Review ArticlePart AR

The Impact of 1,25(OH)2D3 and its Structural Analogs on Gene Expression in Cancer Cells - A Microarray Approach

CARSTEN KRIEBITZSCH, LIEVE VERLINDEN, GUY EELEN, BIAUW KENG TAN, MARK VAN CAMP, ROGER BOUILLON and ANNEMIEKE VERSTUYF
Anticancer Research September 2009, 29 (9) 3471-3483;

Abstract

The active form of vitamin D3, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], is an important regulator of bone metabolism, calcium and phosphate homeostasis but also has potent antiproliferative and pro-differentiating effects on a wide variety of cell types. To identify key genes that are (directly) regulated by 1,25(OH)2D3, a large number of microarray studies have been performed on different types of cancer cells (prostate, breast, ovarian, colorectal, squamous cell carcinoma and leukemia). The variety of target genes identified through these studies reflects the pleiotropic action of 1,25(OH)2D3. Common cellular processes targeted by 1,25(OH)2D3 in the different cancer cell lines include cell cycle progression, apoptosis, cellular adhesion, oxidative stress, immune function and steroid metabolism. Upon comparison of the lists of genes regulated by 1,25(OH)2D3 in the different microarray studies, only a small set of individual genes were commonly regulated, among which are included 24-hydroxylase, growth arrest and DNA-damage-inducible protein, cathelicidin antimicrobial peptide and multiple cyclins.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D3, is an important regulator of a diverse set of physiological actions in the human body. The absence of 1,25(OH)2D3 is linked to rickets and osteomalacia, which are skeletal disorders characterized by defective bone mineralization. Furthermore, 1,25(OH)2D3 has a significant effect on calcium and phosphate homeostasis (1). Next to these classical effects, 1,25(OH)2D3 has a non-classical role in the regulation of cell proliferation and differentiation. Indeed, in the early 1980s the presence of the vitamin D receptor (VDR) was demonstrated in normal and malignant tissues (2). Further work revealed that 1,25(OH)2D3 has antiproliferative (3) and pro-differentiating (4) effects in a wide variety of malignant and normal cell types. 1,25(OH)2D3 inhibits cell growth of normal and tumor cells by hampering the transition from the G1 to the S phase of the cell cycle, which leads to an accumulation of cells in the G1 phase (5, 6). Interestingly, 1,25(OH)2D3 down-regulates genes that mediate this G1-S transition such as cyclins and cyclin dependent kinases (7, 8) whereas cyclin-dependent kinase inhibitors p21 and p27 are up-regulated by 1,25(OH)2D3 (6). The antiproliferative effects open perspectives for the use of 1,25(OH)2D3 as a possible anticancer agent (9). To study the actions of 1,25(OH)2D3 in more detail and to unravel the underlying effects of 1,25(OH)2D3 on the transcriptome, a broad set of microarray studies have been performed. Since the first report about the use of a well-defined set of cDNA for expression profiling in 1987 (10), microarrays have changed molecular biology. In the following years, the technique developed to a high throughput method. In 1995 Schena et al. (11) reported the first use of a miniaturized microarray for gene expression profiling. Since then and with the sequencing of complete genomes (H. influenzae 1995, H. sapiens 2000), genome expression analysis became feasible. Malignant cells, such as prostate (12-18), breast (19-21), leukemia (22-24), colon (25, 26), ovarian cancer (27) and squamous cell carcinoma cells (28, 29) were used to identify novel 1,25(OH)2D3 target genes and to gain more insight on the antiproliferative and pro-differentiating action of 1,25(OH)2D3 and its analogs. In these studies, a variety of technology platforms were used (spotted arrays, Affymetrix genechips, etc.). Although it is very difficult to compare mutual studies due to the experimental set-up (variable parameters such as cell culture conditions, cell clones and platforms), it has to be acknowledged that microarray experiments provide an ample amount of data, which contributes significantly to a better understanding of the mechanism of action of 1,25(OH)2D3 and its analogs. In this review, microarray studies performed on malignant cells treated with 1,25(OH)2D3 or analogs will be discussed. First, microarray studies in individual cancer cell types will be reviewed. Genes and/or pathways shown to be regulated by 1,25(OH)2D3 in different studies on one particular cancer tissue will be discussed individually. Second, a comparison of all the microarray studies in different cancer cell types (prostate, breast, ovarian, colorectal, squamous cell carcinoma, leukemia) will be made in order to identify commonly regulated genes and/or pathways.

