RAL GTPases Drive Intestinal Stem Cell Function and Regeneration through Internalization of WNT Signalosomes.

Ral GTPases are RAS effector molecules and by implication a potential therapeutic target for RAS mutant cancer. However, very little is known about their roles in stem cells and tissue homeostasis. Using Drosophila, we identified expression of RalA in intestinal stem cells (ISCs) and progenitor cells of the fly midgut. RalA was required within ISCs for efficient regeneration downstream of Wnt signaling. Within the murine intestine, genetic deletion of either mammalian ortholog, Rala or Ralb, reduced ISC function and Lgr5 positivity, drove hypersensitivity to Wnt inhibition, and impaired tissue regeneration following damage. Ablation of both genes resulted in rapid crypt death. Mechanistically, RALA and RALB were required for efficient internalization of the Wnt receptor Frizzled-7. Together, we identify a conserved role for RAL GTPases in the promotion of optimal Wnt signaling, which defines ISC number and regenerative potential.

Targeting the LOX/hypoxia axis reverses many of the features that make pancreatic cancer deadly: inhibition of LOX abrogates metastasis and enhances drug efficacy.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related mortality. Despite significant advances made in the treatment of other cancers, current chemotherapies offer little survival benefit in this disease. Pancreaticoduodenectomy offers patients the possibility of a cure, but most will die of recurrent or metastatic disease. Hence, preventing metastatic disease in these patients would be of significant benefit. Using principal component analysis (PCA), we identified a LOX/hypoxia signature associated with poor patient survival in resectable patients. We found that LOX expression is upregulated in metastatic tumors from Pdx1-Cre Kras(G12D/+) Trp53(R172H/+) (KPC) mice and that inhibition of LOX in these mice suppressed metastasis. Mechanistically, LOX inhibition suppressed both migration and invasion of KPC cells. LOX inhibition also synergized with gemcitabine to kill tumors and significantly prolonged tumor-free survival in KPC mice with early-stage tumors. This was associated with stromal alterations, including increased vasculature and decreased fibrillar collagen, and increased infiltration of macrophages and neutrophils into tumors. Therefore, LOX inhibition is able to reverse many of the features that make PDAC inherently refractory to conventional therapies and targeting LOX could improve outcome in surgically resectable disease.

Mutant p53-associated myosin-X upregulation promotes breast cancer invasion and metastasis.

Mutations of the tumor suppressor TP53 are present in many forms of human cancer and are associated with increased tumor cell invasion and metastasis. Several mechanisms have been identified for promoting dissemination of cancer cells with TP53 mutations, including increased targeting of integrins to the plasma membrane. Here, we demonstrate a role for the filopodia-inducing motor protein Myosin-X (Myo10) in mutant p53-driven cancer invasion. Analysis of gene expression profiles from 2 breast cancer data sets revealed that MYO10 was highly expressed in aggressive cancer subtypes. Myo10 was required for breast cancer cell invasion and dissemination in multiple cancer cell lines and murine models of cancer metastasis. Evaluation of a Myo10 mutant without the integrin-binding domain revealed that the ability of Myo10 to transport ß1 integrins to the filopodia tip is required for invasion. Introduction of mutant p53 promoted Myo10 expression in cancer cells and pancreatic ductal adenocarcinoma in mice, whereas suppression of endogenous mutant p53 attenuated Myo10 levels and cell invasion. In clinical breast carcinomas, Myo10 was predominantly expressed at the invasive edges and correlated with the presence of TP53 mutations and poor prognosis. These data indicate that Myo10 upregulation in mutant p53-driven cancers is necessary for invasion and that plasma-membrane protrusions, such as filopodia, may serve as specialized metastatic engines.

Post-transcriptional regulation of VEGF-A mRNA levels by mitogen-activated protein kinases (MAPKs) during metabolic stress associated with ischaemia/reperfusion.

Angiogenesis is a well-characterised response to the metabolic stresses that occur during ischaemia/reperfusion, but the signalling pathways that regulate it are poorly understood. We tested whether activation of mitogen-activated protein kinases (MAPKs) was involved in regulating the expression of pro-angiogenic growth factors by the metabolic stresses associated with ischaemia/reperfusion in H9c2 rat cardiomyoblasts. Metabolic stress had no effect on vascular endothelial growth factor (VEGF) mRNA levels, but recovery after metabolic inhibition led to a strong induction of VEGF-A mRNA (3.8 ± 0.5-fold at 4 h), a modest rise in VEGF-C mRNA levels (1.7 ± 0.3-fold at 4 h), with no effect on VEGF-B or -D. A VEGF-A promoter reporter construct was unresponsive to metabolic inhibition/recovery and increases in VEGF-A mRNA were not blocked by the transcription inhibitor actinomycin D suggesting that increases in VEGF mRNA were due to enhanced VEGF-A mRNA stability. In addition, studies using reporter constructs demonstrated that regions within the 5' untranslated region (UTR) contributed to enhanced mRNA stability following recovery from metabolic stress. Increases in VEGF-A mRNA were abolished by inhibition of extracellular signal-regulated kinase or c-jun N-terminal kinase MAPKs, suggesting that these kinases may promote angiogenesis in response to metabolic stress during ischaemia/reperfusion by increasing VEGF-A message stability.

Wnt inhibitor screen reveals iron dependence of ß-catenin signaling in cancers.

