Download Carcinoma and stromal enzyme activity profiles associated with

Carcinoma and stromal enzyme activity profiles
associated with breast tumor growth in vivo
Nadim Jessani*, Mark Humphrey*, W. Hayes McDonald*, Sherry Niessen*, Kim Masuda*, Beena Gangadharan†,
John R. Yates III*, Barbara M. Mueller†, and Benjamin F. Cravatt*‡
*The Skaggs Institute for Chemical Biology and Departments of Cell Biology and Chemistry, The Scripps Research Institute, La Jolla, CA 92037; and †Division
of Cancer Biology, La Jolla Institute for Molecular Medicine, San Diego, CA 92121
Edited by Peter K. Vogt, The Scripps Research Institute, La Jolla, CA, and approved August 6, 2004 (received for review July 1, 2004)
Cancer research depends on the use of human cell lines for both the
in vitro (culture) and in vivo (xenograft) analysis of tumor progression and treatment. However, the extent to which cultured preparations of human cancer lines display similar properties in vivo,
where important host factors may influence tumor biology, remains unclear. Here, we address this question by conducting a
functional proteomic analysis of the human breast cancer line
MDA-MB-231 grown in culture and as orthotopic xenograft tumors
in the mammary fad pad of immunodeficient mice. Using a suite of
activity-based chemical probes, we identified carcinoma (human)
enzyme activities that were expressed selectively in culture or in
xenograft tumors. Likewise, distinct groups of stromal (mouse)
enzyme activities were found that either infiltrated or were excluded from xenograft tumors, indicating a contribution by specific
host components to breast cancer development. MDA-MB-231 cells
isolated from tumors exhibited profound differences in their enzyme activity profiles compared with the parent cell line, including
the dramatic posttranscriptional up-regulation of the serine proteases urokinase plasminogen activator and tissue plasminogen
activator and down-regulation of the glycolytic enzyme phosphofructokinase. These altered enzyme activity profiles correlated
with significantly greater tumor growth rates and metastases for
xenograft-derived MDA-MB-231 cells upon reintroduction into
mice. Collectively, these data indicate that the in vivo environment
of the mouse mammary fat pad cultivates the growth of human
breast cancer cells with elevated tumorigenic properties and highlight the value of activity-based protein profiling for identifying
proteomic signatures that depict such changes in cancer cell
biology.
T
umorigenesis involves an intricate crosstalk between cancer
cells and the nontumor (stromal) environment (1–3). Host or
stromal factors can be both supportive and responsive to tumors
and have been shown to play key roles in fostering the development of cancer in several experimental models. For example,
both carcinoma-associated fibroblasts (4) and fibroblasts subjected to irradiation (5) have been found to promote transformation of nontumorigenic epithelial cells. Likewise, the deletion
of specific host proteases (6) or protease inhibitors (7) impedes
tumor progression in vivo. For breast cancer, in particular, the
orthotopic host environment appears to play a uniquely supportive role, as the mouse mammary gland and surrounding
mesenchymal tissue, the mammary fat pad (mfp), allow human
breast cancer cells to form tumors more readily than nonorthotopic sites [e.g., the subcutis (8)]. Although an intimate relationship clearly exists between the growing tumor and surrounding
stromal environment, the molecular changes induced in cancer
cells by host factors remain largely unknown.
Determining the impact of stromal influence on the molecular
and cellular properties of tumor cells is especially relevant for the
study of human cancer cell lines, which are commonly examined
both in culture and in mouse xenograft models to compare, for
example, their proliferation兾growth rates (9) and pharmacological responses (10, 11). Several cancer lines display contextdependent differences in behavior, exhibiting aggressive兾
13756 –13761 兩 PNAS 兩 September 21, 2004 兩 vol. 101 兩 no. 38
invasive properties in culture, but failing to form tumors in mice
(9), which suggests an as of yet ill-defined dependence on the
host environment for growth in vivo. Further confounding the
relationship between the in vitro properties of human cancer
lines and their tumorigenicity in animal models is the fact that
many of these lines are heterogeneous (12), which may result in
different subpopulations of cells dominating growth in distinct
environments. In vivo selection strategies have been used to
identify variants of human cancer lines that show altered gene
expression profiles that correlate with enhanced metastatic
properties in mice (12, 13). In these studies, however, the
molecular characteristics of primary tumors were not examined,
leaving several important questions unanswered. For example,
do specific components of the surrounding stroma infiltrate the
primary tumor, and if so, at what stage during tumor development are such host–cancer interactions observed? Similarly,
when during the course of tumor growth do changes in the
molecular profiles of cancer cells occur and are these alterations
maintained after removal from the host environment? Finally,
do the biological properties of cancer cells explanted from
primary tumors differ from those of the original parental line?
