As the recent article in Eureka notes[1]:
The finding, published in the July 11, 2019 issue of Cell
Metabolism, suggests a potential target for new drugs.
"Cancers are characterized not only by major changes
in their genomes, but also by profound shifts in how they take up and utilize
nutrients to propel rapid tumor growth," said senior author Paul S.
Mischel, MD, professor in the UC San Diego School of Medicine Department of
Pathology and Ludwig member. "How do these diverse aspects fit together
and can they be taken advantage of, for the benefit of patients?"
In the new study, conducted in collaboration with
Benjamin Cravatt, PhD, professor at Scripps Research, and led by first author
Junfeng Bi, PhD, in Mischel's lab, researchers identified an enzyme called
LPCAT1, whose levels increase in cancer and which plays a key role in tumor
growth by changing the phospholipid composition of the cancer cells' plasma
membrane, allowing amplified and mutated growth factor signals to spur tumor
growth.
LPCAT1 is an enzyme involved in a variety of lipid cycles.
As such it finds itself at the heart of energy processing and transfer in
cells. The Eureka article continues:
Without LPCAT1, tumors cannot survive. When
researchers genetically depleted LPCAT1 in multiple types of cancer in mice,
including highly lethal glioblastomas (brain) and an aggressive lung cancer,
malignancies shrank dramatically and survival times improved.
The results, wrote the authors, demonstrate that LPCAT1
is an important enzyme that becomes dysregulated in cancer, linking common
genetic alterations in tumors with changes in their metabolism to drive
aggressive tumor growth."
It is not clear that devoid of LPCAT1 the cell will cease
proliferation. Cells, especially malignant cells have a variety of mechanisms
to avoid assaults on their spread. We also know that cancer cells have multiple
alternative metabolic pathways[2].
They continue:
"Advances in DNA sequencing technologies have
reshaped our understanding of the molecular basis of cancer, suggesting a new
and more effective way of treating cancer patients," said Mischel.
"However, to date, precision oncology has yet to benefit many patients,
motivating a deeper search into understanding how genetic alterations in tumors
change the way cancer cells behave, and potentially unlocking new ways to more
effectively treat patients.
"These results also suggest that LPCAT1 may be a
very compelling new drug target in a wide variety of cancer types."
Indeed, if LPCAT1 is a viable target for a therapeutic then
it cant be used as such. We will investigate this target in this brief note.
Now the authors in the above-mentioned paper by Bi et al
note[3]:
Advances in DNA sequencing technologies have reshaped our
understanding of the molecular basis of cancer, providing a precise genomic
view of tumors. Complementary biochemical and biophysical perspectives of
cancer point toward profound shifts in nutrient uptake and utilization that
propel tumor growth and major changes in the structure of the plasma membrane
of tumor cells. The molecular mechanisms that bridge these fundamental aspects
of tumor biology remain poorly understood.
Here, we show that the lysophosphatidylcholine
acyltransferase LPCAT1 functionally links specific genetic alterations in
cancer with aberrant metabolism and plasma membrane remodeling to drive tumor
growth.
Growth factor receptor-driven cancers are found to depend
on LPCAT1 to shape plasma membrane composition through enhanced saturated
phosphatidylcholine content that is, in turn, required for the transduction of
oncogenic signals. These results point to a genotype-informed strategy that
prioritizes lipid remodeling pathways as therapeutic targets for diverse
cancers.
Clearly this seems to be a complex multistep process. We
examine LPCAT and the various metabolic cycles it is involved in in this note.
We present some of the basic facts regarding LPCAT1 and its
functioning in a cell.
Let us begin with a brief definition and description of the
gene expressed LPCAT1. From NCBI[4]:
This gene encodes a member of the
1-acyl-sn-glycerol-3-phosphate acyltransferase family of proteins. The encoded
enzyme plays a role in phospholipid metabolism, specifically in the conversion
of lysophosphatidylcholine to phosphatidylcholine in the presence of acyl-CoA.
This process is important in the synthesis of lung
surfactant and platelet-activating factor (PAF). Elevated expression of this
gene may contribute to the progression of oral squamous cell, prostate, breast,
and other human cancers.
