Breast tumor stem cells have increased microtentacles that promote reattachment and can be targeted therapeutically with curcumin*.
Monica S. Charpentier1,3, Rebecca A. Whipple1, Michele I. Vitolo1, Amanda E. Boggs1,3 , Jana Slovic1,3, Keyata Thompson1, Lekhana Bhandary1,2 , and Stuart S. Martin1,2,3
University of Maryland School of Medicine, Greenebaum NCI Cancer Center1, Department of Physiology2, Graduate Program in Molecular Medicine3, 655 W. Baltimore St., BRB Room 10-29, Baltimore, MD 21201 410-353-1001 (mobile),
[email protected].
ABSTRACT
Breast cancer is the 2nd leading cause of cancer
cancer-related
related death
among women; however, the majority of deaths arise as
complications from metastasis, rather than the primary tumor.
The cancer stem cell (CSC) hypothesis provides an explanation
for the limited success of current therapies for metastatic breast
cancer. CSCs are defined as a subpopulation of tumor-initiating
cells with the stem cell-like characteristics of self-renewal and
multipotency. CSCs have been shown to enter circulation and
reach distal tissues, where they may remain cell-cycle arrested,
andd therefore
th f
resistant
i t t to
t conventional
ti l chemotherapeutics.
h
th
ti
Recent work in our lab has shown that circulating tumor cells
use dynamic tubulin-based microtentacles (McTNs) to reattach
to distant tissues, a critical step in metastasis. McTNs are novel
cellular structures formed by epithelial cells when detached
from the extracellular matrix and are increased in more
metastatic breast cancer cell lines. Given the proposed
metastatic efficiency of CSCs, we examined McTN incidence
and function in mammary stem cells and breast cancer stem
cells. Flow cytometry for the CSC markers CD44 and CD24
showed that breast tumor cell lines with increased CSC
characteristics display higher McTN frequencies. Given this
correlation, CD44 and CD24 immunofluorescence was used to
separate human mammary epithelial (HMLE) cells into CSC
and non-CSC subpopulations by flow cytometry. Stem-like
HMLE cells (CD44hi/CD24lo) have increased McTN levels
compared to non-CSC cells (CD44lo/CD24hi) from the same
HMLE cell line. The CSC subpopulation also demonstrated
increased cytoskeletal modifications that promote McTN
formation, such as elevated vimentin expression and an increase
in both the amount and the bundling of stabilized, detyrosinated
tubulin. Vimentin and detyrosinated tubulin localize to McTNs
in suspended stem-like HMLEs. The increased McTNs in stemlike HMLEs promote faster initial reattachment of suspended
cells that is inhibited by the tubulin-directed drug, colchicine,
confirming a functional role for McTNs in stem cell
reattachment.
tt h
t Moreover,
M
li
live
cell
ll confocal
f l microscopy
i
demonstrates that McTNs participate in the structure of breast
stem cell mammospheres, encircling adjacent cells and
penetrating between cell-cell junctions. McTNs can be reduced
by the CSC targeting drug curcumin. These studies show that
McTNs contribute to the metastatic potential of breast CSCs as
well as the ability of mammary stem cells to form multicellular
mammospheres. We anticipate that this work will clarify the
molecular mechanisms underlying tubulin alterations in breast
cancer stem cells and identify how these tubulin modifications
may be targeted more specifically to reduce McTNs and the
metastatic reattachment efficiency of breast cancer CSCs.
MODEL
Figure 2: Imbalances in cytoskeletal physical forces increase cell
deformability and McTNs. (1) In attached epithelial cells, the
outward force of microtubules originating from the cell center
(green) is counterbalanced by contraction of the cortical actin
cytoskeleton (red). (2) Upon detachment, many epithelial cells die
b anoikis.
by
iki In some cells,
ll mild
ild relaxation
l
i occurs in
i the
h actin
i cortex
and microtubules are processed to a stabilized detyrosinated form
that promotes microtentacle extension. (3) In some cancer cells, the
physical forces in tumor cells are dysregulated. The cytoskeletal
imbalances that produce extended microtentacles likely arise from a
combination of persistent weakening of the actin cortex (leading to
increased deformability) and increased extension and stability of
microtubules (from increased detyrosination or association with
stabilizingg MAPs or vimentin).
)
HYPOTHESIS
Breast CSCs will have a higher incidence of
microtentacles arising from increased
tubulin detyrosination, leading to greater
metastatic potential compared to non-CSCs.
INTRODUCTION
H
i h li l cells
ll and
d other
h
Human mammary epithelial
transformed mammary cell lines produce unique
protrusions of the plasma membrane when detached
from extracellular matrix.
