MMP-10 (Stromelysin-2) and MMP-21 in human and murine squamous cell cancer

MMP-10 (Stromelysin-2) and MMP-21 in human andmurine squamous cell cancer

Sonja Boyd1, Susanna Virolainen1,2, Jenita Parssinen3, Tiina Skoog4,5, Max van Hogerlinden4, Leena

Latonen6, Lauri Kyllonen7, Rune Toftgard4 and Ulpu Saarialho-Kere3,5

1Department of Pathology, Helsinki University Central Hospital and Haartman Institute, University of Helsinki;2Department of Dermatopathology, Huslab, Helsinki University Central Hospital;3Department of Dermatology, Helsinki University Central Hospital and Biomedicum Helsinki, University of Helsinki, Helsinki, Finland;4Department of Biosciences and Nutrition, Karolinska Institutet, Novum, Huddinge, Sweden;5Department of Clinical Science and Education and Section of Dermatology, Karolinska Institutet at Stockholm Soder Hospital, Stockholm,

Sweden;6Molecular Cancer Biology Program, University of Helsinki, Helsinki, Finland;7Department of Transplantation Surgery, Helsinki University Central Hospital, Helsinki, Finland

Correspondence: Ulpu Saarialho-Kere, MD, PhD, Prof. Department of Dermatology, University of Helsinki, Meilahdentie 2, 00250 Helsinki,

Finland, Tel.: +358 9 4718 6306, Fax: +358 9 4718 6561, e-mail: [email protected]

Accepted for publication 3 April 2009

Abstract: The squamous cell cancers (SCC) of renal transplant

recipients are more aggressive and metastasize earlier than those

of the non-immunocompromised population. Matrix

metalloproteinases (MMPs) have a central role in tumor

initiation, invasion and metastasis. Our aim was to compare the

expression of MMPs-10, -12 and -21 in SCCs from

immunosuppressed (IS) and control patients and the contribution

of MMPs-10 and -21 to SCC development in the FVB ⁄ N-

Tg(KRT5-Nfkbia)3Rto mouse line. Immunohistochemical analysis

of 25 matched pairs of SCCs, nine of Bowen’s disease and timed

back skin biopsies of mice with selective inhibition of Rel ⁄ NF-jB

signalling were performed. Semiquantitatively assessed stromal

MMP-10 expression was higher (P = 0.009) in the control group

when compared with IS patients. Tumor cell-derived MMP-10,

-12 and -21 expression did not differ between the groups but

stromal fibroblasts of the control SCCs tended to express MMP-

21 more abundantly. MMP-10 expression was observed already in

Bowen’s disease while MMP-21 was absent. MMP-10 and -21

were present in inflammatory or stromal cells in ageing mice

while dysplastic keratinocytes and invasive cancer were negative.

Our results suggest that MMP-10 may be important in the initial

stages of SCC progression and induced in the stroma relating to

the general host-response reaction to skin cancer. MMP-21 does

not associate with invasion of SCC but may be involved in

keratinocyte differentiation.

Key words: cyclosporin – immunosuppression – mice – NF-jB –

skin cancer

Please cite this paper as: MMP-10 (Stromelysin-2) and MMP-21 in human and murine squamous cell cancer. Experimental Dermatology 2009; 18: 1044–1052.

Introduction

Squamous cell carcinoma (SCC) is the second most com-

mon cancer in white individuals worldwide (1). It is often

preceded by a dysplastic change of the epidermis, actinic

keratosis (AK), of which approximately 10% lead to SCC

(2). The most important risk factor for SCC is ultraviolet

(UV) radiation. The tumor suppressor gene p53 is usually

mutated in both AK and SCC, whereas p16(INK4a) is only

altered in SCC (3).

Non-melanoma skin cancer (NMSC) is the most com-

mon type of cancer among adult renal transplant recipi-

ents, the risk for other types of cancer being increased

3- to 5-fold (4,5). The incidence of SCC among organ

transplant recipients is 65- to 250-fold higher compared

with the general population (6). Male sex, UV radiation,

cigarette smoking, presence of AKs and older age at

transplantation increase the risk for post-transplant

NMSC (6,7). SCCs of organ transplanted individuals are

more aggressive and have more metastatic potential com-

pared with the non-transplanted population (6,8). Fur-

thermore, human papilloma virus infection may increase

the risk of SCC among renal transplant recipients (9).

Abbreviations: AK, actinic keratosis; BD, Bowen’s disease; EGFR,

epidermal growth factor receptor; HPV, human papillomavirus;

IS, immunosuppressed; MMP, matrix metalloproteinase; NMSC,

non-melanoma skin cancer; SCC, squamous cell carcinoma; UV,

ultraviolet

DOI:10.1111/j.1600-0625.2009.00901.x

www.blackwellpublishing.com/EXDOriginal Article

1044 ª 2009 John Wiley & Sons A/S, Experimental Dermatology, 18, 1044–1052

Immunosuppressive treatment along with sun exposure is

thought to be the most important risk factor for post-

transplant NMSC, and the level of immunosuppression is

related to the risk of post-transplant SCC (10). Calcine-

urin inhibitors and azathioprine, in particular, increase

this risk (5).

Matrix metalloproteinases (MMPs) are a group of 24

human zinc-dependent proteolytic enzymes capable of

degrading practically all extracellular matrix proteins and

basement membrane components. Furthermore, MMPs

release growth factors and are involved in angiogenesis,

inflammation, apoptosis, cancer cell proliferation and

metastasis (11). Tumor cells may produce MMPs or induce

stromal cells to secrete them. Some MMPs, such as MMPs-

8, -12 and -26, have an anti-tumor effect depending on the

stage of cancer progression (12). We have recently shown

that among classical cancer-related MMPs, overexpression

of MMP-1, -7, -8 and -13 does not explain the more

aggressive behaviour of SCCs in renal transplant recipients

nor diminished expression of tissue inhibitors of MMPS,

TIMP-1 or TIMP-3 (13).

Matrix metalloproteinase-10 (stromelysin-2) cleaves

in vitro gelatin, types III–V collagen, elastin, fibronectin

and laminin-5 among others (14) but its true physiological

substrates have not yet been reported. It is not expressed in

normal skin but is upregulated in migrating wound edge

keratinocytes (15). In mice overexpressing MMP-10, its

tightly regulated expression was required for limited matrix

degradation at the wound site, thereby controlling

keratinocyte migration (16). Tumor cells in SCC express

MMP-10 mRNA (17) but MMP-10 is also detected in

proliferating keratinocytes of keratoacanthomas, non-malig-

nant ‘precursors’ to SCC (18).

