Lecture #13.2 | Oncogenes as Transcription Factors c-Myc and NFkB Ubiquitin and Proteasome Chronic Inflammation

c-myc

An Early Response Gene; does not need transcription to be activated

• Activated by growth factors in serum.

• Activity peaks at approximately 1-2 hours after serum addition.

• c-Fos and c-Jun are also early response genes.

• HOWEVER: slower, so not an immediate response gene

• C-Myc is not detectable in quiescent fibroblasts (non-proliferating cells)

Characteristics of c-Myc

• 439 amino acids.

• An early response gene.

• A basic Helix-Loop-Helix (bHLH) protein.

• Belongs to a family of TFs

  • Complex network of homo- and heterodimers

  • Binds DNA as a heterodimer with Max

• Activates critical genes required for cell proliferation.

• Activated by gene amplification, not mutation.

• Synergistically induces tumors with Ras.

• Plays a role in apoptosis and differentiation.

Myc/Max: basic Helix-Loop-Helix

B-basic region that interacts with DNA

• helix makes interactions more flexible

Functional domains of c-Myc and Max

1. Basic

2. HLH

3. Leu Zipper

   1. Also NLS motif: nuclear localization sequence

   2. helps localize to the nucleus as they cause the transcription themselves

4. Myc is longer due to the TAD: transactivation domain binds co-activators (only one of family that have)

Heterodimerization with Max

members of Myc/max family can all heterodimerize with Myc

• Myc:Max heterodimers activate genes important for cell proliferation (e.g., cyclins, CDKs).

• Max:Mxi, Max:Mad, and Max:Max all bind CACGTG elements but repress txn (only Myc has the TAD).

How can Myc:Max activate transcription and compete with Mad-Max

• via recruitment of HATs and HDACs.

• Myc can recruit HATs: Histone Acetyltransferases (cause acetylation of chromatin, loosening up DNA to make it more available for transcription)

• MAD can recruit HDACs: Histone Deacetylases (cause deacetylation becoming more tightly bound)

• Myc-Max and Mad-Max compete for regulation of target genes (cyclins, CDK) via binding E boxes.

Importance of Myc in forming tumors

• Wild-type mouse embryonic stem (ES) cells form large tumors that become vascularized.

• ES cells lacking c-Myc do not, showing c-Myc's essential role in tumor cell proliferation.

  • Lacking → much smaller tumors

How is c-Myc being activated into an oncogenes?

Gene amplification, chromosomal rearrangement, and amplification if the gene itself

• myc is present on chromosome 8

• Myc gets placed on chromosome for Immunoglobin H promoter, which is very active, resulting in Burkitt’s lymphoma.

  • Reciprocal translocation: the exchange of material between two chromosomes

  • Juxtaposition of the strong IgH promoter next to the c-Myc gene results in overexpression.

  • Insertion of a strong viral promoter next to c-Myc also leads to overexpression.

chromosomal rearrangement to make Myc oncogenic

• myc is present on chromosome 8

• Myc gets placed on chromosome for Immunoglobin H promoter, which is very active, resulting in Burkitt’s lymphoma.

  • Reciprocal translocation: the exchange of material between two chromosomes

  • Juxtaposition of the strong IgH promoter next to the c-Myc gene results in overexpression.

  • Insertion of a strong viral promoter next to c-Myc also leads to overexpression.

Activation of Myc through amplification of the gene itself

Amplifies the amount of DNA not just RNA and protein

• through double minutes and homogeneous staining region

• both lead to increased transcription of mic

How are normal c-Myc protein levels maintained

Via ubiquitination (Ub = 76 aa polypeptide) of lysine residues that act as markers for the 26S proteasome pathway.

• Three enzymes involved in ubiquitination:

  • E1: Ub activating enzyme

  • E2: Ub conjugating enzyme

  • E3: Ub ligase

    

• the n-terminal domains

Macrophages

• Phagocytic cells of the immune system.

• They move, phagocytose bacteria, and engulf tumor cells.

• They release inflammatory mediators:

  • Reactive oxygen species (ROS) and nitrogen oxide radicals

    • Act as initiators

    • Produced by enzymes COX2 and iNOS

  • Other enzymes:

    • MMPs: matrix metalloproteases, degrade ECM

  • Cytokines and chemokines:

    • TNFalpha

    • IL1

    • IL6

    • IL8

    • MCP1

• Cancer creates increase of inflammatory mediators

• Cytokines and chemokines trigger a signaling pathway that activates the txn factor NFkB

NFkB

• A transcription factor (TF) and member of the Rel family.

  • v-Rel is a viral oncogene.

• NFkB is located in the cytoplasm in an inactivated form

  • Bound to an inhibitor, IkB.

• IkB is phosphorylated by IkB kinase (IKK) in response to cytokines.

  • Releases NFkB which can then translocate to the nucleus.

  • Activates the transcription of its target genes.

• Plays a role in inflammation and innate immune response.

  • Also cell proliferation, apoptosis, and cell migration.

• Is activated by cytokines in response to viral infections

  • Plays an important role in HIV.

• Overexpressed in many tumors

  • e.g., mammary, gastric, and colon tumors.

Is the link between inflammation and cancer

NFkB Pathway

When IkB is phosphorylated, it disengages from NF-kB which is able to enter the cell and activate

• can be used as a detection for early cancer development

What are the NF-kB target genes

• Four classes of genes:

  • Autoregulation (IkB)

  • Inflammation (cytokines): Positive feedback loop

  • Anti-apoptosis

  • Proliferation (c-Myc & cyclin D1)

Role of NFkB in Gastric and Colon Cancer

• H. pylori infection (ulcers), a risk factor for gastric cancer, activates the NFkB pathway.

• NFkB turns on genes like COX2 and iNOS which generate ROS and RNS and promote inflammation and cause DNA damage.

• It also turns on genes such as growth factors and cytokines that stimulate cell proliferation.

• DNA damage and proliferation -- a lethal combination!!