Moogk et al. Journal of Translational Medicine 2014, 12:342
http://www.translational-medicine.com/content/12/1/342
RESEARCH
Open Access
Melanoma expression of matrix
metalloproteinase-23 is associated with
blunted tumor immunity and poor responses
to immunotherapy
Duane Moogk1,2, Ines Pires da Silva3,4,5,6, Michelle W Ma3,4, Erica B Friedman4,7, Eleazar Vega-Saenz de Miera3,4,
Farbod Darvishian2,4, Patrick Scanlon3,4, Arianne Perez-Garcia1,2, Anna C Pavlick1,3,4,8, Nina Bhardwaj1,4,8,
Paul J Christos9, Iman Osman1,3,4 and Michelle Krogsgaard1,2,4*
Abstract
Background: Matrix metalloproteinase-23 (MMP-23) can block the voltage-gated potassium channel Kv1.3, whose
function is important for sustained Ca2+ signaling during T cell activation. MMP-23 may also alter T cell activity and
phenotype through cleavage of proteins affecting cytokine and chemokine signaling. We therefore tested the
hypothesis that MMP-23 can negatively regulate the anti-tumor T cell response in human melanoma.
Methods: We characterized MMP-23 expression in primary melanoma patients who received adjuvant immunotherapy.
We examined the association of MMP-23 with the anti-tumor immune response - as assessed by the prevalence of
tumor-infiltrating lymphocytes and Foxp3+ regulatory T cells. Further, we examined the association between MMP-23
expression and response to immunotherapy. Considering also an in trans mechanism, we examined the association
of melanoma MMP-23 and melanoma Kv1.3 expression.
Results: Our data revealed an inverse association between primary melanoma MMP-23 expression and the anti-tumor
T cell response, as demonstrated by decreased tumor-infiltrating lymphocytes (TIL) (P = 0.05), in particular brisk TILs
(P = 0.04), and a trend towards an increased proportion of immunosuppressive Foxp3+ regulatory T cells (P = 0.07).
High melanoma MMP-23 expression is also associated with recurrence in patients treated with immune biologics
(P = 0.037) but not in those treated with vaccines (P = 0.64). Further, high melanoma MMP-23 expression is associated
with shorter periods of progression-free survival for patients receiving immune biologics (P = 0.025). On the other hand,
there is no relationship between melanoma MMP-23 and melanoma Kv1.3 expression (P = 0.27).
Conclusions: Our data support a role for MMP-23 as a potential immunosuppressive target in melanoma, as well as a
possible biomarker for informing melanoma immunotherapies.
Keywords: Matrix metalloproteinase-23, Melanoma, Immunotherapy, Kv1.3, Tumor-infiltrating lymphocytes
* Correspondence: Michelle.Krogsgaard@nyumc.org
1
Perlmutter Cancer Center at NYU Langone, New York, NY, USA
2
Department of Pathology, New York University School of Medicine, New York,
NY, USA
Full list of author information is available at the end of the article
© 2014 Moogk et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Moogk et al. Journal of Translational Medicine 2014, 12:342
http://www.translational-medicine.com/content/12/1/342
Background
Melanoma is a highly immunogenic tumor [1], yet tumor
progression nevertheless occurs in immunocompetent patients, which suggests the existence of immune-regulatory
mechanisms within the tumor. Tumors can evade immunemediated destruction through the release of soluble factors
that redirect the immune response as well as via mechanisms that limit or inhibit the infiltration or the function of tumor-infiltrating lymphocytes (TILs) [1-3]. Many
therapeutic strategies for melanoma have therefore been
developed to augment anti-tumor immunity by targeting
immunosuppressive mechanisms. Treatments aimed at these
mechanisms, such as cytotoxic T lymphocyte-associated
antigen-4 (CTLA-4) and programmed cell death 1 (PD-1),
work to unrestrain pre-existing TILs from immunosuppressive checkpoints [1]. Select subsets of patients respond favorably to immune-based therapies, but given the
morbidity associated with these treatments, clinicopathological criteria are needed to better identify those patients
who could benefit and to optimize their immunotherapeutic strategy. Identification of new modulators of immune
resistance may also lead to development of anti-melanoma
therapeutics that are favorable to patients that are unresponsive to other treatments, or that may act as adjuvants
to complement existing therapies to further improve
patient outcomes.
Matrix metalloproteinases (MMPs) are a family of zincand calcium-dependent proteolytic enzymes that may be
either membrane anchored or secreted [4]. The major
function of MMPs is degradation of extracellular matrix
(ECM) components [5], which can play a role in cancer
progression by promoting tumor growth, infiltration and
angiogenesis [6]. All MMPs share the common features
of an N-terminal signal peptide that directs it to the
secretory pathway, a catalytic domain containing a zinc
ion in the active site, and a prodomain that interacts
with the active site to block enzymatic activity until its
removal [4,7]. MMPs also function to cleave non-matrix
proteins, including surface receptors, and to activate chemokines and cytokines [4] – mechanisms that have been
implicated in a number of other diseases, including arthritis, vascular disease, and Alzheimer’s disease [7,8].
