Aruna V. Krishnan

Glucocorticoids can promote androgen-independent growth of prostate cancer cells through a mutated androgen receptor.

The androgen receptor (AR) is involved in the development, growth and progression of prostate cancer (CaP). CaP often progresses from an androgen-dependent to an androgen-independent tumor, making androgen ablation therapy ineffective. However, the mechanisms for the development of androgen-independent CaP are unclear. More than 80% of clinically androgen-independent prostate tumors show high levels of AR expression. In some CaPs, AR levels are increased because of gene amplification and/or overexpression, whereas in others, the AR is mutated. Nonetheless, the involvement of the AR in the transition of CaP to androgen-independent growth and the subsequent failure of endocrine therapy are not fully understood. Here we show that in CaP cells from a patient who failed androgen ablation therapy, a doubly mutated AR functioned as a high-affinity cortisol/cortisone receptor (ARccr). Cortisol, the main circulating glucocorticoid, and its metabolite, cortisone, both equally stimulate the growth of these CaP cells and increase the secretion of prostate-specific antigen in the absence of androgens. The physiological concentrations of free cortisol and total cortisone in men greatly exceed the binding affinity of the ARccr and would activate the receptor, promoting CaP cell proliferation. Our data demonstrate a previously unknown mechanism for the androgen-independent growth of advanced CaP. Understanding this mechanism and recognizing the presence of glucocorticoid-responsive AR mutants are important for the development of new forms of therapy for the treatment of this subset of CaP.

The role of vitamin D in reducing cancer risk and progression

Vitamin D is not really a vitamin but the precursor to the potent steroid hormone calcitriol, which has widespread actions throughout the body. Calcitriol regulates numerous cellular pathways that could have a role in determining cancer risk and prognosis. Although epidemiological and early clinical trials are inconsistent, and randomized control trials in humans do not yet exist to conclusively support a beneficial role for vitamin D, accumulating results from preclinical and some clinical studies strongly suggest that vitamin D deficiency increases the risk of developing cancer and that avoiding deficiency and adding vitamin D supplements might be an economical and safe way to reduce cancer incidence and improve cancer prognosis and outcome.

Mechanisms of the Anti-Cancer and Anti-Inflammatory Actions of Vitamin D

Calcitriol, the hormonally active form of vitamin D, is being evaluated in clinical trials as an anti-cancer agent. Calcitriol exerts multiple anti-proliferative, pro-apoptotic, and pro-differentiating actions on various malignant cells and retards tumor growth in animal models of cancer. Calcitriol also exhibits several anti-inflammatory effects including suppression of prostaglandin (PG) action, inhibition of p38 stress kinase signaling, and the subsequent production of pro-inflammatory cytokines and inhibition of NF-κB signaling. Calcitriol also decreases the expression of aromatase, the enzyme that catalyzes estrogen synthesis in breast cancer, both by a direct transcriptional repression and indirectly by reducing PGs, which are major stimulators of aromatase transcription. Other important effects include the suppression of tumor angiogenesis, invasion, and metastasis. These calcitriol actions provide a basis for its potential use in cancer therapy and chemoprevention. We summarize the status of trials involving calcitriol and its analogs, used alone or in combination with known anti-cancer agents.

The vitamin D receptor gene start codon polymorphism : A functional analysis of FokI variants