1.
Control
2. A.
LA
Colchicine
LA+Col
Stem-like mammary epithelial have
increased microtentacles.
Figure 3: Stem-like mammary epithelial cells have increased
microtentacles and microtentacle-promoting cytoskeletal
alterations. A, HMLE cells have distinct subpopulations of stemlike (CD44hi/CD24lo) and non-stem-like (CD44lo/CD24hi) cells. B,
Phase-contrast images detached HMLE subpopulations where
stem-like HMLEs display increase microtentacles (black arrows)
compared to non-stem-like HMLEs. C, Stem-like subpopulations
of HMLEs display significantly higher microtentacle frequencies
than non-stem-like
non stem like subpopulations.
subpopulations
Columns mean for three
Columns,
blinded experiments where at least 100 CellMask-stained cells
were counted, representative of three independent experiments;
bars, SD (P ≤ 0.00005, t test, black asterisk). D, Representative
western blot analysis of HMLE subpopulations shows that stemlike HMLEs have increased vimentin and detyrosinated tubulin
(Glu) whereas total α-tubulin and GAPDH are comparable.
Stem-like mammary epithelial cells have
microtentacle-promoting cytoskeletal alterations.
Mammary stem-like cells have increased
tubulin-dependent initial reattachment
from suspension.
Figure 5: Mammary stem-like cells have increased tubulindependent initial reattachment from suspension. A, Stem-like
HMLE cells attach at significantly faster rates than non-stem-like
HMLE cells as determined by electrical impedance expressed as Cell
Index. Stem-like HMLE and non-stem-like cells both have significantly
reduced attachment when treated with the microtubule polymerization
inhibitor colchicine (50µM). For all reattachment assay: lines, mean for
quadruplicate wells; bars, SD; representative graph from three
independent experiments is shown. B, Phase contrast images of HMLE
non-stem-like (a-d) and stem-like (e-h) subpopulations reattaching from
suspension. Panels, 10x magnification; Insets, 60x magnification.
Microtentacles Persist in Mammospheres
AIMS
1.
3.
1)
McTNs and metastatic capability increase as the percentage of
CSCs iincrease.
CSC
2)
Stem-like mammary epithelial cells have increased McTNs and
faster reattachment from suspension than non-stem-like cells. They
have increased glu-tubulin and vimentin protein levels, bundling,
and localization in McTNs.
3)
McTNs persist on the surface of mammospheres and penetrate
between cell-cell junctions in the mammosphere wall to encircle
adjacent cells
cells.
4)
Short-term treatment with curcumin reduces the CSC
subpopulation, abolishes McTNs, and prevents reattachment.
Define the role of detyrosinated tubulin and
vimentin in the generation of microtentacles
in breast stem-like subpopulations.
RESULTS
SIGNIFICANCE
McTNs are increased in more stem-like
breast cancer cells
Figure 1: Epithelial cells produce long, dynamic, tubulinbased protrusions upon detachment termed microtentacles
(McTNs). (1) Treatment of detached cells with the actin
depolymerizing agent, Latrunculin-A (LA) promotes extension of
these protrusions compared to control cells. Treatment with the
tubulin depolymerizing agent, Colchicine (Col) disrupts these
protrusions and combining LA and Col fragments protrusions into
a beads-on-a-string morphology. (2) Imaging McTNs with
electron microscopy. A) Confocal microscopy of MCF10A cells
labeled with a WGA-QDot655 displays the complexity and
curvature of these protrusions. B) Human mammary epithelial
cells were settled onto polycarbonate filters and processed for
scanning electron microscopy (SEM). (3) McTNs promote
penetration of endothelial monolayers. A) Live MDA-436 cells
expressing membrane-localized GFP (green) were suspended
over endothelial monolayers expressing mCherry (red). Confocal
imaging as the cell attaches discerns the penetration of the tumor
cell through the layer when viewed from underneath (B, white
arrows) or in cross-section (C, black arrows).
Figure 7: Mammary stem-like cells have increased tubulindependent initial reattachment from suspension. A, Cellular viability
assay using CellTiter 96 AQueous One Solution (Promega) reveals no
significant differences in viability between Bt549 cells treated with
doses of curcumin (5, 10, 25, 40, and 50µM) for 6 hours (P ≥ 0.05 for
each time point). B, Treatment with 50µM curcumin for 6 hours
significantly reduces the proportion of stem-like Bt549s compared to
vehicle. Columns, mean for three independent experiments where at
least 10,000 cells were analyzed; bars, SD (P ≤ 0.005, t test, black
asterisk). C, Treatment with 50µM curcumin prevents the formation of
Bt549 mammospheres compared to vehicle. Columns, mean for three
independent experiments; bars, SD (P ≤ 0.0005, t test, black asterisk).
