Deciphering the role of vitamin D on skin cancers and tumour microenvironment

Introduction

Cancer, the emperor of all maladies, has coexisted with humankind since 1600 BC, when the first documented case of breast cancer was found in Edwin Smith Papyrus.¹ Since then, the disease has co-evolved with us and has caused a significant burden across the globe. Cancer affects almost every part of the body, including the lungs, breasts, skin, liver, colon and rectum. According to the global cancer statistics in 2020, skin cancer accounts for 6.2% of all new cancer cases.² The abnormal growth of cells in the epidermis due to mutations, which arise from unrepaired DNA damage, causes skin cancer. Generally, the head and neck are the sites where most cases of skin cancers occur. Skin cancer can be broadly classified into non-melanoma and melanoma as represented in Figure 1. Non-melanoma skin cancers (NMSC) comprise basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Basal cell carcinoma is generally found in the basal cells of the lower epidermis. Though it grows slowly and rarely spreads to other parts of the body, if left undiagnosed and untreated, it may spread to the surrounding tissues.³

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Figure 1:

Classification of skin cancers

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On the other hand, squamous cell carcinoma is a type of cancer that develops in the squamous cells. Figure 2 shows the classification of skin cancers and the outermost cell layer of the epidermis.⁴ Melanomas are cancers that arise in the melanocytes, pigment-producing cells (melanin), present throughout the skin cells.⁵ Ultraviolet radiations is one of the primary causes of this type of cancer.

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Figure 2:

Types of skin cancer

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Incidence, burden and mortality

According to the global cancer statistics (GLOBOCAN) 2020, the estimates of worldwide incidence and mortality of 36 cancer types in 185 countries were analysed, and it was observed that skin cancer accounted for 1,198,073 new cases every year, which represents 6.2% of the total combined cancer cases in 2020 (Figure 3a).² The number of deaths due to skin cancer globally was estimated to be 63,731, accounting for 0.6% of total deaths reported in the year 2020.² It was also observed that melanoma of the skin was the fifteenth most common cause of cancer in males, with an incidence rate of 5.2 per 100,000 males and 0.4 per 100,000 males in countries with higher and lower human development index (HDI), respectively. Figure 3d represents the percentage incidences of melanoma and non-melanoma skin cancers with respect to other cancers in India. However, the mortality rates were least when compared with other cancers, that is, 0.9 per 100,000 males and 0.2 per 100,000 males in higher and lower human development index countries, respectively.² In the case of females, the incidence rates were approximately two folds lower than the males (i.e. 7.9% in females and 15.1% in males respectively).²

The National Cancer Registry Program and GLOBOCAN 2018 reports were collected and analysed to understand the incidence of skin cancer in India. It is believed that, in the Indian population, the incidence rate of different skin cancers is lower, which is attributed to the protective effects of melanin.⁶ However, the unavailability of reports from national surveys and cross-country data and indirect indications from several small reports indicate an increase in the incidences of skin cancer in the country. The incidence of non-melanoma skin cancers and melanomas in India, specifically among the male population, was observed to be the highest in the north-eastern region. Additionally, among males, the incidence of non-melanoma skin cancers and melanoma was observed highest in Pasighat and Nagaland, respectively. However, melanomas and non-melanoma skin cancers were highest among females in the Northern (Delhi) and north-eastern regions (Pasighat). Amongst melanoma and non-melanoma skin cancers, the latter was found to be more common among the Asian population.⁷ Figure 3b represents the incidence of non-melanoma skin cancers worldwide in both sexes. It can be seen that the incidence in Asian countries is 7.26%. However, the mortality rate due to all cancers is relatively high in Asian countries, that is, 43.5%, as shown in Figure 3c.⁸

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Figure 3a:

Pie charts representing status of skin cancers in India and worldwide

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Figure 3b:

Pie charts representing status of skin cancers in India and worldwide

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Figure 3c:

Pie charts representing status of skin cancers in India and worldwide

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Figure 3d:

