BIMM 134: Discussion

[start wk.1] Cancer is the _____ leading cause of death in the US.

Cancer is the second leading cause of death in the US.

Note: All diseases show a statistically significant decrease in 2023 compared to 2022, except CANCER…

Lifetime risk of men/women getting cancer

1 in 3 (for men & women)

Top three incidences of cancer

• Men:

  • Prostate (30%)

  • Lung & Bronchus (11%)

  • Colon & Rectum (8%)

• Women:

  • Breast (32%)

  • Lung & Bronchus (12%)

  • Colon & Rectum (7%)

Top four cancer deaths

• Men:

  • Lung & Bronchus (20%)

  • Prostate (11%)

  • Colon & Rectum (9%)

  • Pancreas (8%)

• Women:

  • Lung & Bronchus (21%)

  • Breast (14%)

  • Pancreas (8%)

  • Colon & Rectum (8%)

Tobacco use

• Lung cancer death rate: 90%

• All cancer death rate: 1/3

• At diagnosis, ____% of cancers have already metastasized

• _____% of US Budget spent on cancer research (pre-Trump admin)

• At diagnosis, 70% of cancers have already metastasized

• ~0.1% of US Budget spent on cancer research (pre-Trump admin)

What is cancer?

• > 100 forms of the disease

• Patient-specific mutations (inter-patient)

• Genetically unstable

• Variation within (intra) tumor

• Variation between primary and metastasized (inter) tumors

• Etc.

Main processes/pathways responsible of cancer

• Cell division

• Cell death

• Cell differentiation

• Metabolism

• Multi-step process (4-7 mutations for malignant transformation)

  • But still requires at least one “renegade cell” to begin the development process

Classification - Naming, Grading, & Stage

Prefix generally based on originating tissue

adeno-

gland

chrondro-

cartilage

erythro-

red blood cell

hemangio

blood vessels

hepato

liver

lipo

fat

lympho

lymphocyte

melano

pigment cell

myelo

bone marrow

myo

muscle

osteo

bone

Carcinoma

Epithelial cells lining body cavity, glands, skin

Squamous cell carcinoma

Derived from protective layer of cells over other underlying cells (ex: skin. cervix)

Adenocarcinoma

derived from cells lining secretory cells (ex: mucous producing cells within lung, colon, prostate)

Sarcoma

Derived from connective tissues (ex: bone, fat)

Hematopoietic

• Blood producing cells

• Lymphoma: from B- and T-cells (solid mass in lymph tissue)

• Leukemia (circulating malignancy)

Grade: appearance of cells in biopsy

Low vs high

4 features to distinguish grade

• Mitotic rate - how many/fast cells divide

  • Higher mitotic rate = more aggressive cancer (worse grade)

• Nuclear grade - normal/abnormal nuclear shape

  • More abnormal nuclei = worse grade

• Cellular differentiation - state of cell specialization

  • Less differentiated (more abnormal, less specialized) = worse grade

• Surgical margins - proximity of tumor cells to surgical edge

  • Positive margins (cells at the edge) = higher chance cancer remains → worse prognosis

Tumor Stage

how far the cancer has spread

TNM Staging (I - IV)

• T - primary Tumor (T0-4, x)

• N - absence/presence in regional lymph Nodes (N0-3, x)

• M - absence/presence of distant Metastasis (M0-1, X)

OG Hallmarks of Cancer

1. Gain-of-function oncogenes

2. Loss of function tumor suppressor

3. Loss of apoptosis

4. Gain telomerase activity

5. Gain blood supply

6. Spread/Growth at distant sites

Proto-oncogene vs Oncogene vs Tumor Suppressor

• Proto-oncogene: promotes cell division under normal conditions

  • Ex: Ras and Myc

• Oncogene: a proto-oncogene after a gain of function mutation

  • Only requires single mutation to promote cancer development (dominant)

