Recall that the general process internal to the mitochondria
can be written as follows.
Specifically we have:
Pyruvate feeds the TCA cycle, it is a result of glycolysis. In
contrast, aerobic glycolysis, the Warburg effect is a mix of both the TCA plus
anaerobic glycolysis, namely no oxygen. The enzyme PDC enables this process.
This we depict above. Pyruvate is the product of glycolysis and AcetylCoA is
the feeder to the TCA.
Lipids, Warburg and PCa
We know that lipids play a role in carcinogenesis. As Hsu
and Sabatini note:
Although studies in cancer metabolism have largely been
energy-centric, rapidly dividing cells have diverse requirements. Proliferating
cells require not only ATP but also nucleotides, fatty acids, membrane lipids,
and proteins, and a reprogrammed metabolism may serve to support synthesis of
macromolecules. Recent studies have shown that several steps in lipid synthesis
are required for and may even actively promote tumorigenesis. Inhibition of ATP
citrate lyase, the distal enzyme that converts mitochondrial-derived citrate
into cytosolic acetyl coenzyme A, the precursor for many lipid species,
prevents cancer cell proliferation and tumor growth (Hatzivassiliou et al.,
2005).
Fatty acid synthase, expressed at low levels in normal
tissues, is upregulated in cancer and may also be required for tumorigenesis
(reviewed in Menendez and Lupu, 2007). Furthermore, cancer cells may also
enhance their biosynthetic capabilities by expressing a tumor-specific form of
pyruvate kinase (PK), M2-PK. Pyruvate kinase catalyzes the third irreversible
reaction of glycolysis, the conversion of phosphoenolpyruvate (PEP) to
pyruvate. Surprisingly, the M2-PK of cancer cells is thought to be less active
in the conversion of PEP to pyruvate and thus less efficient at ATP production.
A simplified version of this in Bauer et al is shown below:
Flavin et al also noted:
Cancer cells synthesize de novo large amounts of fatty
acids and cholesterol, irrespective of the circulating lipid levels and benefit
from this increased lipid synthesis in terms of growth advantage, self-survival
and drug resistance. Key lipogenic alterations that commonly occur in prostate
cancer include over-expression of the enzyme fatty acid synthase (FASN) and
deregulation of the 5-AMP-activated protein kinase (AMPK). FASN is a key
metabolic enzyme that catalyses the synthesis of palmitate from the condensation
of malonyl-CoA and acetyl-CoA de novo and plays a central role in energy
homeostasis, by converting excess carbon intake into fatty acids for storage.
AMPK functions as a central metabolic switch that governs glucose and lipid
metabolism. Recent interest has focused on the potential of targeting metabolic
pathways that may be altered during prostate tumorigenesis and progression.
Several small molecule inhibitors of FASN have now been described or in
development for therapeutic use; in addition, drugs that directly or indirectly
induce AMPK activation have potential benefit in prostate cancer prevention and
treatment[1][2].
Prostate cancer and lipid metabolism has been studied
extensively. Now in a recent report in Science Daily it states[3]:
For years, attempts have been made to understand the
mechanism behind the proliferation of cancer cells: they need metabolites to
grow and proliferate as much as a vehicle needs gasoline or electricity to
move. However, until now it was not known which metabolites cancer cells
actually need. A team of researchers from the Institute of Oncology Research
(IOR) at the Università della Svizzera Italiana (USI, Faculty of Biomedical
Sciences) led by Prof. Andrea Alimonti has identified one of the mechanisms
behind this process, as published in a recent article in the journal Nature
Genetics.
From a theory dating back to the early 20th century by
Nobel Prize laureate Otto Warburg, it has been believed that, in order to
support their growth, cancer cells needed to increase their glucose
consumption, without using mitochondrial metabolism. The mitochondrion is an
organelle that produces the energy needed for the cell survival, operating as a
sort of power station. "Contrary to what was believed for almost a century
-- says Prof. Alimonti -- we have discovered that cells in prostate cancer need
the mitochondrion, not to produce energy, rather to regulate a specific
metabolic process.
Specifically, the mitochondrion is able to regulate fat
synthesis (lipids) through an enzyme complex called PDC.
Thus the glycolysis is but one part. Lipid metabolism is a
second, and we have discussed this earlier as well. PDC is used on the classic
glycolysis and TCA. Here they argue PDC is used in the mitochondria to regulate
lipid development.
