Chapter 8: Stem cells and differentiation (Secondary details)

Question 1

All cells contain the exact same amount of DNA. So how come we have different type of cells in our body?

Answer 1

Because of the expression of genes (by transcription factors and epigenetic factors)

Question 2

What are different epigenetic modifications?

Answer 2

• Transcription factors
• DNA methylation
• Histone modification
• Chromatin openness

Question 3

Stem cell therapy is already proven useful in a couple of diseases. What are they?

Answer 3

One marrow transplantation, skin injury, stroke, heart attack, neurodegenerative diseases and diabetes

Question 4

What are the Yamanaka factors?

Answer 4

Oct3/4, Sox2, Klf4 and c-Myc

Question 5

What are down-sides of embryonic stem cells?

Answer 5

It is harvested from a human embryo, so there are ethnic discussions. There are also not many embryo’s available, which can be an issue

Question 6

What are the advantages of Nuclear Transfer Embryonic Stem (NT-ES) cells relative to e.g. embryonic stem cells?

Answer 6

The cells are derived from the patient self, so there is no issue of immune responses/rejections (that might occur with embryonic cells). Also, lot’s of stem cells can be generated so there is no issue of having too little available

Question 7

In 2012 John B. Gurdon and Shinya Yamanaka received a Nobel Prize in Medicine. Why did they win this prize?

Answer 7

For the discovery that mature cells can be reprogrammed to become pluripotent

Question 8

What did John B. Gurdon discover?

Answer 8

John B. Gurdon discovered in 1962 that the specialisation of cells is reversible. In a classic experiment, he replaced the immature cell nucleus in an egg cell of a frog with the nucleus from a mature intestinal cell. This modified egg cell developed into a normal tadpole. The DNA of the mature cell still had all the information needed to develop all cells in the frog.

Question 9

What did Shinya Yamanaka discover?

Answer 9

Shinya Yamanaka discovered more than 40 years later, in 2006, how intact mature cells in mice could be reprogrammed to become immature stem cells. Surprisingly, by introducing only a few genes (the Yamanaka factors), he could reprogram mature cells to become pluripotent stem cells, i.e. immature cells that are able to develop into all types of cells in the body.

Question 10

Stem cells are long-life targets compared to differentiated cells. So therefore, accumulation of mutations is more likely to happen in stem cells. How long does it take for the skin to ‘create’ a malignant skin cancer?

Answer 10

18 months

Figure: Diagrammatic representation of skin epithelial histology. Turnover rate of epithelial cells is 60 days

Question 11

What happens in the crypt base columnar (CBC) cells in the colon if you block the WNT pathway?

Answer 11

The cells will differentiate (because they lose their ‘stemness’-feature)

Question 12

True/false: AML is a disease that occurs more often in young patients

Answer 12

False, it is a disease of ‘the elderly’ that increases with age

Question 13

Patients with AML receive combination chemotherapy. What is the complete remission rate (>5% of leukemic cells) of this therapy?

Answer 13

80%. So there is relapse/refraction of 20%, which is very hard to treat

Question 14

What does this graph show?

Answer 14

That the survival of AML patients decreases with increasing cancer stem cell frequencies (LSC = leukemic stem cells)

Side note: CD34+ and CD38- are used as immunophenotypical stem cell markers here

Question 15

Is the retinoid acid treatment limited to PML-patients?

Answer 15

No, recently it was discovered that also other subtypes of AML that overexposes EVI-1 are sensitive to retinoid acid

Question 16

Some stem cells are continually active to replace cells (1), while other stem cells remain dormant until a physiological signal is received (2). Name an example for each type

Answer 16

1: Hematopoietic stem cells (HSCs)
2: Hair follicle stem cells (in response to a wound), breast stem cells (in response to pregnancy hormones)

Question 17

It was found that the proportion of brain CSCs identified in a variety of brain cancers correlates with the course of the disease or prognosis. Can you explain that by use of an example?

Answer 17

Fast-growing tumors such as glioblastomas had more brain CSCs than slow-growing tumors like astrocytomas

Question 18

What happens in mice when, by means of the knock-out procedure, Wnt-regulated transcription factor is deleted?

Answer 18

The resulting phenotype is a lack of stem cells in the intestines

Question 19

If possible, try to draw the pathway of Wnt.