Pie charts representing status of skin cancers in India and worldwide

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Causes and risk factors

Skin cancer is caused due to mutations in the DNA of the skin cells, which leads to uncontrolled growth and formation of tumours. However, the combination of environmental and genetic factors is responsible for the risk of developing skin cancer. Long-term exposure to ultraviolet rays remains one of the most common causes. This chronic ultraviolet exposure has been associated with 90% of cutaneous squamous cell carcinoma and basal cell carcinoma cases and 65% of cutaneous melanomas.⁹

Current treatment strategies

The gold standard strategy for diagnosing non-melanoma skin cancers is primary physical examination, followed by biopsy and histopathologic examination.¹⁰ However, squamous cell carcinoma is susceptible to metastasize to adjacent tissues and therefore a thorough lymph node examination is also required for a complete diagnosis. To avoid invasive procedures for diagnostic purposes, several non-invasive tools have been developed, including dermoscopy, multiphoton microscopy, confocal microscopy, Raman spectroscopy, cross-polarized light and fluorescence photography and coherence tomography with high-frequency ultrasound.¹⁰ These technologies help in characterising the features of non-melanoma skin cancers before going with the biopsy method. Dermoscopy, which is mainly used to identify melanocytic lesions, also screens squamous cell carcinoma and basal cell carcinomas.^11,12 The most commonly used technique for treating non-melanoma skin cancers is the surgical removal of the lesion, which has the highest curing rate among other existing methods.^13,14 Other methods which are employed to treat cases of non-melanoma skin cancers include cryotherapy, laser therapy, pharmacotherapy, radiation therapy and photodynamic therapy (PDT).^15,16 However, a detailed discussion about the current treatment or management strategies is out of scope of this review.

Introduction

Vitamin D, also known as calciferol, belongs to the family of fat-soluble vitamins that promote calcium absorption, regulate bone mineral metabolism and maintain muscle function.¹⁷ In nature, vitamin D exists in two primary forms, that is, vitamin D₂ (ergocalciferol) and vitamin D₃ (cholecalciferol) derived from animal or plant sources. Because the human body is not able to synthesise vitamin D₂, it is generally added to foods. In contrast, vitamin D₃ is synthesised in the skin of the human body from endogenous 7-dehydrocholesterol through photochemical reaction by ultraviolet radiation. However, it can also be taken via the consumption of specific foods.¹⁸ Moreover, because vitamin D₃ is pharmacologically more potent than the D₂ form, it is generally preferred as a treatment option for vitamin D deficiency.¹⁹ However, vitamin D₃ is frequently used for the treatment of medical conditions such as refractory rickets and hypothyroidism. Apart from medical uses, it is also commonly used to supplement individuals suffering from low vitamin D levels in their bodies.

Synthesis and metabolism in the human body

The endogenous synthesis of vitamin D₃ begins with the skin exposure to ultraviolet-B radiation of wavelength between 295 nm and 315 nm.²⁰ Upon exposure to ultraviolet-B, the provitamin D₃ (7-dehydrocholesterol), a compound present in skin, is photo-isomerised into previtamin D₃ (precholecalciferol), which is converted into vitamin D₃ via a heat-dependent reaction.^21,22 Later, the hydroxylation of vitamin D₃ occurs in the liver, which uses 25-hydroxylase to convert vitamin D₃ to 25-hydroxy-vitamin D₃ (25(OH)D)_, also known as calcidiol. The calcidiol is further hydroxylated in the kidneys to form calcitriol, an active metabolite in the hormonal form, with the help of the 1-α-hydroxylase enzyme. The flow chart of vitamin D synthesis and its metabolism is shown in Figure 4.²³ The figure depicts the endogenous synthesis and metabolism pathway in the human body. To quantify vitamin D levels in the body, serum-free calcidiol is the best candidate because it has a half-life of 2–3 weeks, and it can be easily measured as well.^24,25

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Figure 4:

