lecture 26: breast and cancer stem cells

Question 1

What defines stem cells?

Answer 1

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

Question 2

What is development of the breast?

Answer 2

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

Question 3

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

Answer 3

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

Question 4

For what are mammary stem cells required?

Answer 4

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

Question 5

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

Answer 5

• 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

Question 6

What is in vivo characterization using mammary fat pad transplantation?

Answer 6

• 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

Question 7

What are the multiple cell types of the mammary gland?

Answer 7

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

Question 8

How many epithelial subpopulations were defined by cell surface markers?

Answer 8

• 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

Question 9

What was transplantation of subsets of lineage-cells?

Answer 9

• cells double-sorted
• rosa 26 donors (lacZ gene in Rosa-26 locus)
• only one population had repopulating capacity when put into cleared fat pad
  
  • CD29^hi
  • CD24+

Question 10

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

Answer 10

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

Question 11

Can the Lin^-CD24⁺CD29^hi cell self-renew?

Answer 11

• 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

Question 12

Do bipotent cells exist and function in vivo?

Answer 12

• 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

Question 13

What is 3D imaging of the mammary gland?

Answer 13

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

Question 14

What is the strategy for in vivo lineage tracing?

Answer 14

• 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

Question 15

What is confetti?

Answer 15

• 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

Question 16

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

Answer 16

• 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

Question 17

What were they able to show in the end?

Answer 17

• 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

Question 18

What is the search for normal human breast stem cells?

Answer 18

• 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

Question 19

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

Answer 19

• 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?

Question 20

Are MaSCs sensitive to ovarian hormone deprivation?

Answer 20

• 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

Question 21

What happens to numbers of MaSCs during pregnancy?

Answer 21

• 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

Question 22

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

Answer 22

• 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

Answer 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?

Question 24

What pathway mediates steroid hormone signalling to stem cells?

Answer 24

• 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

Question 25

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

Answer 25

• 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

Question 26

What are pathways often deregulated in cancer?

Answer 26

• 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

Question 27

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

Answer 27

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

Question 28

What does constitutive Notch signalling in MaSCs promote?

Answer 28

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

Question 29

What are dual functions for Notch?

Answer 29

• 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

Question 30

What are GATA transcription factors?

Answer 30

• 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

Question 31

What is gata-3?

Answer 31

• 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

Question 32

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

Answer 32

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

Question 33

What does gata-3 promote in tumour cells?

Answer 33

• 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

Question 34

How does breast tumorigenesis occur?

Answer 34

• 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

Question 35

What is heterogeneity within individual solid tumours?

Answer 35

• 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

Question 36

What are two key questions in breast cancer?

Answer 36

• 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?

Question 37

Are the ‘cell of origin’ and cancer stem cell the same thing?

Answer 37

• 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

Question 38

What have gene profiling studies done?

Answer 38

• 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

Question 39

What is true of the four mammary gland subpopulations?

Answer 39

• they have distinct gene signatures

Question 40

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

Answer 40

• 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

Question 41

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

Answer 41

• 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

Question 42

What can lineage tracing be used for re: tumours?

Answer 42

• 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

Question 43

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

Answer 43

• 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

Question 44

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

Answer 44

• 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

Question 45

What has been identified in mouse models of mammary tumourigenesis?

Answer 45

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

Question 46

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

Answer 46

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

Question 47

What are cancer stem cell markers in human tumours?

Answer 47

• 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