Cancer I

Question 1

Define whether a gene that regulate cell division, immortality, apoptosis, angiogenesis, or metastasis is an oncogene or tumor suppressor gene

Answer 1

They can both act on all of these, however, how they act are different. An oncogene tends to increase the progression of the cell cycle through a variety of ways, including promoting cell division, inhibiting apoptosis, promoting immortality, promoting angiogenesis, promoting metastasis. A tumor suppressor gene tends to restrict the cell cycle by inhibiting cell division, promoting apoptosis, inhibiting immortality, inhibiting angiogenesis, and inhibiting metastasis.

Question 2

Explain how oncogenes act in a dominant fashion and tumor suppressor genes in a recessive fashion

Answer 2

Oncogenes tend to act in a dominant fashion because a gain of function mutation in a single copy of the cancer critical gene can drive a cell towards cancer. If you get one mutation on one chromosome in a oncogene, you have a an active oncogene (not protooncogene anymore!). For tumor suppressor genes, however, you need TWO mutated tumor suppressor genes in order to get an cancerous gene! Both need to be present.

Tumor suppressors act recessive at the phenotypic level (both alleles must be mutated/lost for cancer to develop), but the “first hit” germline mutation at the genotypic level is actually inherited in an autosomal dominant fashion.

Question 3

Cancer incidence with ages

Answer 3

• One of the themes is that cancer is a very complex disease that is not just the result of a single mutation
• Incidence is established with age, it increases with age.
• If it was a single mutation then the individual would have an equal chance of occurring at any time in that person’s life. However, it doesn’t work this way suggesting that these mutations occur and accumulate during life not due to a single traumatic experience rather it is due to an accumulation of events

Question 4

Explain the concept of penetrance

Answer 4

Penetrance is the percentage of the carriers who do not show evidence for the disease

Penetrance is usually age related, meaning that the trait is not expressed in most carriers at birth but occurs with increased frequency as the carriers get older. For example, germline mutations in mismatch repair genes associated with hereditary nonpolyposis colorectal cancer (HNPCC) are incompletely penetrant. So not all individuals who carry these mutations will get colorectal cancer, but the risk increases as individuals age. About 20 percent of carriers will never develop colorectal cancer.

• An example of how losing these cancer genes can increase your risk of cancer is shown
• This is a resulting mutation in MMR genes. Individuals who carry or inherit a mutation in these genes has a greater risk
• This line NEVER reaches 100  Not everyone with the mutation in the MMR gene will get cancer! Other factors come into play. Has to do with penetrance, or the frequency at which a phenotype is observed
• A large amount of people will get cancer though due to age!
• Not all mutations are fully penetrate
• If you have one you mutation the chance of getting cancer is drastically higher now

Question 5

Two Hit Hypothesis

Answer 5

In 1971, Dr. Alfred Knudson proposed the two-hit hypothesis to explain the early onset at multiple sites in the body of an inherited form of cancer called hereditary retinoblastoma. Inheriting one germline copy of a damaged gene present in every cell in the body was not sufficient to enable this cancer to develop. A second hit (or loss) to the good copy in the gene pair could occur somatically, though, producing cancer. This hypothesis predicted that the chances for a germline mutation carrier to get a second somatic mutation at any of multiple sites in his/her body cells was much greater than the chances for a noncarrier to get two hits in the same cell.
Tumor suppressors act recessive at the phenotypic level (both alleles must be mutated/lost for cancer to develop), but the “first hit” germline mutation at the genotypic level is actually inherited in an autosomal dominant fashion.

• Two hit hypothesis.
• It takes not one mutation, but a combination of mutations in order to get cancer.
• You can inherit one bad copy and then you have a mutation in your lifetime and then you get cancer

Question 6

What are two ways for cells to die?