Matrix metalloproteinase-12 (macrophage metalloelas-

tase) degrades in vitro elastin, type IV collagen, fibronectin,

laminin-1, gelatin, vitronectin, proteoglycan and chondroi-

tin sulphates among others (19). Besides being expressed

by macrophages, MMP-12 can be produced by transformed

epithelial cells in SCC but may have a dual role in tumor

progression (20). Its expression in cancer cells correlates

with tumor aggressiveness (21), while macrophage-derived

MMP-12 may activate angiostatin and function in host-

response (22).

Matrix metalloproteinase-21 was recently cloned by our

group (23). Its physiological substrates are still not known.

MMP-21 has been implicated in foetal development, tumor

progression, inflammation and stromal remodelling (23–

25), and it is expressed by macrophages, fibroblasts and

leucocytes in various skin disorders as well as in fibroblasts

and monocytic cell lines (23,25,26). MMP-21 was reported

to be expressed by tumor cells in a subpopulation of cuta-

neous SCCs (25) but its function in cancer biology is still

poorly understood.

The aim of this study was to investigate whether differ-

ences in the expression patterns of MMP-10, -12, or -21

would explain the more aggressive behaviour of SCCs of

transplant patients compared with those of immunocompe-

tent controls. We also wanted to elucidate more precisely

the role of these MMPs in human SCC in vivo and in an

established experimental skin cancer mouse model.

Methods

Patients and tissuesFormalin-fixed, paraffin-embedded site-matched SCC speci-

mens from 25 renal transplant patients and their non-im-

munosuppressed (IS) controls were obtained from the

Departments of Dermatopathology and Pathology, Helsinki

University Central Hospital, Finland (Table 1). Nine site-

matched pairs of Bowen’s disease (BD) (SCC in situ) sam-

ples from transplant recipients and their controls were also

studied. Mean age of the IS patients was 64.1 years com-

pared with 79.9 years of the control patients. Diagnoses

were confirmed by an experienced dermatopathologist (SV)

and the SCCs were graded as well, moderately or poorly

differentiated by the degree of anaplasia (Table 1). Histo-

logical changes suggestive of human papillomavirus (HPV)

infection (keratinocytes with coarse keratohyaline granules

in the upper layers of the acanthotic epidermis next to the

malignant SCC areas but not in the tumor cells themselves)

were also recorded (8,27) (Table 1). The inflammatory cell

infiltrate at the invasion front of the tumors was histologi-

cally graded as absent (0), weak (1) (a scant amount of

inflammatory cells seen at the invasion front), moderate

(2) (dense inflammatory infiltrate detected around only

part of the tumor or moderate amounts of inflammatory

cells seen around most of the tumor), or strong (3)

(a dense inflammatory infiltrate surrounding most of the

tumor) (Table 1). The cell types were identified on histo-

logical basis (lymphocytes, plasma cells and neutrophils).

CD163 immunodetection was used to distinguish between

macrophages and fibroblasts. Analyses were performed

independently by two investigators (SB, SV). The study was

approved by the corresponding ethical committee of the

Helsinki University Central Hospital, Helsinki, Finland.

ImmunohistochemistryImmunohistochemical staining was performed using the

Dako StreptABComplex ⁄ HRP Duet kit (Dako A ⁄ S, Glost-

rup, Denmark) or Vectastain (Goat ⁄ Rabbit) kit (Vector

Laboratories, Burlingame, CA, USA) as described previ-

ously (18,28). We used two different polyclonal antibodies

for MMP-21 [RP3-MMP-21; Abcam, Cambridge, UK and

antibodies produced against a synthetic peptide by us (23)]

and MMP-12 (SC-12361; Santa-Cruz Biotechnology, Santa

Cruz, CA, USA). Monoclonal antibodies were used for

MMP-10 and -21 in SCC

ª 2009 John Wiley & Sons A/S, Experimental Dermatology, 18, 1044–1052 1045

MMP-10 (NCL-MMP10; Novocastra Laboratories, Newcas-

tle upon Tyne, UK), epidermal growth factor receptor

(EGFR, 31G7; Zymed, San Francisco, CA, USA) and

CD163 (10D6, Novocastra Laboratories). As the RP3-

MMP-21 antibodies cross-reacted with mouse (23,25), they

were employed on mouse tissues. For MMP-10 on mouse,

sections were pretreated in citrate buffer in 95�C water

bath, and MMP-10 (SC-26697, Santa-Cruz Biotechnology)

was used at 1:100 (29). Diaminobenzidine or aminoethyl-

carbazole were used as chromogenic substrates and Mayer

hematoxylin as counterstain. For negative controls, we used

parallel sections of the same tumors with preimmune sera

or normal immunoglobulin.

Evaluation of immunohistochemistryImmunohistochemical stainings of human tissues was

analyzed independently by three (SB, SV, US-K) investiga-

tors through light microscopic observation. The expression

of MMP-10, -12 and -21 was determined in the SCC epithe-

lium (invasive front, central part and superficial part), in the

intact epidermis adjacent to the tumor as well as in stromal

cells at the invasion front. Three hot spots were selected at

40· magnification, whereas counting was performed at 400·magnification. The total number of positive cells was

counted from three high-power fields, and semi-quantitative

grading is as follows: 0 = 0–9 positive cells, 1 = 10–50

positive cells, 12 = 51–100 positive cells, 3 = over 100

positive cells (Table 2). The expression of EGFR (Table 1)

was determined in the SCC epithelium only and that of

CD163 in stromal cells at the invasion front. Staining results

on mouse tissues were analyzed by one investigator (US-K)

and a mouse pathologist (Bjorn Rozell).

Experimental mouse modelThe FVB ⁄ N-Tg(KRT5-Nfkbia)3Rto (hereafter referred to as

K5-IkBa) mice were generated as described previously (30).

