KIF11 inhibition for glioblastoma treatment: reason to hope or a struggle with the brain?

<p>Abstract</p> <p>Background</p> <p>Glioblastomas (GBM) are typically comprised of morphologically diverse cells. Despite current advances in therapy, including surgical resection followed by radiation and chemotherapy, the prognosis for patients with GBM remains poor. Unfortunately, most patients die within 2 years of diagnosis of their disease. Molecular abnormalities vary among individual patients and also within each tumor. Indeed, one of the distinguishing features of GBM is its marked genetic heterogeneity. Due to the brain location of the tumor, the potential target inhibition for anticancer therapy must exhibit a manageable neurotoxicity profile in the concentration range in which the compounds show anti-proliferative activity.</p> <p>Kinesin KIF11 inhibition by small molecules such as Monastrol or Ispinesib is currently under investigation in the field of malignant tumors. In the current study we have assessed the relevance of the anti-mitotic Kinesin-like protein KIF11 in human GBM cell-lines.</p> <p>Results</p> <p>In this study the target was validated using a set of well characterised and potentially specific small molecule inhibitors of KIF11: an ispinesib analog, Monastrol, a Merck compound and 3 simplified derivatives of the Merck compound. Following an <it>in silico </it>selection, those compounds predicted to bear a favorable BBB permeation profile were assessed for their phenotypic effect on cell lines derived both from primary (U87MG) as well as treated (DBTRG-05-MG) glioblastomas. For some compounds, these data could be compared to their effect on normal human astrocytes, as well as their neurotoxicity on primary rat cortical neurons. The ispinesib analogue 1 showed an anti-proliferative effect on GBM cell lines by blocking them in the G2/M phase in a concentration range which was shown to be harmless to primary rat cortical neurons. Furthermore, ispinesib analog increased caspase 3/7-induced apoptosis in U87MG cells.</p> <p>Conclusion</p> <p>In the area of cell cycle inhibition, KIF11 is critical for proper spindle assembly and represents an attractive anticancer target. Our results suggest that KIF11 inhibitors, when able to permeate the blood-brain-barrier, could represent an interesting class of anticancer drugs with low neurotoxic effects in the treatment of brain tumors.</p

Pancreatic cancer spheres are more than just aggregates of stem marker-positive cells

Pancreatic cancer stem-like cells are described by membrane expression of CD24, CD44 and ESA (epithelial-specific antigen) and their capacity to grow as spheres in a serum-free medium containing well-defined growth factors. The capacity of a panel of four pancreatic cancer cell lines (PANC-1, CFPAC-1, PancTu-1 and PSN-1) to form spheres was tested. All cell lines with the exception of PancTu-1 developed spheres. Phenotypically, the sphere-growing cells showed an increased in vitro invasion capability. Both gene and protein expressions of markers of metastases [CXCR4 (CXC chemokine receptor 4), OPN (osteopontin) and CD44v6] and components of active hedgehog pathway signalling were assessed. Spheres clearly demonstrated increased expression of the above-mentioned markers when compared with their adherent counterpart. With the aim of identifying a minimum set of markers able to separate cells that have the capacity to form spheres from those incapable of forming spheres, a PCA (principal component analysis) of the multidimensional dataset was performed. Although PCA of the ‘accepted’ stemness genes was unable to separate sphere-forming from sphere-incapable cell lines, the addition of the ‘aggressiveness’ marker CD44v6 allowed a clear differentiation. Moreover, inoculation of the spheres and the adherent cells in vivo confirmed the superior aggressiveness (proliferation and metastasis) of the spheres over the adherent cells. In conclusion, the present study suggests that the sphere-growing cell population is not only composed of cells displaying classical stem membrane markers but also needs CD44v6-positive cells to successfully form spheres. Our results also emphasize the potential therapeutic importance of pathways such as CXCR4 and hedgehog for pancreatic cancer treatment

Extensive temporally regulated reorganization of the lipid raft proteome following T-cell antigen receptor triggering.

