S100 proteins in cancer Anne R. Bresnick, Nature Reviews Cancer 15, 96–109 (2015)
Author Manuscript Author Manuscript Author Manuscript Author Manuscript Published in final edited form as:
Nat Rev Cancer. 2015 February ; 15(2): 96–109. doi:10.1038/nrc3893.
S100 proteins in cancer
Anne R. Bresnick1, David J. Weber2, and Danna B. Zimmer2
Anne R. Bresnick: anne.bresnick@https://www.sodocs.net/doc/3217009475.html,; David J. Weber: dweber@https://www.sodocs.net/doc/3217009475.html,; Danna B. Zimmer: dzimmer@https://www.sodocs.net/doc/3217009475.html,
1Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
2Center for Biomolecular Therapeutics and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, Maryland 20102, USA.
Abstract
In humans, the S100 protein family is composed of 21 members that exhibit a high degree of
structural similarity, but are not functionally interchangeable. This family of proteins modulates
cellular responses by functioning both as intracellular Ca2+ sensors and as extracellular factors.
Dysregulated expression of multiple members of the S100 family is a common feature of human
cancers, with each type of cancer showing a unique S100 protein profile or signature. Emerging in vivo evidence indicates that the biology of most S100 proteins is complex and multifactorial, and
that these proteins actively contribute to tumorigenic processes such as cell proliferation,
metastasis, angiogenesis and immune evasion. Drug discovery efforts have identified leads for
inhibiting several S100 family members, and two of the identified inhibitors have progressed to
clinical trials in patients with cancer. This Review highlights new findings regarding the role of
S100 family members in cancer diagnosis and treatment, the contribution of S100 signalling to
tumour biology, and the discovery and development of S100 inhibitors for treating cancer.
The term S100 was first used in 1965 to denote a mixture of the two founding family
members, S100A1 and S100B1. This term alludes to the solubility of these approximately
10,000 Da proteins in 100% saturated ammonium sulphate. Although S100 family members
exhibit a high degree of sequence and structural similarity, they are not functionally
interchangeable and they participate in a wide range of biological processes such as
proliferation, migration and/or invasion, inflammation and differentiation2–4. The structure
and function of the S100 proteins are regulated by Ca2+ binding, which allows them to act as
Ca2+ sensors that can translate fluctuations in intracellular Ca2+ levels into a cellular
? 2015 Macmillan Publishers Limited. All rights reserved
Competing interests statement
The authors declare no competing interests.
FURTHER INFORMATION
RCSB Protein Data Bank: https://www.sodocs.net/doc/3217009475.html,/pdb
S100 gene family: https://www.sodocs.net/doc/3217009475.html,/genefamilies/S100
SUPPLEMENTARY INFORMATION
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Author Manuscript Author Manuscript Author Manuscript Author Manuscript Bresnick et al. Page 2
response5,6. Individual family members show unique affinities for divalent metal ions,
oligomerization properties, post-translational modifications and spatiotemporal expression
patterns. Intracellular S100 proteins bind to and regulate the activity of many targets; in
some cases, multiple S100 family members may regulate one target2–4. Several S100
proteins are present in the extracellular space where they can participate in local intercellular
communication (autocrine and paracrine), enter the systemic circulation and coordinate
biological events over long distances. S100 proteins lack a signal peptide for secretion via
the conventional Golgimediated pathway, and whether extracellular S100 proteins are
actively secreted from living cells or passively released is still debated2,4. Extracellular S100
proteins interact with a variety of cell-surface receptors including receptor for advanced
glycosylation end products (RAGE; also known as AGER), G protein-coupled receptors,
Toll-like receptor 4 (TLR4), scavenger receptors, fibroblast growth factor receptor 1
(FGFR1), CD166 antigen (also known as ALCAM), interleukin-10 receptor (IL-10R),
extracellular matrix metalloproteinase inducer (EMMPRIN; also known as basigin) and the
bioactive sphingolipid ceramide 1-phosphate4,7–10. The functional diversity of S100 proteins
and the unique repertoire of family members expressed in cells and tissues enable individual
cells to generate unique and adaptive responses to changes in intracellular Ca2+ levels and
the extracellular environment.
There are 21 S100 proteins — which are exclusively found in vertebrates — encoded in the
human genome11. As new family members were discovered, the S100 nomenclature evolved
with the consequence that numerous aliases exist for some S100 proteins4,12. Four family
members are dispersed throughout the genome: S100B on chromosome 21, S100G on the X
chromosome, S100P on chromosome 4 and S100Z on chromosome 5. The remaining 17
family members (S100A1–S100A14, the S100A7 genes and S100A16) are encoded in two
tandem clusters within a 2 Mb region on chromosome 1q21 that is referred to as the
epidermal differentiation complex (EDC). The EDC also contains genes encoding the S100-
fused type proteins (SFTPs) trichohyalin (TCHH), TCHH-like 1 (TCHHL1), repetin
(RPTN), hornerin (HRNR), filaggrin (FGL), FGL2 and cornulin (CRNN)13. SFTPs contain
a full-length S100 protein domain fused in-frame to multiple tandem repeats composed of
one or two sequences, for which the function is not well characterized. The five genomic
loci that encode S100 proteins are highly conserved, but there are differences among species
that affect the extrapolation of results from preclinical studies to human cancers. For
example, the mouse genome lacks genes encoding S100A12 and S100P, and the protein
encoded by the single mouse S100a7 locus (S100a7a) differs substantially from the proteins
encoded by the three human S100A7 loci (S100A7, S100A7A and S100A7L2)11.
