The potential of carboxypeptidase M as a therapeutic target in cancer

1. Introduction

2. CPM in cancer pathology

3. CPM is a cancer biomarker

4. CPM in tumorigenesis,

proliferation, angiogenesis,

and metastasis

5. Conclusion

6. Expert opinion

Review

The potential of carboxypeptidaseM as a therapeutic target in cancerCatherine J Denis & Anne-Marie Lambeir†

University of Antwerp, Pharmaceutical Sciences, Laboratory of Medical Biochemistry,

Universiteitsplein 1, B-2610 Antwerp, Belgium

Introduction: In the recent literature, carboxypeptidase M (CPM) emerged as

a potential cancer biomarker. CPM modulates receptor signaling of kinins,

anaphylatoxins, and chemokines. These CPM substrates affect proliferation,

angiogenesis, and apoptosis of cancer cells. What is the evidence that CPM

is a drug target for cancer therapy?

Areas covered: The literature was searched using PubMed with the search

terms “carboxypeptidase M” and/or “chromosome 12q13-15” eventually com-

bined with general terms related to cancer. Information was retrieved from

the GEO database and material of gene expression and proteomic studies.

Expert opinion: CPM is a part of the molecular signature of many cancers.

There is good evidence that it is useful for the discrimination and stratifi-

cation of cancer types, possibly in combination with other markers such as

EGFR and MDM2. Whether it is also a drug target remains to be determined.

Lung, kidney, brain, and the reproductive system contain relatively high levels

of CPM, but its functions in those tissues are largely unknown. CPM is

expressed on tumor-associated macrophages. To facilitate the investigation

of CPM in tumor-associated inflammation and in the other aspects of tumor

biology, it is necessary to develop potent and selective CPM inhibitors.

Keywords: anaphylatoxin, bradykinin, carboxypeptidase M, chemokine, inflammation,

tumor-associated macrophages

Expert Opin. Ther. Targets [Early Online]

1. Introduction

Carboxypeptidase M (CPM, EC 3.4.17.12) is a peptidase, specifically a metallo-carboxypeptidase. CPM is a basic carboxypeptidase; its substrates are peptides orproteins that end with arginine or lysine [1]. Based on its structural homology, itwas assigned to the subfamily M14B in the MEROPS database [2]. Human CPMis a monomeric protein of 443 amino acid residues. It is attached to the cell surfaceby means of a glycosylphosphoinositol (GPI) anchor. It is composed of twodomains (Figure 1) [3]. The N-terminal domain contains the active site ofthe enzyme, characterized by the catalytic Zn2+-ion required to activate a boundwater molecule for hydrolysis of the C-terminal peptide bond of the substrates.The C-terminal domain, resembling the b-barrel found in transthyretin, is locatedclosest to the plasma membrane as it is connected to the GPI anchor through a shortpolypeptide segment [1,4]. The transthyretin-like domain contains the residuesinvolved in an interaction with the bradykinin receptor-1 [5]. The exposed locationof CPM on the cell surface may be the reason why it has been discovered as a cell-surface antigen on at least two separate occasions, for mature macrophages (MAX1/MAX11 antigen) and for the human B-lineage acute lymphoblastic leukemia cellline Pre ALP (M27 antigen) [6,7].

Most hypotheses on the role of CPM in physiology and pathology derive from itsexpression profile and the action of its putative substrates (exhaustively reviewed in

10.1517/14728222.2012.741122 © 2013 Informa UK, Ltd. ISSN 1472-8222, e-ISSN 1744-7631 1All rights reserved: reproduction in whole or in part not permitted

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2009 [8]). Until recently, CPM was proposed to be involved ininflammation. This hypothesis is supported by observationsthat CPM modulates the activity of anaphylatoxins andkinins; and the fact that CPM is present on differentiatedimmune cells and on the interface between host and invadingmicroorganisms. In addition, there is an upregulation ofthe CPM expression in inflammation or in tumors and/ortumor environment. However, CPM is also expressed byseveral kinds of specialized cells in normal tissue, so thatorgan-specific roles can be assumed, independent frominflammation [8]. In recent literature, there are reports thatCPM expression coincides with specific stages of cellulardifferentiation, it is up-regulated by stem cell mobilizingfactors, and it features prominently during the developmentof the human brain [9-11]. Available data on CPM as a markerof cell maturation are given in Table 1.The CPM protein was originally found associated with car-

boxypeptidase activity in human urine [12], but the CPM genewas serendipitously discovered on chromosome 12q15 in anexperiment to identify genes associated with the pathogenesisof benign and malignant solid tumors [13]. Expression profil-ing using DNA microarrays and cancer biomarker researchhas sporadically revealed up- or down-regulation of CPM invarious kinds of malignancies. Combining this with thenotion that CPM substrates -- anaphylatoxins, kinins, hor-mones, and growth factors [8] -- are implicated in processessuch as cell growth, cell migration, angiogenesis, and apopto-sis, it appears relevant to look at CPM in the context of cancerpathology and pathogenesis. Indeed, CPM has been describedas a target for cancer immunotherapy [14].

2. CPM in cancer pathology

2.1 CPM in leukemia cell linesCPM gene and protein expression have been documented fora number of human leukemia cell lines and cells isolated fromleukemia patients (Table 2). This was motivated by the discov-ery of CPM as a marker for mature macrophages [15] and byits expression in specific stages of B-lymphocyte develop-ment [6]. It is generally accepted that the degree of CPMexpression correlates with the degree of monocyte tomacrophage-like phenotype in myeloid leukemia cell lines [10].

Presuming that CPM up-regulation parallels expression ofother macrophage-specific genes, there was no incentive toinvestigate whether the CPM gene or its context might bechanged by ploidy, chromosomal defects, and rearrangementsin these commonly used cell lines. There are no indicationsthat CPM is crucial for immortalization or invasiveness ofthe cell lines listed in Table 2.

