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Cancer Detection and Prevention 29 (2005) 46–53

www.elsevier.com/locate/cdp

The role of folates in squamous cell carcinoma of the head and neck
Madeleine A. Kane MD, PhD*
Division of Medical Oncology, University of Colorado Health Sciences Center and the Denver Veterans Affairs Medical Center,
Denver VA Medical Center (111F), 1055 Clermont Street, Denver, CO 80220, USA
Received 29 July 2004; accepted 10 August 2004

Abstract

The primary objective of this review is to explore the hypothesis that folate insufficiency may be important in the pathogenesis of
squamous cell carcinomas of the head and neck (SCCHN) and that folate repletion may be an effective component of chemoprevention. The
main results are that folate insufficiency disrupts DNA global and specific gene methylation patterns such that the activity of certain tumor
suppressor genes such as p16 and possibly p53 may be lost. Folate pool imbalance and impaired repair mechanisms may contribute to DNA
instability and strand breaks. Sensitive methods exist for identification of individuals with folate insufficiency in contrast to the relatively
insensitive conventional serum or red cell folate assays with broad ‘‘normal’’ ranges. The impact of folate supplementation can thus be
quantified. Folate imbalance may result from alterations in folate cellular uptake by the reduced folate carrier (RFC) and/or the folate receptor
(FR) and polymorphisms in enzymes important in folate retention such as folylpolyglutamate synthetase and in folate modification such as
methylene tetrahydrofolate reductase (MTHFR). Known predisposing factors for SCCHN such as alcohol and tobacco carcinogens may
influence folate balance. Folate supplementation may reduce primary or secondary risk of cancer. Formal studies of folate sufficiency in
persons at risk for or diagnosed and treated for SCCHN are needed to define the role of folate supplementation in the prevention of these
cancers.
# 2004 International Society for Preventive Oncology. Published by Elsevier Ltd. All rights reserved.

Keywords: Folate; Head and neck cancer; Squamous cell carcinoma; Chemoprevention

1. Introduction [2]. Factors which seem to promote this field cancerization


include environmental exposures to tobacco smoke and
1.1. Purpose alcohol, viral infections especially with human papilloma
virus (HPV) subtypes 16 and 18, and deficiencies or
The aim of this review is to explore the hypothesis that imbalances in vitamins and micronutrients such as folic acid,
folate insufficiency may be important in the pathogenesis of vitamins A, C and E, and selenium and zinc [3]. Alterations
squamous cell carcinomas of the head and neck (SCCHN) in DNA integrity and normally regulated transcription may
and that folate repletion may be an effective component of result from: changes in global and specific gene DNA
chemoprevention. methylation patterns, misincorporation of bases due to
Squamous cell carcinomas of the head and neck develop nucleotide pool imbalances, DNA strand breaks and faulty
from an epithelium subject to field cancerization, concep- DNA repair mechanisms [4]. Antony presents a full
tually thought of as widespread biologic alterations that discussion of the complexity of folate metabolism [5]. All
predispose to the development of cancer [1]. Thus, people of these processes require homeostasis of single-carbon
who develop these SCCHN remain at risk for recurrent transfer reactions. Insufficient folate status can increase the
tumors or second primary tumors (SPT) in other sites of this frequency at which these changes to DNA occur, leading to
aerodigestive epithelium, including the lung and esophagus transforming changes in cells [6]. Folate insufficiency
results in global DNA hypomethylation with examples of
* Tel.: +1 303 399 8020x3129; fax: +1 303 393 5050. gene-specific hypermethylation such as p16 [7,8], a tumor
E-mail address: madeleine.kane@uchsc.edu. suppressor frequently inactivated or lost in SCCHN [1].

0361-090X/$30.00 # 2004 International Society for Preventive Oncology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.cdp.2004.08.002
M.A. Kane / Cancer Detection and Prevention 29 (2005) 46–53 47

