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FoxP3 is a transcription factor essential for differentiation and function of T regulatory cells (Tregs). There are two major subsets of Tregs: natural Tregs (nTregs) generated in thymus and inducible Tregs (iTregs) produced in peripheral immune system. It has been documented that iTreg development is dependent on soluble mediators including interleukin 2 (IL2), transforming growth factor β (TGFβ) and all-trans-retinoic acid (ATRA). In our experiments we performed a gene expression array, followed by Real-time PCR experiments to study expression of genes regulated by ATRA in cells of myeloid origin. Our experiments revealed that ATRA alone, but also a cocktail of mediators consisting of IL2, TGFβ and ATRA, upregulate expression of FOXP3 gene in normal and leukemic myeloid cells. Our results indicate that signaling pathways which are used at the late steps of T cell differentiation, are also active in the cells of myeloid lineage.

IntroductionPopulation biobanks are essential for the development of public health screening and improvement of personalized medicine. Since 2012, Biobank of Łukasiewicz Research Network – PORT Polish Center for Technology Development (PORT Biobank) has collected more than 120 000 biological samples from nearly 5000 inhabitants of Lower Silesia, together with a variety of demographic, anthropometric, life style and health information.Material and methodsThe analyzed group consisted of 2274 participants (1398 women, 876 men). Both women and men were further subdivided into five age decades (20+, 30+, 40+, 50+, 60+). For this study, the level of lipids (total cholesterol, high-density lipoprotein [HDL], low-density lipoprotein [LDL], triglycerides) was estimated and correlated with the level of high-sensitivity C-reactive protein (hs-CRP) and biometric parameters.ResultsWe have demonstrated for the first time that biochemical changes that may lead to cardiovascular diseases (CVD) occurred already in the group of people aged 30+. Our observation is based on measurements of lipids, glucose, inflammatory (hs-CRP) and biometric markers such as body mass index (BMI) and waist-to-hip ratio (WHR).ConclusionsPositive correlations with age for these variables suggest the ongoing progress of metabolic changes, which in the end may lead to a fatal outcome such as myocardial infarction or stroke. It suggests that CVD screening programs should be dedicated to a wider group, especially younger citizens, in order to prevent fatal outcomes related to CVD.

The role of vitamin D as a treatment option for neoplastic diseases, once considered to have a bright future, remains controversial. The preclinical studies discussed herein show compelling evidence that Vitamin D Derivatives (VDDs) can convert some cancer and leukemia cells to a benign phenotype, by differentiation/maturation, cell cycle arrest, or induction of apoptosis. Furthermore, there is considerable, though still evolving, knowledge of the molecular mechanisms underlying these changes. However, the attempts to clearly document that the treatment outcomes of human neoplastic diseases can be positively influenced by VDDs have been, so far, disappointing. The clinical trials to date of VDDs, alone or combined with other agents, have not shown consistent results. It is our contention, shared by others, that there were limitations in the design or execution of these trials which have not yet been fully addressed. Based on the connection between upregulation of JNK by VDDs and DNA repair, we propose a new avenue of attack on cancer cells by increasing the toxicity of the current, only partially effective, cancer chemotherapeutic drugs by combining them with VDDs. This can impair DNA repair and thus kill the malignant cells, warranting a comprehensive study of this novel concept. J. Cell. Biochem. 117: 1733-1744, 2016. © 2016 Wiley Periodicals, Inc.

FoxP3 is a transcription factor essential for the differentiation and function of T regulatory cells (Tregs). There are two major subsets of Tregs: natural Tregs (nTregs) generated in thymus and inducible Tregs (iTregs) produced in peripheral immune system. It has been documented that iTreg development is dependent on soluble mediators including interleukin 2 (IL2), transforming growth factor β (TGFβ) and all-trans-retinoic acid (ATRA). In our experiments we performed a gene expression array, followed by Real-time PCR experiments, to study the expression of genes regulated by 1,25-dihydroxyvitamin D (1,25D) or ATRA in cells of myeloid origin. Our experiments revealed that ATRA alone, but also a cocktail of mediators consisting of IL2, TGFβ and ATRA, upregulate the expression of FOXP3 gene in lymphoid cells, but also in normal and leukemic myeloid cells. The FoxP3 expression is followed by a phenotypic changes in cells of myeloid origin. Our results indicate that signaling pathways which are used in the late stages of T cell differentiation, are also active in the cells of myeloid lineage

