Anti-tumor activity of calcitriol: pre-clinical and clinical studies

1,25-Dihydroxycholecalciferol (calcitriol) is recognized widely for its effects on bone and mineral metabolism. Epidemiological data suggest that low Vitamin D levels may play a role in the genesis of prostate cancer and perhaps other tumors. Calcitriol is a potent anti-proliferative agent in a wide variety of malignant cell types. In prostate, breast, colorectal, head/neck and lung cancer as well as lymphoma, leukemia and myeloma model systems calcitriol has significant anti-tumor activity in vitro and in vivo. Calcitriol effects are associated with an increase in G0/G1 arrest, induction of apoptosis and differentiation, modulation of expression of growth factor receptors. Glucocorticoids potentiate the anti-tumor effect of calcitriol and decrease calcitriol-induced hypercalcemia. Calcitriol potentiates the antitumor effects of many cytotoxic agents and inhibits motility and invasiveness of tumor cells and formation of new blood vessels. Phase I and II trials of calcitriol either alone or in combination with carboplatin, taxanes or dexamethasone have been initiated in patients with androgen dependent and independent prostate cancer and advanced cancer. Data indicate that high-dose calcitriol is feasible on an intermittent schedule, no dose-limiting toxicity has been encountered and optimal dose and schedule are being delineated. Clinical responses have been seen with the combination of high dose calcitriol+dexamethasone in androgen independent prostate cancer (AIPC) and apparent potentiation of the antitumor effects of docetaxel have been seen in AIPC. These results demonstrate that high intermittent doses of calcitriol can be administered to patients without toxicity, that the MTD is yet to be determined and that calcitriol has potential as an anti-cancer agent.     

The PPARγ ligand ciglitazone regulates androgen receptor activation differently in androgen-dependent versus androgen-independent human prostate cancer cells

The androgen receptor (AR) regulates growth and progression of androgen-dependent as well as androgen-independent prostate cancer cells. Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have been reported to reduce AR activation in androgen-dependent LNCaP prostate cancer cells. To determine whether PPARγ ligands are equally effective at inhibiting AR activity in androgen-independent prostate cancer, we examined the effect of the PPARγ ligands ciglitazone and rosiglitazone on C4-2 cells, an androgen- independent derivative of the LNCaP cell line. Luciferase-based reporter assays and Western blot analysis demonstrated that PPARγ ligand reduced dihydrotestosterone (DHT)-induced increases in AR activity in LNCaP cells. However, in C4-2 cells, these compounds increased DHT-induced AR driven luciferase activity. In addition, ciglitazone did not significantly alter DHT-mediated increases in prostate specific antigen (PSA) protein or mRNA levels within C4-2 cells. siRNA-based experiments demonstrated that the ciglitazone-induced regulation of AR activity observed in C4-2 cells was dependent on the presence of PPARγ. Furthermore, overexpression of the AR corepressor cyclin D1 inhibited the ability of ciglitazone to induce AR luciferase activity in C4-2 cells. Thus, our data suggest that both PPARγ and cyclin D1 levels influence the ability of ciglitazone to differentially regulate AR signaling in androgen-independent C4-2 prostate cancer cells.     

