See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/305361283

Translational significance of Nodal, Cripto-1 and Notch4 in adult nevi

Article in Oncology Letters · June 2016

DOI: 10.3892/ol.2016.4755

CITATIONS READS

9 130

9 authors, including:

Naira V Margaryan Paul Harms

West Virginia University Research Corporation (WVURC) University of Michigan
82 PUBLICATIONS 3,334 CITATIONS 221 PUBLICATIONS 5,627 CITATIONS

SEE PROFILE SEE PROFILE

Gabriele Madonna Gerardo Botti

Istituto Nazionale Tumori "Fondazione Pascale" Istituto Nazionale Tumori "Fondazione Pascale"
98 PUBLICATIONS 1,876 CITATIONS 776 PUBLICATIONS 21,542 CITATIONS

SEE PROFILE SEE PROFILE

All content following this page was uploaded by Gabriele Madonna on 07 September 2016.

The user has requested enhancement of the downloaded file.

 ONCOLOGY LETTERS 12: 1349-1354, 2016

Translational significance of
Nodal, Cripto-1 and Notch4 in adult nevi
LUIGI STRIZZI1‑3, NAIRA V. MARGARYAN1, PEDRAM GERAMI4,5, ZAHRA HAGHIGHAT4,5, PAUL W. HARMS6,
GABRIELE MADONNA7, GERARDO BOTTI8, PAOLO A. ASCIERTO7 and MARY J.C. HENDRIX1,3,5

1
Cancer Biology and Epigenomics Program, Stanley Manne Children's Research Institute, Ann & Robert H. Lurie
Children's Hospital of Chicago; 2Department of Pathology; 3Robert H. Lurie Comprehensive Cancer Center;
4
Department of Dermatology, Northwestern University Feinberg School of Medicine; 5Northwestern University Feinberg
School of Medicine, Chicago, IL 60611, USA; 6Department of Pathology, University of Michigan Medical School, Ann Arbor,
MI 48109, USA; 7Melanoma, Cancer Immunotherapy and Innovative Therapy Unit; 8Department of Pathology,
Istituto Nazionale Tumori Fondazione Pascale, 80131 Napoli, Italy

