Carcinoma and stromal enzyme activity profiles associated with breast tumor growth in vivo - PubMed (original) (raw)

Carcinoma and stromal enzyme activity profiles associated with breast tumor growth in vivo

Nadim Jessani et al. Proc Natl Acad Sci U S A. 2004.

Abstract

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.

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Figures

Fig. 1.

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.

Fig. 2.

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 acyl-peptide 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.

Fig. 3.

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+/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 α-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 μM (27). See ref. for the structures of OL-48 and OL-53.

Fig. 4.

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).

Fig. 5.

Fig. 5.

Comparison of in vitro and in vivo growth rates of parental MDA-MB-231 and 231mfp cells. (A) In vitro growth rates of cultured parental MDA-MB-231 (□) and 231mfp (♦) cells, with doubling times averaged from three independent experiments. (B) In vivo growth rates of parental MDA-MB-231 (□) and 231mfp (▴) xenograft tumors grown in the mfp of immunodeficient mice (n = 4 per group).

Fig. 6.

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.

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