2.2 Deregulated expression of CPM in cancerDNA microarray studies suggested that CPM is a part of themolecular signature of specific cancers. If one focuses exclu-sively on CPM in gene-expression studies, it becomes appar-ent that CPM is present on many kinds of tumors and that itis modulated by factors that promote tumorigenesis ormetastasis (Table 3). There are also examples where CPM isdown-regulated in the tumor compared to the surroundingtissue. Some highly differentiated or polarized cells expressa high level of CPM, e.g., type I alveolar epithelial cells inthe lung, tubular epithelial cells in the kidney, and myeli-nated nerve cells. Sometimes such cells lose their differen-tiated phenotype concomitantly with CPM when theybecome cancerous. This has effectively been observed inrenal-cell carcinoma [16] and perhaps also in small-cell lungcarcinoma [17].

2.3 CPM as a target for immunotherapyRAV15 is a chimeric (humanized) monoclonal antibodyderived from the mouse parent antibody, KID31 [14].RAV15 binds to CPM. Raven Biotechnologies, the companythat developed RAV15, reported in 2005 that it is active in ahuman tumor model of lung cancer injected into immuno-compromised mice. We did not find any articles on CPMas a target for immunotherapy in the scientific literature.

3. CPM is a cancer biomarker

3.1 The chromosome 12q15

multiple-aberration regionRecent observations suggest that the CPM gene may representan interesting candidate in the search for novel diagnosticmarkers in cancer. The reason for this suggestion is that theCPM gene is located in an unstable region of chromosome12, the 12q13-15 multiple-aberration region.

CPM is located 11 kb downstream from a known oncogenecalled murine double minute-2 (MDM2). MDM2 encodes anuclear phosphoprotein that binds to and inhibits transactiva-tion by the p53 protein. Amplification of MDM2 results infunctional inactivation of p53 and diminished p53-mediatedtumor suppression activity. MDM2 can degrade p53 byacting as an E3 ubiquitin ligase. It exerts a critical role inregulating cell proliferation and apoptosis [18]. A variety ofgenes controlling cell cycle and differentiation, cytokinesis,or signal transduction locate on the 12q13--15 chromosomalregion, e.g., CDK4, TSPAN31, and HMGA2/HMGIC.It was suggested that MDM2 and other close-by genes might

Article highlights.

. CPM is part of the molecular signature of many cancers.

. CPM has potential as a biomarker for discrimination ofcancer types and stratification of patients.

. CPM is expressed on tumor-associated macrophages.

. CPM modulates kinin signaling by a completely novelmechanism of GPCR activation.

. To facilitate the investigation of CPM as a drug target itis imperative to develop selective inhibitors.

This box summarizes key points contained in the article.

C. J. Denis & A.-M. Lambeir

2 Expert Opin. Ther. Targets [Early Online]

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be co-amplified on a large single amplification unit [19].MDM2 and CDK4 amplifications are important molecularevents in the development of parosteal osteosarcoma, alow-grade bone malignancy. Co-amplification of MDM2and CPM was observed in classical osteosarcoma. Theco-amplification frequency was even higher in parostealosteosarcoma [20]. Using mitomycin, genotoxic stress wasinduced in the human U2OS cell line. Global expression pro-filing revealed an activation of the MDM2 and CPM genes.MDM2 up-regulation is consistent with the p53-dependentearly response to DNA damage. The upregulation of CPMmight suggest its implication in this process [21].

The chromosome 12q13--15 bands were associated witha variety of benign and malignant solid tumor types by

cytogenetic studies. Among the benign tumors, uterine leio-myoma, pleomorphic adenoma of the salivary gland, andlipoma all cluster to the 12q13--15 chromosomal region [22-26].Moreover, 12q13--15 involvement has been reported forhemangiopericytoma, endometrial polyps, chondromatoustumors, and pulmonary chondroid hamartoma [27-34].A similar observation was made in a number of case studiesincluding benign epithelial breast tumors, diffuse astrocytomas,and a giant-cell bone tumor [35-38]. Recurrent aberrations in12q13--15 have also been detected in malignancies such asmyxoid liposarcoma, soft tissue clear-cell sarcoma, chronic idi-opathic myelofibrosis, and primary diffuse large B-celllymphomas [39-44]. Using directional chromosome walkingand uterine leiomyoma-derived cell lines, a breakpoint hotspot region was found at 12q13--15 (named Uterine Leio-myoma Cluster Region on chromosome 12, ULCR12) [45].Another breakpoint cluster region of 1.7 Mb was detected onchromosome 12 comprising the breakpoints of uterineleiomyoma -- (including the ULCR12), lipoma, and salivarygland adenoma-cells [46]. This so-called multiple-aberrationregion was assigned to 12q15 by regional fine mapping andcontains essentially all breakpoints of chromosome 12 [47].CPM was localized in the 12q15 region at one of the chromo-somal breakpoints in radiation-transformed epithelial breastcell lines. Complex translocations were detected at this break-point. Since these gene rearrangements could alter CPM geneexpression, CPM is likely to represent a breast cancer-involvedcandidate gene [48]. CPM was found to be part of the genesdefining a cluster of breast cancer with high incidence ofrelapse [49]. Of interest, CPM was identified in silico as a puta-tive fusion gene resulting from chromosome rearrangement incarcinogenesis. CPM participates as the 3¢ translocation partnerin two detected fusion cases [50].

The chromosomal aberrations at 12q13--15 can be associ-ated with clinicopathological features and/or the prognosisof tumors. Noteworthily, the discovery of the High MobilityGroup protein gene family (together with frequent fusionpartners) present in the chromosome 12 breakpoint regionand greatly implicated in the tumors enumerated above waspatented for use in diagnosis and therapy [51].

