Novel Insights Into Phosphatidylinositol-mediated Oncogenesis

By Kelly Macdonald-Ramsahai 

Lipids are organic molecules with diverse and fundamental biological roles in living cells. For example, lipids are signalling messengers, energy depots, molecular markers and dominate biological membranes. Through these roles, lipid species and related homeostatic pathways orchestrate highly regulated signaling pathways, which coordinate cellular events including cell cycle progression, apoptosis, survival and movement (Fernandis and Wenk, 2007). Importantly, aberrations in lipid metabolism cause a loss of regulatory cellular processes that results in inappropriate cellular growth and function. Often, this manifests in the growth and development of tumours. Mounting evidence, from past and present, has identified deregulated lipid metabolism as an established culprit of cancer. 

A fundamental aspect of the eukaryotic plasma membrane (PM) includes the phospholipid bilayer, which forms a stable barrier between two aqueous compartments. Approximately 50% of the total membrane lipid is dominated by five phospholipids, which are asymmetrically distributed within the bilayer. These include: phosphatidylcholine (PC) and sphingomyelin (SM) which dominate the outer PM leaflet; and phosphatidylethanolamine (PE), phosphatidylserine (PS) or phosphatidic acid (PA) with preference for the inner (cytosolic) PM leaflet (Neumann et al. 2017). Phosphoinositides (PI) are minor amphiphilic glycerophospholipids, which compose 5-10% of total mammalian PM-lipids. PI exists in two forms: as a phosphatidylinositol (PtdIns) precursor and as polyphosphoinositides (PPI), its phosphorylated derivatives. Similarly to all phospholipids, PI contains a diacylglycerol (DAG) backbone. A polar myo-inositol cyclic six-carbon polypol head group, containing a phosphodiester and five -hydroxyl (-OH) groups, occupies the sn-3 position of the DAG backbone. 

At the cytosolic faces of the PM, the myo-inositol moiety of PtdIns are reversibly mono-, di- or tri-phosphorylated at hydroxyl positions -3, -4 and -5 by resident PI kinases (PIKs) and phosphates. Consequently, seven unique PPI species are generated from PI phosphorylation: PI(3)P, PI(4)P, PI(5)P, PI(3,4)P, PI(3.5)P, PI(4,5)P and PI(3,4,5)P (Blunsom and Cockcroft, 2020). PPI recruit and activate PH-domain containing sub-cellular adaptor proteins, including AKT, PDK1, PKCζ and small G-protein (RAC1). Multiple, sequential phosphorylation events enable the activation of intracellular signalling cascades, such as PTEN/phosphatidylinositol 3-kinase (PI3K)/AKT/TORC1 pathway and the Phosphoinositide-specific phospholipase C (PLC)/Protein Kinase C (PKC)/Ca2+ pathway, which regulate several important cellular events including cytoskeletal movement, cell growth, cell division and metabolism. 

Whilst PI species constitute only a minor portion of total phospholipid content, PPIs play a significant role during oncogenesis. Species such as PtdIns(3,4,5) and PtdIns(4,5)P2 are often increased in cancerous cells, causing their downstream signaling pathways to be exaggerated, which facilitate cancer cell growth, survival proliferation and invasion. For example, PLC hydrolyses PtdIns(4,5)P2 into DAG and inositol 1,4,5 trisphosphate (InsP3), facilitating Protein Kinase C (PKC) activation and Ca2+ release from the endoplasmic reticulum (ER) reservoir into the cytosol. This cellular event regulates multiple aforementioned biological cellular functions promoting tumour initiation and development (Thapa et al., 2017). In addition to its hydrophilic myo-inositol moiety, PI contains two hydrophobic R1 and R2 acyl chains, which occupy the sn-1 and sn-2 positions of the DAG backbone. Uniquely to PI species, human PI R1 and R2 fatty acyl chains are enriched with saturated stearic acid (SA) containing 18 carbons (18:0), and unsaturated arachidonic acid (AA) containing 24 carbons and four double bonds (24:4), respectively – termed PI(38:4) (Neumann et al. 2017). Whilst the roles of the PtdIns myo-inositol head group phosphorylation status and downstream PPI-related signaling axis during oncological progression have  been considerably studied and are well-defined in the literature, the influence of PI acyl chains remain ill-defined. Specifically, there is ambiguity surrounding the impact of PI acyl chains compositions, such as their carbon content and degree of saturation, in regulating cellular events and pathogenesis.

