Oncogenes

Background

Oncogenes are at the heart of massive research efforts to understand both normal cellular and cancer metabolism. These genes produce proteins that are involved in cellular proliferation and differentiation. When these oncogenes are altered, mutated, or lost, the result is deranged and sometimes neoplastic transformation. No one oncogene is responsible for the various physiological occurrences in normal and cancer cells. Instead, it is an interaction of various oncogenes following many pathways.

Generalized Functions

Function CHARACTERIZATION

Tumor Suppressor Genes Cytoplasmic transduction extracellular signals
Nuclear transcription regulators
Nuclear cell cycle regulators
Nuclear DNA repair

Cytoplasmic transducers of extracellular signals Neurofibromin
Schwannomin
APC gene product
PATCHED T
uberin
PTEN

Nuclear transcription regulators pRB
p53
WT-1
BRCA-1
NF-kappaB

Nuclear cell cycle regulators P16 and p15
Phosphatidyl inositol 3kinase

Nuclear DNA repair Msh2/Mik1 protein products
XP protein product

Specific Oncogenes

NAME CHARACTERIZATION

bax Cell death promotor
Member of the bcl-2 multigene family

bcl-2
Protein product protects against cell death (apoptosis)

Transcriptionally regulated by p53 and appears to function as a heterodimer of bcl-2-BAX to inhibit apoptosis

beta-Catenin
Multifunctional protein involved in cell-cell interaction and transcriptional signalling
Largely regulated by its two major binding partners, E-cadherin present on the membrane and APC protein (Adenomatous polyposis coli) in the cytoplasm

In normal colonic epithelium, it is bound to E-cadherin as part of a cell-cell adhesion complex on the lateral cell membrane

Cytoplasmic beta-catenin levels are regulated by binding to a protein complex consisting of APC, glycogen synthase kinase 3beta, and axin followed by ubiquitin/proteasome degradation

Thought to play critical role in sporadic colonic tumorigenesis

Cyclin D1 Regulates the cell cycle promoting progression from the G1 to S phase of the cell cycle

Fas
(Apo-1/CD95) Belongs to the nerve growth factor/tumor necrosis receptor superfamily
Membrane protein that induces apoptosis when binding to its ligand

CD95 ligand (CD95L) immunohistochemistry: a critical study on 12 antibodies.

Strater J, Walczak H, Hasel C, Melzner I, Leithauser F, Moller P.

Institute of Pathology, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany.
Cell Death Differ 2001 Mar;8(3):273-278 Abstract quote
In recent years, some studies on the expression of CD95(Fas/APO-1) ligand (CD95L) in tissues or cells raised concerns about the specificity of the antibodies used.

We therefore tested 12 CD95L antibodies for their reliability in immunocyto/histochemistry by (i) staining CD95L-transfected and control CV-1/EBNA cells and (ii) comparing staining patterns in immunohistochemically labeled tissue sections with the localization of CD95L(+) cells in in situ hybridization. While G247-4, NOK-1, NOK-2, 4H9, and MIKE-1 stained CD95L-transfected cells and did not significantly bind to controls, G247-4 was the only antibody giving satisfying signals in tissue sections perfectly matching the distribution of CD95L(+) cells by in situ hybridization. MAb 33, C-20, and N-20 comparably stained both transfected and control cells and showed considerable background or falsely positive staining in sections. MIKE-2, 8B8, A11, and 4A5 did not or only very faintly bind to either cells and, thus, were not tested on sections.

We conclude that G247-4 is the only tested antibody that is recommendable for immunohistochemistry.