Here, we address these questions by conducting a comparative
functional proteomic analysis of the human breast cancer line
MDA-MB-231 grown in culture or during and after tumor
formation in the mfp of immunodeficient mice. By using a
chemical proteomic approach known as activity-based protein
profiling (ABPP) (14–16), in which a suite of active site-directed
probes are used to measure changes in the activity of many
enzymes in parallel, we show the following: (i) MDA-MB-231
xenograft tumors possess a discrete set of stromal-derived enzyme activities distinguishable from carcinoma enzymes by mass
spectrometry (MS) based on their respective species of origin
(mouse or human); (ii) MDA-MB-231 cells, after passage in vivo,
exhibit dramatic posttranscriptional changes in the activity of
specific enzymes, including proteases and glycolytic enzymes;
and (iii) these alterations in the enzyme activity profiles of
mfp-cultivated MDA-MB-231 cells correlate with enhanced
tumor growth rates and metastasis upon reintroduction of these
cells in vivo. These findings highlight the profound influence of
host environment on the molecular and cellular properties of
tumor cells and intimate that the behavior of human cancer cell
lines grown in vivo may vary considerably from their characteristics in culture.
Materials and Methods
Human Breast Cancer Xenografts. The parental MDA-MB-231
human breast cancer cell line (American Type Culture Collec-
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: ABPP, activity-based protein profiling; ALDH, aldehyde dehydrogenase; CE,
carboxylesterase; DDH, dihydrodiol dehydrogenase; ECH, enoyl-CoA hydratase; FP, fluorophosphonate; mfp, mammary fat pad; h, human; m, mouse; PFK, phosphofructokinase; SH,
serine hydrolase, SE, sulfonate ester; tPA, tissue plasminogen activator; uPA, urokinase
plasminogen activator.
‡To
whom correspondence should be addressed at: The Scripps Research Institute, 10550
North Torrey Pines Road, La Jolla, CA 92037. E-mail: [email protected].
© 2004 by The National Academy of Sciences of the USA
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0404727101
Proteome Labeling and Quantification of Enzyme Activities. For
proteomic analysis mice were killed at different time points, and
xenograft tumors and control mfp samples (harvested from the
contralateral side) were excised. Tumor and mfp tissue were
Dounce-homogenized in 50 mM Tris䡠HCl (pH 8.0), followed by
centrifugation at 100,000 ⫻ g to provide a soluble proteome
fraction (supernatant) and a particulate fraction (pellet). Proteomic fractions were analyzed with ABPP probes as described
(refs. 18–20; see Supporting Text for more details).
Isolation and Identification of Probe-Labeled Enzyme Activities. Isolation of enzyme activities was achieved by using trifunctional
probes (21) containing both a rhodamine and biotin moiety and
avidin-based affinity purification procedures (see Supporting
Text for more details).
Preparation of Xenograft Tumor-Derived MDA-MB-231 Cells. A well
established (10 weeks postinjection) MDA-MB-231 xenograft
tumor growing in the mouse mfp was removed aseptically and
minced with a razor blade; tumor pieces were transferred into
tissue culture flasks with complete medium. After 1 week of
culture, when abundant adherent tumor cells were visible,
floating tumor debris was removed. Attached cells were then
expanded to constitute a cell population referred to here as
231mfp cells. Northern blotting and Western blotting were
conducted according to standard laboratory procedures. Antitissue plasminogen activator and anti-urokinase plasminogen
activator (anti-uPA) antibodies were purchased from American
Diagnostica (Greenwich, CT) and diluted to 1 ␮g兾ml for Western blotting.
Results and Discussion
ABPP of Human Breast Cancer Line Xenografts. In previous studies
(20), we used ABPP to profile enzyme activities in cultured
preparations of human cancer cell lines, which constitute a
homogeneous and renewable source of cancer cells. These
experiments identified proteomic signatures containing both
known (e.g., uPA) and novel (e.g., KIAA1363) enzyme activities
that classified cancer lines into subtypes based on key biological
properties, such as tissue of origin and兾or state of invasiveness.
