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.

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.

The Emerging and Diverse Roles of Bis(monoacylglycero) Phosphate Lipids in Cellular Physiology and Disease.

Although understudied relative to many phospholipids, accumulating evidence suggests that bis(monoacylglycero)phosphate (BMP) is an important class of regulatory lipid that plays key roles in lysosomal integrity and function. BMPs are rare in most mammalian tissues, comprising only a few percent of total cellular lipid content, but are elevated in cell types such as macrophages that rely heavily on lysosomal function. BMPs are markedly enriched in endosomal and lysosomal vesicles compared to other organelles and membranous structures, and their unique sn-1:sn-1' stereoconfiguration may confer stability within the hydrolytic lysosomal environment. BMP-enriched vesicles serve in endosomal-lysosomal trafficking and function as docking structures for the activation of lysosomal hydrolytic enzymes, notably those involved in the catabolic breakdown of sphingolipids. BMP levels are dysregulated in lysosomal storage disorders, phospholipidosis, metabolic diseases, liver and kidney diseases and neurodegenerative disorders. However, whether BMP alteration is a mediator or simply a marker of pathological states is unclear. Likewise, although BMP acyl chain composition may be altered with disease states, the functional significance of specific BMP species remains to be resolved. Newly developed tools for untargeted lipidomic analysis, together with a deeper understanding of enzymes mediating BMP synthesis and degradation, will help shed further light on the functional significance of BMPs in cellular physiology and pathology.

Single-Molecule Fluorescence Detection of the Epidermal Growth Factor Receptor in Membrane Discs.

The epidermal growth factor receptor (EGFR) is critical to normal cellular signaling pathways. Moreover, it has been implicated in a range of pathologies, including cancer. As a result, it is the primary target of many anticancer drugs. One limitation to the design and development of these drugs has been the lack of molecular-level information about the interactions and conformational dynamics of EGFR. To overcome this limitation, this work reports the construction and characterization of functional, fluorescently labeled, and full-length EGFR in model membrane nanolipoprotein particles (NLPs) for in vitro fluorescence studies. To demonstrate the utility of the system, we investigate ATP-EGFR interactions. We observe that ATP binds at the catalytic site providing a means to measure a range of distances between the catalytic site and the C-terminus via Förster resonance energy transfer (FRET). These ATP-based experiments suggest a range of conformations of the C-terminus that may be a function of the phosphorylation state for EGFR. This work is a proof-of-principle demonstration of single-molecule studies as a noncrystallographic assay for EGFR interactions in real-time and under near-physiological conditions. The diverse nature of EGFR interactions means that new tools at the molecular level have the potential to significantly enhance our understanding of receptor pathology and are of utmost importance for cancer-related drug discovery.

Membrane Protein Quantity Control at the Endoplasmic Reticulum.

The canonical function of the endoplasmic reticulum-associated degradation (ERAD) system is to enforce quality control among membrane-associated proteins by targeting misfolded secreted, intra-organellar, and intramembrane proteins for degradation. However, increasing evidence suggests that ERAD additionally functions in maintaining appropriate levels of a subset of membrane-associated proteins. In this 'quantity control' capacity, ERAD responds to environmental cues to regulate the proteasomal degradation of specific ERAD substrates according to cellular need. In this review, we discuss in detail seven proteins that are targeted by the ERAD quantity control system. Not surprisingly, ERAD-mediated protein degradation is a key regulatory feature of a variety of ER-resident proteins, including HMG-CoA reductase, cytochrome P450 3A4, IP3 receptor, and type II iodothyronine deiodinase. In addition, the ERAD quantity control system plays roles in maintaining the proper stoichiometry of multi-protein complexes by mediating the degradation of components that are produced in excess of the limiting subunit. Perhaps somewhat unexpectedly, recent evidence suggests that the ERAD quantity control system also contributes to the regulation of plasma membrane-localized signaling receptors, including the ErbB3 receptor tyrosine kinase and the GABA neurotransmitter receptors. For these substrates, a proportion of the newly synthesized yet properly folded receptors are diverted for degradation at the ER, and are unable to traffic to the plasma membrane. Given that receptor abundance or concentration within the plasma membrane plays key roles in determining signaling efficiency, these observations may point to a novel mechanism for modulating receptor-mediated cellular signaling.

Regulation of the T-box transcription factor Tbx3 by the tumour suppressor microRNA-206 in breast cancer.