This transgenic mouse line with selective inhibition of

Rel ⁄ nuclear factor kappa B (NF-jB) signalling in skin

developed progressive epidermal dysplasia that led to SCC

development (31). In this model, highly proliferating kerat-

inocytes were subjected to the tumor promoting effects of

inflammation orchestrated by growth factors provided by

infiltrating cells and also subjected to DNA damage by the

same inflammatory cells. Due to the inherent defect in cell

cycle regulation these cells then failed to stop proliferating

and could progress to neoplasia. We used these mice to

examine the expression of MMP-10 and -21 in various

stages of SCC development. Paraffin sections from biopsies

Table 1. Clinical information on the patients and SCCs

IS Sex ⁄ Age Site G I Medication EGFR C Sex ⁄ Age Site G I EGFR

1A M ⁄ 74 Ear 2 2 CyAzaSter 3 1B M ⁄ 81 Ear 2 3 12A M ⁄ 65 Chest 2 3 CyAzaSter 0 2B F ⁄ 65 Neck 2 2 13A M ⁄ 53 Nose 2 2 CyAzaSter 2 3B F ⁄ 81 Nose 2 2 04A F ⁄ 60 Leg 1 1 AzaSter 1 4B M ⁄ 64 Knee 2 1 05A M ⁄ 61 Scalp 2 1 AzaSter 1 5B F ⁄ 84 Scalp 1 3 16A M ⁄ 70 Hand 1 1 CyAzaSter 1 6B M ⁄ 90 Hand 1 2 17A M ⁄ 53 Forehead 2 2 MMFCyAzaSter 2 7B M ⁄ 75 Temple 3 3 28A M ⁄ 63 Scalp 1 2 AzaSter 1 8B M ⁄ 82 Scalp 2 1 19A M ⁄ 63 Hand 1 3 AzaSter 1 9B F ⁄ 58 Hand 2 2 0

10A M ⁄ 45 Cheek 1 1 AzaSter 1 10B F ⁄ 73 Cheek 2 2 111A M ⁄ 61 Arm 2 2 CyAzaSter 1 11B F ⁄ 77 Hand 2 3 112A M ⁄ 71 Nose 3 1 MMFCyAzaSter 1 12B M ⁄ 91 Nose 2 2 013A F ⁄ 59 Arm 1 1 AzaSter 1 13B F ⁄ 88 Hand 1 3 114A M ⁄ 63 Hand 1 2 AzaSter 1 14B M ⁄ 81 Hand 1 2 015A M ⁄ 69 Chin 1 2 CyAzaSter 0 15B F ⁄ 87 Chin 1 3 116A M ⁄ 68 Nose 1 1 AzaSter 2 16B F ⁄ 85 Nose 1 2 017A F ⁄ 61 Chest 1 2 CyAzaSter 0 17B F ⁄ 73 Shoulder 1 1 118A M ⁄ 69 Lip 2 2 CyAzaSter 1 18B F ⁄ 78 Lip 2 3 019A M ⁄ 59 Chest 1 2 CyAzaSter 0 19B F ⁄ 81 Chest 1 2 120A F ⁄ 59 Arm 1 1 CyAzaSter 0 20B F ⁄ 90 Lip 1 3 221A M ⁄ 66 Back 1 1 CyAzaSter 0 21B M ⁄ 71 Back 3 3 022A M ⁄ 80 Forehead 2 1 CyAzaSter 2 22B F ⁄ 97 Chin 1 2 223A M ⁄ 71 Face 3 1 MMFCy 2 23B M ⁄ 74 Chin 2 2 124A M ⁄ 79 Forehead 3 1 CyAzaSter 2 24B M ⁄ 83 Forehead 3 2 025A M ⁄ 71 Arm 1 2 CySter 0 25B F ⁄ 88 Arm 1 3 0

IS, immunosuppressed; C, controls; G, grading; I, Inflammation; EGFR, epidermal growth factor receptor (at the invasion front); SCC, squamous

cell carcinoma; M, male; F, female.

Grading: 1 = well, 2 = moderately, 3 = poorly differentiated.

Inflammation: 1 = weak, 2 = moderate, 3 = strong (see Methods for details).

Medication: Cy, cyclosporin; Aza, azathioprine; Ster, steroid; MMF, mycophenolate mofetil.

Boyd et al.


of the back skin taken at 9, 12, 15, 18, 21 days and 8 and

16 weeks postpartum from both male and female mice of

each genotype (wild-type and transgenic) were processed

for immunohistochemistry. For comparison expression of

MMP-10 was analyzed in neutrophils recruited to skin

wound sites in wild-type mice. The animal experiments

were approved by corresponding ethical committee at the

Department of Biosciences, Novum, Karolinska Institut,

Huddinge, Sweden.

Statistical analysisThe semi-quantitative data from immunohistochemical

studies was statistically analyzed with spss 16.0 (SPSS Inc.,

Chicago, IL, USA) using ANOVA, Mann–Whitney U-test,

Pearson’s chi-squared and Student’s t-tests, as needed.

P-value less than 0.05 was considered as statistically significant.

Results

Histological analysisInflammatory infiltrate around the tumors was significantly

more intense in SCCs of control patients (P = 0.0014) than

in those of transplanted patients (Table 1). The most com-

mon inflammatory cell type was the lymphocyte. The total

amount of tumor-associated macrophages did not differ

significantly between the groups as assessed semiquantita-

tively by CD163 immunostaining (means, 1.84 vs. 1.80).

Altogether 21 ⁄ 50 tumors had histological evidence of HPV

but there was no statistically significant difference between

transplant and control patients (12 vs 9, respectively).

MMP-10 in human SCCsMatrix metalloproteinase-10 protein was detected in cancer

cells of 45 ⁄ 50 tumors (Fig. 1a; Table 2). Stromal cells

expressed MMP-10 in 3 ⁄ 25 samples of the IS and 10 ⁄ 25 of

the control group. Semiquantitatively assessed stromal

expression was significantly higher (P = 0.009) in the con-

trol group (Fig. 1b,b¢; Table 2). In the tumors of IS patients,

MMP-10 positive stromal cells were predominantly fibro-

blast-like, whereas in control patients immunoreactivity for

both macrophage- and fibroblast-like cells was detected.

Endothelium was rarely immunopositive for MMP-10 (5 ⁄ 50

specimens). To further understand the role of MMPs in

cancer initiation, 18 BD specimens were stained; MMP-10

was detected in basal keratinocytes of BD lesions in 8 ⁄ 18

specimens, and there was no difference between the tumors

of the IS and control patients (Fig. 1c,c¢). Stromal cells adja-

cent to BD were negative in all specimens (data not shown).