Nonetheless, how these enzyme activity profiles relate to the
functional state of cancer cells in vivo, where a variety of host
(stromal) factors may affect tumor development, remains unknown. To address this important issue, we established orthotopic xenograft tumors of the human breast cancer line MDAMB-231 in the mfp of immunodeficient SCID mice and
compared their enzyme activity profiles with those of cultured
preparations of these cells. We hypothesized that the mixedspecies nature of this xenograft model might permit the simultaneous discovery of carcinoma (human)- and stromal (mouse)derived enzyme activities associated with breast tumor growth in
vivo. Indeed, on average, mouse and human orthologues share
⬇80% sequence identity (22), which should, in general, enable
their discrimination by MS analysis.
MDA-MB-231 xenograft tumors and control mfp tissue were
collected for ABPP analysis over an 11-week time course (two
to four mice per week). ABPP was carried out with rhodaminecoupled fluorophosphonate (FP) and sulfonate ester (SE)
probes, which target serine hydrolases (SHs) (14, 20, 23) and
several groups of metabolic enzymes [e.g., GSTs, sugar kinases,
and aldehyde dehydrogenases (ALDHs)] (19, 21, 24, 25), reJessani et al.
spectively. Tumor and control mfp samples were separated into
soluble and membrane fractions before treatment with ABPP
probes. Fluorescently labeled proteins were then separated by
1D SDS兾PAGE and visualized兾quantified by direct in-gel fluorescence scanning as described (20). In parallel experiments,
trifunctional (rhodamine- and biotin-coupled) ABPP probes
(21) were used to affinity-isolate enzyme activities showing
expression patterns of interest, which permitted their molecular
identification by liquid chromatography-tandem MS (MS兾MS)
methods.
SH Activity Profiles of Human Breast Cancer Xenograft Proteomes.
Fig. 1 shows a representative in-gel fluorescence analysis of the
SH activity profiles of MDA-MB-231 xenograft tumor and
contralateral mfp-soluble proteomes (see Fig. 7, which is published as supporting information on the PNAS web site, for
profiles of membrane proteomes). To account for the diffuse
labeling of several glycosylated enzyme activities, a portion of
each FP-labeled proteome was deglycosylated by treatment with
peptide: N-glycosidase F (PNGaseF) before separation by SDS兾
PAGE, increasing the resolution of these proteins [for example,
see mouse carboxylesterase-1 (mCE-1); Fig. 2A]. Although many
of the soluble and membrane SH activities expressed in cultured
MDA-MB-231 cells were maintained in xenograft tumors (Fig.
1, enzymes labeled in black, and Fig. 7), several additional
activities were also present in these tumors. For example, two
soluble enzyme activities present in the mfp were also found in
tumor tissue throughout the 11-week time course (Fig. 1, enzymes labeled in green). These ⬇34- and ⬇72-kDa SH activities
were identified by MS analysis as the mouse orthologues of CE-1
and esterase D, respectively. In contrast, the activities of several
other SHs displayed an mfp-restricted distribution, including the
mouse enzymes MGC18894 (an uncharacterized CE) and lysophospholipase A1 (Fig. 1, labeled in magenta). These studies
also detected a ⬇35-kDa soluble SH activity that showed selective expression in xenograft tumors but not in mfp or cultured
preparations of MDA-MB-231 cells. This enzyme activity was
identified as human monoacylglycerol lipase (Fig. 1, labeled in
blue), indicating that the expression of certain carcinoma enzyme activities depends on the in vivo tumor microenvironment.
The designation of enzyme activities as human or mouse was
accomplished by liquid chromatography-electrospray tandem
MS (MS兾MS) analysis of their corresponding tryptic peptide
maps. Fig. 2B shows the labeling profile and tryptic peptides for
three representative SH activities identified in xenograft tumor
and mfp tissue. In each case, at least three unique peptides were
identified that allowed for unambiguous assignment of the
enzymes as stromal兾mouse (MGC18894 and CE-1) or tumor兾
human (APH) in origin. For a more complete list of the tryptic
peptides corresponding to each enzyme identified in this study,
see Table 1, which is published as supporting information on the
PNAS web site.
Metabolic Enzyme Activity Profiles of Human Breast Cancer Xenograft
Proteomes. We next profiled MDA-MB-231 xenograft tumor and
mfp control tissues with a rhodamine-tagged phenyl SE probe
(phenyl SE) that targets several different metabolic enzymes in
an active site-directed manner (25). Many of the SE-reactive
enzyme activities observed in cultured MDA-MB-231 cells,
including platelet-type phosphofructokinase (PFK) (21) and
GST␻ (19), were not detected in MDA-MB-231 xenograft
tumors, even at the earliest time points examined (Fig. 3A),
suggesting that they were down-regulated in vivo.