The vitamin D receptor (VDR) gene contains a start codon polymorphism (SCP) which is three codons upstream of a second start site (ATG). The SCP genotype can be determined with the restriction enzyme FokI, where “f” indicates the presence of the restriction site and the first ATG, while “F” indicates its absence. Recent evidence suggests that the ff genotype is correlated with lower bone mineral density (BMD) in some populations. The SCP results in alternate VDRs that differ structurally, with the F variant (F‐VDR) being three amino acids shorter than the f variant (f‐VDR). To determine whether there are functional differences between the f‐VDR and the F‐VDR, we studied the two VDR forms expressed in COS‐7 cells. The proteins were distinguishable from one another on Western blots by their different mobilities, confirming the larger size of f‐VDR. Ligand binding studies showed no significant differences between the affinities of the two VDR forms for [3H]‐1,25‐dihydroxyvitamin D3 ([3H]‐1,25(OH)2D3) (Kd = 131 ± 78 pM, f‐VDR; Kd = 237 ± 190 pM, F‐VDR; p = 0.24); however, a 2‐fold difference in affinity can not be discriminated by this method. There were no differences in the abilities of the two receptor forms to bind DNA as determined by electrophoretic mobility shift assays. The ability of the two VDR forms to transactivate target genes was investigated using three different vitamin D responsive luciferase reporter constructs: 24‐hydroxylase, osteocalcin, and osteopontin. In these transactivation experiments, 1,25(OH)2D3 dose‐response (0.1–10 nM) curves revealed that the ED50 values for transactivation were indistinguishable between the two VDR forms. Additionally, cultured human fibroblasts with FF,Ff, and ff genotypes had similar sensitivity to 1,25(OH)2D3 with respect to the induction of 24‐hydroxylase mRNA. In summary, we were unable to detect significant differences in ligand affinity, DNA binding, or transactivation activity between f‐VDR and F‐VDR forms. We must emphasize, however, that the sensitivity of the methods used limits our ability to detect minor differences in VDR affinity and function. In conclusion, we cannot define a mechanism whereby the SCP in the VDR might contribute to population differences in BMD.

Regulation of Prostaglandin Metabolism by Calcitriol Attenuates Growth Stimulation in Prostate Cancer Cells

Calcitriol exhibits antiproliferative and pro-differentiation effects in prostate cancer. Our goal is to further define the mechanisms underlying these actions. We studied established human prostate cancer cell lines and primary prostatic epithelial cells and showed that calcitriol regulated the expression of genes involved in the metabolism of prostaglandins (PGs), known stimulators of prostate cell growth. Calcitriol significantly repressed the mRNA and protein expression of prostaglandin endoperoxide synthase/cyclooxygenase-2 (COX-2), the key PG synthesis enzyme. Calcitriol also up-regulated the expression of 15-hydroxyprostaglandin dehydrogenase, the enzyme initiating PG catabolism. This dual action was associated with decreased prostaglandin E2 secretion into the conditioned media of prostate cancer cells exposed to calcitriol. Calcitriol also repressed the mRNA expression of the PG receptors EP2 and FP, providing a potential additional mechanism of suppression of the biological activity of PGs. Calcitriol treatment attenuated PG-mediated functional responses, including the stimulation of prostate cancer cell growth. The combination of calcitriol with nonsteroidal anti-inflammatory drugs (NSAIDs) synergistically acted to achieve significant prostate cancer cell growth inhibition at approximately 2 to 10 times lower concentrations of the drugs than when used alone. In conclusion, the regulation of PG metabolism and biological actions constitutes a novel pathway of calcitriol action that may contribute to its antiproliferative effects in prostate cells. We propose that a combination of calcitriol and nonselective NSAIDs might be a useful chemopreventive and/or therapeutic strategy in men with prostate cancer, as it would allow the use of lower concentrations of both drugs, thereby reducing their toxic side effects.

Inhibition of prostate cancer growth by vitamin D: Regulation of target gene expression

Prostate cancer (PCa) cells express vitamin D receptors (VDR) and 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) inhibits the growth of epithelial cells derived from normal, benign prostate hyperplasia, and PCa as well as established PCa cell lines. The growth inhibitory effects of 1,25(OH)2D3 in cell cultures are modulated tissue by the presence and activities of the enzymes 25‐hydroxyvitamin D3 24‐hydroxylase which initiates the inactivation of 1,25(OH)2D3 and 25‐hydroxyvitamin D3 1α‐hydroxylase which catalyses its synthesis. In LNCaP human PCa cells 1,25(OH)2D3 exerts antiproliferative activity predominantly by cell cycle arrest through the induction of IGF binding protein‐3 (IGFBP‐3) expression which in turn increases the levels of the cell cycle inhibitor p21 leading to growth arrest. cDNA microarray analyses of primary prostatic epithelial and PCa cells reveal that 1,25(OH)2D3 regulates many target genes expanding the possible mechanisms of its anticancer activity and raising new potential therapeutic targets. Some of these target genes are involved in growth regulation, protection from oxidative stress, and cell–cell and cell–matrix interactions. A small clinical trial has shown that 1,25(OH)2D3 can slow the rate of prostate specific antigen (PSA) rise in PCa patients demonstrating proof of concept that 1,25(OH)2D3 exhibits therapeutic activity in men with PCa. Further investigation of the role of calcitriol and its analogs for the therapy or chemoprevention of PCa is currently being pursued. J. Cell. Biochem. 88: 363–371, 2003.

