PTEN redundancy: overexpressing lpten, a homolog of Dictyostelium discoideum ptenA, the ortholog of human PTEN, rescues all behavioral defects of the mutant ptenA-.

Mutations in the tumor suppressor gene PTEN are associated with a significant proportion of human cancers. Because the human genome also contains several homologs of PTEN, we considered the hypothesis that if a homolog, functionally redundant with PTEN, can be overexpressed, it may rescue the defects of a PTEN mutant. We have performed an initial test of this hypothesis in the model system Dictyostelium discoideum, which contains an ortholog of human PTEN, ptenA. Deletion of ptenA results in defects in motility, chemotaxis, aggregation and multicellular morphogenesis. D. discoideum also contains lpten, a newly discovered homolog of ptenA. Overexpressing lpten completely rescues all developmental and behavioral defects of the D. discoideum mutant ptenA-. This hypothesis must now be tested in human cells

<i>lpten⁻</i> cells translocating in buffer in the absence of chemoattractant exhibit defects in velocity, turning and the suppression of lateral pseudopod formation.

<p>Cells were analyzed in a perfusion chamber through which buffer without attractant was pumped. A. 2D motility parameters of Ax2, <i>lpten⁻</i> and <i>ptenA⁻/lpten^oe</i> cells assessed with 2D-DIAS software. B, C, D. 2D-DIAS reconstructions of cell perimeters to generate tracks. Arrows denote net direction, and the blue-filled perimeters represent the last cell positions in the tracks. E, F. 3D-DIAS reconstructions at 0° (top view) and 90° (side view) of representative Ax2 and <i>lpten⁻</i> cells, respectively, denoting pseudopods (red). Note that the multiple lateral pseudopods formed by <i>lpten⁻</i> cells, were primarily off the substrate. a, anterior end of cell; p, posterior end of cell; lps, lateral pseudopod. G. 2D analysis of lateral pseudopod formation. Inst. vel., instantaneous velocity; No. turns per 10 min., number of turns per 10 minutes; Percent mot. cells, percent motile cells. Parameters are presented as the means ± standard deviations. T-test was used to determine p values. Parameters are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone.0108495.s002" target="_blank">Table S2</a>.</p

Overexpressing <i>lpten⁻</i> in the <i>ptenA⁻</i> mutant.

<p>A. The transformation vector used to generate strains <i>ptenA⁻/lpten^oe</i>, in which <i>lpten</i> is under the regulation of the <i>actin 15</i> (<i>act15</i>) promoter, fused in frame at the 3′ end to the red fluorescent protein gene (<i>rfp</i>) and terminating with a 3′ <i>actin 8</i> gene sequence. The positions of the primers P8 and P9, for generating the <i>lpten-rfp</i> cDNA, are denoted. Insert shows verification of the <i>lpten-rfp</i> cDNA by PCR. B. <i>lpten</i> is expressed in <i>ptenA⁻/lpten^oe</i> cells at levels more than 10 times that in the parent <i>ptenA⁻</i> mutant. The positions of the primers (P1, P2) for RT-PCR of the 300 bp <i>lpten</i> fragment (F) are denoted. In the insert to the right of panel B, RT-PCR products of chemotactically responsive <i>ptenA⁻</i> and <i>ptenA⁻/lpten^oe</i> cells reveals overexpression of <i>lpten</i> in the latter. Densitometry measurements revealed>10 fold overexpression. C. Fruiting body formation in Ax2 cultures. D. The absence of fruiting body formation in <i>ptenA⁻</i> cultures. E. Fruiting body formation in <i>ptenA⁻/lpten^oe</i> cultures. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone.0108495.s001" target="_blank">Table S1</a> for description of primers.</p

<i>lpten⁻</i> cells undergo normal chemotaxis in the low cAMP concentration gradient generated in the estimated for that of the natural wave.