Answer 6

1) Apoptosis:
- Key features are blebbing off
- In the body it doesn’t stop at this point, it signals to other cells to phagocytose them and engulf them and they do so without causing an inflammatory response

2) Necrosis:
- This causes an inflammatory response because things go really bad in the cell and cause the cell to burst
- Thus these are two distinctly different cell death responses –> One causes an inflammatory response and one does not

-Apoptosis is NOT a bad thing! Some cells need to be removed because they are abberant or because there are too many! In early development apoptosis is required!

Question 7

p53

Answer 7

It is activated by DNA damage to activate cell death signals

Question 8

Bcl2

Answer 8

Inhibits cell death signals from reaching the caspases

Question 9

Describe the steps leading to the activation if the caspase cascade in the death receptor or Extrinsic Apoptotic Pathway

Answer 9

Death Receptors

Trimeric Fas ligands on the surface of a killer lymphocyte interact with trimeric Fas receptors on the surface of the target cell, leading to clustering of several ligand-bound receptor trimers (only one trimer is shown here for clarity). Receptor clustering activates death domains on the receptor tails, which interact with similar domains on the adaptor protein FADD (FADD stands for Fas-associated death domain). Each FADD protein then recruits an initiator caspase (caspase-8) via a death effector domain on both FADD and the caspase, forming a death-inducing signaling complex (DISC). Within the DISC, two adjacent initiator caspases interact and cleave one another to form an activated protease dimer, which then cleaves itself in the region linking the protease to the death effector domain. This stabilizes and releases the active caspase dimer into the cytosol, where it activates executioner caspases by cleaving them.

EXTRINSIC PATHWAY: USES DEATH RECEPTORS!

• The extrinsic pathway is activated by death receptors
• Starts with a killer lymphocyte with a Fas Ligand that binds to the Fas death receptor and activates a death domain that is on the death receptors cytoplasmic tail.
• Now, a FADD (cytoplasmic adaptor protein) with the death domain binds to the death domain via its own and it has a death effector domain. Then, the caspase 8 can bind to the death effector domain of the FADD
• Then, DISC (death inducing signal complex) is assembled from all of this. Once this pathway is activated, the adapter proteins bind to the receptor and can lead to the recruitment and dimerization of the caspases because they are brought into close proximity of one another.
• They can dimerize and then cleave each other to be an active caspase 8 that leads to apoptosis.
• Its Caspase 8 is then cleaved and dimerizes. Then it is activated and activates executioner caspases

-Triggered by a receptor, triggers the formation of DISC, get active caspase 8 which then activated excutioner caspases to get apoptosis

• Once a signal has been received, it binds to the death receptor. Thus, a death receptor ligand has been received.
• There are then cytoplasmic receptor adaptor proteins (TRADD and FADD). The combination of the ligand bound to the receptor leads to the activation or accumulation of the cytosolic adapter proteins which leads to the activation of Caspase 8.
• Then you get the activation of Caspase 8 which is the FIRST Caspase to be activated in the death receptor pathway!
• Then Caspase 8 can funnel through 2 different pathways:
  1) It can directly result in the activation of effector caspases leading to apoptosis
  2) It can cross-talk to the intrinsic pathway through the cleavage of a Blc2 like protein, pro-apoptotic BH3-only protein called tBID

Question 10

Describe the steps leading to the activation if the caspase cascade in the mitochondrial or Intrinsic Apoptotic Pathway

Answer 10

Mitochondrial Dependent

The mitochondria receives an apoptotic signal and thus results in the release of cytochrome-c. The Cytochrome-c can then associate with the Apaf1, which causes the Apaf1 to unfold partially to expose a CARD or caspase recruiting domain which allows it to interact with other Apaf1s at this site. Then, 7 Apaf1’s that where activated by cytochrome-c can form a ring-like structure called an apoptosome and recruit the Caspase 9 that also has a CARD domain. Now the Caspase 9 is active and can therefore cleave executioner caspases that can then cause apoptosis.

