lecture 26: breast and cancer stem cells Flashcards Preview

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Flashcards in lecture 26: breast and cancer stem cells Deck (47):
1

What defines stem cells?

  • defined by their ability to self-renew and differentiate along multiple lineages 
  • stem cell → common progenitor → committed progenitor → mature cells 
  • stem cell → stem cell 

2

What is development of the breast?

  • newborn → puberty → pregnancy → lactation → involution (→ pregnancy → etc) 
  • proliferation at puberty
  • proliferation and differentition during pregnancy 
  • differentiation during lactation
  • apoptosis during involution 

3

What are the three distinct epitheli cell types seen in breast?

  • 18 day pregnant
  • luminal epithelium
    • alveolar
    • ductal
  • myoepithelium 

4

For what are mammary stem cells required?

  • homeostasis in the mammary gland and growth during pregnancy 
  • remarkable generative capacity of breast tissue 
  • more than 25-fold expansion of breast 'epithelium'

5

How have in vivo strategies been used to define the mammary stem cell (MaSC)?

  • identification of cell surface markers to allow fractionation of mammary cells by flow cytometry 
  • transplantation studies:
    • perform limiting dilution assays to allow comparison of the relative repopulating frequencies of different subpopulations 
    • demonstrate the multilineage differentiation capacity of SCs. Serial transplantation is the 'gold standard' to prove the self-renewing capability 
  • lineage tracing studies in vivo

6

What is in vivo characterization using mammary fat pad transplantation?

  • mammary gland not an essential organ
  • cauterize in a young animal (3 weeks) 
  • remove rudimentary tree attached to nippple
  • leave behind an intact fat pad 
  • inject FRESH cells into this 
  • harvest 8 weeks post-transplantation 
  • ask whether we see a ductal tree that has emerged in this area 

7

What are the multiple cell types of the mammary gland?

  • luminal epithelial cells
  • myoepithelial cells
  • basement membrane (separates epithelial cells from surrounding stroma)
  • fibroblasts 
  • adipocytes 
  • blood vessel
  • lymph node 
  • macrophages 
  • complex microenvironment 

8

How many epithelial subpopulations were defined by cell surface markers?

  • CD24 + or -
    • heat stable antigen
  • CD29 + or -
    • beta1-integrin
  • DP - double positive - smallest population, 4.8% 
  • CD24+/CD29- 23.9% 
  • double negative = 55.4% 
  • CD29+/CD24- = 5/3% 
  • transplant cells in numbers proportional to their frequency in the overall population 

9

What was transplantation of subsets of lineage-cells?

  • cells double-sorted
  • rosa 26 donors (lacZ gene in Rosa-26 locus)
  • only one population had repopulating capacity when put into cleared fat pad 
    • CD29hi
    • CD24+ 

10

Were they able to get generation of a functional mammary gland from a single stem cell?

  • yes
  • beta-galactosidase makes blue
  • capable of multilineage differentiation 

11

Can the Lin-CD24+CD29hi cell self-renew?

  • serial transplantation studies
  • MaSC 
  • primary transplantation
  • primary outgrowth 
  • secondary transplantation ( first generation self-renewed MaSCs) 
  • secondary outgrowth 
  • tertiary transplantation (second gen self-renewed MaSCs)
  • tertiary outgrowth
  • most could be passaged ~8 times

12

Do bipotent cells exist and function in vivo?

  • big controversial issue in the field 
  • they had done research with a team in canada → recapitulated findings independently → found a stem cell that could give rise to all of these cells 
  • in 2011 a paper appeared and said that bipotent stem cells do not exist - only unipotent cells exists 
    • there is a myo-SC → myoepithelial cells
    • luminal SC → ductal and alveolar cells 
    • claimed using lineage tracing
  • at the same time they were also carrying out lineage tracing experiments 

13

What is 3D imaging of the mammary gland?

  • 3 week-old gland prior to puberty
  • this is about a 4mm section of an intact mammary gland 
  • can see elongated myoepithelial cells 

14

What is the strategy for in vivo lineage tracing?

  • Rosa26 is a strong, ubiquitous promoter 
  • dtTomato is a red fluorescence protein 
  • all daughter cells of labeled parental cell are permanently marked
  • single colour for quantification  
  • toxicyclin inducible system 
  • tet operon that contains cre 
  • third cross where cre induces expression of a promoter gene 
  • triple transgenic mice
  • if the promoter is expressed in a specific cell of interest, in the presence of toxicyclin, it will activate the tet operon → cre mediating recombination → expression of that particular promoter or reporter gene in that particular cell type 
  • indelible marking of all daughter cells of that parental cell 

15

What is confetti?

  • a stochastic multicolour cre reporter for clonality studies 
  • 4 fluorescent proteins 
  • if you deliver just a small single pulse of an agent (toxicyclin) → random activation of only one of four colours in that cell 
  • because only one pulse is delivered not flipping backwards and forwards

16

What was population dynamics of K5-expressing cells in puberty?

