<i>C</i>. <i>elegans</i> DAF-16/FOXO interacts with TGF-ß/BMP signaling to induce germline tumor formation via mTORC1 activation

<div><p>Activation of the FOXO transcription factor DAF-16 by reduced insulin/IGF signaling (IIS) is considered to be beneficial in <i>C</i>. <i>elegans</i> due to its ability to extend lifespan and to enhance stress resistance. In the germline, cell-autonomous DAF-16 activity prevents stem cell proliferation, thus acting tumor-suppressive. In contrast, hypodermal DAF-16 causes a tumorous germline phenotype characterized by hyperproliferation of the germline stem cells and rupture of the adjacent basement membrane. Here we show that cross-talk between DAF-16 and the transforming growth factor ß (TGFß)/bone morphogenic protein (BMP) signaling pathway causes germline hyperplasia and results in disruption of the basement membrane. In addition to activating MADM/NRBP/<i>hpo-11</i> gene alone, DAF-16 also directly interacts with both R-SMAD proteins SMA-2 and SMA-3 in the nucleus to regulate the expression of mTORC1 pathway. Knocking-down of BMP genes or each of the four target genes in the hypodermis was sufficient to inhibit germline proliferation, indicating a cell-non-autonomously controlled regulation of stem cell proliferation by somatic tissues. We propose the existence of two antagonistic DAF-16/FOXO functions, a cell-proliferative somatic and an anti-proliferative germline activity. Whereas germline hyperplasia under reduced IIS is inhibited by DAF-16 cell-autonomously, activation of somatic DAF-16 in the presence of active IIS promotes germline proliferation and eventually induces tumor-like germline growth. In summary, our results suggest a novel pathway crosstalk of DAF-16 and TGF-ß/BMP that can modulate mTORC1 at the transcriptional level to cause stem-cell hyperproliferation. Such cell-type specific differences may help explaining why human FOXO activity is considered to be tumor-suppressive in most contexts, but may become oncogenic, e.g. in chronic and acute myeloid leukemia.</p></div

IIS balances anti-proliferating (tumor-suppressive) and pro-proliferating (oncogenic) activities across tissue boundaries.

<p>(A) Model of the cell-autonomous and non-cell-autonomous functions of DAF-16. IIS inhibits nuclear entry and activity of DAF-16/FOXO, BMP promotes nuclear entry and activates R-SMAD proteins SMA-2 and SMA-3. Both DAF-16 and SMA-2/3 have individual roles and regulate transcription of distinct target genes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006801#pgen.1006801.ref019" target="_blank">19</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006801#pgen.1006801.ref042" target="_blank">42</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006801#pgen.1006801.ref050" target="_blank">50</a>]. For example, BMP activity in the hypodermis controls body size, oocyte and germline quality maintenance independently of IIS. We currently have no evidence for germline activity of BMP signaling. In the hypodermis DAF-16 interacts with SMA-2/3 in the nucleus to activate mTORC1 at transcriptional level to promote germline proliferation (green arrow). DAF-16 also activates <i>hpo-11</i> transcription independent of BMP signaling to promote cell proliferation. In addition, BMP signaling in the somatic gonad also positively regulate germline proliferation, while DAF-16 in the germline has been proposed to inhibit cell proliferation (inhibitory activity in red) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006801#pgen.1006801.ref005" target="_blank">5</a>]. (B) Hypodermal and germline DAF-16 activities need to be tightly balanced to prevent hyperproliferation. Signaling activities of at least two tissues contribute to IIS homeostasis and may explain, why DAF-16 hyperactivity (in the hypodermis) may cause germline tumors in <i>daf-2(+)</i> background, whereas <i>daf-2(-)</i> (or starvation) decreases DAF-16 oncogenic potential, although in a simplistic view, it is thought to activate DAF-16.</p

mTORC1 and BMP signaling pathways contribute to the DAF-16 dependent tumorous germline phenotype.