MDM2 Negative regulator of p53 inhibiting transcriptional activity
Has a pRB binding site

MMR
DNA mismatch repair system
Family of genes with homologues to bacterial MutS and MutL proteins

hMSH2
hMSH3
hMSH6

hMLH1
hPMS2
hPMS1

Germline mutations of hMSH2 and hMLH1 account for 50% of all cases of HNPCC colon cancer

NF-kappaB
Transcription factors found in the cytoplasm of most cells
Includes:
p65/Rel A
Rel B
c-Rel
p50/p105
p52/p100

Latent form, it is sequestered in the cytoplasm, bound by the IkappaB family of inhibitory proteins
After cellular stimulation by agents such as TNF-alpha, IL-1, endotoxin, gamma-radiation, and phorbol ester, it is activated by signal-induced specific phosphorylation, ubiquitination, and rapid degradation of IkappaB by proteosomes

This releases it from IkappaB and translocated to the nucleus where it plays a major role in the transcription activation of the kappaB site in promotor region of a large number of inducible target genes

Regulates several genes including:
IL-2
IL-6
TNF-alpha
TGF-beta
IFN-beta

With the exception of B lymphocytes, most cells show constitutive activity

The IkappaB kinase complex (IKK) contains two kinase subunits, IKKalpha and IKKbeta, necessary for IkappaB phosphorylation and NF-kappaB activation.

Zandi E, Rothwarf DM, Delhase M, Hayakawa M, Karin M.

Department of Pharmacology, University of California at San Diego, La Jolla 92093-0636, USA.

Cell 1997 Oct 17;91(2):243-52 Abstract quote

Recently we purified a 900 kDa cytokine-responsive IkappaB kinase complex (IKK) and molecularly cloned one of its subunits, IKKalpha, a serine kinase.

We now describe the molecular cloning and characterization of IKKbeta, a second subunit of the IKK complex. IKKbeta is 50% identical to IKKalpha and like it contains a kinase domain, a leucine zipper, and a helix-loop-helix. Although IKKalpha and IKKbeta can undergo homotypic interaction, they also interact with each other and the functional IKK complex contains both subunits. The catalytic activities of both IKKalpha and IKKbeta make essential contributions to IkappaB phosphorylation and NF-kappaB activation.

While the interactions between IKKalpha and IKKbeta may be mediated through their leucine zipper motifs, their helix-loop-helix motifs may be involved in interactions with essential regulatory subunits.

Raf induces NF-kappaB by membrane shuttle kinase MEKK1, a signaling pathway critical for transformation.

Baumann B, Weber CK, Troppmair J, Whiteside S, Israel A, Rapp UR, Wirth T.

Institut fur Medizinische Strahlenkunde und Zellforschung, Universitat Wurzburg Versbacher Strasse 5, 97078 Wurzburg, Germany.

Proc Natl Acad Sci U S A 2000 Apr 25;97(9):4615-20 Abstract quote

NF-kappaB is regulated by inhibitor proteins (IkappaBs), which retain NF-kappaB in the cytoplasm. Signal-induced phosphorylation by the IkappaB-kinase complex containing the IkappaB-kinases 1 and 2 (IKK-1/2 or IKK-alpha/beta) and subsequent degradation of the IkappaB proteins are prerequisites for NF-kappaB activation. Many signals induce NF-kappaB, one of them being oncogenic Raf kinase. We investigated whether NF-kappaB induction is critical for Raf-mediated transformation.

Here, we demonstrate that inhibition of NF-kappaB interferes with transformation by the Raf-oncogene, and we characterized the mechanism of NF-kappaB induction by activated Raf kinase and the tumor promoter phorbol 12-myristate 13-acetate (PMA). NF-kappaB activation by PMA and Raf critically depends on the IkappaB-kinase complex, most notably on IKK-2. A major signaling pathway induced by Raf is the mitogenic cytoplasmic kinase cascade. However, different inhibitors of this cascade do not affect PMA- and Raf-mediated NF-kappaB activation. Raf does not phosphorylate the IkappaB-kinase proteins directly. Raf rather synergizes with another membrane shuttle kinase MEKK1, and Raf-mediated activation of NF-kappaB is blocked by a dominant negative form of MEKK1.

These results suggest that Raf induction of NF-kappaB is relayed by MEKK1, but not by the classical mitogenic cytoplasmic kinase cascade.

NAK is an IkappaB kinase-activating kinase.

Tojima Y, Fujimoto A, Delhase M, Chen Y, Hatakeyama S, Nakayama K, Kaneko Y, Nimura Y, Motoyama N, Ikeda K, Karin M, Nakanishi M.

Department of Geriatric Research, National Institute for Longevity Sciences, Obu, Aichi, Japan.