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Table I.

Summary of microarray studies in prostate cancer cells treated with 1,25(OH)2D3.

Prostate Cancer Cells

Prostate cancer (PCa) is classified as an adenocarcinoma and is the most common non-cutaneous malignancy in men. Interestingly, 1,25(OH)2D3 inhibits PCa growth and progression (30, 31). These effects are not limited to PCa cell lines as they are also observed in primary cultures derived from normal and cancerous prostatic epithelial tissue. Tumor cells which derive from the stroma and not from epithelial cells react differently to 1,25(OH)2D3 exposure. Krill et al. (32) pointed out that stromal cells undergo an increase in proliferation after incubation with low doses of 1,25(OH)2D3. Different microarray studies were performed on PCa cell lines treated with 1,25(OH)2D3 to unravel the induced signalling pathways (Table I). Most studies were performed using the androgen receptor-positive cell line LNCaP (13, 15, 17, 33-35), but androgen receptor-negative PC-3 (14) cells as well as primary human normal and tumor stromal cell lines were also used (12). In the studies discussed, different microarray platforms as well as different cell culture regimens were used making it very hard to compare results from various microarray experiments. Ikezoe et al. (13) reported that with the exception of one gene (FK506-binding protein 5), the gene expression profiles generated in LNCaP cells by two independent studies (13, 15) did not overlap. Nevertheless, important signalling cascades that are involved in the antiproliferative and pro-differentiating action of 1,25(OH)2D3 were identified. Genes of which the transcription was influenced by 1,25(OH)2D3 were involved in cell growth and apoptosis, cellular adhesion, oxidative stress, immune function, steroid metabolism, and intra- and intercellular signalling.

Insulin-like growth factor-binding protein-3 (IGFBP-3). IGFBPs are factors which bind to insulin-like growth factors (IGFs) and thereby enhance or repress IGF activity (36). IGFs are necessary to guarantee proliferation, survival, apoptosis and differentiation. IGF I and II are especially important for normal growth and developmental processes. Moreover, these growth factors and their binding proteins play a role in the development of cancer (37). IGFBP-3 was the most highly induced gene following 1,25(OH)2D3 treatment in a microarray study performed in LNCaP cells (15). Because IGFBP-3 antisense oligonucleotides abrogated 1,25(OH)2D3-mediated growth inhibition, IGFBP-3 was thought to mediate the cell cycle block by 1,25(OH)2D3 in LNCaP cells. It was further suggested that IGFBP-3 mediated the antiproliferative activity of 1,25(OH)2D3 by the induction of the cell cycle inhibitor p21 (38). However, other microarray studies in LNCaP cells could not confirm the regulation of IGFBP-3 by 1,25(OH)2D3 (13).

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Table II.

Summary on microarray studies in breast cancer cell lines treated with 1,25(OH)2D3 or analog Ro3582.

CCAAT/enhancer-binding protein δ (C/EBPδ). C/EBP is a highly conserved family of leucine zipper type DNA-binding proteins that is implicated in the regulation of growth and differentiation of a wide variety of cells. C/EBPδ was found to be highly induced after treatment of LNCaP cells with 1,25(OH)2D3 (13). By blocking protein synthesis, it was demonstrated that VDR bound directly to the C/EBPδ promoter region. Moreover, 1,25(OH)2D3-induced growth inhibition was suggested to be mediated by C/EBPδ action.

Apoptosis. Investigation of gene expression in primary tumor prostatic stromal cells and ALVA-31 cells after treatment with 1,25(OH)2D3 obtained several up- and down-regulated genes related to apoptosis (12). Heat-shock protein 70 (Hsp70), Hsp90 and apoptotic peptidase activating factor 1 (Apaf1) were up-regulated upon 1,25(OH)2D3 treatment. These genes are critical regulators of apoptosome assembly. Furthermore the expression of the antiapoptotic B-cell leukemia/lymphoma 2 gene (Bcl2) was more than 50% reduced after 1,25(OH)2D3 treatment.

