Cancer cell-derived microvesicles induce transformation by transferring tissue transglutaminase and fibronectin to recipient cells.
ABSTRACT Tumor progression involves the ability of cancer cells to communicate with each other and with neighboring normal cells in their microenvironment. Microvesicles (MV) derived from human cancer cells have received a good deal of attention because of their ability to participate in the horizontal transfer of signaling proteins between cancer cells and to contribute to their invasive activity. Here we show that MV may play another important role in oncogenesis. In particular, we demonstrate that MV shed by two different human cancer cells, MDAMB231 breast carcinoma cells and U87 glioma cells, are capable of conferring onto normal fibroblasts and epithelial cells the transformed characteristics of cancer cells (e.g., anchorage-independent growth and enhanced survival capability) and that this effect requires the transfer of the protein cross-linking enzyme tissue transglutaminase (tTG). We further demonstrate that tTG is not sufficient to transform fibroblasts but rather that it must collaborate with another protein to mediate the transforming actions of the cancer cell-derived MV. Proteomic analyses of the MV derived from MDAMB231 and U87 cells indicated that both these vesicle preparations contained the tTG-binding partner and cross-inking substrate fibronectin (FN). Moreover, we found that tTG cross-links FN in MV from cancer cells and that the ensuing MV-mediated transfers of cross-linked FN and tTG to recipient fibroblasts function cooperatively to activate mitogenic signaling activities and to induce their transformation. These findings highlight a role for MV in the induction of cellular transformation and identify tTG and FN as essential participants in this process.
- [show abstract] [hide abstract]
ABSTRACT: Increasing evidence indicates that tissue transglutaminase (tTG) plays a role in the assembly and remodeling of extracellular matrices and promotes cell adhesion. Using an inducible system we have previously shown that tTG associates with the extracellular matrix deposited by stably transfected 3T3 fibroblasts overexpressing the enzyme. We now show by confocal microscopy that tTG colocalizes with pericellular fibronectin in these cells, and by immunogold electron microscopy that the two proteins are found in clusters at the cell surface. Expression vectors encoding the full-length tTG or a N-terminal truncated tTG lacking the proposed fibronectin-binding site (fused to the bacterial reporter enzyme beta-galactosidase) were generated to characterize the role of fibronectin in sequestration of tTG in the pericellular matrix. Enzyme-linked immunosorbent assay style procedures using extracts of transiently transfected COS-7 cells and immobilized fibronectin showed that the truncation abolished fibronectin binding. Similarly, the association of tTG with the pericellular matrix of cells in suspension or with the extracellular matrix deposited by cell monolayers was prevented by the truncation. These results demonstrate that tTG binds to the pericellular fibronectin coat of cells via its N-terminal beta-sandwich domain and that this interaction is crucial for cell surface association of tTG.Journal of Biological Chemistry 11/1999; 274(43):30707-14. · 4.65 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Transglutaminase-2 is involved in the physiological regulation of cell growth, but has also been associated with a number of cancer-associated features such as cell adhesion, metastasis and extracellular matrix modulation. Despite its importance in tumor cell progression and survival, relatively little is known about its expression in human malignancies. We have therefore investigated the transglutaminase-2 expression pattern in breast and ovarian cancer by using tissue arrays which contained 57 invasive breast cancer biopsies and 62 ovarian cancers, and compared it to transglutaminase-2 protein levels in normal human tissues. By using immunohistochemistry, transglutaminase-2 protein was detected in 48 of 57 breast tumors (84%), with epithelial expression in 26 of 41 (63%) ductal invasive carcinomas and in all 6 (100%) lobular invasive carcinomas. Stromal transglutaminase-2 was present in 14 of 41 (34%) ductal subtypes and in 4 of 6 (67%) lobular subtypes, which is in sharp contrast to the infrequent expression in normal breast stroma (P<0.001, Mann-Whitney test) and somewhat also in normal breast epithelium (P = 0.065, Mann-Whitney test). In most other human tissues, transglutaminase-2 protein was less frequent and usually confined to either the epithelium or in adjacent stroma. In ovarian tumors, the protein was detected in 36 of the 62 cases (58%), and seen in all histological subtypes. Taken together, we have demonstrated increased transglutaminase-2 protein expression in both malignant breast epithelium and surrounding stroma, although its selective spatial expression pattern in normal tissues also indicates a physiological role in stromal-epithelial interactions.Clinical and Experimental Metastasis 02/2006; 23(1):33-9. · 3.46 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Brain tumors are horrific diseases with almost universally fatal outcomes; new therapeutics are desperately needed and will come from improved understandings of glioma biology. Exosomes