MMP-23 is a membrane-anchored MMP, distinguished
from other MMPs in that its N-terminal pro-domain
(MMP-23-PD) lacks the enzymatic inhibitory sequence
and the characteristic C-terminal hemopexin domain is
replaced by an immunoglobulin-like cell adhesion molecule domain [4]. Full-length MMP-23 is found predominantly in perinuclear and endoplasmic reticulum (ER)
membranes [9,10], and a single cleavage results in removal of the MMP-23-PD, activation and secretion from
the cell [10]. Prior to cleavage, the MMP-23-PD may
interact with Kv1.3 potassium channels and regulate
their surface expression [11]. Further, MMP-23 also contains
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a toxin-like domain (MMP-23-TxD) immediately following
the catalytic domain [12], which, upon secretion of active
MMP-23, may block Kv1.3 channels on proximal cells [11].
MMP-23 therefore has the ability to interact with Kv1.3 in
two distinct mechanisms that may affect Kv1.3 membrane
expression or function.
The physiological effects of blocking Kv1.3 channels
on autoreactive T cells have been demonstrated in the
context of autoimmune diseases, including rheumatoid
arthritis, type-1 diabetes mellitus, and multiple sclerosis
[13,14], where selective blocking may reduce unwanted
autoimmune responses. Therefore, MMP-23 has the potential to affect cellular processes through in cis Kv1.3
trapping, in trans extracellular Kv1.3 blocking [11,12], or
through cleavage of proteins affecting cytokine and chemokine signaling [4]. However, it is unclear if MMP-23
expression in melanoma or other cancers can affect disease progression through these mechanisms.
The focus on tumor MMPs in cancers, including melanoma, breast, prostate, lung, and colon cancer, has generally
been on their ability to mediate microenvironmental changes
to the ECM that regulate cancer progression [15-17]. However, MMPs also play a role in the regulation of anti-tumor
immune responses [6]. For example, MMP cleavage of necrosis factor-alpha promotes NF-κβ signaling, resulting in
recruitment of immune cells [18]. Similarly, MMPs can
influence T cell phenotype - active MMP-2 induces
Th2 skewing by blocking IL-12 and inducing OX40L
on dendritic cells [19]. While not generally used as a
diagnostic marker of cancer, MMP expression has been
widely studied as a potential prognostic marker for a
number of cancers [7]. Increased tumor expression of
MMP-1, MMP-2, MMP-7, or MMP-9 in lung cancers
[20-22], and increased expression of MMP-1 and MMP-9
in breast cancer [23,24] is associated with poor patient
survival. MMPs, therefore, may be suitable as both
biomarkers for cancer prognosis and as therapeutic
targets. In melanoma, increased expression of MMP-2
is associated with high invasiveness and melanoma progression [25,26], while expression of MMP-9 was associated with metastasis [27]. However, inhibitors designed for
general targeting of MMPs have not seen clinical success,
and have lead to a number of undesirable side effects [28],
highlighting the need for the development of inhibitors
with specific targeting properties.
In this study, we characterize for the first time MMP-23
expression in human melanomas as it relates to antitumor immunity and clinical response to immunotherapy. Our data support a role for MMP-23 in blunting
the anti-tumor response. Further, our data also support
a role for MMP-23 in diminishing melanoma patient
responses to immune biologic immunotherapies. This
highlights the potential use of MMP-23 melanoma expression as a predictive biomarker for the selection of
Moogk et al. Journal of Translational Medicine 2014, 12:342
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therapeutic adjuvants and as a potential therapeutic
target.
Methods
Research design
Primary melanoma tissues obtained before the start of
immunotherapy were retrieved from patients enrolled in
the Interdisciplinary Melanoma Cooperative Group, a prospectively collected clinicopathologic-biospecimen database
at New York University Medical Center [29], between August 2002 and December 2008. Patients were treated with
immune-based therapeutics after primary resection or at
recurrence. Immunotherapies were categorized as immune
biologics (IFN-α, IL-2, GM-CSF) or vaccines (dendritic
cell-, peptide-). Informed consent was obtained from
all patients at the time of enrollment. Demographic and
clinicopathologic information collected included age at
pathological diagnosis, gender, primary tumor thickness
(mm), ulceration status, mitosis (absent vs. present), histotype, anatomic site, TILs (absent vs. present: non-brisk, brisk
as identified by characteristic lymphocytic morphology on
hematoxylin-and-eosin staining) [30], recurrence status, and
melanoma status at last follow-up (December 2010). TILs
were defined as brisk when present throughout the vertical
growth phase (i.e. large dermal aggregates of melanoma over
15–25 cells wide) or present and infiltrating across the entire
base of the vertical growth phase [30]. All research was approved by the NYU School of Medicine’s Office of Science
and Research Institutional Review Board (“Development of
an NYU Interdisciplinary Melanoma Cooperative Group:
A Clinicopathological Database”, IRB Study # i10362).