Nature 2000 Apr 13;404(6779):778-8 Abstract quote

Phosphorylation of IkappaB by the IkappaB kinase (IKK) complex is a critical step leading to IkappaB degradation and activation of transcription factor NF-kappaB. The IKK complex contains two catalytic subunits, IKKalpha and IKKbeta, the latter being indispensable for NF-kappaB activation by pro-inflammatory cytokines. Although IKK is activated by phosphorylation of the IKKbeta activation loop, the physiological IKK kinases that mediate responses to extracellular stimuli remain obscure.

Here we describe an IKK-related kinase, named NAK (NF-kappaB-activating kinase), that can activate IKK through direct phosphorylation. NAK induces IkappaB degradation and NF-kappaB activity through IKKbeta. Endogenous NAK is activated by phorbol ester tumour promoters and growth factors, whereas catalytically inactive NAK specifically inhibits activation of NF-kappaB by protein kinase C-epsilon (PKCepsilon).

Thus, NAK is an IKK kinase that may mediate IKK and NF-kappaB activation in response to growth factors that stimulate PKCepsilon activity.

p16 (CDK1)
J Biol Chem 1999;274:23358

Cyclin dependent kinase inhibitor is significantly upregulated in cultured keratinocytes that have undergone irreversible growth arrest following onset of replicative senescence or after confluency-induced premature senescence

Senescent keratinocytes are resistant to apoptosis

May truncate or block the differentiation pathway

Conversely, inactivation is commonly present in pre-malignant and invasive SCCA of the skin, oral cavity, bladder cancers, and melanoma patients

p21 Inactivates cyclin-Cdk2 complexes that phosphorylate pRB
Arrests cells in G1 in response to p53 activation

p27
Cyclin-dependent kinase inhibitor downstream of p21 that ultimately leads to cell cycle arrest and serves as a tumor suppressor

Regulated posttranscriptionaly through proteasome-mediated degradation

p53 DNA damage
Upregulation with G1 block with DNA repair
Induction of apoptosis
Sequence specific binding activity
Regulates mdm2 and CKI p21

pRB Phosphorylation state changes with cell cycle
Target of enzymatic activity for cyclin-Cdk complexes
Late G1 and early S phase-highly phosphorylated until G2
Regulatory effect by complex formation with DNA binding proteins such as E2F
Phosphorylate RB release the complexed proteins which act as transcription factors

PTEN
Also known as MMAC1 or TEP1
Isolated on the 10q23-24 region

Homology with conserved sequences of protein tyrosine phosphatases and tensin cytoskeletal proteins

Mouse embryos with disruption of this gene have increased cell proliferation and overgrowth of cephalic and caudal regions

Specialized Oncogene Interactions

NAME CHARACTERIZATION

Interaction of viral proteins with p53 and pRB p53 binders and inactivators:
E6 from HPV
SV40 large T antigen
BZLF1 from EBV
E1B from adenovirus
HBX antigen from Hepatitis B virus

Interaction of viral proteins with p53 and pRB pRB binders and inactivators:
E7 from HPV
SV40 large T antigen
EBNA-3C from EBV
E1A from adenovirus
Cyclin D product from HHV8 increases phosphorylation

Additional Oncogenes and Tumor Suppressor Genes

Function CHARACTERIZATION

Immunohistochemical Detection of the Alternate INK4a-Encoded Tumor Suppressor Protein p14ARF in Archival Human Cancers and Cell Lines Using Commercial Antibodies: Correlation with p16INK4a Expression

Joseph Geradts, M.D., Robb E. Wilentz, M.D. and Helen Roberts, B.Sc.

Nuffield Department of Clinical Laboratory Sciences (JG, HR), University of Oxford, John Radcliffe Hospital, Oxford, UK; and Department of Pathology (REW), The Johns Hopkins University School of Medicine, Baltimore, Maryland

Mod Pathol 2001;14:1162-1168 Abstract quote

The INK4a locus encodes two structurally unrelated tumor suppressor proteins, p16INK4a and p14ARF. Although the former is one of the most common targets for inactivation in human neoplasia, the frequency of p14ARF abrogation is not established.