Fatty acid synthase (FAS). Qiao et al. (35) reported the regulation of five genes by 1,25(OH)2D3 in LNCaP cells, which encode for metabolic enzymes involved in biosynthetic or catabolic pathways. As such, fatty acid synthase (FAS), phosphoribosyl-glycinamide formyltransferase (GART) and stearoyl-CoA desaturase (SCD) were down-regulated, whereas histidine ammonial-lyase (HAL) and dopachrome tautomerase were up-regulated by 1,25(OH)2D3. However, the gene dopachrome tautomerase was reported to be down-regulated by 1,25(OH)2D3 in primary E-CA-15 prostate cancer cells (17). Inhibition of FAS activity resulted in a clear suppression of LNCaP proliferation, suggesting that FAS is involved in the antiproliferative action of 1,25(OH)2D3 in prostate cancer LNCaP cells. However FAS is an indirect 1,25(OH)2D3 and androgen-dependent target gene.

Prostaglandin metabolism. Two genes involved in prostaglandin metabolism were found to be regulated by 1,25(OH)2D3 in LNCaP cells. As prostaglandins play a role in prostate cancer development and progression (39), down-regulation of prostaglandin synthesis may represent a molecular pathway induced by 1,25(OH)2D3 in prostate cells. The expression of the putative tumor suppressor gene NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15PGDH) (40, 41), whose gene product is essential for functional inactivation of prostaglandins (PG), was induced by 1,25(OH)2D3 (15, 17). Furthermore, the expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme during PG synthesis, was more than two-fold suppressed in LNCaP cells after 24 h 1,25(OH)2D3 treatment (15, 16).

Breast Cancer

Breast cancer is a common malignancy and the main cause of death in women (21). Carcinogenesis in the breast is a hormonally dependent process, in which the steroid hormone estrogen plays a crucial role because it may initiate and promote cancer development (42). In the early 1980s, Eisman et al. (2) demonstrated that normal as well as malignant breast tissue expressed a functional VDR. Moreover, 1,25(OH)2D3 is a potent inhibitor of estrogen receptor alpha-positive [ERα(+)] (MCF-7) and -negative [ERα(-)] (MDA MB231) breast cancer cell growth. Several microarray studies were conducted to unravel the actions of 1,25(OH)2D3 on breast cancer cells (Table II). The impact of 1,25(OH)2D3 on apoptosis, growth factors and cell adhesion will be discussed.

Growth factors. As mentioned earlier, IGFBP-3 was up-regulated in LNCaP PCa cells after treatment with 1,25(OH)2D3 (15, 38, 43). In malignant and metastatic MCF10CA1a cells, IGFBP3 transcript levels were also induced after treatment with Ro3582, a Gemini vitamin D analog that contains two six-carbon side chains (44). Furthermore, an increased expression of IGFBP-5 was detected in MCF-7 cells after treatment with 1,25(OH)2D3 (21). The role of 1,25(OH)2D3 on the regulation of transforming growth factor-β1 (TGF)-β1 and -2 is contradictory. TGF-β1 expression was repressed after 1,25(OH)2D3 treatment in mouse mammary carcinoma SC-3 cells (19). Moses et al. reported, however, that TGF-β1 represses cell proliferation by suppressing v-myc myelocytomatosis viral oncogene homolog (c-myc) expression (45). TGF-β2 was up-regulated by 1,25(OH)2D3 in the human cell lines MCF-7, MCT10CA1a and MDA MB231 (21, 44, 46). Induction of TGF-β2 by 1,25(OH)2D3 was suggested to mediate the antiproliferative effects of 1,25(OH)2D3 in breast cancer cells (6). These findings are in line with the hypothesis that TGF-β1 and -2 have potent tumor suppressor activity in the mammary gland (47).

Expression levels of fibroblast growth factor (FGF) family members were also regulated by 1,25(OH)2D3. Transcript levels of FGF-7 and -9 were found to be up-regulated after incubation with 1,25(OH)2D3 (20, 21), whereas other studies reported a down-regulation of FGF-7 and -8 (19, 21). Data regarding the regulation of FGF-7 are contradictory, which may be related to differences in cell culture conditions. In the study of Lyakhovich et al. (20) cells were treated with 1,25(OH)2D3 and a low dose of 17β-estradiol (10-9 M) which may have led to an up-regulation of FGF-7. As FGF-8 is known to support mammary tumor cell growth (48), down-regulation of FGF-8 expression after 1,25(OH)2D3 treatment highlights again the possible beneficial effects of 1,25(OH)2D3 in the treatment of cancer.