Answer 19

Pathway in absence of Wnt

1. The protein ß-catenin is bound by a ‘destruction complex’, which includes protein kinases CK1y and GSK-3ß
2. The protein kinase phosphorylate ß-catenin, targeting it for ubiquitination and degradation in the proteasome (= there is no ß-catenin free to move into the nucleus)
3. Trancriptional co-repressors bind to TCF transcription factors
4. Prevention of the expression of certain genes

Pathway in presence of Wnt

1. The Wnt protein binds to the transmembrane receptor protein Frizzled
2. This causes a signal to be transmitted across the membrane by Frizzeld and LRP, activating them
3. Activation of Frizzled and LRP cause protein kinases CK1y and GSK-3ß (from the destruction complex) to assiciate with the membrane
4. The protein kinases phosphorylate the tail of the activated LRP
5. Next, the intracellular signalling protein Dishevelled and the protein Axin are recruited to the cytoplasmic tails of LRP and Frizzled
6. This prevents the formation of the destruction complex, meaning ß-catenin accumulates in the cytoplasm
7. ß-catenin moves into the nucleus and binds to TCF, displacing the co-repressors
8. Target genes are expressed

https://www.youtube.com/watch?v=oweNT288BXo

Question 20

Is Wnt a suppressor gene or a proto-oncogene?

Answer 20

Proto-oncogene

Question 21

Which two events were shown after the APC-gene was deleted in long-lived intestinal stem cells?

Answer 21

Adenomas were induced within several weeks (1) and the progenitor transit-amplyfing cells or differentiated cells did not trigger tumor formation (2). (meaning that a small subset of cells is able to self-renew and maintain a tumor)

Question 22

True/false: Resoring APC levels in colorectal cancer can revert them to functioning normal cells

Answer 22

True, this was proven in vivo. Even when the cancer cells contain other oncogenic mutations such as K-ras and p53!

Question 23

Mutations in the Wnt signaling cascade can also promote other types of cancers. What type of cancers do each of these mutations lead to?

1. Activating mutations of ß-catenin that effects eh regulatory sequnces essential for its targeted degradation: ____
2. Mutations in the axin gene: ____

Answer 23

1. Activating mutations of ß-catenin that effects eh regulatory sequnces essential for its targeted degradation: skin tumors
2. Mutations in the axin gene: hepatocellular carcinoma

Question 24

True/false: The Hedgehog (Hh) signaling pathway is mostly active in adults

Answer 24

False, it is mainly inactive (except for its function in tissue repair and maintenance)

Question 25

Can you give a simplified overview of the Hedgehog pathway?

Answer 25

SMO is the key transducer of the Hh pathway. **In the absence of the Hh ligands**, the putative Hh receptor PTC is localized in the cilium and inhibits SMO signaling. Gli molecules are processed with the help of Su(Fu)/KIF7 molecules into repressor forms, which deactivate the Hh signaling pathway. **In the presence of Hh**, Patched is displaced out of the cilium and unable to inhibit SMO. Hh reception facilitates conformational changes in SMO, promoting Gli activation (GliA) and stimulation of Hh target gene expression. Su(Fu) and KIF7 can inhibit this process.

Question 26

FIll in: ___ mutations in Patched and ___ mutations in Smoothened were identified in sporadic human BCC (skin) tumors, and Gli-1 expression was found in ___ BCCs.

Answer 26

**Inactivating** mutations in Patched and **activating** mutations in Smoothened were identified in sporadic human BCC (skin) tumors, and Gli-1 expression was found in **nearly all** BCCs. *Note: all sporadic BCCs posess an activated Hh signaling pathway*

Question 27

The role of Hh signaling has been extended to chronic myeloid leukima (CML). Can you explain some of their findings *just roughly read it, don't think it's that necessary*

Answer 27

Loss of Smoothened inhibits HSC (hematopoietic stem cell) renewal and decreases the induction of CML by the oncogenic BCR-ABL chromosomal translocation. It was also shown that inhibition of the Hh pathway inhibits the growth of Gleeve-resistant (imatinib) mica and human CML

Question 28

The degree of differentiation of a cell may also affect teh outcome of oncogenic activation. Can you explain the study where this was shown?

