Final Report Summary - HIPPOGAILITE (Regulation of the Hippo tissue growth pathway by nutrient sensing mechanisms in Drosophila melanogaster)
The correct regulation of tissue growth in developing organisms is essential for functional organ formation. The evolutionarily conserved transcriptional co-activator Yorkie (Yki, YAP in mammals) responds to a variety of upstream inputs to promote tissue growth. Yki/YAP is known to regulate stem cell proliferation, thus affecting final organ size. The Hippo/Warts kinase cascade is a key inhibitor of Yki proliferative and anti-apoptotic activity. Although initially discovered in Drosophila, Hippo pathway is highly conserved in mammals, rendering the results obtained in Drosophila system relevant for our understanding of the pathway function in mammals. Increased YAP activity is often found in tumours, thus our understanding of its regulation is important for the understanding of tumour formation and generation of potential cancer treatments. In the host lab, salt inducible kinases (SIKs) were identified as upstream regulators of Yki activity via the Hippo/Wts cascade in the fruit fly Drosophila melanogaster. SIKs belong to the AMP-activated protein kinase (AMPK) family. AMPKs respond to nutrient availability to regulate a variety of cellular and metabolic processes. In this project, I investigated role of nutritionally regulated inputs and other AMPK family members in regulation of Yki activity.
During this project, I have identified Liver Kinase B1 (LKB1), a master kinase that activates AMPK family kinases, as an inhibitor of Yki activity in a tissue-specific manner. LKB1 is a tumour suppressor kinase that regulates multiple processes such as cell polarity and proliferation. In humans, heterozygous mutation of lkb1 leads to an increased benign and malignant tumour predisposition (Peutz-Jeghers syndrome), while sporadic mutations have also been linked to a variety of cancers. lkb1 mutant Drosophila larvae exhibit brain overgrowth, and die at early pupal stages. The Drosophila larval brain is composed of neural stem cells or neuroblasts (NBs) and their progeny, which differentiates into neurons or glial cells. Drosophila NBs have been extensively studied as a model for stem cell proliferation and differentiation. I found that loss of lkb1 function in the larval brain results in increased Yki target gene expression both in the NBs and their progeny, and causes increased cell proliferation. RNAi against yki in lkb1 mutant cells brought Yki target levels and cell proliferation back to wild type levels. Conversely, LKB1 overexpression results in smaller organ size, inhibits cell proliferation and decreases Yki target gene expression.
AMPK is one of the best-characterised downstream targets of LKB1, and has been shown to act as a sensor of intracellular energy levels to inhibit anabolic processes with high energy cost and to promote energy-generating catabolic processes. Similarly to lkb1 mutant phenotype, loss of ampk function in the larval brain induced increased Yki target gene expression. Additionally, RNAi-mediated knockdown of ampk in cells that overexpress LKB1 inhibited the decrease of Yki target gene expression, positioning AMPK downstream of LKB1 in regulation of Yki activity.
The developing Drosophila larval brain consists of two compartments of a different developmental origin: the optic lobe and the central brain. Interestingly, I found that mutations in hpo and wts, core Hpo kinase pathway members, increases transcription of Yki target genes in the optic lobe, but not in the central brain. Similarly, activated form of Yki that cannot be inhibited by Wts activity does not induce overgrowth in the central brain. These results indicate that LKB1/AMPK is the main inhibitory input on Yki activity in the larval central brain. Additionally, I found that LKB1 overexpression inhibits Yki activation in wts mutant cells in the optic lobe. Therefore AMPK/LKB1 form a Hpo/Wts independent pathway to inhibit Yki activity.
In summary, I have shown that Yki is inhibited by the nutrient-sensing LKB1/AMPK cascade independently of Hippo/Warts in the developing Drosophila larval brain. These results suggest a potential tissue specific nutrient-dependent mode of Yki activity regulation, which could represent an adjustment to the proliferation requirements of different tissue types.
During this project, I have identified Liver Kinase B1 (LKB1), a master kinase that activates AMPK family kinases, as an inhibitor of Yki activity in a tissue-specific manner. LKB1 is a tumour suppressor kinase that regulates multiple processes such as cell polarity and proliferation. In humans, heterozygous mutation of lkb1 leads to an increased benign and malignant tumour predisposition (Peutz-Jeghers syndrome), while sporadic mutations have also been linked to a variety of cancers. lkb1 mutant Drosophila larvae exhibit brain overgrowth, and die at early pupal stages. The Drosophila larval brain is composed of neural stem cells or neuroblasts (NBs) and their progeny, which differentiates into neurons or glial cells. Drosophila NBs have been extensively studied as a model for stem cell proliferation and differentiation. I found that loss of lkb1 function in the larval brain results in increased Yki target gene expression both in the NBs and their progeny, and causes increased cell proliferation. RNAi against yki in lkb1 mutant cells brought Yki target levels and cell proliferation back to wild type levels. Conversely, LKB1 overexpression results in smaller organ size, inhibits cell proliferation and decreases Yki target gene expression.
AMPK is one of the best-characterised downstream targets of LKB1, and has been shown to act as a sensor of intracellular energy levels to inhibit anabolic processes with high energy cost and to promote energy-generating catabolic processes. Similarly to lkb1 mutant phenotype, loss of ampk function in the larval brain induced increased Yki target gene expression. Additionally, RNAi-mediated knockdown of ampk in cells that overexpress LKB1 inhibited the decrease of Yki target gene expression, positioning AMPK downstream of LKB1 in regulation of Yki activity.
The developing Drosophila larval brain consists of two compartments of a different developmental origin: the optic lobe and the central brain. Interestingly, I found that mutations in hpo and wts, core Hpo kinase pathway members, increases transcription of Yki target genes in the optic lobe, but not in the central brain. Similarly, activated form of Yki that cannot be inhibited by Wts activity does not induce overgrowth in the central brain. These results indicate that LKB1/AMPK is the main inhibitory input on Yki activity in the larval central brain. Additionally, I found that LKB1 overexpression inhibits Yki activation in wts mutant cells in the optic lobe. Therefore AMPK/LKB1 form a Hpo/Wts independent pathway to inhibit Yki activity.
In summary, I have shown that Yki is inhibited by the nutrient-sensing LKB1/AMPK cascade independently of Hippo/Warts in the developing Drosophila larval brain. These results suggest a potential tissue specific nutrient-dependent mode of Yki activity regulation, which could represent an adjustment to the proliferation requirements of different tissue types.