are endosomally derived 30-100 nm membranous vesicles released from many cell types into the extracellular milieu; surprisingly, exosomes are virtually unstudied in neuro-oncology. These microvesicles were used as vaccines in other tumor settings, but their immunological significance is unevaluated in brain tumors. Our purpose here is to report the initial biochemical, proteomic, and immunological studies on murine brain tumor exosomes, following known procedures to isolate exosomes. Our findings show that these vesicles have biophysical characteristics and proteomic profiles similar to exosomes from other cell types but that brain tumor exosomes have unique features (e.g., very basic isoelectric points, expressing the mutated tumor antigen EGFRvIII and the putatively immunosuppressive cytokine TGF-beta). Administration of such exosomes into syngeneic animals produced both humoral and cellular immune responses in immunized hosts capable of rejecting subsequent tumor challenges but failed to prolong survival in established orthotopic models. Control animals received saline or cell lysate vaccines and showed no antitumor responses. Exosomes and microvesicles isolated from sera of patients with brain tumors also possess EGFR, EGFRvIII, and TGF-beta. We conclude that exosomes released from brain tumor cells are biochemically/biophysically like other exosomes and have immune-modulating properties. They can escape the blood-brain barrier, with potential systemic and distal signaling and immune consequences.The FASEB Journal 01/2009; 23(5):1541-57. · 5.70 Impact Factor
Cancer cell-derived microvesicles induce transformation
by transferring tissue transglutaminase and
Marc A. Antonyaka,1, Bo Lia,b,1, Lindsey K. Boroughsa, Jared L. Johnsonb, Joseph E. Drusoa, Kirsten L. Bryantb,
David A. Holowkab, and Richard A. Cerionea,b,2
Departments ofaMolecular Medicine andbChemistry and Chemical Biology, Cornell University, Ithaca, NY 14853
Edited by Joan S. Brugge, Harvard Medical School, Boston, MA, and approved January 28, 2011 (received for review December 2, 2010)
Tumor progression involves the ability of cancer cells to communi-
cate with each other and with neighboring normal cells in their
microenvironment. Microvesicles (MV) derived from human cancer
cells have received a good deal of attention because of their ability
to participate in the horizontal transfer of signaling proteins be-
tween cancer cells and to contribute to their invasive activity. Here
we show that MV may play another important role in oncogenesis.
In particular, we demonstrate that MV shed by two different
human cancer cells, MDAMB231 breast carcinoma cells and U87
glioma cells, are capable of conferring onto normal fibroblasts and
epithelial cells the transformed characteristics of cancer cells (e.g.,
anchorage-independent growth and enhanced survival capability)
and that this effect requires the transfer of the protein cross-linking
enzyme tissue transglutaminase (tTG). We further demonstrate
that tTG is not sufficient to transform fibroblasts but rather that it
must collaborate with another protein to mediate the transforming
actions of the cancer cell-derived MV. Proteomic analyses of the MV
derived from MDAMB231 and U87 cells indicated that both these
vesicle preparations contained the tTG-binding partner and cross-
inking substrate fibronectin (FN). Moreover, we found that tTG
cross-links FN in MV from cancer cells and that the ensuing MV-
function cooperatively to activate mitogenic signaling activities and
to induce their transformation. These findings highlight a role for
MV in the induction of cellular transformation and identify tTG and
FN as essential participants in this process.
cancer progression|extracellular matrix|oncosomes|shedding vesicles|
into their surroundings is becoming increasingly recognized as
a feature of tumor biology (1–3), but how these structures are
generated and their importance in cancer progression are only just
beginning to be appreciated. Of particular interest are the recent
findings that at least some of the MV generated by aggressive
brain, breast, and prostate cancer cells involve the direct budding
or shedding of vesicles from their plasma membranes. These
membrane-bound structures range in size up to ~2.0 microns in
diameter and contain a variety of cell-surface receptors, intra-
cellular signaling proteins, and cytoskeletal components, as well as
cellular origin (4–8). Interestingly, they are taken up by recipient
cancer cells where the transferred cargo can promote the activa-
tion of survival and mitogenic signaling proteins, including protein
kinase B (PKB/AKT) and ERK (5, 7, 9).
The finding that MV provide a means for cancer cells to share
intracellular proteins and genetic information with each other has
begun to shed some light on a poorly understood but potentially
important mechanism underlying the onset and development of
human cancers. However, numerous questions regarding MV
biogenesis and function need to be addressed to understand more
thoroughly the significance of this unconventional form of cell
he release of microvesicles (MV) or oncosomes from different
types of high-grade or aggressive forms of human cancer cells
communication in oncogenesis. Here we describe findings that
suggest a unique role for MV in cancer progression and identify
the proteins that are essential to this function.
