Sildenafil use and increased risk of incident melanoma in US men: a prospective cohort study

Abstract

Importance

The RAS/RAF/MEK/ERK signaling pathway plays a crucial role in melanoma cell proliferation and survival. Sildenafil (Viagra) is a phosphodiesterase (PDE) 5A inhibitor commonly used for erectile dysfunction. Recent studies have shown that BRAF activation down-regulates PDE5A levels, and low PDE5A expression by BRAF activation or sildenafil use increases the invasiveness of melanoma cells, which raises the possible adverse effect of sildenafil use on melanoma risk.

Objective

To evaluate the association between sildenafil use and risk of incident melanoma among men in the United States.

Design, Setting and Participants Design

In 2000, participants in the Health Professionals’ Follow-up Study (HPFS), an ongoing prospective study. were inquired regarding sildenafil use for erectile dysfunction. We excluded baseline cancers, type 2 diabetes, coronary heart disease, stroke, and hypertension. A total of 14,912 men were included.

Main Outcome Measure

Incidence of skin cancers, including melanoma, squamous cell carcinoma (SCC), and basal cell carcinoma (BCC) was obtained in the self-reported questionnaires biennially. The diagnosis of melanoma and SCC was pathologically confirmed.

Results

We identified 79 melanoma, 305 SCC, and 1,720 BCC cases during the follow-up (2000–2010). Sildenafil use was significantly associated with an increased risk of subsequent melanoma with a multivariate-adjusted hazard ratio (HR) of 2.24 (95% confidence interval (CI): 1.05–4.78). In contrast, we did not observe an increase in risk of SCC (HR = 0.80, 95% CI: 0.46–1.37), or BCC (HR = 1.05, 95% CI: 0.84–1.30) associated with sildenafil use. Moreover, erectile function itself was not associated with an altered risk of melanoma.

Conclusions

Sildenafil use may be associated with an increased risk of developing melanoma.

Introduction

Melanoma has become a major public health problem particularly in the Western world,¹ with 76,000 estimated new cases in the US in 2012.² The biology underlying melanoma development is complex, with the RAS/RAF/MEK/ERK signaling pathway playing a key role in melanoma cell proliferation and survival.³ Hyperactivation of ERK has been found in most human melanomas, commonly regulated through BRAF or NRAS somatic mutations.^3–4 Approximately 50% of melanoma tumors have BRAF mutations leading to elevated kinase activity, among which nearly 90% have the V600E mutation.^5–6 Drugs inhibiting this pathway, particularly those targeting BRAF, have indicated therapeutic efficacy.^6–7 The cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase (PDE) 5A was recently demonstrated as a downstream target of BRAF.^8–9 Through a MEK/ERK cascade, activated BRAF down-regulates PDE5A, which lowers cGMP degradation and leads to an increase in intracellular Ca²⁺, triggering invasion and metastasis of melanoma cells.^8–11 In contrast, rescuing expression of PDE5A in melanoma cells decreased their invasiveness.⁸ Down regulation of PDE5A was also seen in NRAS mutant cell lines, indicating that activation of the MAPK pathway leads to PDE5A downregulation of melanoma cell lines, irrespective of genetic background 8. PDE5A is the target of the drug sildenafil, commercially known as Viagra, which has been widely prescribed for erectile dysfunction (ED) in recent years.^12–13 Treatment with PDE5A inhibitors, such as sildenafil, can promote melanoma cell invasion, particularly in the BRAF-mutated melanoma cell lines.⁸ This indicates that PDE5A suppression by sildenafil use mimics one effect of BRAF/NRAS activation and thus may potentially function as one of the ‘hits’ for melanomagenesis. Most recently, two PDE5 inhibitors were shown to promote melanin synthesis,¹⁴ which may exacerbate melanoma development.¹⁵ These pieces of evidence prompted our hypothesis regarding the potential link between sildenafil use and melanoma development.¹⁶