BACKGROUND: The Tbx3 transcription factor is over-expressed in breast cancer, where it has been implicated in proliferation, migration and regulation of the cancer stem cell population. The mechanisms that regulate Tbx3 expression in cancer have not been fully explored. In this study, we demonstrate that Tbx3 is repressed by the tumour suppressor miR-206 in breast cancer cells. METHODS: Bioinformatics prediction programmes and luciferase reporter assays were used to demonstrate that miR-206 negatively regulates Tbx3. We examined the impact of miR-206 on Tbx3 expression in breast cancer cells using miR-206 mimic and inhibitor. Gene/protein expression was examined by quantitative reverse-transcription-PCR and immunoblotting. The effects of miR-206 and Tbx3 on apoptosis, proliferation, invasion and cancer stem cell population was investigated by cell-death detection, colony formation, 3D-Matrigel and tumorsphere assays. RESULTS: In this study, we examined the regulation of Tbx3 by miR-206. We demonstrate that Tbx3 is directly repressed by miR-206, and that this repression of Tbx3 is necessary for miR-206 to inhibit breast tumour cell proliferation and invasion, and decrease the cancer stem cell population. Moreover, Tbx3 and miR-206 expression are inversely correlated in human breast cancer. Kaplan-Meier analysis indicates that patients exhibiting a combination of high Tbx3 and low miR-206 expression have a lower probability of survival when compared with patients with low Tbx3 and high miR-206 expression. These studies uncover a novel mechanism of Tbx3 regulation and identify a new target of the tumour suppressor miR-206. CONCLUSIONS: The present study identified Tbx3 as a novel target of tumour suppressor miR-206 and characterised the miR-206/Tbx3 signalling pathway, which is involved in proliferation, invasion and maintenance of the cancer stem cell population in breast cancer cells. Our results suggest that restoration of miR-206 in Tbx3-positive breast cancer could be exploited for therapeutic benefit.

Oligomerization of the Nrdp1 E3 ubiquitin ligase is necessary for efficient autoubiquitination but not ErbB3 ubiquitination.

Overexpression of the ErbB3 receptor tyrosine kinase protein in breast and other cancers contributes to tumor malignancy and therapeutic resistance. The RBCC/TRIM family RING finger E3 ubiquitin ligase Nrdp1 mediates the ubiquitination of ErbB3 in normal mammary epithelial cells to facilitate receptor degradation and suppress steady-state receptor levels. Post-transcriptional loss of Nrdp1 in patient breast tumors allows ErbB3 overexpression and receptor contribution to tumor progression, and elevated lability through autoubiquitination contributes to the observed loss of Nrdp1 in tumors relative to normal tissue. To begin to understand the mechanisms underlying Nrdp1 protein self-regulation through lability, we investigated the structural determinants required for efficient autoubiquitination and ErbB3 ubiquitination. Using mutagenesis, chemical cross-linking, size exclusion chromatography, and native polyacrylamide gel electrophoresis, we demonstrate that Nrdp1 self-associates into a stable oligomeric complex in cells. Deletion of its coiled-coil domain abrogates oligomerization but does not affect Nrdp1-mediated ErbB3 ubiquitination or degradation. On the other hand, the presence of the coiled-coil domain is necessary for efficient Nrdp1 autoubiquitination via a trans mechanism, indicating that Nrdp1 ubiquitination of its various targets is functionally separable. Finally, a GFP fusion of the coiled-coil domain stabilizes Nrdp1 and potentiates ErbB3 ubiquitination and degradation. These observations point to a model whereby the coiled-coil domain plays a key role in regulating Nrdp1 lability by promoting its assembly into an oligomeric complex, and raise the possibility that inhibition of ligase oligomerization via its coiled-coil domain could be of therapeutic benefit to breast cancer patients by restoring Nrdp1 protein.

Leucine-rich repeat and immunoglobulin domain-containing protein-1 (Lrig1) negative regulatory action toward ErbB receptor tyrosine kinases is opposed by leucine-rich repeat and immunoglobulin domain-containing protein 3 (Lrig3).

Lrig1 is the founding member of the Lrig family of transmembrane leucine-rich repeat proteins, which also includes Lrig2 and Lrig3. Lrig1 is a negative regulator of oncogenic receptor tyrosine kinases, including ErbB and Met receptors, and promotes receptor degradation. Lrig1 has recently emerged as both a tumor suppressor and a key regulator of epidermal and epithelial stem cell quiescence. Despite this, little is known of the mechanisms by which Lrig1 is regulated. Lrig3 was recently reported to increase ErbB receptor expression suggesting that it may function in a manner opposite to Lrig1. In this study, we explore the interaction between Lrig1 and Lrig3 and demonstrate that Lrig1 and Lrig3 functionally oppose one another. Lrig3 opposes Lrig1 negative regulatory activity and stabilizes ErbB receptors. Conversely, Lrig1 destabilizes Lrig3, limiting Lrig3's positive effects on receptors and identifying Lrig3 as a new target of Lrig1. These studies provide new insight into the regulation of Lrig1 and uncover a complex cross-talk between Lrig1 and Lrig3.