• It is using the mitochonria –> It is mitochondrial dependent, not death receptors
• Upon activation of the death receptor pathway, the tBID will activate the BAX and BAD which then trigger the mitochondria to release cytochrome-c and SMAC.
• Then, cytochrome-c interacts with APAF1 to activate caspase 9, which activates the effector caspases and triggers apoptosis
• There is also a parallel mechanism with SMAC which inactivates XIAP to prevent it from inactivating Caspase 9 and allow the Caspase 9 to remain active and activate the effector caspases
• The effector caspases can then trigger the activation of the endonucleases, result in the cell surface alterations and the cytoskeletal reorganization. Then you get apoptosis and phagocytosis

Question 11

Understand the Mechanisms of Caspase Activation

Answer 11

• There are the initiator caspases (8 and 9) that exist in the cytosol as procaspases which are not active and need to be activated. They have an adaptor binding domain and protease domain and within the protease domain there is a cleavage site
• When the apoptotic signal is received, whether extrinsic or intrinsic pathway, you get dimerization, activation and cleavage. They activate themselves via the cleavage.
• They are inactive monomers that dimerize, they process each other to produce an active caspase that are activating the executioner caspases that are already dimers. The executioner caspase is then cleaved and now activated and lead to apoptosis
• They dimerize and become active
• Executioner caspases already exist as inactive dimers and they are activated via cleavage and then cause apoptosis by cleaving substrates
• They auto-cleave themselves to get activated

Question 12

DNA Fragmentation During Apoptosis via endonuclease CAD

Answer 12

In healthy cells, the endonuclease CAD associates with its inhibitor, iCAD. Activation of executioner caspases in the cell leads to cleavage of iCAD, which unleashes the nuclease. Activated CAD cuts the chromosomal DNA between nucleosomes, resulting in the production of DNA fragments that form a ladder pattern (see B on next slide) upon gel electrophoresis.

Just know that part of the process of apoptosis DNA cleavage

Question 13

BCL-2 family members are related by regions

of sequence and structural homology

Answer 13

They all have the BH3 domain but that is the only BH domain the 3 of them share! It mediates the direct interactions between pro-apoptotic and anti-apoptotic family members. They also share the transmembrane domain.

Bcl2 protein regulate the intrinsic pathway of apoptosis!

Question 14

Anti-Apoptotic Bcl2 Family Protein

Answer 14

Guardians (pro-survival). They include Bcl2 and BclXL

Question 15

Pro-Apoptotic Bcl2 Family Protein

Answer 15

Effectors (pro-apoptotic). They include Bax and Bak

Question 16

Pro-apoptotic BH3-only protein

Answer 16

Initiators (pro-apoptotic). They include Bad, Bid, Bim, Puma and Noxa.

Question 17

Explain how Bcl2 Proteins Regulate the Intrinsic Pathway of Apoptosis

Answer 17

When activated by an apoptotic stimulus, the effector Bcl2 family proteins (Bax, Bak) aggregate on the outer mitochondrial membrane and release cytochrome c and other proteins from the intermembrane space into the cytosol by an unknown mechanism.

• Bax and Bak
• Here is an example of how the process occurs
• Within the mitochondria are the inactive Blc2 family proteins such as Bak and Bax.
• Bax and Bak are located within the membrane of the mitochondria and upon apoptotic stimulus, these Bax and Bak proteins will homodimerize and lead to a process that is not too well understood to create a type of channel which then allows for the release of cytochome-c into the cytosol.
• These allow the formation of dimers within the mitochondrial membrane which allow for the release of cytochrome-c and other proteins such as SMAK.

In the absence of an apoptotic stimulus, anti-apoptotic Bcl2 family proteins (e.g., Bcl-2, BclXL) bind to and inhibit the effector Bcl2 family proteins on the mitochondrial outer membrane (and in the cytosol—not shown).

