Cancer Progression and Metastasis

Question 1

Cancer is generally seen in older patients, with a couple of exceptions, what does this tell us?

Answer 1

That mutations are acquired over time and lead to cancers, where the exceptions arise when a person is born with the mutations, making it easier to actually get cancer.

Question 2

What evidence is there that cancer is caused by successive mutations?

Answer 2

Epidemiology data: a plot of deaths vs age of epithelial cancers show the presence of 5-6 rate limiting steps.

Direct sequencing: different tumor sequencing showed that this number varies for different cancers

Question 3

True or False (and explain)

mutation rates are the same during tumor progression.

Answer 3

False, this varies for every type of tumor and depends on which mutations were already acquired (some mutations can lead to faster mutagenesis)

Question 4

what is the evidence pointing towards a stepwise progression of cancer?

Answer 4

1. Observational: when looking at histology, we can sometimes see a malignant tumor that appears to grow out of a more benign lesion
2. Clinical Intervention: the removal of “early” benign lesions (suhc as polyps or small breast tumors) decreases incident of later development of cancer
3. Longitudinal studies: occationally you can follow lesions and observe progression at a site, making them useful since we can often obtain prospective samples (can only do these when early lesions are at a place where we cannot operate – e.g. bronchi).

Question 5

True or false

Cancers can only have one mutation in the MAPK pathway.

Answer 5

true, if there were 2 or more, it wold lead to senescence, preventing tumor growth

Question 6

How do mutations accumulate in cancer?

Answer 6

via colonal expansion (successive mutations with selection): so there is first an initiating mutation which is selected for, then you get more mutations that are selected for… each expansion is thought to aqcuire because of a selective advantage (so it is not selection itself that causes alterations but instead simply selects the alterations that are the most fit)

Question 7

what is a truncal mutation?

Answer 7

the first mutations that is present in all descendants of tumor

Question 8

what sort of mutations generally occur in cancer formation?

Answer 8

mutations that lead to the loss of genome surveillance over time

Question 9

what is evidence to clonal selection in cancer (using breast cancer as an example)?

Answer 9

• spacially-resolve single cell DNA sequencing showes that there are differences in copy number profile between normal cells, carcinoma in situ, and invasive cancer
• the same sorta things was seen when doing genmonic hybridization (scans the genome for amplifications and deletions)
• in every case, they saw that generally more than one clone escaped the ducts to migrate and establish invasive carcinoma

Question 10

what are the different models of clonal invasion/evolution?

Answer 10

1. independent evolution: independent selection and evolution leading to two populations, in situ and onvasive
2. evolutionary bottleneck: in situ, the population evolves and gains more mutations until it gets enough mutations that it can go outside the outside membrane (leading to a single popultion of cells identical cells
3. multiclonal invasion: in situ, the cells gain multiple mutations which can all independently become invasive, leading to heterogenous population of invasive cancer cells.

Question 11

Tru or false

Stem cells rapidly divide.

Answer 11

False, they undergo occasional mitosis. It is the differentiated cells (called transit-amplifying cells) which undergo frequent mitosis , which can then furtehr differentiate into highly differentiated cells.

Question 12

Are stem cells important in normal devlopemnt?

Answer 12

stems cells retain the potential to develop into many different types of cells in the body. Often for repair or growth. (eg colon, skin epidermis, hair follicles etc)

Question 13

What is symmetric vs asymmetric division of stem cells?

Answer 13

symmetric: produce more stem cell numbers (usually seen in context of organ growth)

asymmetric: produce a stem cell and a non-stem cells whihc has a finite proliferative lifespan (usually seen in context of organ maintenance)

Question 14

How can you detect stem cells in vivo?

Answer 14

stem cells retain original DNA (conserved stand) – so we can administer radiolabelled thymidine and measure nucleotide analog rentention, whoch indicate that those cells are stem cells (non-stem cells will contain nonconserved strand, so will not be labelled)

Question 15

what is some evidence of the presence of Cancer stem cells?