Furthermore, although 1q21–25 is a hotspot for chromosomal alterations, mutations and/or
translocations in S100 genes are rare. The only reported event involving chromosomal
deletions of S100 family members is oral cancer (in which there is a deletion of S100A1–
S100A16)14. Of the four S100A14 polymorphisms reported in oesophageal squamous cell
carcinoma, only the mutation 461G>A is associated with increased cancer susceptibility due
to diminished binding of S100A14 to p53 (REFS 14,15). S100A2 polymorphisms have been
reported in non-small-cell lung cancer (NSCLC), but they are not associated with altered
S100A2 expression or function16.
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Nonetheless, dysregulation of S100 protein expression is a common occurrence in many
human cancers. In vivo studies have shown that altered expression of ten family members
contributes to the growth, metastasis, angiogenesis and immune evasion of numerous
tumours (TABLE 1). Inhibitors directly targeting two family members, S100B and S100A9,
are in clinical trials for melanoma and prostate cancer, respectively. This Review focuses on
new advances regarding the role of S100 proteins in cancer diagnosis and treatment, the
contribution of S100 signalling to cancer cell biology and the development of new S100
protein inhibitors for treating cancer.
Conformation and structure
The S100 proteins are typically symmetric dimers with each S100 subunit containing four α-
helices4. Although there are several reports of in vitro heterodimerization among family
members, and mixtures of S100A1 homodimers, S100B homodimers, and S100A1–S100B
heterodimers can be isolated from brain17, only the S100A8–S100A9 heterodimer has been
documented to have physiologically relevant functions in vivo18–20. Each of the two S100
subunits contains two Ca2+-binding domains: a carboxyterminal canonical EF-hand (a
helix–loop–helix domain) composed of 12 amino acids, and an amino-terminal ‘pseudo’ or
‘S100’ EF-hand that is unique to S100 proteins and is composed of 14 amino acids4. These
motifs are connected by a ‘hinge’ region (loop 2), which consists of 10–12 residues and is
crucial for target interactions. In the absence of target, the Ca2+-binding affinity of most
S100 proteins is low, but when bound to target, the Ca2+-binding affinity increases by 5–
300-fold21–23. This biochemical coupling can be understood in terms of structural transitions,
as upon binding Ca2+, S100 proteins undergo a substantial conformational rearrangement
that reorients helix 3 to expose a hydrophobic cleft that is required for target binding (FIG.
1). Recent studies have suggested that in the absence of a target, Ca2+-bound S100 proteins
exist as an equilibrium population of dynamic conformers with an overall weak Ca2+-
binding affinity, and that target binding narrows the distribution to favour S100 sub-states
with high affinities for Ca2+ ions24. As a consequence, target binding is typically Ca2+-
dependent. Importantly, the three-dimensional structures of S100–target complexes have
revealed that individual family members exhibit distinct modes of target recognition
owing, in part, to differences in surface geometries, hydrophobic residue distribution and
charge density25. In addition, some family members undergo a variety of post-translational
modifications, such as oxidative modification and sumoylation, which can modulate S100–
target complex formation and/or intracellular localization26–29. Structural and biochemical
considerations are particularly relevant to the development of therapies targeting S100
family members. Importantly, the 3D structures of S100 proteins permit an extensive
analysis of target selectivity, which can be exploited for drug discovery, as described later in
this Review.
Expression in cancer
Cancers exhibit a distinctive S100 protein profile that can be both stage-specific and subtype-
specific. In gliomas, S100B expression positively correlates with proneuronal, neuronal and
classic — but not mesenchymal — subtypes, whereas S100A8 and S100A9 expression
positively correlate with mesenchymal subtypes30. Despite the availability of
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protein signatures from breast31, head and neck32, prostate33, melanoma34 and
colorectal35,36 cancers, comprehensive analyses of S100 protein expression have not been
carried out. Nonetheless, discernible trends and notable exceptions emerge when the S100
protein expression profiles in human cancers are compared (see Supplementary information
S1 (table)). Dysregulation of multiple S100 family members occurs in most cancers and
typically involves upregulation. One family member, S100P, is upregulated in all cancers
that have been examined and the remaining S100 family members are upregulated in most
but not all cancers. Two exceptions are head and neck, and ocular cancers, in which 11 out
of 13, and 6 out of 12 dysregulated S100 proteins are downregulated, respectively (see
Supplementary information S1 (table) and references therein). The contradictory expression
profiles reported for some family members may be attributable to cancer subtype, disease
stage, cellular distribution, or issues associated with S100 protein and/or mRNA detection.
For example, S100A11 expression is increased in NSCLC, but is decreased in small-cell
lung cancer37. S100A7 is expressed in pre-invasive, well-differentiated and early-stage oral
squamous cell carcinomas, but not in non-invasive, poorly differentiated, late-stage
tumours38. For S100A7, it is important to note that studies carried out before the
development of paralogue-specific PCR primers and antibodies probably report on multiple
loci39. As the S100A7 paralogues are differentially expressed and regulated, and exhibit
diverse functions, it is important to discriminate between them in order to understand their
distinct functional roles in tumorigenesis. In addition, only a limited number of commercial
S100 antibodies have been tested for cross-reactivity against multiple family members40,
and the generic term anti-S100 indicates potential cross-reactivity with multiple family
members.