Excessive signaling from the Wnt pathway is associated with numerous human cancers. Using a high throughput screen designed to detect inhibitors of Wnt/ß-catenin signaling, we identified a series of acyl hydrazones that act downstream of the ß-catenin destruction complex to inhibit both Wnt-induced and cancer-associated constitutive Wnt signaling via destabilization of ß-catenin. We found that these acyl hydrazones bind iron in vitro and in intact cells and that chelating activity is required to abrogate Wnt signaling and block the growth of colorectal cancer cell lines with constitutive Wnt signaling. In addition, we found that multiple iron chelators, desferrioxamine, deferasirox, and ciclopirox olamine similarly blocked Wnt signaling and cell growth. Moreover, in patients with AML administered ciclopirox olamine, we observed decreased expression of the Wnt target gene AXIN2 in leukemic cells. The novel class of acyl hydrazones would thus be prime candidates for further development as chemotherapeutic agents. Taken together, our results reveal a critical requirement for iron in Wnt signaling and they show that iron chelation serves as an effective mechanism to inhibit Wnt signaling in humans.

Naked1 antagonizes Wnt signaling by preventing nuclear accumulation of ß-catenin.

Cyto-nuclear shuttling of ß-catenin is at the epicenter of the canonical Wnt pathway and mutations in genes that result in excessive nuclear accumulation of ß-catenin are the driving force behind the initiation of many cancers. Recently, Naked Cuticle homolog 1 (Nkd1) has been identified as a Wnt-induced intracellular negative regulator of canonical Wnt signaling. The current model suggests that Nkd1 acts between Disheveled (Dvl) and ß-catenin. Here, we employ the zebrafish embryo to characterize the cellular and biochemical role of Nkd1 in vivo. We demonstrate that Nkd1 binds to ß-catenin and prevents its nuclear accumulation. We also show that this interaction is conserved in mammalian cultured cells. Further, we demonstrate that Nkd1 function is dependent on its interaction with the cell membrane. Given the conserved nature of Nkd1, our results shed light on the negative feedback regulation of Wnt signaling through the Nkd1-mediated negative control of nuclear accumulation of ß-catenin.

Activation of ASK1, downstream MAPKK and MAPK isoforms during cardiac ischaemia.

p38 MAPK is activated potently during cardiac ischaemia, although the precise mechanism by which it is activated is unclear. We used the isolated perfused rat heart to investigate the signalling pathways activated upstream of p38 during global cardiac ischaemia. Ischaemia strongly activated p38alpha but not the JNK pathway. The MAPKKs, MKK3, MKK4 and MKK6 have previously been identified as potential upstream activators of p38; however, in the ischaemic perfused heart, we saw activation of MKK3 and MKK6 but not MKK4. MKK3 and MKK6 showed different temporal patterns of activity, indicating distinct modes of activation and physiological function. Consistent with a lack of JNK activation, we saw no activation of MKK4 or MKK7 at any time point during ischaemia. A lack of MKK4 activation indicates, at least in the ischaemic heart, that MKK4 is not a physiologically relevant activator of p38. The MAPKKK, ASK1, was strongly activated late during ischaemia, with a similar time course to that of MKK6 and in ischaemic neonatal cardiac myocytes ASK1 expression preferentially activated MKK6 rather than MKK3. These observations suggest that during ischaemia ASK1 is coupled to p38 activation primarily via MKK6. Potent activation of ASK1 during ischaemia without JNK activation shows that during cardiac ischaemia, ASK1 preferentially activates the p38 pathway. These results demonstrate a specificity of responses seldom seen in previous studies and illustrate the benefits of using direct assays in intact tissues responding to physiologically relevant stimuli to unravel the complexities of MAPK signalling.

The Hippo pathway regulates Wnt/beta-catenin signaling.

Several developmental pathways contribute to processes that regulate tissue growth and organ size. The Hippo pathway has emerged as one such critical regulator. However, how Hippo signaling is integrated with other pathways to coordinate these processes remains unclear. Here, we show that the Hippo pathway restricts Wnt/beta-Catenin signaling by promoting an interaction between TAZ and DVL in the cytoplasm. TAZ inhibits the CK1delta/epsilon-mediated phosphorylation of DVL, thereby inhibiting Wnt/beta-Catenin signaling. Abrogation of TAZ levels or Hippo signaling enhances Wnt3A-stimulated DVL phosphorylation, nuclear beta-Catenin, and Wnt target gene expression. Mice lacking Taz develop polycystic kidneys with enhanced cytoplasmic and nuclear beta-Catenin. Moreover, in Drosophila, Hippo signaling modulates Wg target gene expression. These results uncover a cytoplasmic function of TAZ in regulating Wnt signaling and highlight the role of the Hippo pathway in coordinating morphogenetic signaling with growth control.

Application of an integrated physical and functional screening approach to identify inhibitors of the Wnt pathway.

Large-scale proteomic approaches have been used to study signaling pathways. However, identification of biologically relevant hits from a single screen remains challenging due to limitations inherent in each individual approach. To overcome these limitations, we implemented an integrated, multi-dimensional approach and used it to identify Wnt pathway modulators. The LUMIER protein-protein interaction mapping method was used in conjunction with two functional screens that examined the effect of overexpression and siRNA-mediated gene knockdown on Wnt signaling. Meta-analysis of the three data sets yielded a combined pathway score (CPS) for each tested component, a value reflecting the likelihood that an individual protein is a Wnt pathway regulator. We characterized the role of two proteins with high CPSs, Ube2m and Nkd1. We show that Ube2m interacts with and modulates beta-catenin stability, and that the antagonistic effect of Nkd1 on Wnt signaling requires interaction with Axin, itself a negative pathway regulator. Thus, integrated physical and functional mapping in mammalian cells can identify signaling components with high confidence and provides unanticipated insights into pathway regulators.