Novel sorafenib-based structural analogues: in-vitro anticancer evaluation of t-MTUCB and t-AUCMB.

In the current work, we carried out a mechanistic study on the cytotoxicity of two compounds, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-N-methyl-benzamide (t-AUCMB) and trans-N-methyl-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzamide (t-MTUCB), that are structurally similar to sorafenib. These compounds show strong cytotoxic responses in various cancer cell lines, despite significant differences in the induction of apoptotic events such as caspase activation and lactate dehydrogenase release in hepatoma cells. Both compounds induce autophagosome formation and LC3I cleavage, but there was little observable effect on mTORC1 or the downstream targets, S6K1 and 4E-binding protein. In addition, there was an increase in the activity of upstream signaling through the IRS1/PI3K/Akt-signaling pathway, suggesting that, unlike sorafenib, both compounds induce mammalian target of rapamycin (mTOR)-independent autophagy. The autophagy observed correlates with mitochondrial membrane depolarization, apoptosis-inducing factor release, and oxidative stress-induced glutathione depletion. However, there were no observable changes in the endoplasmic reticulum-stress markers such as binding immunoglobulin protein, inositol-requiring enzyme-α, phosphorylated eukaryotic initiation factor 2, and the lipid peroxidation marker, 4-hydroxynonenal, suggesting endoplasmic reticulum-independent oxidative stress. Finally, these compounds do not have the multikinase inhibitory activity of sorafenib, which may be reflected in their difference in the ability to halt cell cycle progression compared with sorafenib. Our findings indicate that both compounds have anticancer effects comparable with sorafenib in multiple cell lines, but they induce significant differences in apoptotic responses and appear to induce mTOR-independent autophagy. t-AUCMB and t-MTUCB represent novel chemical probes that are capable of inducing mTOR-independent autophagy and apoptosis to differing degrees, and may thus be potential tools for further understanding the link between these two cellular stress responses.

Co-opted integrin signaling in ErbB2-induced mammary tumor progression.

Although almost two decades of study point to a central role for aberrant ErbB2 activation in breast cancer, many cellular and biochemical mechanisms underlying ErbB2-induced tumor initiation and progression remain to be resolved. A study by Guo et al. published recently in Cell indicates that the signaling function of beta4 integrin actively contributes to the initiation, growth, and invasion of ErbB2-induced mammary tumors in transgenic mice by promoting the activation of c-Jun and STAT3. These observations offer novel mechanistic insight into ErbB2 action and highlight the notion that ErbB2 co-opts the functions of other signaling proteins to elicit tumor progression.

5-Benzylglycinyl-amiloride kills proliferating and nonproliferating malignant glioma cells through caspase-independent necroptosis mediated by apoptosis-inducing factor.

5'-Βenzylglycinyl-amiloride (UCD38B) and glycinyl-amiloride (UCD74A) are cell-permeant and cell-impermeant derivatives of amiloride, respectively, and used here to identify the cellular mechanisms of action underlying their antiglioma effects. UCD38B comparably kills proliferating and nonproliferating gliomas cells when cell cycle progression is arrested either by cyclin D1 siRNA or by acidification. Cell impermeant UCD74A inhibits plasmalemmal urokinase plasminogen activator (uPA) and the type 1 sodium-proton exchanger with potencies analogous to UCD38B, but is cytostatic. In contrast, UCD38B targets intracellular uPA causing mistrafficking of uPA into perinuclear mitochondria, reducing the mitochondrial membrane potential, and followed by the release of apoptotic inducible factor (AIF). AIF nuclear translocation is followed by a caspase-independent necroptotic cell death. Reduction in AIF expression by siRNA reduces the antiglioma cytotoxic effects of UCD38B, while not activating the caspase pathway. Ultrastructural changes shortly following treatment with UCD38B demonstrate dilation of endoplasmic reticulum (ER) and mitochondrial swelling followed by nuclear condensation within hours consistent with a necroptotic cell death differing from apoptosis and from autophagy. These drug mechanism of action studies demonstrate that UCD38B induces a cell cycle-independent, caspase-independent necroptotic glioma cell death that is mediated by AIF and independent of poly (ADP-ribose) polymerase and H2AX activation.