S100 protein expression profiles can be used to facilitate diagnosis and/or prognosis, inform
on treatment options and monitor patient response to therapy (see Supplementary
information S2, S3 (tables) and references therein). S100B expression in the primary tumour
has been used as a diagnostic marker for malignant melanoma in human and veterinary
medicine since the 1980s. S100P levels have diagnostic utility in a variety of human cancers
as S100P can be detected in primary tumours (breast, colorectal, pancreatic and ovarian
cancer), metastatic lesions (lung cancer), serum (breast and colorectal cancer), saliva (head
and neck cancer), bile (cancer of the bile duct) and faeces (colorectal cancer) (see
Supplementary information S2 (table) and references therein). The combined expression
levels of S100A2 and S100A10 are used for prognosis in recurrent colon cancer after
adjuvant 5-fluorouracil (5-FU) therapy and radical surgery5 (also see Supplementary
information S3 (table) and references therein). In addition, autoantibodies directed against
S100A7 have been reported in ovarian cancer and may facilitate diagnosis24(also see
Supplementary information S2 (table) and references therein). It should be noted that the
utility of S100 proteins as cancer biomarkers may be limited by their elevated expression in
other pathologies, including cardiovascular, neurological and inflammatory diseases.
The expression of S100 family members in human cancers is controlled by a complex
regulatory network, which includes epigenetic mechanisms and signal transduction
pathways, including pathways that are activated in response to chemotherapeutic agents.
Epigenetic changes regulate S100P expression in prostate, pancreatic and cervical cancer;
Author Manuscript Author Manuscript Author Manuscript Author Manuscript Bresnick et al. Page 5 S100A4 expression in pancreatic, endometrial, gastric, breast, ovarian, renal and brain
tumours; S100A2 in prostate and breast cancer; S100A6 in prostate and gastric cancer; and
S100A10 expression in pituitary cancer41–45. In colon cancer, seven S100 genes are direct
targets of histone-lysine methyltransferase MLL2 (also known as KMT2B and KMT2D)46;
however, coordinated regulation of S100 proteins within a given cancer is atypical. For
example, in colon cancer, S100A4 expression is controlled by WNT–β-catenin signalling47,
whereas S100P expression is regulated by activation of both the prostaglandin E2 (PGE2)
receptor EP4 subtype (PTGER4) and the MEK–ERK–cAMP-responsive element-binding
protein (CREB) pathway48. In addition, the regulatory mechanisms modulating S100
expression can be cancer type-specific. For instance, the expression of S100A8 and S100A9
is regulated by hypoxia-inducible factor 1 (HIF1) and PGE2–protein kinase A catalytic
subunit (PKA-C)– CCAAT/enhancer-binding protein-β(CEBPβ) signalling in prostate
cancer49,50; by nuclear factor-κB (NF-κB) signalling in liver cancer51; and by ultraviolet
radiation, intrinsic ageing and photo-ageing in skin cancer52. Finally, the modulation of
S100 expression is a common downstream event in S100 signalling cascades, resulting in
feedback loops that can sustain and exacerbate tumour progression. This is exemplified by
the expression of S100A8 and S100A9 in skin carcinogenesis, in which RAGE activation by
extracellular S100A8 and S100A9 upregulates the expression of these ligands, resulting in a
feedforward loop that promotes tumorigenesis53.
S100 signalling in cancer biology
Ten S100 family members actively contribute to in vivo tumour growth, metastasis,
angiogenesis and immune evasion (TABLE 1). Although S100 proteins can also act as
tumour suppressors, examples are rare and cancer type-specific. S100A2 functions as a
tumour suppressor in oral cancer and as a tumour promoter in lung cancer174,178. S100A7
acts as a tumour suppressor in oestrogen receptor-α(ERα)-positive breast cancer but
promotes ERα-negative breast tumour growth54. The roles of S100 proteins have been most
widely examined in breast cancer and melanoma. As cell lines do not mimic the complex
pathology or the S100 protein signatures that are observed in tumours in vivo55,56, our
discussion of S100 signalling in melanoma and breast cancer focuses on in vivo studies.
S100 signalling in breast cancer
Overexpression of several S100 family members (such as S100A1, S100A4, S100A6,
S100A7, S100A8, S100A9, S100A11, S100A14 and S100P) has been reported in breast
cancer57–65. Although alterations in S100 protein expression levels have been correlated
with aggressive disease, the mechanistic contribution of individual family members to
disease progression has only been evaluated for S100A4, S100A7 and the heterodimer
S100A8–S100A9.
S100A7 expression is not detected in epithelial cells in the normal breast. However, high
levels of S100A7 are observed in ductal carcinoma in situ, as well as in a subset of invasive
breast cancers66. With respect to invasive breast carcinomas, S100A7 overexpression is
associated with aggressive, high-grade, ERα-negative lesions with lymphocytic infiltration,
and is an independent prognostic indicator of poor outcome in patients with these tumours67.
In vitro studies indicate that S100A7 promotes the survival of ERα-negative breast tumour Nat Rev Cancer. Author manuscript; available in PMC 2015 March 23.
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cells under conditions of anchorage-independent growth68. In vivo, S100A7 induces ductal
hyperplasia in the mammary glands of transgenic mice69 and enhances tumour growth in
orthotopic breast cancer models68,70,71.