3.2 Liposarcoma and lipomatous tumorsConsistent CPM gene amplification was detected in well-differentiated liposarcomas versus atypical lipomatous tumorsusing array comparative genomic hybridization performed ona large series of lipomatous neoplasms [52]. Using FISH andchromogenic in situ hybridization, amplification of the CPMgene was shown to discriminate well-differentiated liposarcomafrom lipomas [52].These well-differentiated liposarcomas typi-cally show telomeric associations, supernumerary ring chromo-somes, and giant rod marker chromosomes [19]. The abnormalchromosomes consist of amplified genomic sequences derivedfrom chromosome bands 12q13--15 and comprise several genes,including the MDM2 gene. MDM2 was consistently amplifiedin well-differentiated liposarcomas but not in lipomas [53].

Figure 1. Crystal structure of human CPM. Ribbon drawing of

CPM with a-helices and b-strands respectively shown in

orange and blue, and the residual chain in grey. The catalytic

carboxypeptidase domain is shown on top and the cup-

shaped C-terminal domain on bottom of the structure. The

disordered linker connecting the C-terminal domain and

the GPI-anchoring segment is not visible in the structure.

The catalytic zinc ion is depicted as a pink sphere,

whereas the three zinc ligands (sidechains of His66,

Glu69 and His173) are shown as dark blue sticks. The cysteine

residues involved in disulfide bridges (Cys121-Cys268,

Cys225-Cys267 and Cys324-Cys393) are depicted in red.

PDB-code: 1UWY [3]. Drawn with MOE 2009.10.

The potential of carboxypeptidase M as a therapeutic target in cancer

Expert Opin. Ther. Targets [Early Online] 3

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Table

1.CPM

asamarkerin

humancellularmaturation.

Cellulardifferentiationprocess

CPM

Celltype

Level

Ref.

Hematopoieticstem-celldifferentiation

CD34+stem

celldifferentiation

-CD34+hematopoieticprogenitorstem

cells

P+

CD34+hematopoieticprogenitorstem

cells

from

mPB,BM,CB

P,T

[10]

Myeloid

differentiation

-CD34+-derivedim

mature

myeloid

cells

P+

CFU

-GM,CFU

-MegandBFU

-EP,T

[10]

"exvivo

expansionofCFU

-GM

andCFU

-Meg

P,T

[10]

+BM

orPBCD13+myeloid

cells

PMonocyte/m

acrophagematuration

(+)/+

Freshly

isolatedbloodmonocytes(healthydonor)

A,P,T

+BM

mononuclearcells

P,T

[10]

+(m

)PBmononuclearcells

P,T

[10]

+CD14+tonsilcells

P+/+++

Invitromonocyte-derivedmacrophages(healthydonor)

A,P,T

-Tissuemacrophages

P-

Inflammatory

macrophagesin

situ§

P+(var)

Exudate

macrophages(pleural,peritoneal,alveolar)

P(+)

Isolatedalveolarmacrophages:

A,P

++

Culturedin

vitro

P++

From

extrinsicallergicbronchiolitis

P++

From

activepulm

onary

sarcoidosis

P++

From

isolatedpulm

onary

Langerhans’

cellhistiocytosis

P[99]

Dendriticcellmaturation

-Monocyte

P++

Monocyte

PGranulocyticmaturation

-/+

BM

granulocyticcells

(CD15+)

P+

Peripheralgranulocytes(CD15+)

PLymphoid

differentiation

Tlineagedifferentiation

-Tlineageprecursors

P+

13%

ofmature

CD4+Tcells

P-

Mature

CD8+Tcells

P++

38%

ofmature,PHA-activatedCD4+Tcells

P++

Anti-CD3activatedCD4+Tcellclones

P+

3%

ofmature,PHA-activatedCD8+Tcells

PBlineagedifferentiation

-/+

Mature

Blymphocytes

P-

TonsillarBlymphocytes

P

Thecellulardifferentiationprocess,theexpressionand/orregulationofCPM,thecelltype,andtheinvestigatedlevel(s)

ofCPM

(transcript,protein,activity)

are

provided.

Referencesare

only

includedforinform

ationthatwasnotcitedin

[8],whereto

wereferfortheremainingreferences.

*Meanfold

upregulationresultingfrom

differentiationexperiments

startingfrom

BMSCorAMSC.

z Proximalordistalpart

ofthecellcolumnofanchoringvilli.

§Exceptforrejectedrenalallograft

macrophages.

A:Basiccarboxypeptidase

activity;

BM:Bonemarrow;BFU

-E:Erythroid

burst-form

ingunits;

CB:Cord

blood;CFU

:Colony-form

ingunits;

CFU

-F:Fibroblast

colony-form

ingunits;

CFU

-GM:Granulocyte-

macrophagecolony-form

ingunits;

CFU

-Meg:Megakaryocyte

colony-form

ingunits;

ER:Estrogenreceptor;mPB:Mobilizedperipheralblood;MSC:Mesenchym

alstem

cells;P:Protein;PHA:Phytohemagglutinin;

T:Transcript;var:Variable

expressionin

differentpatients.



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Table

1.CPM

asamarkerin

humancellularmaturation(continued).


CPM

Celltype

Level

Ref.