The role of Vitamin D3 metabolism in prostate cancer

Vitamin D deficiency increases risk of prostate cancer. According to our recent results, the key Vitamin D hormone involved in the regulation of cell proliferation in prostate is 25(OH) Vitamin D3. It is mainly acting directly through the Vitamin D receptor (VDR), but partially also through its 1alpha-hydroxylation in the prostate. A deficiency of 25(OH) Vitamin D is common especially during the winter season in the Northern and Southern latitudes due to an insufficient sun exposure, but Vitamin D deficient diet may partially contribute to it. A lack of Vitamin D action may also be due to an altered metabolism or Vitamin D resistance. Vitamin D resistance might be brought up by several mechanisms: Firstly, an increased 24-hydroxylation may increase the inactivation of hormonal Vitamin D metabolites resulting in a Vitamin D resistance. This is obvious in the cancers in which an oncogenic amplification of 24-hydroxykase gene takes place, although an amplification of this gene in prostate cancer has not yet been described. During the aging, the activity of 24-hydroxylase increases, whereas 1alpha-hydroxylation decreases. Furthermore, it is possible that a high serum concentration of 25(OH)D3 could induce 24-hydroxylase expression in prostate. Secondly, Vitamin D receptor gene polymorphism or defects may result in a partial or complete Vitamin D resistance. Thirdly, an overexpression or hyperphosphorylation of retinoblastoma protein may result in an inefficient mitotic control by Vitamin D. Fourthly, endogenous steroids (reviewed by [D.M. Peehl, D. Feldman, Interaction of nuclear receptor ligands with the Vitamin D signaling pathway in prostate cancer, J. Steroid Biochem. Mol. Biol. (2004)]) and phytoestrogens may modulate the expression of Vitamin D metabolizing enzymes. In summary, the local metabolism of hormonal Vitamin D seems to play an important role in the development and progression of prostate cancer.     

Characterization of Vitamin D insensitive prostate cancer cells

The antitumor effects of 1,25-dihydroxyvitamin D₃ (calcitriol) are being exploited for prevention and treatment of prostate cancer (CaP). These studies examined the antiproliferative effects of calcitriol in primary cell cultures derived from transgenic adenocarcinoma of mouse prostate (TRAMP) mice chronically treated with calcitriol (20 μg/kg) or vehicle 3×/week from 4 weeks-of-age until palpable tumors developed. This is a report on the response of two representative control (Vitamin D naïve, naïve) and calcitriol-treated (Vitamin D insensitive, VDI) cells to calcitriol. VDI cells were less sensitive to calcitriol based on less cell growth inhibition and less inhibition of DNA synthesis as measured by MTT and BrdU incorporation assays. Similarly, VDI cells were less sensitive to growth inhibition by the vitamin analog, 19-nor-1,25-dihydroxyvitamin D₂ (paricalcitol). There was no change in apoptosis following treatment of naïve and VDI cells with calcitriol. Vitamin D receptor (VDR) expression was up-regulated by calcitriol in both naïve and VDI cells. In addition, calcitriol induced the Vitamin D metabolizing enzyme, 24-hydroxylase (cyp24) mRNA and enzyme activity similarly in naïve and VDI cells as measured by RT-PCR and HPLC, respectively. In summary, VDI cells are less responsive to the antiproliferative effects of calcitriol. Understanding Vitamin D insensitivity will further clinical development of Vitamin D compounds for prevention and treatment of CaP.     

Hormone-dependent transformation and nuclear localisation of 1,25-dihydroxyvitamin D3 receptors from human breast cancer cell lines and chick duodenum

Recent studies on the 1,25-dihydroxyvitamin D3 (calcitriol) receptor have shown association of unoccupied receptor with isolated nuclei, thus suggesting that hormone is not required for transformation and nuclear localisation of this receptor. In the present work calcitriol receptors from cultured breast cancer cells were studied for evidence of hormone-dependent activation and compared to those from chick duodenum. Unlike other steroid receptors changes in receptor mobility on ion exchange and gel filtration were not found for occupied and unoccupied receptors. Furthermore no changes in affinity were observed on DNA-cellulose with both hormone-bound and unoccupied receptor having equally high affinity, eluting at 0.25 M KCI. However, a substantial hormone-dependent increase in receptor affinity for nuclei was seen. Thus calcitriol receptors do appear to undergo hormone-dependent transformation which is detected by their increased affinity for nuclei, without any accompanying gross changes in charge density, size or affinity for DNA-cellulose. Previously, we have reported that fractionation of T-47D cells in a low salt buffer resulted in recovery of unoccupied receptors in the cytosol, whereas occupied receptors were associated with purified nuclei. The data presented in this paper and our previous work suggest that calcitriol receptors do undergo a hormone-dependent increase in their affinity for nuclei. Furthermore in all this work calcitriol receptors from cultured human breast cancer cells displayed identical physicochemical characteristics to those of chick duodenal receptors.     