Answer 28

The use of selective gene promoters to drive the expression of a Ras oncogene in different cell populations with different degrees of differentiation (e.g.. stem cells or committed progeny cells) resulted in tumors with different malignant potential (e.g. malignant carcinomas vs. benign papillomas). ## Footnote *This suggests that that the activation of an oncogene may be carcinogenic in some states of differentiation, and not in others, within a particular cell lineage (more research is needed)*

Question 29

Fill in: Transformed **committed** **progenitor/stem** cells are likely to be _malignant_, while transformed **committed** **progenitor/stem** cells are likely to be _benign_

Answer 29

Transformed **stem** cells are likely to be _malignant_, while transformed **committed** **progenitor** cells are likely to be _benign_

Question 30

Stem cell express high levels of ATP-binding casette (ABC) transporters, members of the mulit-drug resistance gene family. How can this be used in the clinic?

Answer 30

The ability of the ABC transporters in stem cells to inhibit the accumulation of fluorescent dyes (Hoechst 33342 and rhodamine 123) provides a means of helping to sort stem cells. Anso. therapeutic strategies invloving the administration of ABC inhibitors, along with chemotherapies are being investigated ## Footnote *Note: the populations of tumor cells that do not accumulate these dyes are referred to as 'side population' cells*

Question 31

What mechanism in the Wnt pathway can be targeted for targeted therapy?

Answer 31

Disruption of the protein-protein interaction between ß-catenin and the Tcf transcription factors

Question 32

In what type of cancers has the inhibitition of the protein-protein interaction between ß-catenin and the Tcf transcription factors proven effective (PLEASE note that this is studied very limitedly)

Answer 32

* Colorectal cancer: Tcf inhibition induces the differentiation of colerectal cells into epithelial villi, and also inhibition of expression of two Tcf target genes and inhibited proliferation was proven (from 2 studies) * Liver cancer (pre-clinical) * Leukemia (pre-clinical) ## Footnote *These drugs have a greater chance of tumor eradication, rather than just tumor regression (because they target a molecular pathway that is important in self-renewal)*

Question 33

Fill in: Sprouting of pre-existing vessels requires major reorganization involving ___ of the mature vessel, ___ and ___ of endithelial cells, and \_\_\_.

Answer 33

Sprouting of pre-existing vessels requires major reorganization involving **destabilization** of the mature vessel, **proliferation** and **migration** of endithelial cells, and **maturation**.

Question 34

Can you explain what happens in the VEGF-A signal transduction pathway?

Answer 34

The VEGF-A signal transduction pathway. One molecule of VEGF-A binds to two VEGFR-2 receptors, facilitating dimerization and autophosphorylation. Proteins contains SH2 domains (shaded in grey) bind to the phosphorylated receptro and trigger activation of RAS adn the Taf-MEK-MAPK cascade. In addition, PI3K is activated. AKT leads to inhibition of apoptosis. AKT also stimulates endothilial nitric oxide (NO) synthase and stimulates vascular permeability via NO production. Src is one of several other molecules that are activated by VEGFR-2

Question 35

Explain how each of these angiogenic inhibitors can be activated: * Plasminogen * Angiostatin * Endostatin

Answer 35

* Plasminogen can be cleaved by proteinases, including several MMPs, to release the angiogenic inhibitor engiostatin * Angiostatin binds to its endothelial cell surface receptor, annexin II, to exert its inhibitory effects * Endostatin is a fragment of collagen XVIII and can be proteolytically released by elastase and cathepsin. It blocks MAPK activation in endothelial cells and also MMPs

Question 36

It has been observed that sometimes, when a tumor is removed by surgery or irradiation, dormant metastases are often activated, and growh and angiogenesis are initiated. How is this penomenon been termed?

Answer 36

**Concomitant resistance** *Evidence suggests that the production of angiogenic inhibitors, such as angiostatin and endostatin, by certain tumors prevents the growth of remote micrometastases via the blood. When the primary tumor is removed (along with it's inhibitors) the angiogenic switch is activated for the micrometastases*

Question 37

Which type of cancer is characterized by increased HIF-1 activity and how?

Answer 37

**Kaposi's sarcoma**, a highly vascularized tumor that is caused by a herpesvirus. THree protein products of the viral genome increase HIF-1a half-life, nuclear localization, and transactivation under non-hypoxic condition, thus mimicking the effects of hypoxia

Question 38

\_\_% of tumor endothelial cells originated from circulating endothelial progenitor cells (**vasculogenesis**)

Answer 38

40

Question 39

Aberrant production of growth factor can act in a paracrine manner to stimulate growth of endothelial cells. Which "star" oncogenic proteins have been shown to upregulate the "star" angiogenic inducer VEGF?