• Tumor suppressor: gene that normally prevents excessive cell division

  • Requires loss of function mutation in both alleles of gene to promote cancer development (recessive)

  • Ex: Rb and p53

Apoptosis: destruction of cell

• Chromatin condensation

• DNA fragmentation

• Cell shrinkage

• Cell protrusions “blebbing”

• Necrotic cell death

• Membrane disruption

• Scattered cell debris

Decrease of apoptosis in cancer cells

• Gain of anti-apoptotic proteins (ex. Bcl-2)

• Loss of pro-apoptotic proteins (ex. BAX or p53)

Telomeres

• repeat sequences at the ends of chromosomes

• Normally protect from DNA damage, especially from the typical shortening of chromosomes after each DNA replication

Telomerase

• protein that maintains telomere length in embryonic stem cells

• After differentiation, telomerase activity is silenced in cells, which helps prevent uncontrolled cell division.

  • Differentiation = cells becoming specialized for a specific job.

Gain of Telomerase Activity

Cancer cells lose differentiation state → regain telomerase activity → unlimited lengthening of telomeres → cell immortality

Angiogenesis

• Tumors are normally limited in growth to 1-2 mm

  • Require diffusion of oxygen/nutrients and removal of waste

• Angiogenesis: formation of new blood vessels

  • Normally, the process is activated for new cell growth such as healing

• Cancer cells initiate an angiogenic switch to promote cancer growth

  • Increases release of pro-angiogenic molecules (ex. VEGF)

  • Decreases release of anti-angiogenic molecules (ex. thrombospondin)

  • Allows invasion of cancer cells into the bloodstream → metastasis

Metastasis

• Spread of tumor to distant sites

• Up to 70% of patients with invasive cancers have metastasized

• Metastasis leads to 95% of cancer related deaths

• Example mutations:

  • Loss of E-cadherin

  • Gain of N-cadherin

Next Gen Hallmarks of Cancer

1. Reprogramming energy metabolism

2. Tumor-promoting inflammation

3. Avoiding immune destruction

4. Genome instability and mutation

1. Reprogramming energy metabolism - Warburg Effect

• Cancer cells switch from oxphos to glycolysis even in the presence of oxygen

  • Faster than oxphos

  • Produces other intermediate biomolecules necessary for cell growth (ex. amino acids, lipids, nucleic acids)

  • Intermediates used in anabolic processes

  • Lactic acid creates an acidic tumor environment

    • Promotes tumor growth

    • Suppresses immune system

    • etc.

1.b. Reprogramming energy metabolism - Reverse Warburg effect

• Reverse Warburg effect: cancer cells changing the behavior of surrounding normal cells

• Cancer cells release ROS and other factors, which switch healthy stromal cells to aerobic glycolysis

• Lactate produced by stromal cells is transported to cancer cells to use for energy and cell growth

• Heterogeneity of tumor: Warburg and Reverse Warburg effect

2. Tumor-promoting inflammation

• Inflammatory cells attracted to tumor

• Factors from cancer cells induces release of carcinogen and promoters from inflammatory cells into the tumor microenvironment

3. Avoiding immune destruction

• Disable immune response

• Cancer cells evade immune response

4. Genome instability and mutation

• Loss of DNA damage repair, detection and resistance mechanisms

Cell Signaling - Tyrosine Kinase Receptor

• Ligand binding to surface receptor activates the tyrosine kinase → phosphorylates tyrosine residue on proteins → activating phosphorylation cascade

• Various growth factors regulated by TKR

  • Important for cell growth and division regulation

• Kinases/phosphatases in signal transduction pathways are mutated in cancer cells

Normal TKR Mechanism

1. 1st messenger (EGF) binds and activates TKR (EGF receptor)