LPCAT1 is thus a critical element in the lipid process in a
cell and given the cell walls being fundamentally lipid in nature the
activation can result in the changing of the receptors and thus the activation
of various genes. Some of these changes in receptors and pathway activation
result subsequently in malignant transformations. This the lipid metabolism and
its effects and transitions are of significant interest.
As Matsumoto et al note:
Lysophosphatidylcholine (LPC) is a bioactive
proinflammatory lipid generated by pathological activities. LPC is also a major
phospholipid component of oxidized low-density lipoprotein (Ox-LDL) and is
implicated as a critical factor in the atherogenic activity of Ox-LDL. LPC is
believed to play an important role in atherosclerosis and inflammatory diseases
by altering various functions in a number of cell-types, including endothelial
cells, smooth muscle cells, monocytes, macrophages, and T-cells. LPC activates
several second messengers -- including protein kinase C,
extracellular-signal-regulated kinases, protein tyrosine kinases, and Ca(2+) --
implicating the engagement of transduction mechanisms in its observed actions.
Moreover, recent evidence suggests that in several cell-types, cloned orphan
G-protein-coupled receptors may serve as the specific receptors via which LPC
modulates second messenger pathways (although LPC may not be a direct ligand of
such receptors).
In addition, current evidence suggests that LPC impairs
the endothelium-dependent relaxations mediated by endothelium-derived relaxing
factors and directly modulates contractile responses in vascular smooth muscle.
However, despite all this, and although elevated levels of LPC have been linked
to the cardiovascular complications associated with atherosclerosis, ischemia,
and diabetes, the precise pathophysiological roles played by LPC in several
states remain to be established. In this review, we focus in some detail on the
entirety of the signal-transduction system for LPC, its pathophysiological
implications, and the vascular abnormalities associated with it.
In comparison Li and Vance discuss phosphatidylcholine:
Phosphatidylcholine (PC) is an essential phospholipid in
mammalian cells and tissues and is made in all nucleated cells via the choline
pathway. Choline was first identified in ox bile by Strecker in 1862. The Greek
word for bile is chole. After a long interlude, in 1932, Best and Huntsman
discovered the choline deficiency that results in fatty liver in rodents when
insufficient choline is provided in the diet. In animals, choline can be
acquired from the diet and via de novo biosynthesis: choline is produced
through the methylation of phosphatidylethanolamine (PE) to PC catalyzed by
phosphatidylethanolamine N-methyltransferase (PEMT).
Choline can then be generated from PC via the action of
phospholipases. The PEMT/phospholipase reactions constitute the only known
endogenous pathway for choline biosynthesis in animals, whereas in plants and
some microbes, choline can be made from the methylation of phosphoethanolamine
(5–7). Thus, choline is made from the methylation of the ethanolamine moiety of
phosphoethanolamine or PE. Both exogenous and endogenous choline is converted
into PC, which accounts for 95% of the total choline pool in most animal
tissues. The remaining 5% includes choline, phosphocholine,
glycerophosphocholine, CDP-choline, and acetylcholine. In animals, PEMT is
quantitatively significant only in the liver, and it accounts for 30% of
hepatic PC biosynthesis in rodents. The other 70% of hepatic PC is made via the
choline pathway.
The figure below shows the collection of lipid bilayer elements
appearing in cells. The PC elements is depicted along with other similar
elements.
Li and Vance demonstrate this PC action in cells in the
following figure[5]:
Note the central function of PC and its impact of major
functions of a cell. The use of choline is a central part of this effort. But
as previously demonstrated it is but one of several elements in the cell wall. Kisko
presents the sectioned pathway as shown below (Note: Lyso-PhosphatidylCholine
(PC) and Lyso-PhosphatidylCholine AcylTransferase 1 (LPCAT1))
The PC molecule is shown below. Note the hydrophobic is the
dual tail lipid and the hydrophilic is the top end with the choline. This is
one of the collection of molecules which make up the cell wall. We show the PC
molecule below[6]:
Thus, LPCAT1 facilitates the production of the PC as shown
above, then PC produces the dual lipids in cell walls, the lipids reconfigure
the cell receptors and intensifying various receptor and growth factor effects
and this accelerates the malignant state of the cell[7].
The idea then is if one blocks LPCAT1 does one then block the sequellae states
and mitigate against malignancies? We demonstrate this conjecture below.