Folate supplementation can correct folate insufficiency [9] interest, exposure of cultured SCCHN cells to retinoids
which may reduce the risk for SCCHN. This paper reviews resulted in decreased epidermal growth factor receptor
the role of folates in the carcinogenic process in SCCHN and expression [15].
the potential chemopreventive impact of folate repletion. Vitamin E was effective in prevention of experimental
oral cancers in the dimethylbenzanthrene (DMBA)-treated
hamster buccal pouch model of oral carcinogenesis, and
2. Materials and methods expression of wild type p53 was improved [16]. Selenium
may also help prevent SCCHN. Age-adjusted death rates for
2.1. Search methods head and neck cancers are lower in states where the soil and
forage crops contain higher levels of selenium. Selenium
The information methods used to obtain published works levels were observed to be significantly lower in the sera of
for this review were keyword searches of Medline using patients with SCCHN compared with healthy controls [17].
Ovid Technologies. Approximately one-third of published Another study reported higher serum selenium levels, but
references found were cited in this review. Various lower erythrocyte selenium levels in 50 SCCHN patients
combinations were searched using the keywords folate, [18]. No clinical chemoprevention studies using folates or
folic acid, head and neck cancer, individual anatomic sites selenium have been reported in SCCHN.
combined, squamous cell carcinoma, chemoprevention,
DNA methylation, epidermal growth factor receptor 3.1.2. Postulated mechanisms of carcino
(EGFR), p16, p53, carcinogenesis. genesis for SCCHN
Increased risk for SCCHN is associated with smoking,
ethanol consumption, certain HPV subtypes, dietary factors
3. Results and possibly several genetic factors (family history, certain
polymorphisms in metabolizing enzymes such as glu-
3.1. Criteria for evaluation of validity of studies tathione S-transferase, sensitivity of DNA in peripheral
lymphocytes to agents causing DNA strand breaks and
Because the goal of this review is synthesis of a efficiency of repair) [19–21]. As in other cancers such as
hypothesis, studies were selected for citing based on their colon cancer, sequential changes in genomic DNA precede
relevance to this purpose. The evidence is summarized and accompany development of frank invasive cancer.
descriptively into a logical framework looking at each Sidransky [1] has adapted Vogelstein’s model of colon
relevant component for the foundation of this hypothesis. carcinogenesis to be used for further study in SCCHN
Nutritional studies were selected based on same assessment although he points out that it is the accumulation of changes
of effect (usually odds ratio (OR)). and not necessarily the sequence of changes which leads to
neoplasm.
3.1.1. Natural history of SCCHN Some of the biologic changes frequently observed in
In the United States, more than 40,000 new cases of SCCHN involve both growth-promoting biomolecules and
SCCHN will be diagnosed this year, and 12,000 people will tumor suppressors. EGFR and its ligand are frequently over-
die from this malignancy [10]. Almost two-thirds of patients expressed [22], as is Cyclin D [23]. High levels of cox-2
will have advanced disease (stage III or IV) at diagnosis, and have been reported [24,25]. Diminished activity of the tumor
even with aggressive combined modality treatment, 5-year suppressors p53 and/or p16 activity is also frequently
survival rates are less than 50%, and SPT are not uncommon observed [26,27]. For example, approximately 50% of
[2]. The annual incidence of SPT in patients followed for 5 SCCHN harbor mutations in the conserved region (exon 5–
years was observed to be 4.3% in patients with original stage 9) of p53. Although only 10–15% of SCCHN show
I tumors and 6.4% in stage II. mutations in the p16 gene, homozygous deletion of 9p21,
Chemoprevention for SCCHN is an area of great interest. where p16 gene is located, occurs in about 25% of invasive
Randomized trials have reported effectiveness of 13-cis- tumors and is one of the changes also detected with high
retinoic acid in the treatment of oral pre-malignancy frequency in dysplasia and carcinoma in situ. Hypermethy-
(leukoplakia and dysplasia), but relapse occurred at a high lation of the intact p16 gene promoter also can reduce p16
rate (50%) once treatment was stopped [11]. In a small expression in SCCHN [28].
single institution study, 13-cis-retinoic acid also signifi-
cantly reduced incidence of SPTs compared with placebo in 3.1.3. Folate influence on common molecular
patients who had previously been treated for a SCCHN, but changes in SCCHN
no significant improvements in survival were observed Adequate levels and forms of folates are essential for
because there was no reduction in local, regional or distant intracellular methylation reactions [6]. These reactions in
recurrences [12]. More recent, larger multi-institutional turn are key steps in purine and pyrimidine biosynthesis and
studies from Europe employing retinyl palmitate [13] and thus impact nucleotide pool balance for DNA and RNA
from the U.S. [14] did not observe this benefit, however. Of synthesis. Insufficient folate levels result in competition
48 M.A. Kane / Cancer Detection and Prevention 29 (2005) 46–53