The study published by Nature Genetics shows that without
the ability to efficiently produce lipids, prostate cancer cells are not able
to grow and metastasize, even in the presence of increased glycolysis. "We
noticed -- continues Alimonti -- that in prostate cancer cells the activity of
the enzyme complex PDC is 10 times that of a normal proliferating cell, and
that as a result the cells store several lipids."
As noted above, PDC is the enzyme in the connection between
glycolysis and the TCA.
It is known that a diet rich in fat can increase the risk
of developing prostate cancer, and that obese people are more prone to develop
this type of tumour. However, the fact that the metabolism of lipids acts as a
fuel to support the tumour has never been clarified in detail and this
discovery opens up new and unexpected scenarios in cancer therapy.
"We have identified a number of pharmaceutical
compounds that selectively inhibit -- in different experimental models -- the
mitochondrial enzyme responsible for the tumour growth, thus limiting fat
synthesis and without harming normal cells." "I would like to point
out, however -- concludes Alimonti -- that our discovery does not imply that
cancer patients must undergo a strict dietary regime, which might in fact hurt
them: a reduction of fat in cancer cells can only be obtained by blocking the
cancer cells metabolism through specific drugs."
The authors of the above references paper, Chen et al, note:
The mechanisms by which mitochondrial metabolism supports
cancer anabolism remain unclear. Here, we found that genetic and
pharmacological inactivation of pyruvate dehydrogenase A1 (PDHA1), a subunit of
the pyruvate dehydrogenase complex (PDC), inhibits prostate cancer development
in mouse and human xenograft tumor models by affecting lipid biosynthesis.
Mechanistically, we show that in prostate cancer, PDC localizes in both the
mitochondria and the nucleus.
Whereas nuclear PDC controls the expression of sterol
regulatory element-binding transcription factor (SREBF)-target genes by
mediating histone acetylation, mitochondrial PDC provides cytosolic citrate for
lipid synthesis in a coordinated manner, thereby sustaining anabolism.
Additionally, we found that PDHA1 and the PDC activator pyruvate dehydrogenase
phosphatase 1 (PDP1) are frequently amplified and overexpressed at both the
gene and protein levels in prostate tumors.
Together, these findings demonstrate that both
mitochondrial and nuclear PDC sustain prostate tumorigenesis by controlling
lipid biosynthesis, thus suggesting this complex as a potential target for
cancer therapy.
Now PDHA1 is a part of the PDC. From NCBI[4]:
The pyruvate dehydrogenase (PDH) complex is a
nuclear-encoded mitochondrial multienzyme complex that catalyzes the overall
conversion of pyruvate to acetyl-CoA and CO(2), and provides the primary link
between glycolysis and the tricarboxylic acid (TCA) cycle. The PDH complex is
composed of multiple copies of three enzymatic components: pyruvate
dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2) and lipoamide
dehydrogenase (E3). The E1 enzyme is a heterotetramer of two alpha and two beta
subunits. This gene encodes the E1 alpha 1 subunit containing the E1 active
site, and plays a key role in the function of the PDH complex. Mutations in
this gene are associated with pyruvate dehydrogenase E1-alpha deficiency and
X-linked Leigh syndrome.
There are two SREBF genes. Again from NCBI:
SREBF1[5](SREBP1):
This gene encodes a basic helix-loop-helix-leucine zipper (bHLH-Zip)
transcription factor that binds to the sterol regulatory element-1 (SRE1),
which is a motif that is found in the promoter of the low density lipoprotein
receptor gene and other genes involved in sterol biosynthesis. The encoded
protein is synthesized as a precursor that is initially attached to the nuclear
membrane and endoplasmic reticulum. Following cleavage, the mature protein
translocates to the nucleus and activates transcription. This cleaveage is
inhibited by sterols. This gene is located within the Smith-Magenis syndrome
region on chromosome 17. Alternative promoter usage and splicing result in
multiple transcript variants, including SREBP-1a and SREBP-1c, which correspond
to RefSeq transcript variants 2 and 3, respectively.
and
SREBF2[6]
(SREBP2): This gene encodes a member of the a ubiquitously expressed
transcription factor that controls cholesterol homeostasis by regulating
transcription of sterol-regulated genes. The encoded protein contains a basic helix-loop-helix-leucine
zipper (bHLH-Zip) domain and binds the sterol regulatory element 1 motif.
Alternate splicing results in multiple transcript variants.
Extensions
We do know the impact of the TCA on androgen receptor as
shown below:
Alaso as detailed below we have:
As McFate et al note:
High lactate generation and low glucose oxidation,
despite normal oxygen conditions, are commonly seen in cancer cells and tumors.
