The ‘waves of metabolic reprogramming’ model

Wave 1: oncogene-mediated reprogramming

 During wave 1, a pro-oncogenic stimulus

leads to the deregulation of activated
oncogenes-mediated signalling – e.g. MYC;

 Additional stimuli are necessary/possible,

involving the deactivation of tumor suppressor
genes (e.g. TP53);

 Oncogenic stimuli may involve the activation

of cancer stem cells;

Cancer cells gain a partial Warburg glycolytic phenotype, in

which pyruvate is redirected only in part away from the Krebs
cycle and from OXPHOS, allowing a high growth rate.
Wave 2: hypoxia-dependent reprogramming

During wave 2, the initial tumor growth leads

to the formation of hypoxic areas;

As a consequence, the hypoxia-inducible

factor (HIF) is activated, which further
stimulates glycolysis, through the trascriptional
activation of pro-glycolytic genes;

Pyruvate is re-directed nearly completely

towards LDH, and mitochondrial function is
shut off.

Hypoxic adaptation is not sufficient, on the long term, to

support proliferation without an adequate nutrient supply.
Wave 3: aglycemia-dependent reprogramming

 During wave 3, high glycolytic flux leads to a

condition of aglycemia;

 A strong selective pressure drives an

additional metabolic rewiring towards
malignant phenotypes, along with the
activation of different oncogenes;

Glutaminolysis is promoted, in both modes,

and PDH activity is partially restored;

 Mitochondrial biognesis is also partially

restored, to favor glutaminolysis via respiratory
complex II.

The tumor mass recovers both oxygen and nutrient supply

through neoangiogenesis, following hypoxia response.
Wave 4: recovery of mitochondrial activity

 During wave 4, reoxygenation and the need

to carry out aerobic glutaminolysis lead to
recovery of mitochondrial activity;

 A series of retrograde metabolic signals by

mitochondria aims at modifying nuclear gene
expression;

 In this stage, both glycolysis and

mitochondrial respiration cooperate to
increase efficiency for ATP production.

During each of these highly selective processes and

metabolic changes, the disease is shaped by which cells
survived and how they adapted – giving rise to most diverse
phenotypes.
 According to the growth phase, to genetic lesions, to the capacities of
the tissue of origin, to tumor microenvironment and to the cancer cells’
abilities to adapt, neoplasms may be categorized according to their
bioenergetic phenotype as follows:

 highly glycolytic

 partial OXPHOS

 enhanced OXPHOS

Mitochondria play a central role in cancer progression, as they are the hub of
energy metabolism, from the rewiring of which depends a cancer cell’s fate.
Oncogenes and TSGs: their metabolic role

MYC
 One of the main mutated oncogenes in gastric carcinoma; its
deregulation appears sufficient to cause neoplastic transformation.

HIF

Myc interacts with MAX, in competition with MAD1 and 2, hence regulating
glycolytic genes expression, and represses miR-23a/b, inducing
glutaminase and stimulating aerobic glycolysis.
MYC works in equilibrium with HIF and dependently on
the availability of oxygen.

It may be responsible for the recovery of proliferation

after wave 2, when hypoxia redirects metabolism towards
glycolysis (energy-save mode).

MYC determines glutamine-dependence of certain

tumors.

NRF1
POLG
p53

 One of the most commonly mutated TSGs in human tumors. Germ-line

mutations cause the Li-Fraumeni syndrome.

P53 regulates aerobic metabolism, so that a p53 deficit implies a

respiratory deficit too. AMPK activates p53 upon phosphorylation during
nutrient shortage, and p53 triggers a metabolic checkpoint.
 P53 regulates OXPHOS through two main mechanisms:

 By overregulating transcription of mitochondrial cytochrome oxidase 2 (COX2), necessary

for the assembly of complex IV of the respiratory chain;

 By mediating the transcription of TIGAR (TP53-induced glycolysis and apoptotic regulator),

an inducible isoform of phosphofructokinase that inhibits glycolysis by reducing F2,6BP
levels.

 Besides its role in regulating cell cycle and DNA damage repair following genotoxic stress, the
tumor suppressor role of p53 is indeed metabolic, as its loss of function favors a highly glycolytic
metabolism.
PTEN and the PI3K-Akt pathway

 PTEN is a non-canonical TSG that follows a haploinsufficiency model and must not
necessarily lose the wild type allele to trigger neoplastic progression. PTEN germline
mutations cause Cowden syndrome.

 Akt is an oncogene. Activating mutations of Akt are infrequent, although its activation is
common in human neoplasms, where it depends on PTEN dysfunction or on the
activation of the RAS oncogene. Akt hyperactivation is common in leukemia.
 An imbalance in reducing equivalents with NADH accumulation may cause
PTEN inactivation, with an ultimate activation of the mTOR pathway and
stimulation of glucose catabolism via OXPHOS;

 Akt favors glucose uptake and promotes aerobic glycolysis.

 The Akt pathway promotes HIF stabilization and stimulates fatty acids
synthesis.
RAS

 RAS is an oncogene involved in the transduction of mitogenic signals through

tyrosine kinase receptors. Germline mutations in components of the RAS
pathway cause the so-called RASopathies such as Noonan, Costello, and
LEOPARD syndromes.

