BIO 3315 - Module 2

Cancer is a step-wise process

Cancer develops when genetic mutations alter DNA instructions that control how cells grow, divide, and die

• Progression of mutations

Recepie Analogy

• DNA is the cookbook

• RNA is a recepie re-written down

• The protein is the baked cookies

Overview of cancer formation

There are many different causes of cancer - radiation, chemicals, infectious agents, heredity

• There create mutations in DNA

Hallmarks of Cancer

1. Self-sufficiency in growth signals

2. Insensitivity to antigrowth signals

3. Evasion of apoptosis

4. Limitness replicative potential

5. Sustained angiogenesis

6. Tissue invasion and metastasis

Which cells acquire mutations matters

Somatic DNA changes

• Acquired over a persons lifetime in single cells

• Can lead to cancer

• Can NOT be inherited

• Somatic = body cells

  • Most cancer-causing mutations arise during a person’s life in a body cell and are not inherited

Germline DNA changes

• Present in every cell of the body including egg and sperm

• Can increase cancer susceptibility

• Can be inherited

• Germline = reproductive cells

  • Inherited variants can raise risk, but additional changes are usually needed for cancer to develop

Quick Genetics Overview

• Almost every nucleated cell in your body has the entire genome

  • Genome = all the DNA required to be that individual

DNA mutation = a change in sequence

Normal DNA Sequence

• TCGA

Point Mutation

• TGGA

Deletion

• T--GA

Insertion

• TCAGA

Effects of Mutations:

• NO effect

• Loss-of-function (LOF)

• Gain-of-function (GOF)

DNA mutation → not every mutation matters

Cells acquire random spontaneous mutations all the time

• Passenger mutation = most of the time, no clear effect on cancer growth

• Driver mutation = if starts to promotw hallmarks of cancer (give a cell a growth or survival advantage)

Gene Structure and Expression

• Gene = DNA sequence for encoding a function (usually a protein)

• Gene expression = turning the encoded information in DNA into functional protein

• Promoter = how much to make

• RNA-coding region = what to make

Consequence of Mutation: Silent

Silent mutation

• Changing a single base pair in DNA has no change in the amino acid in protein

• Ex: Instead of two cups of flour, too cups

Consequence of Mutation: Missense

Missense mutation

• Changing a single base pair in DNA changes a single amino acid in protein

• Ex: Instead of two cups of flour, two cups of sour

Consequence of Mutation: Nonsense

Nonsense mutation

• Creates an early stop signal and a shortened protein

• Ex: Instead of two cups of flour, two cups of

Consequence of Mutation: Frameshift

Frameshift mutation

• Insertion or deletaion of a nucleotide of a nucleotide causes the rest of the sequence to be translated incorrectly, likely destroying function

Ex: Instead of two cupe of flour, twi ocu psof

Consequences of changing a recipe

Instead of normal

• Make worse or better, too many or too few

Table

What kinds of “recipes” (genes) are usually mutated in cancer

Homeostasis

• Cells need to regulated their growth and death and have genes for those proteins

Proto-oncagenes

Controls growth

• Directs proteins that tell cells when to grow and divide

Acts as an accelerator

• Works like the gas pedal in a car to promote necessary cell multiplication during development and healing

If mutated by cancer = oncogene

• Overstimulated growth, uncontrolled proliferation, gas pedal is stuck down

• Gain-of-function (GOF)

Tumor Suppressors

Gatekeeper genes

• These direclty stop the cell cycle or tell abnormal cells to self-destruct through programmed cell death (apoptosis)

Caretaker genes

• These fix damaged DNA and keep the genome stable so future mutations do not pile up

If mutated in cancer

• Reduced DNA repair causing increased mutations, damaged cells continue to live and divide

• Loss-of-function (LOF)

Cells must aquire new proteries to become cancerous

Different routes all lead to cancer

• Evading apoptosis

• Self-sufficiency in growth signals

• Insensitivity to anti-growth signals

• Sustained angiogenesis

• Limitless replicative potential

• Tissue invation and metastasis

Review Question 1

CDC73 gene encodes a protein celled parafibromin that functions to inhibit cell growth and division - 70% of parathyroid cancers have a mutation in the CDC73 gene - in some cases the mutation changes a single base pair of DNA but produces a shorter parafibromin protein

• Is the CDC73 an ocogene or tumor supressor gene:

  • Tumor suppressor gene

• The mutation in CDC73 is a:

  • Nonsense mutation

• The mutation in CDC73 is most likely to be a:

  • Loss-of-function

Review Question 2

The CDK4 gene encodes a protein that promotes cell growth and division - some cancers have a single base pair mutation in the promoter of the CDK4 gene

• This type of mutation is a:

  • Point mutation

• This mutation most likely:

  • Increases the activity of the protein

Conclusions

• Mutations can activate oncogenes or inactivate tumor suppressors to start acquisition of hallmarks

  • Multiple hallmarks must be obtained - in many different orders

• Mutations in promoters = too much or too little protein made

• Mutation in coding region = disrupts function of protein, sometimes reducing or eliminating it, and sometimes making it overactive

Hallmarks of cancer - Self-sufficiency in growth signals

Cells must have the ability to make decisions and act on those decision based on internal and external signals

• To make a decision, cells rely on signals

Growth factors signals activate receptors to promote cell division and growth

Different cells express different receptors to be responsive to different signals

Transmitting information in cells

Extracellular signaling molecule attaches to receptor protein

• Intracellular signaling proteins are triggered

• Signals given to target proteins which are the last effector proteins

• Target protein stimulate cellular or physiological responses

Transmitting information in cells - Epidermal growth factor (EGF)

EGF attaches to EGF receptor on cell

• Signal is transferred through signal transduction proteins

• Signal goes into nucleus and promotes cell division

  • Produced in the kidneys, circulates to signal growth in epithelial tissues

  • This is a proto-oncagene

Transmitting information in cells - Platelet derived growth factor

PDGF attaches to PDGF receptor

• Signal is transferred through signal transduction proteins

• Signal goes into nucleus and promotes cell division

  • Produced in platelets, ciruclates to signal growth in connective tissues, mucles, and blood vessels

  • Is PDGF more likely to promote growth of a carcinoma or a sarcoma?

    • Sarcoma - connective tissue

Growth Factor signaling activates Ras-MAPK pathway

• GF attached to receptor

• Signal from receptor causes Ras to grab high energy GTP and flips to its on state

• Active Ras grabs Raf and brings Raf to membrane

• Active Raf → transcription of genes → cell division

How do cancer cells become self-sufficient for growth signals?

Table

Self-sufficiency mechanisms

Autocrine secretion

• Cell starts making its own signal

Overexpress or mutate receptor

• Cell receptor is always activated, regardless of signal

RAS genes are among the most commonly mutated oncogenes in cancer

Are these mutations GOF or LOF

• GOF

RAS can be activated by a single base pair mutation

Mutation prevents turning off of RAS

• Driver mutations

Case Report

Mutation in EGFR - Nucleotide changed from T to G, replacing amino acid leucine with arginine

• Missense mutation

• Gain of function

EGFR is a cell-surface receptor that can activate Ras-MAPK pathway and promote cell division

• Signaling state of EGFR = always on

• EGFR inhibitor is reasonable treatment because it will stop the adaptive proteins from docking and giving Ras the signal to switch from GDP to GTP

Inhibitor seems to control cancer

• Initial shrinkage of tumor suggests the mutant EDGR was a driver mutation

• Drug acts between EGFR and Ras

  • EGF → EGFR x Ras → MAPK pathway → cell division

When cough returns, tests show a KRAS mutation that prevents Ras from efficiently turning off

• KRAS mutation most consistent with gain-of-function

  • Amplifies normal function

• Ras is likely constantly on

• Ras won’t need a signal from EGFR to stay on

• Possible that a small KRAS-mutant subpopulation existed before treatment and expanded after EGFR-sensitive cells were eliminated

• also possible a new mutation hit the same pathway

Conclusion

Cells typically are very reliant on signals and receptors to regulate cell proliferation decisions

• Mutations that increase the activity of signal, receptor, or downstream pathway can lead to unregulated proliferation, a critical hallmark of cancer

• This will cause cells to divide too quickly - but not cancer yet

Hallmark of cancer - Immortality

Three main mechanisms of cellular mortality:

Brake 1: Don’t divide when you shouldn’t

• RB/growth-suppressive signaling

Brake 2: Self-destruct when badly damaged

• p53/apoptosis

Brake 3: Don’t divide forever

• telomeres/telomerase

Restriction Point

Critical decision-making stage located within the G1 phase of the cell cycle

• Once a cell passes through, it is fully committed to completing the cell cycle and no longer requires external growth factors to proceed to S phase (DNA synthesis)

• Before this point, cells depend on external signals (growth factors) to grow and adhance