Immunohistochemical analysis
Immunohistochemistry was performed using rabbit polyclonal anti-human MMP-23 - carboxyterminal end
(ab39087, Abcam, Cambridge, MA, USA), anti-Kv1.3
(APC-002, Alomone Labs, Ltd., Jerusalem, Israel), and
mouse monoclonal anti-human Foxp3 (clone 236A/E7)
(eBioscience, San Diego, CA, USA) antibodies on formalinfixed, paraffin-embedded primary melanoma tissues to
detect MMP-23 expression by melanoma cells, Kv1.3
potassium channel expression on tumor cells, and Foxp3+
Tregs, respectively. In brief, after deparaffinization and rehydration, heat-induced epitope retrieval for MMP-23,
Kv1.3, and Foxp3 were performed in 0.01 M citrate buffer,
pH 6.0, in a 1,200-watt microwave oven at 100% power
for 20, 10, and 10 minutes, respectively. Sections were
cooled in tap water for 5 minutes, quenched in 0.3%
hydrogen peroxide for 30 minutes, washed with PBS, and
incubated for 30 minutes with diluted normal blocking
serum prepared from goat serum for MMP-23 and horse
serum for Foxp3 while a blocking solution containing 5%
bovine serum albumin, 0.1% sodium azide, and 5% goat
serum was used for Kv1.3. Slides were then incubated with
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each primary antibody diluted in buffer (MMP-23, 1:100;
Kv1.3, 1:50; Foxp3, 1:500) at room temperature for 1 hour
and at 4°C overnight, after which they were washed in buffer and incubated with diluted biotinylated secondary antibodies (goat anti-rabbit at 1:500 for both MMP-23 and
Kv1.3-stained sections; horse anti-mouse at 1:500 for
Foxp3-stained sections, Vector Laboratories, Burlingame,
CA, USA) for 1 hour. Avidin-biotinylated horseradish
peroxidase complexes diluted at 1:500 (ABC reagent,
Vector Laboratories) were added. MMP-23 and Kv1.3
staining were both visualized with peroxidase (ImmPACT™
NovaRED™ Peroxidase Substrate, Vector Laboratories) and
diaminobenzidine (DAB substrate kit, Vector Laboratories)
was used to visualize Foxp3 staining. Sections were washed
in distilled water, counterstained with hematoxylin, dehydrated, and then mounted with permanent media. Appropriate positive and negative controls were included with
study sections as well. Specificity of the MMP23 antibody
was shown by competition experiment with immunizing
peptide (Abcam ab41122) (Additional file 1: Figure S1).
MMP23 antibody at a dilution of 1 μg/ml was pre incubated with 2 μg/ml of immunizing peptide for 1 h at
room temperature before the application to the tissue,
as described above. MMP23 antibody was also assessed
by Western blot using 10 μg of protein extracted from
melanoma tissues, melanoma cell line, or placenta as
positive control, and probed with anti-MMP-23 antibody at 1:5000 dilution (Additional file 2: Figure S2).
An attending pathologist (F.D.) blinded to all clinical data
scored the slides for MMP-23, Foxp3, and Kv1.3 expression. Tumor MMP-23 expression was scored for staining
intensity (0 = none, 1 = faint, 2 = intense, 3 = very intense)
and distribution (0 = none, 1 = focal (<50%), 2 = diffuse
(≥50%)), which were summed to generate a composite
score for each case as illustrated in Figure 1. Foxp3 expression was scored as the absolute number of positively
stained cells with characteristic lymphocytic morphology
in a representative high-power field (0.2 mm2), and Kv1.3
expression was scored as the absence or presence of Kv1.3
staining on melanoma cells. Each representative highpower field was selected by scanning each slide at 100x to
identify the field with the highest antibody expression.
Statistical analysis
Descriptive statistics were calculated for MMP-23 expression, Kv1.3 expression, and clinicopathologic variables.
Univariate associations between MMP-23 expression and
continuous clinicopathologic variables were assessed by
the ANOVA test or Kruskal-Wallis test, as appropriate.
Univariate associations between MMP-23 expression and
categorical clinicopathologic variables (including recurrence)
were assessed by the chi-square test or Fisher’s exact test, as
appropriate. Progression-free survival was defined as
the time from immunotherapy to recurrence. Event-time
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Table 1 Demographic and clinicopathologic
characteristics of melanoma patients treated with
immunotherapy
Variable
Number of patients (n = 71)
Age at diagnosis (years)
Median (Range)
55 (21–80)
Gender
Male
40 (56.3%)
Female
31 (43.7%)
AJCC stage at diagnosis
Figure 1 Representative melanoma MMP-23 immunohistochemical
scoring. (A) Composite score = 0; (B) Composite score = 3 (intensity = 1,
distribution = 2) with faint cytoplasmic immunopositivity; (C) Composite
score = 4 (intensity = 2, distribution = 2) with intense cytoplasmic reactivity;
(D) Composite score = 5 (intensity = 3, distribution = 2) with very intense
cytoplasmic reactivity (400X).
distributions were estimated with the use of the Kaplan–
Meier method. Categories of high and low MMP-23 expression were explored in various analyses. All p-values
are two-sided with statistical significance evaluated at the
0.05 alpha level. All analyses were performed in SPSS Version 21.0 (SPSS Inc., Chicago, IL).