<p>The gradient was generated in BSS buffer, in which K⁺ and Na⁺ are the facilitating cations. <i>lpten⁻</i> cells, however, are still defective in suppressing lateral pseudopod formation. A. 2D motility and chemotaxis parameters, assessed by 2D-DIAS software of Ax2, <i>lpten⁻</i> and <i>lpten⁻/lpten^oe</i> cells undergoing chemotaxis in a low cAMP concnetration gradient. B, C, D. 2D-DIAS-reconstructed perimeter tracks of representative cells. The large arrows at panel bottoms denote the net direction of the increasing cAMP gradient. “Sink”, trough with buffer alone; “Source”, trough with buffer plus 1 µM cAMP. E. 2D analysis of lateral pseudopod formation. Direct. Persist, directional persistence; chem. index, Chemotactic Index (CI); Percent pos. chem., percent cells with a positive CI. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone-0108495-g002" target="_blank">Figure 2</a> for additional definitions and details. Parameters are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone.0108495.s002" target="_blank">Table S2</a>.</p

Overexpression of <i>lpten</i> rescues the behavioral defects exhibited by homogeneous populations of <i>ptenA⁻</i> cells undergoing chemotaxis in natural aggregation territories in submerged cultures on glass.

<p>A, B, C. The centroid tracks of four neighboring cells representative of the general behavior of Ax2, <i>ptenA⁻</i> and <i>ptenA⁻/lpten^oe</i> populations, respectively, are presented in relation to the aggregation centers of Ax2 and <i>ptenA⁻/lpten^oe</i> cells, and the interpreted aggregation center of <i>ptenA⁻</i>, deduced retrospectively by the direction of net translocation of groups of cells, in the upper half of each panel. The first (1) and last (150) centered in the centroid tracks are noted. In lower half of each panel, the velocity plots are presented for two respective cells. For normal cells, the peaks of velocity have been shown to correlate with the front of each relayed natural wave.</p

Overexpression of <i>lpten</i> rescues the basic behavioral defects of <i>ptenA⁻</i> cells that are translocating in buffer, and both the behavioral and chemotactic defects in a cAMP gradient generated in the concentration range of the natural wave.

<p>A. 2D motility parameters of cells translocating in buffer, assessed by 2D-DIAS software. B, C, D. 2D-DIAS reconstructions of perimeter tracks of Ax2, <i>ptenA⁻</i> and <i>ptenA⁻/lpten^oe</i> cells, respectively, translocating in buffer. E. 2D analysis of lateral pseudopod formation in buffer. F. 2D motility and chemotaxis parameters assessed by 2D-DIAS software during chemotaxis in a low cAMP concentration gradient. G, H, I. Perimeter tracks of cells in a low cAMP concentration gradient. J. 2D analysis of lateral pseudopod formation during chemotaxis in a low cAMP concentration gradient. See the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone-0108495-g002" target="_blank">Figure 2</a> for explanations of panels A through E, and the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone-0108495-g002" target="_blank">Figure 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone-0108495-g003" target="_blank">3</a> for explanations of panels F through J.</p

Chemotactic behavior of <i>ptenA⁻</i> cells pulsed with cAMP to achieve chemotactic competence: a comparison of four different studies involving either “low cAMP concentrations” in the estimated range for natural cAMP waves or “high cAMP concentration gradients”, at concentrations 10 times that of natural cAMP waves.

<p>a. Reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone.0108495-Iijima1" target="_blank">[29]</a>: Iijima and Devreotes, 2002; reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone.0108495-Hoeller1" target="_blank">[32]</a>: Hoeller and Kay, 2007; reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone.0108495-Wessels1" target="_blank">[30]</a>, Wessels et al., 2007.</p><p>b. Complete composition of buffers used in chemotaxis assay. DB buffer: 5 mM Na₂HPO₄, 5 mM NaH₂PO_4, 2 mM MgSO₄, 0.2 mM CaCl₂ (15 mM Na⁺, 0.2 mM Ca²⁺). KK₂ buffer: 3.9 mM K₂HPO₄, 16.5 mM KH₂PO₄, 2 mM Mg SO₄, 0.1 mM CaCl₂ (24.3 mM K⁺, 0.1 mM CA²⁺, 0.1 mM Ca²⁺). BSS buffer: 20 mM KH₂PO₄, 5 mM NA₂HPO₄, 20 mM KCL, 2.5 mM MgCl₂ (45 mM K⁺/Na⁺, 0 mM Ca²⁺<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone.0108495-Lusche3" target="_blank">[77]</a>.</p><p>c. When the source of the cAMP gradient was 1 µM, it generated a “low cAMP concentration gradient”, in the concentration range estimated for the natural wave <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone.0108495-Tomchik1" target="_blank">[44]</a>, and when it was 10 µM, it generated a high cAMP concentration gradient 10 times that estimated for the natural.</p><p>Chemotactic behavior of <i>ptenA⁻</i> cells pulsed with cAMP to achieve chemotactic competence: a comparison of four different studies involving either “low cAMP concentrations” in the estimated range for natural cAMP waves or “high cAMP concentration gradients”, at concentrations 10 times that of natural cAMP waves.</p