2. TKR adds phosphates to various intracellular signal proteins (S) to activate them

3. This activates proteins in nuclear

   1. Transcription factors → turning genes on/off for protein production

4. Proteins created for cell division

5. Phosphatases remove phosphates from signal proteins (S) → inactivate TKR (EGF receptor)

6. Cancer cells: proteins for cell division made w/o including growth signals

Types of Kinases

• Tyrosine (Y) Kinase

  • adds phos to tyrosine residues

• Serine(S)/Threonine(T) Kinase

  • adds phos to serine and/or threonine residues

• Dual Kinase

  • has activity of both tyrosine and serine/threonine kinases

• Lipid Kinase

  • adds phos to lipids

Tyrosine Kinase Receptors (TKR)

• Many types of TKRs - share various protein domains between one another

• Some ligands can activate various receptors

• Some receptors can be activated by various ligands

• The different receptor pathways can have some downstream pathway overlap

  • Ex: EGFR, HER2, etc.

EGF-RAS-MAPK Pathway (1 to 3)

1. EGF ligand binds to EGFR

2. Binding causes conformational change to enable dimerization

3. Dimerization initiates autophosphorylation of tyrosine residues on EGFR

   1. EGFR is a tyrosine kinase

EGF-RAS-MAPK Pathway (4 to 5)

4. Autophosphorylation recruits GRB2 signal molecule to EGFR (interacting with SH2 domain of the signal molecule)

• SH2: binding domain; key for messenger binding to TKS

• SH2 Domain- allows activated receptors and GRB2 to bind

5. SH3 domain of GRB2 binds to SOS (intracellular messengers)

• proteins bind to one another through SH3 domain

• SH3: binding domain; key for messenger binding to TKS

EGF-RAS-MAPK Pathway (6)

6. SOS recruitment activates RAS

• SOS gets rid of GDP and allows GTP to bind so that RAS becomes active

• SOS is a GEF protein

  • GEF proteins transfer GTP to other proteins

EGF-RAS-MAPK Pathway (7)

7. Farnesyl transferase adds farnesyl lipid and methylation to RAS-GTP and anchors it to the membrane = fully activating RAS

EGF-RAS-MAPK Pathway (8 to 10)

8. RAS activation recruits RAF

• RAF = ser/thr kinase

9. Active RAF phosphorylates MEK

• MEK = dual kinase

  • Dual kinases have tyrosine and serine/threonine kinase activity

10. Active MEK phosphorylates MAPK/ERK to activate it

• MAPK = ser/thr kinases

EGF-RAS-MAPK Pathway (11 to 13)

11. Activated MAPK/ERK is translocated to the nucleus

12. Once in the nucleus, MAPK/ERK activates various transcription factors — promoting cell cycle and division.

• MAPK/ERK is Ser/Thr kinase

• Ex: AP-1, Fos/Jun,

  • or Myc — transcription factor that helps change gene expression/ increases proliferation.

