Stem Cells and Cancer Flashcards
How do we get accumulations of mutations in stem cells?
• It is mutations in genes for cellular growth, division and DNA damage response elements
• Most common mutated gene is p53
• Examples of insults causing mutation include → irradiation, chemical, sporadic error in DNA replication
• If by chance one of these mutations occurs in a gene such as p53, could be the start of cancer development → if this is a stem cell, it will divide and replace the tissue, and all these cells will contain the mutant.
− Therefore more likely that a second insult will occur on an already mutated cell
What are the lessons learned from leukemia about cancer stem cells?
- Differentiated haematopoetic cells don’t last in the body very long – unlikely they will accumulate mutations
- First mutation likely in a stem cell, next either in a stem cell or the progenitor
- So can get leukemia resulting from mutations in stem cells, or progenitors.
What are the two aspects of cancer stem cells
- So the cell of origin is one aspect of stem cell cancer (the fact that cancer arises from mutations in stem cells)
- Another aspect is the tumour having its own population of stem cells.
What are the two models for tumour recurrence?
Current tumour model:
• All cells are equally capable of producing a tumour, so they should all be equally susceptible to chemotherapy
• If one cell survived and re-grew to form a new tumour, maybe that cell had another mutation, or maye it was hidden outside of the radiation field
Cancer Stem Cell tumour model:
• Tumours grow from stem cells like normal tissues grow from stem cells
• Chemotherapy kills the tumour but leaves the cancer stem cell (they may be more inherently resistant – therapies tend to target rapidly proliferating cells, and SCs are quiescent)
• Leaving behind the stem cell gives tumour recurrence.
• CSC inhibitors therefore needed to prevent recurrence.
what is the history of CSCs?
- 1875 → Cohnheim proposes that misplacement of stem cells during embryonic development can lead, in later life, to the development of tumours (so stem cells life behind from development)
- 1964 → Kleinsmith and Pierce show that a single ECC can produce a heterogenous assortment of offspring containing up to 14 different tissue types – implying that teratocarcinomas arise from a malignant stem cells
- 1997 → Bonnet and Dick show that only a small % of AML cells can transfer disease when transplanted into rodent hosts, showing that this 1% had something special about them
What is the gold standard for identifying CSCs?
• The ‘gold standard’ is the growth of human tumours in immunodeficient mice from a CSC isolated by antibodies to cell surface makers using flow cytometry.
− Human breast cancer: ESA+/CD44+/CD24lo
− Human brain and colon cancer: CD133+
• Gold standard is growth of tumours, but sphere colonies will grow in vitro
What is the evidence for CSCs?
Al-Hajj et al, PNAS 2003
Prospective Identificaiton of Tumorigenic Breast Cancer Cells:
• Tumorigenic cells in 8/9 primary breast tumour samples CD44+, CD24-, ESA+, Lin-
• Sorted by FACS
− Transplanted them into mice and see if they formed tumours
− With unsorted breast cancer cells:
− tumour formation every time you inject 500,000-50,000 cells
− tumour formation only 25% of the time when you inject 10,000 cells
− unlikely to get tumour formation with 1000 cells
− With surface marker sorted cells:
− 100% tumour formation with only 1000-200 cells
Singh et al, Nature 2004
Identification of a CSC in Human Brain Tumours
• Used cell surface marker to identify brain cancer tumorigenic cells
• CD133+ cells were the tumorigenic brain CSC population
• Demonstrated by :
− CD133+ cells producing neurosphere colonies in culture
− Transplantation into mice and formation of brain tumours only from CD133+
− In some tumour samples, CD133+ expression was as high as around 40% → almost as high as expression in control mouse neural stem cells
Ricci-Vitiani et al, Nature 2007
Identification and Expansion of Human Colon-Cancer Initiating Cells
• When you transplant unsorted cells that have a % of CD133+ cells, you get a tumour more or less 100% of the time
• When you transplant CD133- cells, you never see a tumour
• Agaim, showing a small % of cells are tumorigenic
What is the evidence against
• Andrease Strasser, Science, 2007
− Showed as few as 10 unsorted cells can initiate mouse lymphomas
− However, he never did it with 1 cell
− This suggests that there are CSCs – but it may be more like 10-20% of cells, rather than 1.
− Just because they might not be rare, doesn’t mean they don’t exist
• Sean Morrison, Nature, 2008
− 1 in 4 human melanoma cells form tumours, and there are no melanoma markers
− Argued that people thought CSCs were rare is because they didn’t follow up the tumour growth for long enough
− Many tumours take 2-5 years to develop, and mice don’t live that long
− Melanoma is fast growing, and said that 1 in 4 times, 1 cell can induce a melanoma as long as you follow it up for at least 12 months
− He argued there was no marker, but now been published that CD271 is a marker
What is the link between EMT and CSCs?