We have developed an immunohistochemical assay that allows the evaluation of p14ARF expression in formalin-fixed, paraffin-embedded tissues, using commercially available antibodies. p14ARF positive cells showed nuclear/nucleolar staining, which was absent in all cell lines and tumors with homozygous deletions of the INK4a gene. The assay was applied to 34 paraffin-embedded cell buttons, 30 non-small cell lung cancers and 28 pancreatic carcinomas, and the staining results were correlated with p16INK4a expression. Loss of p14ARF expression was common but less frequent than down-regulation of p16INK4a (53% versus 76% of all specimens). The p14ARF and p16INK4a expression pattern was concordant in 65 of 92 cases (71%). Significantly, 24 cases were p16INK4a-/p14ARF+, while the opposite staining pattern was observed in three cases, consistent with the notion that the two proteins have nonredundant functions.

The immunohistochemical assay described here may facilitate studies on the prevalence and significance of aberrant p14ARF expression in human tumors.

Am J Dermatopathol 1998;20:302-313
Henry JB. Clinical Diagnosis and Management by Laboratory Methods. Twentieth Edition. WB Saunders. 2001.
Rosai J. Ackerman's Surgical Pathology. Eight Edition. Mosby 1996.
Sternberg S. Diagnostic Surgical Pathology. Third Edition. Lipincott Williams and Wilkins 1999.
Fitzpatrick's Dermatology in General Medicine. 5th Edition. McGraw-Hill. 1999.
Robbins Pathologic Basis of Disease. Sixth Edition. WB Saunders 1999.

