Vangl-dependent Wnt/planar cell polarity signaling mediates collective breast carcinoma motility and distant metastasis.

BACKGROUND: In light of the growing appreciation for the role of collective cell motility in metastasis, a deeper understanding of the underlying signaling pathways will be critical to translating these observations to the treatment of advanced cancers. Here, we examine the contribution of Wnt/planar cell polarity (Wnt/PCP), one of the non-canonical Wnt signaling pathways and defined by the involvement of the tetraspanin-like proteins Vangl1 and Vangl2, to breast tumor cell motility, collective cell invasiveness and mammary tumor metastasis. METHODS: Vangl1 and Vangl2 knockdown and overexpression and Wnt5a stimulation were employed to manipulate Wnt/PCP signaling in a battery of breast cancer cell lines representing all breast cancer subtypes, and in tumor organoids from MMTV-PyMT mice. Cell migration was assessed by scratch and organoid invasion assays, Vangl protein subcellular localization was assessed by confocal fluorescence microscopy, and RhoA activation was assessed in real time by fluorescence imaging with an advanced FRET biosensor. The impact of Wnt/PCP suppression on mammary tumor growth and metastasis was assessed by determining the effect of conditional Vangl2 knockout on the MMTV-NDL mouse mammary tumor model. RESULTS: We observed that Vangl2 knockdown suppresses the motility of all breast cancer cell lines examined, and overexpression drives the invasiveness of collectively migrating MMTV-PyMT organoids. Vangl2-dependent RhoA activity is localized in real time to a subpopulation of motile leader cells displaying a hyper-protrusive leading edge, Vangl protein is localized to leader cell protrusions within leader cells, and actin cytoskeletal regulator RhoA is preferentially activated in the leader cells of a migrating collective. Mammary gland-specific knockout of Vangl2 results in a striking decrease in lung metastases in MMTV-NDL mice, but does not impact primary tumor growth characteristics. CONCLUSIONS: We conclude that Vangl-dependent Wnt/PCP signaling promotes breast cancer collective cell migration independent of breast tumor subtype and facilitates distant metastasis in a genetically engineered mouse model of breast cancer. Our observations are consistent with a model whereby Vangl proteins localized at the leading edge of leader cells in a migrating collective act through RhoA to mediate the cytoskeletal rearrangements required for pro-migratory protrusion formation.

The Cationic Amphiphilic Drug Hexamethylene Amiloride Eradicates Bulk Breast Cancer Cells and Therapy-Resistant Subpopulations with Similar Efficiencies.

The resistance of cancer cell subpopulations, including cancer stem cell (CSC) populations, to apoptosis-inducing chemotherapeutic agents is a key barrier to improved outcomes for cancer patients. The cationic amphiphilic drug hexamethylene amiloride (HMA) has been previously demonstrated to efficiently kill bulk breast cancer cells independent of tumor subtype or species but acts poorly toward non-transformed cells derived from multiple tissues. Here, we demonstrate that HMA is similarly cytotoxic toward breast CSC-related subpopulations that are resistant to conventional chemotherapeutic agents, but poorly cytotoxic toward normal mammary stem cells. HMA inhibits the sphere-forming capacity of FACS-sorted human and mouse mammary CSC-related cells in vitro, specifically kills tumor but not normal mammary organoids ex vivo, and inhibits metastatic outgrowth in vivo, consistent with CSC suppression. Moreover, HMA inhibits viability and sphere formation by lung, colon, pancreatic, brain, liver, prostate, and bladder tumor cell lines, suggesting that its effects may be applicable to multiple malignancies. Our observations expose a key vulnerability intrinsic to cancer stem cells and point to novel strategies for the exploitation of cationic amphiphilic drugs in cancer treatment.

Dispatched uses Na+ flux to power release of lipid-modified Hedgehog.

