Cancer Stem Cells

Why study cancer cells and stem cells synonymously?

There are similarities between their characteristics.

Why is cancer unlikely to originate from mature differentiated cells?

Mature cells do not proliferate (cancer is a disease of proliferating cells) and have a short/finite lifespan, making it unlikely for them to accumulate the >3 mutations required to become cancerous (cancer is caused by an accumulation of mutations within the cell).

What is the clonal cancer theory?

Cancer originates from a single cell that has acquired mutations, leading to abnormal proliferation.

If tumors are clonal in origin, why do they exhibit cellular heterogeneity in differentiation?

Tumours originate from a single mutated cell (clonal origin), but they display heterogeneity in differentiation due to the presence of cancer stem cells (CSCs). CSCs possess the ability to self-renew and differentiate into various cell types within the tumor, leading to a hierarchy of cells with diverse appearances and functions.

Why can only a small number of tumor cells recolonize a tumor, and how does this support the cancer stem cell (CSC) hypothesis?

Only cancer stem cells (CSCs) possess the ability to self-renew and differentiate into multiple cell types within the tumour. Most tumor cells are more differentiated and lack long-term proliferative capacity. The fact that only a small subset of cells can initiate new tumours supports the CSC hypothesis, which proposes that CSCs are solely responsible for sustaining and regenerating the tumour.

How could stem cells be the origin of cancer?

Stem cells are long-lived, capable of self-renewal (explain the proliferative nature of tumours), and proliferate asymmetrically, allowing mutations to accumulate and cause tumour heterogeneity.

What is the Cancer Stem Cell (CSC) hypothesis?

The CSC hypothesis proposes that a small population of cancer cells with stem-like properties is responsible for tumour initiation, maintenance, and relapse. Most of the mutations occur in the CSCs and are then passed down onto daughter cells.

Explain de-differentiation.

Specialised cells revert to a more primitive, stem-like state through gene expression, acquiring characteristics such as increased self-renewal and the potential to contribute to tumour formation.

How does the stem cell niche influence CSCs?

Mutations in the niche can promote CSC expansion, with secondary niches failing to suppress proliferation as effectively as primary niches.

How do CSCs arise?

Expansion of the niche (supports more SCs and thus more progeny), CSCs adapt to a different niche allowing their expansion if the secondary niche doesnʻt suppress proliferation as well, niche-independence (self-renewal is cell-autonomous), failure to switch off proliferation pathways in progenitor cells as they differentiate.

What historical breakthroughs supported the CSC theory?

In 1997, CD34+CD38- leukemia stem cells were identified in AML. In 2003, CD44+CD24-/low CSCs were found in breast tumors, supporting CSC presence in solid tumors.

What evidence supports tumour heterogeneity in CSCs?

Tumours show varied surface markers (Both CD38±) and a range of differentiation (a continuum of stemless), indicating phenotypic plasticity and microenvironmental influence.

Can differentiated cells become CSCs?

Differentiated cells can dedifferentiate due to mutations in growth/developmental signalling pathways (e.g., Wnt pathway) or environmental factors (e.g. hypoxia-HIF), cytokines).

What transcription factors are associated with CSC formation through dedifferentiation?

Oct4, Sox2, KLF4, Nanog.

What cells in the TME have the ability to drive dedifferentiation?

CAFs, MSCs, MDSCs, M2 macrophages.

Can epigenetic changes drive dedifferentiation?

Yes, promoter demethylation of TFs can reactivate promoter regions and facilitate dedifferentiation in hepatocellular cancer cells.

Which signalling pathways are involved in dedifferentiation?

WNT, HIF, NOTCH (Stat3 and SMAD4 activation), TGF-B (EMT), JAK/STAT, ERK inhibition, PI3K/AKT.

What is a common features of all signalling pathways involved in dedifferentiation?

All target gene expression.

Why is AML an important model for studying dedifferentiation?

AML develops in a system where HSCs differentiate via progenitors regulated by transcription factors (TFs). Mutations in TFs like PU.1 (early & late myeloid) and C/EBPα (granulocytes) block differentiation, causing cells to revert to or remain in stem-like states. Chromosomal translocations disrupt differentiation genes, leading to cellular heterogeneity as cells get "stuck" at various stages. This makes AML ideal for studying how dedifferentiation drives cancer.

Where was the Wnt pathway discovered?

Stem cells.

What happens in the absence of Wnt?

GSK3 (part of the inhibitory complex) phosphorylates B-catenin and makes it for degradation.

What happens in the presence of Wnt?

Wnt binds to Frizzled/LRP receptor complex, which sequesters axon and breaks apart the inhibitory complex. GSK3 cannot phosphorylate B-catenin, which translocates to the nucleus to remove TCF4 suppression, recruits Bcl9/pygopus and drives expression of cyclin D (cell cycle re-entry).