To understand the complete workings of lipids it is useful
to summarize. This is done by Moessinger et al who note the complex
interactions and lipid elements:
Lipids are important components of cells, with a function
in cellular structure, regulation, signaling and as energy source, in
particular neutral lipids. The cellular location of storage of neutral lipid is
the lipid droplet (LD). LDs consist of a core of neutral lipids that is
surrounded by a monolayer of phospholipids, mainly phosphatidylcholine.
Different proteins are associated with the LDs, including
several enzymes of lipid metabolism. Many metabolic disorders like diabetes and
cardiovascular diseases are associated with defects in lipid metabolism and
derive from additive defects in different pathways, often described as
metabolic syndrome that can gradually progress into more severe diseases. The
first step is usually the excess storage of lipids within different body
tissues resulting in the development of obesity.
Therefore, it is crucial to understand how the storage of
lipids is regulated under normal conditions. Lipids are in a constant flux and
are continuously converted into each other. Within cells they can move within
membranes and between different cellular compartments. Furthermore, lipids are
exchanged between different tissues.
Extracellularly, the bulk of lipids is transported in
lipoproteins. These lipoproteins are soluble complexes of proteins
(apolipoproteins) and lipids that are transported in the circulation of
vertebrates and insects and that are synthesized in the liver and intestine.
They are classified into chylomicrons (CM), very low density (VLDL), low
density (LDL) and high density (HDL) lipoproteins based on their apolipoprotein
component and their density, which is determined by the lipid composition.
The major neutral lipid, triacylglycerol (TAG), is
secreted from the liver and intestine in apolipoproteinB (apoB) containing
lipoproteins (CM and VLDL). In contrast to other apolipoproteins, apoB is not
exchangeable between lipoproteins and resides in the plasma in a
lipid-associated form only.
While VLDL and CM contain apoE, apoC and apoB, LDL
harbors exclusively apoB. In the absence of loaded lipids apoB cannot be
secreted and is rapidly degraded. The TAG secreted as CM and VLDL mainly
derives from TAG stored in cytosolic LDs…In the Lands cycle phospholipase A2
(PLA2) removes fatty acids at the sn-2 position of PC, which results in the
formation of lysophosphatidylcholine (LPC). This can be used in a reverse
reaction, the addition of a fatty acid at the sn-2 position, to yield PC.
This re-acylation is catalyzed by lysophosphatidylcholine
acyltransferases (LPCATs).
Recently, four LPCATs were cloned and characterized. They
are all reported to localize to the ER compartment.
Due to their structure they divide into two subgroups
with LPCAT1 and LPCAT2 in one and LPCAT3 and LPCAT4 in the other group.
LPCAT1 is reported to function in lung surfactant production,
while LPCAT2 seems to be important in inflammatory reactions…
Essentially, our data now show that reduction of LPCAT1/2
results in unchanged balance between PC and TAG synthesis, along with a
remodeling of LD morphology towards larger LDs, while reduction of the de novo
pathway enzyme CT alpha changes the balance between PC and TAG synthesis
towards the latter, accompanied by larger LDs and higher TAG content. Reduction
of ER-localized LPCAT3/4 apparently does not influence neutral lipid storage.
Also, knockdown of LPCAT1 decreases lipoprotein secretion by hepatoma cells.
As Zhou notes, as do many other authors, that malignancies
often present with high LPCAT1 expression. This is explained in the following:
Lipid profiles provide useful information to determining
the metabolic pathways of altered lipids in prostate cancer. In our previous
studies, we found that the concentrations of all 14 detected
lysophosphatidylcholine species are higher in both plasma and prostatic samples
from patients with prostate cancer, as compared with samples from controls.
Further, we found that expression level of secretory
phospholipase A2 (sPLA2) is increased in cancerous prostate as compared with
benign prostate, which may contribute to the accumulation of lysophospholipid
species in cancer tissues and in plasma (data not published).
Meanwhile, we also found that the expression level of
lysophospholipid acyltransferase 1 (LPCAT1) is significantly higher in
cancerous prostate as compared with benign prostate. Elevated expression of
LPCAT1 also correlated with prostate cancer pathologic grade and clinical
chemical recurrence in prostate cancer.