• -Within the inactive mitochondrial pathway, you have the inactive effector Bcl2 family proteins present within the membrane surface.
• They are not allowed to dimerize because of the action of the active anti-apoptotic Bcl2 family protein that prevents the dimerization of the inactive effector Bcl2 family protein.
• Bcl2 anti-apoptotic proteins block the dimerization which leads to the cytochrome-c
• This is how the Bcl2 proteins block the pathway so that apoptosis cannot occur
• active anti-apoptotic Bcl2 family protein blocks the dimerization and thus activation
• JUST KNOW THAT THE Bcl2 FAMILY PROTEINS (ACTIVE ANTI-APOPTOTIC OR GUARDIANS) BLOCK THE DIMERIZATION OF THE EFFECTORS OR THE PRO-APOPTOTIC EFFECTORS

In the presence of an apoptotic stimulus, BH3-only proteins (e.g., Bid, Bad) are activated and bind to the anti-apoptotic Bcl2 family proteins (e.g., Bcl2, BclXL) so that they can no longer inhibit the effector Bcl2 family proteins ((Bak, Bax); the latter then become activated, aggregate in the outer mitochondrial membrane, and promote the release of intermembrane mitochondrial proteins into the cytosol. Some activated BH3-only proteins may stimulate mitochondrial protein release more directly by binding to and activating the effector Bcl2 family proteins. Although not shown, the antiapoptotic Bcl2 family proteins are bound to the mitochondrial surface.

-Upon an apoptotic stimulus, it activates the BH3-only proteins which include such things as Bad, Bim, Bid, Puma and Noxa.
-Upon activation the BH3-only proteins can activate the apoptotic pathway through 2 mechanisms:
It can bind to and inactive the Bcl2 family protein (anti-apoptotic). These will come in through the BH3 domain and bind to it and inactive it, allowing for the dimerization of the effectors. It can bind to the anti-apoptotic Bcl2 family protein and pull it away from Bax and Bak and allow them to dimerize
It can directly interact and activate Bax and Bak in the mitochondrial membrane.

-Some of these pathways are not fully understood but just keep in mind that there are two pathways in which you can

Question 18

Describe the role of p53 in apoptotic responses

Answer 18

The activation of p53 triggers apoptosis via upregulation of BH3-only expression. DNA damage signals activate a set of kinases (ATM/ATR and Chk1/Chk2) that phosphorylate p53, leading to its stabilization and activation. The Mdm2 protein normally binds to p53 and promotes its ubiquitylation and destruction in proteasomes. Phosphorylation of p53 blocks its binding to Mdm2, allowing for increased levels of p53 and the stimulation of transcription of numerous genes. Among the targtes are the BH3-only genes Puma and Noxa. The BH3-only proteins then bind to Bcl2, allowing for the activation of the apoptotic pathway.

• Another way in which you can activate this pathway is through the upregulation of p53 to create the apoptotic response.
• You get double stranded break which triggers kinase activation. The kinase then phosphorylates p53.
• p53 is normally held inactive by Mdm2, but upon phosphorylation of p53, it releases the Mdm2 and now get activation of the p53
• p53 actually forms a tetramer
• Once p53 is active, it serves as a transcription factor and it turns up the BH3-only genes.
• p53 activated the BH3-only proteins which then inhibit the Bcl2 proteins leading to the activation of the intrinsic pathway.

Question 19

Describe the roles of other protein factors released from the mitochondria in addition to cytochrome-c

Answer 19

The mitochondria also releases anti-IAPs such as Hid or Diablo. These anti-IAPs then inhibit and IAP which can then cause apoptosis. Without the anti-IAPs, apoptosis would be inhibited by the IAP

Question 20

Understand how different survival factors inhibit apoptosis

Answer 20

(A): Increased production of anti-apoptotic Bcl2 family protein

• JUST KNOW THAT SURVIVAL FACTORS CAN ACTIVATE A TRANSCRIPTION FACTOR WHICH LEADS TO THE UPREGULATION OF Bcl2 OR THE ANTI-APOPTOTIC PROTEINS WHICH THEN EFFECTIVELY BLOCK APOPTOSIS
• For example, cancer cells can secrete these survival factors that can upregulate Bcl2 to block apoptosis because they don’t want to kill themselves