What happens in the absence of Hh?

Patched suppresses smoothened.

What happens in the presence of Hh?

Releases the suppression of smoothened and activates the Gli transcription factors which can translocate into the nucleus and activate transcription of target proliferation genes.

How does the Wnt pathway regulate normal intestinal stem cells?

In the normal intestinal crypt, Wnt signaling maintains the stemness and self-renewal of LGR5+ intestinal stem cells. This is important in intestinal tissue as it is highly regenerative (differentiate as they move up the villi to replace the dead cells at the top).

How does Wnt pathway dysregulation contribute to colorectal cancer (CRC)?

Mutations in APC or β-catenin lead to constitutive activation of Wnt signalling, even without Wnt ligands. This causes continuous stabilisation of β-catenin, driving sustained expression of stemness-associated genes and promoting tumourigenesis.

How is TGF-β signaling dysregulated in colon cancer?

TGF-β signalling is often mutated or disrupted, leading to the loss of its tumour-suppressive effects/SMAD4 and promoting tumour progression and conversion of cancer cells into CSCs. TGF-β can also promote epithelial-mesenchymal transition (EMT) through the activation of TWIST1.

How does NF-κB contribute to the dedifferentiation of villus cells?

It can enhance Wnt signalling (increase the expression of key Wnt pathway components). Side note: β-catenin physically interacted with and inhibited NF-κB function in human colon cells.

How is gli involved in CRC?

EMT is regulated by Gli1, which upregulates Snail, Slug. The Hedgehog pathway is normally inactive in adult tissues. Also, upregulate genes like Cyclin D and Myc.

What pathway initiates colon cancer growth?

Wnt pathway/loss of Smad4 drives dedifferentiation of intestinal epithelium to stem cell-like status.

Why does APC mutation not cause tumors in progenitor or differentiated intestinal cells?

These cells lack self-renewal capacity and are epigenetically committed, so they cannot sustain tumor growth despite WNT activation.

Why is APC mutation considered an early trigger in colorectal cancer?

Because it transforms normal intestinal stem cells into cancer stem cells by activating WNT signaling in a context-dependent manner.

What are polycomb proteins also called?

Guardians of stemness.

What are polycomb proteins?

Epigenetic regulators of gene expression. They repress tumour suppressor genes (e.g., silence the INK4a/ARF locus which encodes p16 and p14^ARF) by PRC2).

Why are polycomb proteins involved in cancer?

This silencing mimics a gene mutation as the tumour suppressor is functionally lost.

What are the 2 polycomb complexes?

PRC1 (Bmi) and PRC2 (EZH1, SUZ12)

What is the role of PRC2?

Adds methyl groups to histones at the tumour suppressor gene promoter. This histone mark acts as a repressive signal and recruits PRC1 (has histone methyltransferase activity - adds three methyl groups to histone H3 at lysine 27).

What is the role of PRC1?

PRC1 modifies chromatin leading to tight chromatin packing (heterochromatin) and blocked transcriptional machinery (adds ubiquitin to histone H2A at lysine 119).

What is the function of BMI-1 in DNA repair?

BMI-1 promotes repair of DNA double-strand breaks via homologous recombination.

How does BMI-1 regulate stemness and tumor progression?

It promotes self-renewal (SR) in both normal and cancer cells by repressing tumor suppressor gene p16 through chromatin modification.

What is the significance of BMI-1 overexpression in cancer?

BMI-1 is overexpressed or amplified in many cancers, including leukaemias, contributing to uncontrolled proliferation and maintenance of cancer stem cells. DNA repair means that damaged DNA can be passed onto daughter cells so they are more likely to acquire mutations.

What happens in vitro when BMI-1 is lost in AML cells?

AML cells arrest in G1 phase and undergo differentiation, losing their self-renewing capacity.

What is the effect of BMI-1 knockout in vivo in healthy animals?

There is a depletion of blood cells due to loss of hematopoietic stem cell (HSC) self-renewal.

What happens in vivo when BMI-1 is knocked out in AML models?

Leukemic cells are significantly reduced in circulation, suggesting BMI-1 is essential for leukemia maintenance.

What role do Polycomb group (PcG) proteins play in regulating BMI-1?

PcG proteins repress BMI-1 to regulate stem cell self-renewal and prevent oncogenic transformation.

What is the role of PML in normal cells?

PML promotes p53 activity, leading to cell cycle arrest and apoptosis.

What happens when RAR dimerizes with RXR in normal cells?

RAR and RXR form a dimer that regulates gene expression involved in differentiation.

What happens in the absence of retinoic acid (RA)?

HDAC is recruited to the RAR-RXR complex, leading to gene repression and no differentiation (chromatin compaction), promotes proliferation.

What happens in the presence of retinoic acid (RA)?

