Dysregulation of cellular membrane homeostasis as a crucial modulator of cancer risk

Abstract

Cellular membranes serve as an epicentre combining extracellular and cytosolic components with membranous effectors, which together support numerous fundamental cellular signalling pathways that mediate biological responses. To execute their functions, membrane proteins, lipids and carbohydrates arrange, in a highly coordinated manner, into well-defined assemblies displaying diverse biological and biophysical characteristics that modulate several signalling events. The loss of membrane homeostasis can trigger oncogenic signalling. More recently, it has been documented that select membrane active dietaries (MADs) can reshape biological membranes and subsequently decrease cancer risk. In this review, we emphasize the significance of membrane domain structure, organization and their signalling functionalities as well as how loss of membrane homeostasis can steer aberrant signalling. Moreover, we describe in detail the complexities associated with the examination of these membrane domains and their association with cancer. Finally, we summarize the current literature on MADs and their effects on cellular membranes, including various mechanisms of dietary chemoprevention/interception and the functional links between nutritional bioactives, membrane homeostasis and cancer biology.

Keywords: cancer therapy; chemoprevention; dietary bioactives; endomembranes; intracellular trafficking; lipid rafts; membrane order; nanoclusters; oncogenes; plasma membrane.

Figure 1.. Cellular membrane domain structure and organization.

A) Schematic overview of the plasma membrane and several membrane-bound organelles that make up part of the cellular endomembrane system. Membranes are highly dynamic structures that can move intracellularly from the plasma membrane as part of the endocytic pathway. Endocytosis leads to the formation of membrane vesicles, i.e., early endosomes (1), whose lipid bilayer composition resembles that of the plasma membrane. These vesicles mature to form multivesicular structures or late endosomes (2), which can fuse to lysosomes (endolysosome system) (3), or traffic back to the plasma membrane via recycling endosomes (4 and 5). Other key members of the endomembrane system are the nucleus and endoplasmic reticulum (ER). Interestingly, these two organelles work closely together as evidenced by the fact that they share a common continuous membrane bilayer (6). The ER membrane also interacts with the outer mitochondrial membrane (OMM) and a series of proteins, which form a dynamic bridge structure. This dynamic connection is referred to as mitochondria-associated endoplasmic reticulum membranes (MAMs) (7). MAMs domains are indispensable for the execution of a series of cellular functionalities. B) Compartmentalization of proteo-lipid complexes into specialized domains in the plasma membrane. Multiple proteins and lipids assemble in an orchestrated manner to form distinct specialized membrane domains (purple, orange, green and gray regions), thus creating lateral (horizontal axis) heterogeneity in the plasma membrane. Moreover, plasma membrane heterogeneity is displayed across the lipid bilayer vertical axis, resulting in highly organized domains in both the plasma membrane exofacial and cytofacial leaflets. The latter is not disconnected from the former rather they are coupled, thereby facilitating bilayer crosstalk between inner and outer proteolipid components, which is essential for cellular signaling. Liquid ordered phase (Lo) (purple and orange membrane regions) and liquid disordered phase (Ld) (green and gray membrane regions) domains display distinct biochemical, i.e., protein, lipid and carbohydrate, compositions and biophysical, e.g., membrane rigidity, characteristics. The Lo domains are enriched in cholesterol, sphingolipids, glycolipids, and saturated glycerophospholipids as well as lipidated and glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs), thus generating lipid raft and raft-like domains. In contrast, the Ld domains, also known as non-rafts, are enriched in polyunsaturated and branched lipids. Additionally, formation, organization and stabilization of these membrane domains are believed to involve cortical actin (cytoskeleton). Finally, these diverse proteolipid domains, shown as purple, orange, green and gray regions, exclude one another, yet they all coexist to form the plasma membrane. Consequently, together these domains form a multifunctional highly complex and dynamic signaling platform. C) MAMs and their functionalities. 1. Lipid synthesis and transfer. MAMs are involved in the synthesis of PS. These structures can generate PS through PSS. Subsequently, PS localized in the ER is transported to the mitochondria and later converted to PE. MAMs can also mediate the trafficking of cholesterol between organelles. In this case, ER-localized caveolin-1 transfers cholesterol from the ER into the mitochondria membrane. 2. Ca²⁺ trafficking and apoptosis. Another functionality of MAMs is the modulation of programmed cell death. To initiate the apoptosis signaling cascade, IP3R1, localized in the ER membrane, transfers Ca²⁺ from the ER to mitochondria via VDAC1, which leads to Ca²⁺ trafficking into the mitochondrial matrix via MCU. The resulting increased mitochondrial Ca²⁺ levels, triggers the opening of mPTP. Stimulation of Casp8 leads to the activation of downstream caspases and modulation of BCL and PTEN. These proteins, i.e., BCL and PTEN, maintain intraorganellar Ca²⁺ trafficking. Additionally, SERCA, another MAM-associated protein, removes Ca²⁺ from the cytosolic space. 3. Mitochondria dynamics. MAM domains are involved in mitochondria fission. The fission by mitochondria involves MFN1/2 and MFN2, which are localized in the outer mitochondrial membrane (OMM). The recruitment of DRP1, a mitochondrial fission protein, to MAMs involves Mff, Fis1 and Syntaxin-17. 4. Autophagy. Under normal conditions, Syntaxin-17 interacts with DRP1 in MAMs. However, in the absence of nutrients, DRP1 is replaced by the pre-autophagosome marker ATG14L, which upregulates mTOR and AKT, and triggers the formation of the autophagosome. 5. ROS and ER stress. MAMs are able to modulate cellular oxidative stress. Under oxidative conditions, p66Shc is phosphorylated, which leads to the relocalization of p66Shc into MAMs. This triggers ROS production, which in turn induces ER stress via IRE1 and PERK. 6. Inflammation. Finally, MAMs play a key role in the modulation of the inflammasome. To initiate this process, NLRP3 traffics from the ER to MAMs where it binds its protein adaptor ASC and TXNIP, which subsequently leads to the formation of the inflammasome. PSS, phosphatidylserine synthase; inositol 1,4,5-trisphosphate receptor type 1; VDAC1, voltage-dependent anion-selective channel protein 1; MCU, mitochondrial calcium uniporter; mPTP, mitochondrial permeability transition pore; CASP8, caspase 8; BCL, B cell lymphoma; PTEN, phosphatase and tensin homolog; SERCA, sarco/endoplasmic reticulum Ca²⁺-ATPase; MFN1, mitofusin 1; MFN2, mitofusin 2; DRP, dynamin-related protein 1; Mff, mitochondrion fission factor; Fis1, fission 1 protein; ATG14L, autophagy-related 14-like; mTOR, mammalian target of rapamycin; AKT, serine/threonine kinase; ROS, reactive oxygen species; p66Shc, member of Src homologous-collagen homologue family of adapter proteins; IRE1, inositol-requiring enzyme 1; PERK, inositol-requiring enzyme 1; NLRP3, pyrin domain-containing 3 protein; ASC, apoptosis-associated speck-like protein; TXNIP, thioredoxin interacting protein.