(B): Inactivation of the pro-apoptotic BH3-only protein

• THE SURVIVAL FACTOR BINDS TO A RECEPTOR AND THEN CAUSES ITS ACTIVATION. THE ACTIVATED RECEPTOR THEN ACTIVATES A SERIES OF KINASES SUCH AS Akt KINASE HERE.
• Bcl2 can be held inactive by one of the BH3-only proteins, in this case Bad. When Bad is active, it is blocking the function of Bcl2. However, Akt can phosphorylate Bad causing it to release Bcl2 which is now active and can inhibit apoptosis.

(C): Inactivation of anti-IAPs

• A survival factor binds to the receptor, activating it. Then the receptor activates MAP kinase which acts to inactivate the Hid by phosphorylating it. Now Hid can no longer inhibit the IAPs and now apoptosis is blocked.
• In this particular case, it is activated by a MAP Kinase so again using a kinase pathway.
• Hid normally acts to block the inhibitors of apoptosis but when Hid is inhibited it will allow for apoptosis to be blocked

Question 21

List the different processes that may activate the mitochondrial apoptotic pathway

Answer 21

The intrinsic or mitochondrial pathway can be activated via its own signaling of BH3-only proteins, which are pro-apoptotic proteins that can activate the pro-apoptotic proteins Bak and Bax, causing them to dimerize and release the cytochrome-c which will in turn associate with the Apaf1, exposing a CARD site for 7 of them to bind together and form an apoptosome which will allow it to bind the caspase 9 and activate it. There can also be cross-talk from the extrinsic pathway as well though in which the tBID (a BH3-only protein that is pro-apoptotic) can also activate the Bak and Bax and result in the same effects.

Question 22

Benign Tumor

Answer 22

A benign glandular tumor (adenoma) remains inside the basal lamina that marks the boundary of the normal structure (e.g., duct)

Question 23

Malignant or Invasive Tumor

Answer 23

malignant glandular tumor (adenocarcinoma) can develop from a benign tumor cell that degrades the integrity of the tissue and travels from the original site to colonize other tissues (metastasis).

Question 24

Carcinoma

Answer 24

cancers arising from epithelial cells (most common, ~80%)

Question 25

Sarcoma

Answer 25

arise from connective tissue or muscle cells.

Question 26

Explain the concept of tumor clonality

Answer 26

Each cell, when it divides, generates two identical new ones. So, when a cell acquires a mutation, it passes that mutation on to its progeny during cell growth and division. Because cells with cancer-linked mutations tend to proliferate more than normal cells, cellular candidates for additional mutations grow in number. Mutations continue to accumulate and are copied to descendant cells. If one cell finally acquires enough mutations to become cancerous, subsequent cancer cells will be derived from that one single transformed cell. So all tumors are clonal, which means that they originate from a single parent cell, whether that first mutant cell was of germline or somatic origin.

• Tumors are clonal –> That is they start from a normal cell which acquires a mutation
• If there is only a single mutation then that cell may continue to divide and only carry that single mutation.
• Cancer doesn’t arise from a single mutation! Cancer cells acquire multiple mutations and as they begin to acquire mutation things don’t go bad right away
• There is a critical point in which the cancer cells will create “cancer”
• Cancers don’t arise from one single mutation, they acquire multiple mutations. And as they acquire mutations, they don’t necessarily go bad.
• Clonal simply means that they share a common ancestor
• Such an example is seen with the philadelphia chromosome in which all the tumor cells had this mutation in common, showing that they all came from this.

One thing to keep in mind is that although cancers are clonal in nature, they are heterogeneous, meaning they are different from tumor cells on the other side of the tumor generally.

