Seminars in Cancer Biology 14 (2004) 63–69

Targets for molecular therapy of skin cancer

Cheryl L. Green b , Paul A. Khavari a,b,∗
a VA Palo Alto Healthcare System, Palo Alto, CA 94304, USA
b Program in Epithelial Biology, Stanford University School of Medicine, 269 Campus Drive, Room 2145, Stanford, CA 94305, USA

Abstract

Cancers of the skin encompass the first and second most common neoplasms in the United States, epidermal basal cell carcinoma (BCC)
and squamous cell carcinoma (SCC), respectively, as well as the melanocytic malignancy, malignant melanoma (MM). Recently identified
alterations in the function of specific genes in these cancers provide new potential therapeutic targets. These alterations affect conserved
regulators of cellular proliferation and viability, including the Sonic Hedgehog, Ras/Raf, ARF/p53, p16INK4A /CDK4/Rb and NF-␬B
pathways. New modalities designed to target these specific proteins may represent promising approaches to therapy of human skin cancers.
Published by Elsevier Ltd.

Keywords: Skin; Cancer; Melanoma; Squamous cell carcinoma; Basal cell carcinoma

1. Introduction over a third of the cancers occur on the face. Histologi-

Cancers of the skin arise predominantly from cells located
within the outermost compartment of the tissue, the epi-
dermis. The majority of epidermal cells are keratinocytes,
epithelial cells that generate the cutaneous barrier to the
external environment. These cutaneous epithelial cells pop-
ulate both the follicular and interfollicular regions of the
skin and give rise to the structure of the epidermis as well as
its appendages, namely hair and sweat glands. A number of
neoplasms originate from cutaneous epithelial cells [1], the
most common of which are basal cell carcinoma (BCC) and
squamous cell carcinoma (SCC). Interspersed among epithe-
lial cells are pigment-producing melanocytes, which give
rise to malignant melanoma (MM). Although widespread
and increasing in incidence, BCC, SCC and MM have been
poorly understood at the level of molecular pathogenesis
until recently. Progress over the past decade has identified
an array of molecular alterations in these neoplasias that
may provide opportunities for new molecular therapeutics.
cally, BCCs lack precursor lesions and can be subdivided
into a number of sub-types, including superficial, nodu-
lar and aggressive-growth or morpheaform [3,4]. Classic
histologic features include palisaded nuclei at the tumor
periphery, clefting at tumor–tissue interfaces and formation
of peri-tumor stromal tissue [5]. Based on morphologic
observations in tissue sections, it is believed that a sub-
stantial proportion of all BCCs may arise from hair follicle
keratinocytes. Clinically, BCCs are characterized by local
invasion and contiguous spread. While reports of metastatic
BCC exist in the literature, it is widely recognized that BCC
metastasis is an extremely rare event, in contrast to SCC
[3–5]. Thus, BCC exerts its major damage through destruc-
tion of local tissue and, when arising on the head as it com-
monly does, BCC growth can lead to loss of vital structures
and mutilating disfigurement. As noted below, BCC occurs
both sporadically and as part of heritable syndromes. Cur-
rent therapies for BCC are non-specific and the majority of
approaches rely primarily on surgical removal of the tumor.

1.1. Basal cell carcinoma 1.2. Squamous cell carcinoma

BCC is the most common cancer in fair-skinned pop-
ulations, with an estimated yearly US incidence of ap-
proximately 1 million [2]. Lightly pigmented individuals
are most commonly affected on sun exposed surfaces and
∗ Corresponding author. Tel.: +1-650-725-5266; fax: +1-650-723-8762.
E-mail address: khavari@CMGM.stanford.edu (P.A. Khavari).
SCC is the second most common cancer in the US
with approximately 250,000 new cases diagnosed annually.
In contrast to BCC, SCC is a biologically aggressive tu-
mor and may metastasize at frequencies reported between
1 and 12.5% [6–8]. Following local invasion and tissue
destruction, SCC commonly metasizes to lymph nodes.
Unlike BCC, there is no compelling evidence for a hair