- May be that there is something unique about melanoma – melanocytes are odd in that they don’t form a tissue
- They are part of the skin epithelium, but don’t form the tissue themselves, they are just interspersed → this may make them could at migrating when they form a melanoma
- It has been shown that if you induce the EMT this seems to generate cells with properties of stem cells.
Evidence for a connection between EMT and CSCs
Mani et al, Cell 2008
Epithelial-mesenchymal transition generates cells with properties of stem cells
• EMT transcription factors SNAIL and TWIST induce EMT and stem cell activity
• CD49hi CD24med and CD44hi CD24lo normal and malignant breast stem cells have EMT markers
• EMT in breast cancer cells produces CSC activity
Induction of EMT by extrinsic signals:
• Can be induced in mammary cells by TGF-B and Wnt ligands
• Induces migration and mammosphere formation
• Inhibition of these extrinsic factors in transformed cells prevents tumorigensis and metastatic spread in vivo.
Are CSCs a stable phenotype?
Gupta et al, Cell 2011
Stochastic phenotype state transitions produce an equilibrium in breast cancer cell lines
• 61% of the time it remains as a stem cell after division
• 30% of the time it becomes a differentiated ell, expressing CD24
• 9% of the time becomes a more basal cell – still differentiated but without EpCAM
• Very rarely (1%) a differentiate cell can revert to a stem cell
− We normally think of stem cells as eventually differentiating
− This paper asks, in cancer, is it always one-directional?
− 1% can de-differentiate
− Could be important for treatment → treat the stem cells, but then a more differentiated cell could rever to a stem cell
Does induction of EMT and the CSC phenotypic state really occur in patient tumours?
• Yu et al, 2013 → circulating breast tumour cells exhibit dynamic chances in epithelial and mesenchymal composition
• Baccelli et al, 2013 → identification of a population of blood circulating tumour cells from breast cancer patients that initiates metastasis in a xenograft assay
− Well known that cancer cells metastatise in the blood stream
− These cells have properties of EMT
− If EMT cells are also CSCs, it suggests these may be seeding in other tissues, leading to metastases.
What are mechanisms of CSC resistance to therapies?
Brain Cancer Stem Cells
Bao et al, 2006
Gliomal stem cells promote radioresistance by preferential activation of the DNA damage response
• Normal treatment of gliomas is radiation
• CD133+ celsl are radio-resistance and enriched by treatment
• DNA damage response is more effect in CD133+ cells – preferentially activated
• Radio-resistance can be reversed by inhibitor of Chk1 and Chk2 checkpoint kinases
Breast Cancer Stem Cells
Williams et al, 2015
Focal adhesion kinase and Wnt signaling regulate ‘human ductal carcinoma in situ’ stem cell activation and response to radiotherapy
• Breast cancer stem cells preferentially survive irradiation
− Normally at 8 Gys of radiation, very few cells survive
− In a CSC enriched population, even at 10Gys, you have a high fraction surviving
• Targeting FAK or Wnt could re-sensitise the cells to radiotherapy
What is the mechanism of radioresistance?
Diehn et al, 2009
Association of ROS levels and radioresistance in cancer cells
• Normally, it is the ROS after radiation exposure that binds to DNA and causes breaks
• CSCs have lower levels of ROS – so when you irradiate them they don’t actually get much damage
→ So brain cells have increased ability to repair damage
→ Breast cells don’t get as much damage in the first place
How can differentiation therapy be used to target CSCs and treat cancer
Piccirillo et al, 2006
• BMP4 induced neural cell differentiation
• BMP4 treatment in vitro or in vivo prevents human brain cancer growth in mice
• BMP 4 treatment increases neural differentiation markers and reduces tumourigenic CD133+ cells
How can targeting Wnt signalling be used to treat CSCs and cancer?
Wnt Signalling
- Wnt binds to membrane Frizzled family receptor
- Passes the biological singal to the Dishevelled protein
- Leads to inhibition of proteins that normally bind b-catenin
- Wnt binding therefore frees b-catenin, and allows it to translocate to the nucleus and bind to TFs TCF/LEF1 → causes transcription of SC genes.
Natural inhibitors of the pathway
• sFRP is a secreted version of the frizzled receptor. Competes for binding to Wnt
• DKK1 inhibits the co-receptor LRP5
• Without Wnt signaling, Dishevelled is not inhibitory. b-catenin binding complex is active, and you cant get activation of SC genes
Evidence of involvement in cancer:
• sFRP and DKK1 lost in colorectal and breast cancer
• Wnt1 activation causes increase in mouse mammal SC number and cancer
• APC mutations (part of the b-catenin binding complex) common in CRC
Drugs for the Wnt pathway under development
• Frizzled inhibitors
• b-catenin inhibitors
• Porcupine inhibitors – stop secretion of Wnt
How can Hedgehog signalling be targeted to treat CSCs adn cancer?
