Does testosterone mediate the relationship between vitamin D and prostate cancer? A systematic review and meta-analysis protocol

Prostate cancer is the second most commonly diagnosed cancer and the fifth leading cause of cancer death in men worldwide [1]. The causes of prostate cancer are not well understood, but well-established risk factors are older age [2], Afro-Caribbean or West African origin [3], and having a first-degree relative (father, brother) diagnosed with the disease [4]. None of these factors are modifiable or controllable through intervention. Other putative risk factors for prostate cancer include obesity, physical activity, and diet. These lifestyle factors are amenable with intervention but definitive evidence that they are causally associated with prostate cancer is lacking.

The World Cancer Research Fund Continuous Update Project [5] reviewed the literature on certain nutrients associated with prostate cancer including vitamin D, and found the evidence was limited and inconclusive regarding their effects on prostate cancer. However, the vitamin D receptor is active in many tissues, including prostate tissue [6], and ecologic studies suggest that in regions where sun exposure (the major source of vitamin D) is high there is a reduced risk of prostate cancer [7, 8]. Individual-level observational studies report inconsistent results, with both high and low vitamin D concentrations associated with an increased risk of prostate cancer [9, 10]. These inconsistencies may be explained by small sample sizes or confounders of the vitamin D-prostate cancer relationship. With regards to prostate cancer progression (rather than incidence), there is randomised controlled trial evidence suggesting a modest effect of interventions to increase vitamin D using supplements on measures of disease progression (PSA levels, decrease in number of positive cores, and Gleason score), among men with low and intermediate stage prostate cancer [11, 12].

Vitamin D is a precursor to the steroid hormone calcitriol, which regulates calcium and phosphate and has an important role in bone mineralisation [13]. The majority of vitamin D is obtained from exposure to sunlight which is synthesised through the skin in the form of cholecalciferol (vitamin D₃). A smaller proportion of vitamin D comes from diet as ergocalciferol (vitamin D₂). Vitamin D is converted into calcitriol through two hydroxylation steps: first, cholecalciferol is hydroxylated in the liver into 25-hydroxy vitamin D; second, circulating 25-hydroxy vitamin D is hydroxylated in the kidney to produce calcitriol (1,25(OH)₂D) [13].

Vitamin D increases differentiation and apoptosis, and decreases proliferation and metastasis in prostate cancer cells [14]. However, identifying which mechanism explains the association between vitamin D and prostate cancer, and systematically reviewing the evidence for this mechanism is complex. We, therefore, plan to use a recently developed two-stage methodology (the WCRF International/University of Bristol methodology) to synthesise the evidence from mechanistic studies [15]. We have already carried out step 1 of this process, in which we have used two web-based text mining tools to generate our mechanistic hypothesis. TeMMPo (Text Mining for Mechanism Prioritisation) [16] uses MeSH descriptors to quantify the literature on predefined intermediate concept terms between an exposure and an outcome through co-occurrence (i.e. two concepts that occur frequently together in the same article). TeMMPo provides a priority score to assist with identifying novel intermediate concept terms that have a substantial literature resource. MELODI (Mining Enriched Literature Objects to Derive Intermediates) [17] uses MeSH and free text terms to identify overlapping concept terms between two custom article sets. We first used MELODI to yield novel intermediate concept terms, which were then entered into TeMMPo to assist with prioritising terms.

Two co-authors reviewed the first 20 highest ranked intermediate terms produced by TeMMPo and agreed on a potential mechanism using a process of elimination. Seven of the highest ranked terms were eliminated; four terms were too broad (i.e. ribonucleic acid (RNA), RNA messenger, CD4 (gene), nicotinamide adenine dinucleotide), and three terms related to endogenous retroviruses, which have no well-known drug targets nor are amenable with behavioural intervention. The highest ranked term was a gene (i.e. HSD3B1) involved in androgen metabolism followed by testosterone (ranked ninth) after eliminating the other seven terms. Testosterone was, therefore, chosen as the mechanism for investigation.

Testosterone is a male sex hormone that is produced by the testis and adrenal glands, with a critical role in driving cell division in the prostate. Testosterone binds to proteins (sex hormone-binding globulin and albumin) in the blood whilst a small proportion of it is unbound (free testosterone); total testosterone concentrations refer to the combination of bound and free testosterone. Normal total testosterone levels in healthy men are between 300 and 1050 ng/dl. The androgen hypothesis [18] suggests prostate cancer onset or progression is driven by androgens, which is supported by animal models [19] and the observation that castration or high-dose oestrogen therapy to reduce serum testosterone subsequently reduces metastatic prostate cancer. Therapeutic or surgical (castration) androgen deprivation is used clinically to reduce testosterone production in the treatment of prostate cancer.

The relationship between vitamin D and testosterone is not well-understood, although there is randomised placebo controlled trial [20] and observational [21, 22] evidence of a positive relationship between vitamin D and both total and free testosterone, suggesting testosterone is a plausible biological intermediate on the mechanistic pathway between vitamin D and prostate cancer progression. A review of mechanistic studies is, therefore, warranted to support the existence of this relationship.

Our systematic review aims to synthesise the evidence from human and animal studies to investigate whether testosterone is a causal intermediate in the mechanistic pathway between vitamin D and prostate cancer.

As the potential importance of this relationship is only just emerging, few studies are likely to assess both vitamin D and testosterone in relation to prostate cancer. Therefore, relevant indirect evidence will be drawn from studies of the two pathways: (1) studies linking vitamin D to testosterone; and (2) studies linking testosterone to prostate cancer.

The aim of this review is to determine whether testosterone is a causally relevant intermediate mechanism underpinning the relationship between vitamin D and prostate cancer progression.

To our knowledge, this is the first review examining this mechanistic pathway in accordance with systematically synthesising the evidence of the relationship between vitamin D and testosterone. Other reviews have investigated the relationship between testosterone and prostate cancer outcomes, including increased risk in prostate cancer in hypogonadal men. However, many of these reviews tended to focus on the therapeutic use of testosterone to assess treatment efficacy for cancer [31, 32]. Here, we will review evidence from studies involving humans and experimental studies with rodents according to the WCRF International/University of Bristol methodology [15]. The synthesis of this evidence will suggest whether increase in vitamin D is a potential preventive measure of prostate cancer progression.

The search strategy and study inclusion criteria are relatively broad to capture the wealth of evidence which may exist. For human studies, no restrictions will be placed on population characteristics (i.e. co-morbidities, age), except where applicable (i.e. testosterone-prostate cancer relationship). Different modes of administration of the exposure variables (vitamin D, testosterone) and multiple outcomes of prostate cancer will be considered for inclusion in this review.

The review will exclude in vitro studies. These studies are beneficial to the understanding of the mechanistic pathway in terms of cellular and molecular activity. However, it is difficult for in vitro studies to mimic the complicated process that would occur within a natural biological context. They would also increase the heterogeneity of the synthesised results due to the number of different models that exist in these studies [33]. Therefore, human and in vivo animal studies will be considered for inclusion as their data will have more clinical relevance.

This review seeks to establish whether vitamin D is a potential therapeutic intervention for the prevention and/or progression of prostate cancer via its effects on testosterone levels. Based on its findings, the review may help inform future clinical intervention trials and other types of research, such as public health messages.