1044-579X/$ – see front matter. Published by Elsevier Ltd.

doi:10.1016/j.semcancer.2003.11.007
64 C.L. Green, P.A. Khavari / Seminars in Cancer Biology 14 (2004) 63–69
follicle-derived origin for SCC, and SCC is thus believed
to largely represent a neoplasm of interfollicular epidermis.
Histologically, SCC displays a number of differences from
BCC, including a series of well-defined precursors, a lack
of well-demarcated tumor periphery and the presence of
features of epidermal differentiation [8]. SCC precursors
include actinic keratosis, which represents a focal dysplasia
of epidermal keratinocytes, and SCC in situ, or Bowen’s
disease. The incidence of SCC is greatly increased in
patients receiving immunosuppressive therapy, a process
that may be enhanced by concomitant human papilloma
virus (HPV) infection. In individuals with intact immune
systems, however, clear evidence for a widespread role
for HPV in SCC pathogenesis is lacking and ultraviolet
(UV) radiation-mediated mutagenesis is believed to be the
major environmental factor promoting this cancer. A num-
ber of heritable conditions are associated with increased
incidences of SCC, including xeroderma pigmentosum,
recessive dystrophic epidermolysis bullosa, dyskeratosis
congenital, oculocutaneous albinism and epidermodysplasia
verruciformis [8]. In contrast to BCC, however, the genetic
defects in these syndromes are not specific for SCC. Thus,
beyond underscoring the roles of genomic integrity and
chronic wounding injury in carcinogenesis, their underlying
defects are believed to shed little light on the precise medi-
ators of its pathogenesis. As is the case for BCC, there are
no specifically targeted therapies for SCC and the primary
therapeutic modality is surgery.
1.3. Melanoma
In addition to keratinocytes, the epidermis contains a
number of other cell types, among which are melanocytes.
Derived embryologically from the neural crest, melanocytes
synthesize the melanin pigment that provides cells of the
skin with photoprotection from mutagenic UV rays. MM is
less common than either BCC or SCC, with 87,000 cases
estimated in the US for 2002 [9]. Like BCC and SCC, the
incidence of MM appears to be increasing, with predicted
lifetime risk in the US rising to 1:75 for those born in 2000
[9]. Although less common, MM accounts for a greater
number of deaths than BCC and SCC combined. The risk
factors for MM include light pigmentation, UV exposure, a
tendency to sunburn and multiple nevi. Regarding the latter,
atypical nevi may represent both a marker and a precursor
for MM. Although MM may arise in a pre-existing nevus, it
may also arise de novo. While there are a number of variants
of MM, tumor depth appears to be among the most potent
prognostic factors, with deeper primary tumors associated
with a less favorable prognosis [10]. Families with increased
incidence of MM exist and often display large numbers
of atypical nevi in affected members. Because of the poor
prognosis of metastatic MM and the curative nature of MM