<i>ptenA⁻</i> cells pulsed in suspension with cAMP to induce chemotactic competence are defective in assessing the direction of a low cAMP concentration gradient in the range of a natural wave, but they can efficiently assess the direction of a cAMP gradient in a concentration range 10 times higher.

<p>Pulsing <i>ptenA⁻</i> cells with cAMP also up-regulates <i>lpten</i>. A, B. 2D-DIAS reconstructions of perimeter tracks of Ax2 and <i>ptenA⁻</i> cells, respectively, in a low cAMP concentration gradient, generated by adding 1 µM cAMP to the source well of the gradient chamber. Motility and chemotaxis parameters assessed by 2D-DIAS software are presented in the lower left hand corner of each panel. C, D. Perimeter tracks of representative Ax2 and <i>ptenA⁻</i> cells, respectively, in a high cAMP concentration gradient, generated by adding 10 µM cAMP to the source well of the gradient chamber. Motility and chemotaxis parameters are displayed in the lower left corner of each panel. E, F. Up-regulation of <i>lpten</i> expression in cAMP pulsed Ax2 and <i>ptenA⁻</i> cells, respectively. In each strain, cells were analyzed by RT-PCR using primers P1 and P2 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone.0108495.s001" target="_blank">Table S1</a>), prior to cAMP pulsing (1 hr), after cAMP pulsing for six hours (6 hr) and after cAMP pulsing with buffer for six hours (6 h). The constitutively expressed large subunit ribosomal RNA (<i>rnlA</i>) was assessed for comparability (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone-0108495-g001" target="_blank">Figure 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone-0108495-g004" target="_blank">4</a>). No RT, no reverse transcriptase added; IV, instantaneous velocity; CI, chemotactic index; %+, percent cells with a positive CI; N, number of cells assessed. Parameters in panels A, B, C and D are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108495#pone.0108495.s002" target="_blank">Table S2</a>.</p

Melanoma cells undergo aggressive coalescence in a 3D Matrigel model that is repressed by anti-CD44

<div><p>Using unique computer-assisted 3D reconstruction software, it was previously demonstrated that tumorigenic cell lines derived from breast tumors, when seeded in a 3D Matrigel model, grew as clonal aggregates which, after approximately 100 hours, underwent coalescence mediated by specialized cells, eventually forming a highly structured large spheroid. Non-tumorigenic cells did not undergo coalescence. Because histological sections of melanomas forming in patients suggest that melanoma cells migrate and coalesce to form tumors, we tested whether they also underwent coalescence in a 3D Matrigel model. Melanoma cells exiting fragments of three independent melanomas or from secondary cultures derived from them, and cells from the melanoma line HTB-66, all underwent coalescence mediated by specialized cells in the 3D model. Normal melanocytes did not. However, coalescence of melanoma cells differed from that of breast-derived tumorigenic cell lines in that they 1) coalesced immediately, 2) underwent coalescence as individual cells as well as aggregates, 3) underwent coalescence far faster and 4) ultimately formed long, flat, fenestrated aggregates that were extremely dynamic. A screen of 51 purified monoclonal antibodies (mAbs) targeting cell surface-associated molecules revealed that two mAbs, anti-beta 1 integrin/(CD29) and anti-CD44, blocked melanoma cell coalescence. They also blocked coalescence of tumorigenic cells derived from a breast tumor. These results add weight to the commonality of coalescence as a characteristic of tumorigenic cells, as well as the usefulness of the 3D Matrigel model and software for both investigating the mechanisms regulating tumorigenesis and screening for potential anti-tumorigenesis mAbs.</p></div