Background

1
(No Transcript)
2
Background

• Cancer stem cell hypothesis
• cancers are maintained in a hierarchical
  organization of
• rare, slowly dividing cancer stem cells or
  tumor-initiating cells
• rapidly dividing amplifying cell early
  precursor cells
• differentiated tumor cells

Tumor-initiating cells - self renewing -
can differentiate into multiple lineages -
highly tumorigenic in mice Also important for
- progression - metastasis - resistance to
therapy, and tumor recurrence
3
miRNA
NEJM 2008 358
4
Mechanisms of miRNA-mediated gene silencing
Translation is not inhibited, but the polypeptide
chain is degraded during translation

• Blocking translation elongation
• - Promoting premature dissociation of ribosomes
  (ribosome drop-off)

Argonaute proteins recruit eIF6, which prevents
the large ribosomal subunit from joining the
small subunit
Argonaute proteins compete with eIF4E for binding
to the cap structure
Argonaute proteins prevent the formation of the
closed loop mRNA configuration by a mechanism
that icludes deadenylation
mRNA decay
Cell 132, Jan 11, 2008
5
Expression of miRNA in cancer
CCR 2008 14(2)
6

• Rationale for this study
• miRNAs regulate differentiation and can function
  as oncogenes or tumor suppressors to regulate
  tumor development and prognosis.
• Loss of Dicer1 is embryonic lethal and loss of
  stem cell population
• Argonaute family members are required for
  maintaining germline stem cells.
• Distinct sets of miRNAs are specifically
  expressed in ES cells but not adult tissue.
• Hypothesis
• There are differences in miRNA expression between
  BT-IC/EPC and there more differentiated progeny.

• Breast tumor-initiating cells can be enriched by
• Sorting for CD44CD24- cells
• Selecting side population cells that efflux
  Hoechst dyes
• Isolating clusters of self-replicating cells
  Mammospheres from suspension cultures
• These methods purify both tumor-initiating cells
  and some early precursor cells.

7
- Resistance to chemotherapy distinguishes T-IC
from other cancer cells
Q Does chemotherapy enrich for BT-IC?
Cells from primary breast tumors were cultures to
generate mammospheres
5.8
0.4
Passaged for 2-3 8-10 generations
(figure S1)
Paired samples
5.9
0.5
Chemotherapy selectively enhances the survival of
BT-IC.
8
Q Can we enrich for BT-IC by consecutively
passaging breast cancer cells in SCID mice
treated with chemo?

• - Mice were injected with SKBR3 cells in the
  mammary fat pad.
• Mice were treated with epirubicin weekly for
  10-12 weeks until 2cm
• Cells from the 3rd passage were cultured in
  suspension to generate mammospheres.
• of mammospheres reflect the quantity of cells
  capable of in vitro self renewal
• of cells/sphere measures the self-renewal
  capacity of each sphere-generating cell

- SK-3rd formed 20x more spheres then SKBR3 16.3
vs 0.8 - Same amount of spheres from 2nd and
3rd
Day 5 vs day 15
SK-3rd generates bigger spheres then SKBR3
Freshly isolated cells
- Plated on collagen - 10 FCS no growth factors
SK-3rd has more self-renewing capacity then SKBR3
9
Q Are these SK-3rd cells multipotent?

• SK-3rd cells do not express any differentiation
  markers
• Cells are round
• After further differentiation for 10 days SK-3rd
  do express differentiation markers
• Cells are elongated
• Only 1.5 could form spheres
• 11 fold reduction

T-IC are believed to be resistant to chemo. This
correlates with the ability to expel dyes.
SK-3rd contained 26x more side population cells
then SKBR3
10
- Important feature of T-IC is efficient
xenograft formation

• primary had to reach 2cm before looking for
  metastasis

• Mammospheric SK-3rd cells were at least 100x
  more tumorigenic then SKBR3
• SK-3rd cells from xenograft could be serially
  transplanted.
• Only SK-3rd cells metastasize.
• This suggests that SK-3rd cells have an in vivo
  self-renewal capacity

11
Q Is chemo needed to maintain a stable
percentage of self-renewing cells?
SK-3rd cells were passaged in SCID mice and
treated or not with chemo
Table S3

• SK-4th() contained an equal percentage of
  sphere-forming cells as SK-3rd.
• SK-4th(-) contained 8x fewer spheres.