Results
Patient selection and treatment
Table 1 illustrates the demographic and clinicopathological characteristics of the cohort of primary melanoma
patients studied. Primary melanoma specimens acquired
prior to the initiation of immunotherapy were examined
for each of these patients (n = 71). Immunotherapy was
given after primary resection (n = 40) or at recurrence
(n = 31) (Table 1). Patients were treated with a variety of
immunotherapies, categorized as immune biologics (n = 38;
IFN-α, IL-2, GM-CSF) or vaccines (n = 33; dendritic
cell-, peptide-). Informed consent was obtained from all
patients at the time of enrollment. Median follow-up time
from the date of pathological diagnosis was 6.3 years
(range: 1–10 years). Melanoma was the cause of death
for 38/39 patients who died during follow-up.
Melanoma MMP-23 expression does not correlate with
melanoma Kv1.3 expression
Tumor-derived MMPs in melanoma and other cancers
can mediate microenvironmental changes regulating cancer progression [15-17]. Melanoma cells can express Kv1.3
[31], although the role of tumor Kv1.3 expression is unclear. However, in prostate cancer, reduced tumor cell
Kv1.3 expression is associated with poor clinical outcome
[32]. We therefore hypothesized that melanoma Kv1.3
I
5 (7.1%)
II
26 (36.6%)
III
40 (56.3%)
Primary TILs
Absent
16 (22.6%)
Present
52 (73.2%)
Non-brisk
29
Brisk
23
Unclassified
3 (4.2%)
Immunotherapy settinga
Adjuvant
40 (56.3%)
At Recurrence
31 (43.7%)
Type of immunotherapy
Immune biologic
38 (53.5%)
IFN-α
21
IL-2, IL-18
4
GM-CSF
12
Other
1
Vaccine
33 (46.5%)
Dendritic cell
11
Peptide
22
Abbreviations: AJCC American joint committee on cancer, TILs Tumor-infiltrating
lymphocytes, NOS Not otherwise specified.
a
Patients who received immunotherapy in both settings (n=6).
surface expression may be inhibited by in cis MMP-23
trapping of Kv1.3 at the ER [12]. To examine the possible
effect of melanoma MMP-23 expression on tumor Kv1.3
expression, 20 primary melanomas were evaluated for
both MMP-23, as measured by a composite score of
MMP-23 staining intensity and distribution (Figure 1),
and Kv1.3 expression. Kv1.3 expression was absent in
10/15 (67%) primary tumors that had high MMP-23
expression as well as in 5/5 (100%) primary melanomas
that had absent or low MMP-23 expression (P = 0.27)
(Table 2). Our data therefore suggest that tumor MMP-23
expression does not directly affect tumor Kv1.3 expression
as would occur via an in cis mechanism. Our data did not
demonstrate an association between tumor Kv1.3 expression
and clinical outcome as assessed by recurrence following
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Table 2 MMP-23 expression and recurrence according to Kv1.3 expression.
MMP-23 composite scorea
Recurrenceb
Kv1.3 expression
0-2
3-4
Yes
No
Yes
0 (0%)
5 (33%)
5 (100%)
4 (27%)
1 (20%)
5 (100%)
No
5 (100%)
10 (67%)
15 (100%)
11 (73%)
4 (80%)
15 (100%)
Total
5 (100%)
15 (100%)
15 (100%)
5 (100%)
a
P = 0.27; bP = 0.99.
immunotherapy. Recurrence was observed in 11/15 (73%)
patients whose primary tumors were absent Kv1.3 expression, and similarly in 4/5 (80%) patients whose primary
melanomas stained positive for Kv1.3 (P = 0.99) (Table 2).
Therefore, there does not appear to be a link between
melanoma MMP-23 and Kv1.3 expression, and further,
melanoma Kv1.3 expression does not correlate with immunotherapy outcome.
Higher melanoma MMP-23 expression is associated with
a blunted anti-tumor immune response
In the absence of in cis regulation of melanoma Kv1.3,
MMP-23 also has the potential to act in trans through
cleavage of surface protein or blocking Kv1.3 channels
on nearby cells, including TILs. Blocking of Kv1.3 on T cells
can diminish the driving force for calcium influx, thereby
reducing activation-induced proliferation and motility [33].
Furthermore, prolonged inhibition of Kv1.3 can prevent T cells from receiving activation or survival signals, resulting in death due to cytokine deprivation
[14]. Cleavage of non-ECM proteins may affect cytokine and chemokine signaling, and has the potential to
skew T cell phenotype [18,19]. We therefore considered that melanoma MMP-23 expression could have a
role in regulating anti-tumor immune responses.