Analysis of serum-starved cultures of the highly aggressive human
breast cancer cell line MDAMB231 by scanning electron mi-
croscopy (SEM) (Fig. 1A, Left) or by fluorescent microscopy
MV ranging from ~0.2–2.0 microns in diameter were present on
the surface of ~35% of these cells (Fig. 1B). MV also were
detected on ~25% of serum-deprived U87 human glioma cells,
and their formation was induced in HeLa cervical carcinoma cells
by EGF stimulation (Fig. 1 B and C). In contrast, MV were not
detected on the surface of normal NIH 3T3 fibroblasts cultured
under serum-starved or EGF-stimulated conditions, indicating
that some cell types may not generate MV. Moreover, we de-
termined that MV were actively shed from these cancer cells, as
demonstrated by time-lapse images of the release of a GFP-
labeled MV from the plasma membrane of an MDAMB231 cell
transfected with an EGFP-labeled plasmid (pEGFP) encoding
the plasma membrane targeting sequence (GFP-PM) of the Lyn
tyrosine kinase (Fig. 1D and Movie S1), as well as through the
detection of MV containing GFP in the culturing medium col-
lected from transfectants expressing only pEGFP by immunoblot
(Fig. 1E) and FACS (Fig. S1 A and B) analysis.
Although previously MV have been reported to share their
cargo with cells, we were interested in seeing whether MV might
be capable of conferring some of the transformed characteristics
of the donor cancer cells onto normal (nontransformed) recipient
cells. Thus, we isolated MV constitutively shed by MDAMB231
breast cancer cells and U87 brain tumor cells from their serum-
free culturing medium (Fig. 2A) and added them to cultures of
nontransformed NIH 3T3 fibroblasts. MV generated by either
of these cancer cell lines were capable of stimulating the activities
of the signaling protein kinases AKT and ERK in the recipient
fibroblasts (Fig. 2B), similar to observations when cancer cell-
derived MV were incubated with other cancer cells or endothelial
cells (5, 7, 9). Moreover, when NIH 3T3 fibroblasts were in-
cubated with MV derived from MDAMB231 cells or U87 cells,
they exhibited two phenotypes characteristic of cancer cells,
namely an enhanced survival capability (Fig. 2C) and an ability to
grow under low-serum conditions (Fig. 2D). We then asked
Author contributions: M.A.A., B.L., and R.A.C. designed research; M.A.A., B.L., L.K.B., J.L.J.,
J.E.D., and K.L.B. performed research; J.L.J., J.E.D., and K.L.B. contributed new reagents/
analytic tools; M.A.A., B.L., D.A.H., and R.A.C. analyzed data; and M.A.A., B.L., and R.A.C.
wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1M.A.A. and B.L. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| March 22, 2011
| vol. 108
| no. 12www.pnas.org/cgi/doi/10.1073/pnas.1017667108
whether the cancer cell-derived MV, when added to normal cells,
could induce cellular transformation as read out by anchorage-
independent growth (i.e., colony formation in soft agar). Fig. 2 E
form colonies in soft agar, sustained treatment of fibroblasts with
MV collected from either MDAMB231 cells or U87 cells con-
independent conditions. MDAMB231 cell-derived MV similarly
the normal human mammary epithelial cell line MCF10A. Thus,
normal cells is indeed capable of endowing normal cells with the
characteristics induced by oncogenic transformation.
We then asked what MV-associated protein(s) is responsible
for mediating the transfer of transforming capability. We initially
considered the EGF receptor as a possible candidate protein, be-
cause activated forms of this receptorcan be shared between brain
cancer cells via MV (5, 6, 9). However, it is unlikely that the EGF
receptor accounts for the similar transforming abilities associated
with the MV derived from MDAMB231 breast cancer cells and
U87 glioblastoma cells (Fig. 2 C–E), because activated EGF
receptors cannot be detected in the MV shed from U87 cells (Fig.
2A). This notion was supported further by the finding that the
anchorage-independent growth advantage imparted to NIH 3T3
cells by U87 cell-derived MV is insensitive to treatment with the
EGF receptor tyrosine kinase inhibitor AG1478 (Fig. S2C).
To identify proteins potentially involved in the transforming
listed in Fig. S3. Notably, among the MV-associated proteins was
tissue transglutaminase (tTG), a protein cross-linking enzyme
that has been linked to the chemoresistance and aberrant cell
growth exhibited by some cancer cells (10–15) and that is secreted
from cells by an unknown mechanism (15–17). We confirmed by
immunoblot analysis that tTG is a component of MV derived
from MDAMB231andU87cells (Fig.3A)and demonstrated that
the MV on the surfaces of MDAMB231 cells were detectable
when immunostained with a tTG antibody (Fig. 3B, Upper) but
Left). Likewise, the MVgenerated by U87cells andHeLa cervical
carcinoma cells stimulated with EGF also contained tTG (Fig.