Coactivators are recruited to the RAR-RXR complex, leading to transcription of differentiation genes and promotion of differentiation.

What causes Acute Promyelocytic Leukemia (APL)?

The fusion of PML protein to RAR, forming the PML-RAR fusion protein, resulting from a chromosomal translocation.

What does the PML-RAR fusion protein do?

PML-RAR recruits HDAC and represses differentiation even in the presence of retinoic acid (RA), blocking differentiation and promotes constitutive proliferation.

How does PML-RAR affect p53 function?

The p53 function of PML is disrupted by PML-RAR, leading to no cell cycle arrest or apoptosis.

What is the consequence of the PML-RAR fusion protein in APL?

The PML-RAR fusion protein causes repression of differentiation even in the presence of retinoic acid, disrupted p53 function, and oncogenesis, leading to the development of APL.

What is the actual situation regarding the differentiation of cells in cancer?

The actual situation is likely a continuum of cells at varying stages of differentiation. CSC-like cells are thought to be hierarchical and capable of self-renewal, differentiation, and propagation of the tumour. Accumulated mutations and epigenetic changes are likely to alter differentiation pathways in cancer cells.

What does the altered differentiation pathway mean for cancer cells?

Any cell can potentially become transformed into a cancer cell due to changes in differentiation pathways.

What does the resolution of hypotheses about CSCs suggest?

Cancer likely involves a continuum of cells at various differentiation stages, with mutations and epigenetic changes altering potential for transformation.

What role does the stage of differentiation play in cancer progression?

It can influence malignant potential, with mutations in early-stage cells potentially leading to more aggressive/malignant cancers.

What is metastasis?

The ability to spread around the body.

Why is the study of cancer metastasis relevant for stem cell research?

Because both processes share fundamental biological mechanisms, including cell migration (EMT), plasticity (ability to switch states), and the ability to self-renew and differentiate (signalling pathways).

What is angiogenesis?

Growth of new blood vessels to provide the tumour with oxygen and nutrients, and provide an escape route to break away and recolonise around the body.

What are the characteristics of epithelial cells?

Polarity, adhesion to the ECM, stationary, high level of E-cadherin (adhesive), low level of N-cadherin (migratory).

What are the characteristics of mesenchymal cells?

No polarity, loss of cell adhesion, some interaction with the ECM, migratory and invasive phenotype, low level of E-cadherin (adhesive), high level of N-cadherin.

When is EMT and MET important for normal stem cells?

During embryogenesis and tissue repair.

How do cancer cells metastasise?

They hijack the pathway of EMT.

How is EMT initiated?

By signals from tumour stroma/TME that trigger migratory phenotypes (e.g., HGF and TGF-B that bind to tyrosine kinase receptors).

How does TGF-b activate MET?

It activates SMAD complex which translocates to the nucleus to upregulate TF genes that promote epithelial characteristics.

What TFs does TGF-b upregulate?

Twist, snail, slug, ZEB-1, N-cadherin

How is E-Cadherin repressed during EMT?

Polycomb epigenetic mechanisms.

Why has targeting metastasis been unsuccessful in cancer therapy?

Redundancy in signalling, pathway involvement in normal processes like wound healing, often occurs before diagnosis. However, mature stem cells are less migratory when cancers typically start developing so therapies are less likely to be toxic to these quiescent stem cells.

What is tumour-mediated immune evasion?

Avoidance of immune destruction is a hallmark of cancer.

What are the ways tumours evade the immune system?

Loss of tumour antigens, downregulates MHC, over express immune checkpoint marks (PD-L1) to cause T cell exhaustion, over express anti-apoptotic molecules, secrete immunosuppressive molecules, M2-type macrophage switch.

What stem cells do cancer cells link to?

MSCs. They have the potential to differentiate into many cell types, can secrete factors to promote tumour growth/angiogenesis/metastasis, promote macrophage polarisation and an immunosuppressive microenvironment (IL-10 and TGF-B).

What is the limitation of current cancer therapeutics?

They target the bulk of the tumour successfully (cells in a stage of differentiation) but leave the initating cause untouched (the residual CSCs which are more resistant) which results in secondary growths.

What are CSCs more resistant to cancer therapy?

ABC transporters, slow-division, enhanced DNA repair mechanisms.

Why do stem cells express ABC transporters?

For protection against foreign/exogenous material to maintain a functional long-lived stem cell pool.

How do cancer cells exploit ABC transporters?

Expression is typically lost upon differentiation through the down regulation of stemless signalling, as they can be replaced. However, tumour cells over express ABC transporter and do not switch off the expression. They can efflux chemotherapy, which has led to ABC inhibitors as adjuvant therapy.

What is the issue with ABC inhibitors?

WT stem cells are susceptible.

What are the names of initial ABC inhibitors and why were they unsuccessful?

Verapamil and cyclosporin A (toxic). Progress still with flavonoids and terpenoids.

