Lecture 30 - Radiation Biology and the Hallmarks of Cancer

ONCOL 335 - Radiobiology. University of Alberta

What were the six original hallmarks of cancer?

1. resisting cell death

2. sustaining proliferative signalling

3. evading growth supression

4. activating invasion and metastasis

5. enabling replicative immortality

6. inducing angiogenesis

what were the four expanded hallmarks?

1. deregulating cellular energetics

2. avoiding immune destruction

3. tumor promoting inflammation

4. genome instability and mutation

what do cancer cells require to form a macroscopic tumor?

the ability to replicate unlimitedly, uninhibited by normal space restrictions

what limits the amount of cell divisions in normal cells

the Hayflick limit

what happens after the Hayflick limit is reached?

normal cells either face senscence or will undergo cell death

how do cancer cells break the Hayflick limit to allow for immortality?

Cancer cells often will first down regulate P53 to prevent the cancer cell from dying, and then they will activate telomerase, an enzyme that extends telomeres, thus circumventing the normal limits on cell division.

what normal cells have an excess of telomerase

germ cells

• so cancer cells essentially will mimic germ cells

what was Theodore Puck’s feeder cell experiment

Theodore Puck's feeder cell experiment demonstrated that normal human cells could be cultured indefinitely by using a layer of feeder cells to provide essential growth factors, illustrating the importance of cellular microenvironments in regulating cell behavior.

• this showed that cells need growth factors to grow

• now we don’t need feeder cells, we just use growth factors

examples of discoveries that were found with the use of clonogenic survival assays

• split dose experiments —> sublethal damage repair

• dose rate effects

• OER

what future cancer research is under the category of enabling replicative immortality

understand the causes of intrinsic radiosensitivies and taking our knowledge of in vitro killing to the clinic

which cell cycle checkpoint was discovered first? G2 or G1

G2

why was G2 checkpoint discovered first

research was done on HeLa cells, who had G1 inhibited due to HPV inhibiting p53 cell cycle arrest

G1, G2, and G2/M checkpoints are all activated by …

DNA damage

M checkpoint is activated by ….

chromosome misalignment

what gene is required to inhibit or check DNA after IR

ATM

• without ATM, the cell can keep proliferating and accumulate additional DNA damage, leading to genomic instability.

what future cancer research is under the category of evading growth supression

research on ATM inhibitors and other drugs that abrograte cell cycle checkpoints

what is the most common type of cell death observed after irradiation

mitotic catastrophe

what caused the discovery that p53 can induce apoptosis

the discovery that radiation to cells increased p53 levels

describe the P53 independent form of apoptosis

IR causes the rapid hydrolysis of sphingomyelin to produce ceramide which activates apoptosis

what other form of cell death can radiation induce

autophagy

what future cancer research is under the category of resisting cell death

can we modeulate p53 response: make drugs that force a mutated p53 to recreate a wildtype p53 response

what happens to chromosomes that have damage by radiation

abnormal chromosomal rearrangements (genome instability)

the study of radiation induce chromosome aberrations led to identifying genes involved in

the DNA damage response and DNA repair

What family are RAD51 and RAD50 genes in

RAD sensitivity genes involved in HR

What family is XRCC1 part of

X-ray repair cross complementing proteins

• that play a crucial role in single-strand break repair and cellular responses to DNA damage.

when does the choice between NHEJ and HR occur?

if the DSB gets it’s ends resected

• end resection = HR

which DNA repair pathway is faster? which one is more faithful?

NHEJ is faster

HR is more faithful due to strand template

what gene was found to control both HR and NHEJ

ATM

who discovered mutations as a result of radiation of cells, and developed the non-threshold linear model for cancer?

Hermann Joseph Muller

How do PARP inhibitors work for BRCA mutant cancers

PARP is inhibited so DNA damage cannot be repaired in cancer cells

• PARP is one of the first enzymes that binds to DSB, is thought to recruit other repair enzymes

• Acts even before ATM does

what two types of cancers are BRCA mutants

Breast and ovarian cancer

what is synthetic lethality?

