Mob Family Proteins: Regulatory Partners in Hippo and Hippo-Like Intracellular Signaling Pathways

Studies in yeast first delineated the function of Mob proteins in kinase pathways that regulate cell division and shape; in multicellular eukaryotes Mobs regulate tissue growth and morphogenesis. In animals, Mobs are adaptors in Hippo signaling, an intracellular signal-transduction pathway that restricts growth, impacting the development and homeostasis of animal organs. Central to Hippo signaling are the Nuclear Dbf2-Related (NDR) kinases, Warts and LATS1 and LATS2, in flies and mammals, respectively. A second Hippo-like signaling pathway has been uncovered in animals, which regulates cell and tissue morphogenesis. Central to this emergent pathway are the NDR kinases, Tricornered, STK38, and STK38L. In Hippo signaling, NDR kinase activation is controlled by three activating interactions with a conserved set of proteins. This review focuses on one co-activator family, the highly conserved, non-catalytic Mps1-binder-related (Mob) proteins. In this context, Mobs are allosteric activators of NDR kinases and adaptors that contribute to assembly of multiprotein NDR kinase activation complexes. In multicellular eukaryotes, the Mob family has expanded relative to model unicellular yeasts; accumulating evidence points to Mob functional diversification. A striking example comes from the most sequence-divergent class of Mobs, which are components of the highly conserved Striatin Interacting Phosphatase and Kinase (STRIPAK) complex, that antagonizes Hippo signaling. Mobs stand out for their potential to modulate the output from Hippo and Hippo-like kinases, through their roles both in activating NDR kinases and in antagonizing upstream Hippo or Hippo-like kinase activity. These opposing Mob functions suggest that they coordinate the relative activities of the Tricornered/STK38/STK38L and Warts/LATS kinases, and thus have potential to assemble nodes for pathway signaling output. We survey the different facets of Mob-dependent regulation of Hippo and Hippo-like signaling and highlight open questions that hinge on unresolved aspects of Mob functions

Bunched, the Drosophila homolog of the mammalian tumor suppressor TSC-22, promotes cellular growth

<p>Abstract</p> <p>Background</p> <p>Transforming Growth Factor-β1 stimulated clone-22 (TSC-22) is assumed to act as a negative growth regulator and tumor suppressor. TSC-22 belongs to a family of putative transcription factors encoded by four distinct loci in mammals. Possible redundancy among the members of the TSC-22/Dip/Bun protein family complicates a genetic analysis. In <it>Drosophila</it>, all proteins homologous to the TSC-22/Dip/Bun family members are derived from a single locus called <it>bunched </it>(<it>bun</it>).</p> <p>Results</p> <p>We have identified <it>bun </it>in an unbiased genetic screen for growth regulators in <it>Drosophila</it>. Rather unexpectedly, <it>bun </it>mutations result in a growth deficit. Under standard conditions, only the long protein isoform BunA – but not the short isoforms BunB and BunC – is essential and affects growth. Whereas reducing <it>bunA </it>function diminishes cell number and cell size, overexpression of the short isoforms BunB and BunC antagonizes <it>bunA </it>function.</p> <p>Conclusion</p> <p>Our findings establish a growth-promoting function of <it>Drosophila </it>BunA. Since the published studies on mammalian systems have largely neglected the long TSC-22 protein version, we hypothesize that the long TSC-22 protein is a functional homolog of BunA in growth regulation, and that it is antagonized by the short TSC-22 protein.</p

R-Smad Competition Controls Activin Receptor Output in Drosophila

Animals use TGF-β superfamily signal transduction pathways during development and tissue maintenance. The superfamily has traditionally been divided into TGF-β/Activin and BMP branches based on relationships between ligands, receptors, and R-Smads. Several previous reports have shown that, in cell culture systems, “BMP-specific” Smads can be phosphorylated in response to TGF-β/Activin pathway activation. Using Drosophila cell culture as well as in vivo assays, we find that Baboon, the Drosophila TGF-β/Activin-specific Type I receptor, can phosphorylate Mad, the BMP-specific R-Smad, in addition to its normal substrate, dSmad2. The Baboon-Mad activation appears direct because it occurs in the absence of canonical BMP Type I receptors. Wing phenotypes generated by Baboon gain-of-function require Mad, and are partially suppressed by over-expression of dSmad2. In the larval wing disc, activated Baboon cell-autonomously causes C-terminal Mad phosphorylation, but only when endogenous dSmad2 protein is depleted. The Baboon-Mad relationship is thus controlled by dSmad2 levels. Elevated P-Mad is seen in several tissues of dSmad2 protein-null mutant larvae, and these levels are normalized in dSmad2; baboon double mutants, indicating that the cross-talk reaction and Smad competition occur with endogenous levels of signaling components in vivo. In addition, we find that high levels of Activin signaling cause substantial turnover in dSmad2 protein, providing a potential cross-pathway signal-switching mechanism. We propose that the dual activity of TGF-β/Activin receptors is an ancient feature, and we discuss several ways this activity can modulate TGF-β signaling output

Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society

Adding 6 months of androgen deprivation therapy to postoperative radiotherapy for prostate cancer: a comparison of short-course versus no androgen deprivation therapy in the RADICALS-HD randomised controlled trial

Background Previous evidence indicates that adjuvant, short-course androgen deprivation therapy (ADT) improves metastasis-free survival when given with primary radiotherapy for intermediate-risk and high-risk localised prostate cancer. However, the value of ADT with postoperative radiotherapy after radical prostatectomy is unclear. Methods RADICALS-HD was an international randomised controlled trial to test the efficacy of ADT used in combination with postoperative radiotherapy for prostate cancer. Key eligibility criteria were indication for radiotherapy after radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to radiotherapy alone (no ADT) or radiotherapy with 6 months of ADT (short-course ADT), using monthly subcutaneous gonadotropin-releasing hormone analogue injections, daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as distant metastasis arising from prostate cancer or death from any cause. Standard survival analysis methods were used, accounting for randomisation stratification factors. The trial had 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 80% to 86% (hazard ratio [HR] 0·67). Analyses followed the intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov, NCT00541047. Findings Between Nov 22, 2007, and June 29, 2015, 1480 patients (median age 66 years [IQR 61–69]) were randomly assigned to receive no ADT (n=737) or short-course ADT (n=743) in addition to postoperative radiotherapy at 121 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 9·0 years (IQR 7·1–10·1), metastasis-free survival events were reported for 268 participants (142 in the no ADT group and 126 in the short-course ADT group; HR 0·886 [95% CI 0·688–1·140], p=0·35). 10-year metastasis-free survival was 79·2% (95% CI 75·4–82·5) in the no ADT group and 80·4% (76·6–83·6) in the short-course ADT group. Toxicity of grade 3 or higher was reported for 121 (17%) of 737 participants in the no ADT group and 100 (14%) of 743 in the short-course ADT group (p=0·15), with no treatment-related deaths. Interpretation Metastatic disease is uncommon following postoperative bed radiotherapy after radical prostatectomy. Adding 6 months of ADT to this radiotherapy did not improve metastasis-free survival compared with no ADT. These findings do not support the use of short-course ADT with postoperative radiotherapy in this patient population

Profile of Transforming Growth Factor-β Responses During the Murine Hair Cycle

Transforming growth factor-β (TGF-β) appears to promote the regression phase of the mammalian hair cycle, in vivo in mice and in organ culture of human hair follicles. To assess the relationship between TGF-β activity and apoptosis of epithelial cells during the murine hair cycle, we identified active TGF-β responses using phospho-Smad2/3-specific antibodies (PS2). Strong, nuclear PS2 staining was observed in the outer root sheath throughout the anagen growth phase. Some bulb matrix cells were positive for PS2 during late anagen. Extensive, but weak, staining was observed in this region at the anagen-catagen transition. We also examined expression of TGF-β-stimulated clone-22 (TSC-22), which is associated with TGF-β-induced apoptosis of some cell lines. Recombinant rat TSC-22 was used to generate a rabbit anti-TSC-22 antibody useful for immunohistochemistry. TSC-22 RNA accumulation and immunoreactivity were observed in follicles throughout the murine hair cycle, including the dermal papilla and lower epithelial strand of late-catagen hair follicles. Neither the expression pattern nor the presence of nuclear TSC-22 correlated with the sites of apoptosis, suggesting that TSC-22 is not an effector of apoptosis in mouse catagen hair follicles. These studies support a complex role for TGF-β in regulating the regression phase of the cycle, with potential for indirect promotion of apoptosis during the anagen–catagen transition