If you were inhibit self-renewal of cancer cells, why would you target downstream of the wnt (APC/axin) pathway?

In colorectal cancer, APC or Axin mutations cause constitutive activation of β-catenin (the APC/Axin complex inhibitory complex is disruptive). Therefore, targeting must occur downstream, such as inhibiting β-catenin/tcf interaction, to be effective because targeting Wnt has no impact on abnormal activation.

What happens when tcf is inhibited in colorectal cancer cells?

Inhibition of tcf leads to differentiation of cancer cells into epithelial villus-like structures, reducing their stemness and malignant potential.

What does BMI expression cause?

Chemo/radiotherapy resistance from DNA repair, enhanced EMT under hypoxia by HIF-1a (TF expression), activation of PI3K and NF-KB to promote cell growth.

What are the therapeutic effects of inhibiting Bmi-1?

Sensitises CSCs to chemo and radiotherapy, enhances immune responses, promotes CD8⁺ T cell recruitment, improving anti-PD-1 checkpoint therapy by recruitment of CD8+ cells.

What is an example of a BMI inhibitor and how does it work?

PTC-596, an orally active, selective Bmi-1 inhibitor. It induces p53-independent mitochondrial apoptosis in AML progenitor cells, and is in trials for myeloma and glioma.

What happens during high-dose retinoic acid (ATRA) treatment in leukemia?

ATRA induces hematological remission in some patients with leukemia, but relapses occur over time due to resistant cells that undergo self-renewal (SR) and resist differentiation. More of the fusion-protein is generated and no more solo-RAR not complexed to PML.

How does arsenic trioxide (ATO) help reverse resistance to ATRA?

Arsenic trioxide (ATO) targets and degrades the PML-RAR fusion protein, frees RAR, and reverses resistance by restoring differentiation. This works in combination with high levels of ATRA to eradicate tumor cells.

How does ATRA affect the PML-RAR complex?

ATRA converts the PML-RAR complex from a repressor to a transactivator, driving the expression of differentiation genes (e.g., C/EBP). It also promotes the degradation of the transcription factor complex, controlling negative feedback in transcription.

What is the mechanism of action of arsenic trioxide in combination with ATRA?

Arsenic trioxide binds to and targets PML and PML-RAR fusion proteins for degradation, frees RAR, and restores the ability of ATRA to drive differentiation in resistant leukemia cells.

What are MMP inhibitors, and why have they not been successful in cancer therapy?

MMP inhibitors (e.g., Marimastat) target enzymes that break down the ECM to prevent migration. However, they have shown lack of efficacy and toxicity, largely due to their lack of specificity.

Why is the timing of cancer diagnosis critical in targeting metastasis therapeutically?

By the time cancer is diagnosed, the tumor has often already migrated, making it a missed opportunity to prevent or treat metastasis effectively.

What are C-MET inhibitors, and how do they relate to metastasis?

C-MET inhibitors (e.g., cabozantinib) target the C-MET receptor (HGFr), which drives EMT and metastasis. They inhibit tyrosine kinase activity, preventing signaling that promotes migration and cancer spread.

How do steroid inducers of transcription help in restoring metastatic suppressors?

Steroid inducers of transcription work by restoring metastatic suppressors

What are theranostics in cancer therapy?

A combined approach of therapeutics and diagnostics, where diagnostic agents (e.g., quantum dots, gold particles) are used for imaging while also delivering therapeutic treatments like chemotherapy or gene therapy to target cancer cells.

What are the roles of gold particles or magnetic metal oxides in theranostics?

Gold particles or magnetic metal oxides are used in theranostics for imaging purposes. When exposed to an alternating magnetic field, they heat up and can ablate surrounding cancer tissue, providing a way to visualize and treat the tumor.

How can theranostic nanoparticles (NPs) be enhanced for cancer therapy?

Theranostic NPs can be enhanced by complexing them with genes or chemotherapeutic agents, allowing them to deliver therapeutic treatments directly to cancer cells while also being visualized for diagnostics.

How can mesenchymal stem cells (MSCs) be used in theranostic cancer therapy?

MSCs can be loaded in vitro with theranostic nanoparticles (NPs). These MSCs can then home to tumors, delivering targeted diagnostics and therapies directly to cancer cells.

What are the tumour suppressive functions of TGFbeta?

TGFbtea/SMAD pathway functions at the G1/S checkpoint to induce cell cycle arrest and apoptosis. TGFbeta/Smad3/E2F/p107 complex represses c-myc.

What are the tumour promoting functions of TGF-beta?

TGFbeta/Smad complex upregulate SNAIL/SLUG/ZEB1/TWIST1 expression. Promotes angiogenesis. Induces differentiation (AKT) to evade the immune system and produces more TGFbeta, activating CAFs, and secrete anti-inflammatory factors.