between de novo nucleotide synthesis and other biologic linked in SCCHN. EGFR is currently a target for several
methylation reactions, perturbing normal DNA synthesis, novel agents being tested in the clinic in SCCHN patients.
and in particular increasing the likelihood that uridine will
be incorporated into DNA in place of thymidine. In addition, 3.1.3.4. Tobacco carcinogens. A significantly reduced rate
post-synthetic methylation of cytidine residues (in 50 CpGs) of DNA excision repair was observed in isolated colonocytes
in DNA is critical to normal control of gene expression and from folate-deficient rats compared with rats fed a replete-
also maintenance of DNA integrity. In the absence of folate diet whether or not dimethylhydrazine (DMH) was
adequate folates, both non-specific and specific changes in administered [37]. Thus, folate insufficiency may result in
DNA methylation contribute to aberrant promoter function. an increase in the DNA strand breaks and other alterations
DNA strand breaks, especially in so-called fragile sites, may produced by tobacco carcinogens such as benzo[a]pyrene
occur more frequently as well [29,30]. and 4-(methylnitrosamine)-1(3-pyridyl)-1-butanone (NNK)
[38,39].
3.1.3.1. p53. After dietary folate depletion in a rat colon
tumor model, the promoter region of the p53 tumor 3.1.3.5. Ethanol effects. Ethanol significantly lowered the
suppressor gene (the most frequently mutated gene in levels of the reduced folate carrier (RFC) in the hepatocytes
human solid tumors) was hypomethylated in the same region of ethanol-fed micropigs [40]. The mechanism of this effect
as the highest number of mutations has been found [31]. has not been further explored, and whether a similar effect
Dietary folate deficiency produced progressive accumula- was seen in the cells of the aerodigestive epithelium was not
tion of DNA strand breaks in exons 5–8 of the p53 gene. studied [41]. If such a reduction in RFC is seen in epithelial
Decreased p53 transcripts were also observed compared cells, this could begin to explain a possible role for folate
with p53 transcripts in rats supplemented with folate. The insufficiency in the increased risk of SCCHN in heavy
single site methylation of 450 nt upstream in the p53 drinkers, especially those with poor nutritional status.
promoter region resulted in 85% suppression of promoter
activity in a reporter construct. Further de novo methylation 3.1.4. Folate status and SCCHN
of this promoter sequence was observed 48 h after Diets high in fresh vegetables seem to be protective
transfection into cultured mammalian cells [32]. against SCC of the aerodigestive epithelium. Nutritional
studies have suggested a role for folates as a major nutrient
3.1.3.2. p16. Methylation of p16 gene in its 50 CpG region in many fruits and vegetables in SCC in multiple sites of the
has been observed in one-quarter or more of invasive upper aerodigestive tract mucosal continuum as summarized
SCCHN and is associated with complete block of p16 in Table 1. Early studies reported protein–caloric malnutri-
transcription [27,33,34]. Lack of p16 staining by immuno- tion in almost 50% of patients diagnosed with SCCHN,
histochemistry was observed in most primary lesions, as especially in those with a history of heavy alcohol
well as in the most advanced pre-malignant lesions; thus, consumption and smoking [53].
loss of p16 activity likely plays a major role in SCCHN Clinically low folate levels have been associated with
progression. Since suboptimal folate levels result in cancers of the colon, lung, esophagus, cervix, breast and
abnormal methylation patterns, a possible link to altered pancreas [54–60]. Several case-control and prospective
folate status may be postulated for lack of p16. For example, cohort studies have shown significant protection against
in the rat folate/methyl-deficient model of carcinogenesis, colorectal cancers in patients with higher folates [61].
site-specific de novo methylation of 50 CpG islands in the p16 Similar studies in cervical SCC have been less consistent.
gene preceded actual tumor development [7]. Extensive p16 Folate levels as well as vitamin B12 were significantly lower
promoter methylation was found in 100% of tumors. in samples of squamous cell carcinoma of the lung compared
with adjacent histologically normal tissue samples from the
3.1.3.3. EGFR and transforming growth factor alpha same patient [62]. Global radiolabeled methylation of DNA
(TGF-alpha). Overexpression of EGFR and TGF-alpha was significantly higher in the SCC samples and was
has been linked to SCCHN prognosis and tumor-free inversely correlated with folate levels in both cancerous and
survival. Higher than usual extracellular folate concentra- normal tissues, suggesting folate might be the limiting
tions resulted in reductions in levels of EGFR phosphoryla- vitamin for proper DNA methylation in SCC and tissues at
tion, EGFR protein and TGF-alpha protein levels in cultured risk [63]. Mean folate levels in 42 newly diagnosed SCCHN
human colon carcinoma cell lines; the molecular mechan- cancer patients were recently reported to be significantly
isms for these observations have not been further reported lower (mean, 5.8  2.1 ng/ml) compared with smoking or
[35]. However, if EGFR/TGF alpha are overexpressed in low non-smoking controls (9.1  2.7 ng/ml and 9.7  2.2 ng/
folate conditions, the frequent overexpression of EGFR seen ml, respectively) [64].
in SCCHN suggests that folate repletion might help reverse The concept of folate sufficiency has been defined as
this growth-promoting situation. Amplification of EGFR distinct from the conventional notion of normal folate levels
gene in association with loss of p16 expression has been [9]. The impact of folate supplementation on tissue folate
reported in malignant brain tumors [36] and may also be levels was studied to determine whether supraphysiologic
M.A. Kane / Cancer Detection and Prevention 29 (2005) 46–53 49