Apoptosis and oxidative stress response. The expression level of a set of caspases, which are important for the execution phase of cell apoptosis, was reported to be changed by 1,25(OH)2D3 action. Transcript levels of the effector caspases (caspase-3, -6 and -8) and the inflammatory caspase-4 were induced (21). The only caspase to be down-regulated was the inflammatory caspase-1 (21), having an impact not only on apoptosis but also on cytokine maturation (interleukin (IL)-1) (49, 50). The relevance of caspase-1 regulation is highlighted by the fact that apoptosis resistant human hepatocellular carcinoma cells (HCCs) showed decreased expression levels of caspase-1 (51). Furthermore, thioredoxin reductase (TrxR) expression was elevated by 1,25(OH)2D3 and Ro3582 (21, 44). A functional thioredoxin system seems to be required for the induction of apoptosis by p53, at events of apoptotic p53-inducing stimuli (52). The fact that p53 may be necessary for apoptosis induction is confirmed by the concomitant up-regulation of p53 by 1,25(OH)2D3 in breast cancer microarray studies (21, 46).

Cell adhesion genes. A diverse set of genes involved in cell adhesion and extracellular matrix composition were regulated in breast cancer cells after 1,25(OH)2D3 and Ro3582 treatment, respectively. Gene transcript levels of zyxin, laminin b3, E-cadherin, CD44 and α1-catenin were up-regulated after incubation with 1,25(OH)2D3 (21, 44). On the contrary, expression of other adhesion-related molecules, including cadherin K and integrin α1, was down-regulated by 1,25(OH)2D3 (21, 44, 46). These data confirm the impact of 1,25(OH)2D3 on cell adhesion. However, the exact function of 1,25(OH)2D3 on the global process of adhesion and extracellular matrix composition remains elusive and needs further investigation.

Ovarian Cancer

The leading cause of death from gynecological malignancy is ovarian cancer (53). Supraphysiological doses of 1,25(OH)2D3 and EB1089, a steroidal side chain analog of 1,25(OH)2D3, inhibit the proliferation of multiple ovarian cancer cell lines, including OVCAR3, and reduce the growth of OVCAR3 tumor xenografts in nude mice (54).

In Table III, the number of genes regulated by 1,25(OH)2D3 in the OVCAR3 ovarian cancer cell line is indicated. Regulated genes are not only involved in cell growth and cell death but also in immunity, motility, cell-cell adhesion and extracellular matrix interactions.

Ovarian cancer growth and progression. 1,25(OH)2D3 regulated the expression of a number of genes that have previously been associated with the growth and progression of ovarian cancer such as endothelial differentiation, lysophosphatidic acid G-protein-coupled receptor (Edg2) and the CXC chemokines GRO-β, GRO-γ and IL-8 (55-57). The up-regulation of Edg2 by 1,25(OH)2D3 is in line with the function of Edg2 in growth inhibition. On the other hand, transcript levels of GRO- β, GRO- γ and IL-8 were down-regulated by 1,25(OH)2D3, which is consistent with the fact that these chemokines are shown to promote tumor growth and motility.

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Table III.

Summary on microarray studies in ovarian and colon cancer cells as well as squamous carcinoma cells treated with 1,25(OH)2D3 or analog EB1089.

Apoptosis. The expression levels of a group of apoptosis-related genes were modified in OVCAR3 cells upon treatment with 1,25(OH)2D3. Induction of the pro-apoptotic genes TGF-β, c-abl oncogene 1, receptor tyrosine kinase (ABL1), and the growth arrest and DNA-damage-inducible protein (GADD45A), together with the down-regulation of the antiapoptotic gene apoptosis inhibitor 5 (API5L1) agreed with the finding that induction of cell death represented an important mechanism of 1,25(OH)2D3-induced growth suppression (27). However, whereas persistent 1,25(OH)2D3 treatment induced apoptosis in ovarian cancer cells, pre-treatment with 1,25(OH)2D3 suppressed death receptor-mediated apoptosis. Consistent with this, the decoy receptor that inhibits tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TRAIL receptor 4 were induced after incubation with 1,25(OH)2D3. On the contrary, transcription levels of TNF receptor superfamily, member 6 (Fas), which is crucial to mediate cell death was down-regulated. The protective effects of 1,25(OH)2D3 against TRAIL-induced apoptosis was confirmed in breast and prostate cancer cells (27).