Table 2. Expression of MMP-10 and -21 in SCCs of immunosuppressed and immunocompetent control patients

IS

MMP-10 MMP-10 MMP-21 MMP-21

C

MMP-10 MMP-10 MMP-21 MMP-21

Ca Str Ca Fib Ca Str Ca Fib

1A 11 0 0 0 1B 31 1 0 02A 21 0 0 1 2B 0 1 0 13A 31 1 11 2 3B 11 0 0 14A 1 0 0 1 4B 0 1 1 05A 31 1 0 0 5B 21 1 11 06A 11 0 0 1 6B 11 1 0 27A 11 0 0 0 7B 31 0 0 08A 11 0 0 1 8B 21 0 11 09A 11 0 0 0 9B 0 0 0 1

10A 11 0 0 1 10B 2 1 0 011A 0 0 0 0 11B 21 0 0 112A 11 0 0 0 12B 1 0 0 013A 11 0 0 0 13B 21 0 0 114A 21 1 0 0 14B 21 2 1 015A 21 0 0 0 15B 11 0 1 216A 11 0 0 0 16B 11 1 0 117A 11 0 0 2 17B 31 2 11 118A 11 0 11 1 18B 11 0 0 019A 21 0 0 0 19B 11 0 0 220A 11 0 11 0 20B 0 0 0 021A 11 0 11 1 21B 11 2 0 122A 11 0 11 0 22B 11 0 11 223A 11 0 0 0 23B 21 0 0 224A 31 0 0 1 24B 31 0 0 125A 11 0 0 1 25B 11 0 0 0Sum 34 3 5 13 36 13 7 19

IS, immunosuppressed; C, controls; Ca, carcinoma cells; Str, stromal fibroblasts and macrophages; Fib, fibroblasts; MMP, matrix metalloproteinase;

SCC, squamous cell carcinoma.1Invasion front.

Semi-quantitative grading of immunosignal is described in Methods.



When patients using cyclosporin were compared as a group

with all other patients, there was no significant difference in

the expression of MMP-10 in carcinoma or stromal cells of

SCCs. The inflammation score or differentiation of the

tumors did not correlate with MMP-10 expression in tumor

or stromal cells.

EGFR is present in the same regions as MMP-10Because EGFR has been implicated as a prognostic marker

in SCC (32) and its overexpression correlates strongly with

the expression of MMP-10 (33), we examined whether it

differs among SCCs of IS and control patients. It was

detected in 18 ⁄ 25 specimens of IS and 15 ⁄ 25 specimens of

control patients at the invasion front of SCCs (Fig. 1e;

Table 1). Semiquantitatively assessed staining was, in gen-

eral, more intense among the IS patients, but this differ-

ence did not reach statistical significance (means, 1.04 vs.

0.72) (Table 1). EGFR and MMP-10 colocalized in 20 ⁄ 50

tumors (Fig. 1e,f).

MMP-12 in human SCCMatrix metalloproteinase-12 was detected in tumor cells in

16 ⁄ 50 SCCs (Fig. 1d,d¢) and in occasional macrophages at

the invasion front but no statistically significant difference

was noted in semi-quantitative grading of immunosignal

between the groups (data not shown).

MMP-21 in human SCCsMatrix metalloproteinase-21 protein was detected in cancer

cells focally inside the tumors or at the invasion front in

12 ⁄ 50 SCCs (Fig. 1g; Table 2), and there was no significant

difference between the groups. No positive cancer cells were

observed in the poorly differentiated tumors while eight

well-differentiated and four moderately differentiated SCCs

had MMP-21-expressing tumor cells (Tables 1 and 2).

Peritumoral fibroblasts of the control patients tended to

express MMP-21 more abundantly as assessed semiquanti-

tatively (Fig. 1h; Table 2) but this did not reach statistical

significance. Staining results were similar with both MMP-

21 antibodies used. BD lesions were generally negative for

MMP-21 (data not shown). Interestingly, MMP-21 was

seen in carcinoma cells of the IS group only in patients

using cyclosporin.

MMP-10 and MMP-21 in K5-IkBa miceMatrix metalloproteinase-10 protein was consistently seen

in stratum granulosum in mice at all ages in normal and

acanthotic skin (Fig. 2a). With increasing epidermal hyper-

plasia, the amount of inflammatory infiltration increased

and MMP-10 was also detected in suprabasal keratinocytes

as the mice aged (Fig. 2b). In dysplastic epithelium MMP-

10 was never expressed in basal keratinocytes (Fig. 2c,d)

nor was it associated with keratinocyte apoptosis or atypia.

Invasive islands of SCCs of 16-week-old mice were always

MMP-10 negative (Fig. 2d). The number of MMP-10 posi-

tive polymorphonuclear (PMN) leucocytes increased sur-

rounding hyperplastic epithelium and SCC islands as the

mice aged (Fig. 2e). In age-matched wild-type mice, MMP-

10 was not expressed by keratinocytes (Fig. 2g) and no

positive inflammatory infiltrations were detected. However,

in wounded wild-type mice, neutrophils of 6-day wounds

were positive for MMP-10 (Fig. 2f,f¢).

Epidermis with mild dysplasia and acanthosis in young

transgenic mice did not express MMP-21. Beginning in 21-

day-old mice, MMP-21 protein was seen in suprabasal lay-

ers (stratum spinosum and granulosum) in association with

(a) (b) (b′)

(c′)(c) (d)

(d′)

(e) (f)

(g) (h)

Figure 1. (a) Matrix metalloproteinase (MMP)-10 expression in

squamous cell carcinoma (SCC) tumor cells of a renal transplant

patient. (b) MMP-10 expression in stromal cells of an SCC of an

immunocompetent patient. (b¢) MMP-10 positive fibroblasts (arrow). (c)

MMP-10 expression in Bowen’s disease. Inset c¢: higher magnification

of the positive area. (d) MMP-12 expression in SCC. Inset d¢: higher

magnification of MMP-12 positive tumor cells. (e) epidermal growth

factor receptor positive cells at the invasion front of an SCC. (f) MMP-

10 staining in a nearby section. (g) MMP-21 positivity seen in SCC

tumor cells (arrows) and in peritumoral fibroblasts in a SCC of an

immunosuppressed patient. (h) SCC of a control patient with no MMP-

21 at the invasive front but positive stromal fibroblasts (arrows). Scale

bars: (a–d,g,h) 40 lm; (e,f) 20 lm; (b¢–d¢) 10 lm.