The phenomenon where the simultaneous loss or disruption of two or more genes (or genes and small molecules) leads to cell death, while the loss or disruption of any single gene alone does not

what future cancer research is under the category of genome instability and mutation

developing inhibitors of DNA repair enzymes and radiosensitizers

• ex: ATM/ATR inhibitors

what was Thomlinson and Gray’s hypoxia experiment?

see how oxygen levels decrease in tumors located away from blood vessels

how far can arteries and veins diffuse oxygen

arteries: more than 70 um

veins: less than 70 um

what two reasons are hypoxic cells problematic for radiotherapy

1. they are more resistant to radiation as photons rely on indirect damage with ROS

2. hypoxia can induce factors that create stem cell like properties

   • stem cells are more radioresistant and metastatic

what two factors levels are raised in tumors after radiotherapy

VEGF

HIF1-a

why do hypoxic tumors reoxygenate after radiotherapy

hypoxic tumors reoxygenate as oxygen consumption rate decreases as a result of cell death of radiosensitive oxygenated tumor cells and increase in perfusion

if you neutralize VEGF before RT, whay happens to tumor shrinkage

Tumor shrinkage may be enhanced, as neutralizing VEGF can improve oxygen delivery and reduce hypoxia, making cancer cells more susceptible to radiation.

what is a possible explanation as to why HIF1-a increases after radiation treatment

• HIF1-a may be stored in stress granules and after irradiation it is released in response to cell stress

• ROS stabilize HIF1-a and cause gene transcription/translation

what future cancer research is under the category of inducing angiogenesis

manipulating angiogenesis and tumor vasculature to facilitate perfusion and oxygenation

learning more about role of HIF

What is EMT?

polar epithelial cells lose polarity and connection to eachother to go into lymph and blood vessels to spread

ineffective irradiation of solid tumors can result in ….

enhancement of metastasis

• thus we often combine treatments to try to increase outcome

Howcome hypoxia is a strong driver of metastasis

hypoxia upregulates HIF1-a, and is further upregulated with irradiation stress, which may transcribe genes associated with invasion and metastasis

what future cancer research is under the category of activating invasion and metastasis

learning more about IR influencing cell plasticity

creating inhibitors of tumor cell invasion to use as adjuvants to radiotherapy

what is inflammation

the complex biological response of body tissues to harmful stimuli such as pathogens and irritants

what three substances are involved in inflammation

1. immune cells

2. blood vessels

3. molecular mediators

what is the purpose of inflammation

to eliminate the initial cause of cell injury, clear out necrotic cells, and tissues damaged from the injury to initiate tissue repair

how does the inflammatory response contribute to tumorigenesis

The inflammatory response can contribute to tumorigenesis by creating an environment that promotes cell proliferation, survival, and the accumulation of mutations, ultimately leading to cancer development.

when macrophages are irradiated, what two factors did they release

1. TNF (tumor necrosis factor)

2. Free radical nitric oxide

has significant consequences for vascular function, inflammation, and cell survival

what is the radiation-induced bystander effect (RIBE)

Cells that are not irradiated but are affected by “stress signal factors” released from irradiated cells. These cells, as well as directly irradiated ones, express DNA damage-related proteins and display excess DNA damage, chromosome aberrations, mutations, and malignant transformation.

What is pyroptosis

Pyroptosis is a highly inflammatory form of programmed cell death that is typically triggered by infection or cellular stress. It involves the formation of pores in the cell membrane, leading to cell lysis and the release of pro-inflammatory cytokines

how does pyroptosis affect the RIBE

Pyroptosis enhances the radiation-induced bystander effect (RIBE) by releasing pro-inflammatory cytokines and “stress signal factors” that can impact neighboring non-irradiated cells, leading to increased DNA damage and potential tumorigenesis.

what future cancer research is under the category of tumor promoting inflammation

taking advantage of RIBE effect for radiation therapy

finding ways to modulate inflammatory effects to optimize radiosensitvity of tumors

what factors regulate cellular proliferation and differentiatiation in normal tissues

growth factors

two examples of proteins regulating cell proliferation and survival

• EGFR (epidermal growth factor receptors)

• Transforming growth factor alpha (TGFa)

what two reasons allow cancer cells to show abnormal growth

1. they are less dependent on exogenous factors

2. they are capable of autonomous activation of autocrine and paracrine growth pathways

tumors with higher expressions of EGFR are more or less radioresistant

more radioresistant, leading to treatment challenges.

why are tumors with more EGFR more radioresistant

we get increased repopulation of tumors leading to decreased TCP

the more EGFR are the _____ the TCD₅₀

higher

what future cancer research is under the category of sustaining proliferative signalling

create targeted therapies that target proliferative singalling pathways

• ex: we use herceptin to target HER2 EGFR pathway

what are the two hallmarks of cancer not included by the 5 R’s of radiobiology?