Question 27

How many cells does it take for a tumor to be palpable? Cause death?

Answer 27

-It takes a long time for a tumor to show up because it has to undergo many cell divisions in order for the cancer cells to accumulate.
-10^9 cells the tumor becomes palpable
10^12 cells, it generated death
-Tumor cell has to undergo massive amounts of cell division which gives it an advantage but it takes a long time

Question 28

Genetic instability is an enabling hallmark of cancer. Explain the mechanisms of genetic instability, including how changes in telomerase activity lead to genetic instability and immortality

Answer 28

Genomic instability in cancers. As cells age, they decrease the expression of telomerase and increase the expression of p53, which leads to irreversible senescence. In cancer cells, there is often a loss of p53, allowing fro mutant cells to continue to proliferate. This leads to a condition where chromosomes fuse then break-resulting in chromosome loss and translocations. Telomerase is reactivated in some of these cells leading to stabilization of the aberrant chromosomes and continued proliferation.

• Lets start by looking at a normal endothelium cell which undergoes repeated cell division.
• So what happens in many normal cells is that telomeres shorten and eventually uncap which leads to p53 cell cycle control and senescence which means the cells undergo an irreversible stage of shutting down and stop dividing
• The loss of p53 though allows the cells to grow and proliferate. Because they lack p53 they can continue to divide and cant fix all the mutations that are occurring, thus acquiring different mutations which is referred to as chromosome breakage fusion-bridge cycle.
• JUST KNOW THAT YOU UNDERGO A PROCESS OF ADDITIONAL CHROMOSOME BREAKAGES WHICH LEAD TO CHROMOSOMAL INSTABILITY
• If the damage is too severe, in some cases the cell will die.
• In some cancers, telomerase is reactivated and now the cancer cells can continue to divide and keep the chromosomes.
• High expression of telomerase leads to immortality

-Just know that p53 has a role in chromosome instability and p53 plays an important role in this. Also know that telomerase allows these cancer cells to survive even with unstable chromosome due to the upregulation of telomerase

Question 29

Explain loss of contact inhibition by cancer cells

Answer 29

Transformed cells (grown in culture) are abnormal in shape, motility, responses to growth factors, and loss of contact inhibition. This loss of contact inhibition leads to this rounding up of cancer cells because they no longer associate with their neighboring cells

Question 30

Understand the concept of the Warburg Effect in altered glucose metabolism associated with cancer cells

Answer 30

• One of the ways that cancer cells do continued growth and division is through an altered metabolism.
• Normally cells have a very low metabolic rate when they have stopped dividing.
• In nonproliferating cells, O2 is present and glucose is converted to pyruvate in glycolysis. Then the pyruvate mainly goes to oxidative phosphorylation to generate energy, CO2 and H2O. Only a limited amount goes to form lactate.  THIS IS OXIDATIVE PHOSPHORYLATION

-In a cancer cell though, or in early development and highly proliferating tissues, a slightly different process occurs because of the massive intake of glucose. The glucose is converted to pyruvate in glycolysis but only a small fraction of pyruvate is shunted into oxidative phosphorylation! Majority goes to form lactate. You need the cell to produce more and more molecules, energy, building blocks in order for the cell to continue to proliferate. WANT TO PRODUCE THE BUILDING BLOCKS NECESSARY FOR CELL GROWTH!!!

• This is known at the Warburg Effect where cancer cells undergo this aerobic glycolysis and convert massive amounts of glucose to lactate.
• This was postulated in 1974 and Warburg said that this was the main cause of cancer, that these cells will have defective mitochondria and as a result will use this process instead but Warburg wasn’t really correct. This lactate process just seems to take over but the other process still occurs.
• If the cancer cell shuts down the mitochondria, it will block the intrinsic pathway of apoptosis. Now there are drugs being tested that reactivate the mitochondria to reactivate the mitochondria to reactivate apoptosis
• Normally cells have a low metabolic rate
• In a cancer cell or in highly proliferating tissue, the glucose uptake is massive. Also, a vast amount of the product goes into lactate. Converts glucose to lactate.
• The oxidative phosphorylation process still occurs in the mitochondria