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Last Updated 4/1/2002

NAME	CHARACTERIZATION
bax	Cell death promotor Member of the bcl-2 multigene family
bcl-2	Protein product protects against cell death (apoptosis) Transcriptionally regulated by p53 and appears to function as a heterodimer of bcl-2-BAX to inhibit apoptosis
beta-Catenin	Multifunctional protein involved in cell-cell interaction and transcriptional signalling Largely regulated by its two major binding partners, E-cadherin present on the membrane and APC protein (Adenomatous polyposis coli) in the cytoplasm In normal colonic epithelium, it is bound to E-cadherin as part of a cell-cell adhesion complex on the lateral cell membrane Cytoplasmic beta-catenin levels are regulated by binding to a protein complex consisting of APC, glycogen synthase kinase 3beta, and axin followed by ubiquitin/proteasome degradation Thought to play critical role in sporadic colonic tumorigenesis
Cyclin D1	Regulates the cell cycle promoting progression from the G1 to S phase of the cell cycle
Fas (Apo-1/CD95)	Belongs to the nerve growth factor/tumor necrosis receptor superfamily Membrane protein that induces apoptosis when binding to its ligand
CD95 ligand (CD95L) immunohistochemistry: a critical study on 12 antibodies. Strater J, Walczak H, Hasel C, Melzner I, Leithauser F, Moller P. Institute of Pathology, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany.	Cell Death Differ 2001 Mar;8(3):273-278 Abstract quote In recent years, some studies on the expression of CD95(Fas/APO-1) ligand (CD95L) in tissues or cells raised concerns about the specificity of the antibodies used. We therefore tested 12 CD95L antibodies for their reliability in immunocyto/histochemistry by (i) staining CD95L-transfected and control CV-1/EBNA cells and (ii) comparing staining patterns in immunohistochemically labeled tissue sections with the localization of CD95L(+) cells in in situ hybridization. While G247-4, NOK-1, NOK-2, 4H9, and MIKE-1 stained CD95L-transfected cells and did not significantly bind to controls, G247-4 was the only antibody giving satisfying signals in tissue sections perfectly matching the distribution of CD95L(+) cells by in situ hybridization. MAb 33, C-20, and N-20 comparably stained both transfected and control cells and showed considerable background or falsely positive staining in sections. MIKE-2, 8B8, A11, and 4A5 did not or only very faintly bind to either cells and, thus, were not tested on sections. We conclude that G247-4 is the only tested antibody that is recommendable for immunohistochemistry.
MDM2	Negative regulator of p53 inhibiting transcriptional activity Has a pRB binding site
MMR	DNA mismatch repair system Family of genes with homologues to bacterial MutS and MutL proteins hMSH2 hMSH3 hMSH6 hMLH1 hPMS2 hPMS1 Germline mutations of hMSH2 and hMLH1 account for 50% of all cases of HNPCC colon cancer
NF-kappaB	Transcription factors found in the cytoplasm of most cells Includes: p65/Rel A Rel B c-Rel p50/p105 p52/p100 Latent form, it is sequestered in the cytoplasm, bound by the IkappaB family of inhibitory proteins After cellular stimulation by agents such as TNF-alpha, IL-1, endotoxin, gamma-radiation, and phorbol ester, it is activated by signal-induced specific phosphorylation, ubiquitination, and rapid degradation of IkappaB by proteosomes This releases it from IkappaB and translocated to the nucleus where it plays a major role in the transcription activation of the kappaB site in promotor region of a large number of inducible target genes Regulates several genes including: IL-2 IL-6 TNF-alpha TGF-beta IFN-beta With the exception of B lymphocytes, most cells show constitutive activity
The IkappaB kinase complex (IKK) contains two kinase subunits, IKKalpha and IKKbeta, necessary for IkappaB phosphorylation and NF-kappaB activation. Zandi E, Rothwarf DM, Delhase M, Hayakawa M, Karin M. Department of Pharmacology, University of California at San Diego, La Jolla 92093-0636, USA.	Cell 1997 Oct 17;91(2):243-52 Abstract quote Recently we purified a 900 kDa cytokine-responsive IkappaB kinase complex (IKK) and molecularly cloned one of its subunits, IKKalpha, a serine kinase. We now describe the molecular cloning and characterization of IKKbeta, a second subunit of the IKK complex. IKKbeta is 50% identical to IKKalpha and like it contains a kinase domain, a leucine zipper, and a helix-loop-helix. Although IKKalpha and IKKbeta can undergo homotypic interaction, they also interact with each other and the functional IKK complex contains both subunits. The catalytic activities of both IKKalpha and IKKbeta make essential contributions to IkappaB phosphorylation and NF-kappaB activation. While the interactions between IKKalpha and IKKbeta may be mediated through their leucine zipper motifs, their helix-loop-helix motifs may be involved in interactions with essential regulatory subunits.