Boyd et al.


epidermal hyperplasia (Fig. 3a,b). MMP-21 positivity was

also detected in suprabasal epithelium of 8-week-old mice

(Fig 3c,d) but not in atypical keratinocytes of the basal epi-

dermal layer (Fig. 3d). MMP-21 protein, however, was not

seen in the invasive islands of 16-week-old mice (Fig. 3e,f).

Beginning from day 15, MMP-21 protein was seen in stro-

mal stellate cells resembling activated fibroblasts in mice at

all ages (Fig. 3g): the amount of MMP-21 positive stromal

infiltrate did not markedly increase from epidermal hyper-

plasia to well-differentiated carcinoma. In age-matched

wild-type mice, MMP-21 positive keratinocytes were not

detected. However, beginning from day 15, stromal stellate

cells resembling activated fibroblasts expressed MMP-21

(Fig. 3h).

Discussion

In renal transplant patients, 88% develop a new skin cancer

5 years after transplantation (34). A portion of these SCCs

tend to be aggressive in behaviour and to metastasize early.

The aim of this study was to elucidate whether the profile

of MMP-10, -12 or -21 expression could explain the biol-

ogy of SCCs of IS patients. Furthermore, we wanted to

study their contribution to skin cancer in more detail using

a transgenic mouse model with progressive epidermal

hyperplasia and a strong inflammatory response eventually

leading to SCC development.

Matrix metalloproteinase-10 was more abundantly

expressed by stromal cells in SCCs of immunocompetent

patients compared with renal transplant recipients. Interest-

ingly, cell-cell contact activation of fibroblasts increased

MMP-10 in vitro (35). A transforming growth factor

(TGF)-b1 enriched tumor environment coupled with

amplified EGFR levels or signalling correlates with

increased expression of MMP-10 (36). Our recent results

(13) on the higher number of MMP-9 positive macrophag-

es in SCCs of non-IS compared with IS patients, and the

presence of angiogenesis-promoting MMP-9 positive neu-

trophils in SCCs of tranplanted patients, further emphasize

(a)

(b)

(c)

(d)

(e)

(f) (g)

Figure 2. Tail skin of K5-IjB-a mouse at 12 dpp (a) with positive

staining for matrix metalloproteinase (MMP)-10 in stratum granulosum

and occasional stromal cells. With increasing epidermal hyperplasia at

15 dpp MMP-10 is also detected in suprabasal keratinocytes as the

mice aged (b). The MMP-10 positive stromal infiltrate increases as the

mice age but MMP-10 is absent from dysplastic epithelium (c). Invasive

islands of squamous cell carcinoma (SCCs) of 16-week-old mice are

negative for MMP-10. (d) Higher magnification of c from the area

depicted by lines is shown. (e) The number of MMP-10 positive

polymorphonuclear leucocytes (arrows) increased surrounding

hyperplastic epithelium and SCC islands as the mice aged. (f) MMP-10

is detected in neutrophils of a 6-day-old wound of a wild-type mice. (f¢)Higher magnification of MMP-10 positive cells with trilobular nuclei

(neutrophils) (arrows). (g) Tail skin of wild-type mouse at 15 dpp

stained for MMP-10. Scale bars: (c) 100 lm; (d,f) 40 lm; (a,b,g) 20 lm;

(e,f¢) 10 lm.

(a)

(b)

(c) (d)

(e)

(f)

(g)

(h)

Figure 3. Beginning at 21 dpp, matrix metalloproteinase (MMP)-21

protein is seen in suprabasal keratinocyte layers of tail skin in

association with epidermal hyperplasia in K5-IjB-a mice (a,b). MMP-21

positivity is also detected in suprabasal epithelium of 8-week-old mice

(c) but not in atypic keratinocytes of the basal epidermal layer (d).

MMP-21 is not present in the invasive islands of 16-week-old mice (e,f).

Beginning at 15 dpp, MMP-21 was seen in stromal stellate cells

resembling activated fibroblasts in mice at all ages (g). (h) Tail skin of a

wild-type mouse at 21dpp with MMP-21 positive stromal stellate cells

resembling activated fibroblasts (arrows). Scale bars: (a) 100 lm; (c,e)

40 lm; (b,d,f,h) 20 lm; (g) 10 lm.



the role of stromal cells in the biological behaviour of skin

tumors. Moreover, in our recent study comparing basal cell

carcinomas (BCCs) of IS and control patients, MMP-10

expression was detected in BCC tumor cells and stromal

fibroblasts and macrophages as well (37) but no essential

differences between the two groups studied were noted.

The role of MMP-10 in the progression of cancer is not

well-known. It was not more abundantly expressed in can-

cer cells of SCCs of IS patients and, thus, may not explain

the more aggressive behaviour of their tumors. This is sup-

ported by previous in vitro findings on MMP-10 expression

not correlating with invasiveness of SCC cells (38) and is

also substantiated by our results in the mouse model, in

which MMP-10 was not detected in invasive cancer cells.

Compared with benign keratinocyte hyperproliferation

occurring in psoriasis, MMP-10 was upregulated in biop-

sies of well-differentiated SCCs as assessed by Affymetrix

(Santa Clara, CA, USA) and RT-PCR analyses, suggesting

that its upregulation is typical for cancer cells (39,40). Ye

et al. (41) have recently shown, by genome-wide transcrip-

tomic profiling, that MMP-10 was nearly 9-fold upregulat-

ed when 53 primary tongue SCCs were compared with

matching normal tissues. MMP-10 may function in the

early stages of cancer initiation based on our current results

of a subset of BD lesions being MMP-10 positive as well as

our previous data on keratoacanthomas (18). In cultured

keratinocytes, MMP-10 is induced by important cancer-

associated cytokines such as tumor necrosis factor (TNF)-

a, EGF and TGF-b1 (15). Furthermore, MMP-10 activates

several MMPs important in cancer progression such as

MMP-1, -7, -9 and MMP-13 (36). No correlation with

MMP-10 levels and patient prognosis was found in a study

on oesophageal SCC and the authors speculated that

although MMP-10 expression is characteristic of cells

involved in stromal or basement membrane remodelling

and not necessarily tumor specific, it might be involved in

early stages of degradation of extra-cellular matrix (42).

MMP-10 was also detected within fibroblasts, endothelial

and neoplastic cells in head and neck SCC, while no corre-

lation was found to any clinico-pathological variable (33).

