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Theodoros N. Teknos, M.D.,
Department of Otorhinolaryngology, Head and Neck Surgery Division
Angiogenesis is the process of new blood vessel formation.
It is encountered in essential physiologic processes (e.g.,
endometrial proliferation, embryogenesis, wound healing),
as well as in countless pathologic conditions (e.g., rheumatoid
arthritis, diabetic retinopathy, neoplastic disease) (1).
Dr. Judah Folkman pioneered the concept that primary or metastatic
tumors must become vascularized to exceed 2 mm in size (2).
Based on extensive animal data, Folkman et al., found that
tumor angiogenesis must precede tumor growth and thus, tumor
progression can only occur when neoplasms switch from a prevascular
to an angiogenic phase (3). The process
of angiogenesis, however, is complex and is mediated by the
delicate balance between pro-angiogenic and anti-angiogenic
molecules. The activation of the angiogenic "switch" relies
on a relative preponderance of pro-angiogenic molecules, such
as vascular endothelial growth factor (VEGF), basic fibroblast
growth factor (bFGF), interleukin-8 (IL-8), platelet derived
growth factor (PDGF), angiogenin and hepatocyte growth factor,
to name a few (4,5).
Angiogenesis in Head and Neck Cancer
The requirement of angiogenesis
for tumor growth is rarely debated; however, the utility in
using the level of angiogenesis as a prognostic indicator
is point of great debate. Specifically in head and neck squamous
cell carcinoma, most studies to date have attempted to correlate
intratumoral microvessel density with tumor recurrence and
survival. Although not incontrovertible, a preponderance of
evidence suggests that increased tumor vascularity is associated
with loco-regional recurrence, distant metastases and poor
prognosis (6-12). Several studies in our
laboratory have shown that advanced laryngeal squamous cell
carcinomas with a high level of vascularity tend to behave
more aggressively, be more resistant to cytotoxic therapy
and have decreased overall survival compared to those with
a low level of vascularity (13).
The degree of expression of various
pro-angiogenic cytokines has also provided valuable prognostic
information on various solid tumors. Overexpression of VEGF,
for instance, has been correlated with poor survival rates
in esophageal, gastric, colorectal, pancreatic, lung and breast
carcinomas (14). Our laboratory recently
studied serum VEGF levels in a large number of normal control
subjects and patients with advanced laryngeal carcinoma treated
in the Veterans Administration Laryngeal Cancer Study Group
Protocol #268. In this protocol, patients with stage III or
IV laryngeal cancer were randomized to receive either conventional
therapy (total laryngectomy with postoperative radiation therapy)
or induction chemotherapy followed by radiation therapy. In
the latter arm, surgical salvage was performed on those patients
who failed to respond to two cycles of chemotherapy or on
those with persistent or recurrent disease at the completion
of treatment (15). Not surprisingly,
the serum levels of VEGF were significantly different between
the healthy volunteers vs. the patients with advanced laryngeal
carcinoma (47.8pg/ml vs. 317pg/ml, p>0.001). More importantly,
however, high serum VEGF levels were predictive of poorer
overall survival regardless of the treatment employed (p=0.0018).
A multivariate analysis was also performed comparing VEGF
levels to all known risk factors for recurrence including:
T stage, N stage, tumor growth pattern, site of tumor, treatment
used and patient age. This analysis revealed that serum VEGF
levels are the best independent predictor of poor survival
and patients with levels above the mean had a 47% increased
risk of death (p=0.065).
Copper Suppression as
an Anti-angiogenic Approach
Based on all the available data,
the level of angiogenesis may play a critical role in the
development and progression of human squamous cell carcinoma.
Utilization of an anti-angiogenic compound, therefore, may
prove very beneficial to patients suffering from this relentless
form of cancer. To date, there have been no reports in the
literature of a single agent, anti-angiogenic compound effective
against head and neck squamous cell carcinoma. There is, however,
a growing list of angiogenesis inhibitors under investigation
in a variety of tumor types. These include angiostatin, endostatin,
thalidomide, linomide, TNP-470, SU1444, SU1498 and monoclonal
antibodies against fibroblast growth factor, VEGF and avB3
integrin, to name a few. Due to the numerous steps involved
in angiogenesis, any disruption of this process will require
innovative strategies that inhibit multiple steps in the angiogenesis
pathway. Copper suppression therapy may prove to be one such
Copper plays a key role in multiple
steps along the angiogenesis pathway. It is an essential co-factor
for such molecules as bFGF, VEGF and angiogenin (16-18).