Historically known as the Warburg effect, this altered metabolic phenotype has
long been correlated with malignant progression and poor clinical outcome.
However, the mechanistic relationship between altered
glucose metabolism and malignancy remains poorly understood.
Here we show that inhibition of pyruvate dehydrogenase
complex (PDC) activity contributes to the Warburg metabolic and malignant
phenotype in human head and neck squamous cell carcinoma. PDC inhibition occurs
via enhanced expression of pyruvate dehydrogenase kinase-1 (PDK-1), which
results in inhibitory phosphorylation of the pyruvate dehydrogenase (PDH) subunit.
We also demonstrate that PDC inhibition in cancer cells is associated with
normoxic stabilization of the malignancy-promoting transcription factor
hypoxia-inducible factor-1 (HIF-1) by glycolytic metabolites.
Knockdown of PDK-1 via short hairpin RNA lowers PDH
phosphorylation, restores PDC activity, reverts the Warburg metabolic
phenotype, decreases normoxic HIF-1 expression, lowers hypoxic cell survival,
decreases invasiveness, and inhibits tumor growth. PDK-1 is an HIF-1-regulated
gene, and these data suggest that the buildup of glycolytic metabolites,
resulting from high PDK-1 expression, may in turn promote HIF-1 activation,
thus sustaining a feed-forward loop for malignant progression.
In addition to providing anabolic support for cancer
cells, altered fuel metabolism thus supports a malignant phenotype. Correction
of metabolic abnormalities offers unique opportunities for cancer treatment and
may potentially synergize with other cancer therapies.
As Fan et al note:
The mitochondrial pyruvate dehydrogenase complex (PDC)
plays a crucial role in regulation of glucose homoeostasis in mammalian cells.
PDC flux depends on catalytic activity of the most
important enzyme component pyruvate dehydrogenase (PDH).PDH kinase inactivates
PDC by phosphorylating PDH at specific serine residues, including Ser-293,whereasdephosphorylation
of PDH by PDH phosphatase restores PDC activity. The current understanding
suggests that Ser-293 phosphorylation of PDH impedes active site accessibility
to its substrate pyruvate.
Here, we report that phosphorylation of a tyrosine
residue Tyr-301 also inhibits PDH 1 (PDHA1) by blocking pyruvate binding
through a novel mechanism in addition to Ser-293 phosphorylation. In addition,
we found that multiple oncogenic tyrosine kinases directly phosphorylate PDHA1
at Tyr-301, and Tyr-301 phosphorylation of PDHA1 is common in EGF-stimulated
cells as well as diverse human cancer cells and primary leukemia cells from
human patients.
Moreover, expression of a phosphorylation-deficient PDHA1
Y301F mutant in cancer cells resulted in increased oxidative phosphorylation,
decreased cell proliferation under hypoxia, and reduced tumor growth in mice.
Together, our findings suggest that phosphorylation at distinct serine and
tyrosine residues inhibits PDHA1 through distinct mechanisms to impact active
site accessibility, which act in concert to regulate PDC activity and promote
the Warburg effect.
The Warburg effect is clear in the above. Its presence
disappears when examining lipid metabolism, however. This point is further
emphasized as Zhong et al note:
Cells generate adenosine-5′-triphosphate (ATP), the major
currency for energy consuming reactions, through mitochondrial oxidative
phosphorylation (OXPHOS) and glycolysis. One of the remarkable features of
cancer cells is aerobic glycolysis, also known as the “Warburg Effect”, in
which cancer cells rely preferentially on glycolysis instead of mitochondrial
OXPHOS as the main energy source even in the presence of high oxygen tension.
One of the main players in controlling OXPHOS is the
mitochondrial gatekeeper pyruvate dehydrogenase complex (PDHc) and its major subunit
is E1α (PDHA1). To further analyze the function of PDHA1 in cancer cells, it was
knock out (KO) in the human prostate cancer cell line LnCap and a stable KO
cell line was established. We demonstrated that PDHA1 gene KO significantly
decreased mitochondrial OXPHOS and promoted anaerobic glycolysis, accompanied
with higher stemness phenotype including resistance to chemotherapy, enhanced
migration ability and increased expression of cancer stem cell markers. We also
examined PDHA1 protein expression in prostate cancer tissues by
immunohistochemistry and observed that reduced PDHA1 protein expression in
clinical prostate carcinomas was significantly correlated with poor prognosis.