Matrix metalloproteinase-21 protein has been detected

immunohistochemically in colon, ovarian, breast and pros-

tate cancers (23). In pancreatic cancer, MMP-21 is often

detected in central areas of well-differentiated adenocarci-

nomas, the invasion front being mostly negative (43). Our

current results in human and mouse SCCs and BD did not

support a role for MMP-21 in cancer initiation or invasion.

However, our recent data suggest that MMP-21 is associ-

ated with keratinocyte differentiation but not with prolifer-

ation, apoptosis or epithelial–mesenchymal transition (44).

In oesophageal SCC, MMP-21 was a marker of differentia-

tiated tumor areas (45) and its location in the samples of

this study was fairly similar. In HaCaT cells MMP-21 tran-

scription is very tightly regulated by retinoic acid being one

of the few agents that induces its expression (44). Retinoic

acid normalizes keratinocyte differentiation, a feature used

in the therapy of premalignant skin lesions, and may thus

exert some of its beneficial effects via modulation of MMP-

21, that, like various other MMPs, could eventually have

anti-tumor effects (12).

The progression sequence for cutaneous cancers may

vary between the human disease and its corresponding

mouse models, although several genetic events are common

to both. Analogously to the model used in this study,

human sporadic SCC may show a block in NF-jB signal-

ling based on nuclear exclusion of the RelA NF-jB subunit

(46) and inflammation is known to promote SCC develop-

ment in various skin disorders such as chronic venous

ulcers, lichen planus or lupus. The mouse model we used

is characterized by upregulated TNF-a expression, inflam-

mation, hyperproliferation, increased apoptosis and sponta-

neous early development of SCC with a 100% penetrance

(31). Neither MMP-10 nor MMP-21 was detected in inva-

sive cancer cells. This basically agrees with the current find-

ings on their expression pattern in our human SCCs. In

another mouse model of epithelial carcinogenesis, the

K14-HPV16 mouse, MMP-10 can be identified in dysplas-

tic tissues as well as in SCC, unlike MMP-7 and -13 that

are only produced in SCCs (47), suggesting that MMP-10

is important in tumor initiation but not invasion or metas-

tasis. This is further supported by our finding on MMP-10

expression by proliferating keratinocytes in keratoacan-

thomas (18), precursor lesions of SCCs, that represent a

distinct stage in the process of skin carcinogenesis with

increased expression of p16 and cyclin D1 (18,35). MMP-

10 was upregulated in PMNs in our mouse model as the

SCC evolved and indeed has been linked to regulation of

inflammation based on knock-out mouse studies (48).

PMN MMP-10 may have a critical role in driving the

keratinocyte hyperproliferation in the murine model used

(46). MMP-21 in contrast was not generally upregulated in

inflammatory cells during the evolution of tumors but was

mostly associated with the dermal fibrosis surrounding

hyperplastic ⁄ dysplastic epidermis seen in our mouse model.

Immunosuppressive therapy to prevent allograft rejection

plays a major role in the development of NMSC in trans-

planted patients. Cyclosporin decreases DNA repair and

apoptosis in UVB-irradiated keratinocytes (49). Agreeing

with our data on tissue level demonstrating differences

mainly in stromal expression of MMP-10 and -21 between

control and IS specimens, neither of these MMPs are

upregulated by cyclosporin in cultures of HaCaT cells

(J. Parssinen and U. Saarialho-Kere, unpublished data).

Interestingly, cyclosporin downregulates MMP-10 expres-

sion in SCC-015 cells while the keratinocyte transformation

specific MMP-7 and -13 are upregulated (50). We have

Boyd et al.


recently shown that corticosteroids, also frequently present

in immunosuppressive drug combinations, do not modify

MMP-21 gene transcription (44), while they increase

MMP-10 in keratinocytes in vitro (51).

Ultraviolet radiation and cumulative life-time UV dose

are the most important risk factors for NMSC in trans-

planted patients (7). MMP-10 is induced in keratinocytes

by UVA ⁄ B irradiation (52,53) and UV radiation is known

to induce the expression of MMP-12 in skin (54). MMP-

21, however, is not upregulated in keratinocytes by UVA ⁄ Birradiation, unlike many previously characterized MMPs (S.

Suomela, L. Latonen and U. Saarialho-Kere, unpublished

data).

Staining for EGFR tended to be more intense in the

SCCs of IS patients. Its overexpression was associated with

advanced pathological stages of head and neck SCCs and

correlated strongly with the expression of MMP-10 (33).

While EGFR overexpression correlates with poor prognosis

in head and neck SCC (32), this matter is still unclear in

cutaneous SCC as very few studies with limited patient

numbers currently exist (55).

The precise role of MMP-12 in SCC is still unclear,

although it has been suggested to possess anti-tumor effects

in several cancer types due to its ability to cleave angiosta-

tin (12). In this study, MMP-12 was expressed by tumor

cells in a subset of SCCs in agreement with previous data

on oesophageal SCC (56), but we cannot conclude that its

expression in cancer or stromal macrophages would con-

tribute to more aggressive behaviour of tumors of IS indi-

viduals.

In conclusion, our results suggest that MMP-10 is more

strongly expressed in the stroma of SCCs of immunocom-

petent patients which may relate to the general host-

response reaction to skin cancer. Based on our results on

the murine cancer model and human in situ SCCs, MMP-

10 seems to be important already in the initial stages of

SCC progression. Our in vivo human and mouse data sug-

gest that MMP-21 does not promote invasion of SCC but

may relate to tumor differentiation.

Acknowledgements

We thank Dr. Bjorn Rozell (Department of Laboratory Medicine, Karo-

linska Institutet) for his expertise in murine histopathology and Ms Alli

Tallqvist and Ms Jonna Jantunen for skilful technical assistance. This study

was supported by the Academy of Finland (grants no. 115590 to US-K and

no. 108828 to LL), Sigrid Juselius Foundation, Finska Lakaresallskapet, Hel-

sinki University Central Hospital Research Fund (TYH6241), Finland and

Cancerfonden (US-K), Swedish Research Council (US-K) and Welander-

Finsen Foundation (TS), Sweden.

References

1 Goldman G D. Squamous cell cancer: a practical approach. Semin Cutan MedSurg 1998: 17: 80–95.

2 Fuchs A, Marmur E. The kinetics of skin cancer: progression of actinic keratosisto squamous cell carcinoma. Dermatol Surg 2007: 33: 1099–1101.

