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Invadopodia are actin-based proteolytic membrane protrusions required for invasive behavior and tumor growth. In this study, we used our high-content screening assay to identify kinases whose activity affects invadopodia formation. Among the top hits selected for further analysis was TAO3, an STE20-like kinase of the GCK subfamily. TAO3 was overexpressed in many human cancers and regulated invadopodia formation in melanoma, breast, and bladder cancers. Furthermore, TAO3 catalytic activity facilitated melanoma growth in three-dimensional matrices and in vivo. A novel, potent catalytic inhibitor of TAO3 was developed that inhibited invadopodia formation and function as well as tumor cell extravasation and growth. Treatment with this inhibitor demonstrated that TAO3 activity is required for endosomal trafficking of TKS5α, an obligate invadopodia scaffold protein. A phosphoproteomics screen for TAO3 substrates revealed the dynein subunit protein LIC2 as a relevant substrate. Knockdown of LIC2 or expression of a phosphomimetic form promoted invadopodia formation. Thus, TAO3 is a new therapeutic target with a distinct mechanism of action.Significance:An unbiased screening approach identifies TAO3 as a regulator of invadopodia formation and function, supporting clinical development of this class of target.

Effect of SBI-581 on TKS5alpha vesicle trafficking

Effect of SBI-581 on Rab11 vesicle trafficking

Includes Sup. Figures 1-7, Tables 1-6 and Methods.

Effect of SU11333 on TKS5alpha vesicle trafficking

Trafficking of TKS5alpha vesicles

Invadopodia are actin-based proteolytic membrane protrusions required for invasive behavior and tumor growth. In this study, we used our high-content screening assay to identify kinases whose activity affects invadopodia formation. Among the top hits selected for further analysis was TAO3, an STE20-like kinase of the GCK subfamily. TAO3 was overexpressed in many human cancers and regulated invadopodia formation in melanoma, breast, and bladder cancers. Furthermore, TAO3 catalytic activity facilitated melanoma growth in three-dimensional matrices and in vivo. A novel, potent catalytic inhibitor of TAO3 was developed that inhibited invadopodia formation and function as well as tumor cell extravasation and growth. Treatment with this inhibitor demonstrated that TAO3 activity is required for endosomal trafficking of TKS5α, an obligate invadopodia scaffold protein. A phosphoproteomics screen for TAO3 substrates revealed the dynein subunit protein LIC2 as a relevant substrate. Knockdown of LIC2 or expression of a phosphomimetic form promoted invadopodia formation. Thus, TAO3 is a new therapeutic target with a distinct mechanism of action. Significance: An unbiased screening approach identifies TAO3 as a regulator of invadopodia formation and function, supporting clinical development of this class of target.

Prenatal androgens are largely responsible for growth and differentiation of the genital tract and testis and for organization of the control mechanisms regulating male reproductive physiology and behavior. The aim of the present study was to evaluate the impact of inappropriate exposure to excess testosterone (T) during the first trimester of fetal development on the reproductive function, sexual behavior, and fertility potential of rams. We found that biweekly maternal T propionate (100 mg) treatment administered from Day 30-58 of gestation significantly decreased (P < 0.05) postpubertal scrotal circumference and sperm concentration. Prenatal T exposure did not alter ejaculate volume, sperm motility and morphology or testis morphology. There was, however, a trend for more T-exposed rams than controls to be classified as unsatisfactory potential breeders during breeding soundness examinations. Postnatal serum T concentrations were not affected by prenatal T exposure, nor was the expression of key testicular genes essential for spermatogenesis and steroidogenesis. Basal serum LH did not differ between treatment groups, nor did pituitary responsiveness to GnRH. T-exposed rams, like control males, exhibited vigorous libido and were sexually attracted to estrous females. In summary, these results suggest that exposure to exogenous T during the first trimester of gestation can negatively impact spermatogenesis and compromise the reproductive fitness of rams.

Invadopodia are actin-based proteolytic membrane protrusions required for invasive behavior and tumor growth. We used our high-content screening assay to identify kinases impacting invadopodia formation. Among the top hits we selected TAO3, a STE20-like kinase of the GCK subfamily, for further analysis. TAO3 was over-expressed in many human cancers, and regulated invadopodia formation in melanoma, breast and bladder cancers. Furthermore, TAO3 catalytic activity facilitated melanoma growth in 3-dimensional matrices andin vivo. We developed potent catalytic inhibitors of TAO3 that inhibited invadopodia formation and function, and tumor cell extravasation and growth. Using these inhibitors, we determined that TAO3 activity was required for endosomal trafficking of TKS5α, an obligate invadopodia scaffold protein. A phosphoproteomics screen for TAO3 substrates revealed the dynein subunit protein LIC2 as a relevant substrate. Knockdown of LIC2 or expression of a phosphomimetic form promoted invadopodia formation. Thus, TAO3 is a new therapeutic target with a distinct mechanism of action.SIGNIFICANCETargeting tumor invasive behavior represents an understudied opportunity. We used an unbiased screening approach to identify kinases required for invadopodia formation and function. We validated TAO3, both genetically and with a novel inhibitor, and determined TAO3 function. Our data support clinical development of this class of target.