3C). These findings, when coupled with the fact that a GFP-
tagged form of tTG is incorporated more efficiently than GFP
alone into MV shed by MDAMB231 (Fig. 3D), demonstrate that
tTG is targeted to MV generated by distinct types of cancer cells
and in response to specific cell-culturing conditions.
As shown in Fig. 3B, tTG frequently was enriched in the
membranes of MV, as indicated by the ring-shaped staining pat-
terns detected with a tTG antibody in cells actively forming MV.
The same tTG antibody labeled MV that protruded from the
plasma membranes of nonpermeabilized MDAMB231 cells (Fig.
3E) and detected tTG on the surfaces MV of individually isolated
from MDAMB231 cells by immuno-SEM (Fig S4A). The upper
panel in Fig. 3F shows that tTG expressed in whole-cell lysates
was enzymatically active as read out by its ability to catalyze the
incorporation of biotinylated pentylamine (BPA) into casein.
Pretreatment of the intact MDAMB231 cell-derived MV with the
cell-permeable tTG inhibitor monodansylcadaverine (MDC)
greatly diminished the levels of BPA-labeled casein detected in
the assay. Interestingly, the cell-impermeable tTG inhibitor T101
(Fig. S4 B and C) also effectively blocked the cross-linking activity
associated with MV derived from MDAMB231 cells (Fig. 3F),
suggesting that tTG is predominantly localized and activated on
the outer leaflet of MV membranes.
The MVderivedfromMDAMB231breast cancer cellswerenot
sensitive tothetraditional secretoryinhibitorsBFA orExoI,which
block Arf GTPase activation (18), as indicated by monitoring MV
formation by immunofluorescence staining of vesicle-associated
tTG (Fig. S5A). We then considered the possibility that the ability
of tTG to cross-link proteins is important for the formation and/or
shedding of MV by cancer cells. Immunofluorescent analysis with
a tTG antibody revealed that exposing MDAMB231 cells to the
tTG inhibitors MDC and T101 had no effect on MV formation
(Fig. S5A). The shedding of MV by MDAMB231 cells also did not
as shown by the detection of nearly equivalent amounts of the MV
marker flotillin-2 (5) and tTG in MV isolated from the culturing
medium of control cells or cells treated with different inhibitors
(Fig. S5B, Left and Right). Correspondingly, knocking down tTG
cells generate MV. (A) MDAMB231 cells
were analyzed by SEM (Left) and immu-
nofluorescent microscopy (IF) using rho-
damine-conjugated phalloidin to detect
F-actin (Right). Some of the largest MV are
indicated by arrows. (B) Quantification
of MV production by various cell lines
stimulated conditions. Cells generating
MV were detected by labeling the sam-
ples with rhodamine-conjugated phalloi-
din. The data shown represent the mean ±
SD from three independent experiments.
(C) Images of cells from the experiment
performed in B. Some of the MV are de-
noted by arrows. (D) MDAMB231 cells
transiently expressing a GFP-tagged form
of the plasma membrane-targeting se-
quence (GFP-PM) from the Lyn tyrosine
kinase were subjected to live-imaging
fluorescent microscopy. Shown are a series
of time-lapse images of a transfectant
taken at 2-min intervals. The arrow indi-
cates an MV that forms and is shed from
a cell. (E) Serum-deprived MDAMB231
cells that were mock transfected or trans-
fected with pEGFP were lysed, and the MV shed into the medium by the transfectants were isolated and lysed as well. The WCL and the MV lysates
then were immunoblotted with antibodies against GFP, the MV marker flotillin-2, and the cytosolic-specific marker IκBα.
Distinct types of human cancer
Antonyak et al.PNAS
| March 22, 2011
| vol. 108
| no. 12
in MDAMB231 cells, which depleted the expression of tTG in the
MV, caused little change in the amount of MV shed by these cells
as read out by the flotillin-2 marker (Fig. S5B, Center). Moreover,
tTG mutants defective in their ability to cross-link substrates (tTG
C277V) or to bind GTP (tTG R580L) when ectopically expressed
in MDAMB231 cells were targeted to MV as efficiently as ec-
topically expressed wild-type tTG (Fig. S5C). Thus, these results
indicate that tTG is not essential for the ability of cancer cells to
form or shed MV, nor is the enzymatic activity of tTG needed for
its targeting to MV.
We then examined whether tTG might function as MV cargo
and be transferred to recipient cells. NIH 3T3 fibroblasts were
incubated for 30 min with MV derived from serum-starved cul-
tures of either MDAMB231 cells or U87 cells and then analyzed
for tTG expression by immunoblot analysis (Fig. 4A and Fig.