removal when the cancer is caught prior to metastasis, clini-
cal efforts have been focused on early detection and surgical
excision [11].
2. Molecular defects in skin cancer
Over the past decade, a number of genetic alterations
have been characterized in BCC, SCC and MM (Fig. 1).
These alterations involve both gain-of-function changes in
oncogenes as well as loss-of-function of genes that act as
suppressors of tumor formation. The spectrum of changes
differs among these tumor types, with BCC and MM per-
haps best understood currently.
2.1. Sonic Hedgehog pathway
In addition to its sporadic occurrence, BCC is a feature
of a number of inherited syndromes, including the nevoid
basal cell carcinoma syndrome (NBCCS, or Gorlin’s syn-
drome), Bazex syndrome and Rombo syndrome [5]. The
former was instrumental in the identification of mutations
in the PATCHED1 (PTCH1) gene, an important component
of the Sonic Hedgehog (SHH) signal transduction pathway,
in both NBCCS and sporadic BCC [12,13]. The PTCH1
gene encodes a transmembrane protein that binds SHH and
inhibits SHH pathway action by blocking the function of
another transmembrane protein, Smoothened (SMO), a G
protein-coupled receptor. SMO signals to GLI family tran-
scription factors, which regulate genes involved in tissue
morphogenesis. Subsequent studies have shown that alter-
ations at a number of points in the SHH pathway in addition
to PTCH1 loss-of-function are relevant to BCC, including
gain-of-function in SHH, SMO and GLI proteins. In spo-
radic BCC, pathogenic mutations have been found in genes
encoding SHH, SMO and PTCH1 [12–15]. In epidermis,
SHH pathway activation promotes proliferation and renders
cells resistant to stimuli promoting their cell cycle exit and
terminal differentiation [16]. In mice, overexpression of
SHH, active SMO, GLI1 or GLI2 promotes development
of BCC-like tumors [14,15,17,18]. The potency of SHH
pathway activation in inducing hallmark BCC features in
human tissue was confirmed using human skin regener-
ated on immune deficient mice, where SHH overexpression
alone triggered cardinal features of BCC within 3 weeks
[19]. Thus, compelling genetic and functional data points
to induction of the SHH pathway as a major factor in BCC
and suggests that its inhibition may represent a promising
therapeutic opportunity (Fig. 1 and Table 1).
2.2. p16INK4A , p14ARF and CDK4
The CDKN2A locus on 9p21 is widely mutated in a variety
of human cancers [20,21]. This locus is commonly mutated
in both sporadic and familial melanoma and encodes both
a cyclin-dependent kinase (CDK) inhibitor, p16INK4A , and
a p53 regulator that is translated via an alternative reading
frame (ARF) [22,23]. p16INK4A acts to inhibit cell cycle pro-
gression in the G1 phase by binding and inhibiting CDK4/6
kinases. CDK4/6 phosphorylate Rb proteins to remove the
mid-G1 Rb block to cell cycle progression [24]. Loss of
 C.L. Green, P.A. Khavari / Seminars in Cancer Biology 14 (2004) 63–69 65