• Proportion of BT-IC had already plateaued by the
  3rd passage.
• Selective pressure from chemo is required to
  maintain the proportion of self-renewing cells in
  vivo.
• Chemo is not responsible for BT-IC tumor-forming
  or metastatic capacity.

12
Summary Figure 1 Chemotherapy selectively
enriches for self-renewing breast cancer cells
(BT-IC)

• At least 16 of SK-3rd cells displayed all the
  expected properties of T-IC
• in vitro stable mammosphere formation
• growth under non-adherent conditions
• multipotent differentiation
• CD44/CD24- phenotype
• drug-expelling side population
• High rate of forming tumors capable of serial
  transplantation

13

• Compare miRNA expression in mammospheric SK-3rd
  and SKBR3
• Most of the 52 miRNAs had reduced expression in
  mammospheric SK-3rd
• During differentiation most of the miRNA are
  increased to the level of SKBR3

Figure 2
SK-3rd 10 days
SK-3rd 1 day
SK-3rd 8hrs
SKBR3
SK-3rd
Most significant change Let-7 family
Northern Blot for all let-7 homologs
qRT-PCR for Let-7a
Reduced let-7 is not a consequence of
chemotherapy or anchorage-independent growth
Let-7 is low in cells with self-renewal capacity
and increases during differentiation
14
Let-7

• Initially identified as a miRNA that regulates C.
  Elegans development
• One of the key target genes is Let-60 a RAS
  homolog
• Mammalian studies
• 11 let-7 family members
• Differentially expressed in different tissues
• Redundant targets and functions
• Human studies
• Downregulated in some human cancers
• Associated with poor prognosis in lung cancer
• Targets are RAS and HMGA2
• Focused on let-7 in this paper, because
• Known tumor suppressor

15
Q What is the function of Let-7?
Figure 2
Transfection of luc-reporter containing a let-7
target 3UTR sequence
Let-7 represses the reporter

• H-RAS and HMGA2 proteins were highly expressed in
  mammospheric SK-3rd cells
• greatly reduced in differentiated SK-3rd cells
  and SKBR3
• mRNA levels did not change

RAS and HMGA2 are known let-7 targets

• Let-7 silences RAS and HMGA2 expression by
  inhibiting translation.
• Reduced Let-7 leads to RAS and HMGA2
  overexpression

16
Q Is let-7 also reduced in BT-IC of primary
breast cancers?
Figure 2
Sorted for BT-IC
qRT-PCR
Northern Blot

• Let-7 is reduced in BT-IC
• This is independent of chemotherapy
• Reduced let-7 is an intrinsic property of
  BT-IC/EPC

17
Q Is let-7 important for self renewal?
Figure 3
let-7 in SK-3rd cells
let-7a ASO
of spheres is 5.3x reduced
of spheres is 6x increased
of cells/ sphere 2-3x less
of spheres is reduced 3x of spheres is
reduced with each passage
let-7 in primary BC
Reduced let-7 is required to maintain mammospheres
18
Property of self-renewing cells potential to
expand under differentiating conditions
Figure 3
3H incorporation

• SK-3rd proliferates ½ the rate of SKBR3
• Under differentiating conditions, SK-3rd
  increased 7x
• SK-3rd Let-7 peak declined by 58

Let-7 reduces the proliferative potential of
differentiating precursor cells
T-IC hallmark they are undifferentiated and have
the potential for multilineage differentiation
Figure S11
10 days

• Most cells expressed diff. markers
• 15 remained neg, Let-7 expression 6

SK-3rd did not express CK14 nor CK18
Low Let-7 helped to maintain the undifferentiated
status and proliferative potential of
mammospheric cells
19
Q Are the effects of reduced Let-7 on promoting
self renewal and multilineage differentiation
because of RAS?
Figure 3E
Figure 3B
Figure 3A
H-RAS shRNA has ½ of control 3x more
then Let-7
Intermediate reduction in proliferation
Intermediate size
Figure S11B
Did not reduce the proportion of undifferentiated
cells
Let-7 inhibits self renewal in part by regulating
RAS
20
Q Are the effects of reduced Let-7 on promoting
self renewal and multilineage differentiation
because of HMGA2?
Figure 4
Did not alter mammosphere formation
spheres
adherent
Slightly reduced proliferation
Reduced proportion of undifferentiated cells
Let-7 causes differentiation by silencing HMGA2
21
Q What is the effect of Let-7 expression on
tumor formation?