Received November 2, 2015; Accepted May 24, 2016

DOI: 10.3892/ol.2016.4755

Abstract. The TGF‑β associated growth factor Nodal is highly Introduction

expressed in aggressive metastatic melanoma. Determining
the risk for melanomagenesis from Nodal expression in nevi Previous work has determined that aggressive melanomas
prior to the development of melanoma may be useful for both express Nodal, an embryonic morphogen belonging to the
the screening and prevention of melanoma. Tissue sections of TGF‑ β family of growth factors (1). It was demonstrated
human adult nevi with or without a history of melanoma were that Nodal expression increases during the progression of
stained by immunohistochemistry (IHC) for Nodal, the Nodal melanoma, from superficial, radial growth phase to deeper,
co‑receptor Cripto‑1, and Notch4, which have previously been vertical growth phase to advanced stage, metastatic mela-
shown to be associated with Nodal expression in melanoma. noma (1). A subsequent report confirmed initial observations
The degree of Nodal, Cripto‑1 and Notch4 staining was scored of lower Nodal expression in superficial melanoma compared
and correlated with available clinical data. Median IHC scores to robust expression in deep melanoma and metastatic mela-
for Nodal, Cripto‑1 and Notch4 expression were significantly noma (2). Furthermore, it has been demonstrated that normal
higher in nevi removed from patients who eventually developed and dysplastic nevi expressed lower levels of Nodal (2). A
melanoma compared with nevi from patients with no history well‑known risk factor for melanoma is the number and types
of melanoma. In addition, the degree of Nodal expression in of melanocytic nevi, indicating that these may represent
nevi from patients who eventually developed melanoma corre- precursor lesions to melanoma (3). In fact, patients diagnosed
lated significantly with the Breslow depth of the melanoma. with familial dysplastic nevi syndrome who exhibit a large
Expression of Nodal and components of its signaling pathway number of melanocytic nevi, sometimes >50, have shown an
in nevi may represent a biomarker for selecting a unique subset increased risk for developing melanoma (4). It is desirable,
of patients requiring increased surveillance for screening and therefore, to characterize the molecular profile of excised
prevention of melanoma. nevi and determine unique expression pattern(s), which may
facilitate screening of patients with an increased likelihood for
developing melanoma.
Since Nodal is a secreted molecule, it may have the
potential for paracrine effects by influencing the homeostasis
of cells that surround or are distant to the secretory source
of this powerful morphogen. This is in agreement with the
Correspondence to: Dr Luigi Strizzi or Dr Mary J.C. Hendrix, phenomenon known as ‘field cancerization’ whereby abnor-
Cancer Biology and Epigenomics Program, Stanley Manne
malities, such as genetic or epigenetic alterations or changes
Children's Research Institute, Ann & Robert H. Lurie Children's
in growth factor and receptor expression profiles, have been
Hospital of Chicago, Northwestern University Feinberg School of
Medicine, 225 East Chicago Ave, Box 222, Chicago, IL 60611, USA documented in histologically normal tissues distant from
E‑mail: lstrizzi@luriechildrens.org areas of overt cancer (5). Given the positive ‘feed‑forward’
E‑mail: m‑hendrix@northwestern.edu inductive characteristics of Nodal signaling (6), we hypoth-
esize that Nodal secreted from a melanoma induce its own
Key words: Nodal, Cripto‑1, Notch4, immunohistochemistry, expression in otherwise nonmalignant nevi located in regional
nevus, biomarker, screening, melanoma or distant sites causing phenotypic changes in these benign
lesions, which may lead to increased propensity of these
lesions towards malignant transformation. In this retrospective
1350 STRIZZI et al: NODAL PATHWAY IN ADULT NEVI

study, the immunohistochemical expression patterns of Nodal

were determined, together with Cripto‑1, the co‑receptor for
Nodal (7), and Notch4, a stem cell‑associated molecule with
two RBPJ binding sites on the Nodal gene shown to be associ-
ated with Nodal expression in melanoma (8). These findings
were correlated with available clinical data, including history
of subsequent melanoma development.