Taken together, cycle: tumor cells upregulate the
expressions of both sPLA2 (which generate adequate lysophospholipid species,
substrates for LPCAT1) and LPCAT1, in order to secure de novo synthesis of
various phospholipid species for building cellular membranes of newly
proliferated cancer cells.
By combining data of lipid profiles in individual
species, cluster, group and class of lipids with lipid MAPS, more lipid
metabolic pathways critical to prostate cancer could be identified.
Thus, LPCAT1 is a central figure as an enzyme. It produces
PC and the excess production of PC facilitates the malignant status. PC
apparently be suppressed by the suppression of LPCAT1 as noted earlier.
We demonstrated briefly the place of LPCAT1 in the
production of the lipids in cell walls. Wei et al have noted:
Big data analysis can help us acquire more information
about the mechanisms of the development and progression of tumors.
By searching tumor-related online databases and examining
the gene expression in primary loci and adjacent tissues in healthy subjects
and lung-cancer patients, we found that LPCAT1 was highly expressed in
pulmonary tissues and its over-expression was correlated with the poor
prognosis of NSCLC. LPCAT1 is a cytosolic enzyme that catalyzes the conversion
of lysophosphatidylcholine (LPC) into phosphatidylcholine (PC) in remodeling
the pathway of PC biosynthesis.
The high expression levels of LPCAT1 was not just with NSCLC
but clearly across a wide spectrum of malignancies. They continue:
To date, LPCAT1 overexpression has been reported in clear
cell renal cell carcinoma, oral squamous cell carcinoma, gastric cancer and
breast cancer. LPCAT1 has been found to be a contributor to the progression,
metastasis, and recurrence of cancer. However, reports on the role and the
underlying mechanism of LPCAT1 in NSCLC have been scanty.
LPCAT1 was essential for the proliferation, migration
and invasion of NSCLC in vitro. Given that substantially higher
LPCAT1 expression in LUAD tissues than in normal lung tissues according to TCGA
LUAD and GEO datasets, we first looked into whether the elevated expression of
LPCAT1 is associated with the development of NSCLC. Analysis of the TCGA
datasets revealed that the copy number of LPCAT1 was directly proportional to
its mRNA expression.
In this presentation there is still an issue regarding just
what LPCAT1 accomplishes to make is a accelerator of malignancies. The paper in
question makes the argument for lipid layer production, yet even there the full
nexus is left open for discussion. They continue:
Moreover, the expression of LPCAT1 in LUAD was
significantly higher than in normal lung tissues and in lung squamous cell
carcinoma. Additionally, we searched the THPA database to further examine the
expressions of LPCAT1 in patients with various cancers. We found that LPCAT1
expression was relatively higher in patients with lung cancers than in those
with other 16 tumors.
Moreover, search of the THPA dataset showed the
positive rate of LPCAT1 was up to 80% in lung cancer tissues. Together,
these findings suggested LPCAT1 level increased in NSCLC tissues. Next, we
performed PCR and Western blotting to assess LPCAT1 expression in NSCLC cell
lines. As expected, both LPCAT1 mRNA expression and protein expression were
found to be highly expressed in NSCLC cell lines.
Understanding the overall effects and their causes has been
a focus by many for a while. For example, Kisko et al note:
All living organisms require a variety of essential
elements for their basic biological functions. While the homeostasis of
nutrients is highly intertwined, the molecular and genetic mechanisms of these
dependencies remain poorly understood.
Here, we report a discovery of a molecular pathway
that controls phosphate (Pi) accumulation in plants under Zn deficiency. Using
genome-wide association studies, we first identified allelic variation of the
Lyso- PhosphatidylCholine (PC) AcylTransferase 1 (LPCAT1) gene as the key
determinant of shoot Pi accumulation under Zn deficiency.
We then show that
regulatory variation at the LPCAT1 locus contributes significantly to this
natural variation and we further demonstrate that the regulation of LPCAT1
expression involves bZIP23 TF, for which we identified a new binding site
sequence. Finally, we show that in Zn deficient conditions loss of function of
LPCAT1 increases the phospholipid Lyso-PhosphatidylCholine/PhosphatidylCholine
ratio, the expression of the Pi transporter PHT1;1, and that this leads to
shoot Pi accumulation.
