Acentrosomal spindle organization renders cancer cells dependent on the kinesin HSET

Centrosomes represent the major microtubule organizing centres (MTOCs) of animal somatic cells and orchestrate bipolar spindle assembly during mitotic cell division. In meiotic cells, the kinesin HSET compensates for the lack of centrosomes by focusing acentrosomal MTOCs into two spindle poles. By clustering multiple centrosomes into two spindle poles, HSET also mediates bipolar mitosis in cancer cells with supernumerary centrosomes. However, although dispensable in non-transformed human cells, the role of HSET in cancer cells with two centrosomes has remained elusive. In this study, we demonstrate that HSET is required for proper spindle assembly, stable pole-focusing and survival of cancer cells irrespective of normal or supernumerary centrosome number. Strikingly, we detected pronounced acentrosomal MTOC structures in untreated mitotic cancer cells. While in most cancer cells these acentrosomal MTOCs were rapidly incorporated into the assembling bipolar spindle, some cells eventually established bipolar spindles with acentrosomal poles and free centrosomes. These observations demonstrate that acentrosomal MTOCs were functional and that both centrosomal and acentrosomal mechanisms were required for bipolar spindle organization. Our study shows that HSET is critical for clustering acentrosomal and centrosomal MTOCs during spindle formation in human cancer cells with two bona fide centrosomes. Furthermore, we show that in checkpoint-defective cancer cells, acentrosomal spindle formation and HSET-dependence are partially mediated by a constitutive activation of the DNA damage response. In summary, we propose that acentrosomal spindle assembly mechanisms are hyperactive in cancer cells and promote HSET, a key driver of acentrosomal spindle organization, as an attractive target for cancer therapy

Partial deficiency of sphingosine-1-phosphate lyase confers protection in experimental autoimmune encephalomyelitis

Background: Sphingosine-1-phosphate (S1P) regulates the egress of T cells from lymphoid organs; levels of S1P in the tissues are controlled by S1P lyase (Sgpl1). Hence, Sgpl1 offers a target to block T cell-dependent inflammatory processes. However, the involvement of Sgpl1 in models of disease has not been fully elucidated yet, since Sgpl1 KO mice have a short life-span. Methodology: We generated inducible Sgpl1 KO mice featuring partial reduction of Sgpl1 activity and analyzed them with respect to sphingolipid levels, T-cell distribution, and response in models of inflammation. Principal Findings: The partially Sgpl1 deficient mice are viable but feature profound reduction of peripheral T cells, similar to the constitutive KO mice. While thymic T cell development in these mice appears normal, mature T cells are retained in thymus and lymph nodes, leading to reduced T cell numbers in spleen and blood, with a skewing towards increased proportions of memory T cells and T regulatory cells. The therapeutic relevance of Sgpl1 is demonstrated by the fact that the inducible KO mice are protected in experimental autoimmune encephalomyelitis (EAE). T cell immigration into the CNS was found to be profoundly reduced. Since S1P levels in the brain of the animals are unchanged, we conclude that protection in EAE is due to the peripheral effect on T cells, leading to reduced CNS immigration, rather than on local effects in the CNS. Significance: The data suggest Sgpl1 as a novel therapeutic target for the treatment of multiple sclerosis

Sphingolipid concentration in selected tissues of inducible Sgpl1-deficient mice.

<p>Two weeks after tamoxifen induction, tissues of Sgpl1^Flox/Flox Cre^+/− mice (open bars) and of Sgpl1^Flox/Flox Cre^−/− controls (filled bars) were obtained (n = 5/group). Tissues were extracted and sphingolipids were quantified by LC/MS. <i>A, B,</i> S1P; <i>C, D</i>, Sph; <i>E,</i> C16-ceramide. <i>A, C,</i> and <i>E</i> show absolute concentrations per weight of tissue; <i>B and D</i> show fold increase in inducible KO mice.</p

Foxp3⁺ Treg are overrepresented in LN and spleen of in inducible Sgpl1-deficient mice.

<p>Two weeks after tamoxifen treatment, cells from blood, LN and spleen were stained for T cell markers and Foxp3. <i>A,</i> Mean percentage and <i>B</i>, absolute numbers of Foxp3⁺ cells among CD4⁺ T cells (n = 4/group).</p

Normal T cell development, reduced splenic cellularity, and increased LN cell number in inducible Sgpl1-deficient mice.

<p>B and T cell subpopulations in tamoxifen-treated Sgpl1^Flox/FloxCre^+/− (open bars) and Sgpl1^Flox/Flox Cre^−/− mice (closed bars) (n = 4/group), were enumerated based on total live cell counts and cell proportions as established by flow cytometry. <i>A</i>, Thymus; <i>B, C</i>, spleen; <i>D, E</i> lymph nodes. In <i>C</i> and <i>E</i>, CD8 and CD4 T cells were analysed for co-expression of CD44 and CD62L to define populations of naive and memory T cells; the insert in <i>C</i> provides a gating example for naïve/memory type T cells.</p

Protection of inducible Sgpl1-deficient mice in EAE.

<p>Tamoxifen-induced Sgpl1^Flox/Flox Cre^+/−, Sgpl1^Flox/Flox Cre^−/−, Cre^+/−, and Cre^−/− mice (n = 6–10/group) were immunized with MOG emulsified in Complete Freund’s Adjuvans. Data from one representative experiment out of three independent studies are shown. <i>A</i>, Incidence of mice with a clinical EAE score ≥1; <i>B</i>, clinical score; <i>C</i>, body weight. For histological analysis thoracic sections of spinal cord tissue from Sgpl1^Flox/Flox Cre^+/− and Sgpl1^Flox/Flox Cre^−/− mice undergoing EAE (day 24) were stained (<i>D</i>) with H&E to visualize CNS-invading cells (scale bar is 500 µm, arrows highlight areas of inflammation); <i>E</i>, for CD3⁺ T cells (scale bar is 500 µm, rectangles indicate area of magnification, where scale bar represents 100 µm); and <i>F</i>, with solochrome to assess the integrity of the myelin sheath (scale bar is 500 µm, arrows highlight areas of beginning demyeliniation).</p

Histology of inducible Sgpl1-deficient mice in EAE.

<p>For histological analysis thoracic sections of spinal cord tissue from Sgpl1^Flox/Flox Cre^+/− and Sgpl1^Flox/Flox Cre^−/− mice undergoing EAE (day 24) were stained (<i>A</i>) with H&E to visualize CNS-invading cells (scale bar is 500 µm, arrows highlight areas of inflammation); <i>B</i>, for CD3⁺ T cells (scale bar is 500 µm, rectangles indicate area of magnification, where scale bar represents 100 µm); and <i>C</i>, with solochrome to assess the integrity of the myelin sheath (scale bar is 500 µm, arrows highlight areas of beginning demyeliniation).</p