Answer 15

A group isolated cells from human breast cancer and injected them into mice, which then developed tumors. They then took the tumor that formed in the mice and FACS sorted them for stem cell markers, and injected the cells that contained and didn’t contain stem cell markers into other mice. They found that only the mice that go injected the cells that contain stem cell markers developed cancer.

and needed very little cells to actually form tumors

Question 15

True or false

all cancer cells give rise to other cancer cancer cells.

Answer 15

False, only a subset of cancer cells can produce cancer cells, called cancer stem cells (CSCs)

Question 16

what are markers of breast cancer stem cells?

Answer 16

CD44+/CD24-

Question 17

define cancer stem cells.

Answer 17

a subset of cancer-derived cells that have the ability to perpetuate the growth of a malignant cell population indefinitely

Question 18

does age in which women have thier first pregnancy have an efefct on risk of developing breast cancer?

Answer 18

yes, women who ahve their first child “early” have decreased risk and those that have their first “later” have increased risk

Question 19

what is the effect of DMBA and hCG treatment in virgin mice?

Answer 19

• DMBA only: ductal carcinoma in situ (DCIS) formation
• DMBA in mice that had a preganancy: much less susceptible
• DMBA in mice pre-treated with hCG (mimics pregancy-induced differentiation): susceptible to cancer
• DMBA + hCG treatment: less susceptible to cancer formation

tells us that differentiation of cells caused by pregancy is what is preventing mutagens from causing tumor induction

Question 20

what properties do cancer stem cells have? (what makes them hard to target?)

Answer 20

They regulate ABC transporters, leading to increased chemotherpy efflux.
And, they are more radio-resistant due to increase checkpoint adherence

Question 21

If mutation in stem cells are rare how is cancer so prevalent?

Answer 21

well, there is evidence that is consistent with that transit-amplyfying cells can de-differentiate back into cancer stem cells (TA cells are much more numerous, proliferate frequently, and aren’t as protected, so can rapidly develop cancer and lead to more mutations)

leads to “new mutations” in cancer stem cells

Question 22

what are the highest causes of death due to lung cancer?

Answer 22

• tumor burder
• infection
• metastatic complications

Question 23

which metastasis regions lead to the “best” and worse outcomes?

Answer 23

best: bone (painful but can cut it off)
worse: brain

Question 24

what are the different models for differential tissue colonization (i.e. region of metastasis)?

Answer 24

1. patterns of blood flow (can explain some of them) 2. seed and soil hypothesis 3. Tissue tropisms of metastasizing cells –tissues may secrete chemokines that recruit cancer cells 4. Vascular zip code model – chemical signal or proteins on endothelial cells differ in different tissue and may offer specialized docking sites for cancer cells

Question 25

what metastatic patterns can be explained by blood flow?

Answer 25

about 50%: pancreas to liver prostate to bone marrow colon to liver breast to bone marrow and lung

Question 26

what is the seed an dsoil hypothesis of differential metastasis?

Answer 26

the idea that cancer cells can disseminate throughout the body but they only colonize where they find hospitable conditions

Question 27

what are the 7 steps required for tumor cells to metastasize?

Answer 27

1. angiogenesis at primary tumor site (required for tumor formation) 2. localized invasion (across the basment membrane) 3. intravasation 4. transport through circulation and arrest in microvessels of various organs 5. extravasation 6. formation of micrometastasis 7. eventually macrometastasis (with the help of angiogenesis)

Question 28

what is the role of neo-vascularization in metastasis?

Answer 28

new blood vessels must provide oxygen and remove waste from growing tumors: i) oxygen has a diffusion limit that solid tumors are limited ii) migrate to invade locally and distantly

Question 29

what is the role of intravasation in metastasis and what phenomena/properties are seen here?

Answer 29

i) cancer cell enters the blood stream or lymphatics, where they are refered to as Circulating Tumour Cells (CTCs) -- can be either as single cells of a chunk of cells ii) interact with cells in the vasculature/lymphatics (e.g. cna intercat with platelets who will mask the cancer cells from the immune system)

Question 30

what is the half-life of circulating cancer cells?

Answer 30

about 30 mins, but can be higher if they bind to something

Question 31

when does extravasation of a circulating cancer cell occur?

Answer 31

CTCs stop at a location (trapped or targeted through endothelial addresses)

Question 32

what is the effect of shear force on circulating cancer cells?