Figure 2.. Modulation of lipid and protein organization in cellular membrane domains and their functions.

A) Membrane structure, proteolipid organization and signaling. Highly dynamic interactions between protein and lipid molecules shape features displayed by specialized membrane domains. The preferential interactions between specific proteins and lipids, e.g., cholesterol, can induce precise spatial compartmentalization of defined membrane components, thus creating molecularly well-defined domains or compartments. This, in turn, modulates cellular membrane-associated signaling by facilitating the recruitment of specific signaling protein and lipid effectors at an exact location and time. Another important feature of membrane domains is transbilayer coupling. In this scenario, proteins and lipids, e.g., PS and GPI-anchored proteins, are organized in membrane domains within a bilayer with distinctive exofacial and cytofacial leaflets. These leaflets differ in terms of their lipid and protein compositions. Accordingly, membrane domains localized in the exofacial leaflet can induce the formation of proteolipid assemblies in the cytofacial leaflet. Inner leaflet effector organization is regulated by complex interactions between the cytoskeleton, lipids and other proteins. These proteolipid assemblies localized at the cytofacial leaflet can influence other effectors located in the exofacial leaflet and engage in transbilayer coupling. This is noteworthy, because transbilayer coupling is a mechanism by which membrane domain components are brought together at opposing sides of the membrane bilayer to efficiently signal. Finally, another fundamental feature of membrane domains is proteolipid clustering. In multiple cases, formation of protein and lipid clusters requires a stimulus to initiate the rearrangement of cluster forming molecules between different membrane domains, resulting in the activation and oligomerization of these molecules. Cell membrane clusters contain a variety of protein functionalities that can originate from different cellular compartments, including the plasma membrane and cytosol. Clustering of multiple functionalities at membranes modulate membrane molecule diffusion, which in turn enhances signaling robustness. B) Cholesterol uptake and efflux. (1) Low density receptor (LDLR) can bind VLDL, LDL and chylomicrons to mobilize cholesterol inside cells. (2) These particles are endocytosed via clathrin-mediated endocytosis as part of clathrin-coated vesicles. Subsequently, (3) endosomes fuse with lysosomes and (4) unesterified “free” cholesterol is transported to various cell compartments, including ER, PM and mitochondria according to cellular needs. The ER regulates most of the cholesterol homeostasis-associated processes in the cell. In the ER, (5) unesterified cholesterol downregulates the nuclear translocation of SREBP, decreasing HMGCR activity and transcription. (6) Excess free cholesterol can be esterified by the enzyme ACAT and stored in lipid droplets to avoid cell lipotoxicity. Alternatively, cells can transport excess free cholesterol to the extracellular space via the activity of the ATP binding cassette (ABC) family of cholesterol transporters in a process referred as cholesterol efflux. ABCA1 and ABCG1 are the most studied members of the family. (7) ABCA1 transports free cholesterol to lipid poor ApoA1 or nascent HDL particles while (8) ABCG1 lipidates mature HDL particles. ACAT, acyl-coenzyme A (CoA):cholesterol acyltransferase; SREBP, sterol regulatory-element binding protein; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; ABCA1, ATP binding cassette subfamily A member 1; ABCG1, ATP binding cassette subfamily G member 1; ApoA1, apolipoprotein A1.