In ERα-positive breast cancer cells, S100A7 inhibits proliferative capacity by mediating the
degradation of β-catenin through a mechanism that involves glycogen synthase kinase 3β
(GSK3β) and E-cadherin signalling54. However, in ERα-negative breast cancer cells,
S100A7 activates several pro-survival pathways by interacting with the transcription
cofactor COPS5 (also known as JAB1), including upregulation of the activator protein 1
(AP-1) and NF-κB pathways, increased phospho-AKT and downregulation of cyclin-
dependent kinase inhibitor CDKN1B (also known as p27 or Kip1)68,70. In addition to
mediating pro-survival effects, S100A7 enhances the invasive capabilities of ERα-negative
breast cancer cells by augmenting epidermal growth factor receptor (EGFR) signalling and
matrix metalloproteinase 9 (MMP9) secretion72,73. S100A7 is also secreted by tumour
cells69,74, and extracellular S100A7 may facilitate tumour angiogenesis75 and the
recruitment of tumour-associated macrophages69 through interactions with RAGE on
endothelial cells and macrophages, respectively (FIG. 2).
Several studies have suggested that in early-stage breast cancer, expression of S100A4 in
combination with expression of either hepatocyte growth factor receptor (HGFR; also
known as MET) or osteopontin is a predictive indicator of metastatic disease and poor
survival58,59,76. Consistent with these observations, engineered overexpression of S100A4
in non-metastatic rat mammary Rama 37 tumour cells induces a metastatic phenotype in
orthotopic mammary tumours77. Although S100A4 overexpression in the mammary
epithelium is not tumorigenic78, it significantly enhances tumour metastasis in pre-existing
tumorigenic backgrounds, such as those in the mouse mammary tumour virus (MMTV)-Neu
transgenic79 and GRS/A78 mouse models of breast cancer. In both transgenic and orthotopic
models, S100A4 expression has a negligible effect on tumour latency, suggesting that
S100A4 specifically modulates tumour metastasis rather than tumour growth.
In breast cancer cells, S100A4 overexpression is also associated with increased migratory
capacity. Accordingly, S100A4 is found in the pseudopodia of migrating cells80,81, and
Ca2+-bound activated S100A4 localizes to the leading edge of breast cancer cells that are
undergoing polarized migration82. Furthermore, the Ca2+-dependent interaction of S100A4
with non-muscle myosin IIA regulates the formation and stability of lamellipodia to enhance
chemotactic migration83–85. S100A4 also interacts with the Rhotekin–RHOA complex to
promote membrane ruffling and invasion in EGF-stimulated breast cancer cells86. In
addition to the interactions with myosin IIA and Rhotekin, S100A4 can bind several other
cytoskeletal and adhesion proteins, including F-actin, non-muscle tropomyosin and liprin β1.
However, the regulation of these putative S100A4 targets is not well characterized.
Not only does S100A4 drive metastasis when expressed in the tumour, but S100A4
expression in the host stroma also contributes to metastatic dissemination. The metastatic
potential of orthotopic mammary tumours is significantly reduced in S100a4?/?mice
compared with in S100a4+/+ mice87, and metastasis to the lungs is inhibited in MMTV-
polyoma middle T antigen (MMTV-PyMT)–S100a4?/?transgenic mice88,89. Despite these Nat Rev Cancer. Author manuscript; available in PMC 2015 March 23.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript Bresnick et al. Page 7 observations, the specific stromal cell types that contribute to S100A4-mediated tumour
metastasis have not been well characterized. In the breast tumour microenvironment, the
majority of S100A4+ stromal cells originate from the bone marrow90 and in MMTV-PyMT–
S100A4?/?transgenic mice, the recruitment of CD45+ leukocytes and CD3+ T lymphocytes
to tumours is significantly reduced89, suggesting that stromal cell-derived S100A4
modulates the tumour immune response. In addition, recent studies indicate that non-bone-
marrow-derived S100A4+ cells, such as fibroblasts, are required for meta-static colonization
of breast tumour cells to the lungs87. Currently, a molecular understanding of how stromal
cell-derived S100A4 promotes metastasis is lacking and it remains to be determined whether
this is mediated by intracellular and/or extracellular S100A4. Both macro-phages and
fibroblasts can secrete S100A4 (REFS 91,92). S100A4 monoclonal antibodies significantly
limit breast tumour invasion and metastasis in vivo, consistent with an extracellular function
for S100A4 (REFS 93,94). Extracellular S100A4 has been shown to stimulate production of
MMP13 in endothelial cells and may contribute to tumour angiogenesis95,96, and recent
studies suggest that extracellular S100A4 induces the expression and secretion of pro-
inflammatory cytokines in tumour cells to elicit a pro-tumoural response in the
microenvironment97,98. Although the cell-surface receptors responsible for S100A4 binding
and its associated signal transduction pathways remain largely unknown, the identification
of these molecules will provide the foundation for a mechanistic understanding of the
signalling cascades that are crucial to the metastatic process.
