Tumor infiltrated type II (M2) macrophages promote tumorigenesis by suppressing immune clearance, promoting proliferation, and stimulating angiogenesis. Interestingly, macrophages were also found to enrich in small foci of altered hepatocytes containing liver tumor-initiating cells (TICs). However, whether and how TICs specifically recruit macrophages and the function of these macrophages in tumor initiation remain unknown due to technical difficulties. In this study, by generating genetically defined liver TICs, we demonstrate that TICs actively recruit M2 macrophages from as early as the single-cell stage. Elimination of TIC-associated macrophages (TICAMs) abolishes tumorigenesis in a manner dependent on the immune system. Mechanistically, activation of the Hippo pathway effector Yes-associated protein (YAP) underlies macrophage recruitment by TICs. These results demonstrate for the first time that macrophages play a decisive role in the survival of single TICs in vivo and provide a proof of principle for TIC elimination by targeting YAP or M2 macrophages.

The Hippo pathway regulates cell proliferation, apoptosis, and stem cell self-renewal, and its inactivation in animal models causes organ enlargement followed by tumorigenesis. Hippo pathway deregulation occurs in many human cancers, but the underlying mechanisms are not fully understood. Here, we report tyrosine phosphorylation of the Hippo pathway tumor suppressor LATS1 as a mechanism underlying its regulation by cell adhesion. A tyrosine kinase library screen identified Src as the kinase to directly phosphorylate LATS1 on multiple residues, causing attenuated Mob kinase activator binding and structural alteration of the substrate-binding pocket in the kinase domain. Cell matrix adhesion activated the Hippo pathway effector transcription coactivator YAP partially through Src-mediated phosphorylation and inhibition of LATS1. Aberrant Src activation abolished the tumor suppressor activity of LATS1 and induced tumorigenesis in a YAP-dependent manner. Protein levels of Src in human breast cancer tissues correlated with accumulation of active YAP dephosphorylated on the LATS1 target site. These findings reveal tyrosine phosphorylation of LATS1 by Src as a novel mechanism of Hippo pathway regulation by cell adhesion and suggest Src activation as an underlying reason for YAP deregulation in tumorigenesis. .

Organ size determination is one of the most intriguing unsolved mysteries in biology. Aberrant activation of the major effector and transcription co-activator YAP in the Hippo pathway causes drastic organ enlargement in development and underlies tumorigenesis in many human cancers. However, how robust YAP activation is achieved during organ size control remains elusive. Here we report that the YAP signaling is sustained through a novel microRNA-dependent positive feedback loop. miR-130a, which is directly induced by YAP, could effectively repress VGLL4, an inhibitor of YAP activity, thereby amplifying the YAP signals. Inhibition of miR-130a reversed liver size enlargement induced by Hippo pathway inactivation and blocked YAP-induced tumorigenesis. Furthermore, the Drosophila Hippo pathway target bantam functionally mimics miR-130a by repressing the VGLL4 homolog SdBP/Tgi. These findings reveal an evolutionarily conserved positive feedback mechanism underlying robustness of the Hippo pathway in size control and tumorigenesis.

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The Hippo pathway plays important roles in controlling organ size and in suppressing tumorigenesis through large tumor suppressor kinase 1/2 (LATS1/2)-mediated phosphorylation of YAP/TAZ transcription co-activators. The kinase activity of LATS1/2 is regulated by phosphorylation in response to extracellular signals. Moreover, LATS2 protein levels are repressed by the ubiquitin-proteasome system in conditions such as hypoxia. However, the mechanism that removes the ubiquitin modification from LATS2 and thereby stabilizes the protein is not well understood. Here, using tandem affinity purification (TAP), we found that anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase complex, and USP9X, a deubiquitylase, specifically interact with LATS2. We also found that although APC1 co-localizes with LATS2 to intracellular vesicle structures, it does not regulate LATS2 protein levels and activity. In contrast, USP9X ablation drastically diminished LATS2 protein levels. We further demonstrated that USP9X deubiquitinates LATS2 and thus prevents LATS2 degradation by the proteasome. Furthermore, in pancreatic cancer cells, USP9X loss activated YAP and enhanced the oncogenic potential of the cells. In addition, the tumorigenesis induced by the USP9X ablation depended not only on LATS2 repression, but also on YAP/TAZ activity. We conclude that USP9X is a deubiquitylase of the Hippo pathway kinase LATS2 and that the Hippo pathway functions as a downstream signaling cascade that mediates USP9X's tumor-suppressive activity.

Yes-associated protein (YAP) is a transcriptional co-activator and a major effector of the Hippo pathway that promotes cell proliferation and stemness, while inhibiting apoptosis. YAP plays a central role in organ size control, and its deregulation strongly promotes cancer initiation and progression. However, the mechanisms by which YAP promotes cell invasion and metastasis are not fully understood. Here, we report that YAP induces leukocyte-specific () expression in cancer cells, thereby promoting cell invasion through the endothelium in a manner mimicking leukocytes. Through independent biochemical purification and a functional screen, we further identified PR/SET domain 4 (PRDM4) as a transcription factor interacting with the WW domains of YAP to mediate expression and cell invasion. Consistently, and mRNA levels are significantly increased in metastatic prostate cancer. In addition, PRDM4 contributes to YAP-induced tumorigenesis possibly via mediating the expression of other YAP target genes. Our results demonstrate that YAP promotes cell invasion by inducing leukocyte-specific integrin expression, and identify PRDM4 as a novel transcription factor for YAP targets.

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The Hippo pathway restricts organ size during development and its inactivation plays a crucial role in cancer. Yes-associated protein (YAP) and its paralog transcriptional coactivator with PSD-95/Dlg/ZO-1 (PDZ)-binding motif (TAZ) are transcription co-activators and effectors of the Hippo pathway mediating aberrant enlargement of organs and tumor growth upon Hippo pathway inactivation. It has been demonstrated that genetic inactivation of YAP could be an effective approach to inhibit tumorigenesis. In order to identify pharmacological inhibitors of YAP, we screened a library of 52,683 compounds using a YAP-specific reporter assay. In this screen we identified cyclopeptide RA-V (deoxybouvardin) as a specific inhibitor of YAP and TAZ but not other reporters. Unexpectedly, later experiments demonstrated that RA-V represses the protein but not mRNA levels of YAP target genes. Nevertheless, RA-V strongly blocks liver enlargement induced by Mst1/2 knockout. Furthermore, RA-V not only inhibits liver tumorigenesis induced by YAP activation, but also induces regression of established tumors. We found that RA-V inhibits dedifferentiation and proliferation, while inducing apoptosis of hepatocytes. Furthermore, RA-V also induces apoptosis and inhibits proliferation of macrophages in the microenvironment, which are essential for YAP-induced tumorigenesis. RA-V is thus a drug candidate for cancers involving YAP/TAZ activation.