Colorectal Cancer

Colorectal cancer is a common non-cutaneous malignancy. As a model of colon cancer different cell lines have been used in microarray studies (Table III). CaCo-2 cells and SW480ADH (58), a subline of SW480 cells, express endogenous VDR and are 1,25(OH)2D3 responsive. LS-174T cells are less responsive to 1,25(OH)2D3 and do not differentiate after 1,25(OH)2D3 exposure (26). Nevertheless, 24-hydroxylase (CYP24), responsible for the catabolism of 1,25(OH)2D3, was highly induced in this cell line after treatment with 1,25(OH)2D3. Comparable with other microarray studies, 1,25(OH)2D3 affected the expression of a wide variety of genes involved in multiple processes such as regulation of transcription, cell adhesion, DNA synthesis, apoptosis, redox control and intracellular signalling. Some genes have been found to be regulated by 1,25(OH)2D3 in different colon cancer cell lines and may therefore represent important mediators of the effects of 1,25(OH)2D3.

JNK regulation by 1,25(OH)2D3. The mitogen-activated protein kinase 8 (JNK), is a member of the mitogen-activated protein kinase (MAPK) family, which plays an important role in the regulation of basic cellular processes such as development, differentiation, proliferation, regulation of transcription and apoptotic cell death. The JNKs are encoded by three different JNK loci and are activated by various stimuli (59). One of the down-stream targets of JNK is the transcription factor AP-1. AP-1 is a multiprotein complex composed by the gene products of the jun, fos and ATF genes (60-62). The microarray studies in colon cancer cell lines showed that different jun family members (c-jun, JunD, JunB) were up-regulated after 1,25(OH)2D3 exposure (25, 26). These findings agree with other studies, which reported that expression of JNK and members of the jun family increased after 1,25(OH)2D3 or analog treatment (29, 63, 64). Qi et al. (65) suggested that crosstalk between MAPK signalling cascades might mediate the action of 1,25(OH)2D3 in cancer cells.

GTP-binding protein overexpressed in skeletal muscle (Gem) up-regulated by 1,25(OH)2D3. Gem is a member of the RGK family of GTP-binding proteins within the Ras superfamily (66). The up-regulation of Gem by 1,25(OH)2D3 action has also been demonstrated in other cancer cell types (29, 46). The known cellular function of Gem is the inhibition of Rho kinase (ROK)-mediated cytoskeletal rearrangement and the inhibition or expression of voltage-gated calcium channels at the cell surface (66, 67). Gem is able to inhibit cell proliferation by reducing calcium-mediated cell growth (66). The antiproliferative effects of 1,25(OH)2D3 in CaCo-2 cells may be further promoted by an interplay of Gem and Sorcin (68). Sorcin, which is also up-regulated by 1,25(OH)2D3 action (25), is a calcium-binding protein modulating intracellular calcium release. The interplay between these two new 1,25(OH)2D3 target genes may affect calcium channel activity and thereby cell growth and physiology.

Other 1,25(OH)2D3 target genes in colon cancer cells. Transcript levels of transient receptor potential-vanilloid 6 (TRPV6/CaT1/ZFAB) were induced by 1,25(OH)2D3 in SW480-ADH as well as in LS-174T colon cancer cells. TRPV6 is a major calcium transporter in intestinal epithelial cell membranes and is involved in 1,25(OH)2D3-mediated active calcium absorption in the intestine. This calcium channel is not only highly important in intestine but also in other organs with high calcium transport requirements (69). Previous studies already reported the induction of TRPV6 by 1,25(OH)2D3 (70) and identified TRPV6 as a direct vitamin D target gene (71). A gene commonly up-regulated by 1,25(OH)2D3 in SW480 ADH and LS-174T cells is kallikrein-related peptidase 6 (KLK6/protease M). KLK6 is a member of the kallikrein gene family, which are serine proteases implicated in various physiological processes ranging from cellular homeostasis to tissue remodelling (72). Expression of KLK6 is often dysregulated in cancer (73), although its physiological role is still unknown. Its basal expression is down-regulated in breast cancer whereas transcript levels of KLK6 are higher in colorectal cancer tissue (74, 75). The KLK6 gene is transcriptionally regulated by steroid hormones. Treatment with 1,25(OH)2D3 and analogs was also shown to increase expression of KLK6 (26, 29, 44). The gene bilirubin UDP-glucuronosyltransferase isozyme 2 was also shown to be induced by 1,25(OH)2D3 in SW480-ADH and LS-174T cells. Bilirubin UDP-glucuronosyltransferase isozyme 2 is part of the glucuronidation pathway. The enzymes in the pathway transform lipophilic molecules (drugs, bilirubin or steroids) into water soluble and excretable metabolites. A study conducted in 1,25(OH)2D3-deficient rats showed that 1,25(OH)2D3 regulates a set of biotransformator genes (76). The biotransformator gene UDP-glucuronosyltransferase (UGT1A) was up-regulated by 1,25(OH)2D3, suggesting that 1,25(OH)2D3 is a factor necessary in detoxification and protection against environmental toxins (76). In concordance with these data, the regulation of genes involved in the oxidative stress pathway [gluthationine peroxidase, glutathione-S-transferase, alpha 4 (GSTA4)] has been described in 1,25(OH)2D3-deficient rats as well as in colon cancer cells (26, 76).