Expression of both S100A8 and S100A9 is upregulated in invasive ductal carcinoma of the
breast63. Notably, S100A9 upregulation is associated with basal type tumours, high-grade
lesions, ERα- and progesterone receptor (PR)-negative status, and HER2- and EGFR-
positive tumours99. The increased levels of S100A8 and S100A9 observed in breast tumour
samples are due, in part, to the recruitment of S100A8- and S100A9-expressing myeloid-
derived suppressor cells (MDSCs) to the tumour stroma100,101. S100A9 expression is down-
regulated during the normal differentiation of myeloid precursors to macrophages and
dendritic cells. However, in cancer, tumour-derived factors upregulate S100A9 expression in
myeloid precursors, which inhibits macrophage and dendritic cell differentiation, and
promotes MDSC accumulation100. The expression of S100A9 is strictly required for MDSC
recruitment, as MDSC accumulation in the tumour is ablated in S100A9-null mice100. In
addition, MDSC-secreted S100A8–S100A9 heterodimer binds to carboxylated N-glycans on
RAGE on the MDSC cell surface102. Thus, S100A8 and S100A9 maintain an autocrine
feedback loop that sustains MDSC recruitment and the maintenance of immune suppression
within the tumour microenvironment.
S100A8 and S100A9 heterodimers and homodimers also have paracrine functions through
interactions with RAGE and TLR4 on tumour cells18,19,103. In tumour cells, RAGE binding
activates MAPK and NF-κB signalling pathways, and upregulates the expression of genes
associated with tumour growth and invasion18,103. With respect to intracellular functions, in
phagocytes, S100A8 and/or S100A9 bind arachidonic acid and activate NADPH oxidase
through the direct interaction of S100A8 with cytochrome b558, resulting in the formation of
reactive oxygen species (ROS) and the activation of NF-κB signalling20,104. In addition, the
S100A8–S100A9 complex is prone to oxidative modifications (such as nitrosylation,
Nat Rev Cancer. Author manuscript; available in PMC 2015 March 23.
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glutathionylation and oxidation) by various forms of ROS27,105,106, but how these
modifications modulate the tumour-promoting functions of the complex has not been
determined.
Extracellular S100A8 and/or S100A9 also contribute to the formation of the pre-metastatic
niche. The release of tumour necrosis factor-α(TNFα), transforming growth factor-β
(TGFβ) and vascular endothelial growth factor A (VEGFA) from the primary tumour
promotes expression of S100A8 and/or S100A9 in pre-metastatic lung endothelium and
lung-associated myeloid cells107. Furthermore, extracellular expression of S100A8 and
S100A9 induces the expression of serum amyloid A3 (SAA3), which potentiates its own
secretion via a TLR4-mediated NF-κB signalling cascade that recruits CD11b+ myeloid
cells to the pre-metastatic lung108. This produces a pro-inflammatory milieu that mobilizes
circulating tumour cells and promotes pulmonary metastasis108. Signalling via extracellular
S100A8 and S100A9 also supports the establishment of a pre-metastatic niche in the brain.
The expansion of bone marrow-derived CD11b+GR1+ cells, which express high levels of
S100A8 and S100A9, creates a local inflammatory environment that mediates the further
recruitment of CD11b+GR1+ myeloid cells and tumour cells through TLR4 signalling to
promote brain metastasis109. These observations indicate that for breast cancer, S100A8 and
S100A9 are crucial factors for establishing the pre-metastatic niche at multiple organ sites.
However, it is unknown whether the upregulation of S100A8 and S100A9 within each of
these target organs occurs through similar signalling pathways or whether organ-selective
factors mediate S100A8 and/or S100A9 expression, as well as the recruitment of bone
marrow-derived cells. Lastly, the requirement for S100A8 and/or S100A9 in creating a
permissive environment at other organ sites that are relevant to breast cancer metastasis
(such as bone) requires further investigation.
In addition to functions in the tumour microenvironment and the pre-metastatic niche,
S100A8 and/or S100A9 also mediate chemoresistance and subsequent metastasis of breast
cancer cells101. Chemotherapy induces the release of cytokines and chemokines, including
TNFα, by the tumour stroma. Stromal cell-derived TNFαcan then boost the expression and
secretion of CXC-chemokine ligand 1 (CXCL1) and CXCL2 by breast tumour cells,
resulting in the recruitment of CD11b+GR1+ myeloid cells and the release of S100A8 and
S100A9 within the tumour microenvironment101. Activation of the pro-survival ERK1,
ERK2 and ribosomal protein S6 kinase β1 (S6K1; also known as P70S6K) pathways by
S100A8 and/or S100A9 facilitates the expansion of chemoresistant breast cancer cells both
within the primary tumour and at distant metastatic sites101, thus providing a survival
advantage to breast cancer cells that are under chemotherapeutic stress. Clinical targeting of
multiple aspects of the TNFα–CXCL1/CXCL2– S100A8/S100A9 signalling axis may
provide a mechanism for limiting drug resistance and metastatic dissemination.
S100 signalling in melanoma
Malignant melanoma is a highly proliferative and heterogeneous cancer that is resistant to
conventional chemotherapy34. In melanoma, unlike most cancers, mutations in TP53 are
rare. Instead, driver mutations that activate oncogenes (such as BRAF and NRAS) and
inactivate cell cycle regulators (such as CDKN2A (which encodes p16) and PTEN) prevent Nat Rev Cancer. Author manuscript; available in PMC 2015 March 23.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript Bresnick et al. Page 9
wild-type TP53 from activating downstream target genes and inducing cell cycle arrest
and/or apoptosis. Twelve S100 family members are expressed in melanoma: four exhibit no
change in expression (S100A8, S100A9, S100A10 and S100A11); one is downregulated
(S100A2); and seven are upregulated (S100A1, S100A4, S100A6, S100A13, S100B and
S100P; see Supplementary information S1 (table)). The considerable number of S100 family
members that are expressed in melanoma is consistent with the localization of S100A1–
S100A16 genes to the EDC on human chromosome 1, and with previous observations that
the skin expresses the largest number of S100 family members110. However, there is little
information regarding the cellular distribution of S100 family members in melanoma.