• After passing this point, the machinery runs autonomously

  • Usually a cell exits the cell cycle if conditions are poor or growth factors are missing before the restriction point

• Final output of cell division is

E2F is a transcripton factor for S phase

• E2F already present inside the cell, but is held hostage and inactivated by the Rb protein

• E2F activates genes required for DNA replication and cell division

• E2F gene expression = transcription

IGNORE

How Growth Factor (GF) controls transition of cell from a resting state to preparing for cell division during the G1 phase

E2F is a transcription factor - tells RNA polymerase to transcribe genes from DNA that are translated into proteins and enzymes required for S phase and DNA replication

GF → Ras-MAPK pathway → make sure to go through slides

Main Points

• E2F is a transcription factor for S phase (master regulator)

• Cyclin & Cdk work together

• Cdk activity depends on the amount of cyclin

• E2F is inhibited by Rb protein

• Rb protein controls the restriction point

• Growth factor signaling turns off restriction point

• Active Rb protein prevents passage through restriction point

• Active E2F turns on gene expression of S phase protins, promotes division

Normal cells respect anti-growth signals

• Density-dependent inhibition of growth

• Anchorage-dependent inhibition of growth

Anti-growth signals use the restriction point

Transforming growth factor → TGF receptor → chain reaction → Cdk inhibitors → Cdk-cyclin can’t phosphorylate Rb protein → cell cycle arrest (E2F stuck to Rb)

• Rb is tumor suppressor

Types of mutations in Rb gene

All cause loss-of-function

• Nonsense

• Frameshift

• Missense

• Chromosomal deletion

Whole genes are lost in chromosomal deletion

Great way to cause LOF mutations

Evasion of Apoptosis (Programmed cell death)

Used during development

• Used during normal processes

• Removes infected cells

• Removes cells in DNA damage

Apoptosis dismantles a cell

• Cell shrinkage

• DNA digested into small pieces

• Mitochondrial pores

• Blebs - eaten by immune cell

External signals regulate apoptosis

Death signal → death receptor → etc.

Internal signals - DNA damage checkpoints in cell cycle

p53 - guardian of the genome

• Recognizes DNA damage

• Pauses cell cycle via blocking Cdk/Cyclin or induces apoptosis by making Bax

• Mitochondria = hub for regulating apoptosis - Bax causes the mitochondria to leak and turn on death pathways

• p53 causes Bcl2 to release Bax

• Increased expression mutation of Bcl2 would prevent apoptosis

Chromosome translocation mutation can increase expression

Driver mutation:

• Movement of entire Blc2 in front of a promoter or enhancer sequence

Henrietta Lacks

• cervical cancer biopsied in 1951

• able to discover telomeres

Clock for # cell divisions = telomere length

• Telomeres: ends of chromosomes

• Telomeric DNA: short sequence repeated ~2500x

Telomeres shorten every cell division, biological “clock” for cell age

• telomere shortening - tells cell to stop dividing

In stem cells and germ cells - more than 30 cell cycles

Cancer cells turn in expression of telomerase

Point mutation in promoter → increased transcription of telomerase

A cell has - lost functional RB, acquired a p53 mutation and activated telomerase

Lost functional RB

• Solves problem of cell cycle arrest at the G1/S checkpoint

• RB normally acts as a brake on cell proliferation by holding back E2F transcription factors

• Losing RB allows the cell to constantly skip past this checkpoint and divide without needing growth signals

Acquired a p53 mutation

• Solves problem of apoptosis (programmed cell death) and DNA repair arrest

• When a cell undergoes stress or DNA damage, normal p53 stops division to repair the damage or forces the cell to self-destruct

• A mutated p53 allows the cell to survive and continue dividing despite massive genetic damage

Activated telomerase

• Solves the problem of normal cells only dividing a limited number of times before their chromosome tips (telomeres) become too short, triggering cell death

• Teloerase elongates these tips, giving the cell immortality so it can divide indefinitely

Why having all three is more dangerous than just one

• Having only one can trigger safety backups

• When all three are present - cell creates a perfect storm for cancer

  • Permanently ordered to divide (no RB)

  • Cannot be forec to commit suicide despite being damaged (no p53)

  • Can replicate forever without running out of telomere length

Summary of hallmarks of cancer

So far, cells can keep dividing and never die - but not yet cancer

• A primary tumor must solve two problems to become life-threatening:

  • Gain a blood supply

  • Successfully spread and grow elsewhere

Formation of blood vessels

Vasculogenesis = formation of blood vesels from undifferentiated cells

Angiogenesis = formation of blood vessels from pre-existing vessels

Angiogenesis rarely used in adults

• Default state of angiogenesis in adulta is off

• Two exceptions:

  • Wound repair

  • Uterus (menstrual cycle and pregnancy)

Angiogenesis is regulated by balance of activators and inhibitors

Activators

• Vascular endothelial growth factor (VEGF)

Inhibitors

• Thrombospondin-1 (TSP-1)

VEGF stimulates blood vessel formation

VEGF = Vascular Endothelial Growth Factor

• Binds to receptor →

• Signal transuction proteins →

• Genes activated in nucleus →

• Cell proliferation, etc.