Fig. 1. Schema of a selected of proteins altered in skin carcinogensis. Color denotes proteins for which defined mutations have been confirmed in specific
skin cancer types: gain-of-function is shown in green, loss in red. The approximate percentage of primary tumors with alterations indicated are noted
in parentheses, however, in some cases these figures are either controversial (i.e., RAS in SCC) or not clearly quantitated (i.e., CDK4 in MM). For
presentation, proteins have been diagrammed in pathways but this simplification does not reflect either confirmed epistatic relationships in these cancers or
the likely complexity in actual tumor tissues. CyD: D-type cyclins, MM: malignant melanoma, SCC: squamous cell carcinoma, BCC: basal cell carcinoma.

p16INK4A leads to deregulated CDK4/6 action and promo- in MM commonly abrogate function of both p16INK4A and
tion of cell division. An additional subset of patients with p14ARF , lesions that only affect one or the other have also
familial MM display mutations in CDK4 that render the ki- been reported, consistent with a role for both proteins in in-
nase insensitive to p16INK4A inhibition [25], indicating that hibiting metanocytic neoplasia [29–32]. Extensive work in
multiple mechanisms to bypass the Rb block are operative in animal models of MM has demonstrated that impairing the
MM. In addition to MM, CDK4 has been implicated in SCC function of p16INK4A and ARF along with activating Ras sig-
development by the recent demonstration of its overexpres- naling can facilitate the development of this tumor [33–36].
sion in this tumor and the fact that it is sufficient to induce Data on the sufficiency of altering these genes in human tis-
this neoplasm in human epidermis when it is co-expressed sue to induce MM, however, has not yet been published. Ef-
with oncogenic Ras [26]. ARF (p14ARF in humans, p19ARF forts to preserve the function of both the Rb (p16INK4A and
in mice) acts to stabilize p53 by inhibiting MDM2-mediated CDK4) and p53 (ARF) pathways, then, may be of utility in
degradation and also appears to induce anti-proliferative the prevention and treatment of MM and, possibly, of SCC.
genes on its own [27,28]. While CDKN2A mutations found
2.3. Ras and Raf
Table 1 Ras GTPases regulate proliferation, angiogenesis, apop-
Potential molecular targets in skin cancer
tosis and cellular morphology and may represent the most
Protein Pathway Skin cancer widely altered oncoproteins in human neoplasia [37]. Ras
SHH SHH BCC exerts its effects via three main downstream pathways initi-
PTCH1 SHH BCC ated by binding of GTP-bound Ras to families of pathway
SMO SHH BCC effectors including the Raf kinases, type I phosphatidyl-
PKA SHH BCC inositol-3 kinases (PI3Ks) and Ral guanine nucleotide
GLI1/2 SHH BCC
Ras Ras SCC, BCC
exchange factors (RalGEF) [38]. The best-characterized
Raf Ras MM, SCC, BCC Ras effector pathway proceeds via a kinase cascade that
ERK Ras MM, SCC, BCC includes Raf, two mitogen activated protein kinase kinases
p53 p53 SCC, BCC (MAPKKs or MAP/ERK kinases (MEKs): MEK1 and
p14ARF p53 MM, SCC, BCC MEK2, also called MKK1 and MKK2), and two MAPKs
p16INK4A Rb MM, SCC
CDK4 Rb MM, SCC
(ERK1 and ERK1) [39]. ERK has been observed to be
IKK, I␬B␣ NF-␬B SCC constitutively active in MM cells and sequencing of RAS
NF-␬B subunits NF-␬B SCC and RAF genes in melanoma tissue and cell lines has iden-
SHH: Sonic Hedgehog, PTCH1: PATCHED1, SMO: smoothened, PKA: tified activating RAS mutations in approximately 10% and
protein kinase A, ERK: extracellular; IKK: I␬B kinase, CDK: cyclin- kinase-activating BRAF mutations in approximately 70%
dependent kinase. of MM [40–42]. Thus, Ras/Raf gain-of-function appears
66 C.L. Green, P.A. Khavari / Seminars in Cancer Biology 14 (2004) 63–69
characteristic of MM, however, the presence of mutations
that activate B-Raf in a majority of benign nevi indicates
that induction of the latter alone is not sufficient for tumori-
genesis of melanocytes. In addition, to melanoma, activation
of Ras/Raf signaling has been implicated in both BCC and
SCC. In non-transformed epidermis, Ras and Raf inhibit
differentiation, stimulate cell division and augment inte-
grin expression, changes all characteristic of SCC [43–45].
The PDGFR␣ has been shown to be upregulated in BCC,
with associated activation of the Ras/MAPK pathway [46].
While activating mutations and amplification of RAS genes
have been reported in a minority of SCC [47], such genetic
changes are not required for the increased levels of active
GTP-bound Ras observed in the majority of spontaneous
human SCC tissue [48]. This suggests the possible exis-
tence of an analogous situation to that seen in breast cancer
where RAS mutations are rarely seen but where Ras is acti-