Materials and methods

De‑identified slides with tissue sections of human malignant

and nonmalignant melanocytic lesions for immunohistochem-
istry (IHC) were collected in compliance with Institutional
Review Board ethics committee approval of study design and
protocols for patient consent from Lurie Children's Hospital,
Northwestern University, Chicago, IL, USA (approval
no. 2010‑14126), University of Michigan, Ann Arbor, MI,
USA (approval no. HUM00045834) and Istituto Nazionale
Tumori (INT) Fondazione Pascale, Naples, Italy (approval
no. DMTMT‑OMTI‑34‑2011). Slides from a total of 225 indi-
vidual cases were collected from the following sources:
University of Michigan (nonmalignant negative history of
melanoma, n=12; nonmalignant positive history of mela-
noma, n=16; malignant melanoma, N=33); INT Fondazione
Pascale (nonmalignant negative history of melanoma, n=65);
histological slides purchased from Northwestern University
Department of Dermatology Tissue Core (nonmalignant nega-
tive history of melanoma n=50; nonmalignant positive history
of melanoma, n=49). The de‑identified slides were stained by
IHC for Nodal, Cripto‑1 and Notch4, following the procedure
previously described (8). Briefly, following antigen retrieval and
blocking steps, sections were incubated in primary antibody
for 60 min [mouse monoclonal anti‑Nodal (1:200 dilution;
cat no. ab55676; Abcam, Cambridge, MA, USA); rabbit
polyclonal anti‑Notch4 (1:80 dilution; cat no. sc‑5594; Santa
Cruz Biotechnology, Inc., Dallas, TX, USA); rabbit polyclonal
anti‑Cripto‑1 (1:1500 dilution; cat no. 600‑401‑997; Rockland
Scientific International Inc., Limerick, PA, USA)], followed
by ready‑to‑use anti‑rabbit (cat no. GR602H) or anti‑mouse
(cat no. GM601H) biotinylated secondary antibody (Biocare
Medical Inc., Concord, CA, USA), and then streptavidin‑horse- Figure 1. Immunohistochemistry results. Representative images of immuno-
radish peroxidase (ThermoFisher Scientific Inc., Waltham, histochemistry staining results showing weak and strong staining for Nodal,
MA, USA). Color was developed with 3,3'‑diaminobenzidine Cripto‑1 and Notch4 in nevi. IS, index score.
substrate (ThermoFisher Scientific Inc.) and sections were
counterstained with hematoxylin (Biocare Medical Inc.). As a
negative control, adjacent serial sections were incubated with as nevi from patients with no history of melanoma (negative
species appropriate irrelevant IgG (Jackson ImmunoResearch Hx of melanoma) or nevi from patients that subsequently
Labs) at the same concentration as primary antibodies. The developed melanoma (positive Hx of melanoma). Mean IHC
quality and intensity of staining were analyzed and scored IS were calculated for Nodal, Cripto‑1 and Notch4 and overall
at low power and high power in order to calculate an Index comparative analysis was performed to determine differences in
Score (IS), as previously described (2). Student's t‑test, median expression levels among 4 groups for each marker, as summa-
test and Pearson correlation were used for statistical analyses rized in Table I. The mean level for Nodal IS progressively
of the appropriate data. P<0.05 was considered to indicate a increased from nevi with a negative history of melanoma to nevi
statistically significant difference. with a positive history of melanoma to cutaneous melanoma to
metastatic melanoma. The level of Nodal IS was significantly
Results different among these groups (Fig. 2: all P<0.05 versus negative
history of melanoma). The same trend of progressive increase
The IHC results demonstrated a broad range of staining from nevi with a negative history of melanoma to nevi with a
intensities for Nodal, Cripto‑1 and Notch4 (Fig. 1). After IHC positive history of melanoma to cutaneous melanoma to meta-
intensity scores (IS) were assigned, cases were grouped either static melanoma was observed for Notch4 IS with a statistically
 ONCOLOGY LETTERS 12: 1349-1354, 2016 1351

Table I. Results of intensity of immunohistochemistry staining for Nodal, Cripto‑1 and Notch4, represented as mean index score,
in nevi from patients with a negative or positive history of melanoma, and cutaneous and metastatic melanoma

Gene and type of lesion Mean IS N P‑valuea

Nodal
Nevi, negative Hx of melanoma 3.14+/‑2.1 129
Nevi, positive Hx of melanoma 4.77+/‑2.45 61 <0.01
Cutaneous melanoma 5.42+/‑2.17 12 <0.01
Metastatic melanoma 7.52+/‑3.57 21 <0.01
Cripto‑1
Nevi, negative Hx of melanoma 3.38+/‑2.65 128
Nevi, positive Hx of melanoma 4.3 +/‑2.98 61 0.03
Cutaneous melanoma 2.17+/‑2.41 12 NS
Metastatic melanoma 3.57+/‑3.03 21 NS
Notch4
Nevi, negative Hx of melanoma 1.55+/‑1.96 121
Nevi, positive Hx of melanoma 2.08+/‑1.87 61 NS
Cutaneous melanoma 2.58+/‑2.71 12 NS
Metastatic melanoma 3.81+/‑3.03 21 <0.01

t‑test vs. nevi with negative Hx melanoma. IS, index score; Hx, history.
a

Figure 2. Immunohistochemistry IS. Histograms representative of mean immunohistochemistry IS for Nodal, Cripto‑1 and Notch4 staining in nevi from
patients with or without a Hx of melanoma and in cutaneous and metastatic melanoma (*P<0.05; t‑test). IS, index score; Hx, history.