Tissue-Selective Regulation of Aromatase Expression by Calcitriol: Implications for Breast Cancer Therapy

Aromatase, the enzyme that catalyzes estrogen synthesis, is critical for the progression of estrogen receptor-positive breast cancer (BCa) in postmenopausal women. We show that calcitriol, the hormonally active form of vitamin D, regulates the expression of aromatase in a tissue-selective manner. Calcitriol significantly decreased aromatase expression in human BCa cells and adipocytes and caused substantial increases in human osteosarcoma cells (a bone cell model exhibiting osteoblast phenotype in culture) and modest increases in ovarian cancer cells. Calcitriol administration to immunocompromised mice bearing human BCa xenografts decreased aromatase mRNA levels in the tumors and the surrounding mammary adipose tissue but did not alter ovarian aromatase expression. In BCa cells, calcitriol also reduced the levels of prostaglandins (PGs), major stimulators of aromatase transcription, by suppressing the expression of cyclooxygenase-2 (which catalyzes PG synthesis) and increasing that of 15-hydroxyprostaglandin dehydrogenase (which catalyzes PG degradation). The mechanism of aromatase down-regulation by calcitriol in BCa cells is therefore 2-fold: a direct repression of aromatase transcription via promoter II through the vitamin D-response elements identified in this promoter and an indirect suppression by reducing the levels of PGs. Combinations of calcitriol with three different aromatase inhibitors (AIs) caused enhanced inhibition of BCa cell growth. The combination of calcitriol and an AI may have potential benefits for BCa therapy. In addition to augmenting the ability of AIs to inhibit BCa growth, calcitriol acting as a selective aromatase modulator that increases aromatase expression in bone would reduce the estrogen deprivation in bone caused by the AIs, thus ameliorating the AI-induced side effect of osteoporosis.

TWO MUTATIONS IDENTIFIED IN THE ANDROGEN RECEPTOR OF THE NEW HUMAN PROSTATE CANCER CELL LINE MDA PCA 2A

PURPOSE We have characterized the androgen receptor (AR) in a new human prostate cancer cell line, MDA PCa 2a, that has recently been established from a bone metastasis of a patient whose cancer exhibited androgen-independent growth. MATERIALS AND METHODS Androgen responsiveness of these cells was assessed by measuring the effect of DHT and R1881 on cell growth and PSA secretion. Scatchard analysis was used to characterize the affinity and abundance of AR protein. Using a PCR based strategy, genomic DNA of the entire coding region of AR gene was sequenced to identify possible mutations. RESULTS These cells express abundant AR (Nmax = 685 +/- 149 fmol./mg. protein), but the AR binding affinity (Kd) for DHT is only 25 nM, approximately 50-fold lower affinity than the mutated AR in LNCaP prostate cancer cells (Kd = 0.5 nM) or the wildtype AR in MCF-7 breast cancer cells (Kd = 0.4 nM). Two mutations, L701H and T877A, were identified in the ligand binding domain of the AR gene. Compared with LNCaP cells, the new cell line is significantly less responsive to DHT and R1881 as well as to other androgens such as testosterone, androstenedione, and DHEA. Similar to LNCaP cells, the ligand specificity of the AR in MDA PCa 2a cells appears to be relaxed and non-androgens such as progesterone and estradiol act as agonists although with less potency than in LNCaP cells. Interestingly, in the absence of androgens, the new cell line expresses 15-fold higher baseline levels of PSA than LNCaP. CONCLUSIONS Two mutations were identified in the AR gene of the MDA PCa 2a cell line that are likely responsible for the decreased androgen sensitivity and altered ligand specificity observed in these cells. Thus, this new cell line with partial androgen responsiveness and PSA expression can serve as a functionally relevant model system of bone metastatic prostate cancer, and can be used to investigate the role of AR mutations in prostate cancer and its progression to androgen independence.