Endogenous synthesis of vitamin D and its metabolism

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Source

Vitamin D can be obtained via three resources, namely, via conversion of 7-dehydrocholesterol into vitamin D3 (cholecalciferol) upon sun exposure, from food and supplements. Dietary sources include natural food such as fish, beef liver, egg yolk, cheese and mushrooms. The Food and Drug Administration (FDA) has also approved ultraviolet-treated mushroom powders as a source of vitamin D₂ and as a food additive.²⁶ Table 1 shows the different food sources of vitamin D.²⁷ Sun exposure is one of the prominent sources of vitamin D, which fulfills its daily requirement. Specifically, ultraviolet-B, which falls in the wavelength range of 290–320 nm, is required to synthesise vitamin D₃. This radiation has poor penetration through glass, and therefore, the sun’s exposure indoors through a window does not help in vitamin D synthesis.²⁸ According to the literature, approximately 5–30 mins of sun exposure, twice a week or daily without sunscreen, is crucial for the synthesis of vitamin D that is adequate for the body.^28,29 Various other factors such as latitude, color and season also affect vitamin D synthesis in the body.²⁹ However, it is important to note that its overall synthesis can occur within the epidermis in order to associate this potentially with skin cancer.³⁰ Induction of 25-hydroxylase mRNA release by vitamin D3 and ultraviolet-B radiation leads to the activation of 1,25(OH)₂D that causes local effects within the cell.³¹ Within the epidermis, the 2D action takes place via vitamin D receptor (VDR), which helps in the regulation of proliferation in the basal cells and also promotes the differentiation of keratinocytes as they start to form the upper layer of the epidermis.^30,32

Protective effects of vitamin D from ultraviolet rays

The long wave ultraviolet-A and short wave ultraviolet-B are the types of ultraviolet rays in sunlight that damage DNA. In the case of ultraviolet-A, it penetrates into the deeper layers of skin and leads to production of free radicals, which then cause oxidative and DNA damage. Whereas ultraviolet-B usually affects the superficial layers of the skin, causing DNA damage leading to driver and bystander mutations followed by cellular transformations and malignancies.⁴⁸ Despite the photocarcinogenic⁴⁹ and immunosuppressive effects⁵⁰ of ultraviolet-B components, it is also crucial for the synthesis of vitamin D for its various functions in the body. In order to repair DNA damage post-ultraviolet exposure, the growth of the cell is arrested, wherein faulty repair or improper removal of damaged cells results in clonally expanded cells that constitute tumours.^37,51–54 In vivo studies, trying to understand the role of vitamin D receptors upon short-term ultraviolet exposure, observed a decrease in the proliferation of normal mouse keratinocytes, however, the vitamin D receptor–ablated cells showed more proliferation. These results suggest the role of vitamin D receptor in promoting growth arrest upon short ultraviolet treatment.³⁷ Additionally, fewer apoptotic cells were observed in vitamin D receptor–null skin than in normal skin post-ultraviolet exposure, demonstrating the critical role of vitamin D receptor in eliminating severely damaged cells. Apart from ultraviolet exposure, the application of either oral or topical agents containing carcinogens also increases skin tumours in vitamin D receptor–null mice.⁵⁴

Dixon et al.⁵⁵ studied the inhibitory effects of 1α,25(OH)-vitamin D, and non-genomic vitamin D analogue against ultraviolet-induced skin carcinogenesis.⁵⁵ They demonstrated that both vitamin D₃ and 1α,25(OH)₂-lumisterol, a conformational analogue, were effective inhibitors of ultraviolet damage in an immunocompetent mouse (Skh:hr1) model susceptible to ultraviolet-induced tumours. Additionally, in mice, they observed inhibition of the development of skin cancer in the case of squamous cell carcinoma. These observations suggest the protective role of these compounds in the prevention of skin carcinogenesis.