miR-103a-2-5p/miR-30c-1-3p inhibits the progression of prostate cancer resistance to androgen ablation therapy via targeting androgen receptor variant 7

Androgens and androgen receptors are vital factors involved in prostate cancer progression, and androgen ablation therapies are commonly used to treat advanced prostate cancer. However, the acquisition of androgen ablation therapy resistance remains a challenge. Recently, androgen receptor splicing variants lacking the ligand-binding domain have been reported to play a critical role in the acquisition of androgen ablation therapy resistance. In the present study, we revealed that the messenger RNA expression and the protein levels of an androgen receptor variant 7 (AR-V7) were higher in prostate cancer tissue samples and in the AR-positive prostate cancer cell line, VCaP. In contrast, microRNA (miR)-30c-1-3p/miR-103a-2-5p expression was significantly downregulated in tumor tissues and cells. miR-30c-1-3p/miR-103a-2-5p overexpression could inhibit AR-V7 expression, suppress VCaP cell growth, and inhibit AR-V7 downstream factor expression by directly targeting the 3′-untranslated region of AR-V7. Under enzalutamide (Enza) treatment, the effects of AR-V7 overexpression were the opposite of those of miR-103a-2-5p/miR-30c-1-3p overexpression; more importantly, the effects of miR-103a-2-5p/miR-30c-1-3p overexpression could be significantly reversed by AR-V7 overexpression under Enza. In summary, we demonstrated a novel mechanism of the miR-30c-1-3p/miR-103a-2-5p/AR-V7 axis modulating the cell proliferation of AR-positive prostate cancer cells via AR downstream targets. The clinical application of miR-30c-1-3p/miR-103a-2-5p needs further in vivo validation.     

Evidence for DNA-binding domain--ligand-binding domain communications in the androgen receptor

DNA binding as well as ligand binding by nuclear receptors has been studied extensively. Both binding functions are attributed to isolated domains of which the structure is known. The crystal structure of a complete receptor in complex with its ligand and DNA-response element, however, has been solved only for the peroxisome proliferator-activated receptor γ (PPARγ)-retinoid X receptor α (RXRα) heterodimer. This structure provided the first indication of direct interactions between the DNA-binding domain (DBD) and ligand-binding domain (LBD). In this study, we investigated whether there is a similar interface between the DNA- and ligand-binding domains for the androgen receptor (AR). Despite the structural differences between the AR- and PPARγ-LBD, a combination of in silico modeling and docking pointed out a putative interface between AR-DBD and AR-LBD. The surfaces were subjected to a point mutation analysis, which was inspired by known AR mutations described in androgen insensitivity syndromes and prostate cancer. Surprisingly, AR-LBD mutations D695N, R710A, F754S, and P766A induced a decrease in DNA binding but left ligand binding unaffected, while the DBD-residing mutations K590A, K592A, and E621A lowered the ligand-binding but not the DNA-binding affinity. We therefore propose that these residues are involved in allosteric communications between the AR-DBD and AR-LBD.     

Non-genomic effects of the androgen receptor and vitamin D agonist are involved in suppressing invasive phenotype of prostate cancer cells