1. avoiding immune destruction

2. deregulating cellular energetics

Immune cells theoretically have the ability to recognize cancer cells based on the cancer associated antigens the cells express on their surface. Why does the immune system fail from catching cancer cells?

• tumors have the abiloty to hide their tumor antigens

• have secretions that inhibit immune response

what are the most radiosensitive immune cells? what form of cell death do they undergo?

lymphocytes

undergo apoptosis

what type of radiation therapy has been used as a form of immunosupression?

Total body irradiation (TBI)

• done before a bone marrow transplant

Based on the radiosensitivity of immune cells at the tumor, it was orginially thiought that irradiation was immunosuppressive. However, later research has found certain doses can lead to the upregulation of tumor associated antigens on cancer cells, resulting in immunogenic cell death. what level of doses cause increased immunogeneicity?

above 10 Gy we seem to get better immune response

what type of radiation therapy may result into making tumor cells more immunogenic?

SBRT (Stereotactic Body Radiation Therapy)

• high doses, hypofractionated

what is an abscopal effect

damage to the primary site may release tumor associated antigens into the environment to be picked up by immune cells. this may result in the regression of disease in metastatic sites

combining radiation therapy with _____ can trigger anti-tumor immuniry and lead to regression of the disease

immunotherapy: monoclonal antibodies agaisnt immune checkpoint inhibitors

• CTLA-4, PD1

what is the ideal abscopal effect situation

An ideal abscopal effect situation occurs when localized radiation treatment not only reduces the primary tumor but also induces systemic anti-tumor immune responses that lead to the regression of untreated metastases.

what are four issues that may inhibit an abscopal response

1. hypoxia may supress immune cells

2. tumor death may release chemocines to transform immune cells to M2 phenotype

3. TGF-B signals may transform immune cells to M2 phenotype

4. tumor death releases ATP, and adenosine can inhibit immune cells

what future cancer research is under the category of avoiding immune destruction

developing immunomodulators to improve radioresponse and anti-tumor immunity

learn more about influence of immune system on TME

does the way a cell die matter for an immune response

yes, some cell death mechanisms lead to better immune response

• apoptosis leads to poor immune response

what three ways does the TME differ from a normal cell environment

1. hypoxia

2. lower pH

3. nutrient deprivation

under aerobic conditions, what does metabolism look like

Normal metabolism relies on oxygen for energy production, primarily through oxidative phosphorylation, resulting in efficient ATP generation and low lactate production.

what cycle will aerobic metabolism undergo

Krebs cycle (Citric Acid Cycle)

under anaerobic conditions, what does metabolism look like?

Metabolism shifts to anaerobic glycolysis, resulting in less efficient ATP generation and increased lactate production, often leading to acidosis.

what process will anaerobic metabolism undergo

fermentation: to generate ATP and produce byproducts like ethanol or lactic acid.

what did Otto Warburg win a nobel prize for?

Discovering the Warburg Effect

what is the Warburg Effect?

a phenomenon where cancer cells preferentially produce energy through aerobic glycolysis rather than the more efficient oxidative phosphorylation, even in the presence of ample oxygen. This means they convert glucose to lactate instead of fully breaking it down in the mitochondria.

Three reasons why cancer will choose aerobic glycolysis over OxPhos

1. It provides intermediates for biosynthesis (nucleotides, amino acids, lipids)

2. it alows for faster ATP production (though less efficient)

3. it creates an acidic tumor microenvironment, which may aid in invasion and immune evasion

what role does HIF1 have in aerobic glycolysis?

increases glycolytic metabolism and increase glucose transporter expression

how do we know HIF1 affects aerobic glycolysis

in HIF1 depleted tumors, ATP levels are lower

what future cancer research is under the category of deregulating cellular energetics

developing glycolysis inhibitors to add to RT treatments to potentially improve outcome

learning more how metabolism influences radiation response