To evaluate the relationship between primary melanoma
MMP-23 expression and anti-tumor immunity, we compared melanoma MMP-23 expression with two separate
measures of the strength of the intrinsic anti-tumor immune response – the presence and the degree of TIL
infiltration [30] – at the time of primary resection (Figure 2).
We observed that increased melanoma MMP-23 expression is inversely associated with the presence of TILs
(presence = 79.4% for melanomas with low MMP-23
expression and 53.8% for melanomas with high MMP23 expression, P = 0.05). Quantification of the intensity
of lymphocytic infiltration also showed a significant inverse correlation between MMP-23 expression and a
brisk lymphocytic response (brisk TILs = 65.0% for melanomas with low MMP-23 expression and 25.0% for melanomas with high MMP-23 expression, P = 0.04). These
results suggest a role for tumor-derived MMP-23 in the
suppression of anti-tumor immune responses.
In addition to tumor-specific T-lymphocytes, the TIL
population is also comprised of immunosuppressive Foxp3+
regulatory T cells (Tregs) that play an important role in
immune evasion [34,35]. The accumulation of Tregs in
the tumor microenvironment can be attributed to a
number of factors, including the local expression and
secretion of factors affecting Treg migration and retention, the expansion of naturally occurring Tregs , or the
de novo generation of induced Tregs [36]. To investigate
the potential role of melanoma MMP-23 in contributing
to conditions favorable to Tregs, we assessed the relationship between MMP-23 expression and Treg prevalence, as
determined by the number of Foxp3+ cells (Figure 3). We
observed a trend towards an increased number of Tregs
in primary melanomas with higher MMP-23 expression
(53.1 ± 33.8 in melanomas with high MMP-23 expression and 35.0 ± 25.1 in melanomas with low MMP-23,
P = 0.07). This suggests a potential role for MMP-23 in
skewing TIL phenotype. Combined, these results suggest that MMP-23 plays a role in blunting the immune
response to melanoma as it affects the prevalence, distribution, and composition of TILs in favor of tumor
immune evasion.
Figure 2 Intensity of lymphocytic infiltration decreases with
increased melanoma MMP-23 expression. Hematoxylin-andeosin-stained primary melanoma specimens showing a representative
brisk (A) and absent (C) lymphocytic infiltrate and the corresponding
consecutive MMP-23-stained sections (B and D, respectively) with
MMP-23 composite scores of 2 and 4, respectively (80X).
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Figure 3 Foxp3+ Tregs in the primary tumor increases with
higher melanoma MMP-23 expression. (A) Heterogeneous
nuclear positivity for Foxp3 in TILs from a primary melanoma with a
high MMP-23 composite score and 100 Foxp3+ Tregs/high-power
field. (C) Nuclear reactivity for Foxp3 limited to a few TILs (5 Foxp3+
Tregs/high-power field) (arrow) from a primary melanoma with no
MMP-23 expression (200X). (B, D) Corresponding consecutive
MMP-23-stained sections of A and C, respectively.
Higher MMP-23 expression is associated with resistance
to immune biologic immunotherapy
To further evaluate the potential role of MMP-23 in tumor
immune escape, we examined the relationship between primary melanoma MMP-23 expression and clinical response
to immunotherapy, as measured by the rate of recurrence.
Considering the entire cohort of patients – receiving
immunotherapy after primary resection or at recurrence –
subsequent recurrence was detected in 11/19 (59%)
patients with low melanoma MMP-23 expression (composite score = 0–2) compared with 42/52 (81%) patients
high melanoma MMP-23 expression (composite score =
3–4) (P = 0.067) (Table 3). This trend holds when considering only patients who had received immunotherapy after
primary resection (vaccines (n = 21), immune biologics
(n = 19)), where the recurrence rate was lower in patients
Table 3 Analysis of melanoma recurrence according to
MMP-23 expression in the entire cohort and the subset
of patients who received immunotherapy after primary
resection
MMP-23 composite score
Entire cohorta
Immunotherapy after
primary resectionb
0-2
0-2
3-4
Table 4 Stratified analysis of melanoma recurrence risk
by type of immunotherapy (immune biologics and
vaccines) in the patients who had primary adjuvant
immunotherapy
MMP-23 composite score
3-4
Recurrence
Yes
with low melanoma MMP-23 expression (5/11; 46%) compared with patients with high melanoma MMP-23 expression (23/29; 79%) (P = 0.056) (Table 3). These trends suggest
that MMP-23 may play a role in regulating immune
responses in the context of immunotherapy.
Different classes of immunotherapies target different
components of the anti-tumor immune response. Vaccines
target CD8+CCR7+ central memory T cells (TCM) and
immune biologics target CD8+CCR7− effector memory
T cells (TEM) [37-39]. Therefore, we evaluated the clinical outcomes of primary melanoma patients treated
with vaccines and immune biologics separately to determine if the observed relationship between tumor
MMP-23 expression and recurrence could be attributed to an effect on a specific T cell subset (Table 4).