+Germ

inalcentercells

PMesenchym

aldifferentiation

+MSC

T[10]

+CFU

-FT

[10]

Osteogenesis

"Osteocytes,

early(3

days,27-fold*)andlate

(14days,31-fold*)

T[100]

++

Osteoblasts-likecellmembranes

A+

Primary

osteoblastsfrom

humanfetallongbone

T"

InvitrohumanMSC-derivedosteocytes

PChondrogenesis

"Chondrocytes,

early(3

days,3.8-fold*)andlate

(14days,5-fold*)

TAdipogenesis

"Adipocytes,

early(3

days,30-fold*)andlate

(14days,47-fold*)

T"

Differentiatedadipocytes

P"

Adipocytes,

earlydifferentiation

TStromalcellform

ation

+Fibroblasts

P,A

[10]

Thereproductivesystem

MENSTRUALCYCLE

Follicularmaturation

+Cumulusgranulosa

cells

P,T

"FO

XL2

granulosa-likecells

(2.1-fold)

T-

PAntralfollicles

+to

++/-

Theca

internacells/granulosa

cells

PGrowingandleadingfollicles

++/-

Theca

internacells/granulosa

cells

PPre-ovulatory

follicles

+Cumulusgranulosa

cells

P,T

Ovulation

Lutealmaturation

Earlylutealcorpusluteum

++/+++

Theca

lutealcells/granulosa

lutealcells

P,T

Midlutealcorpusluteum

++/+++

Theca


lutealcells

P,T

Late

lutealcorpusluteum

++/+++

Theca


lutealcells

PPREGNANCY

Lutealmaturation

+++/+++

Theca


lutealcells

PPregnancy

corpusluteum

(6--1

4weeksofgestation)

Endometrialmaturation

#Late

proliferative

phase

endometrium,byprogesterone(2.1-fold)

TDecidualization

+Decidualcells

TTrophoblast

differentiation

-Cytotrophoblastsin

floatingchorionic

villi

P-

Extravilloustrophoblasts(proximalz )

P-/+

Extravilloustrophoblasts(distalz )

P


ofCPM


are

provided.

Referencesare

only

includedforinform


[8],whereto


*Meanfold



startingfrom

BMSC

orAMSC.




macrophages.


activity;

BM:Bonemarrow;BFU

-E:Erythroid

burst-form

ingunits;

CB:Cord

blood;CFU

:Colony-form

ingunits;

CFU

-F:Fibroblast

colony-form

ingunits;

CFU

-GM:Granulocyte-


ingunits;

CFU

-Meg:Megakaryocyte

colony-form

ingunits;


alstem



expressionin

differentpatients.



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Table

1.CPM

asamarkerin

humancellularmaturation(continued).


CPM

Celltype

Level

Ref.

++

Syncytiotrophoblasts

P++

Extravilloustrophoblastsin

thetrophoblastic

shell

P++

Interstitialtrophoblastsofthedecidua

P++

Endovascularextravilloustrophoblasts

P"

Isolatedextravilloustrophoblasts,

by20%

O2

P,T

Therespiratory

system

TypeIalveolarepithelialcellgeneration

+++

TypeIalveolarepithelialcells

P-

TypeIIalveolarepithelialcells

PMuscle

Smooth

muscle

cellmaturation

+Blood-derivedsm

ooth

muscle

progenitorcells

P[11]

-/+

Skeletalmuscle

T-

Carotidsm

ooth

muscle

cells

PBrain

Development

Mature

++

Pons,

frontalcortex

T A,P

[9]


ofCPM


are

provided.

Referencesare

only

includedforinform


[8],whereto


*Meanfold



startingfrom

BMSC

orAMSC.




macrophages.


activity;

BM:Bonemarrow;BFU

-E:Erythroid

burst-form

ingunits;

CB:Cord

blood;CFU

:Colony-form

ingunits;

CFU

-F:Fibroblast

colony-form

ingunits;

CFU

-GM:Granulocyte-


ingunits;

CFU

-Meg:Megakaryocyte

colony-form

ingunits;


alstem



expressionin

differentpatients.



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12q15 rearrangements were detected in retroperitoneal lipoma-tous tumors without cytologic atypia. However, these tumorswere not characterized by the presence of a ring or giant markerchromosomes, nor by the amplification ofMDM2 or CPM [54].Neither CPM nor MDM2 amplification was detected in pleo-morphic lipomas, pleomorphic liposarcomas, lipoblastomas,or normal fat. Loss of the CPM/MDM2 loci was detected intwo myxoid liposarcomas [52]. Accordingly, MDM2/CPMamplification assessment was proposed as a complementarytool for better histological classification of lipomatous tumorsand evaluation of the impact of surgical procedures on the riskof local recurrence [55].

3.3 Co-expression with EGFR is a prognostic marker

for clear-cell lung carcinomaIn lung adenocarcinoma, CPM and epidermal growth factorreceptor (EGFR) protein expression appeared to be heteroge-neous. CPM and EGFR were mainly restricted to tumor cellmembranes. CPM expression was not limited to a specific histo-type, and did not correlate with the tumor grade or stage. Thepresence of CPM negatively correlated with disease survival.Eighty percent of the CPM-positive adenocarcinoma wereEGFR-positive. Interestingly, the co-existence of CPM andEGFR strongly predicted a poor outcome (9.5% vs. 60.4% of5-year survival for CPM+-EGFR+ and CPM--EGFR- tumors

Table 3. Deregulation of CPM expression in cancer.

CPM Level

Adenocarcinoma (tumor marker) + mRNAEndometrium and myometrium tumor tissue up mRNAInvasive ductal breast carcinoma up mRNAClear cell ovarian cancer up mRNASerous cystadenocarcinoma (ovarian carcinoma) compared to clear-cell tumors down mRNAPrimary cutaneous squamous cell carcinoma up mRNASoft-tissue carcinomas (synovial sarcoma, gastrointestinal stromal tumors, dedifferentiated-pleomorphicliposarcomas)

up mRNA

Lung cancer (bronchoalveolar lavage fluid) up activityRapidly growing hepatoma up activityHepatic cell lines response to benzopyrene up mRNAPancreatic ductal adenocarcinoma up mRNAOsteosarcoma cells (U-2 OS) after DNA damage up mRNALung cancer cell line down mRNASmall cell lung cancer down mRNALeukemic mantle cell lymphoma down mRNAPrimary breast cancer cells with complete response to therapy with gemcitabine, epirubicin and docetaxel + mRNAWell differentiated liposarcoma versus lipoma + FISHLipomatous tumors + HistologyLung adenocarcinoma (co-expressing EGFR) with poor survival outcome + histology

The information was taken from [8], whereto we refer for the original references.