Signalling by immunoreceptors is orchestrated at specific plasma membrane microdomains, referred to as lipid rafts. Here we present a proteomics approach to the temporal analysis of protein association with lipid rafts following T-cell antigen receptor (TCR) triggering. We show that TCR engagement promotes the temporally regulated recruitment of proteins participating in the TCR signalling cascade to lipid rafts. Furthermore, TCR triggering results in profound modifications in the composition of lipid rafts involving a number of proteins associated either directly or indirectly with both plasma and intracellular membranes. Raft-associated proteins can be clustered according to their temporal profile of raft association. The data identify lipid rafts as highly dynamic structures and reveal a dramatic impact of surface TCR triggering not only on components of the TCR signalling machinery but also on proteins implicated in a number of diverse cellular processes

Copper binding to R1 and R2 fragments of Tau protein

Tau is a 441-mer peptide present in significant amounts in neurons, where it contributes to the stabilization of microtubules. Insoluble amyloid aggregates of tau are associated with over 20 neurological disorders known as tauopathies, among which is Parkinson.[1] In neurons, tau binds tubulin through its microtubule binding domain which comprises four repeats (R1-R4) characterized by the presence of histidine residues. These regions are potential binding sites for metal ions.[2] The elucidation of the binding capacities toward metal ions, especially those redox active such as copper(II), may shed light on the biomolecular processes that underlie the progression of tauopathies.[3] In this contribution we examine the stability of Cu(II) and Cu(I) adducts with two peptide fragments which are encompassed in the R1 and R3 repeats of tau (Fig. 1). R1 (HL): Ac-257VKSKIGSTENLKHQGGG273-NH2 R3 (L): Ac-323GSLGNIHHKPGGG335-NH2 Copper(II) binding to R1 (HL) starts at pH 4. The relevant species at pH 7.4 is [CuLH]2+, where the imidazole ring, two amidic nitrogen atoms, and a water molecule occupy the equatorial coordination positions of copper(II). As for the R3 peptide, at pH 7.4 [CuL]2+ and [CuLH-1]+ are the two most abundant species (in a ratio of ca. 1:2). In the case of [CuL]2+, the two imidazole groups of R3 and one deprotonated amidic nitrogen atom are bound to the equatorial plane. In [CuLH-1]+, a further amidic nitrogen bounds the metal ion in the equatorial plane, most likely pushing one imidazole group to the axial position. Copper(II) binding to R1 (HL) starts at pH 4. The relevant species at pH 7.4 is [CuLH]2+, where the imidazole ring, two amidic nitrogen atoms, and a water molecule occupy the equatorial coordination positions of copper(II). As for the R3 peptide, at pH 7.4 [CuL]2+ and [CuLH-1]+ are the two most abundant species (in a ratio of ca. 1:2). In the case of [CuL]2+, the two imidazole groups of R3 and one deprotonated amidic nitrogen atom are bound to the equatorial plane. In [CuLH-1]+, a further amidic nitrogen bounds the metal ion in the equatorial plane, most likely pushing one imidazole group to the axial position. Copper(I) adducts with the R1 and R3 tau fragments were investigated via spectrophotometric competition titrations with the metallochromic ligand ferrozine (Fz).[4] The chromophoric complex [CuI(Fz)2]3- (having two characteristic absorption bands at 470 nm and 600 nm) is formed by titrating a Cu(I) solution with ferrozine. The back titration of this solution with the R1 and R3 fragments, led to a decrease in the absorbance values (Fig. 2). A significative change in the absorbance, which decreases of almost 0.40 units, is observed upon the addition of R3 to [CuI(Fz)2]3-. On the contrary, in the case of the R1 peptide, the absorbance decrease of only 0.20 units can be fully accounted by dilution effects. Data treatment using HypSpect program yields a log β value of 10.1(2) for the Cu(I)-R3 complex, while for the back titration of [CuI(Fz)2]3- with R1 it confirms the absence of significant interactions of Cu(I) with R1. NMR data suggest that the binding of Cu(I) to R3 occurs at the tandem HH site, as it occurs for Cu(II). The redox behavior of these complexes will be discussed in terms of their speciation. Also, an insight of the role of the copper adducts with R1 and R3 in catecholase activity will be given. REFERENCES [1] M. Goedert, D. S. Eisenberg, R. A. Crowther, Annu. Rev. Neurosci. 2017, 40, 189-210. [2] M. G. Savelieff, S. Lee, Y. Liu, M. H. Lim, ACS Chem. Biol. 2013, 8, 856-865. [3] A. Soragni, B. Zambelli, M. D. Mukrasch, J. Biernat, S. Jeganathan, C. Griesinger, S. Ciurli, E. Mandelkow, M. [3] Zweckstetter, Biochemistry 2008, 47, 10841-51. [4] Z. Xiao, L. Gottschlich, R. Meulen, S. R. Udagedara, A. G. Wedd, Metallomics, 2013, 5, 501-513. ACKNOWLEDGEMENTS The authors acknowledge MIUR for financial support through the project "Metal ions, dopamine, and oxidative stress in Parkinson's disease” (PRIN 2015T778JW)

