Riassunto esame Molecular oncology, Prof. Spallotta Francesco, libro consigliato Molecular Biology of Cancer, Lauren Pecorino

RAS.

- class II unknown functions.

→

- class III known for roles in cell

→

growth and autophagy.

These proteins are generally constituted by regulatory subunits (p85s) and a catalytic

domain (p110s). Several RTK, like EGFR, PDGFR, IGFR but also adaptor in T and B

lymphocytes contain in their intracellular tail a specific YXXM-based motif, that, once

phosphorylated, binds the SH2 domain of p85 regulatory subunits, activating PI3K’s

catalytic domain.

Thanks to the activation by PI3K, Akt regulates cell survival:

a. it phosphorylates FOXO3A, thus blocking its nuclear translocation and the

expression of pro-apoptotic BIM,

suppressing the intrinsic apoptotic pathway.

b. it phosphorylates and inactivates pro-

apoptotic BAX and BAD, suppressing the

intrinsic apoptotic pathway.

c. it induces the expression of anti-apoptotic

BCL-XL.

d. it favours Mdm2-mediated degradation of

the oncosuppressor protein p53.

In addition, by activating Akt pathway, PI3K takes part in the

negative regulation of GSK3 (glycogen synthase kinase 3),

which is phosphorylated by Akt, thus inhibiting its functions

in proliferation, migration, inflammation and glucose

metabolism.

Finally, through Akt, PI3K also controls mTOR activation.

mTOR is the Ser/Thr kinase catalytic subunit of two distinct complexes: mTORC1 and

mTORC2.

In resting cells mTORC1 is maintained inactive, but when Akt pathway is triggered,

mTOR phosphorylates and activates the ribosomal kinase “p70S6K” and the

eucaryotic translation initiation factor 4E (eIF4E)-binding protein, “4EBP1”, resulting

in pushing of protein synthesis.

Most found mutations: nd

- the gene PI3KCA encoding for the catalytic subunit p110α is the 2 most

mutated in cancer.

- PI3K and mTOR promote tumor metastasis and epithelial-mesenchymal cell

transition (EMT).

- loss of PTEN results in constitutive signalling by the PI3K pathway.

Inhibitors of mutated PI3K: →

PI3K Isoform specific inhibitors can be used at low doses and have low toxicity

→

PERIFOSINE acts against Akt, preventing AKT/PIP3 binding

→

RAPAMYCIN and analogues act against mTOR, have no therapeutic benefits but

allow to extend the survival of patients with renal cell carcinoma, mantle cell

lymphoma and neuroendocrine tumors.

D. Proteins that control the cell cycle

Cell cycle is fine regulated by Cyclin/Cdk turnover.

Mutations in proteins that regulate Cyclin/Cdk

turnover result in protein hyperactivation, thus

leading to tumorigenesis.

For some tumors inhibitors

that slow down, or block

cell cycle activity have

been developed.

E. Transcription Factors

They are proteins that bind to DNA-regulatory sequences, called enhancers if they

make the transcription increase and silencers if they contribute to diminish it. Usually

localized in the 5’-upstream region of target genes, TFs are able to modulate the rate

of gene transcription, resulting in increased

or decreased gene transcription, protein

synthesis, and subsequent altered cellular

function.

Transcription factors contain a set of independent protein modules or domains, such as

DNA-binding domains (usually an α-helix that binds the major groove), transcriptional

activation domains, dimerization domains and ligand-binding domains. Some TFs

work in a dimer and so require a dimerization domain, while others only function upon

binding of a ligand and therefore require a ligand-binding domain.

Usually TFs recognize consensus sequences, consisting of very common nucleotides

located in high conserved regions of DNA.

The binding of TFs to DNA is only possible when chromatin has already been made

accessible through epigenetic

modifications (i.e. histone acetylation by

the “histone acetyltransferase” enzyme

(HAT), that adds acetyl groups to specific

histone tails). Note that enzymes like

HATs are also capable of modify non-

histone proteins, including transcription

factor itself, as occurs for E2F and p53.

Because these proteins are able to contact directly the DNA entering the nucleus, they

are strictly associated to tumor growth. In particular, the genes codifying for TFs are

proto-oncogenes. Myc

The Myc family of transcription factors, consisting of Myc, Max, Mad, Mxi, plays

several roles in different biological pathways, like MAPK cascade. Myc gene was

identified in 1982 as the cellular homologue to the viral oncogene v-myc of the “avian

myelocytomatosis” retrovirus.

Myc protein promotes proliferation by regulating the expression of target genes,

including N-Ras and p53. Myc protein is found highly expressed during embryogenesis

and in tissue compartments of the adult that possess high proliferative capacity (i.e.

skin and gut).

Myc requires the constitutively expressed Max to function, as that they form

heterodimers capable of recognizing the regulatory sequence E-box (CACGTG) in

their target genes.

By inducing transcription, Myc is able to push cells into S phase, making the cycle

progress:

- Myc/Max dimer promotes transcription of cyclin D2 and CDK4 kinase, which

are the regulators of early G1 phase.

- This results in the sequestration and proteasomal degradation of p27, one cyclin

E–CDK2 inhibitor.

