Lipid droplet evolution gives insight into polyaneuploid cancer cell lipid droplet functions

Archaea are a domain of single cell organisms that lack a cell nucleus. These microorganisms’ membrane lipids differ widely from bacteria and eukaryotes as they are composed of glycerol ether lipids rather than glycerol esters [1]. Additionally, archaea do not synthesize fatty acyl esters which are the most common components of LDs in other phyla. Instead, archaeal lipids are based on isoprenoid chains. All known LD-containing archaeal species are capable of accumulating a range of polymeric lipids, the most common being polyhydroxyalkanoates (PHAs). Additionally, LDs in archaea appear to act exclusively as energy stores (Table 1). A large accumulation of lipids can occur in response to specific forms of nutrient limitations (most commonly a low carbon/nitrogen ratio) [1]. These instances of accumulated lipids frequently mark a stressful period leading to the interruption of growth and division, and the entry of these unicellular organisms into a non-proliferative state [1].

Bacteria constitute a large domain of prokaryotic unicellular microorganisms. These microorganisms have cell walls but lack an organized nucleus. Bacteria differ from archaeal prokaryotes as they contain peptidoglycan in their cell walls, and their membranes are lipid bilayers compared to the archaeal membranes which can be a lipid bilayer or monolayer. Additionally, bacteria contain fatty acids on their cell membranes while archaea contain phytanyl. Similar to archaea, the majority of bacteria store carbon in the form of PHAs within LDs. However, bacteria are the only known prokaryote to have evolved the ability to also accumulate triacylglycerols (TAG) or wax esters within their LDs [1]. The highest levels of TAG accumulation in bacteria have been reported in mainly nocardioforms such as Mycobacterium, Nocardia, Rhodococcus, Micromonospora, Dietzia, Gordonia, and some streptomycetes [31, 32]. LDs in bacteria are used exclusively as energy stores (Table 1). When hydrocarbonoclastic bacteria lack suitable growth substrates they will enter a dormant phase and live off their accumulated LD reserves until a suitable growth substrate is reintroduced in the environment [33].

Protists are any eukaryotic single-celled organism that is not an animal, plant, or fungus. A large variety of protists act as parasites or pathogens. The ability to accumulate cytosolic LDs is key for many protists’ success as infectious agents, an example is the malarial parasite Plasmodium falciparum [1]. An essential factor for the proliferation of P. falciparum within infected human erythrocytes is the ability to accumulate and mobilize large amounts of TAGs [34]. Most of the host-derived acyl groups get transferred to the parasite and accumulate in the cytosol as TAGs are released into the infected erythrocyte. The sudden release of fatty acids in the erythrocyte causes membrane lysis and cell rupture, resulting in the release of merozoites that are now capable of infecting new cells [34]. Therefore, in P. falciparum, LDs have two functions: a nutrient source, and a mechanism to enable P. falciparum cells to escape from host cells and enter the next phase of their life cycle (Table 1) [1, 34].

Fungi are eukaryotic, heterotrophic organisms that contain chitin in their cell walls and acquire food by absorbing dissolved molecules. Similar to other eukaryotes, fungal LDs appear to arise from the endoplasmic reticulum [35]. The main protein involved in LD biogenesis in fungi is the fat storage inducing transmembrane protein (FIT) [36]. Most fungi have cytosolic LDs, but the function of these LDs can vary according to species, developmental stage, or environmental conditions [1]. For example, LDs typically form during vegetative growth in saprophytic fungi (e.g., mushrooms, mold). However, LDs can also increase during the formation of their reproductive structures [1, 37]. Throughout evolution, fungal LDs have moved from only functioning as energy stores to also functioning to trap toxins within the organism to prevent it from harm (Table 1). In the endolichenic fungus Phaeosphaeria sp., phototoxic perylenequinones (PQs) are sequestered in LDs following exposure to light irradiation, providing resistance to the phototoxins, and the survival of the fungus [38]. This evolved trait allows fungi to utilize LDs in more than one way, improving their chances of survival in stressful conditions.