Answer 39

Receptor tyrosine kinases (e.g. EGFR), intracellular tyrosine kinases (e.g. Src), intracellular transducers (e.g. Ras) and transcription factors (e.g. Fox, Jun)

Question 40

Anti-angiogenic drugs are cystostatic, rather than cytotoxic. What does this mean for the duration of the treatment?

Answer 40

That long-term continuous administration is needed

Question 41

One strategy (for targeting VEGF/VEGFR) involves targeting angiogenic factors such as VEGF. Explain how this works

Answer 41

Bevacizumab (Avastin) is a recombinant humanized monoclonal antibody that recognizes VEGF-A and was first approved for treatment of colorectal cancer. Its use has broadened to several other cancers

Question 42

Earlier we talked about Bevacizumab (Avastin), which is an antibody that recognizes VEGF-A. The drug failed to show a consistent effect on ovorall survival in breast cancer trials (but it is used in e.g. colorectal cancer). Why was a different response observed in the two different cancers? ## Footnote *I don't think you have to know this but personally thought it was very interesting so enjoy :)*

Answer 42

The answer lies in the angiogenic swtich: colon tumors are more dependent on VEGF for the induction of angiogenesis, while this is true only for the early stages of breast cancer. Advanced breast cancer utilizes a broader arsenal of angiogenic inducers, thus inhibition of just one inducer does not have an effect

Question 43

One strategy (for targeting VEGF/VEGFR) involves creating a fusion protein that acts as a decoy to prevent VEGF from interacting with its receptor. How does this work?

Answer 43

A drug called Aflibercept consists of VEGF-1 and VEGF-2 ligand-binding domains fused to an IgG constant region (Fc), and can therefore bind more than one VEGF ligand. Aflibercept was approved for colorectal cancer ## Footnote *(we discussed Bevacizumab/Avastin earlier, which can only bind to VEGF-A)*

Question 44

One strategy (for targeting VEGF/VEGFR) involves antibodies that target against the extracellular domain of growth factor inhibitors. Can you give an example?

Answer 44

Ramucirumab, a fully humanized antibody target the extracellular domain of VEGFR-2, and has been approved for advanced gastric cancer

Question 45

One strategy (for targeting VEGF/VEGFR) involves a small-molecule tyrosine kinase inhibitor. Explain a few and how they work

Answer 45

The mechanism of action is that it inhibits receptor autophosphorylation Semaxanib was the first VEGFR-inhibitor to enter phase III clinical trials, but is not used anymore. Axitinib, sunitinib and sorafenib have been approved for the treatment of advanced renal cell carcinoma. All three drugs target both VEGFRs and PDGF-Rs *Intersting to know: there are other multi-kinase inhibitors, whose targets include VEGFR and FGFR or VEGFR and Tie, which have been approved (very effective, probably because they target multiple angiogenic regulators, next to VEGF)*

Question 46

In this figure you see the anti-vascular effects of combretstatin on tumor neovasculature. Can you explain what is seen

Answer 46

(a) shows the primary tumor before, and (b) shows the tumor after treatment with combretastatin. Strong anti-vascular effects are seen in the core of teh tumor after treatment, but a small, viable rim of tumor tissue can be seen at the periphery. Regrowth of tumor cells from the rim indicates the need for combination therapy (with ionizing radiation/chemotherapy)

Question 47

Nanoparticle technology, drug delivery and vascular targetin have come together in an exciting report with hings at future applications in tumor neovasculature. What is this report? ## Footnote *Really don't think this is relevent, however almost halve a page of the book is dedicted to this so that's why i made a card about it*

Answer 47

Knowledge of several biochemical areas has been combined specifically to target a drug to the neovasculature of tumors in mice. Liposome-based nanoparticles were coated with (NGR) peptides as ligand to a tumor endothelial cell marker (animopeptidase N). These tagged liposomes encapsulated a promising drug for neuroblastoma. IV-delivery to neuroblastoma-bearing mice resulted in living longer than controls and lower toxicity was demonstrated

Question 48

How are peptides that target av integrins that are specifically expressed on tumor endothelial cells used in the clinic? ## Footnote *Honestly, i would skip this, not that relevant in my opinion*

Answer 48

To enhance the penetration of drugs into a tumor in mice, without coupling of the drug to the peptide or other carrier (the peptide binds to the integrins and is cleaved). This strategy targets tumor vasculature in combination with cancer drugs and enhances tumor-specific delivery in mice may increase the therapeutic index of already approved cancer drugs for humans