3 Mortier L, Marchetti P, Delaporte E et al. Progression of actinic keratosis tosquamous cell carcinoma of the skin correlates with deletion of the 9p21region encoding the p16(INK4a) tumor suppressor. Cancer Lett 2002: 176:205–214.

4 Kyllonen L, Salmela K, Pukkala E. Cancer incidence in a kidney-transplantedpopulation. Transpl Int 2000: 13 (Suppl 1): S394–398.

5 Vasudev B, Hariharan S. Cancer after renal transplantation. Curr Opin NephrolHypertens 2007: 16: 523–528.

6 Euvrard S, Kanitakis J, Claudy A. Skin cancers after organ transplantation.N Engl J Med 2003: 348: 1681–1691.

7 Ramsay H M, Fryer A A, Reece S, Smith A G, Harden P N. Clinical risk factorsassociated with nonmelanoma skin cancer in renal transplant recipients. Am JKidney Dis 2000: 36: 167–176.

8 Harwood C A, Proby C M, McGregor J M, Sheaff M T, Leigh I M, Cerio R.Clinicopathologic features of skin cancer in organ transplant recipients: a ret-rospective case-control series. J Am Acad Dermatol 2006: 54: 290–300.

9 Meyer T, Arndt R, Nindl I, Ulrich C, Christophers E, Stockfleth E. Associationof human papillomavirus infections with cutaneous tumors in immunosup-pressed patients. Transpl Int 2003: 16: 146–153.

10 Jensen P, Hansen S, Moller B et al. Skin cancer in kidney and heart transplantrecipients and different long-term immunosuppressive therapy regimens. J AmAcad Dermatol 1999: 40: 177–186.

11 Chang C, Werb Z. The many faces of metalloproteases: cell growth, invasion,angiogenesis and metastasis. Trends Cell Biol 2001: 11: S37–43.

12 Lopez-Otin C, Matrisian L M. Emerging roles of proteases in tumour suppres-sion. Nat Rev Cancer 2007: 7: 800–808.

13 Kuivanen T, Jeskanen L, Kyllonen L, Isaka K, Saarialho-Kere U. Matrix metallo-proteinase-26 is present more frequently in squamous cell carcinomas ofimmunosuppressed compared to immunocompetent patients. J Cutan Pathol(in press).

14 Vihinen P, Kahari V M. Matrix metalloproteinases in cancer: prognostic mark-ers and therapeutic targets. Int J Cancer 2002: 99: 157–166.

15 Rechardt O, Elomaa O, Vaalamo M et al. Stromelysin-2 is upregulated duringnormal wound repair and is induced by cytokines. J Invest Dermatol 2000:115: 778–787.

16 Krampert M, Bloch W, Sasaki T et al. Activities of the matrix metalloproteinasestromelysin-2 (MMP-10) in matrix degradation and keratinocyte organizationin wounded skin. Mol Biol Cell 2004: 15: 5242–5254.

17 Kerkela E, Ala-aho R, Lohi J, Grenman R, M K V, Saarialho-Kere U. Differentialpatterns of stromelysin-2 (MMP-10) and MT1-MMP (MMP-14) expression inepithelial skin cancers. Br J Cancer 2001: 84: 659–669.

18 Kuivanen T T, Jeskanen L, Kyllonen L, Impola U, Saarialho-Kere U K. Transfor-mation-specific matrix metalloproteinases, MMP-7 and MMP-13, are presentin epithelial cells of keratoacanthomas. Mod Pathol 2006: 19: 1203–1212.

19 Chandler S, Cossins J, Lury J, Wells G. Macrophage metalloelastase degradesmatrix and myelin proteins and processes a tumour necrosis factor-alphafusion protein. Biochem Biophys Res Commun 1996: 228: 421–429.

20 Kerkela E, Ala-Aho R, Jeskanen L et al. Expression of human macrophage me-talloelastase (MMP-12) by tumor cells in skin cancer. J Invest Dermatol 2000:114: 1113–1119.

21 Yang X, Dong Y, Zhao J et al. Increased expression of human macrophagemetalloelastase (MMP-12) is associated with the invasion of endometrialadenocarcinoma. Pathol Res Pract 2007: 203: 499–505.

22 Kerkela E, Ala-aho R, Klemi P et al. Metalloelastase (MMP-12) expression bytumour cells in squamous cell carcinoma of the vulva correlates with invasive-ness, while that by macrophages predicts better outcome. J Pathol 2002: 198:258–269.

23 Ahokas K, Lohi J, Lohi H et al. Matrix metalloproteinase-21, the human ortho-logue for XMMP, is expressed during fetal development and in cancer. Gene2002: 301: 31–41.

24 Marchenko G N, Marchenko N D, Strongin A Y. The structure and regulationof the human and mouse matrix metalloproteinase-21 gene and protein. Bio-chem J 2003: 372: 503–515.

25 Ahokas K, Lohi J, Illman S A et al. Matrix metalloproteinase-21 is expressedepithelially during development and in cancer and is up-regulated by trans-forming growth factor-beta1 in keratinocytes. Lab Invest 2003: 83: 1887–1899.

26 Skoog T, Ahokas K, Orsmark C, Jeskanen L, Isaka K, Saarialho-Kere U. MMP-21 is expressed by macrophages and fibroblasts in vivo and in culture. ExpDermatol 2006: 15: 775–783.

27 Grayson W, Calonje E, McKee P H. Infectious diseases of the skin. In: McKeeP, Calonje E, Granter S, eds. Pathology of the Skin. Philadelphia: ElsevierMosby, 2005, pp840–841.

28 Impola U, Jeskanen L, Ravanti L et al. Expression of matrix metalloproteinase(MMP)-7 and MMP-13 and loss of MMP-19 and p16 are associated withmalignant progression in chronic wounds. Br J Dermatol 2005: 152: 720–726.

29 Takaishi S, Wang T C. Gene expression profiling in a mouse model of Helicob-acter-induced gastric cancer. Cancer Sci 2007: 98: 284–293.

30 van Hogerlinden M, Rozell B L, Ahrlund-Richter L, Toftgard R. Squamous cellcarcinomas and increased apoptosis in skin with inhibited Rel ⁄ nuclear factor-kappaB signaling. Cancer Res 1999: 59: 3299–3303.