The scaffold protein Tks5α is required for invadopodia-mediated cancer invasion both in vitro and in vivo. We have previously also revealed a role for Tks5 in tumor cell growth using three-dimensional (3D) culture model systems and mouse transplantation experiments. Here we use both 3D and high-density fibrillar collagen (HDFC) culture to demonstrate that native type I collagen, but not a form lacking the telopeptides, stimulated Tks5-dependent growth, which was dependent on the DDR collagen receptors. We used microenvironmental microarray (MEMA) technology to determine that laminin, collagen I, fibronectin and tropoelastin also stimulated invadopodia formation. A Tks5α-specific monoclonal antibody revealed its expression both on microtubules and at invadopodia. High- and super-resolution microscopy of cells in and on collagen was then used to place Tks5α at the base of invadopodia, separated from much of the actin and cortactin, but coincident with both matrix metalloprotease and cathepsin proteolytic activity. Inhibition of the Src family kinases, cathepsins or metalloproteases all reduced invadopodia length but each had distinct effects on Tks5α localization. These studies highlight the crosstalk between invadopodia and extracellular matrix components, and reveal the invadopodium to be a spatially complex structure.

Evidence suggests that the hypothalamic–pituitary–gonadal (HPG) axis is active during the critical period for sexual differentiation of the ovine sexually dimorphic nucleus, which occurs between gestational day (GD) 60 and 90. Two possible neuropeptides that could activate the fetal HPG axis are kisspeptin and neurokinin B (NKB). We used GD85 fetal lambs to determine whether intravenous administration of kisspeptin-10 (KP-10) or senktide (NKB agonist) could elicit luteinizing hormone (LH) release. Immunohistochemistry and fluorescent in situ hybridization (FISH) were employed to localize these peptides in brains of GD60 and GD85 lamb fetuses. In anesthetized fetuses, KP-10 elicited robust release of LH that was accompanied by a delayed rise in serum testosterone in males. Pretreatment with the GnRH receptor antagonist (acyline) abolished the LH response to KP-10, confirming a hypothalamic site of action. In unanesthetized fetuses, senktide, as well as KP-10, elicited LH release. The senktide response of females was greater than that of males, indicating a difference in NKB sensitivity between sexes. Gonadotropin-releasing hormone also induced a greater LH discharge in females than in males, indicating that testosterone negative feedback is mediated through pituitary gonadotrophs. Kisspeptin and NKB immunoreactive cells in the arcuate nucleus were more abundant in females than in males. Greater than 85% of arcuate kisspeptin cells costained for NKB. FISH revealed that the majority of these were kisspeptin/NKB/dynorphin (KNDy) neurons. These results support the hypothesis that kisspeptin–GnRH signaling regulates the reproductive axis of the ovine fetus during the prenatal critical period acting to maintain a stable androgen milieu necessary for brain masculinization.

Prenatal exposure to excess androgen may result in impaired adult fertility in a variety of mammalian species. However, little is known about what feedback mechanisms regulate gonadotropin secretion during early gestation and how they respond to excess T exposure. The objective of this study was to determine the effect of exogenous exposure to T on key genes that regulate gonadotropin and GnRH secretion in fetal male lambs as compared with female cohorts. We found that biweekly maternal testosterone propionate (100 mg) treatment administered from day 30 to day 58 of gestation acutely decreased (P < .05) serum LH concentrations and reduced the expression of gonadotropin subunit mRNA in both sexes and the levels of GnRH receptor mRNA in males. These results are consistent with enhanced negative feedback at the level of the pituitary and were accompanied by reduced mRNA levels for testicular steroidogenic enzymes, suggesting that Leydig cell function was also suppressed. The expression of kisspeptin 1 mRNA, a key regulator of GnRH neurons, was significantly greater (P < .01) in control females than in males and reduced (P < .001) in females by T exposure, indicating that hypothalamic regulation of gonadotropin secretion was also affected by androgen exposure. Although endocrine homeostasis was reestablished 2 weeks after maternal testosterone propionate treatment ceased, additional differences in the gene expression of GnRH, estrogen receptor-β, and kisspeptin receptor (G protein coupled receptor 54) emerged between the treatment cohorts. These changes suggest the normal trajectory of hypothalamic-pituitary axis development was disrupted, which may, in turn, contribute to negative effects on fertility later in life.