13. Active transcription factors will initiate transcription of various cell growth genes (ex: cyclins)

RAS-PI3K-AKT

1. Active RAS can also activate PI3K via direct binding

   1. PI3K is lipid kinase

1. Active PI3K will phosphorylate PIP2, turning it into PIP3

2. PIP3 binds to both PDK1 and AKT

   1. PDK1 and AKT is Ser/Thr kinase

3. PDK1 activates AKT via phosphorylation

4. Activated AKT activates mTOR

   1. mTOR is a Ser/Thr kinase

mTOR

• Increased survival

• Increased proliferation

• Increased motility

• Angiogenesis

• Decreased apoptosis

RAS-PI3K-AKT-mTOR

• Mutations in PI3K pathway are most common in cancers

• Loss of PTEN (phosphatase), a common tumor suppressor mutation

  • Converts PIP3 back to PIP2 → inhibiting the pathway

Oncogenic mutations

1. Point mutations

   1. abnormal protein

2. Gene amplification

   1. excess normal protein

3. Chromosomal translocation

   1. When a piece of one chromosome breaks off and attaches to a different chromosome.

4. Local DNA rearrangements

5. Insertional mutagenesis

Oncogenic Mutations: Tyrosine Kinase Receptors

• Point mutation/deletion

  • Ligand-independent dimerization and activation

• Gene amplification

  • Overexpression of receptors

  • Ligand-independent dimerization and activation

• Inducing conversion of paracrine to autocrine signals

  • Normally differentiated cells transition to paracrine signaling

  • Autocrine signals will activate TKRs → promoting continued growth

• Ex: EGFR (HER1), HER2

Point Mutation - RAS

• Single DNA base mutation

  • → change 1 amino acid

    • → abnormal protein

• Normally, RAS is deactivated (GAP) after the signal pathway has been activated

• Point mutation prevents its interaction with GAP

  • → prevents hydrolysis of GTP

    • → Ras stays activated

“Point mutations are prevalent in all three RAS isoforms (H-Ras, N-Ras, K-Ras)”

• H-Ras (hematopoietic)

• K-Ras most commonly mutated

  • (pancreatic, lung, and colon cancer)

• N-Ras

  • (Neuroblastoma, Melanoma)

• Isoforms have strong homology at the GAP binding site

Point Mutation - RAS

• Most common mutations in codon 12, 13, 61

• Glycine (G) to aspartic acid (D)/valine(V)/cysteine(C)

  • Adding bulky side groups that interfere with GAP-activation at the catalytic site on RAS

• G12C - lung adenocarcinoma

  • Tobacco use

• G12D - pancreatic and colorectal cancer

RAS mutation therapeutics

• Originally considered undruggable bc

  • The GTP binding pocket is relatively inaccessible

  • High affinity for GTP

  • High levels of cytoplasmic GTP

Two main types of RAS mutation therapeutics

• Off inhibitors: bind to inactive mutant

• On inhibitors: bind to active mutant

RAS mutation therapeutics

1. Inhibitors target G12C mutation: Sotorasib & Adagrasib (RAS off)

2. Inhibitors target G12D mutation: RMC-6236 (RAS on)

3. Farnesyltransferase inhibitors

4. RAS mimic: Rigosertib

Inhibitors target G12C mutation: Sotorasib & Adagrasib (RAS off)

• covalent binding to cysteine in the GTP binding pocket preventing/decreasing Ras activation

• Don’t affect WT RAS or other RAS mutations

• Eventual resistance to these drugs (inc RAS activation via other pathways and mutations)

Inhibitors target G12D mutation: RMC-6236 (RAS on)

• Non-covalent binding to any G12 mutation

• Potentially treat various RAS mutant driven cancers

• Could avoid drug resistance seen with other RAS off therapeutics

Farnesyltransferase inhibitors

• Prevents RAS localization to membrane

• Not successful as stand alone therapeutic

RAS mimic: Rigosertib

• Binds to normal RAS binding partners and inhibits their activity

• RAF to inhibit downstream MAPK/ERK pathway

• Also binds PI3K inhibiting downstream PI3K/AKT/mTOR pathway

Gene Amplification - Myc

• Results in many copies of the gene (100-1000)

• Excess protein is present in cells (protein itself is normal)

• Amplified Myc is seen as extrachromosomal elements or intrachromosomal areas

• Myc: transcription factor that form hetero dimer with the related TF Max

  • Binds 100s of gene promoters (esp those involved in cell cycle progression like cyclin D; CDK4/6)

• Overexpression can activate Myc

• Normal Myc expression is initiated when there is cellular stress, which subsequently activates Arf and p53 to pause cell cycle and induce apoptosis

  • For a full oncogenic effect, loss of p53 is also needed

Myc Therapeutic - OMOMYC

• Small molecule inhibitor

• Prevents Myc and Max from binding to DNA

  • → blocks Myc turning proliferation genes on (ex: cyclin D; CDK4/6)

Chromosomal Translocation

• A piece of a chromosome moves to another chromosome

• Results in loss of normal protein control

  • Protein is made constantly instead of being initiated by a specific signal cascade

Chromosomal Translocation - BCR-ABL

• ABL gene is normally part of chromosome 9

• Translocation occurs on chromosome 22 downstream of the BCR gene

• Resulting in the Philadelphia chromosome

  • → BCR-ABL fusion protein

  • 95% of chronic myelogenous leukemia cases have the Philadelphia chromosome

“The fusion protein contains the dimerization domain of BCR.”