S6A) and immunofluorescent microscopy (Fig. S6B). The results
from these experiments show that tTG levels were increased
significantly in fibroblasts that had been incubated with the
cancer cell-derived MV relative to the barely discernible levels of
tTG in control fibroblasts.
These findings raised the question of whether the MV-
be important for conferring these cells with enhanced survival
capability and the characteristics of transformation. To address
this question, we took advantage of our earlier findings that tTG
is localized on the surfaces of MV so that its cross-linking activity
is susceptible to inhibition by the cell-impermeable, irreversible
forming normal fibroblasts. (A) WCL of serum-starved MDAMB231 and U87
cells, as well as lysates of the MV shed by these cells, were immunoblotted
with antibodies against the MV markers actin and flotillin-2, the cytosolic-
specific marker IκBα, and the activated (phospho)-EGF receptor. (B–D) Mul-
tiple sets of serum-deprived NIH 3T3 fibroblasts were incubated with serum-
free medium, medium containing 2% CS, or medium supplemented with
intact MV derived from either MDAMB231 or U87 cells as indicated. (B) One
set of cells was lysed after being exposed to the various culturing conditions
for the indicated lengths of time and then was immunoblotted with anti-
bodies that recognize the activated and total forms of AKT and ERK. Two
additional sets of fibroblasts were evaluated for their abilities to undergo
serum deprivation-induced cell death (C) and to grow in low serum (2% CS)
(D). For the growth assays, the culturing medium (including the MV) was
replenished daily. (E) NIH 3T3 fibroblasts incubated with or without MV
derived from MDAMB231 or U87 cells were subjected to anchorage-in-
dependent growth assays. The soft agar cultures were re-fed (including
adding freshly prepared MV) every third day. NIH 3T3 cells expressing Cdc42
F28L were used as a positive control for these experiments. (F) Images of the
resulting colonies that formed in E. The data shown in C–E represent the
mean ± SD from at least three independent experiments.
MDAMB231 and U87 cancer cell-derived MV are capable of trans-
MDAMB231 and U87 cells, as well as lysates of the MV shed by these cells,
were immunoblotted with several antibodies, including one against tTG. (B)
(Upper Left) MDAMB231 cells immunostained with a tTG antibody. (Upper
Right) The boxed area was enlarged, and arrows indicated certain MV.
(Lower) An MDAMB231 cell costained with only the secondary antibody
(Left) and with rhodamine-conjugated phalloidin to label the MV (Right). (C)
Images of serum-starved U87 glioma cells and HeLa cervical carcinoma cells
that were left untreated or were stimulated with EGF for 15 min as indicated
and then were immunostained with a tTG antibody. Pronounced MV are
indicated by arrows. (D) WCL of MDAMB231 cells ectopically expressing ei-
ther GFP only or GFP-tTG, as well as lysates of the MV shed by these trans-
fectants into their culturing medium, were immunoblotted with antibodies
against GFP, the MV marker flotillin-2, and the cytosolic-specific marker IκBα.
(E) Fluorescent images of permeabilized and nonpermeabilized samples of
MDAMB231 cells stained with antibodies against tTG, the intracellular pro-
tein Ras homolog enriched in brain (Rheb), and DAPI to label nuclei. (F) WCL
of serum-starved MDAMB231 cells and intact MV generated by these cells
treated with or without the tTG inhibitors T101 (cell impermeable) or MDC
(cell permeable), were assayed for transamidation activity as read out by the
incorporation of BPA into casein. The samples then were immunoblotted
with antibodies against tTG, flotillin-2, and IκBα.
MV shed by cancer cells contain tTG. (A) WCL of serum-starved
| www.pnas.org/cgi/doi/10.1073/pnas.1017667108 Antonyak et al.
inhibitor T101 (Fig. 3 B, E, and F). By pretreating cancer cell-
derived MV with T101 before adding them to fibroblast cultures,
we were able to inhibit the cross-linking activity of the MV-
associated tTG selectively and irreversibly (Fig. 4B and Fig. S6C).
Using this approach, we compared how the survival advantage
afforded to NIH 3T3 fibroblasts by MV collected from cancer
cells was affected by the inhibition of tTG activity. Fig. 4C and
Fig. S7A show that pretreatment of the MV derived from
MDAMB231 or U87 cells with T101 severely compromised their
ability to protect the recipient fibroblasts from serum deprivation-
induced cell death. Importantly, the extent of cell survival ach-
ieved by culturing NIH 3T3 cells in medium supplemented with
a nominal amount (2%) of calf serum (CS) was unchanged by the
addition of T101, indicating that the ability of this small-molecule
inhibitor to abolish the protection afforded by the cancer cell-
fibroblasts to apoptosis. Analogous experiments then were per-
formed in which MDAMB231 cell-derived MV were incubated
with serum-starved NIH 3T3 cells in the presence of the cell-
permeable tTG inhibitor MDC (Fig. 4C) or MV collected from
MDAMB231 cells in which tTG had been knocked down (Fig.