S100A4 is primarily expressed in stromal cells, whereas S100B, S100A6 (REFS 111, 112)
and S100A10 (REF. 113) are expressed in tumour cells. Tumour and serum levels of S100B
have been used as a diagnostic marker for melanoma for many years and more recently,
urinary S100A7 levels have been reported as a potential diagnostic tool114. Decreased levels
of serum S100B are associated with the dramatic initial clinical responses to targeted
therapies for melanoma. Nonetheless, durable responses to these and other agents are rare
owing to the rapid development of multifactorial resistance within individual tumours and
patients. To date, increased expression of only one S100 family member, S100A13, has been
associated with melanoma resistance to chemotherapy (specifically, resistance to the DNA-
modifying agents dacarbazine and temozolomide; see Supplementary information S3
(table)). In vivo studies have confirmed that S100A4, S100A9 and S100B contribute to
melanoma progression and may be therapeutic targets.
In melanoma, signalling via extracellular S100A4, S100A8 and S100A9 culminates in the
expression of cytokines, chemokines, MMPs, and angiogenic and anti-apoptotic factors
(FIG. 3). However, their molecular mechanisms of action are different. Extracellular
S100A9, but not S100A8, binds the EMMPRIN receptor and requires the adaptor protein
TNF receptor-associated factor 2 (TRAF2) to upregulate the expression of TNFα, IL-1, IL-6
and other factors9. Whether extracellular S100A8 homodimers and S100A8–S100A9
heterodimers also mediate cytokine expression has not been examined. Moreover,
upregulation of S100A8 and S100A9 expression by these cytokines generates a feedforward
mechanism that can drive tumour progression9. The interaction of stromal cell-derived
S100A4 with RAGE on tumour cells also activates NF-κB-dependent gene expression97.
The subsequent release of pro-inflammatory cytokines and paracrine factors by tumour cells
stimulates endothelial cells and monocytes to promote angiogenesis and protumour immune
responses, respectively97. On the basis of the well-established link between chronic
inflammation and cancer115, the role of S100B in chronic inflammation116 and the presence
of S100B in the systemic circulation of patients with melanoma, it is likely that signalling
mediated by extracellular S100B also contributes to melanoma progression. Finally,
melanoma-derived exosomes induce the expression of S100A8–S100A9 at pre-metastatic
sites117.
There is interest in identifying the mechanisms in melanoma that prevent wild-type TP53
from activating cell cycle arrest and/or apoptosis in response to DNA-damaging agents or
UV radiation118–120. In melanoma cell lines, the regulation of growth and survival by
intracellular S100B involves a feedback loop that inactivates TP53 (REFS 121–125). Wild- Nat Rev Cancer. Author manuscript; available in PMC 2015 March 23.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript Bresnick et al. Page 10 type TP53 upregulates S100B expression by directly binding to the S100B promoter121.
However, S100B downregulates TP53 levels and activity by directly binding to and
dissociating the TP53 tetramer126, and stimulating TP53 polyubiquitylation and
degradation122,126. Additionally, S100B blocks covalent modification of TP53 (such as
phosphory lation and acetylation)127. Notably, a reduction in S100B levels or activity by as
little as twofold elevates TP53 and phospho-TP53 levels, reduces survival and restores UV
sensitivity125,128. There are a number of additional TP53-binding proteins that directly
compete with S100B and modulate the TP53-S100B interaction. For example, TP53 and
S100B both bind MDM2 and MDM4, which may allow for synergy in TP53
downregulation127. As is typical for S100 proteins, the effects of S100B expression on
melanoma cell growth are not limited to a single pathway. S100B binding to the p90
ribosomal S6 kinase (RSK) blocks ERK-dependent phosphorylation and results in
cytoplasmic sequestration of RSK124. However, it is not known how the shift in the
subcellular distribution of RSK affects cell growth. Ascertaining the pathways that regulate
both anti-tumorigenic and protumorigenic melanoma responses will require a careful
assessment of the number, relative abundance and cellular distribution of S100 family
members and their target proteins. Nonetheless, the beneficial effects of S100B inhibition on
multiple pathways that contribute to the melanoma cell phenotype make it an excellent
target for drug discovery.
Other S100 family members
Although there is a considerable amount of literature on the contributions of S100A4,
S100A7, S100A8, S100A9 and S100B to tumour growth and metastasis in a number of
cancers (see TABLE 1 and references therein), the role of the other S100 family members in
modulating tumorigenesis is less well defined. In murine models of cancer, S100A1,
S100A2, S100A3, S100A6, S100A10, S100A11, S100A14 and S100P are all reported to
affect tumour growth. However, with the exception of S100P, the contribution of these S100
family members to promoting a cancerous phenotype has only been examined in one or two
model systems (TABLE 1) and the mechanistic basis for the observed effects on tumour
progression has not been delineated. By contrast, S100P has been shown to enhance tumour
growth in several cancers, including lung, prostate, colorectal and pancreatic cancer
(TABLE 1). Although S100P enhances proliferation in all models that have been examined,
transcriptional regulation of S100P expression is highly dependent on the type of cancer. In
colon cancer, expression is mediated by PGE2–PTGER4 signalling, which activates CREB
via the ERK–MEK pathway48; in prostate cancer by IL-6 (REF. 129); and in breast and
cervical cancer by glucocorticoids130. Consistent with the observation that S100P enhances
the migratory capabilities of tumour cells in vitro131 and metastasis in vivo132–134, the
majority of S100P targets are cytoskeletal regulators. S100P–mediated activation of ezrin135
(a membrane–cytoskeleton linker protein) and Ras GTPase-activating-like protein 1
(IQGAP1; a juxtamembrane scaffolding protein that links plasma membrane receptors with
downstream signalling pathways)136provides a direct mechanism for the regulation of
tumour cell migration by S100P. In addition, the binding of extracellular S100P to RAGE
upregulates NF-κB activity to enhance cell survival137. Currently, extracellular S100P is
thought to stimulate tumour cell proliferation through an autocrine signalling mechanism.