Molecular pathways mediating the anti-inflammatory effects of calcitriol: implications for prostate cancer chemoprevention and treatment

Calcitriol, the hormonally active form of vitamin D, exerts multiple anti-proliferative and pro-differentiating actions including cell cycle arrest and induction of apoptosis in many malignant cells, and the hormone is currently being evaluated in clinical trials as an anti-cancer agent. Recent research reveals that calcitriol also exhibits multiple anti-inflammatory effects. First, calcitriol inhibits the synthesis and biological actions of pro-inflammatory prostaglandins (PGs) by three mechanisms: i) suppression of the expression of cyclooxygenase-2, the enzyme that synthesizes PGs; ii) up-regulation of the expression of 15-hydroxyprostaglandin dehydrogenase, the enzyme that inactivates PGs; and iii) down-regulation of the expression of PG receptors that are essential for PG signaling. The combination of calcitriol and nonsteroidal anti-inflammatory drugs results in a synergistic inhibition of the growth of prostate cancer (PCa) cells and offers a potential therapeutic strategy for PCa. Second, calcitriol increases the expression of mitogen-activated protein kinase phosphatase 5 in prostate cells resulting in the subsequent inhibition of p38 stress kinase signaling and the attenuation of the production of pro-inflammatory cytokines. Third, calcitriol also exerts anti-inflammatory activity in PCa through the inhibition of nuclear factor-kappaB signaling that results in potent anti-inflammatory and anti-angiogenic effects. Other important direct effects of calcitriol as well as the consequences of its anti-inflammatory effects include the inhibition of tumor angiogenesis, invasion, and metastasis. We hypothesize that these anti-inflammatory actions, in addition to the other known anti-cancer effects of calcitriol, play an important role in its potential use as a therapeutic agent for PCa. Calcitriol or its analogs may have utility as chemopreventive agents and should be evaluated in clinical trials in PCa patients with early or precancerous disease.

Vitamin D: Biology, Actions, and Clinical Implications

Abstract Vitamin D is the major regulator of calcium homeostasis in the body and is critically important for normal mineralization of bone to prevent rickets in children and osteomalacia in adults. The active hormone, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3, also called calcitriol), is produced by sequential hydroxylations of vitamin D in the liver (25-hydroxylation) and the kidney (1α-hydroxylation). 1,25(OH)2D3 acting via the vitamin D receptor (VDR), functions by a genomic mechanism similar to the classical steroid hormones to regulate target gene transcription. The traditional actions of 1,25(OH)2D3 are to enhance calcium and phosphate absorption from the intestine in order to maintain normal mineral concentrations in the circulation and to provide adequate amounts of these minerals to the bone-forming sites to allow mineralization of bone to proceed normally. However, since the early 1990s, it has become increasing clear that vitamin D has many additional extraskeletal functions that implicate the hormone in a wide array of actions unrelated to bone or mineral metabolism. It is now known that 1,25(OH)2D3 modulates the activity of hundreds, if not thousands, of genes in essentially every tissue in the body making it a potent regulator of many critical processes. Some of these actions can be categorized as antiproliferative, prodifferentiating, immunosuppressive, and anti-inflammatory. In this chapter, we will describe the basic biology of vitamin D including its metabolism, physiology, mechanism of action, and its diverse functions in the body including those actions that relate to mineral metabolism as well as the newer extraskeletal actions. Several reviews of vitamin D mechanism of action and function have been published as well as a new edition of a comprehensive book addressing all areas of vitamin D biology. Specific issues relating to vitamin D and osteoporosis are discussed in Chapter 73.