Table 2. CPM expression in leukemia cells.

System CPM Cell type Level Refs.

Myeloid- (P)/++ (T) chronic myelogenous leukemia in blast crisis (K562) P, T [6]

- acute myelogenous leukemia cells (KG-1a) P, T [10]

+ acute myelogenous leukemia cells (KG-1) P, T [10]

-/(+) acute promyelocytic leukemia cells (HL-60) P, T [6,7,15,64]

(+) histiocytic lymphoma cells (U937) P [7,15,64]

(+) myelomonocytic cells (MonoMac-6) P [7,15,64]

+++ acute monocytic leukemia cells (THP-1) P, T [10]

- acute myeloid leukemia cells (7/8 cases) P [6]

LymphoidT lineage - to + acute T cell leukemia cell line (Jurkat) P, T [6,10]

B lineage + pre-B acute lymphoblastic leukemia cell line P [6]

+ acute lymphoblastic leukemia samples (6/7 cases) P [6]

+ Burkitt lymphoma samples (2/4 cases) P [6]

- Raji cells P, T [10]

-/+ chronic lymphocytic leukemia cells (1/4 cases) P [6]

P: Protein; T: Transcript.



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respectively). An unfavorable role for CPM-EGFR co-expres-sion was suggested in early tumor stages. Two cases of CPM--EGFR+ primary lung adenocarcinoma became CPM+-EGFR+

when metastasized to the brain, suggesting CPM represents aninducible molecule. CPM genemutation or alteration and splicevariants were not analyzed in this study. Since only a limitednumber of patients were included, the results should be inter-preted carefully. EGF represents a good endogenous substratefor CPM. Therefore, CPM could modulate receptor--ligandinteraction and activity on tumor cells, thereby affecting tumorbiology in EGFR+ tumors [56].

3.4 Renal-cell carcinomaImmunohistochemistry indicates a broad distribution ofCPM along the renal tubular structures in a healthy kidney.CPM was identified at the parietal epithelium beneath theBowman’s basement membrane and in glomerular mesangialcells. Capillaries, podocytes, and most interstitial cells wereCPM-negative. Tumor cells of renal-cell carcinoma subtypeslose CPM expression upon dedifferentiation. In a studyof seven clear-cell renal carcinoma specimens and 1 chromo-phobe renal-cell carcinoma CPM was co-localized withCD31 (endothelium), vimentin (tumor marker) and CD68(macrophages) [16].Denis et al. (2012) studied the co-expression of CPM and

EGFR by immunohistochemistry using a tissue microarray con-taining 104 cases of various renal tumors and diseased renaltissue [16]. An association between the CPM histology-score(H-score) and tumor grade was observed for clear-cell carcinoma.Cluster analysis of the CPM and EGFR H-scores in this studyshowed co-existing high scores for CPM and EGFR only forpapillary renal carcinoma. Although co-existence of CPM andEGFR was detected in papillary renal-cell carcinoma, the differ-ent subcellular localization argues against an interaction of bothproteins. In this cancer type, expression of CPM is up-regulated along with tumoral dedifferentiation. Moleculargenetic analysis of papillary renal-cell tumors revealed loss ofthe chromosome Y markers together with trisomy of chromo-somes 3q, 7, 8, 12, 16, 17, and 20. Trisomy of 12, 16, and20 possibly are related to tumor progression [57]. Allelic duplica-tions were detected at the 12q12--14 chromosomal regions (towhich the CPM gene maps), among others [58]. Other renal-cell carcinomas that were tested in the tissue microarray analysisshow genetic alterations of specific chromosomes, but chromo-some 12does not seem to be implicated in their development [57].Possibly, transitional cell carcinoma forms an exception sinceMDM2 and (sometimes) CDK4 amplification were describedin 4--6% of this cancer type.

4. CPM in tumorigenesis, proliferation,angiogenesis, and metastasis

4.1 CPM in the tumor environmentThe connection between cancer and inflammation has beenestablished for a long time. Tumor-associated macrophages

represent a major component of the leukocytic infiltrate oftumors and are key regulators of the cancer-inflammationlink. They mainly originate from circulating monocyteswhich selectively migrate into the tumor microenvironmentin response to locally produced chemotactic factors. Tumor-associated macrophages accumulate into tumoral necroticregions. Generally exhibiting the M2 phenotype (also called“alternatively activated” phenotype), tumor-associated macro-phages are thought to promote tumor growth (by producinggrowth factors such as EGF, and cytokines), tissue remodeling(via matrix metalloproteinases) and angiogenesis (through, e.g., CXC chemokines) while subverting adaptive immunityresponses. It is in this type of macrophages that CPM ismost prominently expressed [16,59].The adaptive immunity isinvolved in immuno-editing, a process of surveillance andelimination of nascent tumors. In contrast to their pro-tumor activities, activated macrophages can also inhibittumor growth, destroy neoplastic cells, and elicit cancer-destructive inflammatory responses. Inflammatory reactions,particularly mediated by macrophages, thus exert an ambiva-lent effect on tumor proliferation, progression, and metastaticspread [60].

In two patients with lung adenocarcinoma, Tsakiris et al.found a high number of CPM+ tumor-associated macro-phages infiltrated in EGFR+ tumor tissue [56]. The necroticareas of cerebral gliomas arising after I-125 interstitial brachy-therapy were lined by a dense layer of end-stage macrophagesthat showed CPM positivity. CPM could be important in theremoval of necrotic debris that is achieved by these infiltratingmacrophages [61,62]. An atypical, sarcoid-like (without second-ary changes such as necrosis) granulomatous reaction wasobserved in a case of conventional clear-cell type renal cancer.Tumor-associated granulomas are a host-versus-tumorresponse against a cancer antigen that serve to inhibit localtumor growth. They mainly consist of epithelioid macro-phages [63]. Elevated CPM levels were found on alveolarmacrophages in active pulmonary sarcoidosis [64]. CPM+

macrophages were also observed in the renal-cell carcinomastudy described in the previous section [16].