Answer 32

can leads to a chnage in properties, such as becoming more stem cell like

Question 33

do formation of micrometastasis occur often?

Answer 33

yes, but they often remain dormant for years before some sort of trigger allows them to grow and reengage in the cell cycle | e.g. 30% of breast cancer have bone marrow micrometastasis

Question 34

what does the corneal pocket assay show us?

Answer 34

allows us to study new blood vessel formation (by adding an angiogenic, or a suspected angiogenic, factor and measuring vessel formation on the cornea, which is avascualar) | e.g. can put tumor cell on cornea

Question 35

Why the need for angiogenesis in tumour?

Answer 35

because there is a certain range in which oxygen can travel when it leaves the vessel, so cells far away from the vessels become hypoxic and undergo necrosis

Question 36

Cells measure oxygen tension through the _________ protein

Answer 36

Von Hippel Lindau (VHL)

Question 37

what is the role/function of VHL protein?

Answer 37

* Tumor suppressor * Cytoplasmic protein involved in ubiquitination and proteosomal degradation. * Acts as a target recruitment subunit in the E3 ubiquitin ligase complex * Ubiquitinates in an oxygen-responsive manner | so leads to degradation in normoxia and doesn't in hypoxia

Question 38

what is the substrate of VHL protein and how does it work?

Answer 38

Hypoxia-inducible transcription factor-1 (HIF-1), which is a **heterodimeric transcription factor made of HIF1a (or HIF2a) and HIF1b**. **normoxia**: HIF-1 is hydroxylated (OH on proline), so is recognized by pVHL, which binds to it and leads to degradation of HIF-1 **hypoxia**: HIF-1 is not hydroxylated, so can dimerize, leading to formation of a functional transcription factor, which induced transcription of genes such as VEGF and Epo.

Question 39

what was found to be the ‘tumour angiogenesis factor’ (TAF)?

Answer 39

VEGF (vascular endothelial growth factor) | but also include PDFG, FGF

Question 40

what is the effect of VEGF inhibition?

Answer 40

Inhibition of VEGF directly or its receptors prevents neo-vascularization and tumour growth (doesn't kill vessels but stops growth of new vessels)

Question 41

which VEGF receptors and ligands are important in the context of angiogenesis?

Answer 41

VEGFA which binds to VEGFR1 and 2

Question 42

what signal can lead to induction of VEGF expression?

Answer 42

RAS

Question 43

what are properties of VEGFs?

Answer 43

* Capillary and lymph duct formation (both important for tumor spread) * recruitment of hematopoietic progenitor cells from the bone marrow to sites where more vessels are needed * capillary permeability (capillaries are more leaky)

Question 44

what is the role of FGF (fobroblast growth factor)?

Answer 44

bFGF may be critical for vasculogenesis through the FGFR1 receptor ## Footnote FGFs are often sequestered in the extracellular matrix and are released with protease activity / damage

Question 45

what is TSP-1, and how is it dysregulated in cancer?

Answer 45

* it is produced by many cell types and binds to endothelial cells (e.g. CD36) leading to inhibition of their proliferation * TSP1 is a p53 transcriptional target (goes up in response to p53 signal, so induction lost in mutant p53) * downregulated in mutant Ras (via high signaling in the Ras pathway) | so is downregulated in many cancers

Question 45

what are negative regulators of new blood vessel formation?

Answer 45

Thrombospondin-1 (TSP-1)

Question 46

how does TSP-1 work?

Answer 46

* Newly made endothelial cells produce FAS receptor * TSP1 induces production of FASligand in endothelial cells causing autocrine-induced extrinsic cell apoptosis in newly formed vessels/sprouts -- so prevents vessel growth in places where they are not needed * BUT, mature endothelial cells lack the FAS receptor

Question 47

what are the steps leading new vessel growth in tumors?

Answer 47

1. Hypoxia in tumor leads to stabilization of HIF-1a/b dimer, leading to transcription of VEGF/FGF. 2. Also, it leads to upregulation of protease (e.g. MMP), leading to basement membrane breakdown. 3. Then, a tip cell migrates along the angiogenic factor gradient. 4. endothelial cells differentiate into stalk cells, whihc proliferate and lead to vessel formation 5. pericytes attach to the outisde, providing support

Question 48

inhibition of PDGFR and VEGFR causes what?