Other SE-labeled enzyme activities were detected in mfp tissue,
the majority of which were not observed in xenograft tumors (Fig.
3A). The identification of these presumably stromal enzyme activities by standard avidin enrichment-MS analysis proved challenging
because of the low quantities of mouse mfp proteome. As an
PNAS 兩 September 21, 2004 兩 vol. 101 兩 no. 38 兩 13757
BIOCHEMISTRY
tion) was cultured in Leibovitz L-15 medium supplemented with
10% FCS (complete medium). Cells were injected orthotopically
into the mfp of 6-week-old female C.B-17 SCID mice (Taconic
Farms) as described (ref. 17; see Supporting Text, which is
published as supporting information on the PNAS web site, for
more details).
Fig. 1. SH activity profiles of MDA-MB-231 xenograft tumors and mfp. Representative in-gel fluorescence analysis of SH activity profiles obtained from reactions
of soluble proteomes from MDA-MB-231 xenograft tumors and contralateral mfp samples with a rhodamine-tagged FP probe. The identities of enzymes are listed
on either side of the gel and are color-coded based on their respective expression patterns (see Table 1 for full names of enzymes). Each proteome sample was
analyzed both before and after deglycosylation by treatment with peptide: N-glycosidase F (PNGaseF) to enhance resolution. The double arrowhead highlights
mfp-restricted stromal activities that were not identified. Asterisks indicate enzyme activities that were only observed by MS analysis in the mfp, but their absence
in xenograft tumors could not be unambiguously determined because of comigration with carcinoma-derived enzyme activities. NS, nonspecific target of FP
probes. For a Coomassie blue-stained gel image of these proteomes, see Fig. 11, which is published as supporting information on the PNAS web site. mACAT,
mouse acyl-CoA thioesterase; mFAS, mouse fatty acid synthase; mLyso-PL1, mouse lysophospholipase A1; hMAG-lipase, human monoacylglycerol lipase.
alternative strategy to ascertain the identity of enzyme activities in
the mfp, we tested the ability of various cofactors兾inhibitors to
compete the SE labeling of specific proteins in this tissue. The SE
Fig. 2. General MS-based strategy to distinguish stromal (mouse) and carcinoma (human) enzyme activities in xenograft tumors. (A) Expanded view of
representative FP-labeled tumor and mfp SH activities showcasing the increased resolution that is achieved for glycosylated enzyme activities (e.g.,
mCE-1) after treatment with peptide: N-glycosidase F (PNGaseF). Identities of
enzyme activities are shown on either side of the gel: hAPH, human acylpeptide hydrolase; and mMGC18894, uncharacterized mCE. (B) Tryptic peptide maps for representative SH activities shown in A. Blue, peptides unique to
human orthologue; magenta, peptides unique to mouse orthologue; green,
peptide shared between mouse and human orthologues. The asterisk highlights a peptide that is shared between CE-1 and MGC18894 enzymes.
13758 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0404727101
reactivity of a 55-kDa enzyme activity was blocked by NAD⫹ or
NADP⫹ (Fig. 3B), indicating that it is likely a member of the ALDH
family, which is labeled by SE probes (19, 24). Likewise, the SE
labeling of a 36-kDa enzyme activity was specifically inhibited by
NADP⫹ (Fig. 3B), indicating that this protein is the SE target
dihydrodiol dehydogenase (DDH), which selectively utilizes this
cofactor for catalysis (26). Finally, a 30-kDa SE target was identified
as enoyl-CoA hydratase-1 (ECH-1) by blocking its labeling with an
ECH-1 selective inhibitor (27) (Fig. 3C). Together, these findings
highlight the value of competitive ABPP methods, which enabled
the molecular classification of three SE targets by using ⬍100 ␮g of
total protein from mouse mfp. In contrast to these enzymes, which
were restricted in expression to the mfp, an additional 37-kDa SE
target was observed in both mfp and tumor samples and was
identified as long-chain acyl-CoA dehydrogenase-1.
ABPP of mfp-Cultivated MDA-MB-231 Cells. MDA-MB-231 cells
grown in vivo in the mouse mammary fad pad were isolated from
xenograft tumors and recultured to provide a propagatable
subpopulation of cells referred to as 231mfp cells. Comparative
ABPP of 231mfp and parental MDA-MB-231 cells revealed
dramatic differences in the levels of discrete enzyme activities.