Squamous Cell Carcinoma

Squamous cell carcinoma is a malignant tumor derived from epithelium or mucous membranes. Proliferation of the SCC25 head and neck squamous carcinoma cell line is inhibited by 1,25(OH)2D3 and the analog EB1089. Moreover, EB1089 reduced tumor growth in a mouse model of head and neck squamous cell carcinoma (77). An overview of microarray studies conducted in the SCC25 squamous carcinoma cell line after EB1089 treatment is shown in Table III.

Microarray analysis of EB1089-treated SCC25 cells revealed that treatment with EB1089 drove SCC25 cells towards a less malignant phenotype (29). EB1089-regulated genes controlled different cellular processes such as cell cycle progression, cell adhesion, extracellular matrix composition, inter- and intracellular signalling, G protein-coupled receptor function, intracellular redox balance and steroid metabolism. EB1089 was found to target the same genes as the parent molecule 1,25(OH)2D3, albeit in a more sustained manner. Upon comparison of the lists of genes regulated by EB1089 in the two independent microarray experiments, only a few genes were found in common: galectin (cell adhesion), COX-2 (steroid metabolism) and amphiregulin (growth factor).

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Table IV.

Summary on microarray studies in HL-60 leukemia cells treated with 1,25(OH)2D3.

Growth factor signalling. Amphiregulin is a member of the epidermal growth factor family of peptide growth factors. Transcript levels of amphiregulin have been induced by 1,25(OH)2D3 and EB1089 (28, 29, 78). Moreover, amphiregulin itself was able to partially repress proliferation of squamous cancer cells (78), which suggested that amphiregulin may act as a component of the antiproliferative response to 1,25(OH)2D3 and its analogs by inhibiting proliferation in an autocrine or paracrine manner. Interestingly, transcript levels of amphiregulin were also modified upon 1,25(OH)2D3-treatment in colon cancer (25).

Steroid metabolism. COX-2 transcript levels were induced after treatment with EB1089 in squamous cell carcinoma whereas the expression of COX-2 was suppressed by 1,25(OH)2D3 in prostate cancer cells (15, 16, 29, 34). Nevertheless, these findings indicate once again that the 1,25(OH)2D3-induced signalling pathway affects prostaglandin metabolism.

Leukemia

Acute myeloid leukemia (AML) is a heterogeneous group of leukemias that result from clonal transformation of hematopoietic precursors through the acquisition of chromosomal rearrangements and multiple gene mutations (79). As acute myeloid leukemia cells are proliferatively immature and not fully differentiated, differentiation therapy is potentially effective for initiation of terminal differentiation and may represent a supplementary approach for the treatment of neoplastic disease in combination with existing treatment modalities (22, 80). 1,25(OH)2D3 is a potent initiator of differentiation in human AML cell lines such as HL-60, which are predominantly neutrophilic promyelocytes (81). Treatment of HL-60 cells with 1,25(OH)2D3 resulted in a reduced proliferation and enhanced differentiation along the monocyte-macrophage pathway (82, 83). To understand the changes on the transcriptional level accounting for the differentiation process in HL-60 cells upon treatment with 1,25(OH)2D3, different microarray studies have been conducted (Table IV). Microarray studies of HL-60 leukemia cells treated with 1,25(OH)2D3 show that transcription of a diverse set of genes was regulated. Affected cellular processes included cell differentiation, cell cycle regulation, protein synthesis and nuclear transport.

Oncogene-related genes. Oncogenes, which have been reported to be down-regulated by 1,25(OH)2D3, are c-myc (24), v-myc (the viral homolog of c-myc) (23), Myb-related protein B (22) and v-myb myeloblastosis viral oncogene homolog (23). These findings confirmed earlier studies in which the decrease in myc expression after 1,25(OH)2D3-treatment was shown (84, 85). The inhibitory effect of 1,25(OH)2D3 on oncogene expression highlights the possible role of 1,25(OH)2D3 in cancer therapy.