No epidemiologic data has been reported on the effect of sildenafil use on melanoma risk. We sought to investigate the association between sildenafil use for ED and risk of incident melanoma in the Health Professionals’ Follow-up Study (HPFS). We compared the sildenafil use-associated risks for melanoma and non-melanoma skin cancers (predominantly comprised of squamous cell carcinoma (SCC) and basal cell carcinoma (BCC)), which are the most common human malignancies.¹⁷

Methods

Study population

The HPFS began in 1986 when 51,529 US male health professionals aged 40–75 years, completed a baseline questionnaire on medical history and lifestyle practices. Biennially, participants received a questionnaire and a response rate exceeding 90% has been achieved in the follow-up. The study was approved by the Human Research Committee at Brigham and Women’s Hospital (Boston, MA). Participants’ completion and return of the questionnaire was considered informed consent.

Assessment of exposure

The primary exposure was sildenafil use for erectile function problems. In 2000, the HPFS participants were queried whether they had had surgery or treatment to correct problems with erections during the past 3 months, including use of sildenafil (Viagra), shots or penile injection, vacuum suction, MUSE (alprostadil), and others, and whether they had had any treatment for ED ever before, including sildenafil and other medications, penile implant, vacuum suction, testosterone, MUSE, and others. The participants were also asked to rate their ability before 1986, 1986–1989, 1990–1994, 1995–2000, and during the past 3 months, without treatment, to have and maintain an erection adequate for intercourse. Response options included very poor, poor, fair, good and very good. Men with poor or very poor ability were considered to have ED.^18–19

Information on skin cancer risk factors was obtained in the self-reported questionnaires. Number of moles on arms with ≥3 mm diameter was asked in 1987. Natural hair color at age 18 years was collected in 1988. State of residence at birth, age 15, and 30 years, lifetime numbers of sunburn that blistered, and adolescent tendency to sunburn were asked in 1992. According to the state of residence reported, the ultraviolet (UV) index at birth, age 15, and 30 years was categorized into three categories respectively (≤5, 6, or ≥7). Family history of melanoma in first-degree relatives was collected in 1990 and 1992. Information on outdoor sun exposure in the middle of the day during summer months (between 10 am and 3 pm) was collected for high school/college, age 25–35, 36–59, and ≥60 years in 2008. Information on smoking, body mass index (BMI), and physical activity was collected biennially since the baseline.

Assessment of outcome

Since 1986, participants have reported diagnoses of melanoma, SCC, BCC, and other primary cancers on biennial surveys. When a diagnosis was reported, related medical records were requested with the participant’s permission and reviewed by physicians blinded to exposure to confirm their self-reported diagnosis. We excluded participants who denied the diagnosis or permission to review their records. For melanoma, SCC, and the other non-skin primary cancers, only those pathologically confirmed invasive cases were included in this study. We did not seek to obtain the medical records for all the self-reported cases of BCC, but previous studies have indicated a high validity of BCC self-reports, with more than 90% confirmed by histopathology records.^20–21

Statistical analysis

Men who answered the questionnaire in 2000 served as the base population for our analyses (n=39,006). We excluded those with missing date of birth (n=7), diagnosed with cancers (including melanoma, SCC, BCC, and non-skin cancers, n=10,323) at baseline, diagnosed with other major chronic diseases (including diabetes, coronary heart disease, stroke, and hypertension, n=12,972), and users of the other therapies but not sildenafil for ED (n=97). We further excluded all non-Caucasians (n=695) and 14,912 participants remained in our study. We excluded those reporting cancer, cardiovascular disease, type 2 diabetes, and hypertension at baseline because the sexual intercourse and sildenafil use may have been affected by the health status and PDE5A inhibitors have shown their efficacy in diseases other than ED, such as pulmonary hypertension, cardiac hypertrophy, and some other diseases.^12,22–24