For example, several FP-labeled enzyme activities were highly
up-regulated in the conditioned media of 231mfp cells (Fig. 4A).
These enzymes were identified as uPA and the single- and
double-chain forms of tissue plasminogen activator (tPA). Additionally, multiple cytosolic SE-labeled enzyme activities were
down-regulated in 231mfp cells, including PFK (Fig. 8, which is
published as supporting information on the PNAS web site),
indicating that the reduced expression of these enzyme activities
in xenograft tumors (Fig. 3A) was maintained in culture. Notably, these differentially expressed enzyme activities stood out
against an otherwise similar background of enzyme activity
profiles for the 231mfp and parental MDA-MB-231 cells, as
Jessani et al.
Fig. 3. Metabolic enzyme activities labeled by a phenyl SE probe in xenograft tumor and mfp tissue. (A) Representative in-gel fluorescence analysis of enzyme
activity profiles obtained from reactions of soluble proteomes from MDA-MB-231 xenograft tumors and contralateral mfp samples with a rhodamine-tagged
phenyl SE probe (two independent tumors shown per week). Identities of enzymes are listed on either side of the gel and are color-coded based on their respective
expression patterns [black, parental MDA-MB-231 enzyme activities; magenta, mfp-restricted enzyme activities; green, enzyme activities expressed in both mfp
and tumor (double arrowhead denotes unidentified SE-reactive protein)]. mLCAD, mouse long-chain acyl-CoA dehydrogenase-1. NS refers to nonspecific SE
reactivities as determined by lack of heat-sensitive labeling (data not shown). (B) Indirect identification of SE-labeled enzymes by competitive profiling with
cofactors. The inhibition of labeling of 55- and 36-kDa enzymes by NAD⫹兾NADP⫹ and NADP⫹, respectively, indicates that these proteins correspond to a mALDH
and mDDH, respectively. (C) Indirect identification of an SE-labeled enzyme by competitive profiling with inhibitors. The inhibition of labeling of a 30-kDa enzyme
by the trifluoromethyl ketone agent OL-53, but not the related ␣-keto heterocycle agent OL-48, provides evidence that this protein is mECH, which has been
shown to be inhibited selectively by the former agent with an IC50 value of 0.63 ␮M (27). See ref. 27 for the structures of OL-48 and OL-53.
Fig. 4. Posttranscriptional up-regulation of secreted serine protease activities in mfp-cultivated MDA-MB-231 cells (231mfp cells). (A) Representative
in-gel fluorescence analysis of SH activity profiles of the secreted (conditioned
media) proteomes of parental MDA-MB-231 and 231mfp cells showing the
dramatic elevation in the activity of the serine proteases uPA and tPA [both
single chain (sc) and double chain (dc) forms] in 231mfp cells. (B and E)
Quantification of levels of active tPA and uPA as measured by in-gel fluorescence scanning. Data reported as averages ⫾ standard errors (SE); n ⫽ 3 per
group. (C and F) Relative mRNA levels for tPA and uPA as measured by
Northern blotting (n ⫽ 3 per group; values expressed in arbitrary units
normalized to an internal 7S RNA control). (D and G) Relative protein levels for
tPA and uPA as measured by Western blotting (n ⫽ 3 per group).
Jessani et al.
including the EGF receptor and integrin-␤1, as determined by
flow cytometry (data not shown). Finally, it is noteworthy that
the 231mfp and parental MDA-MB-231 cells also displayed
equivalent levels of GST␻ (Fig. 8), an SE target that was not
observed in xenograft tumors, indicating that the expression of
this enzyme activity was reconstituted upon culturing 231mfp
cells.
Northern blotting revealed that the parental MDA-MB-231
and 231mfp lines expressed similar levels of tPA and uPA
transcripts (Fig. 4 C and F), whereas Western blotting showed
that the expression of both proteases (Fig. 4 D and G), like their
activity (Fig. 4 B and E), was highly elevated in 231mfp cells.