Cytokine expression. Several microarray studies in HL-60 cells reported the upregulation of the pro-inflammatory cytokines IL-1β, IL-8 and TNF-α after treatment with 1,25(OH)2D3 (22-24, 86, 87). IL-1β and IL-8 were suggested to be involved in the monocytic differentiation of normal hematopoietic progenitors (88). Furthermore, IL-1β was able to induce the expression of hematopoetic growth factors, which are important for the regulation of cellular function and traffic (89). The up-regulation of monocyte differentiation CD14 antigen has also been reported (23, 24). CD14, already known to be induced upon treatment with 1,25(OH)2D3, is a membrane-associated glycosyl-phosphatidylinositol-linked protein expressed at the surface of cells and is often used as a monocyte/macrophage marker (23, 24, 90). Elevated levels of CD14 confirmed the differentiation of HL-60 cells along the monocyte-macrophage pathway after 1,25(OH)2D3 treatment.

S-100 calcium-binding proteins. The expression of the genes for S-100 calcium-binding proteins S-100 A8, A9 (91) and A4 (86) was up-regulated in HL-60 cells treated with 1,25(OH)2D3. Court et al. (92) showed that the proteins S-100 A8 and S-100 A9, which inhibit cell migration and differentiation, are the most highly expressed in non-malignant cells. Furthermore, the involvement of S-100P protein in isopentenyladenine (IPA)-driven cell differentiation was shown by Ishii et al. (86). These data suggest that S-100 proteins may play a role during differentiation of HL-60 cells into a less malignant and more differentiated cell population. Interestingly, in different breast epithelial cells S-100 A7 expression was considerably higher in premalignant than in fully malignant and metastatic cells after treatment with the 1,25(OH)2D3 Gemini analog Ro3582 (44).

Eukaryotic translation initiation factors. The down-regulation of different eukaryotic translation initiation factors (eIF), including the factors eIF-2, subunit beta and eIF-3, has been reported in a number of microarray studies (22, 23, 87). Those factors consist of several different subunits and form stable complexes with the 40S ribosomal subunit (93). A down-regulation of these factors by 1,25(OH)2D3 culminated in a down-regulation of translation and concordantly in reduced cellular function and activity.

Importin and exportin expression. Importins and exportins are karyopherin nuclear transport factors of the importin-beta superfamily. Exportins are crucial for the nuclear export of RNAs (94, 95) and encompass proteins such as eukaryotic translation elongation factor 1A (96), interleukin enhancer binding factor 3 (97), amongst others (98). Suzuki et al. (23) demonstrated that exportins 5 and 7, exportin-tRNA and exportin 1/CRM1 were down-regulated in 1,25(OH)2D3-treated HL-60 cells. Furthermore, it has been previously reported that inhibition of exportin 1/CRM1 inhibited cell proliferation and cell cycle progression (99, 100). Interestingly, gene expression of most importins was down-regulated when HL-60 cells were treated with 1,25(OH)2D3 (23). The suppression of importins and exportins by the same substance is coherent exemplifying an equilibrium in nuclear transport.