<p>(A) Representative pictures of MitoTracker stained gonadal basement membrane. The white arrows point to disrupted gonadal basement membrane. (B) RNAi knock-down of <i>sma-6</i>, <i>hpo-11</i> and the components of mTORC1 signaling suppresses the defects in the gonadal integrity in <i>shc-1;Is[daf-16</i>::<i>GFP]</i> animals. (C) Anti-PGL-1 germline antibody staining of <i>shc-1;Is[daf-16</i>::<i>GFP]</i> and <i>shc-1;sma-6;Is[daf-16</i>::<i>GFP]</i> L3 larvae. The white arrow points to hyperplasia in the anterior gonad arm of a representative <i>shc-1;Is[daf-16</i>::<i>GFP]</i> animal (top). In <i>sma-6</i> mutant background, hyperproliferation is suppressed (bottom). (D) <i>sma-6</i> mutation significantly decreases the elevated number of the germ cells in the anterior gonad arms of <i>shc-1;Is[daf-16</i>::<i>GFP]</i> L3 animals. Numbers of germ cells per gonad arm ± SEM: wild-type: 47 ± 2 (n = 13); <i>shc-1;Is[daf-16</i>::<i>GFP]</i>: 67 ± 4 (n = 23); <i>shc-1;sma-6;Is[daf-16</i>::<i>GFP]</i>: 49 ± 2 (n = 21) (P<0.0001 compared to <i>shc-1;Is[daf-16</i>::<i>GFP]</i> animals). (E) RNAi knock-down of the components of mTORC1 signaling and <i>hpo-11</i> significantly decreases the elevated number of the germ cells in <i>shc-1;Is[daf-16</i>::<i>GFP]</i> L3 animals. Numbers of germ cells per gonad arm ± SEM: L4440 control: 90 ± 6 (n = 32); <i>let-363</i> RNAi: 64 ± 4 (n = 28); <i>rheb-1</i> RNAi: 57 ± 6 (n = 20); <i>rsks-1</i> RNAi: 54 ± 5 (n = 22); <i>hpo-11</i> RNAi: 61 ± 4 (n = 32) (P<0.0001 compared to <i>shc-1;Is[daf-16</i>::<i>GFP]</i> + RNAi L4440 control animals). (F) Inactivation of genes in the BMP signaling pathway completely abolishes gonad disruption in <i>shc-1;Is[daf-16</i>:: <i>GFP]</i> L3 animals. (G) Inactivation of genes in the BMP signaling pathway strongly suppresses gonad disruption in <i>shc-1;Is[daf-16</i>:: <i>GFP]</i> day one adult animals. Scale bar: 10 μm. In this and the following figures, percentages of basement membrane defects are presented as mean ± SD; the mean values and statistical analysis are summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006801#pgen.1006801.s012" target="_blank">S2 Table</a>; the asterisks represent: * P<0.01, ** P<0.001, and *** P<0.0001.</p

Comparison of expression of DAF-16 and BMP signaling regulated genes.

<p>(A) Overlap in the transcriptional outputs of DAF-16 and BMP signaling. 2,006 genes are significantly regulated by DAF-16 by comparing <i>shc-1(ok198);Is[daf-16</i>::<i>GFP]</i> and <i>shc-1(ok198) daf-16(mu86)</i> animals; 1,975 genes respond significantly to SMA-6 by comparing <i>shc-1(ok198);Is[daf-16</i>::<i>GFP]</i> and <i>shc-1(ok198);sma-6(wk7);Is[daf-16</i>::<i>GFP]</i>. 777 genes among them are regulated by both DAF-16 and SMA-6. (B) Classification of the response patterns of 777 genes responding to both DAF-16 and BMP signaling. Most genes are either upregulated or downregulated in response to both factors; opposing transcriptional effects were observed for only 57 genes. (C) Genes responding to DAF-16 in a SMA-6 independent manner are more strongly enriched in the known class I and II DAF-16 targets than genes responding to both DAF-16 and SMA-6.</p

DAF-16 and BMP signaling positively regulate germline proliferation.

<p>(A) DAF-16 promotes germline proliferation in a non-cell-autonomous way. (B) BMP signaling promotes germline proliferation in a non-cell-autonomous way. (C) Hypodermis-specific RNAi against <i>rsks-1</i>, <i>rheb-1</i>, <i>daf-15</i> and <i>hpo-11</i> reduces mitotic germ cell numbers. (D) Hypodermis-specific RNAi against <i>rsks-1</i>, <i>rheb-1</i>, <i>daf-15</i> and <i>hpo-11</i> in <i>daf-16</i> mutant animals. (E) Hypodermis-specific RNAi against <i>rsks-1</i>, <i>rheb-1</i>, <i>daf-15</i> and <i>hpo-11</i> in <i>sma-6</i> mutant animals. The average numbers of germ cell nuclei in the proliferation zone of day one adult animals ± SEM and P-values are summarized in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006801#pgen.1006801.s019" target="_blank">S9 Table</a>.</p

DAF-16 and BMP signaling do not cooperate in regulation of lifespan or body size.

<p>(A) Lifespan extension resulting from DAF-16 activation upon reduced IIS does not require SMA-6. Since <i>sma-6</i> mutant animals have a strong egg-laying defect, lifespan was performed in a temperature sensitive <i>fer-15</i> mutant background, in which animals become sterile at 25°C due to defective sperm formation [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006801#pgen.1006801.ref033" target="_blank">33</a>]. Mean lifespan and statistical analysis are summarized in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006801#pgen.1006801.s013" target="_blank">S3 Table</a>. (B) DAF-16 and BMP signaling regulate body size independently. Average body length are presented as mean ± SD. N2: 1.10 ± 0.09 mm (n = 17); <i>daf-2(e1370)</i>: 1.22 ± 0.06 mm (n = 14); <i>daf-16(mu86);daf-2(e1370)</i>: 1.08 ± 0.06 mm (n = 19); <i>sma-6(wk7)</i>: 0.70 ± 0.06 mm (n = 15); <i>sma-6(wk7);daf-2(e1370)</i>: 0.94 ± 0.07 mm (n = 10); <i>daf-16(mu86);sma-6(wk7);daf-2(e1370)</i>: 0.59 ± 0.04 mm (n = 21). (C) <i>sma-6</i> mutation does not affect nuclear localization of DAF-16(4A)::GFP in <i>shc-1;Ex[daf-16(4A)</i>::<i>GFP]</i> animals. Arrows: DAF-16::GFP in the hypodermal (h), the muscle (m) and intestinal nuclei (i). (D) <i>sma-6</i> mutation strongly suppresses the gonadal basement membrane defect in <i>shc-1;Ex[daf-16(4A)</i>::<i>GFP]</i> animals.</p