These findings indicate that tPA and uPA expression兾activity are
regulated by a posttranscriptional or posttranslational mechanism in MDA-MB-231 cells. In this regard, mRNA levels for the
endogenous tPA兾uPA inhibitor, plasminogen activator inhibitor-1 were ⬇4-fold decreased in 231mfp cells (Fig. 10, which is
published as supporting information on the PNAS web site),
suggesting that the elevated tPA兾uPA expression and activity in
these cells may reflect, at least in part, the down-regulation of
this protease inhibitor. Indeed, both tPA and uPA are rapidly
cleared by cells after complexation with plasminogen activator
inhibitor-1 (28, 29). Discordance was also observed between the
quantity of activity and transcript for the SE target PFK, for
which equivalent mRNA levels were detected in parental MDAMB-231 and 231mfp cells, but activity was observed only in the
parental cells (Fig. 9). Together these findings demonstrate that
posttranscriptional and兾or posttranslational events play a major
role in determining the functional state of enzymes in cancer cell
proteomes.
231mfp Cells Exhibit Enhanced Tumor Growth Rates and Metastasis In
Vivo. We next compared the biological properties of parental
MDA-MB-231 and 231mfp cells in culture and in vivo. Under
standard cell culture conditions, these MDA-MB-231 variants
exhibited similar growth rates, with doubling times of 36.3 ⫾
3.7 h and 35.0 ⫾ 2.9 h, respectively (Fig. 5A), and equivalent
invasiveness as determined by Matrigel assays (data not shown).
In contrast to these in vitro results, 231mfp cells were found to
show a dramatic increase in tumor growth rates in vivo. Whereas
PNAS 兩 September 21, 2004 兩 vol. 101 兩 no. 38 兩 13759
BIOCHEMISTRY
exemplified by their nearly identical soluble and membrane
FP-reactivity profiles (Fig. 9, which is published as supporting
information on the PNAS web site). Also consistent with the
general similarity of these two MDA-MB-231 cell variants, they
did not differ in the expression of the uPA receptor or other cell
surface molecules implicated in tumor stroma interactions (2),
352 ⫹ 116 metastatic foci per tumor; MDA-MB-231 ⫽ 108 ⫹ 75
metastatic foci per tumor; P ⫽ 0.01, planned comparison; n ⫽ 4
tumors per group). Together, these data indicate that the
environment of the mouse mfp selected for, or supported the
growth of, a subpopulation of MDA-MB-231 cells that display
enhanced tumorigenic properties in vivo.
Fig. 5. Comparison of in vitro and in vivo growth rates of parental MDAMB-231 and 231mfp cells. (A) In vitro growth rates of cultured parental
MDA-MB-231 (䊐) and 231mfp (䉬) cells, with doubling times averaged from
three independent experiments. (B) In vivo growth rates of parental MDAMB-231 (䊐) and 231mfp (Œ) xenograft tumors grown in the mfp of immunodeficient mice (n ⫽ 4 per group).
parental MDA-MB-231 tumors underwent a lag time of 5–7
weeks after injection before growing progressively, 231mfp cells
produced fast-growing tumors without any apparent lag time
(Fig. 5B). Furthermore, tumors from 231mfp xenografts were
larger, weighing 1,245 ⫾ 292 mg at 6 weeks after injection
compared with a weight of 882 ⫾ 681 mg at 12 weeks after
injection for parental MDA-MB-231 tumors. Finally, 231mfp
tumors also produced significantly more lung metastases than
parental MDA-MB-231 tumors of equivalent weight (231mfp ⫽
Conclusions
Using a suite of chemical probes for ABPP, we have comparatively profiled human breast cancer cells grown in culture and
during and after in vivo passage as orthotopic xenograft tumors
in the mfp of immunodeficient mice. These functional proteomic
studies identified at least seven types of enzyme activities with
distinct expression patterns (Fig. 6): (i) tumor-associated activities derived from parental MDA-MB-231 cells (e.g., hCathepsin
A); (ii) tumor-specific activities (absent in both parental cells and
mfp; e.g., human monoacylglycerol lipase); (iii) stromal activities
excluded from tumors (e.g., mMGC18894); (iv) stromal activities
present in tumors (e.g., mCE-1); (v) carcinoma activities expressed selectively in the parental line (e.g., hPFK); (vi) carcinoma activities expressed selectively after in vivo passaging (e.g.,
huPA and htPA); and (vii) carcinoma activities reconstituted
after in vivo passaging (e.g., hGST␻). Together, the myriad
changes observed in the MDA-MB-231 proteome after cultivation in the mouse mfp point to the existence of a subpopulation
of breast cancer cells (231mfp cells) that exhibits increased
tumor growth rates and metastasis in vivo. Although it remains
to be determined whether the differentially expressed enzyme
activities identified by ABPP contribute to the enhanced tumorigenic properties of 231mfp cells, it is nonetheless significant that
many of the most dramatic alterations in enzyme activities
occurred as a result of posttranscriptional events (e.g., the
up-regulation of tPA and uPA activity in 231mfp cells). These
findings indicate that large-magnitude changes in cancer cell
proteomes may occur without corresponding alterations in transcript expression and thus underscore the value of ABPP for
measuring the functional outcome of posttranscriptional and
posttranslational events that ultimately govern enzyme activity
in vivo.