In another study by Zhang et al.,⁵³ they investigated the role of DNA damage-inducible transcript 4 (DDIT4) in human squamous cell carcinoma.⁵² They observed a significant suppression of DDIT4 in the case of squamous cell carcinoma and A431 cell lines. Moreover, the reduction of DDIT4 accelerated the proliferation of keratinocytes but obstructed the autophagy flux through the mTORC1 pathway by affecting the downstream S6 kinase1, 4E-BP1, beclin1 and LC3 II/I. The in vivo studies revealed the anti-carcinogenic effect of vitamin D in case of xenograft tumor–bearing mice treated with different concentrations of 1,25(OH)₂D₃ and DDIT4.

Apart from environmental factors, genetic factors also play a role in the development of skin cancer. For example, people with fairer skin are at an increased risk of developing skin cancer. Moreover, individuals with freckling and red hair carry two copies of the R allele variant of the MC1R gene and are also at increased risk of developing skin cancer. Xeroderma pigmentosum, a rare autosomal recessive disease, is a condition where the individuals suffering from this disease cannot repair the ultraviolet-induced damage leading to mutations in the genes that form an integral part of the nucleotide excision repair pathway. This pathway plays a vital role in repairing the damage caused due to ultraviolet rays.¹⁸ This abnormal condition predisposes an individual to an increased risk of developing freckling, sunburn and early childhood malignancies.

Daily requirement of vitamin D and preventive strategies

The daily requirement of vitamin D is dependent on an individual’s age, but the standard concentration required for daily intake has been a topic of discussion. The Institute of Medicine, an independent, non-profit organisation, has provided data on daily vitamin D intake for individuals of different age groups, as shown in Table 2.¹⁸ The daily intake of vitamin D varies with age of an individual. For example, infants require a daily intake of 400 IU vitamin D₃, whereas individuals in the age group 1–70 years require 600 IU and for individuals above 70 years of age, the recommended intake is 800 IU/day.

Seamans et al.⁵⁶ validated the reliability and robustness of circulating levels of vitamin D as a marker of vitamin D status in the human body.⁵⁶ According to the American Endocrine Society, serum levels of vitamin D under 20 ng/mL are considered deficient. In contrast, serum levels in the range of 20–30 ng/mL are considered insufficient and levels above 30 ng/mL are considered essential for maintaining good health.⁵⁷

The Institute of Medicine has specified 20 ng/mL as the lower limit indicating healthy and sufficient vitamin D levels [Table 3].¹⁸ A higher level of vitamin D in serum, particularly above 200 ng/mL, is considered toxic, and the associated symptoms include weight loss, polyuria, anorexia and cardiac arrhythmia. Moreover, vitamin D toxicity also increases the risks of calcification of tissues and blood vessels and formation of kidney stones.²⁷

A few lifestyle changes in daily life could help maintain healthy levels of vitamin D in the body and prevent its deficiency. This includes getting enough sun exposure as it is the primary source for vitamin D synthesis, daily intake of foods rich in vitamin D and being proactive to maintain overall health. However, for patients who have skin cancer and cannot get sensible sun exposure, the administration of vitamin D as a supplement could be employed as an alternative way to meet the daily requirements.

Effects of vitamin D on tumor microenvironment

Studies also demonstrated that vitamin D inhibits the growth of cancer cells and cancer stem cells⁵⁸ and plays an essential role in regulating the tumor microenvironment. Various studies suggest regulating tumor initiation and metastasis by influencing cell-microenvironment interactions by vitamin D.⁵⁹ A large number of tumor cells and a small portion of cancer stem cells exist in the stromal microenvironment, along with vasculature for nourishment, cancer-associated fibroblasts, immune cells and extracellular components. It is well demonstrated that chronic inflammation is the main contributor to the initiation of tumours.⁶⁰ In this regard, vitamin D is known to exert its anti-inflammatory effects with tumor-specific outcomes. For example, vitamin D₃ is responsible for the inhibition of the prostaglandin pathway that is involved in pro-inflammatory responses via inhibition of cyclooxygenase-2, prostaglandin receptors and degradation of prostaglandin in prostate^61,62 and breast⁶³ tumor cells.