significant difference for the highest mean levels of Notch4 IS other groups (Fig. 2). Since calculation of mean values can be
in the metastatic melanoma group (Fig. 2; P<0.05 metastatic distorted by outliers, the median IS was also determined among
melanoma versus negative history of melanoma). Interestingly, the different groups for each marker. In fact, this analysis shows
mean Cripto‑1 IS level was significantly greater only in the a significantly higher median IS for Nodal, Cripto‑1 and Notch4
group of nevi from patients with a positive history of melanoma in the group of nevi from patients with a positive history of
compared to the group of nevi from patients with a negative melanoma compared with the group of nevi from patients with
history of melanoma (Fig. 2; P<0.05). There was no significant no history of melanoma (Fig. 3).
difference observed in the mean Cripto‑1 IS between melanoma Pearson's correlation analysis was performed to determine
and the other groups. In addition, the cutaneous melanoma associations between Nodal, Cripto‑1 and Notch4 expression
group showed the lowest mean Cripto‑1 IS compared with the levels for each group of nevi. Nodal levels did not correlate with
1352 STRIZZI et al: NODAL PATHWAY IN ADULT NEVI

Figure 4. Correlation analyses. Linear correlation analysis between Nodal,

Cripto‑1 and Notch4 immunohistochmistry IS in nevi from patients with or
Figure 3. Median IS. Box plots representative of the distribution and median without melanoma Hx. IS, index score; Hx, history.
value of immunohistochemistry IS for Nodal, Cripto‑1 and Notch4 staining
in adult nevi (*P<0.05; median test). IS, index score; Hx, history.

Discussion

Cripto‑1 or Notch4 in either group of nevi (data not shown). The present study demonstrated that there is a wide range of
Notably, a significant positive correlation was observed for variability in the intensity of IHC staining of Nodal, Cripto‑1
Cripto‑1 and Notch4 expression levels in the group of nevi and Notch4 in nonmalignant melanocytic lesions regardless
from patients with no history of melanoma (R2= 0.041; P= 0.03; of whether or not the nevi were from patients with or without
n=121) and in the group of nevi from patients with a positive a history of melanoma, as reflected by the means of the IHC
history of melanoma (R 2= 0.141; P= 0.003; n=61) (Fig. 4). In scores calculated for the different groups analyzed. Calculating
the group of nevi from patients with a positive history of the mean from a grading system of low to high expression
melanoma, no significant correlation was observed between levels, however, can be distorted by outlier cases having
IS for Nodal, Cripto‑1 or Notch4 and patient age, time elapsed values at either end of the spectrum. Calculation of median
between surgical removal of the nevus and diagnosis of mela- levels with corresponding interquartile ranges provides an
noma, and anatomical site (data not shown). When analyzing improved representation of the distribution of IHC scores for
high Nodal expression and anatomical sites in the group of nevi individual cases. In fact, this method showed a significantly
from female patients that subsequently developed melanoma, higher median value for all three markers in the group of
the highest Nodal IS (9) for both the nevi and the subsequent nevi from patients with a history of melanoma compared with
melanomas was predominantly associated with lesions located nevi from patients with no melanoma history. However, on a
on the posterior torso (Fig. 5). In contrast, male patients showed case‑by‑case basis, no significant correlation was observed
the highest Nodal IS (9) for both the nevi and the subsequent in IS between Nodal and Cripto‑1 or Nodal and Notch4 for
melanomas in lesions located on the limbs (Fig. 5). either group of nevi. This was unexpected since Cripto‑1 is a
Finally, in patients with a positive history of melanoma, a well‑established co‑receptor for canonical Nodal signaling (7)
significant positive correlation was observed between Nodal and Notch4 has been shown to be associated with Nodal
IS in the preceding nevus and Breslow depth in the subsequent expression in aggressive melanoma (8), and thus one would
melanomas (R²= 0.1067; P= 0.02; n=48) (Fig. 6A). The same anticipate a relationship between these two protein markers.
significant positive correlation was found between Nodal IS These data may indicate that either Nodal biological effects,
and Breslow depth in the cutaneous melanoma cases (R²=0.44; assuming that nevi are responsive to Nodal, may be mediated
P=0.02; n=12) (Fig. 6B). independently of Cripto‑1 or Notch4, or that Nodal's effect may
 ONCOLOGY LETTERS 12: 1349-1354, 2016 1353

Figure 5. Anatomical site of Nevus and Nodal expression. Nodal immunohistochemistry IS shown for nevi from patients with a positive history of melanoma
and for the melanoma that subsequently developed according to anatomical sites and gender. IS, index score.