In a model for in vitro generation of tumor-associated mac-rophages, tumor cells were capable of modifying the pheno-type (and thus differentiation state) and effector function ofinfiltrating monocytes. CPM expression on in vitro generatedtumor-associated macrophages was lost inside spheroidsderived from the invasive, poorly differentiated J82 cell line,whereas it remained when cultured with a highly diffe-rentiated tumor cell line or a non-tumorigenic cell line. Thissuggests an association between tumor malignancy and sup-pressive influence on macrophage maturation [65,66]. Theseand other observations (not included in this study) lead tothe hypothesis that tumors expressing high levels of CPMwould be more accessible for the immune system and mighthave a better clinical outcome.

One could speculate that CPM expression on cells inthe tumor environment might have similar effects. In the



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renal-cell carcinoma study, CPM staining was occasionallyobserved in stromal cells and on endothelial cells in the bloodvessels of the tumor but not in the normal surroundingtissue [16].

4.2 CPM substratesBy virtue of its catalytic activity, CPM is believed to modulatethe action of bioactive peptides present in the tumor environ-ment. In addition to reducing the binding of cationic peptidesto glycosylaminoglycans in the extracellular matrix, removalof C-terminal basic amino acids is known to affect receptorbinding and/or triggering for specific peptides that havebeen studied in their own right as targets for cancer therapy.

4.2.1 KininsKinins are a group of bioactive peptides generated by the pro-teolytic action of plasma and tissue kallikreins on their sub-strates, the low and high molecular weight kininogens.Kinins work in an autocrine or paracrine fashion, i.e., onthe cells surrounding the site of production. High levels ofkinins are found in biological fluids of cancer patients, pro-duced by tumor cells or other cells in the tumor environment.Many cancers produce a high level of kallikreins and expresseither or both of the B1 and B2 kinin receptors. Kinin recep-tors are regarded as targets for cancer therapy and kinin recep-tor antagonists have been developed that inhibit tumorgrowth (reviewed in this journal [67]).

The products of kallikrein action are the decapeptidekallidin (Lys-bradykinin) or the nonapeptide bradykinin, themajor agonist of the kinin B2 receptor. Once formed, andby proteolytic cleavage of the C-terminal arginine, these pep-tides may be converted into Lys-des[Arg9]bradykinin or des[Arg9]bradykinin, agonists of the kinin B1 receptor. By stim-ulating these two G-protein-coupled receptors, kinins increasecell proliferation, chemotaxis, and release of matrix metallo-proteinases from cancer cells. In addition, transactivation ofEGFR signaling cascades by kinin receptors has also beenreported. These effects are enhanced by kinin action on cellsin the tumor environment.

CPM converts B2 receptor agonists into B1 receptor ago-nists and activates the B1 receptor cascade by an interactionindependent from its catalytic activity [5,68]. This interactionis blocked by a monoclonal antibody that recognizes an epitopein the transthyretin-like domain of CPM. Noteworthily,disruption of the CPM-B1 receptor complex in cytokine-treated human lung microvascular endothelial cells by a specificpeptide targeting the C-terminal domain of CPM impeded B1receptor-dependent NO release stimulated by bradykinin inthe presence of a B2R antagonist. The increase in endothelialpermeability caused by bradykinin and pyrogallol was alsoinhibited [5].

4.2.2 AnaphylatoxinsThe anaphylatoxins, C3a and C5a, are produced by proteo-lytic cleavage of the complement proteins C3 and C5 upon

complement activation or other proteolytic cascades in thecirculation or the tissues. Although originally assigned to theancient innate immune system, we now know that anaphyla-toxins are immunoregulatory peptides also of the humoral,adaptive immune system [69]. C3a and C5a have pleiotropiceffects that they exert by activation of the G-protein-coupledreceptors C3aR, C5aR and C5L2. Apart from a role in acuteand chronic inflammation, Klos et al. (2009) described rolesin the CNS during neurogenesis, differentiation, and neuro-degeneration [70]. Several companies developed inhibitors ofanaphylatoxin action [71,72], targeting primarily C5a which isthe most potent in inflammation. C3a and C5a are rapidlyinactivated by carboxypeptidases, notably CPN in the blood,but CPM can also quite effectively remove the C-terminal arginine that is crucial for receptor binding and acti-vation [8,73,74]. The anaphylatoxins are not only chemotacticfactors attracting immune cells to the sites of inflammation,but they also have a role in wound healing and stem cellmobilization, where they connect with the CXCL12/CXCR4 axis (see below) [75,76]. It has been shown that C5aregulates the adaptive immunity in tumor immunology. In amodel of cervical cancer, C5aR targeting resulted in decreasednumbers of myeloid-derived suppressor cells -- myeloid cellsin intermediate stages of differentiation toward mononuclearcells or polymorphonuclear granulocytes -- within thetumor [77]. This was associated with high numbers of CD8+effector T cells and decreased tumor growth.