The Dispatched protein, which is related to the NPC1 and PTCH1 cholesterol transporters1,2 and to H+-driven transporters of the RND family3,4, enables tissue-patterning activity of the lipid-modified Hedgehog protein by releasing it from tightly -localized sites of embryonic expression5-10. Here we determine a cryo-electron microscopy structure of the mouse protein Dispatched homologue 1 (DISP1), revealing three Na+ ions coordinated within a channel that traverses its transmembrane domain. We find that the rate of Hedgehog export is dependent on the Na+ gradient across the plasma membrane. The transmembrane channel and Na+ binding are disrupted in DISP1-NNN, a variant with asparagine substitutions for three intramembrane aspartate residues that each coordinate and neutralize the charge of one of the three Na+ ions. DISP1-NNN and variants that disrupt single Na+ sites retain binding to, but are impaired in export of the lipid-modified Hedgehog protein to the SCUBE2 acceptor. Interaction of the amino-terminal signalling domain of the Sonic hedgehog protein (ShhN) with DISP1 occurs via an extensive buried surface area and contacts with an extended furin-cleaved DISP1 arm. Variability analysis reveals that ShhN binding is restricted to one extreme of a continuous series of DISP1 conformations. The bound and unbound DISP1 conformations display distinct Na+-site occupancies, which suggests a mechanism by which transmembrane Na+ flux may power extraction of the lipid-linked Hedgehog signal from the membrane. Na+-coordinating residues in DISP1 are conserved in PTCH1 and other metazoan RND family members, suggesting that Na+ flux powers their conformationally driven activities.

CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy.

Ovarian cancer and triple-negative breast cancer are among the most lethal diseases affecting women, with few targeted therapies and high rates of metastasis. Cancer cells are capable of evading clearance by macrophages through the overexpression of anti-phagocytic surface proteins called 'don't eat me' signals-including CD471, programmed cell death ligand 1 (PD-L1)2 and the beta-2 microglobulin subunit of the major histocompatibility class I complex (B2M)3. Monoclonal antibodies that antagonize the interaction of 'don't eat me' signals with their macrophage-expressed receptors have demonstrated therapeutic potential in several cancers4,5. However, variability in the magnitude and durability of the response to these agents has suggested the presence of additional, as yet unknown 'don't eat me' signals. Here we show that CD24 can be the dominant innate immune checkpoint in ovarian cancer and breast cancer, and is a promising target for cancer immunotherapy. We demonstrate a role for tumour-expressed CD24 in promoting immune evasion through its interaction with the inhibitory receptor sialic-acid-binding Ig-like lectin 10 (Siglec-10), which is expressed by tumour-associated macrophages. We find that many tumours overexpress CD24 and that tumour-associated macrophages express high levels of Siglec-10. Genetic ablation of either CD24 or Siglec-10, as well as blockade of the CD24-Siglec-10 interaction using monoclonal antibodies, robustly augment the phagocytosis of all CD24-expressing human tumours that we tested. Genetic ablation and therapeutic blockade of CD24 resulted in a macrophage-dependent reduction of tumour growth in vivo and an increase in survival time. These data reveal CD24 as a highly expressed, anti-phagocytic signal in several cancers and demonstrate the therapeutic potential for CD24 blockade in cancer immunotherapy.

Cellular and molecular mechanisms underlying planar cell polarity pathway contributions to cancer malignancy.

While the mutational activation of oncogenes drives tumor initiation and growth by promoting cellular transformation and proliferation, increasing evidence suggests that the subsequent re-engagement of largely dormant developmental pathways contributes to cellular phenotypes associated with the malignancy of solid tumors. Genetic studies from a variety of model organisms have defined many of the components that maintain epithelial planar cell polarity (PCP), or cellular polarity in the axis orthogonal to the apical-basal axis. These same components comprise an arm of non-canonical Wnt signaling that mediates cell motility events such as convergent extension movements essential to proper development. In this review, we summarize the increasing evidence that the Wnt/PCP signaling pathway plays active roles in promoting the proliferative and migratory properties of tumor cells, emphasizing the importance of subcellular localization of PCP components and protein-protein interactions in regulating cellullar properties associated with malignancy. Specifically, we discuss the increased expression of Wnt/PCP pathway components in cancer and the functional consequences of aberrant pathway activation, focusing on Wnt ligands, Frizzled (Fzd) receptors, the tetraspanin-like proteins Vangl1 and Vangl2, and the Prickle1 (Pk1) scaffold protein. In addition, we discuss negative regulation of the Wnt/PCP pathway, with particular emphasis on the Nrdp1 E3 ubiquitin ligase. We hypothesize that engagement of the Wnt/PCP pathway after tumor initiation drives malignancy by promoting cellular proliferation and invasiveness, and that the ability of Wnt/PCP signaling to supplant oncogene addiction may contribute to tumor resistance to oncogenic pathway-directed therapeutic agents.