Suppression of invasive phenotype is essential in developing new therapeutic tools to treat prostate cancer (PC). Evidence indicates that androgen-dependent (AD) prostate cancer cells are characterized by a lower malignant phenotype. We have demonstrated that transfection with an androgen receptor (AR) expression vector of the androgen-independent (AI) prostate cancer cell line PC3 decreases invasion of these cells through modulation of alpha6beta4 integrin expression, indicating a genotropic effect of androgens in inhibiting invasion ability of AD PC cells. Later on, we have shown that also a non-genotropic mechanism is involved in such an effect. By using immunoconfocal fluorescent microscopy, we demonstrated that AR in PC3-AR cells co-localizes with the EGFR receptors (EGFR) in PC3-AR cells. Co-immunoprecipitation studies both in PC3-AR cells and in the AD cell line LNCaP that physiologically express both receptors, confirm the occurrence of an interaction between of the two proteins. In PC3-AR cells, we demonstrated a disruption of EGFR signalling properties (reduced EGF-induced EGFR autotransphosphorylation, reduced EGF-stimulated PI3K activity as well as EGFR-PI3K interaction) contributing to the lower invasive phenotype of these cells. In another study, we investigated the effects of a new Vitamin D analogue, BXL628, on invasion in response to KGF in the androgen-independent PC cell line DU145. We found that the compound was able to reduce proliferation and invasion of the cells in response to the growth factor. In addition, we found that KGF-induced autotransphosphorylation of KGF receptor (KGFR) and PI3K activation were suppressed after short-term (5min) pre-treatment with the analogue before addition of KGF. Collectively, these studies demonstrate that a non-genotropic effect due to a direct interaction of the androgen receptor with EGFR and to a rapid effect of a Vitamin D agonist on KGFR may disrupt signalling of GF leading to decreased tumorigenicity and a less malignant phenotype of PC cells in vitro.     

A New Formulation of Calcitriol (DN-101) for High-Dose Pulse Administration in Prostate Cancer Therapy

Although the antineoplastic activity of calcitriol in prostate cancer has been known for many years, the agent's use in oncology has been prevented because of the occurrence of hypercalcemia with daily administration. High-dose pulse administration of calcitriol has the potential to improve the therapeutic index of calcitriol. Results of a phase II study of calcitriol and docetaxel (Taxotere(R)) suggest that this combination may have utility in androgen-independent prostate cancer (AIPC). DN-101, a high-dose (15 mug) formulation of calcitriol suitable for use in oncology, is now being tested in a randomized trial (AIPC Study of Calcitriol Enhancing Taxotere). This formulation of calcitriol could become an important new tool for improving the efficacy of docetaxel in the treatment of AIPC and would join the ranks of other nuclear receptor ligands in cancer treatment. Investigations of DN-101 in the treatment of a broad range of tumor types and in combination with a variety of agents are an exciting new area of research.     

Androgen regulation of androgen receptor content and distribution in the ventral and dorsolateral prostates of aging AXC rats

Total androgen receptor content of ventral or dorsolateral prostate of intact, aged (730–740 day old) rats is decreased 50% when compared to intact, young mature (150–170 day old) rats. Treatment with exogenous testosterone increased ventral and dorsolateral prostate androgen receptor content per cell in aged rats to values identical to those of prostates of young mature rats. The increase in prostate receptor content was not attributable to testosterone mediated cellular hypertrophy or hyperplasia. At 24 hr post-orchiectomy ventral prostate cytoplasmic androgen receptors are depleted of endogenous androgen, without any decrease in number of receptors per cell, and nuclear androgen receptors are undetectable. During 30 to 60 min after a single 200 μg testosterone injection, ventral prostate nuclear receptor content increased to the level of intact control rats without producing any reduction in total cytoplasmic androgen receptor content. Although dorsolateral prostate is devoid of cytoplasmic androgen receptor, the effects of orchiectomy and testosterone treatment upon nuclear androgen receptor are comparable to those seen in ventral prostate. These effects of orchiectomy and testosterone injection upon prostatic receptor content and distribution were identical in prostates of young and aged rats. Our studies show that receptor processing in prostates of young and aged rats does not involve a process by which nuclear receptor is derived by depletion of cytoplasmic receptor. Moreover, our studies of the effect of short-term (48 hr) exogenous testosterone treatment upon androgen receptor content in prostates of aged rats are the first demonstration that androgen receptor content may be enhanced independent of generalized androgen mediated anabolic effects in prostate.