Nat Rev Cancer. Author manuscript; available in PMC 2015 March 23.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript Bresnick et al. Page 11
However, a complete examination of potential paracrine functions and the cell types
involved has been limited by the absence of the S100P gene in the mouse genome. Targeting S100 proteins
S100 family members are excellent targets for cancer treatment as mouse models suggest
that genetic deletion has minimal effects on normal physiology. In addition to cancer, some
members of the S100 family represent attractive targets for the treatment of other diseases.
For example, S100B and S100A1 inhibitors may delay the progression of Alzheimer’s
disease116,138. However, S100A1 inhibitors may be contraindicated in patients with heart
disease, as S100A1 delays the development of cardiomyopathy139.
A number of pharmacological approaches have been used to modulate S100 signalling in
models of and in patients with cancer. Calcimycin (a calcium ionophore), niclosamide (an
antihelminth drug) and sulindac (a non-steroidal anti-inflammatory drug) have all been
identified as inhibitors of S100A4 transcription140,141. However, the efficacy of this
approach may be limited owing to the long half-life of S100 proteins, which could prevent
the achievement of sufficiently low steady-state protein levels to elicit a therapeutic effect.
Transcriptional modulators may also have clinically significant toxic off-target effects
owing to their ability to regulate the expression of numerous proteins under the control of
the same or related transcriptional regulatory assemblies. Although gene therapy has not
been used to modulate the expression of S100 family members in patients with cancer, it has
been used in preclinical animal models, in which it beneficially upregulates S100A1
expression in heart disease142. Other approaches to modulate S100 protein activity include
S100A4- and S100P–neutralizing antibodies93,94,143, and peptibodies (peptide–Fc fusion
proteins) directed against S100A8 and S100A9 (REF. 144); both approaches reduce tumour
growth in murine cancer models. Although the specificity of antibody-based therapies may
reduce toxicity and off-target effects, their efficacy may be limited by their ability to target
only extracellular S100 proteins. However, conformationally constrained inhibitory peptides
directed against S100B, which are capable of penetrating cells, have been shown to reduce
tumour growth in a melanoma xenograft model145.
The most common strategies for inhibiting S100 proteins exploit small molecules that block
the hydrophobic cleft required for the recognition of S100 targets, and for eliciting
biological effects5. Examples include paquinimod (also known as ABR-215757) and
tasquinimod (also known as ABR-215050), which are quinoline-3-carboxamide derivatives
that block the interaction of S100A8 and S100A9 with RAGE and TLR4, respectively
(REFS 19,146). Paquinimod exerts anti-inflammatory effects in a number of in vivo disease
models, but has not been specifically tested in cancer models. However, tasquinimod
improves progression-free survival in patients with metastatic castration-resistant prostate
cancer, possibly by reducing the recruitment of MDSCs and inhibiting metastasis147,148.
Cromolyn, an anti-histaminic drug, disrupts the S100P– RAGE interaction, reducing
pancreatic tumour growth and increasing the effectiveness of the chemotherapeutic drug
gemcitabine149,150. Cromolyn binds several other S100 family members (S100A1, S10012
and S100A13), but the biochemical and biological consequences of these interactions have
not been examined. Amlexanox, another anti-inflammatory anti-allergic immunomodulatory
Bresnick et al. Page 12
Author Manuscript Author Manuscript Author Manuscript Author Manuscript drug, interacts with several S100 proteins (S100A1, S100A4 and S100A13)151–153. Amlexanox inhibits S100A13 secretion, disrupts the interaction of S100A13 with fibroblast growth factor 1 (FGF1), and antagonizes the mitogenic and angiogenic effects of FGF1 (REF. 152). Phenothiazines also interact with multiple S100 family members. In the case of S100A4, phenothiazine-mediated S100A4 oligomerization results in the sequestration of
S100A4 away from its protein targets154,155. It should be noted that all of these drugs were developed for other indications and their lack of selectivity within the S100 family is not surprising given the high degree of structural similarity between S100 proteins. Ongoing efforts to target S100 proteins are focused on improving selectivity and other pharmacological properties of S100 inhibitors such as affinity and biological half-life.