  • equi-expression of all four fluorescent proteins 
  • K5 marks long myoepithelial cells 
  • shows that many progenitor cells are involved in morphogenesis of the gland during puberty 

17

What were they able to show in the end?

  • K5 marks both luminal and myoepithelial cells after induction in the adult 
  • able to show that after an 8 week chase 
  • unicoloured clonal regions 
  • both myoepithelial and cuboidal luminal
  • proved that bipotent stem cells exist 
  • capable of giving rise to all the epithelial cells of the mammary gland
  • also luminal progenitor cell exists 

18

What is the search for normal human breast stem cells?

  • not able to do lineage tracing but did do cell surface marker analysis 
  • found that there were four distinct populations 
  • only one of these, if you transplant back into the mouse fat pad, has the ability to give rise to ductal outgrowths 
  • same pattern for human and mouse mammary tissue  

19

What are functional similarities between mammary epithelial subpopulations in mouse and human?

  • can identify bipotent stem cells in both 
  • two types of luminal progenitors 
  • can prospectively isolate all the mature cells as well
  • don't know about all the precursors leading up to myoepithelial cells 
  • both the mouse and human MaSCs lack receptors for the ovarian hormones oestrogen and progesterone 
  • important because increased progesterone and oestrogen are linked with increased breast cancer risk 
  • could they still be influenced?

20

Are MaSCs sensitive to ovarian hormone deprivation?

  • yes - highly sensitive 
  • stem cells appear to retain a 'memory' of prior steroid hormone deprivation 
  • repopulating frequency:
    • control: 1/58
    • ovariectomy: 1/247
  • also when they could generate tissue, it was only very little 

21

What happens to numbers of MaSCs during pregnancy?

  • pregnancy is accompanied by an 11-fold increase in the number or activity of MaSCs 
  • the augmented MaSC pool drives secretory cell expansion 
  • seen in mid-pregnancy 
  • transplantation assay measures function 
  • stem cell highly receptive to hormonal signalling 

22

What is evidence that ovarian hormones (oestrogen and progesterone) profoundly influence stem cell activity?

  • hormone deprivation and anti-oestrogens decrease MaSC pool/activity 
  • excess hormones increase MaSC function
  • ageing is associated with increased MaSC function 
  • pregnancy dramatically increases MaSC pool 

23

  • ductal luminal cells express ER and PR - remain the most important prognostic markers for breast cancer to date 
  • 70% of breast cancers are ER positive and if they are there is a much better prognosis 
  • how is this signalling to the stem cell when the stem cell doesn't have receptors for the hormones?

24

What pathway mediates steroid hormone signalling to stem cells?

  • The RANKL/RANK pathway 
  • signals to NFkB 
  • steroid hormones like progesterone stimulate ductal cell to make RANKL 
  • signals to the stem cell pool
  • drives proliferation 
  • probably other molecules involved as well

25

What is a strategy for assessing gene function in mammary stem and progenitor cells?

  • retrovirus-mediated manipulation of gene expression in discrete populations 
  • harvest virgin mammary gland 
  • single cell suspension
  • cell sorting into subpopulations 
  • plate subpopulation on feeder cells
  • transduce with retrovirus 
  • sort GFP+ cells after single cell suspension 
    • colony forming assay on feeder cells
    • colony forming assay in matrigel
    • transplant into cleared fat pad 
      • puberty or pregnancy 
      • mammary outgrowth

26

What are pathways often deregulated in cancer?

  • self-renewal pathways in stem cells 
  • decided to look at notch - associated with the most aggressive subtype of cancer (triple negative) 
  • wanted to understand the normal role of notch 
  • wnt, notch and hedgehog are all very important pathways → embryonic and normal development
    • always see them reemerging in cancer 

27

Can short-term cultured MaSCs reconstitute a functional mammary gland?

  • yes
  • 5 or 6 days of culture did not abrogate this ability 

28

What does constitutive Notch signalling in MaSCs promote?

  • luminal cell commitment and induces hyperplasia in vivo
  • deregulated notch1 can go on to produce luminal tumours 

29

What are dual functions for Notch?

  • restricts MaSC proliferation and directs luminal cell fate determination 
  • complex pathway
  • normally it represses proliferation of the mammary stem cell 
  • but when activated it promotes formation of luminal progenitors 

30

What are GATA transcription factors?

  • six family members (GATA-1-GATA-6)
  • bind DNA at the consensus sequence (A/T)GATA(A/G) 
  • play critical roles in development, cell-fate specification and differentiation 

31

What is gata-3?

  • gata-3 is a master regulator of mammary gland development in the embro and adult 
  • in the embryo:
    • essential for placode formation (E11.5) → bud (E13.5) → nipple sheath (E16.5) 
  • in puberty: 
    • important for development of the tree 
    • pivotal for differentiation into a milk producing cell
  • gata 3 is essential for luminal cell differentiation during different developmental stages 
    • gata-3f/f: tree can only grow a bit, no milk producing cells during pregnancy 

32

What happens with a loss of gata-3 re:mammary tumours?