Table 1
Nutritional studies implicating high consumption of fresh vegetables and reduction in risk of SCC cancer of the aerodigestive epithelium
Primary site N (S/C) Criteria Effect References Comments
Larynx 148/444 Raw vegetables (heavy smoking + low OR 0.29 (OR 9.2) [42] Case-control; Uruguay
fruits/vegetables)
Hypopharynx + 346/11936 Raw vegetables Decreased OR [43] Case-control; Japan
esophagus (all men)
Nasal cavity + 60/414 Allium vegetables + oranges/tangerines 50% decrease [44] Case-control; Shanghai
sinuses
SCCHN 167/177 Mutagen sensitivity, Bleomycin test Increased OR [45] Case-control
(OR 4.95)
SCCHN/lung 19/– Dietary modification after curative Rx N/A [46] Feasible
SCC esophagus 206/687 Folates (Fiber, b-carotene, vitamins B6, C) Decreased OR [47] Adenocarcinoma esophagus +
stomach also decreased OR
SCC esophagus 159/159 Green salads OR 0.42 [48] Case-control; UK
(all women)
SCC esophagus 830/1779 Hot mate tea Increased OR [49] Case-control; South America
Vegetables Decreased OR
(Fruits, cereals, tea) (Decreased OR)
SCC esophagus 165/815 High b-carotene, vitamin C, alpha 40–50% decrease [50] Case-control; Sweden
tocopherol (? folates) (adenocarcinoma esophagus
also decreased)
SCC esophagus 304/743 Raw vegetables (pasta/rice 0.7; poultry 0.4; OR 0.3 [51,52] Case-control; Italy validated
citrus 0.4; other fruits 0.5; mono UFA 0.5; food-frequency questionnaire
carotene 0.3; lutein + zeaxanthin 0.4;
vitamin C 0.4; niacin 0.5)