become relevant only when Cripto‑1 and Notch4 reach critical

levels. In fact, Nodal, Cripto‑1 and Notch4 have been shown
to reach significantly high expression levels in advanced stage
melanoma (8,9). Similarly, the data in the present study also
show that Nodal, Cripto‑1 and Notch4 reach higher levels in
metastatic melanoma. Notably, a significant correlation was
not observed between Cripto‑1 and Notch4 expression in
nevi regardless of the previous history of melanoma. Indeed,
an earlier study demonstrated the close association between
Cripto‑1 and all 4 Notch proteins by describing the role for
Cripto‑1 in facilitating the post‑translational maturation
of Notch receptors (10). Perhaps this maturation process
must occur in these nevi for Nodal to exert a more effective
biological role. Further studies are required to determine the
combined roles of Cripto‑1 and Notch4 during melanomagen-
esis and how this may relate to Nodal expression and function
in melanoma.
Since the nevi in the present study were surgically excised,
a direct association between the high levels of Nodal, Cripto‑1
and Notch4 and potential melanomagenesis in the excised
nevus cannot be experimentally established. Nevertheless, a
proportion of these patients eventually developed a melanoma
following excision of the nevus, i.e. in the group of patients
with a positive history of melanoma. Perhaps in this group,
the melanomas may have developed from the malignant trans-
formation of other high Nodal expressing nevi that were not
Figure 6. Nodal expression and melanoma depth. (A) Correlation between yet excised. Alternatively, high Nodal expressing melanomas
Nodal immunohistochemistry IS in nevi removed from patients prior to a
may secrete a level of Nodal capable of inducing additional
diagnosis of melanoma and the Breslow depth of their melanomas that sub-
sequently developed. (B) Correlation between Nodal IS and Breslow depth in Nodal expression in other nevi at locoregional sites in the
cutaneous melanoma. IS, index score; Hx, history. same patient, since feedforward autoinduction from paracrine
1354 STRIZZI et al: NODAL PATHWAY IN ADULT NEVI