• Let-7 expression
• Less tumors
• Slower growing tumors

Figure 5
Let-7 inhibits self-renewing capacity
22
No big differences in tissue structure and cell
morphology of tumors
Figure 5

• H-RAS was more highly expressed in SK-3rd then in
  SKBR3 tumors
• Let-7 tumors had reduced H-RAS expression like
  the SKBR3 tumors

• More cells of the SK-3rd tumors expressed PCNA
• Let-7 reduced PCNA staining, but not as in SKBR3
• RAS-shRNA reduced PCNA staining, but not as in
  Let-7 tumors

Lack of Let-7 enhanced SK-3rd cell
tumorigenicity, in part by modulating H-RAS
23
Q Is let-7 reduction also important for
tumorigenesis of primary cancer cells?

• CD44CD24- cells could be serial transplanted
  without reduced tumorigenicity
• Let-7 transduction reduced tumorigenicity and
  also tumor formation upon serial transplantation

Enforced let-7 expression in primary BT-IC
interferes with both tumor initiation and in vivo
self renewal
24
Q Does enforced let-7 or RAS-shRNA expression
affect metastasis?
Figure 6
-Tumor 2cm in size

• Let-7 infection reduced the of mice with mets,
  and the average lung weight
• Mets were not only smaller, but also dispersed
  among alveoli reduced clinical severity.
• qRT-PCR for hHPRT showed reduction of lung and
  liver mets in mice with - let-7 infected cells
• - RAS-shRNA infected cells (less then let-7)

• Let-7 reduced metastasis to both lung and liver.
• Only partially due to RAS
• Could result from slower growth of primary tumor
  OR altered metastatic potential of let-7
  expressing cells

25
Summary

• Chemotherapy selectively enriches for
  self-renewing breast cancer cells
• Mammospheric cells have reduced let-7
• Let-7 is reduced in BT-IC from clinical cancer
  specimens
• Reduced let-7 is required to maintain
  mammospheres
• Reduced let-7 maintains proliferation, but
  inhibits differentiation
• Silencing RAS or HMGA2 partially recapitulates
  the effect of let-7
• Let-7 expression inhibits tumor formation in
  NOD/SCID mice
• Let-7 expression inhibits tumorigenesis of
  primary cancer cells

26
Discussion

• Isolation BT-IC
• -Tumors of chemotherapy-treated patients are
  enriched for cells with BT-IC properties
• -early precursors dividing fast higher
  percentage
• -SK-3rd cell line generation has all properties
  of BT-IC
• 16 T-IC rest is EPC (based on mammosphere assay
  and SP cells
• -This technique can be used on any cancer cell
  line, but can accumulate mutations
• Neoadjuvant chemotherapy
• Selects for the survival of BT-IC and EPC
• Selective outgrowth of less-differentiated cells
• miRNA
• Global reduction of miRNA in BT-IC reduced
  miRNA processing? embryonic development
• Reduced let-7 in an intrinsic property of BT-IC,
  not a consequence of chemo or anchorage-independen
  t growth also cells from chemotherapy-naïve
  patients have reduced let-7
• RAS HMGA2
• H-RAS is increased in 60 of breast tumors, but
  mutations are rare maybe post-transcriptional
  regulation?
• HMGA2 overexpression due to translocation other
  mechanism is reduced let-7 (this study)

27
Discussion, cont.

• RAS HMGA2
• Regulate different aspects of stemness
• RAS is important for self renewal
• HMGA2 maintains multipotency overexpressed in
  embryos and poorly differentiated tumors
• Let-7
• Other targets CDC25a, CDK6 and Cyclin D
• Regulates multiple oncogenes and more then 1 T-IC
  pathway attractive target for therapy
• Let-7 as a target Let-7 mimics - useful to
  target differentiation resistant T-IC
• more potent then silencing 1 oncogene
• Could be used a single agent or in combination
  with chemo-radiation
• Should not trigger unintended toxicity to
  noncancerous cells.
• Let-7 as a marker- measuring let-7 reduction in
  breast cancers could be
• - a readout for the frequency of BT-IC or other
  poorly differentiated cells in the tumor
• - provide prognostic information about the
  likelihood of response or relapse.
• (low let-7 and high HMGA2 expression strongly
  correlates with poor
• prognosis in advanced ovarian cancer)