Considering all patients who received vaccine therapy
(after primary resection or at recurrence), no significant difference in recurrence was detected between patients with low melanoma MMP-23 expression and
those with high melanoma MMP-23 expression. This
is also true when considering all patients who received
immune biologics (after primary resection or at recurrence). However, when we focused our analysis on patients treated with immune biologics only after primary
resection, and therefore as primary adjuvant therapy,
higher primary melanoma MMP-23 expression was associated with increased recurrence, as recurrence was
detected in 1/4 (25%) patients with low melanoma
MMP-23 expression, compared with 13/15 (87%) patients
with high melanoma MMP-23 expression (P = 0.037). In
contrast, the level of tumor expression of MMP-23 in patients treated with vaccine immunotherapies at primary
resection was not associated with recurrence (P = 0.64).
To further investigate the affect of MMP-23 on patients receiving immune biologics, we next compared
the progression free survival between patients with high
versus low melanoma MMP-23 expression (Figure 4).
These data revealed that high melanoma MMP-23 expression is associated with shorter periods of progression-free
survival (P = 0.025). Together, these results suggest that,
Immune biologicsa
Vaccinesb
0-2
3-4
0-2
3-4
Recurrence
11 (59%)
42 (81%)
5 (46%)
23 (79%)
Yes
1 (25%)
13 (87%)
4 (57%)
10 (71%)
No
8 (41%)
10 (19%)
6 (54%)
6 (21%)
No
3 (75%)
2 (23%)
3 (43%)
4 (29%)
Total
19 (100%)
52 (100%)
11 (100%)
29 (100%)
Total
4 (100%)
15 (100%)
7 (100%)
14 (100%)
a
b
P = 0.067; P = 0.056.
a
b
P = 0.037; P = 0.64.
Moogk et al. Journal of Translational Medicine 2014, 12:342
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Figure 4 Progression-free survival of melanoma patients
receiving immune biologics. Patients treated with immune
biologics were monitored for recurrence from the time treatment,
and grouped based on expression of MMP-23 prior to initiation of
treatment (dashed line – MMP-23 composite score = 0–2; solid
line – MMP-23 composite score = 3–4). P = 0.025.
in patients treated with immune biologics, high MMP-23
expression levels augment the anti-tumor immune response to both increase the likelihood of recurrence and
shorten the time to recurrence. Although statistically
significant, we acknowledge that larger sample sizes
are required to test these results with greater statistical
rigor. These results suggest that high tumor expression
of MMP-23 confers a level of resistance to immune
biological therapy when given as a primary adjuvant. Interestingly, tumor MMP-23 seems to affect specifically TEM
cells, as immune biologics including IFN-α, IL-2, and
GM-CSF function by targeting TEM expansion.
Discussion
We examined the expression of MMP-23 in melanoma
and our data suggest that MMP-23 represents an immune
escape mechanism and potential immunotherapeutic
target. Our data show that melanoma MMP-23 expression
correlates with a diminished anti-tumor T cell response
and higher numbers of Tregs in the TIL population. These
data together support a role for tumor MMP-23 in modulating both the infiltration and activation of tumor-reactive
lymphocytes.
Anti-tumor T cells comprise the majority of the tumor
lymphocytic infiltrate, and our study shows that the number, intensity of infiltration, and composition of TILs are
negatively affected by melanoma MMP-23 expression. Increased tumor expression of MMP-23, therefore, mediates
intrinsic tolerance with the potential to confer resistance
against immunotherapy. As such, an evaluation of melanoma MMP-23 expression may be particularly useful in
selecting candidates for adoptive T cell therapy, the success of which depends on the harvest, expansion and
immunophenotype of TILs [40]. Furthermore, our results
suggest the clinical relevance of assessing primary melanoma MMP-23 expression prior to initiating adjuvant treatment with immune biologics, as melanoma MMP-23
Page 7 of 10
expression negatively correlates with response to adjuvant
immune biologic therapy. Immune biologics, such as IFN-α,
IL-2, and GM-CSF, preferentially expand TEM cells, whereas
vaccines induce TCM responses. The dependence of immune
biologics but not vaccines on MMP-23 expression, suggests
that MMP-23 specifically affects anti-tumor TEM responses.
When activated, TEM express an increased number of Kv1.3
channels [37,38] and sustained and complete activation is
dependent of their function. On the other hand, TCM
are characterized by low Kv1.3 expression and are not
dependent on Kv1.3 function for activation [14]. Therefore, MMP-23 may inhibit anti-tumor TEM responses in
adjuvant immune biologic therapies through in trans
blocking of TEM Kv1.3 channels. This could explain
the lack of an observed association between melanoma
MMP-23 expression and recurrence among patients
treated with adjuvant vaccine therapy. Patient stratification based on MMP-23 expression, type of adjuvant
therapy, and timing of treatment (i.e. at the time of
primary resection) significantly reduced the number of patients in specific groups. The statistical analysis of these
data, specifically patients with low MMP-23 expression
receiving immune biologic therapy at primary resection
(n = 4), is presented with the acknowledgement that the
conclusions drawn from these data would benefit from
larger sample sizes.