vated due to a number of factors, including overexpression
of upstream receptor tyrosine kinases [49]. Inhibition of
Ras/Raf cascade signaling, then, may constitute a poten-
tially promising approach in the management of MM, BCC
and SCC.
2.4. p53
While p53 dysfunction has been reported in a wide array
of cancers, including BCC and MM, a number of studies sug-
gest a significant role for it in the pathogenesis of SCC. For
example, p53 plays a central role in the induction of epider-
mal cell apoptosis following UV radiation-mediated DNA
damage [50]. Since UV serves as both a genotoxic initiator
as well as tumor promoter, p53-dependent death of these
cells is believed to function as a protective mechanism pre-
venting SCC development. Over 90% of SCOs display p53
mutations [51]. Consistent with a significant role of p53 in
SCC development, SCC precursors display p53 mutations in
over 70% of actinic keratoses [52]. While these data strongly
implicate p53 abnormalities in SCC development, the degree
of p53 impact in this setting is not yet firmly established.
For example, TP53 knockout mice display a surprising re-
sistance to cutaneous carcinogenesis induced by overexpres-
sion of certain oncogenes [53]. However, these mice appear
more susceptible to SCC induction by UV, a likely more
biologically relevant stimulus [54]. In short-term studies
in human skin tissue, in contrast, mutant p53 protein itself
fails to promote invasive neoplasia when co-expressed with
oncogenic Ras, suggesting that its anti-neoplastic effects
operate through protecting genomic integrity rather through
direct promotion of oncogenesis. These findings regarding
the functional actions of p53 in SCC are complicated by the
experimental approaches to alter p53 function, including
differences between genetic deletion versus overexpression
of well-characterized mutant p53 proteins. Further studies
are required to address these issues but preserving p53
function remains an attractive possibility for the prevention
of SCC.
2.5. NF-κB/Rel
NF-␬B/Rel proteins are conserved transcription factors
implicated in regulating inflammation, morphogenesis,
apoptosis, differentiation and proliferation. These proteins
contain a characteristic 300 amino acid Rel homology do-
main (RHD) involved in DNA binding and protein–protein
interactions [55,56]. NF-␬B can be activated by an upstream
cascade involving I␬B kinases (IKKs) and inhibited by I␬B
proteins [57,58]. In contrast to its oncogenic role in other
settings, NF-␬B may act as an important anti-proliferative
safeguard in stratified epithelium because its blockade facil-
itates hyper-proliferation in both murine [59,60] and human
epidermis [60]. Consistent with this, mice with inhibited
epidermal NF-␬B function display increased susceptibility
to induction of SCC [59]. Moreover, NF-␬B blockade in
concert with oncogenic Ras can transform human epidermis
into a highly aggressive invasive neoplasia indistinguishable
from SCC [48]. Recent analysis of a series of spontaneous
SCCs demonstrated evidence of NF-␬B blockade with I␬B␣
upregulation and cytoplasmic sequestration of NF-␬B RelA
[48]. Therefore, while less well established than other al-
terations in common skin cancers, NF-␬B dysfunction may
constitute a significant feature of SCC and thus represent a
potential target for therapy.
3. Potential molecular therapeutics approaches for
skin cancer
The expanding knowledge of the pathogenesis of com-
mon cutaneous malignancies offers the opportunities to
develop new therapies directed at the specific molecules
altered in these cancers (Table 1). In addition to surgery,
current therapeutic approaches include topical measures
such as cryosurgery, immunomodulation with imiquimod,
5-Fluorouracil, photodynamic therapy and radiation [5].
While surgical excision remains the practical modality of
choice due to its definitive nature, the increasing incidence
of skin cancers and the enormous resources involved in
their diagnosis and treatment has propelled efforts to de-
velop more specific new strategies for their prevention and
therapy. Because early treatment, especially in the case
of MM and SCC, is essential to reducing disease mortal-
ity and morbidity, potent topical agents for skin-localized
cancers and their precursors are of especial interest. Cur-
rent challenges facing efforts towards this goal include
(a) development of effective, specific agents targeted at
molecules of interest and (b) transcutaneous delivery of
these therapeutics across the formidable skin permeability
barrier.
3.1. Gene transfer
The field of cutaneous gene transfer has seen significant
recent progress in the field of monogenic skin disorders and
an array of genetic approaches may be envisioned to either