In the absence of copper, these cytokines cannot function
and neovascularization is abated. A number of intracerebral
tumor models have utilized this strategy, employing penicillamine
and a low copper diet to successfully reduce tumor growth
(19, 20). A low-copper diet is not
possible in humans due to ubiquitous nature of copper in food.
Tetrathiomolybdate (TM), however, is a powerful copper chelator
that is well tolerated when taken orally. Dr. George Brewer
developed TM at the University of Michigan for the treatment
of patients with Wilson's disease, an autosomal recessive
disease of copper transport that results in abnormal copper
accumulation and toxicity. TM's ability to reduce copper stores
involves at least two mechanisms: the first is the formation
of a complex together with food proteins in the gastrointestinal
tract, blocking the absorption of copper (21).
The second is that the absorbed TM forma a tripartite complex
with copper and albumin in blood, rendering the copper unavailable
for cellular uptake and therefore removing it from use in
angiogenesis. This tripartite complex has no biologic activity
and is excreted via the biliary tract and urine (21).
The low toxicity profile of TM is possible because the levels
of copper required for angiogenesis is higher than that required
for essential copper dependent processes, such as heme synthesis,
superoxide dismutase and cytochrome function (21).
Merajver and Brewer et al., showed dramatic efficacy of TM
as an anti-angiogenic compound for the treatment of inflammatory
breast cancer in a HER2/Neu transgenic mouse model (22).
In our studies, we found that mice treated with TM after the
establishment of a very aggressive murine squamous cell carcinoma
had a dramatic decrease in tumor growth rate and significant
reduction in tumor vascularity (Fig. 1) (23).
Clearly, TM was effective after tumors had been established
and the angiogenic "switch" had been thrown. To investigate
its use as a chemopreventative agent, we initially made mice
copper deficient compared to controls then implanted tumor
cells. As Figure 2 shows, tumors remained very small in size
compared to controls up to day 55. When TM was removed from
the mice's drinking water, the tumors rapidly enlarged, approaching
the growth rate of the untreated subjects (24).
Ongoing studies have shown similar effectiveness in human
tumors established in immunodeficient SCID mice (Figure 3)
(25). Based on these preclinical
findings, clearly there is ample evidence to support the use
of TM in humans with squamous cell carcinoma of the head and
neck. In collaboration with Drs. Francis Worden, Susan Urba,
George Brewer, Sofia Merajver and others, a phase II clinical
study using TM in patients with metastatic unresectable squamous
cell carcinoma is presently being organized at the Univer-sity
of Michigan Comprehensive Cancer Center.
Squamous cell carcinoma of the head and neck continues to
be a difficult disease process to treat. Despite dramatic
advances in surgical and non-surgical treatment options, the
cure rates remain unchanged over the past 30 years. Angiogenesis
inhibitors are a novel and very encouraging treatment approach
to these tumors. Continued investigations and the collaborative
effort of all physicians caring for head and neck cancer patients
will be necessary to determine if these approaches will ultimately
1. Folkman J. Angiogenesis in cancer,
vascular, rheumatoid, and other disease. Nat Med. 1995;1:27-31.
2.Folkman J. What is the evidence that
tumors are angiogenesis dependent? J Natl Cancer Inst. 1990;82:4-6.
3. Hanahan D, Folkman J. Patterns
and emerging mechanisms of the angiogenic switch during tumorigenesis.
4.Folkman J. Tumor Angiogenesis. In: Mendelsohn
J, Howley PM, Israel MA, Liotta LA, eds. The Molecular Basis
of Cancer. Philadelphia: W.B. Saunders; 1995:206-32.
5.Folkman J, Klagsburn M. Angiogenic factors
(review). Science. 1987;235;442-7.