The observed trend toward an increased number of
Tregs within tumors that expressed high levels of MMP23 suggests that this may contribute further to suppression of anti-tumor T cell responses. A number of studies
have reported a correlation between Treg infiltration and
patient prognosis, where high percentages of Tregs in
both primary melanomas and lymph node metastases
correlated with increased recurrence and decreased survival rates [41-43], although this correlation was not observed in other studies [44,45]. Treg accumulation in the
tumor may be driven by a number of factors, including
local chemokine and integrin-ligand expression [46], and
immunosuppressive factors promoting expansion of existing Tregs or generation of induced Tregs [47]. How increased
MMP-23 expression might function to increase the tumor
Treg population is unclear. Recent work by Godefroy and
colleagues [19] showed that MMP-2 in melanoma degrades
type I IFN receptor, effectively preventing IL-12p35 production, and skews CD4 T cells towards a Th2 phenotype.
It is possible that MMP-23 activity alters signaling pathways controlling Treg expansion or de novo production of
induced Tregs. A Kv1.3 knock-out induced EAE mouse
model of infection showed that CD4+ T cells expressed
greater levels of IL-10 and lower levels of IL-17 and IFN-γ,
and suppressed proliferation of wild-type CD4+ T cells,
suggesting a skewing toward a regulatory phenotype [48],
although an increase in the number of Foxp3+ cells was
not observed.
Moogk et al. Journal of Translational Medicine 2014, 12:342
http://www.translational-medicine.com/content/12/1/342
While our data suggest a role for melanoma MMP-23
in blunting anti-tumor immunity by selectively blocking
T cell Kv1.3 channels, we also explored the possibility
that melanoma MMP-23 may act in cis by trapping
Kv1.3 in the ER and preventing surface expression. Of
the primary melanomas evaluated for Kv1.3 surface
expression, most were negative, which may reflect the
suppression of Kv1.3 surface expression due to in cis
MMP-23 trapping [12] or an absence of Kv1.3 expression altogether. However, for our limited sample size, we
did not observe a relationship between MMP-23 expression and tumor Kv1.3 surface staining. Kv1.3 channels on
melanoma cells, however, have previously been shown to
be in close proximity to β1-integrins, such that blockade
of Kv1.3 channels dysregulates integrin function and results in loss of cell adherence [31]. Disruption of cell-cell/
cell-matrix adhesion is one of many steps in metastatic
progression, and evidence from prostate cancer support
the association between reduced tumor cell Kv1.3 expression and poor clinical outcome [32]. Studies in breast and
colon cancer, in contrast, suggest that blockade of tumor
cell Kv1.3 expression is protective [49,50] as it prevents
progression through the G1/S checkpoint, which requires
transient hyperpolarization [51]. In trapping Kv1.3 in the
ER, melanoma MMP-23 would likewise alter the tumor
cell membrane potential and facilitate the transition to the
M phase. Further studies are warranted to explore the
possible role of MMP-23 in tumor Kv1.3 suppression.
Inhibition of MMP-23 in combination with other immunotherapies may further augment anti-tumor immunity. With the clinical success of monoclonal antibodies
against inhibitory immune checkpoint proteins in melanoma, including CTLA-4 [52] and PD-1 [53], anti-MMP23 therapy also holds promise as a potential treatment
strategy. Yet, such strategies must consider the possibility
of off-target effects; thus the normal tissue distribution
and physiologic role of MMP-23 must first be understood.
Unlike other MMPs, MMP-23 is widely expressed under
physiologic conditions, in particular at high levels in the
ovary, testis, prostate, and heart and at low levels in the
lung, pancreas, and colon [10,54]. Intralesional delivery of
MMP-23 inhibitors may therefore be preferred over systemic injection to minimize possible off-target effects.
Furthermore, locoregional MMP-23 inhibition has the
potential to alter both the number and the composition
of the TILs, such that adoptive T cell therapy candidates
could benefit from pre-treatment with intralesional MMP23 inhibitors.
In identifying MMP-23 as a novel immunotherapeutic
target, it is important to recognize that inhibition of
MMP-23 may result in immune-related adverse events,
much like the inhibition of CTLA-4 [55]. Ongoing efforts to identify biomarkers to predict the response to
anti-CTLA-4 therapy are under way just as additional
Page 8 of 10
studies to identify those patients most likely to benefit
from MMP-23 inhibition are needed in parallel to those
of the role of MMP-23 in melanoma. Our study provides
the foundation for both as data suggest that an assessment
of melanoma MMP-23 expression by routine immunohistochemistry may prove useful, much like using immunohistochemistry to determine the estrogen/progesterone
receptor status in breast cancer [56]. Yet, the clinical
value of assessing melanoma MMP-23 expression in conjunction with Kv1.3 expression remains to be determined.