They continue:
1. LPCAT1 is involved in regulating shoot Pi concentration in Zn
deficiency…
2. LPCAT1 acts downstream of bZIP23 transcription factor…
3. Allelic variation of LPCAT1 determines natural variation of
Pi content under zinc deficiency…
4. LPCAT1 mutation impacts phospholipid concentrations in -Zn
One of the controlling pathways is the Kennedy Pathway. We
discuss it briefly since understanding it may yield additional insight to control
by therapeutics.
As Gibellini and Smith note:
The glycerophospholipids phosphatidylcholine (PC) and
phosphatidylethanolamine (PE) account for greater than 50% of the total
phospholipid species in eukaryotic membranes and thus play major roles in the
structure and function of those membranes.
In most eukaryotic cells, PC and PE are synthesized by an
aminoalcoholphosphotransferase reaction, which uses sn-1,2-diradylglycerol and
either CDP-choline or CDP-ethanolamine, respectively.
This is the last step in a biosynthetic pathway known as
the Kennedy pathway, so named after Eugene Kennedy who elucidated it over 50
years ago.
This review will cover various aspects of the Kennedy
pathway including:
i.
each of the
biosynthetic steps,
ii.
the functions and
roles of the phospholipid products PC and PE, and
iii.
how the Kennedy
pathway has the potential of being a chemotherapeutic target against cancer and
various infectious diseases. …
Choline phospholipid metabolism is altered in a wide
variety of human cancers. The observed elevated levels of phosphocholine are
caused in part to the growth factor-activated Ras and phosphatidylinositol
3-kinase (PI3K) signaling cascades that stimulate the initial enzyme of the
choline branch of the Kennedy pathway, CK. CKs have been found to be
activated in malignant cells and tumors of the lung, colon, breast, prostate,
cervix, and ovaries.
For this reason, it has been proposed to use CK as a
prognostic marker for cancer progression as well as a molecular target for the
development of novel cancer chemotherapeutic agents. Toward this
goal, the use of small interfering RNA or small hairpin RNA plasmids of CK has
been shown to reduce intracellular phosphocholine, selectively reduces
proliferation, and increases apoptosis in breast adenocarcinoma cells, but not
in normal human mammary epithelial cells.
Returning to Moessinger et al we note:
Lipids are stored within cells in lipid droplets (LDs).
They consist of a core of neutral lipids surrounded by a monolayer of
phospholipids, predominantly phosphatidylcholine (PC). LDs are very dynamic and
can rapidly change in size upon lipid uptake or release. These dynamics require
a fast adaptation of LD surface. We have recently shown that two Lands cycle PC
synthesizing enzymes, LPCAT1 and LPCAT2 can localize to the LD surface….
The major phospholipid of the LD surface,
phosphatidylcholine, can be synthesized by three different pathways:
(i) the de-novo pathway, which is also known as Kennedy
pathway,
(ii) the Lands cycle and
(iii) the phosphatidylethanolamine methyl transferase
(PEMT) pathway, which is restricted to liver cells.
In the Kennedy pathway, phosphocholine is activated
with cytidine triphosphate (CTP) and transferred to diacylglyceride (DAG) to
form PC. These reactions are catalyzed by the cytoplasmic
CTP:phosphocholine cytidylyltransferase (CT alpha) and the membrane-embedded
cholinephosphotransferase or choline/ethanolamine phosphotransferase
(CEPT1/CPT1).
In the Lands cycle phospholipase A2 (PLA2)
removes fatty acids at the sn-2 position of PC, which results in the formation
of lysophosphatidylcholine (LPC). This can be used in a reverse reaction, the
addition of a fatty acid at the sn-2 position, to yield PC. This re-acylation
is catalyzed by lysophosphatidylcholine acyltransferases LPCATs). Recently,
four LPCATs were cloned and characterized.
They are all reported to localize to the ER
compartment. Due to their structure they divide into two subgroups with LPCAT1
and LPCAT2 in one and LPCAT3 and LPCAT4 in the other group. LPCAT1
is reported to function in lung surfactant production, while LPCAT2 seems to be
important in inflammatory reactions.
Overall a great deal is known about PC and the LPCAT1
interaction. However as wel shall note, the specific details of this complex
process is still missing.
We can now make several observations. Our interest is
setting forth questions more than conclusions.