-Just know it occurs in cancer cells and it is an effect on metabolism that gives cancer cells an advantage by creating building blocks. Lactate will be broken down to make other things

Question 31

Explain the role of laminin receptors in the three step mechanism for basement membrane invasion by tumor cells

Answer 31

Three-step hypothesis for tumor cell invasion across basement membrane. Receptors on the surface of tumor cells bind to laminin protein in basement membrane. This induces the secretion of pro-collagenases and other proteolytic enzymes (i.e., uPA or urokinase plasminogen activator), which degrades the collagen and other proteins of the basement membrane. Tumor cell then migrates through the dissolved region of the matrix. The reiteration of the process leads to the invasion of the metastasizing tumor cell through the extracellular matrix and basement membranes in metastasis Non-transformed endothelial cells in angiogenesis invade by a similar mechanism.

To invade through basement membrane, cell expresses the protease enzymes procollagenases and urokinase plasminogen activator (uPA). uPA activates plasminogen (expressed by liver and ubiquitously located in the extra-cellular matrix (ECM)). uPA activates the plasminogen in extracellular matrix by hydrolyzing a central peptide bond that gives the active protease enzyme plasmin. Plasmin will degrade the ECM directly and also activate procollagenase to collagenase. The collagenase is specific to type IV collagen of basement membranes.

• Part of the way it does this is because the tumor cells have a lamin receptor and they bind to lamin which is on the basal lamina. Then the tumor cells release a collagenase which can allow it to break through the basal lamina and allow the cell to move through it.
• In this case, the tumor cells themselves are secreting the enzyme necessary to break through the basal lamina beginning with receptors on the basal lamina
• They need laminin receptors

Question 32

Describe the contribution of EMT to cancer progression and metastasis

Answer 32

The cells will undergo a EMT shift via the TGF-beta and Wnt (Epithelial to Mesenchymal shift) to become more de-differentiated and this will allow the cells to metastasize. Then, once they reach a new location, they undergo and MET shift to become more differentiated again

Progression from normal epithelium to invasive carcinoma goes through several stages. The invasive carcinoma stage involves epithelial cells losing their polarity and detaching from the basement membrane. The composition of the basement membrane also changes, altering cell-ECM interactions and signaling networks. The next step involves EMT and an angiogenic switch, facilitating the malignant phase of tumor growth. Progression from this stage to metastatic cancer also involves EMTs, enabling cancer cells to enter the circulation and exit the blood stream at a remote site, where they may form micro- and macro-metastases, which may involve METs and thus a reversion to an epithelial phenotype.

• Primary cancers occur in the normal epithelial and lose their contact inhibition as well as by the extracellular matrix and basal lamina.
• They acquire metastatic properties by undergoing EMT  They actually change their whole program of gene expression and allows them to escape their normal barriers.

The subset of cells in a tumor that metastasize go through an epithelial to mesenchymal state transition. In the mesenchymal state the cell acquires a stem-like de-differentiated phenotype. In order to colonize its target tissue the metastatic cell reverses its phenotype by transitioning back to a more differentiated epithelial state. EMT, epithelial to mesenchyme transition; MET, mesenchyme to epithelial transition.

• How do cancer cells undergo these transitions from being benign to metastatic?
• There is actually a physical process that has to occur  EMT (epithelial to mesenchymal transition)
• Cancer cells start in an endothelial state and undergo EMT which takes them from a more differentiated state to a less differentiated state (mesochymal)
• This allows the cells to get more motility

Cells lose E-cadherin in their plasma membrane  loss of the ability to form adheren junctions
Cells lose cell-cell and ECM contacts, round-up, and acquire a motility.
They activate a series of kinases or kinase cascades. Activation of PI3K/AKT and classical RAS/MAPK pathways. Pluripotent transcription factors activated which give the cancer cells the ability to de-differentiate and allow the cells to acquire a more stem cell like state
(Slug, Snail, Twist, Nanog, FoxC2 , Myc).
Increased protease secretions (urokinase plasminogen activator and type IV procollagenase) coupled
with decreased protease inhibitor and extracellular matrix secretions.