- Cyclin E/CDK2 complex is phosphorylated and activated, leading to S phase

entry.

- Myc/Max dimers also promote transcription of the “translation initiation

factors” eIF2 and eIF4, important for protein synthesis.

Note that transcriptional activity of Myc/Max heterodimers depend on their association

with the co-activator TRRAP, that contains HAT activity, leading to acetylation of

histones H3 and H4 and DNA transcription.

By contrast, Mad/Max heterodimers antagonize Myc activity by binding its targets’

promoters (they compete for the binding) and repressing the transcription of target

genes. In particular, Mad/Max heterodimers associate with SIN3 that contains HDAC

activity, leading to histones H3 and H4 deacetylation; this results in repression of

transcriptional activity.

Myc deregulation:

Myc chromosomal translocation correlates with Burkitt’s

lymphoma, as result of translocation from chromosome 8 to

chromosome 14, in a location that falls within the regulation

of the strong promoter of immunoglobulin genes, that

increases the amount of expression of myc gene and leads to

oncogenic activation.

In addition, deregulated expression of c-myc is associated with poor prognosis in

several human cancers, like breast, colon, cervical, small-cell lung carcinomas,

osteosarcomas, glioblastomas, melanoma and myeloid leukaemia, leading to tumour

progression.

What’s strange is that when a hyperactivated form of Myc is experimentally

overproduced, in most normal cells it results NOT in excessive proliferation but in

permanent cell-cycle arrest or apoptosis. Thus, normal cells are able to detect

abnormal mitogenic stimulation and they respond by preventing further division, in

order to prevent survival and proliferation of cells with various cancer-promoting

mutations. This is possible thanks to the linkage between the activity of Myc and the

oncosuppressor p53:

- c-Myc indirectly activates p53 via p19ARF, thus leading to transcription of Bax,

one pro-apoptotic member of the Bcl-2 family

- Bax activation leads to the release of cytochrome c from the mitochondria into

the cytosol

This means that, as Myc acts as oncogenic protein, while in normal cells its

hyperactivation results in apoptosis induced by oncosuppressor genes, in cancer cells

it would lead to tumor growth and proliferation, generally correlating with p53

deregulation; thus, by blocking Myc it would be possible to make the tumor regret or

induce cell cycle arrest.

Myc is also involved in glycolysis regulation, by inducing the expression of both

LDHA (Lactate Dehydrogenase A), that converts pyruvate in lactate during anaerobic

respiration (it is part of the metabolic pathway that allows cells to produce energy in

the absence of oxygen), and PKD1 (Pyruvate Dehydrogenase Kinase 1), that

phosphorylates and inhibits PDH (Pyruvate Dehydrogenase complex), thereby

reducing the conversion of pyruvate to acetyl-CoA and promoting anaerobic

metabolism. This implies that when Myc results deregulated, anaerobic energetic

metabolism is pushed on through PDK1 and PDHA induction.

Inhibitors of mutated Myc: →

Small Molecule Inhibitors for example compounds that target the Myc-Max

heterodimer.

→

siRNA used to reduce Myc expression at the mRNA level, effectively silencing its

activity.

Note that many are still in the experimental stages.

Myc can activate mechanisms of Synthetic Lethality, by which one mutation is

tolerable but co-occurring mutations are lethal; thus, it was discovered that the usage

of drugs together with protein overexpression could lead to this phenomenon, that

implies cancer cells death. NF-kB

Its complete name is “Nuclear factor kappa-light-chain-enhancer of activated B cells”;

it is a protein complex that controls transcription

of DNA, cytokine production and cell survival.

NF-κB is found in almost all animal cell types

and is involved in cellular responses to stimuli

such as stress, cytokines, free radicals, heavy

metals, ultraviolet irradiation, oxidized LDL, and

bacterial or viral antigens. In addition, NF-κB

has other downstream effects that contribute to

tumorigenesis, such as the inhibition of

apoptosis and the promotion of metastasis and

angiogenesis.

Thus, NF-κB provides a molecular link between

inflammation and cancer.

NF-κB is a dimeric transcription factor made up of hetero-/homo-dimers of proteins;

the 5 NF-κB family members share an RHD domain, needed for dimerization and

binding to DNA, and are placed into two groups:

→

1. RelA (p65), RelB and c-Rel synthesized as mature products; they contain

transactivation domains (TADs). →

2. NF-κB1 (p50) and NF-κB2 (p52) must

be proteolytically processed; they cannot

activate transcription on their own.

The most predominant NF-κB dimer activated by the classical pathway is p65–p50.

N.B.: an important finding that could link NF-κB to carcinogenesis was the discovery

that c-rel is the proto-oncogene of the v-rel oncogene.

Activation pathway:

I. In normal conditions, NF-κB is

sequestered in the cytoplasm by the

inhibitor IκB.

II. Upon cell activation, IKK, IκB kinase,

phosphorylates IκB and targets it for

degradation via a ubiquitin ligase complex.

III. This causes the release and translocation of

NF-κB to the nucleus, where it can regulate

the transcription of his target genes that are

more than 200.

IKK is the “Inhibitor of nuclear factor-kappa B

kinase”; more precisely, the regulator is its subunit γ, also known as NEMO. IKK is