Raf induces NF-kappaB by membrane shuttle kinase MEKK1, a signaling pathway critical for transformation. Baumann B, Weber CK, Troppmair J, Whiteside S, Israel A, Rapp UR, Wirth T. Institut fur Medizinische Strahlenkunde und Zellforschung, Universitat Wurzburg Versbacher Strasse 5, 97078 Wurzburg, Germany.	Proc Natl Acad Sci U S A 2000 Apr 25;97(9):4615-20 Abstract quote NF-kappaB is regulated by inhibitor proteins (IkappaBs), which retain NF-kappaB in the cytoplasm. Signal-induced phosphorylation by the IkappaB-kinase complex containing the IkappaB-kinases 1 and 2 (IKK-1/2 or IKK-alpha/beta) and subsequent degradation of the IkappaB proteins are prerequisites for NF-kappaB activation. Many signals induce NF-kappaB, one of them being oncogenic Raf kinase. We investigated whether NF-kappaB induction is critical for Raf-mediated transformation. Here, we demonstrate that inhibition of NF-kappaB interferes with transformation by the Raf-oncogene, and we characterized the mechanism of NF-kappaB induction by activated Raf kinase and the tumor promoter phorbol 12-myristate 13-acetate (PMA). NF-kappaB activation by PMA and Raf critically depends on the IkappaB-kinase complex, most notably on IKK-2. A major signaling pathway induced by Raf is the mitogenic cytoplasmic kinase cascade. However, different inhibitors of this cascade do not affect PMA- and Raf-mediated NF-kappaB activation. Raf does not phosphorylate the IkappaB-kinase proteins directly. Raf rather synergizes with another membrane shuttle kinase MEKK1, and Raf-mediated activation of NF-kappaB is blocked by a dominant negative form of MEKK1. These results suggest that Raf induction of NF-kappaB is relayed by MEKK1, but not by the classical mitogenic cytoplasmic kinase cascade.
NAK is an IkappaB kinase-activating kinase. Tojima Y, Fujimoto A, Delhase M, Chen Y, Hatakeyama S, Nakayama K, Kaneko Y, Nimura Y, Motoyama N, Ikeda K, Karin M, Nakanishi M. Department of Geriatric Research, National Institute for Longevity Sciences, Obu, Aichi, Japan.	Nature 2000 Apr 13;404(6779):778-8 Abstract quote Phosphorylation of IkappaB by the IkappaB kinase (IKK) complex is a critical step leading to IkappaB degradation and activation of transcription factor NF-kappaB. The IKK complex contains two catalytic subunits, IKKalpha and IKKbeta, the latter being indispensable for NF-kappaB activation by pro-inflammatory cytokines. Although IKK is activated by phosphorylation of the IKKbeta activation loop, the physiological IKK kinases that mediate responses to extracellular stimuli remain obscure. Here we describe an IKK-related kinase, named NAK (NF-kappaB-activating kinase), that can activate IKK through direct phosphorylation. NAK induces IkappaB degradation and NF-kappaB activity through IKKbeta. Endogenous NAK is activated by phorbol ester tumour promoters and growth factors, whereas catalytically inactive NAK specifically inhibits activation of NF-kappaB by protein kinase C-epsilon (PKCepsilon). Thus, NAK is an IKK kinase that may mediate IKK and NF-kappaB activation in response to growth factors that stimulate PKCepsilon activity.
p16 (CDK1)	J Biol Chem 1999;274:23358 Cyclin dependent kinase inhibitor is significantly upregulated in cultured keratinocytes that have undergone irreversible growth arrest following onset of replicative senescence or after confluency-induced premature senescence Senescent keratinocytes are resistant to apoptosis May truncate or block the differentiation pathway Conversely, inactivation is commonly present in pre-malignant and invasive SCCA of the skin, oral cavity, bladder cancers, and melanoma patients
p21	Inactivates cyclin-Cdk2 complexes that phosphorylate pRB Arrests cells in G1 in response to p53 activation
p27	Cyclin-dependent kinase inhibitor downstream of p21 that ultimately leads to cell cycle arrest and serves as a tumor suppressor Regulated posttranscriptionaly through proteasome-mediated degradation
p53	DNA damage Upregulation with G1 block with DNA repair Induction of apoptosis Sequence specific binding activity Regulates mdm2 and CKI p21
pRB	Phosphorylation state changes with cell cycle Target of enzymatic activity for cyclin-Cdk complexes Late G1 and early S phase-highly phosphorylated until G2 Regulatory effect by complex formation with DNA binding proteins such as E2F Phosphorylate RB release the complexed proteins which act as transcription factors
PTEN	Also known as MMAC1 or TEP1 Isolated on the 10q23-24 region Homology with conserved sequences of protein tyrosine phosphatases and tensin cytoskeletal proteins Mouse embryos with disruption of this gene have increased cell proliferation and overgrowth of cephalic and caudal regions

Function	CHARACTERIZATION
Tumor Suppressor Genes	Cytoplasmic transduction extracellular signals Nuclear transcription regulators Nuclear cell cycle regulators Nuclear DNA repair
Cytoplasmic transducers of extracellular signals	Neurofibromin Schwannomin APC gene product PATCHED T uberin PTEN
Nuclear transcription regulators	pRB p53 WT-1 BRCA-1 NF-kappaB
Nuclear cell cycle regulators	P16 and p15 Phosphatidyl inositol 3kinase
Nuclear DNA repair	Msh2/Mik1 protein products XP protein product

NAME	CHARACTERIZATION
Interaction of viral proteins with p53 and pRB	p53 binders and inactivators: E6 from HPV SV40 large T antigen BZLF1 from EBV E1B from adenovirus HBX antigen from Hepatitis B virus
Interaction of viral proteins with p53 and pRB	pRB binders and inactivators: E7 from HPV SV40 large T antigen EBNA-3C from EBV E1A from adenovirus Cyclin D product from HHV8 increases phosphorylation