The presentations noted above reflect a reasonable set of
observations and reflections on possible therapeutic targets. What seems to be
missing are the details associated with this process. For example, we know that
PC is produced but we do not know the details as to how this reflects in the
activation of more growth factor receptors or others similar effects.
What is the proposed target? Is it LPCAT1 itself and if so
how can it be targeted? What activates LPCAT1, and is it activated through one
of the many kinase paths in the cells. Are there cell surface markers that show
such activation?
In many cancers one can now find multiple markers, often
blood borne, to be used for diagnosis and prognosis. The question here is; are
there any such markers here? One may ask if there are exosomes that contain
such markers.
It appears from the analysis that the "sole"
factor is excess LPCAT1. However, what is driving that excess? The underlying
gene is in overdrive in expression. Are there certain activation via growth
factors or other activators or is the activation internal? If the latter then
what is the genetic mechanism of this overdrive?
We know that many cancers have as a drive a stem cell[8].
Is the LPCAT1 a factor in the stem cell or all the cells.
1. Bi et al, Oncogene Amplification in Growth Factor Signaling
Pathways Renders Cancers Dependent on Membrane Lipid Remodeling, Cell
Metabolism 11 July 2019
2. Bridges et al, LPCAT1 regulates surfactant phospholipid
synthesis and is required for transitioning to air breathing in mice, Jrl Clin
Invest May 2010
3. Farine et al, Phosphatidylethanolamine and phosphatidylcholine
biosynthesis by the Kennedy pathway occurs at different sites in Trypanosoma
brucei, Scientific Reports, Nov 2015
4. Gibellini and Smith, The Kennedy Pathway—De Novo Synthesis of
Phosphatidylethanolamine and Phosphatidylcholine, IUBMB Life, 62(6): 414–428,
June 2010
5. Kisko et al, LPCAT1 controls phosphate homeostasis in a
zinc-dependent manner, eLife 2018;7:e32077.
6. Li and Vance, Phosphatidylcholine and choline homeostasis, Journal
of Lipid Research Volume 49, 2008
7. Luo et al, Regulation of migration and invasion by Toll-like
receptor-9 signaling network in prostate cancer, Oncotarget, Vol. 6, No. 26,
2015
8. Matsumoto et al, Role of lysophosphatidylcholine (LPC) in
atherosclerosis., Curr Med Chem. 2007;14(30):3209-20
9. Moessinger et al, Two different pathways of phosphatidylcholine
synthesis, the Kennedy Pathway and the Lands Cycle, differentially regulate
cellular triacylglycerol storage, BMC Cell Biology 2014, 15:43
10. Wei et al, LPCAT1 promotes brain metastasis of lung
adenocarcinoma by up-regulating PI3K/AKT/MYC pathway, Journal of Experimental
& Clinical Cancer Research (2019) 38:95
11. Zhou et al, Identification of Plasma Lipid Biomarkers for
Prostate Cancer by Lipidomics and Bioinformatics, PLOS ONE, November 2012,
Volume 7, Issue 11
12. Zhou, Prospective Application of Lipidomics in Prostate Cancer, J
Glycomics Lipidomics 4: 114., 2014
[5]
Pathways involved in choline and phosphatidylcholine (PC) homeostasis. E,
ethanolamine; P-E, phosphoethanolamine; CDP-E, CDP-ethanolamine; PE,
phosphatidylethanolamine; SM, sphingomyelin. Enzyme names are indicated by
numbers. 1, choline acetyltransferase; 2, choline kinase; 3, CTP:phosphocholine
cytidylyltransferase; 4, CDP-choline:1,2-diacylglycerol
cholinephosphotransferase; 5, sphingomyelin synthase; 6, phosphatidylserine synthase
1; 7, phosphatidylserine decarboxylase; 8, phosphatidylethanolamine N-methyltransferase;
9, ethanolamine kinase; 10, CTP: phosphoethanolamine cytidylyltransferase; 11,
CDP-ethanolamine: 1,2 diacylglycerol ethanolaminephosphotransferase; 12,
various phospholipase and lysophospholipase activities; 13, sphingomyelinase; 14,
choline oxidase; 15, betaine aldehyde dehydrogenase.
[6]
See p 217, Litwack, Human Biochemistry, Academic, 2019.
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