Copper(I) and Copper(II) Binding to R1 and R3 Fragments of Tau Protein

Tau (τ) is a 441-mer peptide present in significant amounts in neurons, where it contributes to the stabilization of microtubules. Insoluble amyloid aggregates of tau are associated with over 20 neurological disorders known as tauopathies, among which is Parkinson’s [1]. In neurons, tau binds tubulin through its microtubule binding domain which comprises four repeats (R1-R4) characterized by the presence of histidine residues. These regions are potential binding sites for metal ions [2]. The elucidation of the binding capacities toward metal ions, especially those redox active such as copper(II), may shed light on the biomolecular processes that underlie the progression of tauopathies [3]. In this contribution we examine the thermodynamic stability of copper(I) and copper(II) adducts with two peptide fragments which are encompassed in the R1 and R3 repeats of tau, namely Ac- 257VKSKIGSTENLKHQGGG273-NH2 (R1τ, HL1 in its neutral form) and Ac-323GSLGNIHHKPGGG335- NH2 (R3τ, L3 in its neutral form). Copper(II) binding to R1τ (HL1) at pH 7.4 is dominated by the formation of [Cu(HL1)]2+, where (L1)- is tridentate. The copper(II) equatorial coordination positions are occupied by the imidazole ring of His269, two amido nitrogens, and a water molecule. As for the R3τ (L3) peptide, at pH 7.4 the two most abundant species are [CuL3]2+ and [Cu(L3H-1)]+ (in a ratio of ca. 1:4, Figure 1, left). While copper(II) coordination mode in [Cu(L3H-1)]+ is similar to that in [Cu(HL1)]2+, that of [CuL3]2+ is different and possibly most interesting. Spectroscopic data suggest that in [CuL3]2+ two imidazole donors and one amido nitrogen are equatorially coordinated to copper(II), plus a water molecule (Figure 1, right). The presence of this tandem HisHis fragment makes this peptide interesting in view of the stabilization of copper(I). Indeed, spectroscopic competition titration using a metallochromic indicator clearly showed that copper(I) binds significantly to R3τ at neutral pH but not to R1τ. The catalytic activity in reactions of oxidation of catecholes and the NMR features of these complexes will be discussed in terms of the speciation of the thermodynamic stability of these complexes with copper in both oxidation states. The authors acknowledge MIUR for financial support through the project "Metal ions, dopamine, and oxidative stress in Parkinson's disease” (PRIN 2015T778JW). References [1] M. Goedert, D. S. Eisenberg, R. A. Crowther, Annu. Rev. Neurosci. 40 (2017) 189–210. J.A. White "Biological inorganic chemistry" (1973) Oxford University Press, Oxford. [2] M. G. Savelieff, S. Lee, Y. Liu, M. H. Lim, ACS Chem. Biol. 8 (2013) 856–865. [3] A. Soragni, B. Zambelli, M. D. Mukrasch, J. Biernat, S. Jeganathan, C. Griesinger, S. Ciurli, E. Mandelkow, M. Zweckstetter, Biochemistry 47 (2008) 10841–10851

F-actin dynamics controls segregation of the TCR signaling cascade to clustered lipid rafts

Following ligand binding the TCR segregates to plasma membrane microdomains, termed lipid rafts, characterized by a highly ordered lipid structure favoring partitioning of glycosyl phosphatidyl inositol-linked costimulatory receptors and acylated signaling molecules. Here we show that the inducible association of the TCR and key signaling proteins with lipid rafts is dependent on the actin cytoskeleton through a mechanism involving raft coalescence. Although lipid rafts are required for full activation of the TCR-dependent tyrosine phosphory- lation cascade and sustained signaling, triggering of TCR-proximal events, including Fyn activation and a first wave of Vav phosphorylation, is independent of lipid rafts, while a sec- ond wave of raft-dependent Vav phosphorylation occurs after raft coalescence, as also sup- ported by the finding that Vav is phosphorylated in response to lipid raft clustering by GM1 aggregation. The constitutive association found between Vav and the CD3 ́ chain suggests a model whereby the TCR-associated signaling machinery initiates raft aggregation by pro- moting F-actin reorganization, which permits full activation of the tyrosine phosphorylation cascade, further reorganization of the actin cytoskeleton and sustained signaling, leading to cell activatio