We compared the distribution of major characteristics between sildenafil users and non-users using Student’s t-test or chi-square test. Person-years of follow-up for each participant were calculated from the return date of the 2000 questionnaire to the date of diagnosis of melanoma or non-melanoma skin cancers, death, the last questionnaire response, or end of the follow-up (Jan 2010), whichever came first. Cox proportional hazards analysis stratified by age and follow-up interval was performed to calculate the age and multivariate-adjusted hazard ratios (HRs) and 95% confidence intervals (CIs). We evaluated the sildenafil recent use (in the past three months) as the primary exposure. Multivariate models were adjusted for age, BMI, smoking, physical activity, childhood reaction to sun, the number of severe sunburns, mole count, hair color, family history of melanoma, sun exposures at high school, age 25–35, age 36–59, and age≥60, UV index at birth, age 15, and age 30, as well as other treatment for erectile function problems. An indicator variable was created for the missing category of each covariate. We adjusted for these covariates because they are well-defined factors associated with melanoma or sildenafil use. One factor can be a confounder because it changes the effect of the exposure of interest when included in the model or when included with other covariates.²⁵ We evaluated the age-standardized absolute risk associated with sildenafil use. Population-attributable risk and 95% CI were calculated to estimate the percentage of melanoma cases that would be prevented by eliminating sildenafil use.

In the secondary analysis, we evaluated sildenafil use ever before (defined as all current and prior users) as the main exposure and adjusted for the aforementioned covariates. In additional analysis, we excluded all sildenafil users who also used other therapeutic options for ED to eliminate the confounding effects of these therapies. We also performed a lag analysis by excluding the outcomes occurring in the first follow-up period (2000–2002) to clarify the temporal relationship between sildenafil use and the occurrence of skin cancers.

To further explore whether the overall erectile function (very good, good, fair, or ED) was associated with risk of subsequent skin cancers, we conducted the analysis excluding users of sildenafil and other treatments for ED, and adjusting for the aforementioned covariates. In these analyses, men who did not report the erectile function were further excluded. To clarify the effect of sildenafil use on other cancers, we evaluated the risk of incident total non-skin cancers as well as the major individual cancers according to sildenafil use, adjusting for age, BMI, smoking, physical activity, UV index at birth, age 15, and age 30, multi-vitamin use, physical examination in the last two years, and other treatments for erectile function problems.

Analyses were carried out by using SAS (version 9.2; SAS Institute Inc, Cary, NC). All P values were 2-tailed with the significance level set at P <0.05.

Results

Baseline characteristics of 14,912 participants are shown in Table 1. The mean age at baseline was 63.7 (SD 8.6) years. 4.9% (727/14,912) reported taking sildenafil for erectile function problems recently and 5.7% (843/14,912) reported use ever before. Those using sildenafil were more likely to be older and obese, have family history of melanoma, and take physical examinations, but were exposed to less sun light as adults (age 25–59 years). Sildenafil users had a markedly higher proportion of men with ED.

Table 1.

Baseline characteristics of the study population according to the recent use of sildenafil for erectile dysfunction: HPFS (2000)

	Recent sildenafil use		P value
	No (n=14,185)	Yes (n=727)
Age*, mean (SD), year	63.6 (8.6)	65.4 (7.6)	<0.0001
Body mass index, kg/m2, mean (SD)	25.0 (5.3)	25.5 (4.3)	0.01
Physical activity, metabolic equivalent hours/wk, mean (SD)	36.4 (40.3)	35.1 (33.3)	0.40
Current smoking (%)	809 (5.7)	52 (7.2)	0.38
Family history of melanoma (%)	667 (4.7)	47 (6.5)	0.04
Burn or blistering skin reaction to the sun (%)	8401 (68.6)	451 (71.6)	0.91
UV index of residence at birth ≥7 (%)	3370 (28.2)	174 (27.6)	0.62
UV index of residence at age 15 yrs ≥7 (%)	3614 (30.0)	187 (29.3)	0.62
UV index of residence at age 30 yrs ≥7 (%)	4169 (35.4)	243 (39.3)	0.09
Natural red or blonde hair (%)	1513 (13.1)	74 (11.4)	0.28
≥6 moles on the arms, 3+ mm diameter (%)	462 (4.7)	25 (4.3)	0.93
History of ≥6 severe or blistering sunburns (%)	4398 (35.8)	260 (38.6)	0.05
Sun exposure ≥11 hours/wk in college/high school (%)	4998 (51.3)	284 (51.3)	0.43
Sun exposure ≥11 hours/wk at age 25–35 yrs (%)	3137 (32.3)	153 (26.0)	0.009
Sun exposure ≥11 hours/wk at age 36–59 yrs (%)	2724 (28.2)	131 (22.5)	0.01
Sun exposure ≥11 hours/wk at age ≥60 yrs (%)	2654 (27.7)	135 (23.5)	0.05
Erectile dysfunction (%)	2414 (20.9)	421 (56.8)	<0.0001
Physical exam (%)	9869 (81.9)	625 (87.1)	<0.0001
Use of other erectile function treatment (%)	0 (0.0)	43 (5.9)	<0.0001
Ever use of sildenafil in the past (%)	116 (0.8)	727 (100.0)	<0.0001
Ever use of other erectile function treatment in the past (%)	116 (0.8)	249 (34.3)	<0.0001