Collectively, our results show that negative PDHA1 gene
expression is associated with significantly higher cell stemness in prostate
cancer cells and reduced protein expression of this gene is associated with shorter
clinical outcome in prostate cancers…..
We herein demonstrated that PDHA1 gene knockout resulted
in dysfunctional mitochondrial OXPHOS and enhanced glycolysis. We previously
reported that impartial mitochondrial OXPHOS by using mitochondrial pyruvate carrier
(MPC) blocker enhanced stemness phenotype of prostate cancer cells. In keeping
with our previously study, the mitochondrial gatekeeper PDHA1 gene knockout also
leads to dysfunctional mitochondrial and enhanced glycolysis, as well as higher
cell stemness phenotype. And by immuno-histochemical examination of PDHA1
protein expression in prostate cancer samples, it was revealed that negative
PDHA1 protein expression was related with poor clinical outcome in patients
with prostate cancer.
As Li et al note:
Alternative pathways of metabolism endowed cancer cells
with metabolic stress. Inhibiting the related compensatory pathways might
achieve synergistic anticancer results. This study demonstrated that pyruvate
dehydrogenase E1α gene knockout (PDHA1 KO) resulted in alterations in tumor
cell metabolism by rendering the cells with increased expression of
glutaminase1 (GLS1) and glutamate dehydrogenase1 (GLUD1), leading to an
increase in glutamine-dependent cell survival. Deprivation of glutamine induced
cell growth inhibition, increased reactive oxygen species and decreased ATP
production.
Pharmacological blockade of the glutaminolysis pathway resulted
in massive tumor cells apoptosis and dysfunction of ROS scavenge in the LNCaP PDHA1
KO cells. Further examination of the key glutaminolysis enzymes in human
prostate cancer samples also revealed that higher levels of GLS1 and GLUD1 expression
were significantly associated with aggressive clinicopathological features and
poor clinical outcome. These insights supply evidence that glutaminolysis plays
a compensatory role for cell survival upon alternative energy metabolism and
targeting the glutamine anaplerosis of energy metabolism via GLS1 and GLUD1 in
cancer cells may offer a potential novel therapeutic strategy.
As Justus et al note:
There are several molecular mechanisms whereby acidosis
may alter tumor cell metabolism. p53 is an important regulator of the metabolic
response to acidosis. The ability of acidosis to activate p53 and stimulate the
TCA cycle through inhibition of glycolysis has been demonstrated. For example,
acidosis induced p53 expression may transcriptionally inhibit the expression of
glucose transporters GLUT1 and GLUT4 in specific tissues, thereby effectively
reducing glucose availability for glycolysis. In addition, acidosis is reported
to activate p53 and increase expression of glucose 6 phosphate dehydrogenase
(G6PD) and glutaminase 2.
This is suggested to direct glucose towards the pentose phosphate
pathway (PPP) as well as increase glutaminolysis. This may also drive the TCA
cycle through the production of metabolic intermediates and increase the amount
of NADPH in the cell to counteract ROS production. p53 activation may also
induce the expression of Parkin (PARK2), a Parkinson disease-associated gene,
to reduce glycolytic activity.
PARK2 regulates the expression of pyruvate dehydrogenase
alpha 1 (PDHA1), a critical component for the activity of pyruvate
dehydrogenase (PDH). PDHA1 knockdown increases glucose uptake, rate of
glycolysis, and lactate production, facilitating the “Warburg effect”. This
gives PARK2 the ability to effectively reverse the “Warburg effect” by inducing
PDHA1.
Moreover, PARK2 also regulates expression of reduced
glutathione (GSH), a major antioxidant and ROS scavenger in the cell. This is
proposed to occur through activation of p53 and may reduce ROS when oxidative
phosphorylation is increased. Furthermore, γ-irradiation-induced tumorigenesis
is sensitized following the knockout of PARK2 in C57BL/6J mice, indicating the
PARK2 gene as a tumor suppressor.
The ability for p53 to regulate cancer cell metabolism by
reducing glycolysis and increasing oxidative phosphorylation while
simultaneously mitigating ROS is crucial for understanding acidosis induced
metabolic alterations in the tumor.
Observations
We can make a few observations.
1. Warburg effect generally refers to glycolysis and the
reduced production of ATP. It indirectly refers to any lipid processing. Fatty
acid oxidation can produce an estimated 129 ATP per oxidation[7].
This is a large producer of energy and well exceeds that of glycolysis results.