Fig. 6. Enzymes identified by ABPP and their distribution among MDA-MB-231 xenograft and cell culture samples. See Table 1 for a list of the tryptic peptides
that were used to assign the identity of these enzymes.
13760 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0404727101
Jessani et al.
molecular and cellular properties. How might such changes
arise? One possibility is that host (stromal) factors induce
alterations in the proteomic expression pattern of MDA-MB-231
cells. Alternatively, considering that many cancer cell lines are
heterogeneous (12, 13), the in vivo environment may support the
expansion of a rare subpopulation of MDA-MB-231 cells that
comes to dominate the xenograft tumor. Irrespective of the
underlying mechanism(s), the distinct enzyme activity profiles of
MDA-MB-231 cells grown in vitro (culture) and in vivo (xenograft tumors) suggest that these cells represent two fundamentally different populations (designated here as parental and
231mfp, respectively). Considering further that the 231mfp cells
exhibit superior tumorigenic properties in mice, we propose that
these cells may represent a preferred model for future investigations of breast cancer growth in vivo. More generally, these
findings suggest that studies carried out on human cancer cell
lines in culture, such as measurements of proliferation rates,
invasiveness, and drug sensitivity, may not be predictive of the
behavior of these lines in vivo. A potentially more effective
strategy may be to passage cancer lines through mice before
analysis in culture, thus enabling the selection of subpopulations
of cancer cells more capable of forming tumors in vivo.
This work was supported by National Institutes of Health Grants
CA087660 (to B.F.C.), CA085405 (to B.M.M.), and RR11823 (to
J.R.Y.), the California Breast Cancer Research Foundation (N.J. and
B.F.C.), and The Skaggs Institute for Chemical Biology.
1. Wiseman, B. S. & Werb, Z. (2002) Science 296, 1046–1049.
2. Bissell, M. J., Radisky, D. C., Rizki, A., Weaver, V. M. & Peterson, O. W. (2002)
Differentiation (Berlin) 70, 537–546.
3. Tlsty, T. D. & Hein, P. W. (2001) Curr. Opin. Gen. Dev. 11, 54–59.
4. Olumi, A., Grossfeld, G., Hayward, S., Carroll, P., Tlsty, T. D. & Cunha, G.
(1999) Cancer Res. 59, 5002–5011.
5. Barcellos-Hoff, M. & Ravani, S. (2000) Cancer Res. 60, 1254–1260.
6. Shapiro, R. L., Duquete, J. G., Roses, D. F., Numes, I., Harris, M. N., Kamino,
H., Wilson E. L. & Rifkin, D. B. (1996) Cancer Res. 56, 3597–3604.
7. Bajou, K., Noel, A., Gerald, R. D., Masson, V., Brunner, N., Holst-Hansen, C.,
Skobe, M., Fusenig, N. E., Carmeliet, P., Collen, D. & Foidart, J. M. (1998) Nat.
Med. 4, 923–928.
8. Felding-Habermann, B., O’Toole, T. E., Smith, J. W., Fransvea, E., Ruggeri,
Z. M., Ginsberg, M. H., Hughes, P. E., Pampori, N., Shattil, S. J., Saven, A.,
et al. (2001) Proc. Natl. Acad. Sci. USA 98, 1853–1858.
9. Thompson, E. W., Paik, S., Brunner, N., Sommers, C. L., Zugmaier, G., Clarke,
R., Shima, T. B., Torri, J., Donahue, S., Lippman, M. E., et al. (1992) J. Cell
Phys. 150, 534–544.
10. Bissery, M. C., Nohynek, G., Sanderink, G. J. & Lavelle, F. (1995) Anti-Cancer
Drugs 6, 339–355.
11. Sliwkowski, M. X., Lofgren, J. A., Lewis, G. D., Hotaling, T. E., Fendly, B. M.
& Fox, J. A. (1999) Semin. Oncol. 26, 60–70.