6.Albo D, Granick MS, Jhala N, Atkinson
B, Solomon MP. The relationship of angiogenesis to biological
activity in human squamous cell carcinomas of the head and
neck. Ann of Plast Surg. 1994;32(6):588-94.
7.Gasparini G, Weidner N, Maluta S, et
al. Intratumoral microvessel density and p53 protein: correlation
with metastasis in head-and-neck squamous-cell carcinoma.
Int Journal Cancer. 1993;55(5):739-44.
8.Murray JD, Carlson GW, McLaughlin K,
et al. Tumor angiogenesis as a prognostic factor in laryngeal
cancer. Am J Surg. 1997;174(5):523-6.
9.Pazouki S, Chisholm DM, Adi MM, et al.
The association between tumour progression and vascularity
in the oral mucosa. J Pathol. 1997;183(1):39-43.
10.Eisma RJ, Spiro JD, Kreutzer DL. Vascular
endothelial growth factor expression in head and neck squamous
cell carcinoma. Am J Surg. 1997;174(5):513-7.
11.Maeda T, Matsumura S, Hiranuma H,
Jikko A, Furukawa S, Ishida T, Fuchihata H. Expression of
vascular endothelial growth factor in human oral squamous
cell carcinoma: Its association with tumour progression and
p53 gene status. J Clin Pathol. 1998;51(10):771-5.
12.Moriyama M, Kumagai S, Kawashiri S,
Kojima K, Kakihara K, Yamamoto E. Immunohistochemical study
of tumour angiogenesis in oral squamous cell carcinoma. Oral
13.Teknos TN, Cox C-, Barrios MA,
et al. Tumor angiogenesis as a predictive marker for organ
preservation in patients with advanced laryngeal carcinoma.
The Laryngoscope. In press.
14.Tae K, El-Naggar AK, Yoo E, et
al. Expression of vascular endothelial growth factor and microvessel
density in head and neck tumorigenesis. Clin Cancer Res. 2000;6:2821-8.
15.Teknos TN, Cox CC, Yoo SK, Wolf
GT, Fischer SG. Serum vascular endothelial growth factor correlates
with outcome in the V.A. laryngeal cooperative study Group
Protocol #268. (Unpublished data)
16.Watanabe T, Seno M, Sasada R, Igarashi
K. Molecular characterization of recombinant human acidic
fibroblast growth factor produced by E. coli: comparative
studies with human basic fibroblast growth factor. Mol Endocrinol.
17.Patstone G, Maher P. Copper and
calcium binding motifs in the extracellular domains of fibroblast
growth factor receptors. J Biol Chem. 1996;271:3343-6.
18.Ziche M, Jones J, Gullino PM. Role
of prostaglandin E and copper in angiogenesis. J Natl Cancer
19.Brem SS, Zagzag D, Tsanaclis AMC,
Gately S. Inhibition of angiogenesis and tumor growth in the
brain. Am J Pathol. 1990;137:1121-42.
20. Brem S, Tsanaclis AMC, Zagzag D.
Anticopper treatment inhibits pseudopodial protrusion and
the invasive spread of 9L gliosarcoma cells in the rat brain.
21.Brewer GJ, Dick RD, Yuzbasiyan-Gurkin
V, Tankanow R. Initial therapy of patients with Wilson's disease
with tetrathiomolybdate. Arch Neurology. 1991;48:42-7.
22.Merajver SD, Irani J, van Golen
K, Brewer GJ. Copper depletion as an anti-angiogenic strategy
in HER2-neu transgenic mice. Proc AACR Special Conference
on Angiogenesis and Cancer Research. 1998, B11.
23.Cox C, Teknos TN, Barrios M,
Brewer GJ, Dick RD, Merajver SD. The role of copper suppression
as an anti-angiogenic strategy in head and neck squamous cell
carcinoma. The Laryngoscope. In press.
24.Cox CC, Teknos TN, Merajver SD,
Brewer GJ. Tetrathiomolybdate as a chemopreventative agent
in squamous cell carcinoma. (Unpublished data.)
25.Teknos TN, Cox CC, Gupta A, et
al. Copper suppression in the treatment of human squamous
cell carcinoma. (Unpublished data.)
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