Thus considering the lipid elements as conjoint is highly reasonable.
Lipids are also energy rich as noted above. Perhaps cancer
cells can function in this manner if the throughput of lipids can be high.
Targets for therapeutics using the lipid control may have
potential deleterious effects. If the proposal is to target the enzyme in the
production of ultimately ATP from lipids then one must be aware of the
significant downside regarding cross cell contamination.
This is an interesting and useful alternative and again
shows that there are many options about the Warburg process.
References
1. Bauer et al, ATP citrate lyase is an important component of cell
growth and transformation, Oncogene (2005) 24, 6314–6322
2. Cerniglia et al, The PI3K/Akt Pathway Regulates Oxygen
Metabolism via Pyruvate Dehydrogenase (PDH)-E1a Phosphorylation, Mol Cancer
Ther; 14(8) August 2015
3. Chen et al. Compartmentalized activities of the pyruvate
dehydrogenase complex sustain lipogenesis in prostate cancer, Nature Genetics
(2018)
4. Fan et al, Tyr-301 Phosphorylation Inhibits Pyruvate
Dehydrogenase by Blocking Substrate Binding and Promotes the Warburg Effect, The
Journal Of Biological Chemistry Vol. 289, No. 38, pp. 26533–26541, September
19, 2014
5. Ferreira et al, Metabolic reprogramming of the tumor, Oncogene
(2012) 31, 3999–4011
6. Ferrier, Biochemistry, 6th Edition, Lippincott (New
York) 2015.
7. Flavin et al, Metabolic alterations and targeted therapies in
prostate cancer, J Pathol. 2011 January ; 223(2): 283–294
8. Hsu and Sabatini, Cancer Cell Metabolism: Warburg and Beyond, Cell
134, September 5, 2008
9. Jin et al, Androgen receptor genomic regulation, Transl Androl
Urol 2013;2(3):158-177
10. Justus et al, Molecular Connections between Cancer Cell
Metabolism and the Tumor Microenvironment, Int. J. Mol. Sci. 2015, 16,
11055-11086
11. Li et al, PDHA1 gene knockout in prostate cancer cells results
in metabolic reprogramming towards greater glutamine dependence, Oncotarget,
Vol. 7, No. 33 2016
12. McFate et al, Pyruvate Dehydrogenase Complex Activity Controls
Metabolic and Malignant Phenotype in Cancer Cells, THE JOURNAL OF BIOLOGICAL
CHEMISTRY VOL. 283, NO. 33, pp. 22700–22708, August 15, 2008
13. McGarty, Glucose, Warburg, Cancer and Pathways, Jan 2018, http://www.telmarc.com/Documents/White%20Papers/148Warburg.pdf
14. Sugden et al, Fuel-sensing mechanisms integrating lipid and
carbohydrate utilization, Biochemical Society Transactions (200 I) Volume 29,
part 2
15. Wittig and Coy, The Role of Glucose Metabolism and
Glucose-Associated Signalling in Cancer, Perspectives in Medicinal Chemistry 2007:1
64–82
16. Zhong et al, Pyruvate dehydrogenase expression is negatively
associated with cell stemness and worse clinical outcome in prostate cancers, Oncotarget,
2017, Vol. 8, (No. 8), pp: 13344-13356
[1] FASN
from NCBI: The enzyme encoded by this gene is a multifunctional protein. Its
main function is to catalyze the synthesis of palmitate from acetyl-CoA and
malonyl-CoA, in the presence of NADPH, into long-chain saturated fatty acids.
In some cancer cell lines, this protein has been found to be fused with
estrogen receptor-alpha (ER-alpha), in which the N-terminus of FAS is fused
in-frame with the C-terminus of ER-alpha. https://www.ncbi.nlm.nih.gov/gene/2194
[2]
AMPK from NCBI: The protein encoded by this gene belongs to the ser/thr protein
kinase family. It is the catalytic subunit of the 5'-prime-AMP-activated
protein kinase (AMPK). AMPK is a cellular energy sensor conserved in all
eukaryotic cells. The kinase activity of AMPK is activated by the stimuli that
increase the cellular AMP/ATP ratio. AMPK regulates the activities of a number
of key metabolic enzymes through phosphorylation. It protects cells from
stresses that cause ATP depletion by switching off ATP-consuming biosynthetic
pathways. Alternatively spliced transcript variants encoding distinct isoforms
have been observed. https://www.ncbi.nlm.nih.gov/gene/5562
[7]
See Ferrier, pp 192-193
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