The large, shallow and relatively ‘featureless’ interfaces typified by most protein–protein interactions (PPIs) pose unique challenges for the development of high-affinity PPI inhibitors. In contrast to typical protein–protein interfaces, the relatively deep target-binding clefts of S100 proteins can readily accommodate small molecules that have been discovered through traditional high-throughput experimental screening and computer-aided drug design. These approaches have successfully identified new compounds that inhibit the interactions of S100P149, S100A4 (REFS 156,157), S100A9 (REF. 156), S100A10 (REF 158) and
S100B123,159 with their respective targets. An examination of these S100–small-molecule complexes has revealed that most small molecules target one of three distinct pockets within S100 proteins160 (FIG. 4). Site 1 is exposed by Ca2+-mediated conformational rearrangements and involves residues from helices 3 and 4, and loop 2 (the hinge region), as occurs in the S100B– SEN205A interaction, for example161 (FIG. 4a). Interactions at sites 2 and 3 involve residues from loop 2 and helix 4, and the C-terminal loop and helix 1, respectively. In some instances, multiple copies of the same compound occupy both sites 2 and 3 (as occurs in the S100B–pentamidine162and S100A4–trifluoperazine interactions154). However, compounds have also been identified that target only one of these sites (such as
S100B–SBiX-inhibitor complexes160or the S100A13–amlexanox interaction152). A comparison of S100 protein–small-molecule structures reveals that the location and orientation of ligands bound in sites 2 and 3 are less well conserved than those bound in site 1. The diversity of binding positions observed at sites 2 and 3 probably reflects differences in the highly specific interactions that mediate the binding of various targets (such as H- bonds, hydrophobic interactions and other types of interactions) within the S100 protein family and thus provides opportunities for specific S100–small-molecule interactions.
Given that S100 proteins can accommodate small molecules at several sites, it is not unexpected that multiple mechanisms of inhibition are observed, including classic competitive binding between an inhibitor and physiological target to the same or overlapping sites on a S100 protein, such as amlexanox binding to S100A13 or SEN205A binding to S100B152,161 (FIG. 4a). However, some inhibitors of S100 proteins may exhibit subtle allosteric effects as exemplified by pentamidine binding to S100B. Each S100B subunit binds two pentamidines, with one binding site adjacent to the target binding site and the second at the dimer interface162. Although the pentamidine binding sites exhibit minimal overlap with the S100B target-binding cleft, pentamidine disrupts the S100B–TP53 interaction and potently inhibits the growth of malignant melanoma cells163, suggesting that inhibition occurs due to allosteric effects. More recently, structural, biophysical, and protein
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
dynamics studies have shown that the binding of small-molecule inhibitors and peptides can
increase Ca2+-binding affinity upon complex formation in a manner analogous to that
associated with the binding of bona fide targets24.
When considering how small molecules and peptides that bind at some distance from the
S100 EF-hands may affect Ca2+-binding affinities, it is important to take into account the
structural features as well as dynamic properties of S100 proteins24,164–166. In the absence
of a protein target, S100 proteins sample a large ensemble of conformational sub-states that
exhibit a wide range of Ca2+ affinities but, overall, their apparent affinity is low. Target
binding biases the distribution of sub-states towards those with high Ca2+ affinity. This
model, termed the ‘binding and functional folding’ (BFF) model (FIG. 4c), provides a
foundation for understanding how the biochemical activities of S100 proteins fit into overall
cell physiology. The coupling of Ca2+ and target binding allows for high intracellular S100
protein concentrations (>1 μM) without substantial sequestration of free Ca2+ or disruption
of Ca2+ oscillations, and thus cells remain highly responsive to changes in calcium and/or
target availability. S100 inhibitor screens are now incorporating an examination of Ca2+
affinity and protein dynamics24, which should allow for the identification of inhibitors with
increased binding affinities and specificities.
Future directions
In the past decade, important advances regarding the expression, structure and signalling of
S100 proteins have improved our understanding of normal cell physiology as well as the
pathophysiology of cancer. The development of molecular probes — such as antibodies and
small-molecule inhibitors — will be instrumental for deciphering the in vivo functions of
specific S100 proteins, as well as distinguishing the contribution of intracellular and
extracellular S100 proteins. Moreover, these probes may also have therapeutic potential for
cancer or other diseases. Despite considerable progress in S100 protein biology, we
currently have little information on how post-translational modifications or heterodimer
formation affect S100 signalling. A mechanistic examination of both S100 protein biology
and biochemistry will be required to define how each family member contributes to the
proliferation, metastasis, angiogenesis and immune evasion of cancers and other diseases. Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgements
The authors apologize to the numerous colleagues whose important contributions could not be included in this
Review owing to space limitations. The authors thank S. C. Almo and J. M. Backer for critical reading of this
Review. The authors’ research is funded by grants from the New York State Department of Health, Health
Research Science Board (H11R-040; to A.R.B.), the National Cancer Institute (US National Institutes of Health;
CA100324 to A.R.B. and CA107331 to D.J.W.), the Albert Einstein Cancer Center (NCI, NIH; CA013330), the
Marlene and Stewart Greenebaum Cancer Center (NCI, NIH; CA134274) and the Center for Biomolecular
Therapeutics, University of Maryland School of Medicine, USA (to D.J.W. and D.B.Z.).
Author Manuscript Author Manuscript Author Manuscript Author Manuscript Glossary
Sub-states Closely related interconverting conformational states that can be
sampled by a protein under a given set of conditions GRS/A An inbred mouse strain carrying the Mtv2a allele, which controls
the expression of endogenous mouse mammary tumour virus and
the early development of hormone-induced mammary tumours Lamellipodia Transient cellular protrusions that form during cell migration
Chemotactic
migration
Directional cell migration in response to soluble extracellular
ligands
MMTV-polyoma
middle T antigen
(MMTV-PyMT). A murine breast cancer model with expression
of PyMT under the control of the mouse mammary tumour virus
(MMTV) promoter
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