The Hippo Tumor Suppressor Pathway Duojia Pan Iswar Hariharan Georg Halder - The Hippo pathway is one of many tumor suppressor pathways that is used to control the growth of tissues and organs. The founding members of the pathway were discovered by Georg Halder and Iswar Hariharan. Together with Duojia Pan (as well as many others) the entire pathway has been elucidated in both flies and mammals. As you can see in the image to the right, nearly all of the components are conserved across 500 million years of evolution. There are some differences in the number of elements and genetic elements but the overall pathway is conserved. - This pathways was first identified through efforts to understand how a tissue or organ knows when to stop growing. Since then the pathway has been closely associated with tumor formation and cancer. Mutations in several pathway members such as Fat4 and NF2 are the underlying causes of breast cancer and certain schwannomas respectively. Similarly, the main target of the Hippo pathway, the Yorki (Yki) transcription factor, is expressed at higher than normal levels in several breast, colorectal and liver cancers.

How Do You Find a Tumor Suppressor? - In Drosophila there are genetic tricks that one can employ to generate patches of cells that are mutant for both copies of a single gene (-/-). When such mutant clones are generated a twin clone that is wild type for both copies of your gene of interest is also generated (+/+). The growth features of these two clones (relative to each other) can be analyzed. - If the gene that you have mutated is not involved in growth control then the size of the mutant clone will be the same as the wild type twin spot (left middle image). If the gene that you have mutated is required for a cell to grow then the mutant clone will be smaller than the wild type twin spot (left top image). And finally, if the gene that you have mutated is a tumor suppressor then the mutant clone will be larger than the wild type twin spot (left bottom panel). - The Drosophila eye can be used to identify tumor suppressors. In the panels to the left the wild type tissue is marked with the red pigment and the mutant tissue lacks pigment. In panel A there is approximately equal amounts of red and white tissue (control panel). However, in panel A the mutant tissue is completely taken over the entire retina. The gene that is mutant in this example is a member of the Hippo tumor suppressor pathway. - Why do you create mutant clones instead of looking at entire animals? Tumor suppressor genes are usually expressed in all cells of a developing organism (this keeps growth control in check). A mutation that removes a tumor suppressor gene from the entire animal will die early in development due to tumor formation. The generation of mutant clones in the eye allows for the rest of the animal to develop normally. Since the eye is a dispensable organ a fly containing retinal clones that are mutant for a tumor suppressor will survive to be analyzed.

Phenotype of a Hippo Pathway Mutant (shar-pei) - Georg Halder’s research group designed the screen that is depicted on the previous slide and identified the founding member of the Hippo tumor suppressor pathway. Panels C-F (to the left) document the effects of removing this gene from the entire head and retina. In contrast to the wild type control animals which have a flat head cuticle surface (panels C,E) the mutant tissue over-proliferates leaving undulating folds of head capsule tissue. This phenotype resembles the undulating folds of skin on a shar-pei dog – based on this similarity the gene was called shar-pei. - The ability to suppress cell proliferation is not limited to the head and retina. Removal of shar-pei within a clone of cells within the thorax leads to tumor formation (panel H, arrow). Loss of shar-pei throughout the entire haltere leads to a significant increase in size (panel I,J). In every tissue examined shar-pei (and by extension the entire Hippo pathway) controls organ size throughout all developing Drosophila tissues. The same has been shown for the mammalian Hippo pathway. - Tissues can appear larger for two reasons. First, the number of cells in wild type and mutant tissue can be the same but the cells can be bigger in the mutant. Second. The size of wild type and mutant cells can be the same but the number of these cells can be significantly higher in the mutant tissue. In the fly retina each ommatidium is separated from its neighbors by a single cell (panel c below). In the shar-pei mutant there are more cells between the ommatidia (panel b below). These results indicate that the Hippo pathway is a true tumor suppressor pathway and that its role in development is to suppress cellular proliferation.

Phenotype of a Hippo Pathway Mutant (shar-pei) - Georg Halder’s research group designed the screen that is depicted on the previous slide and identified the founding member of the Hippo tumor suppressor pathway. Panels C-F (to the left) document the effects of removing this gene from the entire head and retina. In contrast to the wild type control animals which have a flat head cuticle surface (panels C,E) the mutant tissue over-proliferates leaving undulating folds of head capsule tissue. This phenotype resembles the undulating folds of skin on a shar-pei dog – based on this similarity the gene was called shar-pei. - The ability to suppress cell proliferation is not limited to the head and retina. Removal of shar-pei within a clone of cells within the thorax leads to tumor formation (panel H, arrow). Loss of shar-pei throughout the entire haltere leads to a significant increase in size (panel I,J). In every tissue examined shar-pei (and by extension the entire Hippo pathway) controls organ size throughout all developing Drosophila tissues. The same has been shown for the mammalian Hippo pathway. - Tissues can appear larger for two reasons. First, the number of cells in wild type and mutant tissue can be the same but the cells can be bigger in the mutant. Second. The size of wild type and mutant cells can be the same but the number of these cells can be significantly higher in the mutant tissue. In the fly retina each ommatidium is separated from its neighbors by a single cell (panel c below). In the shar-pei mutant there are more cells between the ommatidia (panel b below). These results indicate that the Hippo pathway is a true tumor suppressor pathway and that its role in development is to suppress cellular proliferation.