Conclusions
Our study suggests that MMP-23 may play a role in
tumor-induced immune escape and that melanoma MMP23 expression represents a novel biomarker and possible
immunotherapeutic target. MMP-23 inhibition in combination with other therapeutic agents may be more effective
than as monotherapy, yet studies are still needed to elucidate the exact mechanism by which melanomas upregulate
MMP-23 to allow the development of a specific MMP-23
inhibitor with a favorable risk-benefit profile.
Additional files
Additional file 1: Figure S1. Demonstration of MMP-23 antibody specificity
by competition with immunizing peptide. Immunostaining by MMP-23 primary
antibody plus secondary antibody (A), preincubated with immunizing peptide
before addition of primary and secondary antibodies (B), and with secondary
antibody only in the absence of primary MMP-23 antibody (C).
Additional file 2: Figure S2. Western blot demonstrating detection of
MMP23 expression using MMP-23 antibody. (A) MMP-23 Western blots
using different MMP-23-specific antibodies to probe 10 μg of placenta
protein per lane: Lane A) - ab39087, immunogen C terminus of MMP-23,
diluted 1:5000; Lane B) ab74215 immunogen peptide derived from the C
terminus of human MMP-23 protein, diluted 1:1000; Lane C) ab39086,
immunogen peptide corresponding to the hinge region of human
MMP-23, diluted 1:5000; and Lane D) ab 53148, immunogen Synthetic
peptide derived from human MMP-23, diluted 1:1000. (B) 10 μg of
protein was extracted from melanoma tissues (Lanes A-D), melanoma
cell line (Lane E) or placenta (Lane F) and assessed by Western blot
using ant-MMP-23 antibody, ab39087.
Abbreviations
MMP-23: Matrix metalloproteinase-23; MMPs: Matrix metalloproteinases;
MMP-23-PD: MMP-23 pro-domain; MMP-23-TxD: MMP-23 toxin-like domain;
TIL: Tumor infiltrating lymphocytes; CTLA-4: Cytotoxic T lymphocyte-associated
antigen-4; PD-1: programmed cell death-1; ECM: Extracellular matrix;
ER: Endoplasmic reticulum; T CM: Central memory T cell; TEM: Effector
memory T cell.
Competing interests
The authors have no competing interests to declare. A patent application
pertaining to cancer treatment methods and the role of MMP-23 as a
therapeutic target (PCT/US13/39022) has been filed by M.K. and I.O.
Authors’ contributions
Conception and design: DM, MWM, AP, IO, MK; Development of
methodology: DM, MWM, AP, EBF, EVM, FD, PJC, IO, MK; Acquisition of data:
DM, MWM, EBF, EVM, FD, PS, AP, ACP, NB, IO, MK; Analysis and interpretation
of data: DM, MWM, IS, EBF, EVM, NB, PJC, IO, MK; Writing, review, and/or
revision of the manuscript: DM, MWM, IS, EBF, EVM, FD, PS, AP, ACP, NB, PJC,
IO, MK; Administrative, technical, or material support: PS, IO, MK; Study
Moogk et al. Journal of Translational Medicine 2014, 12:342
http://www.translational-medicine.com/content/12/1/342
Page 9 of 10
supervision: IO, MK; Review of pathologic samples: FD All authors read and
approved the final manuscript.
13.
Acknowledgements
We thank Dr. Edward Skolnik and Dr. Stefan Feske for their critical reading of
the manuscript. M.K. was a Pew Scholar in the Biomedical Sciences
supported by the Pew Trust. This work was supported by the National
Institute of Health grants NCI 1U01CA137070 and NIGMS 5R01GM085586 (to
M.K.), an American Cancer Society Research Scholar grant RSG-09-070-01-LIB
(to M.K), a Cancer Research Investigator grant from the Cancer Research
Institute (CRI) (to M.K.) and the NYU Cancer Institute, the NYU Cancer
Institute Cancer Center Support Grant 5P30CA016087-27 (to I.O.), and
the Marc Jacobs campaign to support the Interdisciplinary Melanoma
Cooperative Group.
Author details
1
Perlmutter Cancer Center at NYU Langone, New York, NY, USA. 2Department
of Pathology, New York University School of Medicine, New York, NY, USA.
3
Ronald O. Perelman Department of Dermatology, New York University School
of Medicine, New York, NY, USA. 4Interdisciplinary Melanoma Cooperative
Group, New York University School of Medicine, New York, NY, USA. 5Instituto
Português de Oncologia de Lisboa Francisco Gentil, Lisboa, Portugal.
6
Programme for Advanced Medical Education, Lisbon, Portugal. 7Department of
Surgery, New York University School of Medicine, New York, NY, USA.
8
Department of Medicine, New York University School of Medicine, New York,
NY, USA. 9Division of Biostatistics and Epidemiology, Weill Cornell Medical
College, New York, NY, USA.
Received: 24 July 2014 Accepted: 24 November 2014
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Cite this article as: Moogk et al.: Melanoma expression of matrix
metalloproteinase-23 is associated with blunted tumor immunity
and poor responses to immunotherapy. Journal of Translational
Medicine 2014 12:342.
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