2 Points:
They undergo the EMT transition.
They undergo the reverse process once they get to their new location! MET occurs then!

These cells have the ability to undergo both!!!

• They are becoming less differentiated via EMT
• TGFB and Wnt stimulate EMT to get epithelial state to mesenchymal state

Question 33

What are two ways cancer cells avoid senescence?

Answer 33

1) Activate or maintain telomerase: Telomeres do not shorten during cell growth
2) Inactivate p53 to block senescence: p53, when activated, is a pro-apoptotic response which creates BH3-only proteins.

Question 34

Describe the steps and barriers to the metastatic process

Answer 34

Metastasis accounts for 90% of cancer associated deaths. Metastasis is a multi-step process involving invasion of local tissues, movement through the circulation, leaving the vessels, establishing new cellular colonies at distant sites. Only a small portion of invasive cancer cells can function as founders of metastases (less than one in one million cells).

• This is the process of metastasis
• There is an initial growth of a benign tumor in the epithelial cells and they continue to divide
• At some point they acquire invasive properties and invade the capillaries
• Now they can move throughout they body.
• At a certain point, they can adhere to blood vessel walls in the body and then they can escape from the blood vessel to form a micrometastasis
• Where the tumor began in one part of the body, it has now moved and invaded somewhere else and is now in a new microenvironment and can form a second tumor in a different location

Studies of labeled tumor cells leaving a tumor site, entering the circulation, and establishing metastases show which steps in the metastatic process, outlined in Figure 20–16, are difficult or “inefficient,” in the sense that they are steps in which large numbers of cells fail and are lost. It is in these difficult steps that cells from highly metastatic tumors are observed to have much greater success than cells from a nonmetastatic source. It seems that the ability to escape from the parent tissue, and an ability to survive and grow in the foreign tissue, are key properties that cells must acquire to become metastatic. (Adapted from A.F. Chambers et al.,Breast Cancer Res. 2:400–407, 2000. With permission from BioMed Central Ltd.)

• In considering the whole process of metastasis, it is not a simple process
• There are easy parts and hard parts
• Only about 1 in 1,000,000 cancer cells that invade can go one to form and metastasize at another place. But it only takes a couple of cells to do this!
• Out of all of the cells it doesn’t take that many to go out and metastasize.
• It is difficult for the cancer cells to invade the blood vessels  They have to acquire the ability to secrete these molecules in order to invade the barriers that hold the cells where they are. That is a difficult process to occur which is why it typically takes a while to see metastasis occur.
• However, once they get into the blood vessels or lymphatic system they can survival pretty well and exit in a new location easily because they already have these enzymes and such. This is easy to do
• Once they get to the new site though, it is not easy to form a new microcolony! They first have to be able to survive in the foreign tissue which is not as clear cut. And they have to begin to grow and form microcolonies and then invade the normal constraints of growth in the new location

Question 35

Explain how invasion in angiogenesis by non-transformed endothelial cells has a mechanistic similarity to invasion by metastatic cells

Answer 35

In both cases, the cell secretes uPA (urokinase plasmingen activator) which will cleave the plasminogen to plasmin which can then degrade the ECM and also cleave procollagenase to collagenase.

Question 36

Know how cancer cells signal to epithelial cells to initiate new blood vessel growth

Answer 36

Tumor cells express Fibroblast Growth Factor (FGF) and Vascular Endothelial Growth Factor (VEGF) which cause endothelial vascular cells to increase expression of proteases and inhibit expression of protease inhibitors. This assists in the invasion process.