How does VEGF get activated?

Hypoxia

• Hypoxic conditions stimulate expression of VEGF

• Activator = HIF (Hypoxia inductible factor)

• If HIF met with oxygen, it is destroyed

• If HIF not met with oxygen, goes inside cell and binds to transcription sites to make VEGF

Cancer cells stimulate angiogenesis

Tumor cells exploit nearby normal cells to promote tumor growth

• Secrete VEGF

Role of Angiogenesis in Cancer

Angiogenesis helps tumors grow

• It can give tumor cells access to blood vessels which can allow access to metastasize

Steps of Metastasis

Step 1 - Invasion

• Detach from neighbor cells and ECM

• Invade basal lamina

Step 2 - Travel to a vessel

• Degrade ECM

• Move through tissue

• Move through vessel lining

Step 3 - Transport through vessel lining

Step 4 - Colonization

• Leave vessel

• Move through tissue

• Grow in new location

Epithelial cells versus mesenchymal cells

Epithelial cells

• Organized and anchored to each other

Mesenchymal cells

• Disorganized and not connected

Epithelial to mesenchymal transition (EMT)

Normal EMT during early embryogenesis

• Normal EMT during wound healing

Inappropirate EMT enables invation and metastasis

Adhesion to neighbors - parts of epithelial cell identity

E-cadherin = glue that holds epithelial cells together

• An invasive cell will have LESS E-cadherin than normal cells

Extracellular Matric (ECM)

Complex network of marcomolecules outside cells that provides structural and biochemical support to surrounding tissues

Cancer cells express proteases - Plasim and MMPs

Plasminogen

• Already in ECM

Plasminogen activator

• Made by cancer cell

Plasminogen activator turns plaminogen into plasmin → Plasmin reacts with MMP precursors to activate MMPs (Matrix metalloproteases)

• MMPs and Plasmin degrade ECM and basal lamina

Cancer cells also use immune cells to break down ECM

Cancer cells send signals to Immune cells

• Immune cells secrete MMPs → break down ECM

Cancer cells become more motile

Epithelial

• Cell adhesion

• Stationary

• High E-cadherin

Mesenchymal

• Loss of adhesion

• Motile

• Change cell shape

• High N-cadherin

Millions of cells released into circulation each day

<1/1000,000 survive

• Cancer cells in blood stream

  • die with circulation or adhere to endothelial surface

  • die, lie dormant, or penetrate vascular wall

  • die, lie dormal, or grow

  • die, or continue growing

Colonization

Cells must leave the circulatory system by moving through the ECM and vessles

• Establish micrometasis

• Mesenchymal to Epithelial transition (MET)

  • Reverses back to more like original structure to establish macrometastasic colonies

Blood flow influences site of secondary tumor

• Cancer cells usually penetrate in a capillary

• What tissue will breast cancer cells encounter first?

Consequences of Metastasis

1. Disruption of Vital Organs

   • organ overload - tumors multiply inside essential organs like the lungs, liver, and brain, crowing out healthy tissue

   • systemic obstruction - can block digestive tract or ducts, leading to organ rupture or sevre metabolic failure

2. Resistance to Treatment

   • Widespread distribution - scattered microscopic metates throughout the body are much harder to eradicate

   • Drug resistance - migrating cancer cells often mutate or adapt in new tissue environments, making them less responsive to conventional chemotherapy and targeted drugs

3. Severe systemic complications

   • Bone marrow failure - when cancer spreads to bone marrow, it impairs blood cell production, causing fatal anemia, severe infections, and uncontrolled bleeding

   • Cachexia - advanced cancers frequently trigger severe muscle and weight wasting, which compromises heart and respiratory health

Cells within primary tumor differ in metastatic capability

Primary tumor = contains genetically different subpopulations of cells

Hallmark summary

• All hallmarks utilize biological processes - including angiogenesis in response to hypoxia, and invasion and metastasis as seen in early development

• Hallmarks accumulate - easier to get second mutation than the first, third than the second, etc.