signaling is one of several established mechanisms underlying in patients with benign melanocytic lesions could represent
Nodal's biological effects (6,11). In fact, in the group of nevi a powerful tool for screening and prevention of melanoma.
from patients that subsequently developed melanoma, the The results of this study indicate that nevi with high Nodal
majority of these nevi showing highest Nodal expression (i.e. expression are associated with the development of aggressive
posterior torso) were from the same anatomical sites in which melanoma, suggesting that patients from whom these types
the melanoma subsequently developed‑in both female and male of nevi are removed should be closely monitored for potential
patients. Perhaps the surrounding melanocytic cells/lesions in melanomagenesis.
the posterior torso may have become more responsive to Nodal
as a consequence of accumulated effects of other melanogenic Acknowledgements
factors, such as exposure to UV radiation. In fact, the posterior
torso is a common site in both men and women for increased The present study was supported by grants from the
sun exposure. This also raises the possibility of a potential National Institutes of Health (Bethesda, MD, USA; grant
dosage effect of Nodal whereby, more Nodal would presum- nos. RO1CA121205 and R37CA59702) and H. Foundation
ably exert more autoinductive effects. To this regard, it would and Dixon Translational Research Grants to Dr Mary J.C.
be interesting to study the circulating Nodal levels and number Hendrix (Ann & Robert H. Lurie Children's Hospital of
of nevi to determine whether higher levels of Nodal can be Chicago, Northwestern University, Chicago, IL USA); and
detected in patients with increased numbers of nevi. Unfortu- a grant from the American Cancer Society Institutional
nately, this data was not available since an exact count of all Research Grant (grant no. 93‑037‑18) to Dr Luigi Strizzi
nevi present in each patient was not performed during whole (Northwestern University).
body inspection prior to excision of the nevus. Notably, in the
group of nevi from patients with a prior history of melanoma, References
for which accompanying Breslow depths were available for the
1. Postovit LM, Margaryan NV, Seftor EA, Kirschmann DA,
melanomas that subsequently developed, a significant positive Lipavsky A, Wheaton WW, Abbott DE, Seftor RE and
correlation was identified between Nodal expression in those Hendrix MJ: Human embryonic stem cell microenvironment
nevi and thicker melanomas. These observations suggest suppresses the tumorigenic phenotype of aggressive cancer cells.
Proc Natl Acad Sci USA 105: 4329‑4334, 2008.
that high Nodal expression in nevi may have the potential to 2. Yu L, Harms PW, Pouryazdanparast P, Kim DS, Ma L and
predict approximate anatomical site and aggressiveness of a Fullen DR: Expression of the embryonic morphogen Nodal in
subsequent melanoma. No other clinical parameter, including cutaneous melanocytic lesions. Mod Pathol 23: 1209‑1214, 2010.
3. Seykora J and Elder D: Dysplastic nevi and other risk markers for
patient age, anatomical site of the nevus or time elapsed melanoma. Semin Oncol 23: 682‑687, 1996.
between excision of the nevus and subsequent development of 4. Slade J, Marghoob AA, Salopek TG, Rigel DS, Kopf AW and
melanoma, correlated with any of the markers analyzed. Bart RS: Atypical mole syndrome: Risk factor for cutaneous
malignant melanoma and implications for management. J Am
As in all retrospective studies such as this one, it is difficult Acad Dermatol 32: 479‑494, 1995.
to determine direct cause and effect. However, the signifi- 5. Dakubo GD, Jakupciak JP, Birch‑Machin MA and Parr RL:
cantly high levels of Nodal, Cripto‑1 and Notch4 in nevi from Clinical implications and utility of field cancerization. Cancer
Cell Int 7: 2, 2007.
patients that subsequently developed melanoma compared to 6. D'Andrea D, Liguori GL, Le Good JA, Lonardo E, Andersson O,
the levels detected in the nevi from patients with no history of Constam DB, Persico MG and Minchiotti G: Cripto promotes
melanoma strongly suggests that Nodal signaling may serve A‑P axis specification independently of its stimulatory effect on
Nodal autoinduction. J Cell Biol 180: 597‑605, 2008.
a key role during melanoma development. Another limitation 7. Shen MM: Nodal signaling: Developmental roles and regulation.
of the present study is the relatively small sample size due to Development 134: 1023‑1034, 2007.
the difficulty in collecting sufficient numbers of excised nevi 8. Hardy KM, Kirschmann DA, Seftor EA, Margaryan NV,
Postovit LM, Strizzi L and Hendrix MJ: Regulation of the
from patients that subsequently develop melanoma, since the embryonic morphogen Nodal by Notch4 facilitates manifes-
majority of these patients are lost to follow‑up following nevi tation of the aggressive melanoma phenotype. Cancer Res 70:
removal and the establishment of a benign diagnosis. Never- 10340‑10350, 2010.
9. De Luca A, Lamura L, Strizzi L, Roma C, D'Antonio A,
theless, these data provide a strong evidence‑based rationale Margaryan N, Pirozzi G, Hsu MY, Botti G, Mari E, et al:
for developing future, long term, and prospective follow‑up Expression and functional role of CRIPTO‑1 in cutaneous
studies of a large patient cohort with high Nodal expressing melanoma. Br J Cancer 105: 1030‑1038, 2011.
10. Watanabe K, Nagaoka T, Lee JM, Bianco C, Gonzales M,
nevi, which will better determine the likelihood of these Castro NP, Rangel MC, Sakamoto K, Sun Y, Callahan R and
patients, with or without a history of melanoma, to subse- Salomon DS: Enhancement of Notch receptor maturation and
quently develop a recurring or novel melanoma, respectively. signaling sensitivity by Cripto‑1. J Cell Biol 187: 343‑353, 2009.
11. Le Good JA, Joubin K, Giraldez AJ, Ben‑Haim N, Beck S,
Nodal expression in melanoma is associated with aggres- Chen Y, Schier AF and Constam DB: Nodal stability determines
sive, metastatic disease. The ability for early detection of Nodal signaling range. Curr Biol 15: 31‑36, 2005.

View publication stats