Answer 48

regression of vasculature (PDGF inhibition impaires support provided by pericytes, making the endothelial cells sensitive to VEGF inhibition and chemotherapy)

Question 49

where do the new endothelial cells in tumor vascularization come from? | and how do we know this?

Answer 49

from the bone marrow. When mouse gets a bone marrow injection from GFP-labelled bone marrow mouse, we see that in the tumor, there are GFP-endothelial cells. | tumor attracts endothelial cells from the bone marrow

Question 50

Mobilization of bone marrow dervied cells requires which regulators?

Answer 50

Id1 and Id3 | deleting them leads to inhibition of BMC-derived cells

Question 51

what key points are obsered in RIP-Tag mice?

Answer 51

1. Tumour formation and progression occurs only with angiogenesis 2. All of the hyperplastic islets express large amount of VEGF but only a few become angiogenic.

Question 51

what is the RIP-Tag transgenic mice? why is it useful?

Answer 51

RIP-Tag mice express large T antigen in insulin producing beta cells: * large T binds to RB and inactivates RB * large T binds to p53 and inactivates it

Question 52

what is the "angiogenic" switch seen in tumors? | generally

Answer 52

stromal cells (ie not the tumour cells) are responsible for signals leading to angiogenesis (the tumour cells alter the stromal cells and the stromal cells cause release of endothelial/pericyte growth/recruitment factors)

Question 53

how do cancer cells migrate?

Answer 53

they can either disseminate as single cells or as a collective "clump" of cells atatched via junctions

Question 53

In the RIP-Tag model, what leads hyperplastic islet cells to become angiogenic?

Answer 53

Hyperplastic islet cells send signals (chemokines) that go to the bone marrow. This signal causes mast cells to mobilize and when near islets, they secrete MMPs that release VEGF from the ECM (inactive VEGF released from ECM as soluble, active VEGF). This leads to angiogenesis.

Question 54

what happens during single cell motility?

Answer 54

interdependent molecular steps lead to changes in cell shape, position, and tissue structire which it migrates through. Cytoskeleton polarizes by actin polymerization to form a leading protrusion. The leading edge protrusion engages with the extracellular substrates (ECM and other cells), where there are proteases at the leading edge, allowing the cell to move forward.

Question 55

what happens during collective cells migration?

Answer 55

protrusion and retraction are coordinated in a supracellular manner: cytoskeletal protrusion and contractility are mechanically mediated through cell-cell junction (the group behaves as a mega cell)

Question 56

How can epithelial cell migrate?

Answer 56

* epithelial cells are anchored to cells around them via e-cadherens/anchor proteins/and actin. * But under the right conditions epithelial cells can chnage their "state" enabling them to move (EMT (epithelial-mesenchymal transition))

Question 57

What is EMT?

Answer 57

* the signal can be a wound. * the epithelial cells lose epithelial markers (E-cadherin) and start to express mesenchymal markers (e.g. vimentin) * it enhances Rac-dependent mesenchymal migration, contributing to cell growth, cell survival and re-emergence of stem cell characteristics) * EMT is reversible | epithelial-mesenchymal transition

Question 58

what can trigger EMT?

Answer 58

* damage (e.g. a wound) * TGF-b * MMPs (matrix metaloproteases)

Question 59

what is the effect of the loss of e-cadherens?

Answer 59

* frees the epithelial cells * also press b-catenin (which is usuallt bound to e-cadheren) meaning that it can them transloacte to the nucleus to form hetero-oligomers with TFs that regulate EMT (and proliferation)

Question 60

which TFs are considered EMT-TFs? | and what do they do?

Answer 60

* repress e-cad **indirectly**: Twist, TCF4, goosecoid, SIX1, FOXC2 * bind e-cad **directly**: Snail1/2, Zeb1/2, TCF3 KLF8 * **TWIST**: is activated by many signaling pathways: Ras, Akt, MAPK, STAT3, Wnt * they also confer stem cell-like properties to endothelial cells

Question 61

High EMT-TF expression (twist) correlates with what?

Answer 61

poor prognosis