 kinase (RSK) in malignant melanoma

      Alasady, Milad; Weber, David J., Ph.D. (2017)
      The S100B protein, a member of the S100 protein family, is highly elevated during the progression of melanoma. Elevated level of S100B in the serum is used as a marker in melanoma and other cancers. However, the function of S100B in the progression of melanoma is not completely understood. Here we uncovered a regulatory mechanism that defines interplay between S100B, Interleukin-6 (IL-6), STAT3, and CREB. First, we show that S100B inhibits IL-6 mRNA and protein levels. Silencing S100B in melanoma cells induces the expression and secretion of IL-6, which in turn induced STAT3 phosphorylation and activation. S100B exerts its effect on the IL-6/STAT3 pathway via the p90 ribosomal S6 kinase (RSK), and the phosphorylation and activation of transcription factor CREB. High S100B in melanoma cells binds to RSK and sequesters RSK in the cytoplasm. Silencing of S100B enables RSK nuclear translocation, which in turn elevates CREB phosphorylation and its transcriptional activity in the nucleus to induce IL-6 expression. Therefore, high S100B in melanoma suppresses IL-6 expression. Since IL-6 was shown to inhibit the proliferation of melanocytes and early stage melanoma cells, we propose that the suppression of IL-6 by S100B evolved to circumvent the inhibitory effect of IL-6 and STAT3 at early stages of melanoma. It is also possible that suppression of IL-6 by S100B evolved to curb the local immune response, which otherwise would be elevated by the secreted IL-6. Indeed, we show that S100B silencing upregulates the expression of several chemokine ligands, chemokine receptors, and interleukins that are involved in the immune response. For example, we show that S100B depletion induces expression of cytokines, CSF-1 and CSF-2, in STAT3-dependent manner. These results suggest that S100B inhibits interleukins and chemokines to perhaps curb the immune response within the tumor microenvironment. Future experiments in mice models and cell culture systems are necessary to further evaluate the role of S100B in regulating the immune response in malignant melanoma. We are also in search for S100B inhibitors that can potentially prevent S100B-target complex formation and reduce tumor growth. Cellular characterization of pentamidine/heptamidine derivatives, covalently bound inhibitors, and SC0025 were explored in the non-targeting scrambled and stable S100B knockdown WM115 cell line. We show that lower concentrations of SC124, SBi4172, SC1982, and SC0025 inhibitors were needed to inhibit cell growth in the non-targeting scrambled WM115 versus stable S100B knockdown WM115 cells indicating that these compounds have specificity toward S100B-containing cells. We also show that SC1982 restored p53 protein level by more than 2-fold, while SC0025 restored only IL-6 protein level. The characterization of SC1982 and SC0025, which occupy sites 2 and 3 within S100B binding pockets, respectively, reveals that occupying one binding site within S100B binding pocket is not sufficient to restore multiple S100B targets. The goal is to identify S100B inhibitors that can prevent binding of multiple targets and reduce tumor growth as therapeutic intervention for melanoma therapy with elevated S100B.
    • The three-dimensional structure and subunit association of S100B in the apo and Ca(2+)-bound states, and its Ca(2+)-dependent interaction with target proteins

      Drohat, Alexander Clark; Weber, David J., Ph.D. (1997)
      S100B(beta beta), a member of the S100 protein family, is a Ca2+-binding protein with noncovalent interactions at its dimer interface. The solution structure of S100B(beta beta) has been determined, in the apo- and Ca2+-loaded state, using 2D, 3D, and 4D NMR spectroscopy. In both states, the S100beta subunits (91 residue) contain four alpha-helices and a small antiparallel beta-sheet which brings together the two helix-loop-helix Ca2+-binding domains (EF-hands). Both structures are found to be globular and compact with an extensive hydrophobic core and a highly charged surface, consistent with the high solubility of S100B(beta beta). The antiparallel alignment of helix 1 with 1 prime (other subunit) and of helix 4 with 4 prime, and the perpendicular association of these pairs of antiparallel helices forms an X-type four helical bundle at the dimer interface in both structures. However, the orientation of helix 3 relative to helices 1, 2, and 4 in each subunit of apo-S100B(beta beta) differs significantly from that of Ca2+-loaded S100B(beta beta). This conformational change is well illustrated by the difference in interhelical angle (delta omega=112 degrees) of the C-terminal EF-hands (apo-S100beta, omega=140 degrees; Ca2+-loaded S100beta, omega=106 degrees). For comparison, omega ranges from 118 degrees to 145 degrees in the apo-state and from 84 degrees to 128 degrees in the Ca2+-bound states of the EF-hands in calbindin D subscript 9k, calcyclin, and calmodulin. The significant conformational change required of C-terminal EF-hand for it to adopt the Ca2+-bound structure readily explains the dramatic spectral effects previously observed for S100B({beta beta) upon the addition of Ca2+. The conformational change exposes a cleft defined by residues in the loop linking the EF-hands, helix 3, and the C-terminal loop of Ca2+-loaded S100B({beta beta). This cleft is absent in the apo-structure and is therefore likely to play a role in target protein binding. S100B(beta beta) has been shown to interact with the tumor suppressor protein, p53, and this interaction was examined with a peptide derived from the C-terminal regulatory domain of p53 (residues 367-388). Fluorescence and NMR spectroscopy experiments show that the p53 peptide binds to a location of S100B(beta beta) that probably involves residues in the hinge region (S41, L44, E45, E46) and the C-terminal loop (A83, C84, H85, E86, F87, F88) as previously predicted. However, residues in helix 3 (V52, V53, V56, T59) are also effected by p53 peptide binding, which supports the proposal that the conformational change observed in helix 3 plays a role in target protein binding. Finally, S100B(beta beta) was shown to exist (>99%) as a non-covalently associated dimer at concentrations as low as 1 nM (subunit concentration, 500 pM dimer) in apo and Ca2+-loaded state. Therefore, in reducing environments and at physiological concentrations, the noncovalent dimer is most likely the form of S100B presented to target proteins.