 RAS transformation induces

a strong dependence from
glycolysis, and reduces
fatty acids synthesis

 RAS induces a potent metabolic remodeling during cell transformation,

triggering an imbalance in the transcription of the PDH complex, as well as of
respiratory complexes II, IV and V.
Overview of the metabolic role of common
oncogenes and TSGs
Mitochondrial TSGs: SDH

 Paragangliomas are slow-growing tumors of

the paraganglia;

 When they arise in adrenal paraganglia they

are called pheochromocytomas;

 Common mutations detected in

paragangliomas map in the genes MAX, VHL,
SDHC, SDHD and, in 40% of cases, in SDHB,
i.e. the B subunit of succinate dehydrogenase,
or respiratory complex II.
 Complex II acts as a bridge
between the mitochondrial
membrane and the
mitochondrial matrix, hence
participating to both the TCA
cycle and the respiratory
chain.
 Complex II is a four-subunit enzyme: homozygous germline mutations are
responsible for severe neurological conditions;

 Heterozygous mutations predispose to the occurrence of paraganglioma,

where often the loss of the wild type allele is observed (LOH – loss of
heterozygosity);

 Only one case of mutation in SDHC has been reported, involving the first
amino acid shifting the translation start and deleting the signal peptide for
insertion in the mitochondrial membrane.

Mutations in SDH cause disassembly of

complex II.
 Mitochondrial TSGs: FH

 Heterozygous mutations in the fumarate hydratase TCA gene (FH) cause

hereditary leiomyomatosis and type 2 papillary-type renal cell carcinoma
(HLRCC).
 Most reported mutations are frameshift and missense in conserved AA, and
they map within the 5’;

 All mutations associated with HLRCC imply a partial enzymatic activity loss;

 Missense mutations are often more severe than truncating ones, as FH

functions as a homotetramer (dominant negative effect).
 FH, or fumarase, is the enzyme that follows SDH within the
TCA cycle, and converts fumaric acid into malate.
Mitochondrial TSGs and their tumor-promoting mechanism: – HIF1

 The hypoxic response, which triggers a metabolic remodeling towards the

glycolytic shift, is mainly driven by the hypoxia-inducible factor 1 (HIF1);

 HIF1 has a ubiquitous expression. It is a heterodimeric transcription factor,

composed by a constitutively expressed subunit (HIF1b) and a subunit that is
constantly degraded in normoxic conditions (HIF1a);

 During hypoxia, the HIF1a

subunit is stable and may bind HIF1b.
The heterodimer translocates
to the nucleus to activate
a number of pro-glycolytic,
pro-angiogenetic and metastatic
genes.
 HIF1a structure and domains
Oxygen degradation
Hydroxylation of N803 is
dependent domain
crucial for HIF1
transcriptional activity

DNA binding domain

necessary for
transcriptional activity Hydroxylation of prolines
402 and 564 by cytosolic
prolyl-hydroxylases (PHDs)
mediates VHL recognition
 The prolyl-hydroxylase reaction

 PHDs use ascorbic acid, iron and O2 to transfer one oxydril group from a-
ketoglutarate onto HIF1a prolines;

 The main reaction byproduct is succinate, the main PHD allosteric inhibitor;
PHD has several levels of regulation:
 The enzyme affinity of its main substrate, namely molecular oxygen, is
regulated in a directly proportional fashion by a-ketoglutarate
concentration;
 Both succinate and fumarate are allosteric inhibitors;
 ROS inhibit the enzyme, along with nitric oxide;

Conditions that favor succinate or

fumarate accumulation, such as
mutations in SDH or FH, block
PHD activity and favor HIF1a
stabilization.

 Cells with mutations in SDH ed FH, in which the enzymatic activity is lost and
succinate or fumarate accumulate, are subjected to a condition of chronic
pseudohypoxia.
Isocitrate dehydrogenase (IDH) and glioma
 Even if low grade, gliomas are among the most
devastating neoplasms with poor prognosis;

 Survival is often less than two years at diagnosis;

 Molecular causes are still unknown, although some

recurrent genetic lesions have been detected.
 Out of 939 cancer samples, the frequency of IDH1 and IDH2 mutations is
high (in particular R132H);

 Residue 132 and its corresponding 172 of IDH2, similarly mutated at high
frequency, maps in the enzyme catalytic site and modifies its function.
 Cloning and mutagenesis of IDH
according to the variants found in vivo
shows a dramatic decrease of enzyme
activity, when mutations are present.

 Evaluation of enzymatic activity is

carried out by measuring NADPH
production, since this is a product of the
IDH reaction.
 Prognosis of patients with IDH
mutations is better than those with wild
type IDH!
 IDH1 mutations confer a novel function to the
enzyme, i.e. to generate the (R)-HG (hydroxyglutarate)
enantiomer from 2-OG.

 Unlike (S)-HG, the R enantiomer is an activating

substrate of PHD (alias EGLN), which leads to
destabilization of HIF1a.
proton Magnetic Resonance Spectroscopy (MRS)
 A lower HIF1a due to higher PHD activity has been called responsible for a higher
tumorigenicity of astrocytes in culture.

 In brain tumors and leukemia the transforming effect of the (R)-HG

enantiomer may depend on HIF1a inactivation.

 HIF1a may have a pro- or an anti-tumorigenic role ina context-dependent

fashion, or depending on the isoforms mainly expressed (yet all IDH mutated
patients have a better prognosis…).
 Oncometabolites and epigenetics:
one-carbon metabolism

 Fumarate, succinate, hydroxyglutarate are considered oncometabolites due

to their allosteric regulation of proteins involved in epigenetic regulation;

 The role of a-ketoglutarate is controversial, and it is currently considered an

anti-oncometabolite…
 Epigenetic regulators

 Several proteins contribute to epigenetic changes, at the DNA or at the

histone level
- DNMT/TET: DNA methylases and demethylases
- HAT: histone acetyltransferase, and histone deacetylases
- HMT: histone methyltransferase
- …