4.2.3 Chemokines CXCL12 and CCL1Stromal cell-derived factor-1a (SDF-1 a) is the most abun-dant of the CXCL12 isoforms and ends on a lysine.CXCL12’s most important function is to regulate hematopoi-etic stem cell migration into and out of the bone marrow.During development, CXCL12 serves as a powerful chemoat-tractant for hematopoietic cells when they need to passthrough endothelial cell barriers. Several recent reviews high-light the importance of CXCL12 and its receptors, CXCR4,and CXCR7, on tumor growth, tumor neovascularizationand metastasis [78,79], as well as its role in re-emergence oftumors after radiotherapy [80]. It has been observed thatCXCL12 is secreted by stromal cells in the tumor microenvi-ronment where it may attract cancer cells via stimulation ofthe CXCR4 receptor that is up-regulated by tumor cells.CXCL12/CXCR4 activation also potentiates metastasis toorgans expressing high levels of CXCL12, i.e., the bone mar-row. Besides many other effects on tumor development, thisseems to be the primary role together with the recruitmentof hematopoietic cells and perhaps the establishment of thecancer stem-like cell within the tumor microenvironment.CXCL12 produced in the tumor environment by fibroblasts,macrophages, and neutrophils (among others) recruits ahighly tumorigenic population of tumor cells and promotescell survival, proliferation, angiogenesis, and metastasis. Apartfrom CXCR4, it has been demonstrated that CXCR7 is alsoinvolved in tumor cell growth, survival, and metastasis in



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several tumor types. It is highly expressed on tumor-associatedvasculature and may have an important role in tumor neovas-cularization. CXCR4 antagonists, developed for the treatmentof inflammation and to prevent entry of the HIV virus,showed promising results as anti-cancer agents in preclinicaltrials, summarized in [78,79,81] to which we refer fororiginal citations.In serum, conversion of CXCL12a to des[Lys68]CXCL12a

is mediated by carboxypeptidase N [82]. However, CPM alsocatalyzes the C-terminal truncation of CXCL12a, at leastin vitro [10]. Even though binding to the CXCR4 receptorwas not perturbed, loss of Lys68 negatively affected severalfunctional properties of CXCL12a such as heparin andcell-binding ability, cell proliferation, and chemotaxis [10,82,83].Another chemokine, CCL1, ending C-terminally in

--Lys71-Arg72-Lys73, is an equally interesting substrate forCPM. It has been suggested that CCL1 could play a role inangiogenesis and tumor immunity [84-86]. Noteworthily,CCL1 is secreted by monocytes and activated macrophagesand attracts monocytes [87] and other cells that express theCCR8 receptor. Moreover, CCL1 exerts anti-apoptotic andproliferative activity on murine thymic lymphoma cell lineswhen exposed to dexamethasone (DEX) [88]. Removal of threeamino acids from the C-terminus of CCL1 reduces CCR8binding, but turns CCL1 into a more potent CCR8 agonist,resulting in enhanced anti-apoptotic activity [89].

5. Conclusion

CPM is exposed on the outer surface of a variety of cells,where it is optimally located to exert its basic carboxypepti-dase activity on substrates in the extracellular environment.In normal tissues, CPM expression in regulated and restrictedto specific cell types in specific stages of differentiation anddevelopment. In inflammation, CPM is up-regulated duringmacrophage maturation; in vivo it is found mainly onM2-macrophages with foam-cell appearance. CPM is presentin many types of tumors. In some cases, expression is highbecause CPM is up-regulated by factors that stimulate tumorgrowth and metastasis. Sometimes, dedifferentiation of cancercells causes a decrease in CPM expression compared to thesurrounding healthy tissue, for instance, in clear-cell renal car-cinoma. In other cases, CPM expression is high because theCPM gene is amplified due to aberrations in the chromosome12q13--15 region. Amplification of CPM together with theoncogene MDM2 makes CPM a useful marker for well-differentiated lipomas over atypical lipomatous tumors, andpresumably for papillary renal carcinomas, which also expressEGFR. Co-expression of CPM and EGFR was considered tobe an indication of poor outcome in clear-cell lung carci-noma. CPM is often found on tumor-associated macrophagesand occasionally on stromal cells and in the tumor vascula-ture. It is not known whether the presence of CPM on thetumor cells and in the tumor environment is beneficial ornot. There is circumstantial evidence that CPM might affect

proliferation, tumor growth, or metastasis because it is ableto modulate the activity and receptor specificity of bioactivepeptides regulating cell migration, angiogenesis, and apopto-sis. As examples we used kinins, anaphylatoxins, and chemo-kines (CXCL12 and CCL1), that all have been proposed tobe anti-cancer targets.

6. Expert opinion

A key finding in recent CPM research is the observation thatCPM is highly up-regulated during mesenchymal cell differ-entiation and adipocyte maturation. This implies that its pres-ence and/or activity is important for normal functions ofadipocytes or for formation and organization of adipose tis-sue. In well-differentiated liposarcoma, CPM levels are evenhigher because of chromosomal amplification of the CPMgene and it was suggested that CPM could be used as a markerfor distinguishing well-differentiated liposarcomas from othertypes of cancer. The liposarcoma/lipoma study is one of onlya few studies wherein CPM was singled out as a biomarkerand systematically investigated on different levels and in asubstantial number of patients. CPM was also specificallyinvestigated as a potential marker in lung adenocarcinomaand renal carcinoma. This is not a co-incidence; both thelung and the kidney were previously regarded as rich sourcesof CPM. By the same token, it would be worthwhile to sys-tematically study CPM expression, by immunohistochemistryand in situ hybridization techniques, in tumors of the brain,and both the male and female reproductive systems. Thus, itappears that CPM definitely has a potential for the discrimi-nation of different cancers and that it may be used in combi-nation with other markers for the stratification of tumors andfor establishing therapeutic strategies.

Most other evidence of CPM expression in various cancersis indirect as it is based on either transcriptome analysis orchromosome abnormalities. Since these results suggest thatCPM is present on many tumor types, it may have potentialfor targeting tumoral cells for immunotherapy. On the otherhand, some tissues are rich in CPM and very little is knownof the function of CPM and indeed its endogenous substratesin those tissues. Antibody-based therapies could cause inflam-mation of CPM-rich tissues and should be carefullycontrolled. Some caution is advised when extrapolating pre-clinical results obtained in murine models because CPMexpression patterns may vary between human and mouse.