Lipidomics analyses reveal PI in non-cancerous cells such as Mouse Embryonic Fibroblasts (MEFs) and cells derived from primary tissues contain dominantly 38-carbon length acyl chains, indicating that this may represent a ‘normal’ state in cells. It is speculated that PI(38:4) enrichment aids in providing optimal fluidity, rigidity and stereochemistry within the lipid bilayer – facilitating its functionalities such as receptor mediated signal transduction and membrane tracking events. Human embryonic kidney cells grown at high confluence express a more saturated PI-acyl chain profile compared to cells grown under normal confluence, which expressed mainly PI(38:4). This shift is attributed to increased contact inhibition and decreased metabolites which alters lipid metabolism in cells (Bozelli and Epand, 2019). In agreement with the formed observation, common laboratory cancer-derived cell lines lack dominance of PI(38:4) species. For example, NIH3T3 and Human-derived pancreatic cancer (Capan-2) malignant cell lines expressed multiple 38-carbon PI species. Additionally, HCT116, HeLa, and Human-derived pancreatic cancer (MiaPaCa-2) malignant cell lines expressed multiple 34/36-carbon PI species (Naguib et al., 2015). Together these findings suggest a difference between PI acyl chain compositions between normal and transformed cells, indicating alterations in PI acyl chains may induce down-stream tumour promoting events.

Studies conducted by Naguib et al., discovered that PI acyl chain compositions are altered by genetics, such as the tumour-promoting p53 mutation (Trp53). Cancer cells from a genetically-engineered pancreatic neoplasia, induced by Kras and Trp53 genetic lesions, express reduced PI(38:4) species and an increase in PI(36:1/2), but also displayed a significant increase in PI (34:1) and  PI(38:2/3) species, compared to pancreatic cells from WT mice which dominantly PI(38:4) species). Upon further exploration, pancreas-derived tumour cells containing exclusively the Kras mutation retained a PI(38:4) dominated lipid profile, similar to non-cancerous cells – suggesting specifically the p53 mutation was the driver of former observation. Cre-mediated introduction of a Trp53 mutation in heterozygous p53 mutant MEFs (p53+/Trp53) – forming homozygous Trp53 mutant MEFs – caused a 50% increase in 36-carbon PI, supporting this notion. However, Cre-mediated introduction of a Trp53 mutation WT MEFs – forming heterozygous p53 mutant cells – did not increase the proportion of 36-carbon PI-lipids. Therefore, it appears the p53 gene must acquire two Trp53 mutations in order to modulate PI acyl chains, specifically to reduce their carbon content (shorter) and increase saturation. Interestingly, p53 knockout MEFs retained a PI lipid profile similar to WT MEFs, indicating that a mutation in p53, as opposed to the loss of p53 entirely, is responsible for alteration in PI acyl chain composition (Naguib et al., 2015).

Additionally, malignant Prostate Cancer (PCa) specimens express a higher proportion of PI species with 0-2 double bonds within their acyl chains, and lower proportion of species with more than 3 double bonds compared to Benign Prostate Hyperplasia (BPH) patient samples. This initial finding suggests increased saturation of PI acyl chains is a hallmark of PCa development. Similarly, malignant PCa samples from patients with a higher pathological stage have a higher quantity of total PI containing 0-2 double bonds in acyl chains than PCa samples from patients  classified at lower pathological stages. This indicates an increased degree of saturation in PI acyl chains may be an indicator of the extent of metastasis during PCa (Koizumi et al., 2019). In addition, melanoma cells with a higher pathological stage classification are associated with increased saturated and mono-unsaturation in fatty chains of PI species, such as 16:0/18:0, 16:0/18:1, 18:0/18:0 and 18:0/18:1, compared to melanoma cells with lower metastatic potential (Kim et al, 2017). 