doses of folate would raise tissue folate levels to potentially homocysteine (HC). DNA methylation in leukocytes might
toxic ranges and whether subclinical folate deficiency provide another functional indicator of folate sufficiency
(folate ‘‘insufficiency’’) could be identified based upon status.
response to folate supplementation. The subjects studied Polymorphisms in enzymes requiring folate coenzymes
were identified as at risk for folate insufficiency, since they may also influence risk. Altered methylene tetrahydrofolate
were entering a voluntary alcoholism rehabilitation pro- reductase (MTHFR) activity associated with different
gram. Tissue folate levels were determined on whole blood polymorphisms may predispose to elevated HC levels and
using a highly sensitive and reproducible mass spectrometric increased toxicity to antifol chemotherapy [66]. Certain
folate assay [65]. Folate sufficiency was defined by initial MTHFR polymorphisms have been associated with
tissue folate levels which could not be increased by increased risk of certain types of cancer, especially if folate
supplemental folate, indicating saturation of folate tissue status was suboptimal. In Chinese patients diagnosed with
storage mechanisms in these individuals and subsequent SCC of the esophagus, the CGTTT polymorphism was
excretion of excess folates. However, only one-quarter of associated with an increased odds ratio of 6.18 (p < 0.001);
these subjects were folate sufficient. Thus, a high percentage other polymorphisms had little or no association with OR
of these recovering alcoholics were folate insufficient, [67]. A significantly increased risk for gastric adenocarci-
raising the question of whether this finding might noma (OR 1.87) in another Chinese study was also reported
significantly correlate with increased risk of SCCHN in for this polymorphism, with gastric cardia subset with
alcoholics, whether this analysis could identify persons at greatest susceptibility (OR 2.47) [68]. Certain MTHFR
high risk and thus candidates for folate supplementation, and polymorphisms have also been associated with neural tube
what the underlying mechanisms for folate insufficiency defects in newborns [69]; this risk can be reduced by 70% by
might be in individuals who have ‘‘normal’’ folate levels by supplemental folate at 4 mg orally each day [70]. The most
conventional assays. effective dose and form for cancer chemoprevention have
A study of the effects of dietary folate on genomic DNA not been established [71], but 1 mg restored folate
methylation in older women (ages 60–85) also demonstrated sufficiency in most [9] and 4 mg orally is well tolerated
that serum folate levels in the usual normal range may not in pregnant women.
reflect metabolic folate sufficiency [8]. A significant Folate polyglutamate synthetase (FPS) is an enzyme
decrease in leukocyte global DNA methylation was critical to retention of folates within the cell, since in the
observed after 7 weeks of a low folate (118 mg/day) diet. monoglutamated form, they readily diffuse out of the cell,
However, after 7 weeks of a folate-replete (200 mg/day or but are trapped intracellularly once the polyglutamate side
415 mg/day), DNA methylation had not returned to baseline chain is lengthened by this enzyme [72]. Several observa-
pre-study levels despite normalization of serum folate and tions suggest that polymorphisms in this enzyme could be
50 M.A. Kane / Cancer Detection and Prevention 29 (2005) 46–53

important in folate sufficiency as measured by tissue folates. ‘‘insufficiency’’, and no trials of folate supplementation
For example, some individuals experience severe toxicity to have been performed in people at risk or diagnosed with
antifols which cannot be predicted based on conventional SCCHN.
assessments of folate status. Folate insufficiency due to less-
than-saturated tissue folate levels could be the cause. This 4.1.1. Summary of theoretical basis for the
might relate to less active FPS polymorphisms because the role of folates in SCCHN
most common folate, 5-methylene tetrahydrofolate, is not Nutritional observations predict a higher likelihood of
easily glutamated unlike the rest of the folate coenzyme SCC including SCCHN development in people who
forms. Thus, polyglutamated 5-MTHF mostly occurs consume less raw vegetables (which have significant folate
through the folate cycle methylation of polyglutamated content). People diagnosed with SCCHN often have poor
THF to polyglutamated 5-MTHF. If FPS were less active, overall nutritional status and are more likely to be folate
much less of 5-MTHF will be retained intracellularly, and deficient [53]. Lower serum folate levels in the blood of
these individuals would be more susceptible to any patients with SCCHN have been observed [64]. Clinical
additional perturbations of folate status or folate-related case-control and prospective trials have established the
processes. impact of folate on chemoprevention in colon cancer [4], but
no trials have addressed folate impact on SCCHN.
Fig. 1 shows a hypothetical schema for the role of folates
4. Discussion in SCCHN. Biochemically, insufficient folate status results
in reduction in intracellular one-carbon transfer reactions
4.1. Criteria for conclusions and recommendations including production of thymidine from uridine. Lower
intracellular concentration of thymidine results in altered
This model is based on a synthesis of relevant published nucleotide balance and misincorporation of bases into DNA,
works into a testable hypothesis. The recommendation of with increased likelihood of DNA strand breaks. Low folate
clinical testing of folate repletion as a chemopreventive has also been associated with impaired DNA repair
mechanism in SCCHN is based on suggestive correlative processes, again partly a result of altered nucleotide pools.
evidence for other tumors, literature reporting insufficient Global and specific alterations in DNA methylation also
folate status in people at risk or diagnosed with SCCHN, occur, resulting in increased or decreased activation of
evidence that currently used methods to detect folate specific genes. Under low folate conditions, global DNA
‘‘deficiency’’ are not sensitive enough to detect folate hypomethylation is observed, but site-specific de novo

Fig. 1. Hypothetical role for folate in SCCHN development.


M.A. Kane / Cancer Detection and Prevention 29 (2005) 46–53 51

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