^*All values other than age are age-adjusted. Proportions are based on non-missing values.

From 2000 to 2010, a total of 79 cases of melanoma, 305 of SCC, and 1,720 of BCC were documented. Recent sildenafil users had a significantly elevated risk of invasive melanoma with multivariate-adjusted HR (95% CI) of 2.24 (1.05–4.78). In contrast, we did not observe a significantly altered risk of SCC or BCC associated with sildenafil use; the HR (95% CI) was 0.80 (0.46–1.37) for SCC and 1.05 (0.84–1.30) for BCC (Table 2). The age-standardized absolute risk associated with sildenafil use was 90.4 cases per 100,000 person-years. We calculated the population-attributable risk assuming sildenafil use as the exposure that was linked with melanoma, and 3.7% (95% CI: 1.0%-6.5%) of the incident cases of melanoma might have been prevented by the elimination of sildenafil use.

Table 2.

The hazard ratio (HR) of incident melanoma, squamous cell carcinoma, and basal cell carcinoma associated with use of sildenafil, HPFS 2000–2010

	Person-Years	Cases	Age-adjusted HR (95% CI)	Multivariate-adjusted HR* (95% CI)
Melanoma
No-use	113,515	70	1.00	1.00
Use	5,774	9	2.37 (1.17–4.77)	2.24 (1.05–4.78)
SCC
No-use	111,692	291	1.00	1.00
Use	5,676	14	0.80 (0.47–1.37)	0.80 (0.46–1.37)
BCC
No-use	111,692	1,631	1.00	1.00
Use	5,676	89	1.00 (0.80–1.24)	1.05 (0.84–1.30)

^*Adjusted for age (continuous variable), body mass index (<24.9, 25–29.9, or ≥30 kg/m²), smoking (never, past, or current), physical activity (in quintiles, metabolic equivalent hours/wk), childhood reaction to sun (tan without burn, burn, or painful burn/blisters), times of sunburns (0, 1–2, 3–5, or ≥6), mole count (0, 1–2, 3–5, or ≥6), hair color (red, blonde, light brown, or dark brown/black), family history of melanoma (yes or no), sun exposures at high school, age 25–35, age 36–59, and age ≥60 (<5, 6–10, or ≥11 hours for each), UV index at birth, age 15, and age 30 (≤5, 6, or ≥7), as well as other treatment for erectile function problems.

In the secondary analysis, use of sildenafil ever before was associated with a higher risk of melanoma (HR=2.77; 95% CI: 1.32–5.85) (eTable 1). The association between sildenafil use and melanoma remained significant after excluding all users of other treatments for ED (HR=2.24; 95% CI: 1.05–4.78), or excluding the melanoma cases occurring in the first two years (HR=2.81; 95% CI: 1.28–6.19).

We examined the overall erectile function and subsequent risk of melanoma. Compared with those reporting very good function, we did not observe a significant change in risk among those with unfavorable erectile abilities (eTable 2). We further evaluated the association between sildenafil use and risk of total non-skin cancers, as well as the major individual cancers. We did not observe any association with these cancers (eTable 3).