- Ligands include Shh, Ihh and Dhh
- Pathway is mutated in 25% of cancers → particularly medulloblastoma and basal cell carcinoma
Signalling
Active
• Hedgehog binds to the patch receptor
• This leads to activation of co-receptor smoothened
• Sets off an intracellular signaling pathway that ends with Gli binding tp promotes of genes to switch on stem cell genes
Inactive
• In the absence of Hedgehog, Patch acts as a repressor for smoothened
• Intracellular pathway is switched off
Discovery of Cyclopamine and Jervine:
• Plant alkaloids present in Corn Lily
• Shepherds noted that sheep which crazed on Corn Lily had lambs with cyclopic-like faces
➢ Cylopamine binds smoothened directly and inhibits hedgehog signal
Hedgehog in stem cells and cancer
• Prostate cancer
− Normal prostate does not express smoothened, but it may be in SCs are Hedgehog is required for DHT-driven prostate regeneration
− There is a 10-fold increase of Ptc (measure of Hh activity) in metastatic prostate cancer
− Rat prostate cancer mestastais cells are dependent on Hh activity
− Rat prostate cancer is enhanced by Gli and inhibitied by cyclopamine
− Gli can transform primary prostate cells into cancer-like cells
How can Notch signalling be targeted to treat CSCs and cancer?
• Notch is TM receptor with an extracellular and intracellular domain
• Ligands are Delta and Jagged
− Ligand binding to the Notch receptor is unique in that the ligands need to be bound to an adjacent cell → signal sending cell, and signal receiving cell
− When the ligand binds Notch, it disrupts the receptor, opening it up, revealing binding sites for ADAM proteases and y-secretase
− These cleave the receptor, and the intracellular domain is released
− The intracellular domain translocates to the nucleus, binding to TFs that leads to SC gene transcription.
Notch in cancer:
Weng et al, 2004
Activating mutations in NOTCH1 in human T cell ALL
• 56% of all T cell ALL patients have activating Notch mutations
• Must mutations are in the region of enzymatic cleavage – makes it constitutively active
• Also mutations in the intracellular domain which increases its stability in the nucleus
Harrison et al, 2010
• Notch inactivation using y-secretase inhibitors reduces mammosphere formation
How is aldehyde dehydrogenase involved in CSCs and cancer?
Ginestier et al, 2007
Aldehyde dehydrogenase is a marker of normal and malignant breast stem cells, and a predictor or poor clinical outcome
• ALDH may have a role in differentiation of SCs through its role in oxidizing retinol to retinoic acid
• Increasd ALDH activity found in SC populations in multiple myeloma and AML
• ALDH activity may therefore provide a common marker for normal and malignant stem and progenitor cells
ALDH identified by ALDEFLUOR:
• ALDEFLUOR positive cell population from human breast tumours in SCID mice has CSC properties
• The more ALDEFLUOR positive cells, the greater the tumour size
Expression of ALDH1 in breast cancer predicts overall survival:
• Tumours with high ALDH1 respond poorly to treatment
• Having high levels of ALDH only gives 50% chance of surving 10 years, compared to 75% with low ALDH
Charafe-Jauffret, 2009
Breast cancer cell lines contain functional CSCs with metastatic capacity and a distinct molecular signature
• Aldefluor associated genes include retinoic acid signaling pathway
• Aldefluor + cells increases metastatic capacity
• Aldefluor + cells increased CXCR1 expression → so IL-8 must be involved
− Since been found that cytokines including IL-8, IL-6 and CCL5 activate a feedback loop (receptors on Aldefluor + cells)
− This leads to STAT3 and AKT signaling
− This leads to NFkB activation
Other cytokines are also involved
Li et al, 2012
Cancer-stimulated MSCs create a carcinoma stem cell niche via prostaglandin E2 signalling
• CSCs secrete IL-1 dependent on Wnt
• IL-1 activates MSCs to secrete IL-8, IL-6 and prostaglandin
• These all act back on the CSC
Singh et al
Targeting CXCR1 significantly reduces breast CSC activity and increases the efficacy of inhibiting HER2
• CXCR1 is the receptor for IL-8
• Patient metastatic fluid levels of IL-8 positively correlate with mammosphere formation
• Inhibiting CXCR2 at the same time as administering Lapatinib treatment (HER2 inhibitor) increases the efficiency