Figure 3.. The role of membrane domains in signaling pathways associated with cancer.

A) Wnt signaling pathway. In the “off’ state, Wnt signaling receptors, i.e., LRP6 and Frizzled (Fzd), localize into both raft (ordered) and non-raft (disordered) domains. In the presence of Wnt ligands (“on” state), cholesterol bilayer asymmetry is triggered, which leads to enrichment of cholesterol in the cytofacial leaflet. Moreover, Wnt-Fzd-LRP6 complexes preferably localize to cholesterol-enriched membrane domains containing caveolin. This, in turn, leads to the recruitment of the Wnt signaling effector Dvl into Wnt-associated clusters. The ability of Dvl to oligomerize stimulates Fzd and LRP6 clustering and the recruitment of Axin to Wnt signalosomes, leading to LRP6 phosphorylation by Wnt-associated kinases, i.e., GSK3 and CK1, in ordered domains. Simultaneously, lipid kinases, e.g., PI4KII and PIP5KI (not shown), are recruited to rafts and promote the production of PIP₂, which in turn promotes LRP6 and Fzd clustering and phosphorylation. Notably, although active Wnt-LRP6-Fzd complexes can localize to non-raft domains, Lypd6, a GPI-anchored protein that localizes specifically in lipid rafts, guarantees that LRP6 phosphorylation and thus receptor activation and efficient signaling occur in ordered domains in order to enable downstream signaling. B) EGFR/KRas signaling pathway. In the “off” state, EGFR preferably localizes into non-raft domains. In the presence of EGF (“on” state), EGFR signaling is initiated from highly organized nanoscale proteolipid domains, driven by the precise production of specific lipids. For example, EGFR activation by EGF initiates the formation of lipid domains enriched in PIP₂ and PA. PA generated by PLD2 acts as a beacon to recruit KRas and SOS1, and along with PIP₂, acts as a cofactor for SOS1-mediated activation of KRas. Furthermore, this pool of PA stabilizes the actin cytoskeleton, of which KRas nanoclustering is dependent. Together, these steps contribute to efficient ERK activation and downstream signaling. LRP6, LDL receptor related protein 6; Fzd, frizzled; Dvl, dishevelled; GSK3, glycogen synthase kinase 3; CK1, casein kinase 1; EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; PLD2, phospholipase D2; KRas, kirsten rat sarcoma virus proto-oncogene; SOS1, SOS Ras/Rac guanine nucleotide exchange factor 1; ERK, extracellular regulated MAP kinase.

Figure 4.. Cholesterol-dependent signaling pathways associated with cancer.

A) p53 signaling pathway. (1) When DNA damage occurs in a healthy cellular state, p53 interaction with E3 Ubiquitin-Protein Ligase Mdm2 (MDM2) is suppressed, and (2) subsequently accumulates in the nucleus, thereby (3) promoting gene expression. Depending on the extent of DNA damage, (4) p53 can induce the expression of p21 resulting in (5) cell cycle arrest or (6) cell apoptosis by coordinating protein activity in mitochondria. In a cancerous cellular state, (7) mutant p53 (mp53) proteins can acquire new gain of function (GOF) oncogenic properties. (8) Mutant p53 can bind to the promoter of SREBP and (9) increase the expression of its targets, thereby upregulating mevalonate pathway-related genes and de novo cholesterol synthesis leading (10) to the subsequent accumulation of cholesterol in transformed cells. B) Hippo signaling pathway. The Aster B/C protein (11) binds and (12) transports PM cholesterol to the ER by via a non-vesicular mechanism. Once in the ER, (13) cholesterol triggers the activity of soluble adenylate cyclase (sAC) which (14) produces cyclic AMP to (15) activate protein kinase A (PKA). Upon PKA activation, (16) inositol phosphate receptor (IP3R1) is phosphorylated and mediates the release of calcium. (17) Calcium activates the small GTPase, RhoA. (18) RhoA can act as an effective inhibitor of the Hippo pathway kinases LATS 1 and 2. In its active form, (19) LATS1/2 phosphorylates TAZ and (20) induces its proteasomal degradation. In contrast, inactivation of LATS1/2 prevents phosphorylation and degradation of TAZ, which consequently (21) leads to its translocation into the nucleus. Once inside the nucleus, (22) TAZ binds transcription factor TEAD1 and promotes gene expression.