Yeast are eukaryotic unicellular microorganisms. This relatively simple organism has been shown to share many features of LD organization and function found in more complex eukaryotic organisms such as mammals [1, 39]. Yeast LDs can contain either TAGs or sterol esters exclusively, or a mixed composition of the two [39]. LDs in yeast cells have many roles including being energy stores, functioning as a hub for the regulatory network that modulates TAG and SE formation in the endoplasmic reticulum, and fatty acid oxidation in peroxisomes. If LD formation is blocked in S. cerevisiae, exogenous fatty acids become toxic when taken up by the cells [40]. This indicates that one of the constitutive functions of these LDs is to maintain intracellular lipid homeostasis by reducing the fatty acid toxicity via sequestration of the excess acyl groups [1, 40]. The evolution of LDs to function and sequester fatty acids allows yeast cells the capability to survive in stressful situations they may have otherwise perished in (Table 1).

Plants are mainly multicellular photosynthetic eukaryotes in the kingdom Plante. All major groups of plants, from unicellular algae to more complex multicellular flowering plants, are capable of producing LDs in some of their cells or tissues [1]. The mechanism of LD formation in plant cells is similar to that of animal cells as the accumulation of TAGs in the endoplasmic reticulum is followed by the release of small LDs that mature into larger LDs in the cytoplasm [41]. Simple plants like algae that fall in the kingdom Plante range from small unicellular organisms to complex multicellular seaweeds. Many algal species are capable of accumulating a high amount of cytosolic TAG filled LDs as storage reserves in response to stress [42]. These LDs can make up as much as 86% of the cell dry weight, allowing the plant to survive off these stores for long periods of time [1]. In more complex plants, LD accumulation was first thought to be confined to specific tissues with the singular function of energy storage. However, new evidence has shown the presence of dynamic LDs that do not accumulate as long-term lipid stores. In most cases, LDs in plants will accumulate TAGs (one known species, oilseed, accumulates fluid wax esters instead). In addition to cytosolic LDs, some plants accumulate structures similar to LDs known as plastoglobules in their plastid organelles. Platoglobules have a lipidic phase which can include TAGs, sterol esters, and various lipophilic pigments. In terrestrial plants, there is evidence showing that cytosolic TAG rich LDs are mainly detected in the leaf mesophyll cells. Slocombe and colleagues noted that TAG accumulation in leaves is increased following a manipulation of fatty acid breakdown and lipid synthesis indicating that cytosolic LDs in leaves could be acting as a buffer to take up or release acyl moieties in order to maintain metabolic homeostasis in cells [43]. From energy stores in algae to buffering cell toxicity in higher plants, LDs prove themselves as important organelles in plant survival (Table 1).

Drosophila melanogaster has been acknowledged as an invertebrate model organism for observing core functions of LDs [1, 44]. Most Drosophila LDs are prominent organelles in tissues dedicated to TAG (neutral lipid) storage and breakdown [44]. Neutral lipids can be transported between storage sites as lipoprotein particles [44]. Drosophila lipoprotein particles mainly are composed of diacylglycerol (DAG) but can also be composed of lipophorins, phospholipids, and transport lipids [44]. This unique composition suggests that storage TAGs are deacylated before they are loaded onto pre-formed lipoprotein particles [44]. Lipoprotein particles can then be unloaded at their intended site through interactions with lipophorin receptors in an endocytosis-independent manner [44]. Beyond their role in lipid transport, lipoprotein particles also serve important signaling functions in flies (i.e., modulation of the wingless [WNT] and hedgehog [Hh] morphogen activities). The Drosophila LD proteome is similar to that of mammals, indicating evolutionarily conserved functions [1]. This makes Drosophila a good model to investigate LD malfunction in human diseases such as metabolic syndrome and lipodystrophy [1]. LDs in Drosophila serve many purposes allowing these organisms to flourish in many environments while dysregulation of LD formation or utilization can be extremely detrimental (Table 1).