• Can be combination of oncogene GOF and tumor suppressor LOF

• Super hard to acquire all the mutations to become cancer and successfully invade and metastasize

Role Of Immune System in Cancer

Evading and exploiting the immune system potentially viewed as a hallmark for cancer

Does our immune system prevent cancer?

Yes

• Some immune cells attack cancer cells

• But tumor cells overcome to become cancerous

• And some cells evade the immune system

No

• Some immune cells promote cancer formation

• immune cells that promote inflammation

Immune cells often found in the tumor; both helping and hurting

Different types of immune cells

Innate immunity

• Timing = right away or within hours

• Specificity = non-specific, treats all germs the same way

• Memory = has no memory; reacts the exact same way every time

• Key components = physcial barriers (skin, mucous membranes), chemical barriers (stomach acid, saliva), and cells like macrophages and neutrophils

Adaptive immunity

• Timing = takes days or weeks to build a strong response during first infection

• Specificity = highly specific; creates targwted weapons for each exact germ

• Memory = builds an immunological memory bank, allowing a fast and powerful response to future exposures

• Key components = white blood cells called lymphocytes, specifically T cells (destroy infected cells) and B cells (make antibodies)

Immune system preventing cancer

Immune surveillance

• The immune system’s ability to recognize and destroy abnormal or transformed cells before they become malignant

T cells and B cells recognize antigens

• Antigen = molecule that stimulates immune response

• Antigen-presenting cell = macrophages & dendritic cells

• Lymphocyte - T cells and B cells = cytotoxic T cells destroy infected cells with antigen

Immune system is trained to not attack itself

Present self “antigens”

• Only the cells that ignore the body’s own harmless self-antigens - while keeping the ability to spot foreign shapes - are allowed to graduate and circulate into the bloodstream

Some tumors express “foreign” antigens

• Tumor-associated antigens = proteins with higher expression than in normal cells

  • normal proteins expressed in high amounts

• Tumor-specific antigens = protein new sequence not found in normal cells

  • entirely new or mutated proteins

• Attack immune cells can recognize and destroy these tumor cells

For a cytotoxic T cell to kill a tumor cell:

It must recognize an abnormal peptide presented on MHC I**

Cancer cells can overcome immune attack

• Cytotoxic T cells and natural killer cells attack tumor cells that are recognized by the immune system

• Tumor cells not recognized by the immune system will continue to grow

1: Hide

A tumor can be harder to detect by:

• Losing or changing the abnormal antigen that T cells recognize

• Reducing MHC class I expression

• Disrupting antigen-processing machinery, so abnormal peptides never reach MHC I

• Expressing mostly proteins that look normal self proteins, generating only a weak immune response

2: Hit the brakes - I have been recognized by I can stop the attack

Some tumor cells display molecules such as PD-L1 that send inhibitory signal to T cells

• The T cell may recognize the tumor, but it becomes less active, exhausted, or unable to kill effectively

  • Blocking PD-L1/PD1 binding as a targeted treatment

3: Change the neighborhood - make the tumor microenvironment friendly to cancer and hostile to immune attack

Tumors can reshape the tumor microenvironment by recruiting immune cells and sending signals that promote wound healing, blood-vessel growth, tissue remodeling, and immune suppression

• Instead of attacking the cancer, these cells can help it grow instead

Inflammation - used to fight infection and repair injury (wound healing)

Acute

• Onset - minutes to hours - innate immune system

• Duration - hours to days

• Cellular infiltrate - neutrophils, macrophages

• Vascular changes - vasodilation, increased permability

• Tissue injury - self-limited

• Fibrosis - mild

• local and systemic signs - prominent

Chronic

• onset slow - adaptive immune system

• Duration - weeks to months or years

• Cellular - macrophages, plasma cells, lymphocytes

• Vascular changes - angiogenesis

• tissue injury - progressive

• fibrosis - severe

• Local and systemic signs - less

Tumor associated macrophages (TAMs)

• Most malignant tumors have TAMs - up to 50% of tumor mass

• TAMs correlate with poorer prognosis

• Recruited by tumor - secreted signals: CSF 1/CCL2

• Normally used to help recruit macrophages to sites of injury or repair to clean up

TAMs help tumors grow and spread

Secrete VEGF - Secrete EGF - Secrete MMPs - generate reactive ocygen species, etc.

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