Discussion

In this prospective study with 10 years of follow-up, we found that men who used sildenafil for erectile function problems had a significantly elevated risk of melanoma. The association remained fairly robust in the models controlling for the major host characteristics, outdoor sun exposure behavior, and UV index in the state of residence.

Through selective regulation of cGMP, the PDE5A plays an essential role in vascular smooth muscle contraction in the corpus cavernosum.²⁶ PDE5A inhibitors competitively inhibit the hydrolysis of cGMP, thereby leading to smooth muscle relaxation and penile erection.^12,24 Since the approved use of sildenafil (Viagra) in 1998 and vardenafil (Levitra) and tadalafil (Cialis) in 2003, PDE5A inhibitors have remained the first line therapy for most men with ED, and also been used for benign prostate hyperplasia and pulmonary arterial hypertension in recent years.^12,22 Adverse effects are generally mild,¹² although vision-threatening ocular complications and hearing loss have been reported.^22,27 Arozarena et al. recently found that PDE5A inhibition by sildenafil induces invasion of melanoma in vitro.⁸ Moreover, the oncogenic BRAF mutation was shown to down-regulate PDE5A through the RAS/RAF/MEK/ERK signaling pathway and PDE5A down-regulation can stimulate a dramatic increase in melanoma cell contractility and invasion, despite causing a slight decrease in proliferation.^8,11 Among the 10 BRAF-mutant cell lines, the authors did not find down-regulated PDE5A mRNA or protein expression in the 501mel cell line, in which sildenafil use induced invasion. In contrast, the authors did not observe increased invasion of A375 or WM266.4 cell lines by sildenafil use, while both were BRAF mutated and had suppressed PDE5A expression.⁸ These data suggest that either RAS/RAF activation or sildenafil can inhibit PDE5A activity. Sildenafil could act in the MEK/ERK downstream signaling, playing a role similar with one of those by RAS/RAF activation. The effect of sildenafil on melanoma may not involve the mutations or activation of RAS/RAF. It remains to be investigated whether the presence of both RAS/RAF mutations/activations and PDE5A inhibitors could trigger a synergistic or antagonistic effect on melanoma. By increasing intracellular cGMP levels, sildenafil could deregulate diverse physiological functions such as phototransduction, vascular permeability, proliferation and apoptosis,²⁸ which further suggests a biological plausibility underlying our observed associations between sildenafil use and melanoma. Cancer metastasis requires invasion in multiple steps and cellular adhesion to vasculature or anoikis resistance.²⁹ Therefore, sildenafil could induce invasion by fostering other cellular abilities. Braf-V600E mutation has been shown to induce both melanocyte senescence and melanoma in vivo.¹⁰ It would be interesting to examine whether PDE5A inhibition may play a role in either promoting or escaping melanocyte senescence, particularly in BRAF-driven melanomas. The finding that PDE5A downregulation leads to increased invasion in a BRAF specific manner may highlight the so-called “oncogene addiction” that prevails in melanomas with BRAF mutations.⁸ The lack of response of RAS-mutated melanomas to sildenafil in invasion assays may be due to the additional complexity of RAS-mutated melanomas,⁸ since RAS activates many other pathways in addition to the MAPK 30. Arozarena et al provided limited data on melanoma proliferation and other features of carcinogenesis, which requires further functional studies to clarify.⁸

It is worth noting that the previous study tested melanoma cell lines mostly of metastatic origin and did not test the effect of PDE5A inhibitors on invasive potential of cells from primary tumors.⁸ However, since primary tumors expressed substantially higher levels of PDE5A than metastatic melanomas, the effect may be even more marked. Also melanoma is highly heterogeneous in characteristics, unlike homogeneous cell lines,³¹ thus a small population of cells which respond strongly could be highly significant. Our study only included pathologically confirmed invasive melanomas in the radial and/or vertical growth phase, and thus strongly suggests a role for PDE5A inhibitors in primary melanoma development and progression. Given that PDE5A downregulation increased invasiveness, and that PDE5A expression was higher in primary tumors than in metastatic tumors, it is biologically very plausible that PDE5A inhibitors would play a role in promoting invasion of the primary tumors. Biological studies are needed to confirm the role of PDE5A inhibitors in primary melanoma invasion.