Replication Study: The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate.

In 2016, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Fiehn et al., 2016), that described how we intended to replicate selected experiments from the paper "The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate" (Ward et al., 2010). Here, we report the results of those experiments. We found that cells expressing R172K mutant IDH2 did not display isocitrate-dependent NADPH production above vector control levels, in contrast to the increased production observed with wild-type IDH2. Conversely, expression of R172K mutant IDH2 resulted in increased alpha-ketoglutarate-dependent consumption of NADPH compared to wild-type IDH2 or vector control. These results are similar to those reported in the original study (Figure 2; Ward et al., 2010). Further, expression of R172K mutant IDH2 resulted in increased 2HG levels within cells compared to the background levels observed in wild-type IDH2 and vector control, similar to the original study (Figure 3D; Ward et al., 2010). In primary human AML samples, the 2HG levels observed in samples with mutant IDH1 or IDH2 status were higher than those observed in samples without an IDH mutation, similar to what was observed in the original study (Figure 5C; Ward et al., 2010). Finally, we report meta-analyses for each result.

The ER structural protein Rtn4A stabilizes and enhances signaling through the receptor tyrosine kinase ErbB3.

ErbB3 and ErbB4 are receptor tyrosine kinases that are activated by the neuregulin (NRG) family of growth factors. These receptors govern various developmental processes, and their dysregulation contributes to several human disease states. The abundance of ErbB3 and ErbB4, and thus signaling through these receptors, is limited by the E3 ubiquitin ligase Nrdp1, which targets ErbB3 and ErbB4 for degradation. Reticulons are proteins that influence the morphology of the endoplasmic reticulum (ER) by promoting the formation of tubules, a response of cells to some stressors. We found that the ER structural protein reticulon 4A (Rtn4A, also known as Nogo-A) increased ErbB3 abundance and proliferative signaling by suppressing Nrdp1 function. Rtn4A interacted with Nrdp1 and stabilized ErbB3 in an Nrdp1-dependent manner. Rtn4A overexpression induced the redistribution of Nrdp1 from a cytosolic or perinuclear localization to ER tubules. Rtn4A knockdown in human breast tumor cells decreased ErbB3 abundance, NRG-stimulated signaling, and cellular proliferation and migration. Because proteins destined for the plasma membrane are primarily synthesized in the sheet portions of the ER, our observations suggest that Rtn4A counteracts the Nrdp1-mediated degradation of ErbB3 by sequestering the ubiquitin ligase into ER tubules. The involvement of a reticulon suggests a molecular link between ER structure and the sensitivity of cells to receptor tyrosine kinase-mediated survival signals at the cell surface.

Hexamethylene amiloride engages a novel reactive oxygen species- and lysosome-dependent programmed necrotic mechanism to selectively target breast cancer cells.

Anticancer chemotherapeutics often rely on induction of apoptosis in rapidly dividing cells. While these treatment strategies are generally effective in debulking the primary tumor, post-therapeutic recurrence and metastasis are pervasive concerns with potentially devastating consequences. We demonstrate that the amiloride derivative 5-(N,N-hexamethylene) amiloride (HMA) harbors cytotoxic properties particularly attractive for a novel class of therapeutic agent. HMA is potently and specifically cytotoxic toward breast cancer cells, with remarkable selectivity for transformed cells relative to non-transformed or primary cells. Nonetheless, HMA is similarly cytotoxic to breast cancer cells irrespective of their molecular profile, proliferative status, or species of origin, suggesting that it engages a cell death mechanism common to all breast tumor subtypes. We observed that HMA induces a novel form of caspase- and autophagy-independent programmed necrosis relying on the orchestration of mitochondrial and lysosomal pro-death mechanisms, where its cytotoxicity was attenuated with ROS-scavengers or lysosomal cathepsin inhibition. Overall, our findings suggest HMA may efficiently target the heterogeneous populations of cancer cells known to reside within a single breast tumor by induction of a ROS- and lysosome-mediated form of programmed necrosis.

Vangl1 and Vangl2: planar cell polarity components with a developing role in cancer.