Structure-Activity Relationship Study Identifies A Novel Lipophilic Amiloride Derivative That Induces Lysosome-Dependent Cell Death in Therapy-Resistant Breast Cancer Cells.

Amiloride and its derivatives have long attracted attention as potential anticancer therapeutic agents. Several early studies characterized amilorides as inhibitors of sodium-proton antiporter-dependent tumor growth and urokinase plasminogen activator-mediated metastasis. However, more recent observations indicate that amiloride derivatives are specifically cytotoxic toward tumor cells relative to normal cells and have the capacity to target tumor cell populations resistant to currently-employed therapies. A major barrier to clinical translation of the amilorides is their modest cytotoxic potency, with EC 50 values in the high micromolar to low millimolar range. Here we report structure-activity relationship observations that underscore the importance of the guanidinium group and the presence of lipophilic substituents at the C(5) position of the amiloride pharmacophore in promoting cytotoxicity. Moreover, we demonstrate that our most potent derivative called LLC1 is specifically cytotoxic toward mouse mammary tumor organoids and drug-resistant populations of various breast cancer cell lines, and induces lysosomal membrane permeabilization as a prelude to lysosome-dependent cell death. Our observations offer a roadmap for the future development of amiloride-based cationic amphiphilic drugs that engage the lysosome to specifically kill breast tumor cells.

A complex of Wnt/planar cell polarity signaling components Vangl1 and Fzd7 drives glioblastoma multiforme malignant properties.

Targeting common oncogenic drivers of glioblastoma multiforme (GBM) in patients has remained largely ineffective, raising the possibility that alternative pathways may contribute to tumor aggressiveness. Here we demonstrate that Vangl1 and Fzd7, components of the non-canonical Wnt planar cell polarity (Wnt/PCP) signaling pathway, promote GBM malignancy by driving cellular proliferation, migration, and invasiveness, and engage Rho GTPases to promote cytoskeletal rearrangements and actin dynamics in migrating GBM cells. Mechanistically, we uncover the existence of a novel Vangl1/Fzd7 complex at the leading edge of migrating GBM cells and propose that this complex is critical for the recruitment of downstream effectors to promote tumor progression. Moreover, we observe that depletion of FZD7 results in a striking suppression of tumor growth and latency and extends overall survival in an intracranial mouse xenograft model. Our observations support a novel mechanism by which Wnt/PCP components Vangl1 and Fzd7 form a complex at the leading edge of migratory GBM cells to engage downstream effectors that promote actin cytoskeletal rearrangements dynamics. Our findings suggest that interference with Wnt/PCP pathway function may offer a novel therapeutic strategy for patients diagnosed with GBM.

Non-canonical WNT5A-ROR signaling: New perspectives on an ancient developmental pathway.

Deciphering non-canonical WNT signaling has proven to be both fascinating and challenging. Discovered almost 30 years ago, non-canonical WNT ligands signal independently of the transcriptional co-activator ß-catenin to regulate a wide range of morphogenetic processes during development. The molecular and cellular mechanisms that underlie non-canonical WNT function, however, remain nebulous. Recent results from various model systems have converged to define a core non-canonical WNT pathway consisting of the prototypic non-canonical WNT ligand, WNT5A, the receptor tyrosine kinase ROR, the seven transmembrane receptor Frizzled and the cytoplasmic scaffold protein Dishevelled. Importantly, mutations in each of these signaling components cause Robinow syndrome, a congenital disorder characterized by profound tissue morphogenetic abnormalities. Moreover, dysregulation of the pathway has also been linked to cancer metastasis. As new knowledge concerning the WNT5A-ROR pathway continues to grow, modeling these mutations will likely provide crucial insights into both the physiological regulation of the pathway and the etiology of WNT5A-ROR-driven diseases.

Cellular transformation promotes the incorporation of docosahexaenoic acid into the endolysosome-specific lipid bis(monoacylglycerol)phosphate in breast cancer.

Bis(monoacylglycero)phosphates (BMPs), a class of lipids highly enriched within endolysosomal organelles, are key components of the lysosomal intraluminal vesicles responsible for activating sphingolipid catabolic enzymes. While BMPs are understudied relative to other phospholipids, recent reports associate BMP dysregulation with a variety of pathological states including neurodegenerative diseases and lysosomal storage disorders. Since the dramatic lysosomal remodeling characteristic of cellular transformation could impact BMP abundance and function, we employed untargeted lipidomics approaches to identify and quantify BMP species in several in vitro and in vivo models of breast cancer and comparative non-transformed cells and tissues. We observed lower BMP levels within transformed cells relative to normal cells, and consistent enrichment of docosahexaenoic acid (22:6) fatty acyl chain-containing BMP species in both human- and mouse-derived mammary tumorigenesis models. Our functional analysis points to a working model whereby 22:6 BMPs serve as reactive oxygen species scavengers in tumor cells, protecting lysosomes from oxidant-induced lysosomal membrane permeabilization. Our findings suggest that breast tumor cells might divert polyunsaturated fatty acids into BMP lipids as part of an adaptive response to protect their lysosomes from elevated reactive oxygen species levels, and raise the possibility that BMP-mediated lysosomal protection is a tumor-specific vulnerability that may be exploited therapeutically.