Conclusion

To identify key genes that are (directly) regulated by 1,25(OH)2D3 and involved in the growth-inhibitory and pro-differentiating pathway of 1,25(OH)2D3, a broad set of microarray studies have been performed on different types of cancer cells (prostate, breast, ovarian, colorectal, squamous cell carcinoma, and leukemia). Upon comparison of the lists of genes regulated by 1,25(OH)2D3 in the different microarray studies on several cancer cell types, only a small set of individual genes was found to be commonly regulated (Table V). Discrepancies between the number and nature of genes regulated by 1,25(OH)2D3 may have different causes. Although each human cell possesses the same genomic setting, the spectrum of active genes in each tumor cell is diverse and depends on the tissue from which the cancer cell derives. In addition, 1,25(OH)2D3 acts in a cell type- and tissue-specific manner, which may further contribute to differences in the sets of 1,25(OH)2D3-regulated genes. Moreover, the use of different microarray platforms, different cell line subclones and differences in cell culture conditions are supplementary causes of variability. Nevertheless, upon comparison of all microarray data it was possible to distinguish some individual genes or processes that were commonly regulated by 1,25(OH)2D3 (Table V). Not surprisingly, most microarray studies revealed a large induction of CYP24 in cancer cells treated with 1,25(OH)2D3 (Table V). Common cellular processes targeted by 1,25(OH)2D3 in the different cancer cell lines include cell cycle progression, apoptosis, cellular adhesion, oxidative stress, immune function, steroid metabolism, and intra- and intercellular signalling. In view of the growth-inhibitory effect of 1,25(OH)2D3, it was not surprising that a number of genes either directly involved in cell cycle progression or pathways regulating cell growth were affected by 1,25(OH)2D3. A number of genes encoding for cyclin family members, which are important regulators of cyclin-dependent kinases during cell cycle progression, were transcriptionally regulated by 1,25(OH)2D3 in different target tissues (22, 23, 25, 27, 29, 46, 101). Transcript levels of G0/G1 switch 2 (G0S2) were induced by 1,25(OH)2D3 in breast, colon and squamous cell carcinomas (26, 29, 46). Interestingly, G0S2, thought to be involved in the G0/G1 switch, has been previously identified as a direct target gene of other nuclear receptors such as PPAR and RAR (102, 103). GADD45A is one of the few genes of which the expression was regulated by 1,25(OH)2D3 in most discussed cancer cell types (14, 26-28, 44, 46). GADD45 proteins function as stress sensors by mediating a complex interplay of physical interactions with other cellular proteins that are implicated in cell cycle regulation and stress response. As such, GADD45 is able to interact with proliferating cell nuclear antigen (PCNA), p21, cdc2/cyclin B1, and the p38 and JNK stress response kinases (104, 105). These interactions will eventually lead to cell cycle arrest, DNA repair, cell survival and senescence or apoptosis. Induction of GADD45A by 1,25(OH)2D3 has been previously demonstrated and was suggested to mediate G2/M arrest, or apoptosis of cells after incubation with 1,25(OH)2D3 (106). Depending on the cell type and/or growth conditions, 1,25(OH)2D3 was shown to interact with different signalling pathways, which may contribute to its growth-inhibitory effects. Indeed, multiple studies demonstrated the interplay between 1,25(OH)2D3-signalling and the growth factors TGF-β and IGF-I (through IGFBP proteins) (107, 108). Furthermore, various microarray studies demonstrated the interference of 1,25(OH)2D3-induced signalling and prostaglandin metabolism, which may point to an important role of regulation of prostaglandin synthesis in the antiproliferative activity of 1,25(OH)2D3 (16). The immunomodulatory properties of 1,25(OH)2D3 are reflected by the induction of CD14, a correceptor for toll-like receptors, in leukemia cells and by a marked up-regulation of cathelicidin antimicrobial peptide in leukemia and in prostate, breast and ovarian cancer cells (17, 23, 27, 44). Cathelicidin antimicrobial peptide has been previously shown to be a direct vitamin D target gene and to be required for the 1,25(OH)2D3-triggered antimicrobial activity (109, 110). However, the functionality of this up-regulation by 1,25(OH)2D3 in cancer cells remains to be investigated. From Table V, the induction of G-protein-coupled receptor kinase 5 (GPRK5) and GTP-binding protein overexpressed in skeletal muscle (GEM) by 1,25(OH)2D3 in most cancer cell types is evident. However, their role in the pleiotropic effects of 1,25(OH)2D3 still needs further examination.

View this table:
Table V.

Summary on genes commonly regulated by 1,25(OH)2D3 or analog in cancer cells based on microarray studies.

In conclusion, although it is very difficult to compare mutual studies due to differences in cell types and experimental set-up, it has to be acknowledged that microarray experiments provided an ample amount of data which contributed significantly to a better understanding of the mechanism of action of 1,25(OH)2D3 and its analogs.

Acknowledgements

This work was supported by the Fund for Scientific Research (FWOG.0508.05 and FWOG.0553.06), the KU Leuven Research Council (EF/05/007 SymBioSys) and the EU (Marie Curie RTN NucSys). G. Eelen is a postdoctoral fellow of the Fund for Scientific Research.

  • Received January 29, 2009.
  • Revision received March 18, 2009.
  • Accepted April 2, 2009.

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The Impact of 1,25(OH)2D3 and its Structural Analogs on Gene Expression in Cancer Cells - A Microarray Approach
CARSTEN KRIEBITZSCH, LIEVE VERLINDEN, GUY EELEN, BIAUW KENG TAN, MARK VAN CAMP, ROGER BOUILLON, ANNEMIEKE VERSTUYF
Anticancer Research Sep 2009, 29 (9) 3471-3483;
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