Figure 5.. The role of membrane domains and their downstream signaling components in cancer.

The role of rafts in the TCR pathway. TCRα/TCRβ and TCRγ/TCRδ heterodimers form complexes with the CD3/CD8 molecules. The variable region of TCR heterodimers recognize the antigen peptide-loaded on MHC (pMHC). In the absence of pMHC, the cytoplasmic domains of the CD3 molecules form a closed conformation, which is inaccessible to kinases for phosphorylation. At steady state, active Lck, Lat and a fraction of the TCR (not shown) are associated with lipid rafts. The domains of the CD3 cytosolic chains on TCR/CD3 are unphosphorylated as they are not accessible to Lck due to the confinement of TCR/CD3 and Lck in distinct membrane domains; CD3 cytosolic domains are not exposed (closed conformation). Upon TCR activation, raft domains rearrange, TCR/CD3 and active Lck form clusters and the CD3 domains change their conformation (open conformation). Subsequently, Lck phosphorylates tyrosine residues within TCR/CD3 and the phosphorylated regions become docking sites for the tandem SH2 domains of the tyrosine kinase ZAP-70, which is subsequently activated by autophosphorylation and by Lck-mediated phosphorylation. (1) Activated ZAP-70 phosphorylates the transmembrane adapter Lat, which in turn interacts and activates PLCγ1. (2) As a result, PIP₂ synthesis is triggered, which leads to an increase in its levels at the cytofacial leaflet and, simultaneously, (3) CD28 activation leads to the phosphorylation of its cytoplasmic tail and the subsequent recruitment of PI3K as well as the reorganization of the cell cytoskeleton (not shown). (4) Furthermore, TCR/CD3/pMHC and CD28/PI3K signaling pathways can independently activate PKC-θ for the downstream activation and nuclear translocation of NF-κB, and (5) includes the activation of multiple types of cytokines, e.g., IL-1 and TNF-α molecules. Cytokine-mediated programmed cell death or apoptosis is triggered via JAK1/STAT3/caspase-7–dependent pathway. PD-1/PD-L1 signaling. (6) TCR signal activation stimulates the expression of PD-1. Consequently, (7) PD-1 directly antagonizes TCR signaling by recruiting phosphatases to its immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) domains on the PD-1 tail. This consequently prevents Lck-mediated phosphorylation of ZAP70. PD-1 can also inhibit CD28-induced activation of PI3K (not shown). In comparison, (8) PD-L1 expressed on tumor cells interacts with PD-1 triggers inhibitory signals in T cells. PD-L1 can also deliver inhibitory signals to tumor cells to attenuate STAT3/caspase-7–dependent cytotoxicity.

Figure 6.. Dysregulation of membrane domains and the role of MADs as modulators of membrane homeostasis.

Plasma membrane domain biochemical and biophysical homeostasis, i.e., membrane order, proteolipid clustering and spatial compartmentalization of signaling components, regulates intracellular signaling. A) In a healthy cellular state, membrane domains display appropriate levels of membrane rigidity and clustering and signaling-associated effectors temporally relocalize to optimize canonical cell signaling. B) In the context of cancer, alterations in membrane domain features are modified, e.g., increased membrane rigidity, clustering and effector recruitment. This loss of membrane domain homeostasis leads to an abnormal hyperactive cellular state. C) Remarkably, MADs possess the ability to target and reshape membrane domains. In various cases, MADs have been shown to alter membrane fluidity, proteolipid complexes and effector localization, consequently, suppressing oncogenic signaling. In this way, MADs can reinstate, to a significant extent, healthy cell signaling.

Figure 7.. MAD metabolism and their incorporation into cellular membranes.

(1) Dietary bioactives or MADs are orally ingested as part of our regular diet. MADs, e.g., fish oil, curcumin and polyphenols, travel through the digestive system. (2) Upon reaching the small intestine, they are trafficked in different ways. (3) In the case of EPA and DHA (the main membrane-active components in fish oil), they are absorbed by the small intestine and are transported into the bloodstream. (4) In contrast, polyphenols, e.g., procyanidin dimers and trimers, and curcumin, are poorly absorbed by the small intestine and transit into the colon. (5) In the colon, select MADs encounter the microbiome, which can further metabolize these molecules. Finally, curcumin/polyphenols and EPA/DHA are incorporated into the membranes of colonocytes via the (6) colonic lumen or (7) blood, respectively. MADs can modulate membrane homeostasis and serve as chemopreventive cancer agents.