S5B) were added to serum-starved NIH 3T3 cells (Fig. S7B).
Collectively, the results from these experiments point to a critical
role for tTG in mediating the survival advantage imparted to
fibroblasts by cancer cell-derived MV.
We then asked whether the transforming abilities of the cancer
cell-derived MV were dependent on tTG. As shown previously in
Fig. 2E and Fig. S2B, and again in Fig. 4D and Figs. S7C and S8A,
incubating normal NIH 3T3 fibroblasts and MCF10A epithelial
cells with MV derived from MDAMB231 cells or U87 cells in-
independent conditions. However, when recipient fibroblasts or
epithelial cells were incubated with MV preparations that had
been pretreated with T101 (Fig. 4D and Figs. S2B and S8A) or in
which tTG had been knocked down (Fig. S7C), the number of
colonies that formed was reduced. We then verified that T101 did
not generally inhibit cellular transformation by showing that this
inhibitor had no influence on the ability of NIH 3T3 cells
expressing an activated form of the small GTPase Cdc42 (Cdc42
F28L) to grow under anchorage-independent conditions, even
when a fivefold excess of T101 was used (Fig. S8B).
These findings prompted us to consider whether cancer cell-
derived MV might function similarly in vivo and promote tumor
growth by causing normal cells in the tumor microenvironment to
acquire the ability to form a tumor. To investigate this idea, we
took advantage of the fact that exposing MDAMB231 cells to the
mitotic-arresting agent mitomycin C before injecting them into
nude mice inhibited their ability to form tumors under conditions
in which their control counterparts (untreated MDAMB231 cells)
were quite effective at inducing tumor formation (Fig. 4H).
However, when the mitomycin C-treated MDAMB231 cells were
3T3 fibroblasts into mice, four of six mice formed tumors, sug-
gesting that the MV shed by the mitotically arrested cancer cells
were capable of causing the neighboring NIH 3T3 fibroblasts to
become transformed, inducing tumor growth. We then went on to
show that knocking down tTG expression in the mitotically
arrested MDAMB231 cells blocked the ability of the coinjected
consistent with the idea that cancer cells can generate MV in vivo
and that their ability to cause normal cells in the tumor microen-
vironment to promote tumor formation is dependent on tTG.
These findings demonstrate that the MV-mediated transfer of
tTG into recipient cells is necessary for the ability of MDAMB231
cell- and U87 cell-derived MV to transform fibroblasts. However,
is tTG alone sufficient to confer survival and transforming capa-
bilities to the recipient cells? In fact, we found that although NIH
3T3 fibroblasts stably overexpressing Myc-tagged tTG (Fig. 4E)
were indeed resistant to serum deprivation-induced apoptosis, an
effect that was ablated by treating the cells with MDC (Fig. 4F),
they were unable to form colonies in soft agar, unlike vector
control-expressing fibroblasts incubated with MV derived from
MDAMB231 cells (Fig. 4G). This result shows that although
overexpression/overactivation of tTG in normal cells is not suffi-
cient to induce their transformation fully (i.e., NIH 3T3 cells ec-
formation requires the transfer of tTG from MV to recipient cells. (A and B)
Extracts of serum-starved NIH 3T3 fibroblasts that were incubated with se-
rum-free medium or serum-free medium supplemented with MDAMB231
cell-derived MV that had been pretreated for 30 min with or without the tTG
inhibitor T101 were immunoblotted with tTG and actin antibodies (A) and
assayed for transamidation activity as read out by the incorporation of BPA
into lysate proteins (B). (C) Cell death assays were performed on fibroblasts
maintained in serum-free medium, 2% CS medium, or serum-free medium
containing MDAMB231 cell-derived MV. Each culturing medium was left
unsupplemented or was supplemented further with the tTG inhibitors T101
(cell impermeable) or MDC (cell permeable) as indicated. (D) Anchorage-
independent growth assays were performed on fibroblasts incubated with
MDAMB231 cell-derived MV that were untreated or treated with T101, the
RGD peptide, or the control RGE peptide. (E) Lysates of NIH 3T3 cells stably
overexpressing vector alone or Myc-tTG were immunoblotted with Myc and
actin antibodies and were assayed for transamidation activity as read out by
the incorporation of BPA into lysate proteins. (F) Cell death assays were
performed on the NIH 3T3 stable cell lines maintained in serum-free medium
treated with T101, MDC, or 2% CS or left untreated. (G) Anchorage-
independent growth assays were performed on the NIH 3T3 stable cell lines.