  • more aggressive 
  • remove a single allele of gata 3 you get a huge decrease in latency → much more aggressive, came up faster 

33

What does gata-3 promote in tumour cells?

  • gata-3 promotes the differentiation of tumour cells and thereby leads to better patient prognosis 
  • expressed in the stem cell and important for its proliferation
  • most important role is in the differentiation of a luminal progenitor to a mature luminal cell 

34

How does breast tumorigenesis occur?

  • normal → hyperplasia → atypical hyperplasia → carcinoma in situ (DCIS, LCIS) → invasive carcinoma → metastatic disease 
  • genetic and epigenetic alterations 
  • when they break through the basement membrane you have metastatic disease

35

What is heterogeneity within individual solid tumours?

  • e.g. oestrogen receptor expression 
  • 20 different and very distinct pathological subtypes
  • at a molecular level about 6 discreet molecular substypes 
  • any patient with 1% of cells expressing ER is denoted as ER positive 

36

What are two key questions in breast cancer?

  • which cell is the target of transformation in breast cancer - does it result in different tumour subtypes?
  • which cells sustain the tumour i.e. can we identify tumour-propagating cells?

37

Are the 'cell of origin' and cancer stem cell the same thing?

  • no they refer to different concepts 
  • cell of origin = cell that experienced first oncogenic hit 
    • could be any cell along the hierarchy
  • CSC = specific subset of cells within the tumour that have the ability to seropassage the tumour 

38

What have gene profiling studies done?

  • altered the clinical landscape in breast cancer 
  • 5 primary molecular subtypes (probably a few more)
  • luminal A (ER++)
  • luminal B (ER+)
  • basal-like
  • Erb2/HER2-positive 
  • claudin low - metaplastic with high expression of proteins like SNAI2, EMT genes 

39

What is true of the four mammary gland subpopulations?

  • they have distinct gene signatures 

40

Do different epithelial cells give rise to same subtypes of breast cancer?

  • possibly not 
  • stem cell → claudin low 
  • luminal progenitor → basal (surprising)
  • something on the pathway from luminal progenitor to ductal cell → luminal B or HER2
  • ductal cell → luminal A 

41

Are stem or progenitor populations altered in breast cancer prone BRCA1 mutation carriers?

  • breast cancer 65%, often early onset 
  • ovarian cancer 40%
  • BRCA1-mutant luminal progenitors exhibit factor-independent growth 
  • MaSC did not 
  • highly proliferative /aberrant 
  • luminal progenitors are the cell of origin for basal breast cancers arising in individuals with BRCA1 mutation s

42

What can lineage tracing be used for re: tumours?

  • lineage tracing can be used to track tumour-initiating cells in vivo 
  • activate cre-ERT2 in single cell at base of a small intestinal crypt
  • track tagged cell in vivo 
  • e.g. deletion of Apc in stem but not TA cells gives rise to tumours 

43

What is the current status/perspectives on cancer stem cells?

  • definition: refers to a subset of tumour cells that can self-renew and generate the diverse cells that comprise the tumour. CSCs can initiate and sustain tumorigenesis 
  • lie at the apex of the cancer cell hierarchy 
  • distinct from the 'cell of origin' 
  • CSCs do not necessarily originate from the transformation of normal stem cells 
  • their existence is best demonstrated through serial transplantation of subpopulations into the relevant site  
  • drugs that kill tumour cells but not cancer stem cells → tumour shrinks but grows back 
  • they self-renew, multi-potential, relatively quiescent, long-lived 
  • clonal evolution and cancer stem cell models are contradictory 

44

What is prospective isolation of CSC-enriched populations from solid tumours based on transplantation of sorted cells?

  • none of these markers are exclusively expressed by solid tumour CSCs 
  • even for breast cancer CD44/ALDH1 do not characterise CSCs in every breast cancer
  • even of the same type
  • difficult area to move in at the moment 
  • cancer stem cells shift their markers and undergo clonal evolution themselves → not genetically, only epigenetically 

45

What has been identified in mouse models of mammary tumourigenesis?

  • identification of 'cells of origin' in preneoplastic tissue 
  • in Wnt1 model increased number of MaSCs but not MMTV-neu mice 

46

What defines CSCs in MMTV-wnt-1 mammary tumours?

  • CD61
  • CD61+ cells are 20-fold enriched for CSCs

47

What are cancer stem cell markers in human tumours?

  • none of the markers used to isolate CSCs from various cancerous tissues are expressed exclusively by stem cells
  • some markers are common between CSCs from different tumours:
  • CSC phenotype will not necessarily be uniform between cancer subtypes or even tumours of the same subtype 
  • what are the similarities between normal stem cells and CSCs? self-renewal pathways