Is CPM itself an oncogene? Does this enzyme contribute tothe cellular response to DNA damage or otherwise affect cellsurvival, proliferation, or migration? At present, there is ahuge gap in our understanding of even the normal role ofCPM. The greatest challenge in this field will be to uncoverwhat the cellular functions of CPM are and whether or howthey relate to oncology. Perhaps the first aspect to tackle isto establish a role for CPM in tumor-associated inflamma-tion. For that, we need to look at proinflammatory peptidesin the tumor environment that are substrates of CPM and



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find out what their action is on the tumor cells and theinflammatory cells in the tumor environment.

Another key finding in the recent CPM literature relates tothe role of CPM in inflammation, in particular to its abilityto modulate kinin signaling cascades by switching fromB2 to B1 receptor and activating the B1 receptor. This studyrevealed a new mechanism of G-protein- coupled receptoractivation and it will be exciting to discover whether CPMalso interacts with other receptors in a similar fashion. Thereare two potential strategies to interfere with this process: inhi-bition of the catalytic activity and blocking the interactionbetween CPM and the receptor. The latter has already beenexplored [5], and at present, the former is still problematic.Basic carboxypeptidase inhibitors (2 mercaptomethyl-3-guanidinoethylthiopropanoic acid and 2-guanidinoethyl-mercaptosuccinic acid) have been used for many years, butnone has been described that is selective for CPM [1]. Itwould be an enormous boost for CPM research if a selectiveinhibitor were available. A typical metallocarboxypeptidaseinhibitor contains a moiety that recognizes the primary sub-strate- binding site (S1’), a non-cleavable linker and a ligandfor the active site zinc ion. In the case of the basic carboxypep-tidases, there is likely to be some overlap in reactivity betweenCPM, CPN, CPD, and CPE, that all belong to the same sub-family. There have been some successes with the developmentof selective CPU inhibitors, that are currently clinicallyinvestigated for use in anti-thrombotic therapy [90]. Although

not completely within the scope of this study, it might bementioned that other metallocarboxypeptidases are studiedeither as cancer biomarkers (CPE, CPU) or for their involve-ment in tumorigenesis, proliferation, or metastasis (CPE,CPD, CPA, CPA4) [91-98]. As CPM likely shares substrateswith other basic carboxypeptidases, selective inhibitors wouldalso be useful research tools.

The next challenge would be to validate CPM as a drug tar-get in cancer models such as those described for bradykininand anaphylatoxin-targeting drugs. One would expect thatpharmacological or genetic knock-down of CPM has multipleand perhaps contrary effects on different signaling cascades.Blocking the kinin B1 receptor is expected to suppress itssignaling cascades, but the actions of the anaphylatoxins willbe prolonged by CPM inhibitors and the CXCL12 chemotac-tic gradient will be preserved. At present, it seems impossibleto predict whether CPM inhibition will reduce or enhanceinflammatory reactions and whether the net effect willpromote or block cancer progression.

Declaration of interest

CJ Denis was a research assistant of the Research Foundation-Flanders (F.W.O.-Vlaanderen). This paper was supportedby grants from F.W.O.-Vlaanderen and a grant from theUniversity of Antwerp. The authors declare no other conflictsof interest.



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CPM in relationship with cancer.

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tumor-microenvironment interactions

predict metastatic behavior.

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50. Hahn Y, Bera TK, Gehlhaus K, et al.

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the expressed sequence databases.

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Schoenmakers HFPM, Mols R.

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Roth CW, et al. Carboxypeptidase M:

a biomarker for the discrimination of

well-differentiated liposarcoma from

lipoma. Mod Pathol 2009;22:1541-7.. A key paper about the use of CPM as

a biomarker.

53. Weaver J, Downs-Kelly E, Goldblum JR,

et al. Fluorescence in situ hybridization

for MDM2 gene amplification as a

diagnostic tool in lipomatous neoplasms.

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Wang X, et al. Retroperitoneal

lipomatous tumors without cytologic

atypia: are they lipomas?

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of 19 cases. Am J Surg Pathol

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Wang X, et al. Molecular testing for

lipomatous tumors: critical analysis and

test recommendations based on the

analysis of 405 extremity-based tumors.

Am J Surg Pathol 2010;34:1304-11. The use of CPM as a

cancer biomarker.

56. Tsakiris I, Soos G, Nemes Z, et al. The

presence of carboxypeptidase-M in

tumour cells signifies epidermal growth

factor receptor expression in lung

adenocarcinomas: the coexistence predicts

a poor prognosis regardless of EGFR

levels. J Cancer Res Clin Oncol

2008;134:439-51.. A key paper about the use of CPM as

a biomarker.

57. Eble JN, Sauter G, Epstein JI,

Sesterhenn IA. Pathology and genetics of

tumours of the urinary system and male

genital organs. WHO Classification of

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59. Tsakiris I, Torocsik D, Gyongyosi A,

et al. Carboxypeptidase-M is regulated by

lipids and CSFs in macrophages and

dendritic cells and expressed selectively in

tissue granulomas and foam cells.

Lab Invest 2012;92:345-61.. An important paper for the

investigation of a role of CPM in

tumor associated inflammation.

60. Mantovani A, Allavena P, Sica A,

Balkwill F. Cancer-related inflammation.

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Carboxypeptidase M and kinin

B1 receptors interact to facilitate efficient

b1 signaling from B2 agonists.

J Biol Chem 2008;283:7994-8004.. A key publication describing a

functional role of CPM in bradykinin

receptor activation and signaling.

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et al. Targeted disruption of the gene

encoding the murine small subunit of

carboxypeptidase N (CPN1) causes

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AffiliationCatherine J Denis PhD &

Anne-Marie Lambeir† PhD†Author for correspondence

University of Antwerp,

Pharmaceutical Sciences,

Laboratory of Medical Biochemistry,

Universiteitsplein 1,

Antwerp, B-2610, Belgium

Tel: +32 3 2652549;

Fax: +32 3 2652685;

E-mail: [email protected]



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The potential of carboxypeptidase M as a therapeutic target in cancer

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