D, Vehicle-treated Bt549s display significantly higher microtentacle
frequencies than Bt549s treated with 50µM curcumin for 6 hours.
Columns, mean for three blinded experiments where at least 100
CellMask-stained cells were counted, representative of three
independent experiments; bars, SD (P ≤ 0.0005, t test, black asterisk).
E, Vehicle-treated Bt549s attach at significantly faster rates than Bt549s
pretreated with 50µM curcumin for 6 hours and then seeded into Epplates containingg vehicle or drug,
g, respectively,
p
y, as determined byy
electrical impedance expressed as Cell Index. Lines, mean for
quadruplicate wells; bars, SD; representative graph from three
independent experiments is shown.
CONCLUSIONS
Examine the incidence and function of
microtentacles in breast stem-like
subpopulations
B.
2.
Stem-cell targeting agent Curcumin reduces
microtentacles and reattachment
Table 1: McTNs correlate with increased stemness across a
panell off breast
b
t cancer cell
ll lines.
li
A panell off breast
b
cancer cell
ll
lines were compared for their invasive and metastatic potential,
microtentacle levels, and subpopulations of stem-like
(CD44hi/CD24lo) and non-stem-like cells (CD44lo/CD24hi).
Invasiveness, metastatic capability, and microtentacle levels
increased with increasing stem cell character.
Figure 4: Stem-like mammary epithelial cells have
microtentacle-promoting cytoskeletal alterations. A, Attached
non-stem-like HMLE cells show weak, diffuse staining for Glutubulin (b) and a loss of vimentin protein and organization (c)
whereas stem-like HMLE cells show bundling of filamentous Glutubulin (f) and a robust network of vimentin filaments (g).
Hoescht was used to visualize the nuclei (d, h). B, Suspended
immunofluorescence where detached HMLE subpopulations were
spun down onto glass coverslips and fixed. Immunostaining
reveals the presence of Glu-tubulin (f) and vimentin (g) in
membrane protrusions (white arrows) in the stem-like HMLE
subpopulation but not in the non-stem-like subpopulation (b,c).
H
Hoescht
h was usedd to visualize
i li the
h nuclei
l i (a,e).
( )
*This work is in press as : Curcumin targets breast cancer stem-like cells with microtentacles that
persist in mammospheres and promote attachment. Charpentier MS, Whipple RA, Vitolo MI, Boggs
AE, Slovic J, Thompson KN, Bhandary L, Martin SS. Cancer Res. 2013 Dec 26. [Epub ahead of print]
*Supported by the ACEA Biosciences, Inc. Travel Award, MD Stem Cell Research Foundation
Exploratory Award #2008-0081, the National Cancer Institute (R01-CA154624), Susan G. Komen
Foundation (KG100240) and an Era of Hope Scholar award from the Department of Defense,
(BC100675).
Figure 6: Microtentacles Persist in Mammospheres. A, Live cell
confocal imaging of a HMLE mammosphere stained with CellMask
Orange revealing dynamic microtentacles (a, black arrows) that
disappear upon treatment with microtubule polymerization inhibitor
Colchicine (b, 50µM, 10 minutes). B, Live cell confocal imaging of
mammospheres stained with CellMask Orange (red) and transduced
with GFP-membrane (g
(green)) suspended
p
over a gglass surface reveals
McTNs from the GFP-membrane cell encircling other cells in the
mammosphere. Three-dimensional computer-rendered image of a
HMLE mammosphere (a) and a Bt549 mammosphere (d) viewed
from the bottom of the mammosphere. Confocal sectioning of GFPmembrane transduced cells reveals long, flexible protrusions (b, e)
that encircle other cells in the mammosphere (c, f).
Figure 8: Targeting circulating CSCs to reduce metastasis. Of the heterogeneous
cells
ll off the
th primary
i
t
tumor,
f cells
few
ll are able
bl to
t survive
i the
th hostile
h til conditions
diti
off the
th
circulatory system. Breast cancer stem cells (CSCs, in yellow) are primed for
successful metastasis and eventual outgrowth due to their stem cell properties of selfrenewal, multipotency, and drug resistance. CSCs are also more deformable, and
produce greater numbers of McTNs, which facilitate initial reattachment from
suspension to the capillary endothelium, preventing death via fragmentation in
narrow capillaries. Identifying the mechanisms by which CSCs increase McTN
formation will aid in the design in novel therapeutics to reduce metastases by
targeting circulating cancer stem cells.