We carefully addressed the concern regarding the possible differences in health status and lifestyles practices between users and non-users which may confound the observed findings. Sildenafil use was associated exclusively with melanoma but not with either SCC or BCC, which thereby showed that the association with melanoma was less likely due to sun exposure, physical examinations or detection bias. The homogeneity of the cohort decreases the misclassification of work-related sun exposures or health awareness. The effect did not materially change after excluding all users of other therapies for ED. We further examined the risk of melanoma according to categories of erectile function and erection itself was not independently associated with melanoma risk. Together, these results suggest a positive association between sildenafil use and melanoma, regardless of other characteristics.

We acknowledge some limitations. First, sildenafil use was self-reported in 2000 and not updated during the follow-up. However, the healthcare-related professional background was reassuring and the relatively higher accuracy of their reports would have tended to cause non-differential misclassification of the exposure. Sildenafil is usually effective within 30–120 min after administration and may be cleared rapidly such that the PDE5A inhibition could be transient and reversible.^8,12 However, a 2-hour time window is sufficient for some drugs to produce critical transcriptional changes.⁹ PDE5A inhibitors have been the prime option for men with ED since the approval of sildenafil in 1998.¹² Among the recent users of sildenafil in 2000, 78% also used sildenafil ever before. One study in the HPFS has indicated a sharply increased risk of ED with aging.¹⁸ With the massive increase in sildenafil use following its introduction, among the recent users of sildenafil in 2000, the expected proportion of users after 2000 could be much higher than recent non-users, which may lead to a chronic downstream effect of PDE5A inhibition. Nevertheless, laboratory studies are warranted to elucidate whether either temporary or long-term use of sildenafil has the potential to induce irreversible change in host features and elevated risk of melanoma. For example, it remains to be confirmed that a single, or repeated, dose of sildenafil can cause sufficient inhibition of PDE5A in melanocytes to promote transcriptional changes of key genes. The misclassification of exposure would tend to affect those unexposed in 2000, leading to a conservative effect estimate. We do not have information about frequency and dosage of sildenafil use and are not able to indicate a dose-dependent association.

Second, we did not have the information on other PDE5A inhibitors because neither vardenafil (Levitra) nor tadalafil (Cialis) had been approved by FDA in 2000. The observed association between sildenafil use and melanoma might be partly attributed to the later use of vardenafil and tadalafil among recent sildenafil users. However, because of the similar roles that other PDE5A inhibitors may play, this would not materially jeopardize our study implications.

Third, an observational study cannot rule out the possibility of residual confounding by unmeasured or imperfectly measured confounders.

Fourth, our study had a modest sample size and the subgroup analyses by melanoma sites and known risk factors, particularly nevi,³² were underpowered. Previous studies have shown that melanoma on the trunk more frequently harbored BRAF mutations than those on the head and neck.³³ BRAF and NRAS mutations also tend to occur exclusively.⁴ It is unknown whether sildenafil use and BRAF/NRAS activation could have synergistic effects on melanoma risk. Furthermore, additional studies are warranted to investigate the association between sildenafil use and melanoma by BRAF/NRAS mutations and body sites. It also remains to be seen whether the association is stronger for thick melanoma and those that ultimately metastasized.

In conclusion, the results from a well-established, long-term cohort study suggest a positive association between PDE5A inhibitor (sildenafil) use for erectile function problems and risk of subsequent melanoma. Further studies are needed to confirm our findings in other populations, particularly in a dose-dependent manner, and to investigate the underlying biological mechanisms. It would also be very important to examine the possible latency period of exposure to PDE5A inhibitor (sildenafil) use and risk of melanoma. Our results should be interpreted cautiously and are insufficient to alter current clinical recommendations. Nevertheless, our data provide epidemiological evidence on the possible skin adverse effect by PDE5A inhibitors and support a need for continued investigation of this relationship.