Vector-control fibroblasts were incubated with MDAMB231 cell-derived MV
from at least three independent experiments. (H) Tumor-formation assays
were performed in which 5 × 105MDAMB231 cells mitotically arrested using
mitomycin C (Mito-C-MDAMB231) expressing either control siRNA (siCont) or
tTG siRNAs (siTG-1or siTG-2) were injected s.c. alone or combined with 5 × 105
NIH 3T3 fibroblasts into nude mice. As controls, untreated MDAMB231 and
NIH 3T3 cells were injected into nude mice. The resulting tumors that formed
for each condition were counted, and the results are shown in the table.
The ability of MDAMB231 cell-derived MV to induce cellular trans-
Antonyak et al.PNAS
| March 22, 2011
| vol. 108
| no. 12
when grown under anchorage-independent conditions), it does
confer upon normal cells some characteristics of the transformed
state, allowing them to grow in a monolayer under low-serum
conditions and to become less sensitive to serum deprivation-
induced cell death. However, these findings also indicate that for
cancer cell-derived MV to enable recipient cells to exhibit one of
the majorhallmarksofcellulartransformation,namely anchorage-
independent growth, another protein must be transferred along
with tTG. The cytoskeletal component fibronectin (FN) was a
particularly attractive candidate, because it is a known binding
partner of tTG (16, 17, 19) and was identified in our proteomics
screen of MDAMB231 cell- and U87 cell-derived MV (Fig. S3).
We confirmed by immunoblot analysis that FN was expressed in
the MV collected from each of the cancer cell lines (Fig. 3A). We
then assessed the potential role of the MV-associated FN in confer-
ring upon fibroblasts the ability to exhibit anchorage-independent
growth by using the arginine-glycine-aspartic acid (RGD) peptide as
a means to interfere with the ability of FN to bind to and activate
integrins on the surface of the recipient fibroblasts (20, 21). Anchor-
age-independent growth assays performed on fibroblasts cotreated
peptide or the control arginine-glycine-glutamic acid (RGE) peptide
showed that the RGD peptide, like T101, blocked the MV-triggered
induction of cellular transformation, whereas the control peptide did
not (Fig. 4D and Fig. S8A).
cell-derived MV to transform recipient cells, might they work to-
gether to elicit this cellular outcome? To address this question we
first examined whether tTG interacts with FN in MV. Fig. 5A
shows that FNcoimmunoprecipitateswithtTGfromMDAMB231
WCL, as previously reported (16, 17, 19), as well as with tTG from
lysates of MV shed by these cells. In addition to binding the mo-
nomeric form of FN, tTG associated with a larger form of FN with
an apparent molecular mass of ~440 kDa that likely represented
cross-linked FN dimers and was detectable only in the MV lysate.
Pretreating intact MV collected from MDAMB231 cells or U87
cells with the tTG inhibitor T101 before lysing the MV and sub-
of tTG to be coimmunoprecipitated with monomeric FN from the
MV lysates (Fig. S9). However, pretreating the MV with the tTG
kDa FN species detected in the MV lysate samples (Fig. 5B),
cell-derived MVis generated through the ability of tTG to interact
with and cross-link FN.
Thedimerization ofFNstrongly enhancesitsabilitytobindand
activate integrins on the surfaces of cells (22, 23). The ability of
tTG to cross-link FN in MV shed by cancer cells suggested that
this covalently modified form of FN is capable of potentiating
integrin activation. Indeed, we found that preparations of intact
MV isolated from the medium of serum-deprived MDAMB231
cells or U87 cells were capable of stimulating signaling activities
that are well known to be downstream from activated integrins
including focal adhesion kinase (FAK) and ERK (Fig. 5C).
Moreover, the activation of these kinases by MV was blocked by
using either the tTG inhibitor T101 or the RGD peptide that
interferes with integrin signaling, thus further demonstrating the
importance of tTG and FN for the signaling functions of MV.
The findings described here shed important light on an un-
conventional and poorly understood mechanism of cell-to-cell
communication and how that communication may have signifi-
cant consequences in human cancer progression. In particular,
we have shown that exposing normal recipient cells to bioactive
MV that are constitutively shed by certain human cancer cells
can cause the recipient cells to acquire a transformed phenotype
(Fig. 5D). We have identified the protein cross-linking enzyme
tTG and the extracellular matrix protein FN as components of
cancer cell-derived MV that functionally cooperate to mediate
the transforming activities of the MV. The fact that tTG and FN
are aberrantly regulated in several types of human cancers (24–
30) raises exciting possibilities regarding the broad roles played
by MV in cancer progression.