• Fusion of ABL to this dimerization domain promotes dimerization of ABL

  • This dimerization leaves ABL permanently activated

    • In normal conditions, ABL dimerization is controlled via a signal pathway

• The tyrosine kinase domain of the fusion ABL will activate other growth pathways

  • RAS-MAPK and PIP3-AKT

• Mutant protein is not transported to the nucleus (cytosolic)

  • → increased activation of Ras/MAPK and PI3K pathways, and decreased normal apoptosis

BCR-ABL therapeutic: Gleevec (imatinib)

• Gleevec targets the hybrid protein specifically, unlike other chemo and radiation therapies

• Works by outcompeting ATP at the ATP binding site on the fusion protein

  • Prevents BCR-ABL from activating other proteins in signaling pathways

Anti-EGF receptor monoclonal antibodies

• The antibody directly interacts with the receptor, blocking ligands from activating

• Herceptin - Metastatic breast cancer with upregulated HER2

  • Binds and inactivates HER2

• Erbitux - colorectal cancer with upregulation of EGFR (HER1)

  • These will be ineffective in patients with downstream oncogenic mutations (ex: RAS)

Intracellular tyrosine kinase inhibitors

• Compete with ATP-binding site of kinase

• TARCEVA and IRESSA (Gilotrif)

• Developed to target common chemoresistance mutations in patients

mTOR inhibitor

Everolimus - inhibits downstream targets of mTOR

Inhibition of PI3K or tyrosine kinase pathways results in ________ of the other to increase cell survival and reduce ______

Inhibition of PI3K or tyrosine kinase pathways results in compensatory upregulation of the other to increase cell survival and reduce apoptosis

Breast cancer therapeutics

1. Tamoxifen

2. Herceptin

3. IBRANCE

4. LETROZOLE

Tamoxifen

• Small molecule that inhibits estrogen/progesterone from binding to its receptor

• Targets ER+ PR+ breast cancer

• Blocking this slows growth of cancer cells

Herceptin

• Antibody that binds and inactivates HER2

• Used in patients who are HER2+++ (overexpression of HER2)

IBRANCE

• Small molecule intracellular inhibitor of CDK proteins (control cell cycle/cell division)

• Used in combination with anti-estrogen hormonal therapy

LETROZOLE

• Hormone therapy - aromatase inhibitor

• Increased survival compared to hormone therapy alone

• Often used in combo with IBRANCE

• Used to treat ER+ PR+ breast cancer

Three main mechanisms of Herceptin Resistance + Therapeutic Strats

1. Amplification or mutation of EGFR

   1. Combine Herceptin or other EGFR Ab with a TK inhibitor like Iressa or Tarceva

2. Increase in alternative RAS activation

   1. Combine Herceptin with RAS inhibitor (Rigosertib, FTI, Sotorasib, Adagrasib, etc)

3. Upregulation of PI3K/AKT/mTOR pathway

   1. Combine Herceptin with PI3K/AKT/mTOR inhibitor (Everolimus)

RAS inhibitors

• Sotorasib

• Adagrasib

• RMC-6236

• Farnesyltransferase inhibitor (FTI)

• Rigosertib

Myc inhibitor

OMOMYC

BCR-ABL inhibitor

Gleevec (imatinib)

Other kinase inhibitors

• Erbitux (EGFR Antibody)

• TARCEVA (intracellular TKR)

• IRESSA (intracellular EGFR)

• Everolimus (mTOR)

• Vemurafenib (RAF)

Breast Cancer inhibitors

• Tamoxifen

• Herceptin

• IBRANCE

• LETROZOLE