E3 ubiquitin ligases in ErbB receptor quantity control.

Signaling through ErbB family growth factor receptor tyrosine kinases is necessary for the development and homeostasis of a wide variety of tissue types. However, the intensity of receptor-mediated cellular signaling must fall within a precise range; insufficient signaling can lead to developmental abnormalities or tissue atrophy, while over-signaling can lead to hyperplastic and ultimately neoplastic events. While a plethora of mechanisms have been described that regulate downstream signaling events, it appears that cells also utilize various mechanisms to regulate their ErbB receptor levels. Such mechanisms are collectively termed "ErbB receptor quantity control." Notably, studies over the past few years have highlighted roles for post-transcriptional processes, particularly protein degradation, in ErbB quantity control. Here the involvement of ErbB-directed E3 ubiquitin ligases is discussed, including Nrdp1-mediated ErbB3 degradation, ErbB4 degradation mediated by Nedd4 family E3 ligases, and CHIP-mediated ErbB2 degradation. The hypothesis is forwarded that protein degradation-based ErbB quantity control mechanisms play central roles in suppressing receptor overexpression in normal cells, and that the loss of such mechanisms could facilitate the onset or progression of ErbB-dependent tumors.

EGF receptor activation by heterologous mechanisms.

A myriad of growth factor-dependent and -independent mechanisms for activating the EGF receptor and the related ErbB receptors underscore the importance of receptor inhibitors in the treatment of some solid tumors.

Vangl as a Master Scaffold for Wnt/Planar Cell Polarity Signaling in Development and Disease.

The establishment of polarity within tissues and dynamic cellular morphogenetic events are features common to both developing and adult tissues, and breakdown of these programs is associated with diverse human diseases. Wnt/Planar cell polarity (Wnt/PCP) signaling, a branch of non-canonical Wnt signaling, is critical to the establishment and maintenance of polarity in epithelial tissues as well as cell motility events critical to proper embryonic development. In epithelial tissues, Wnt/PCP-mediated planar polarity relies upon the asymmetric distribution of core proteins to establish polarity, but the requirement for this distribution in Wnt/PCP-mediated cell motility remains unclear. However, in both polarized tissues and migratory cells, the Wnt/PCP-specific transmembrane protein Vangl is required and appears to serve as a scaffold upon which the core pathway components as well as positive and negative regulators of Wnt/PCP signaling assemble. The current literature suggests that the multiple interaction domains of Vangl allow for the binding of diverse signaling partners for the establishment of context- and tissue-specific complexes. In this review we discuss the role of Vangl as a master scaffold for Wnt/PCP signaling in epithelial tissue polarity and cellular motility events in developing and adult tissues, and address how these programs are dysregulated in human disease.

Ligand-induced transmembrane conformational coupling in monomeric EGFR.

Single pass cell surface receptors regulate cellular processes by transmitting ligand-encoded signals across the plasma membrane via changes to their extracellular and intracellular conformations. This transmembrane signaling is generally initiated by ligand binding to the receptors in their monomeric form. While subsequent receptor-receptor interactions are established as key aspects of transmembrane signaling, the contribution of monomeric receptors has been challenging to isolate due to the complexity and ligand-dependence of these interactions. By combining membrane nanodiscs produced with cell-free expression, single-molecule Förster Resonance Energy Transfer measurements, and molecular dynamics simulations, we report that ligand binding induces intracellular conformational changes within monomeric, full-length epidermal growth factor receptor (EGFR). Our observations establish the existence of extracellular/intracellular conformational coupling within a single receptor molecule. We implicate a series of electrostatic interactions in the conformational coupling and find the coupling is inhibited by targeted therapeutics and mutations that also inhibit phosphorylation in cells. Collectively, these results introduce a facile mechanism to link the extracellular and intracellular regions through the single transmembrane helix of monomeric EGFR, and raise the possibility that intramolecular transmembrane conformational changes upon ligand binding are common to single-pass membrane proteins.

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.