Bunched and Madm Function Downstream of Tuberous Sclerosis Complex to Regulate the Growth of Intestinal Stem Cells in Drosophila

The Drosophila adult midgut contains intestinal stem cells that support homeostasis and repair. We show here that the leucine zipper protein Bunched and the adaptor protein Madm are novel regulators of intestinal stem cells. MARCM mutant clonal analysis and cell type specific RNAi revealed that Bunched and Madm were required within intestinal stem cells for proliferation. Transgenic expression of a tagged Bunched showed a cytoplasmic localization in midgut precursors, and the addition of a nuclear localization signal to Bunched reduced its function to cooperate with Madm to increase intestinal stem cell proliferation. Furthermore, the elevated cell growth and 4EBP phosphorylation phenotypes induced by loss of Tuberous Sclerosis Complex or overexpression of Rheb were suppressed by the loss of Bunched or Madm. Therefore, while the mammalian homolog of Bunched, TSC-22, is able to regulate transcription and suppress cancer cell proliferation, our data suggest the model that Bunched and Madm functionally interact with the TOR pathway in the cytoplasm to regulate the growth and subsequent division of intestinal stem cells

Excessive Baboon signaling perturbs wing development in a Mad-dependent manner.

<p><i>vestigial-</i>GAL4 (<i>vg</i>) was used to express combinations of Baboon, dSmad2, and RNAi for <i>mad</i> and <i>dSmad2.</i> Normal wing development (A; one copy of <i>vg</i>-GAL4) was disrupted by Babo* expression (B, C; Babo*mod and Babo*strong are UAS insertions with varying activity as characterized in other assays). The Babo* phenotype was abrogated by simultaneous <i>mad</i> RNAi (H) and resembled <i>mad</i> RNAi alone (E). In contrast, the crumpling defect of Babo* wings was enhanced in conjunction with <i>dSmad2</i> RNAi (I) and was more severe than <i>dSmad2</i> RNAi alone (F). Overexpression of FLAG-dSmad2 did not affect wing formation (D), but partially rescued the Babo* overexpression phenotype, producing normal sized flat wings with residual peripheral vein defects (G). For each genotype, a representative wing is shown out of 4–7 wings photographed. Within a genotype there was only slight variation in appearance, except that 3/6 dSmad2 RNAi wings had vein defects and a blister (shown) and 3/6 had vein defects without a blister (not shown).</p

P-Mad elevation in <i>dSmad2</i> null mutant tissues depends on <i>baboon</i>.

<p>The schematic depicts the location of the l(X)G0348 <i>P</i> element insertion in relation to the <i>dSmad2</i> locus (A). The F4 excision product removed the entire coding region of <i>dSmad2</i> and portions of the <i>P</i>-element. The genomic breakpoints are indicated above the <i>dSmad2</i> mRNA; they were determined by sequencing PCR products, indicated by dotted lines. (B–D) P-Mad was detected by IHC of fixed larval tissues from several genotypes. For each image, a merged DAPI (blue) and P-Mad (red) panel is displayed above the isolated P-Mad channel. (B) Single confocal sections of P-Mad staining in the fat body. Under the staining conditions employed, endogenous nuclear P-Mad in a control fat body was barely detected (B1), but was increased in a <i>dSmad2</i> null mutant animal (B2). <i>Baboon</i> single mutants and <i>dSmad2</i>; <i>baboon</i> double mutants had normal P-Mad staining (B3,4). (C, D) P-Mad staining at two representative positions along the digestive tract. Images are Maximal Intensity Projections of 3 micron interval confocal sections through the entire sample. The P-Mad primary antibody was omitted from “No Ab Control” samples to convey any background staining and auto-fluorescence in the red channel. (C) In the gastric caeca near the proventriculus, <i>dSmad2</i> mutants showed elevated P-Mad (C3) compared to wildtype control males (C2). <i>baboon</i> single mutants and <i>dSmad2</i>; <i>baboon</i> double mutants showed wildtype levels (C4 and C5). Distal Malpighian tubule staining (lumpy tubes marked with asterisks) showed the same pattern, with the <i>dSmad2</i> mutant displaying the strongest P-Mad staining (D3 compared to D2, D4 and D5).</p