One aspect of our work that merits further consideration
involves the potential effects of MV on tumor growth. A growing
body of evidence demonstrates that cancer cells are capable of
generating MV in vivo, suggesting that the shedding of MV by
cancer cells is not merely an artifact of tissue culture but rather
represents a tumor-relevant process. A particularly good example
comes from a recent study which showed that brain tumor cell-
derived MV could be detected routinely in blood samples taken
from human patients afflicted with glioblastoma multiforme (4).
actions of MV on recipient fibroblasts. (A) WCL of MDAMB231 and lysates of
the MV shed by these cells were immunoblotted (Input) or were subjected to
immunoprecipitation using a tTG antibody (IP: tTG) and then immuno-
blotted with FN, tTG, and actin antibodies. Note the detection of cross-
linked FN in the MV lanes (FN dimer). (B) Intact MV collected from
MDAMB231 or U87 cells were treated with T101 or were left untreated
before being lysed. The MV extracts then were immunoblotted with FN and
tTG antibodies. Note that the cross-linked forms of FN detected in the MV
samples (FN dimer) were reduced significantly by T101 treatment. (C) Lysates
of fibroblasts that were incubated with or without MV derived from
MDAMB231 and U87 cells that had been pretreated or not with T101 were
immunoblotted with antibodies against FAK and ERK or with antibodies
that specifically recognize the activated forms of these protein kinases. (D)
Diagram depicting how MV transform recipient cells. MV containing tTG and
fibronectin are generated and released from the surfaces of human cancer
cells. The MV then can be taken up by or can directly alter the microenvi-
ronment of neighboring normal cells, where the cotransfer of tTG and FN
function cooperatively on the recipient cells to induce signaling events that
promote cell survival and aberrant cell growth.
tTG cooperates functionally with FN to mediate the transforming
| www.pnas.org/cgi/doi/10.1073/pnas.1017667108 Antonyak et al.
Moreover, it also was shown that when MV shed by cultures of
several different human primary tumor cells or established cancer
these findings are considered in the context of a tumor setting, the
increased proliferative capacity afforded by sharing MV among
cancer cells could be envisioned as a mechanism to augment tu-
mor growth. However, our results now suggest that MV may im-
pact cancer progression in another way, specifically, by conferring
microenvironment (i.e., fibroblasts and epithelial cells), the
transformed characteristics of cancer cells. This effect would be
consistent with the idea that the expansion of a tumor mass would
not necessarily depend solely on the proliferation of the cancer
cells but rather could include as well the aberrant growth in the
tumor microenvironment exhibited by stromal cells (including
fibroblasts) and normalepithelium that have beenexposed toMV
shed by cancer cells. If this notion turns out to be true, we must
adjust our understanding of cancer progression in vivo to include
the potential contribution of MV.
However, the ability of the cancer cell-derived MV to induce
a transformed phenotype in normal (nontransformed) recipient
cell types is not sustained. For MV derived from MDAMB231
breast cancer cells or U87 brain tumor cells to promote the
growth of normal cells (i.e., NIH 3T3 fibroblasts and MCF10A
mammary epithelial cells) in low serum and to induce their
ability to form colonies in soft agar, the recipient cells had to be
treated repeatedly with freshly prepared MV throughout the
duration of these growth assays. This requirement implies that
the proteins and RNA transcripts contained within MV that are
involved in promoting their transforming activity have a finite
lifespan after being added to normal recipient cells and need to
be replenished continuously. In the context of a tumor setting,
the chronic shedding of MV by the cancer cells into their mi-
croenvironment might provide the continuous supply of MV
required by the nearby recipient stroma and normal epithelium
to induce and maintain a transformed phenotype.
The MV field is still in its infancy, and many of the advances
made in our understanding of MV formation, as well as the
identity of their contents and their mechanisms of action (i.e.,
transforming capability), have been largely limited thus far to
tissue culture experiments. However, as our methods for iso-
lating and characterizing cancer cell-derived MV continue to
advance, it ultimately will be important to extend these studies
into animal models. Toward this end, we currently are focusing
our efforts on understanding the signaling events that are cou-
pled to the formation and shedding of MV from the surface of
human cancer cells and on determining whether additional pro-
teins (i.e., aside from tTG and FN) contribute to the abilities of
cancer cell-derived MV to transform recipient cells.
Materials and Methods
The isolation of MV from cancer cells was carried out essentially as described
previously (4, 5). A detailed description of the protocol is provided in SI
Materials and Methods. All reagents and additional procedures used in this
study, including cell culture, cell growth and survival assays, electron and
fluorescent microscopy, immunoprecipitation, immunoblot analysis, flow
cytometry, proteomic analyses, transamidation assays, and mouse xenograft
experiments are described in SI Materials and Methods.
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Antonyak et al.PNAS
| March 22, 2011
| vol. 108
| no. 12