In mammals, the majority of LDs are found in adipose tissue, liver, and muscle [45]. Mammalian cells have similar basic LD systems as other animal groups. However, in higher mammals, such as placental mammals, basic LD mechanisms such as energy storage are accompanied by additional layers of complexity [1]. These complex mechanisms have been adapted throughout the evolution of these long-lived group of eukaryotes [1]. Mammalian LDs are involved in lipid transport, lipid synthesis, lipid degradation, and protein degradation [45] (Table 1). At the metabolic level, mammals have a larger variety of key enzymes to assist with LD formation (DAG acyltransferase) [46] and LD turnover/breakdown (lipases) [47‐49]. Having a larger variety of enzymes for LD formation and degradation allows for one pathway to compensate if another pathway is dysregulated, still ensuring proper LD operations. Conversely, this indicates that to efficiently halt LD formation or breakdown, targeting multiple pathways may be necessary.

One of the hallmarks of cancer is metabolic reprogramming [50, 51]. Beyond the classical metabolomic changes highlighted by the Warburg effect, research has pointed to the metabolism of lipids as critical for tumorigenesis [2, 9, 51, 52]. Cancer cells have remarkable metabolic plasticity that allows them to adapt to adverse conditions and drives their resistant and metastatic potential [50]. Even with remarkable plasticity, cancer cells need a mechanism of survival as they are constantly faced with various stressors (e.g., hypoxia of the tumor microenvironment or applied anti-cancer chemotherapy). When stressed, a subset of cancer cells undergo polyploidization to enter a transient PACC state [16, 18, 29, 53]. Cells in the PACC state contain a higher number of LDs than the cancer cells they are derived from (Fig. 1) [30, 54]. As PACCs are hypothesized to be mediators of tumorigenesis, metastasis, and resistance, understanding their LD biology may be crucial in learning how to target them [18].

LDs have emerged as novel regulators of many metabolic processes in cancer cells [2, 9]. LDs are dynamic organelles capable of responding to nutrient fluctuations and environmental stressors. These responses allow the cell to control trafficking, storage, and use of the neutral lipids within the LDs [2, 55‐59]. An increase in LDs can affect major cancer hallmarks including cell growth, proliferation, metabolism, migration, inflammation, and immunity [2, 8, 57, 60‐62]. LDs are also associated with protein turnover, gene transcription, and nuclear function [9]. LDs act as switches that can coordinate lipid trafficking and consumption for various purposes. In healthy cells, free fatty acids are absorbed directly and used for energy, so their lipid synthesis pathway is typically inactive. In contrast, the lipid synthesis pathway in cancer cells is activated while the lipid breakdown processes are limited. When under metabolic stress, different cancer types have demonstrated different lipid metabolism responses [63, 64]. Under metabolic stress, some cancer cells can upregulate FA synthesis while other cancer cells increase intake of exogenous FAs from the microenvironment for survival [2, 63, 65, 66]. Cancer cells capable of acquiring extracellular FAs or adapting intracellular mechanisms for lipid generation, mobilization, and recycling have a higher chance of survival [2]. Some aggressive cancer types use opportunistic mechanisms of FA acquisition and increase their capacity for FA accumulation in LDs [67‐69]. However, too high levels of free fatty acids or other lipids in a cell can lead to lipotoxicity and cell death unless sequestered. LDs are capable of sequestering toxic lipids to reduce cellular toxicity [1, 2]. LDs can also affect drug efficacy by altering cellular distribution and activation of lipophilic anti-cancer agents (i.e., cisplatin, ponatinib), rendering some anti-cancer therapies ineffective [70, 71]. Learning more about LD function and biogenesis may give insight on how to target them to increase toxicity in cancer cells and eventually lead to their death.