Src activates Abl to augment Robo1 expression in order to promote tumor cell migration

Cell migration is an essential step in cancer invasion and metastasis. A number of orchestrated cellular events involving tyrosine kinases and signaling receptors enable cancer cells to dislodge from primary tumors and colonize elsewhere in the body. For example, activation of the Src and Abl kinases can mediate events that promote tumor cell migration. Also, activation of the Robo1 receptor can induce tumor cell migration. However, while the importance of Src, Abl, and Robo1 in cell migration have been demonstrated, molecular mechanisms by which they collectively influence cell migration have not been clearly elucidated. In addition, little is known about mechanisms that control Robo1 expression. We report here that Src activates Abl to stabilize Robo1 in order to promote cell migration. Inhibition of Abl kinase activity by siRNA or kinase blockers decreased Robo1 protein levels and suppressed the migration of transformed cells. We also provide evidence that Robo1 utilizes Cdc42 and Rac1 GTPases to induce cell migration. In addition, inhibition of Robo1 signaling can suppress transformed cell migration in the face of robust Src and Abl kinase activity. Therefore, inhibitors of Src, Abl, Robo1 and small GTPases may target a coordinated pathway required for tumor cell migration

lin-28 Controls the Succession of Cell Fate Choices via Two Distinct Activities

lin-28 is a conserved regulator of cell fate succession in animals. In Caenorhabditis elegans, it is a component of the heterochronic gene pathway that governs larval developmental timing, while its vertebrate homologs promote pluripotency and control differentiation in diverse tissues. The RNA binding protein encoded by lin-28 can directly inhibit let-7 microRNA processing by a novel mechanism that is conserved from worms to humans. We found that C. elegans LIN-28 protein can interact with four distinct let-7 family pre-microRNAs, but in vivo inhibits the premature accumulation of only let-7. Surprisingly, however, lin-28 does not require let-7 or its relatives for its characteristic promotion of second larval stage cell fates. In other words, we find that the premature accumulation of mature let-7 does not account for lin-28's precocious phenotype. To explain let-7's role in lin-28 activity, we provide evidence that lin-28 acts in two steps: first, the let-7–independent positive regulation of hbl-1 through its 3′UTR to control L2 stage-specific cell fates; and second, a let-7–dependent step that controls subsequent fates via repression of lin-41. Our evidence also indicates that let-7 functions one stage earlier in C. elegans development than previously thought. Importantly, lin-28's two-step mechanism resembles that of the heterochronic gene lin-14, and the overlap of their activities suggests a clockwork mechanism for developmental timing. Furthermore, this model explains the previous observation that mammalian Lin28 has two genetically separable activities. Thus, lin-28's two-step mechanism may be an essential feature of its evolutionarily conserved role in cell fate succession

Genetic interactions of heterochronic mutants.

1<p>All animals examined were homozygous for null alleles of the genes indicated and carry an integrated transgene <i>wIs78(scm::GFP; ajm-1::GFP)</i> to mark seam cells. All alleles are null.</p>2<p>Seam cell counts were performed on L4 animals except where indicated.</p>3<p>Alae formation was assessed in the early L4 stage.</p>4<p>Strains carrying the <i>let-7</i> mutation additionally contained a linked <i>unc-3</i> mutant allele. They were grown at 15°C to limit constitutive dauer formation that results from the <i>unc-3</i> mutation at higher temperatures in these backgrounds.</p>5<p>Seam cell fusion with no alae formation was observed in the other 85% of animals.</p><p>SEM, standard error of the mean; ND, not determined.</p

<i>lin-28</i> mutants can be two stages precocious due to <i>let-7</i> activity.

1<p>All strains are homozygous for null alleles of the genes indicated and carry an integrated transgene of the seam cell marker <i>wIs78(scm::GFP; ajm-1::GFP)</i>. All alleles are null.</p>2<p>Percentage of seam cells synthesizing adult alae by early L3.</p>3<p>n = number of seam cells scored.</p

<i>lin-28</i> positively regulates <i>hbl-1</i> reporter expression.

<p>Nomarski and fluorescence micrographs of <i>hbl-1::GFP::hbl-1 3′UTR</i> reporter expression. Early stages are late L1 or early L2. Late stages are L4 or age-matched post-L3 molt <i>lin-28</i> animals. A, wild type. B, <i>mir-48 mir-241; mir-84 (3 let-7s)</i>. C, <i>lin-28; mir-48 mir-241; mir-84 (lin-28; 3 let-7s)</i>. D, a <i>hbl-1::GFP::unc-54 3′UTR</i> reporter in <i>lin-28; mir-48 mir-241; mir-84 (lin-28; 3 let-7s)</i>. Se, seam nuclei. hyp, hyp7 nuclei. All fluorescence images were captured with a 2 sec. exposure time. Scale bar, 10 microns.</p

Relative contribution of <i>hbl-1</i> and <i>lin-41</i> for the <i>let-7</i> retarded phenotype.

1<p>The <i>let-7</i> mutants were identified by Unc phenotype due to the <i>unc-3</i> mutation.</p>2<p>The precocious alae were assessed at the end of L3–L4 molt or in the early L4 stage of development.</p>3<p>As previously noted, <i>hbl-1(RNAi)</i> causes a proliferation defect in the late L4 which is not interpreted as heterochronic <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002588#pgen.1002588-Lin2" target="_blank">[53]</a>. These divisions were not scored.</p><p>ND, not determined.</p

Seam cell lineages of animals with altered <i>lin-28</i> activity.

<p>Lineage patterns characteristic of lateral hypodermal seam cells V1, V2, V3, V4 and V6. Left to right: Wild type <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002588#pgen.1002588-Sulston1" target="_blank">[56]</a>. Animals lacking <i>mir-48</i>, <i>mir-84</i>, and <i>mir-241</i> (<i>3 let-7s</i>), or animals carrying a transgene constitutively expressing <i>lin-28</i> (<i>lin-28(gf)</i>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002588#pgen.1002588-Moss3" target="_blank">[62]</a>. <i>let-7</i> null mutants, whose defect in these lineages is first visible in the late L4 stage. Two types of seam cell lineages observed in <i>lin-28</i> null mutants <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002588#pgen.1002588-Ambros1" target="_blank">[1]</a>. Seam cell lineages that skip L2 fates in <i>lin-28(low RNAi)</i> animals (see text). Three horizontal lines indicate the time of adult alae formation. Dashed lines indicate variable lineage patterns in <i>lin-28(gf)</i> animals.</p

A model for the two sequential activities of LIN-28 in specifying cell fates.

<p>Top, Genetic formalisms depicting the two <i>lin-28</i> pathways that regulate the L2-to-L3 and the L3-to-L4 fate transitions. Bottom, A schematic time course depicting the regulatory dynamics during the first three larval stages. LIN-14, LIN-28, HBL-1 and LIN-41 are expressed at the start of larval development and are eventually repressed by the microRNAs lin-4, let-7 and the three let-7 family members miR-48, miR-84, and miR-241 (3 let-7s). The approximate times of LIN-14's two activities are indicated with boxed letters. The relevant times of LIN-28's two activities that correspond to the pathways above are depicted with black lines and circled letters.</p

The male tail tip morphogenesis is delayed in let-7 males.

<p>Nomarski images of wild type (A) and <i>let-7</i> null (B) L4 males approximately 8 hours after the L3 molt. The extracellular space between the L4 cuticle and the tail tip in the wildtype indicates the retraction of male tail tip <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002588#pgen.1002588-Nguyen1" target="_blank">[68]</a>. Arrow head, unretracted hypodermis in the <i>let-7</i> mutant.</p