Mechanistic Role of the Hippo Pathway - Over the years a variety of genetic and biochemical screens have led to the isolation of all of the Hippo pathway components that are depicted in the drawing on the right. With one exception (which we will discuss on the next slide) the loss of any member of the Hippo pathway leads to the same phenotypes that are seen in the shar-pei mutants. For example, removal of hippo gene leads to over-proliferation (panel f), tissue undulations (panel a) and an increase in overall organ size (panel c). Additional defects in planar cell polarity, cell junction integrity and cell migration are seen in some pathway members that are associated with the membrane (ie Crumbs, Fat and Dachous). - As you can see in the pathway diagram on the right the entire cytoplasmic Hippo pathway is focused on inhibiting that activity of the Yorki (Yki) transcription factor. Yki encodes a transcriptional co-activator that cannot bind to DNA on its own but rather interacts with a DNA binding protein called Scalloped (Sd). The Sd-Yki composite transcription factor binds to enhancers of genes that are involved in promoting cell proliferation and tissue growth. The Hippo pathway is tasked with regulating Yki so that tissues and organs stop growing when the appropriate size has been achieved.

Yorki (Yki) Promotes Cell Proliferation - A member of the Hippo pathway was isolated in screens that were looking for growth regulators. However, the mutant clones grew poorly when compared with the wild type tissue. In keeping with the dog-naming trend this gene was called Yorki (Yki) since these dogs are very small – below is an image of a yorkie that despite being 12 weeks old is not much bigger than a Pepsi can. Note that this is in contrast to the effects seen with removing other Hippo pathway mutants. The images in the top panels depict what happens when yki is removed from the developing head and retina – both tissues are considerably smaller than wild type. The bottom panels demonstrate what happens when yki is expressed at higher than normal levels in the developing wing disc – the tissue while maintaining its overall shape is considerably larger than its wild type counterpart. This indicates that Yki promotes growth and the Hippo pathway’s role in development is to negatively modulate the activity of Yki.

Yorki Activates Expression of the bantam (ban) microRNA Georg Halder Stephen Cohen - Georg Halder and Stephen Cohen conducted a series of experiments that demonstrated that one of the targets of the Yki-Sd complex is the bantam microRNA gene. If you generate clones of wild type tissue these cells grow happily in a wing disc (panel A - top and bottom). If you over-express Yki these clones are bigger (panel B – top). If you remove yki then these clones hardly grow at all (panel B – bottom). Both results support the idea that Yki promotes cell proliferation. - If you remove the bantam microRNA the clones grow very poorly (panel C – top). If you now remove bantam from a clone of cells that is over-expressing Yki the clones grow more poorly than if bantam is present (panel B and D – top). If you over-express the bantam microRNA in cells that are lacking yki now the clones grow better than clones that just lack yki (panel B and D – bottom). - Both results suggest that bantam is a genetic target of the Yki-Sd complex. The idea is that in cells that are growing, Yki-Sd activates expression of bantam which in turn will bind to the 3`UTR of a set of target mRNAs (that is what microRNAs do). The resulting degradation of the target mRNA or the blockage of translation of that mRNA results in enhanced growth.

bantam binds to the head involution defective (hid) mRNA - microRNAs bind to the 3`UTR of mRNA transcripts and either stimulate the degradation of the transcript or block translation. A search for putative targets identified the head involution defective (hid) mRNA transcript as a putative target. With the 3`UTR of hid are five potential binding sites of the bantam miRNA. - Stephen Cohen developed a miRNA sensor assay to determine if a particular 3`UTR is indeed a true target of any given miRNA. In this experiment he fused the 3`UTR of hid to the coding sequences of GFP. In a wild type wing disc the mRNA is translated normally and the wing disc glows green (panel B). Then using the UAS-GAL4 system he forcibly expressed the bantam miRNA along the A/P axis (arrow). As you can see in the picture the level of GFP expression dropped along the A/P axis. This indicates that the bantam miRNA was able to bind to the 3`UTR and block the production of GFP (panel C).

Hid Promotes Apoptosis (Cell Death) - Hid is a member of the programmed cell death pathway. In response to apoptotic stimuli hid and several other proteins initiate a cascade of events that will ultimately lead to the death of a cell. This pathway comes in many different version depending upon the organism (right panel). In the fly embryo there is a significant amount of programmed cell death (green stain in panel A - top left). This is caused by the overproduction of cells during the early stages of development. These excess cells must be pruned away prior to the hatching of the embryo into a larva. In mutants that lack the hid gene all cell death is blocked (panel B – top left) If you over-express hid within the developing eye high levels of cell death are induced and as a result only a small tiny eye is produced (compare the wild type panel with the GMR-hid panel). - In cells that are growing the Yki-Sd complex activates the expression of the bantam microRNA which in turn binds to the hid mRNA and blocks production of Hid protein. Without this key cell death inducer apoptosis is blocked and cells can proliferate. When it is time for cells to stop growing the Hippo pathway blocks the activity of the Yki-Sd. This results in the loss of bantam expression. Without the bantam microRNA the hid mRNA can be translated. The Hid protein can then induce cell death and prevent a tissue or organ from growing out of control.

Conservation of the Hippo Pathway and Function - As we discussed in the first slide the Hippo pathway is conserved from flies to humans. The above table lists the Drosophila and mouse Hippo pathway genes and the types of proteins that they encode. Several tumors and cancers in human patients are associated with the loss of several different Hippo pathway elements. In the example to the left the loss of Hippo signaling in the mouse liver leads to a dramatic increase in the size of that organ. Mst1 and Mst2 are homologs of the Drosophila Hippo protein. Loss of either Mst1 or Mst2 individually has no effect of organ size. Both genes must be simultaneously eliminated in order to see an effect on cell proliferation. Mst1 and Mst2 are the products of a duplication event that occurred after the split in the insect and vertebrate lineages. Since the individual mutations have no phenotype it is thought that these two genes are completely redundant to each other in function. Similar to that seen in flies, the vertebrate Hippo pathway is tasked with repressing the activity of the Yki homolog (YAP). Thus the Hippo pathway represents a universal mechanism for controlling growth.