Cancers commonly reactivate embryonic developmental pathways to promote the aggressive behavior of their cells, resulting in metastasis and poor patient outcome. While developmental pathways such as canonical Wnt signaling and epithelial-to-mesenchymal transition have received much attention, our understanding of the role of the planar cell polarity (PCP) pathway in tumor progression remains rudimentary. Protein components of PCP, including a subset that overlaps with the canonical Wnt pathway, partition in polarized epithelial cells along the planar axis and are required for the establishment and maintenance of lateral epithelial polarity. Significant insight into PCP regulation of developmental and cellular processes has come from analysis of the functions of the core PCP scaffolding proteins Vangl1 and Vangl2. In particular, studies on zebrafish and with Looptail (Lp) mice, which harbor point mutations in Vangl2 that alter its trafficking and localization, point to roles for the PCP pathway in maintaining cell polarization along both the apical-basal and planar axes as well as in collective cell motility and invasiveness. Recent findings have suggested that the Vangls can promote similar processes in tumor cells. Initial data-mining efforts suggest that VANGL1 and VANGL2 are dysregulated in human cancers, and estrogen receptor (ER)-positive breast cancer patients whose tumors exhibit elevated VANGL1 expression suffer from shortened overall survival. Overall, evidence is beginning to accumulate that the heightened cellular motility and invasiveness associated with PCP reactivation may contribute to the malignancy of some cancer subtypes.

Novel insights of the gastric gland organization revealed by chief cell specific expression of moesin.

ERM (ezrin, radixin, and moesin) proteins play critical roles in epithelial and endothelial cell polarity, among other functions. In gastric glands, ezrin is mainly expressed in acid-secreting parietal cells, but not in mucous neck cells or zymogenic chief cells. In looking for other ERM proteins, moesin was found lining the lumen of much of the gastric gland, but it was not expressed in parietal cells. No significant radixin expression was detected in the gastric glands. Moesin showed an increased gradient of expression from the neck to the base of the glands. In addition, the staining pattern of moesin revealed a branched morphology for the gastric lumen. This pattern of short branches extending from the glandular lumen was confirmed by using antibody against zonula occludens-1 (ZO-1) to stain tight junctions. With a mucous neck cell probe (lectin GSII, from Griffonia simplicifolia) and a chief cell marker (pepsinogen C), immunohistochemistry revealed that the mucous neck cells at the top of the glands do not express moesin, but, progressing toward the base, mucous cells showing decreased GSII staining had low or moderate level of moesin expression. The level of moesin expression continued to increase toward the base of the glands and reached a plateau in the base where chief cells and parietal cells abound. The level of pepsinogen expression also increased toward the base. Pepsinogen C was located on cytoplasmic granules and/or more generally distributed in chief cells, whereas moesin was exclusively expressed on the apical membrane. This is a clear demonstration of distinctive cellular expression of two ERM family members in the same tissue. The results provide the first evidence that moesin is involved in the cell biology of chief cells. Novel insights on gastric gland morphology revealed by the moesin and ZO-1 staining provide the basis for a model of cell maturation and migration within the gland.

Comparative study of ezrin phosphorylation among different tissues: more is good; too much is bad.

In a comparison of three different tissues, the membrane cytoskeleton linker protein ezrin was found to assume high levels of phosphorylation on threonine-567 (T567) in the brush border membranes of renal proximal tubule cells and small intestine enterocytes, in contrast to the apical canalicular membrane of gastric parietal cells. Together with an earlier observation that increased T567 phosphorylation is associated with more elaborate microvilli in parietal cells, this comparative study suggested a higher phosphorylation level requirement for the denser and more uniform distribution of microvilli at brush border surfaces. Using a kinase inhibitor, staurosporin, and metabolic inhibitor, sodium azide, relatively high turnover of ezrin T567 phosphorylation was observed in all three epithelia. Aiming to understand the role of phosphorylation turnover in these tissues, detergent extraction analysis of gastric glands and proximal tubules revealed that an increased phosphorylation on ezrin T567 greatly enhanced its association with F-actin, while ezrin-membrane interaction persisted regardless of the changes of phosphorylation level on ezrin T567. Finally, expression of Thr567Asp mutant ezrin, which mimics the phospho-ezrin state but does not allow turnover, caused aberrant growth of membrane projections in cultured proximal tubule cells, consistent with what had previously been observed in several cell lines and gastric parietal cells. These results fit into a model of surface plasticity, which posits that the turnover of phosphorylation on T567 empowers ezrin to relax and reposition membrane to the underlying cytoskeleton under varying conditions of filament growth or rapid membrane expansion (or depletion).