12. Kang, Y., Siegel, P. M., Shu, W., Drobnjak, M., Kakonen, S. M., Cordon-Cardo,
C., Guise, T. A. & Massague, J. (2003) Cancer Cell 3, 537–549.
13. Clark, E. A., Golub, T. R., Lander, E. S. & Hynes, R. O. (2000) Nature 406,
532–535.
14. Liu, Y., Patricelli, M. P. & Cravatt, B. F. (1999) Proc. Natl. Acad. Sci. USA 96,
14694–14699.
15. Jessani, N. & Cravatt, B. F. (2004) Curr. Opin. Chem. Biol. 8, 54–59.
16. Speers, A. E. & Cravatt, B. F. (2004) Chembiochem 5, 41–47.
17. Price, J. E., Polyzos, A., Zhang, R. D. & Daniels, L. M. (1990) Cancer Res. 50,
717–721.
18. Patricelli, M. P., Giang, D. K., Stamp, L. M. & Burbaum, J. J. (2001) Proteomics
1, 1067–1071.
19. Adam, G. C., Sorensen, E. J. & Cravatt, B. F. (2002) Nat. Biotechnol. 20,
805–809.
20. Jessani, N., Liu, Y., Humphrey, M. & Cravatt, B. F. (2002) Proc. Natl. Acad.
Sci. USA 99, 10335–10340.
21. Adam, G. C., Sorensen, E. J. & Cravatt, B. F. (2002) Mol. Cell. Proteomics 1,
828–835.
22. Mouse Genome Sequencing Consortium (2002) Nature 420, 520–562.
23. Kidd, D., Liu, Y. & Cravatt, B. F. (2001) Biochemistry 40, 4005–4015.
24. Adam, G. C., Cravatt, B. F. & Sorensen, E. J. (2001) Chem. Biol. 8,
81–95.
25. Adam, G. C., Burbaum, J. J., Kozarich, J. W., Patricelli, M. P. & Cravatt, B. F.
(2004) J. Am. Chem. Soc. 126, 1363–1368.
26. Asada, Y., Aoki, S., Ishikura, S., Usami, N. & Hara, A. (2000) Biochem.
Biophys. Res. Commun. 278, 333–337.
27. Leung, D., Hardouin, C., Boger, D. L. & Cravatt, B. F. (2003) Nat. Biotechnol.
21, 687–691.
28. Orth, K., Madison, E. L., Gething, M. J., Sambrook, J. F. & Herz, J. (1992) Proc.
Natl. Acad. Sci. USA 89, 7422–7426.
29. Nykjaer, A., Petersen, C. M., Moller, B., Jensen, P. H., Moestrup, S. K., Holtet,
T. L., Etzerodt, M., Thogersen, H. C., Munch, M., Andreasen, P. A. &
Gliemann, J. (1992) J. Biol. Chem. 267, 14543–14546.
BIOCHEMISTRY
Some of the potential shortcomings of current methods for
ABPP were also revealed during the course of these studies. For
example, ABPP failed to detect any secreted enzyme activities in
xenograft tumors, even though several of these proteins (e.g.,
tPA and uPA) were identified at high levels in the conditioned
media of cultured breast cancer cells. Although it is possible that
these secreted serine protease activities were inactivated in vivo,
thereby precluding detection by ABPP, these enzymes may have
also been diluted into the extracellular milieu of the in vivo tumor
environment, resulting in levels effectively below the sensitivity
limit of 1D gel-based ABPP detection methods. The limited
resolution of 1D gel analysis also prevented, in certain regions of
the gel, the unambiguous determination of the potential presence of stromal enzyme activities in xenograft tumors due to
comigration with carcinoma-derived enzyme activities (e.g.,
mouse fatty acid synthase and mouse acyl-CoA thioesterase in
Fig. 1). Although future efforts to implement gel-free strategies
for ABPP (25) should assist in both the detection and resolution
of low-abundance enzyme activities in complex biological samples, such as xenograft tumors, it should still be noted that 1D gel
analysis possesses several advantages for ABPP, most notably of
which is exceptional throughput, permitting the rapid comparative analysis of large panels of proteomes.
In summary, we have shown, by using functional proteomic
techniques, that the in vivo environment of the mouse mfp
cultivates the growth of human breast cancer cells with distinct
Jessani et al.
PNAS 兩 September 21, 2004 兩 vol. 101 兩 no. 38 兩 13761