Lipidomics analysis reveals the drastic changes displayed by  Non-small cell lung cancer (NSCLC) cells in phospholipid profiles compared to WT cells, including a specific phospholipid signature of 20 species that differed statistically compared to healthy patients. In relation to PI species, the most significant changes included an increase in PI(38:3), PI(40:3) and PI(38:2) in NSCLC patients compared to healthy patients, reflecting how there was an increased proportion of PIs with increased saturation (Marien et al, 2015). These findings suggest increased PI acyl chain saturation is not only specific to PCa, but instead may be a ubiquitous phenotype of metastatic tumours. Literature suggests tumour cells may favour decreased PM fluidity by promoting increased saturation of fatty acids in lipid species of the PM. This may provide protective functions against endogenous and exogenous threats including lipid peroxidation and chemotherapeutic agents (Urbanelli et al, 2020)

Whilst PI contributes to only a minor proportion of membrane phospholipid content, phosphorylated PPI species and its down-stream signalling pathways confer significant roles in oncogenesis. To add to this, novel observations of altered PI acyl chain compositions in cancer models reveal a possibility of an additional layer of regulation in PI-mediated oncogenesis, independent of PPI-mediated oncogenic signalling. At present, the down-stream influence of altered PI acyl chain compositions during oncogenesis have not been addressed – although this would be uninteresting further avenue of research. Understanding these mechanisms could generate novel insights into tumour progression, with a focus on metastasis. Lastly, further exploration into these mechanisms may provide additional avenues for the intervention of cancer-related signaling axis to facilitate the development of novel, effective therapeutic treatments.

References:

Blunsom N, Cockcroft S. Phosphatidylinositol synthesis at the endoplasmic reticulum. Biochimica et Biophysica Acta (BBA) – Molecular and Cell Biology of Lipids. 2020;1865(1):158471.

Bozelli J, Epand R. Specificity of Acyl Chain Composition of Phosphatidylinositols. PROTEOMICS. 2019;19(18):1900138.

Fernandis A, Wenk M. Membrane lipids as signaling molecules. Current Opinion in Lipidology. 2007;18(2):121-128.

Kim H, Lee H, Kim S, Jin H, Bae J, Choi H. Discovery of potential biomarkers in human melanoma cells with different metastatic potential by metabolic and lipidomic profiling. Scientific Reports. 2017;7(1).

Koizumi A, Narita S, Nakanishi H, Ishikawa M, Eguchi S, Kimura H et al. Increased fatty acyl saturation of phosphatidylinositol phosphates in prostate cancer progression. Scientific Reports. 2019;9(1).

Marien E, Meister M, Muley T, Fieuws S, Bordel S, Derua R et al. Non-small cell lung cancer is characterized by dramatic changes in phospholipid profiles. International Journal of Cancer. 2015;137(7):1539-1548.

Naguib A, Bencze G, Engle D, Chio I, Herzka T, Watrud K et al. P53 Mutations Change Phosphatidylinositol Acyl Chain Composition. Cell Reports. 2015;10(1):8-19

Neumann J, Rose-Sperling D, Hellmich U. Diverse relations between ABC transporters and lipids: An overview. Biochimica et Biophysica Acta (BBA) – Biomembranes. 2017;1859(4):605-618.

Thapa, N., Tan, X., Choi, S., Lambert, P. F., Rapraeger, A. C., & Anderson, R. A. (2016). The Hidden Conundrum of Phosphoinositide Signaling in Cancer. Trends in cancer, 2(7), 378–390. https://doi.org/10.1016/j.trecan.2016.05.009

Urbanelli L, Buratta S, Logozzi M, Mitro N, Sagini K, Raimo R et al. Lipidomic analysis of cancer cells cultivated at acidic pH reveals phospholipid fatty acids remodelling associated with transcriptional reprogramming. Journal of Enzyme Inhibition and Medicinal Chemistry. 2020;35(1):963-973.

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