 C.L. Green, P.A. Khavari / Seminars in Cancer Biology 14 (2004) 63–69
interfere with aberrant signaling or restore defective tumor
suppressor genes [61]. Approaches of interest could restore
expression of proteins, such as p16INK4A , or disrupt pathway
function with dominant-negative interference, such as by
expression of transdominant ERKs. Applying gene transfer
to the treatment of skin cancer, however, is hindered by
the difficulty in delivering therapeutic genes to every cell
within a tumor. To date, no approach supports such efficient
delivery to skin or any other tissue. Without such efficiency,
even the most potent genetic sequences would be of limited
value in this setting.
3.2. Small molecule drugs
Development of specific pharmacotherapeutics for skin
cancer is still in early stages, however, a number of poten-
tially useful proteins are being targeted in broader screening
efforts. Small molecule regulators have been identified for
a number of pathways relevant to skin cancer, including el-
ements of the SHH, Ras/Raf, NF-␬B, p16INK4A /CDK4/Rb
and ARF/p53 pathways. For SHH, both agonists and antag-
onists have been identified that extend original observations
made with the original antagonist, cyclopamine [62]. Ef-
forts at Ras/Raf inhibition involve an array of molecules,
including inhibitors of enzymes that mediate Ras activation
as well as Raf and MEK kinase inhibitors [63,64]. Compo-
nents of the IKK1/NF-␬B cascade are also an active focus
of pharmacologic efforts directed at cancer and inflamma-
tory diseases [65,66]. A variety of CDK kinase inhibitors
have also been developed to support maintenance of the Rb
block [67–69]. In the case of p53, novel molecules such
as CP-31398 have been developed that stabilize p53 pro-
tein even in the absence of ARF [70]. A key problem with
many of these efforts to block pleiotropic master regulator
proteins, such as Ras and NF-␬B, has been the undesired
complexity of altering their function throughout the body
when these agents are administered systemically. For exam-
ple, the majority of efforts directed at NF-␬B are designed
to block its function in malignancies of lymphoid and other
tissue [66,71]. Unfortunately, this may end up promoting
cutaneous carcinogenesis [48], a major problem in immune
suppressed cancer patients. By virtue of its tractability to
local topical therapy that could avoid unwanted paradoxical
effects in differing tissues, the skin offers unique advantages
in this regard. Nonetheless, significant obstacles confront
current efforts to capitalize on recent mechanistic insight
into skin neoplasia by developing new molecular therapies.
3.3. Transcutaneous transporters
Topical delivery of new therapeutics across the epidermal
permeability barrier to prevent or treat localized pre-cancers
or cancers of the skin presents a significant challenge given
the skin’s well known capacity to exclude foreign molecules.
The development of new strategies to accomplish this, how-
ever, may permit the transport of a variety of therapeutic
67
molecules into the skin. Among such strategies are protein
transduction [72–74], which relies on arginine-rich peptides
or peptidomimetics that replicate them to deliver macro-
molecules across cell and tissue barriers. This technology
has recently been used to reversibly link and deliver both
peptides and previously non-penetrating drugs across hu-
man and mouse skin [75,76]. Combined with highly specific
new compounds designed to target proteins dysfunctional in
human skin cancer, such new delivery approaches provide
promise for new molecular therapies for these diseases.
4. Conclusion
Insight into the pathogenesis of human skin cancers has
provided potential opportunities for preventive and thera-
peutic intervention. These targets include components of
evolutionarily conserved signaling cascades importance
in tissue morphogenesis and cell fate, namely the SHH,
Ras/Raf, NF-␬B, p16INK4A /CDK4/Rb and ARF/p53 path-
ways. To represent a significant advance in the treatment
of skin-localized non-metastatic tumors, however, new tar-
geted therapies will have to display superior features to the
current gold standard, surgical excision. Given the definitive
nature of the latter approach and its widespread availability,
this will not prove easy to accomplish in the short-term.
The evolution of new approaches to deliver agents that alter
the function of specific molecules involved in skin neo-
plasms, however, may provide new future strategies to the
management of these common human malignancies.
Acknowledgements
We thank N. Griffiths and P. Bernstein for administrative
support. This work was supported by the USVA office of
Research and Development and by NIH AR44012, AR43799
to P.A.K.
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