Vitamin D₃ is also known to suppress the p38 MAPK-regulated pro-inflammatory pathway in prostate cancer cells.⁶⁴ Blocking of pro-inflammatory nuclear factor kappa B (NFκB) pathway was also reported via vitamin D₃, and also the inhibition of Akt within macrophages.⁶⁵ Modulation of cancer and immune cell phenotypes by suppressing pro-inflammatory cytokines such as TNF-α and IL-6 by vitamin D₃ was also demonstrated.⁶⁶ Vitamin D receptor–null mice developed impaired cutaneous wound healing at the expense of F4/80+ macrophages recruited to the wound site.⁶⁷ Whether this impaired innate immune response observed in the skin can also contribute to the tumor microenvironment remains unexplored.

Vitamin D: How much is enough?

Various studies cited above correlated elevated serum levels of vitamin D with higher exposure to the sun. A study by Hart et al.⁶⁸ demonstrated possible independent effects of sun exposure on health, particularly immune-mediated effects not occurring through a vitamin D pathway.⁶⁸ This can be related to an issue linked to skin cancer development and its prognosis. Berwick et al.⁶⁹ were the first to validate the advantageous effect of the sun’s ultraviolet rays on melanoma prognosis. The study reported that high intermittent exposure to the sun is associated with low death risk in patients with melanoma within 5 years of diagnosis.⁶⁹

Arguments have always been there regarding the optimal levels of vitamin D required for good health conditions. Various observational studies associate low levels of vitamin D with an increased risk of internal cancers. However, considering the various reviews of associations with a wide range of disease outcomes, the United States Institute of Medicine suggested a significant causal association of vitamin D levels with good bone health, and recommended serum levels of 50 nmol/L should be considered sufficient.¹⁸

The ultraviolet index, or UVI, is an international standard measurement of the strength of sunburn-producing ultraviolet radiation at a particular place and time. There was a consensus that vitamin D levels less than 25 nmol/L are considered deficient.⁷⁰ The Institute of Medicine concluded that vitamin D levels less than 30 nmol/L should be considered a deficiency risk.¹⁸ Upon diagnosis of skin cancer or in persons with increased risk of skin cancer, it may be appropriate to avoid sun exposure as much as possible and ensure adequate vitamin D status. There is considerable evidence that suggests a fall in vitamin D status following a diagnosis of skin cancer, for example the study by Idorn et al.⁷¹ The findings from this study suggested that patients with cutaneous malignant melanoma had a higher ultraviolet exposure in the summer before the diagnosis than did controls.

The production of vitamin D is primarily induced only by ultraviolet-B radiation. It is thus most effective when the intensity of ultraviolet-B is the highest throughout the day, that is, at noon, with simultaneous minimization of the risk of erythema.⁷² A general notion that directly correlates the production of vitamin D₃ with the surface area of the skin is that the more the skin area, the more will be the production of vitamin D₃.

On the other hand, erythema of skin or epidermal cells induces DNA damage and increases the risk of skin cancer. As a result, there should be maximum exposure to the skin when ultraviolet index is highest but for a short period for adequate vitamin D production.

Although, the absolute difference in time required to achieve the above condition when vitamin D₃ production and erythema being optimal at high ultraviolet index is narrow. For example, based on current dose-response models and International Commission on Illumination (CIE) action spectra, when the ultraviolet index is 12, it takes less than 5 min to produce an acceptable daily dose of vitamin D with only face and arms exposed. On the other hand, it takes about 15 min for somebody to experience erythema.⁷³ While extreme caution would be required to avoid harmful levels of sun exposure, the appropriate strategy for people at high risk of skin cancer may be to apply sunscreen to the hands, face and arms routinely but to deliberately expose less frequently exposed areas of skin for a short time at a high ultraviolet index.