31 van Hogerlinden M, Rozell B L, Toftgard R, Sundberg J P. Characterization ofthe progressive skin disease and inflammatory cell infiltrate in mice with inhib-ited NF-kappaB signaling. J Invest Dermatol 2004: 123: 101–108.

32 Chung C H, Ely K, McGavran L et al. Increased epidermal growth factor recep-tor gene copy number is associated with poor prognosis in head and necksquamous cell carcinomas. J Clin Oncol 2006: 24: 4170–4176.

33 O-charoenrat P, Rhys-Evans P H, Eccles S A. Expression of matrix metallopro-teinases and their inhibitors correlates with invasion and metastasis in squa-mous cell carcinoma of the head and neck. Arch Otolaryngol Head Neck Surg2001: 127: 813–820.

34 Euvrard S, Kanitakis J, Decullier E et al. Subsequent skin cancers in kidney andheart transplant recipients after the first squamous cell carcinoma. Transplan-tation 2006: 81: 1093–1100.

35 Siren V, Salmenpera P, Kankuri E et al. Cell-cell contact activation of fibro-blasts increases the expression of matrix metalloproteinases. Ann Med 2006:38: 212–220.

36 Wilkins-Port C E, Higgins P J. Regulation of extracellular matrix remodeling fol-lowing transforming growth factor-beta1 ⁄ epidermal growth factor-stimulatedepithelial–mesenchymal transition in human premalignant keratinocytes. CellsTissues Organs 2007: 185: 116–122.

37 Boyd S, Tolvanen K, Virolainen S, Kuivanen T, Kyllonen L, Saarialho-Kere U.Differential expression of stromal MMP-1, MMP-9 and TIMP-1 in basal cell car-cinomas of immunosuppressed patients and controls. Virchows Arch 2008:452: 83–90.

38 De Angelis T, Noe A, Chatterjee M, Mulholland J. Stromelysin-1 activation cor-relates with invasiveness in squamous cell carcinoma. J Invest Dermatol 2002:118: 759–766.

39 Choi P, Chen C. Genetic expression profiles and biologic pathway alterationsin head and neck squamous cell carcinoma. Cancer 2005: 104: 1113–1128.

40 Haider A S, Peters S B, Kaporis H et al. Genomic analysis defines a cancer-specific gene expression signature for human squamous cell carcinoma anddistinguishes malignant hyperproliferation from benign hyperplasia. J InvestDermatol 2006: 126: 869–881.

41 Ye H, Yu T, Tenam S et al. Transcriptomic dissection of tongue squamous cellcarcinoma. BMC Genomics 2008: 9: 1–11.

42 Mathew R, Khanna R, Kumar R, Mathur M, Shukla N K, Ralhan R. Stromely-sin-2 overexpression in human esophageal squamous cell carcinoma: potentialclinical implications. Cancer Detect Prev 2002: 26: 222–228.

43 Bister V, Skoog T, Virolainen S, Kiviluoto T, Puolakkainen P, Saarialho-Kere U.Increased expression of matrix metalloproteinases-21 and -26 and TIMP-4 inpancreatic adenocarcinoma. Mod Pathol 2007: 20: 1128–1140.

44 Skoog T, Elomaa O, Pasonen-Seppanen S M et al. Matrix metalloproteinase-21expression is associated with keratinocyte differentiation and upregulated byretinoic acid in HaCaT cells. J Invest Dermatol 2009: 129: 119–130.

45 Ahokas K, Karjalainen-Lindsberg M L, Sihvo E, Isaka K, Salo J, Saarialho-KereU. Matrix metalloproteinases 21 and 26 are differentially expressed in esopha-geal squamous cell cancer. Tumour Biol 2006: 27: 133–141.

46 Lind M H, Rozell B, Wallin R P et al. Tumor necrosis factor receptor 1-medi-ated signaling is required for skin cancer development induced by NF-kappaBinhibition. Proc Natl Acad Sci USA 2004: 101: 4972–4977.

47 van Kempen L C, Rhee J S, Dehne K, Lee J, Edwards D R, Coussens L M. Epi-thelial carcinogenesis: dynamic interplay between neoplastic cells and theirmicroenvironment. Differentiation 2002: 70: 610–623.

48 Page-McCaw A, Ewald A J, Werb Z. Matrix metalloproteinases and the regula-tion of tissue remodelling. Nat Rev Mol Cell Biol 2007: 8: 221–233.

49 Yarosh D B, Pena A V, Nay S L, Canning M T, Brown D A. Calcineurin inhibi-tors decrease DNA repair and apoptosis in human keratinocytes followingultraviolet B irradiation. J Invest Dermatol 2005: 125: 1020–1025.

50 Tiu J, Li H, Rassekh C, van der Sloot P, Kovach R, Zhang P. Molecular basis ofposttransplant squamous cell carcinoma: the potential role of cyclosporine a incarcinogenesis. Laryngoscope 2006: 116: 762–769.

51 Madlener M, Mauch C, Conca W, Brauchle M, Parks W C, Werner S. Regula-tion of the expression of stromelysin-2 by growth factors in keratinocytes:implications for normal and impaired wound healing. Biochem J 1996: 320 (Pt2): 659–664.

52 Dazard J E, Gal H, Amariglio N, Rechavi G, Domany E, Givol D. Genome-widecomparison of human keratinocyte and squamous cell carcinoma responses toUVB irradiation: implications for skin and epithelial cancer. Oncogene 2003:22: 2993–3006.

53 Ramos M C, Steinbrenner H, Stuhlmann D, Sies H, Brenneisen P. Induction ofMMP-10 and MMP-1 in a squamous cell carcinoma cell line by ultraviolet radi-ation. Biol Chem 2004: 385: 75–86.

54 Saarialho-Kere U, Kerkela E, Jeskanen L et al. Accumulation of matrilysin(MMP-7) and macrophage metalloelastase (MMP-12) in actinic damage. JInvest Dermatol 1999: 113: 664–672.

55 Fogarty G B, Conus N M, Chu J, McArthur G. Characterization of the expres-sion and activation of the epidermal growth factor receptor in squamous cellcarcinoma of the skin. Br J Dermatol 2007: 156: 92–98.

56 Ding Y, Shimada Y, Gorrin-Rivas M J et al. Clinicopathological significance ofhuman macrophage metalloelastase expression in esophageal squamous cellcarcinoma. Oncology 2002: 63: 378–384.

Boyd et al.


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MMP-10 (Stromelysin-2) and MMP-21 in human and murine squamous cell cancer