The current standard regimens for the treatment of acute myeloid leukemia (AML) are curative in less than half of patients; therefore, there is a great need for innovative new approaches to this problem. One approach is to target new treatments to the pathways that are instrumental to cell growth and survival with drugs that are less harmful to normal cells than to neoplastic cells. In this review, we focus on the MAPK family of signaling pathways and those that are known to, or potentially can, interact with MAPKs, such as PI3K/AKT/FOXO and JAK/STAT. We exemplify the recent studies in this field with specific relevance to vitamin D and its derivatives, since they have featured prominently in recent scientific literature as having anti-cancer properties. Since microRNAs also are known to be regulated by activated vitamin D, this is also briefly discussed here, as are the implications of the emerging acquisition of transcriptosome data and potentiation of the biological effects of vitamin D by other compounds. While there are ongoing clinical trials of various compounds that affect signaling pathways, more studies are needed to establish the clinical utility of vitamin D in the treatment of cancer.

The physiological form of vitamin D, 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), has a role in normal hematopoiesis, enhancing monocyte–macrophage differentiation. It also has antiproliferative and prodifferentiation effects against various myeloid leukemia cell lines and acute myeloid leukemia (AML) blasts ex vivo. Nevertheless, hematopoiesis in mice with vitamin D receptor (VDR) deletion is essentially normal, indicating that in mammals the vitamin D pathway appears to have a nonessential but perhaps a contributory role in blood formation. This chapter reviews the general mechanisms by which vitamin D derivatives (VDDs) can drive cell differentiation, the associated cell cycle traverse arrest, and alter cell survival. However, in vivo these activities of 1,25(OH) 2 D 3 require supraphysiological levels of 1,25(OH) 2 D 3 . The important distinctions between different mechanisms of 1,25(OH) 2 D 3 -induced differentiation that are cell type and cell context–specific and the different pathways that participate in 1,25(OH) 2 D 3 -induced signal propagation are described here. Clinical trials with 1,25(OH) 2 D 3 have been performed for the treatment of preleukemia/myelodysplastic syndrome and AML, but the doses effective in vitro caused hypercalcemia in vivo. However, numerous vitamin D analogs have been synthesized, which, in model systems, have reduced calcemic activity but have increased ability to induce cell differentiation and to inhibit proliferation of leukemic cells. Perhaps the available or new analogs combined with other differentiating or antiproliferative agents, each working through different pathways, will demonstrate synergistic activity and offer improved therapy for AML.

1,25-Dihydroxyvitamin D3 (1,25D) used to treat human acute myeloid leukemia (AML) cells induces features of normal monocytes, but the mechanisms underlying this response are not fully understood. We hypothesized that one or more microRNAs (miRNA) known to control mouse hematopoiesis and lineage commitment might contribute to the ability of 1,25D to control the malignant phenotype. Here we report that 1,25D markedly induces expression of miR-32 in human myeloid leukemia cells, in which it targets the 3′-untranslated region of the mRNA encoding the proapoptotic factor Bim to reduce its expression. RNAi-mediated suppression of the miRNA-processing enzymes Drosha and Dicer increased Bim levels, in support of the concept that Bim is under miRNA control in AML cells. Antisense-mediated suppression of miR-32 was sufficient to upregulate Bim expression in AML cells. Conversely, ectopic expression of miR-32 downregulated Bim expression and increased the differentiation response to 1,25D treatment in a manner that was associated with increased cell survival. The positive effects of miR-32 on cell survival were confirmed by evidence of increased cell death in AML cells preexposed to antisense miR-32 before treatment with arabinocytosine, a chemotherapeutic drug used to treat human AML. Together, our findings indicate that miR-32 blockade is sufficient to elevate Bim expression and sensitize AML cells to chemotherapy-induced apoptosis. Thus, agents which can inhibit miR-32 expression may offer clinical utility by enhancing therapeutic efficacy in human AML. Cancer Res; 71(19); 6230–9. ©2011 AACR.

Acute Myeloid Leukemia (AML) is a predominant acute leukemia among adults, characterized by accumulation of malignantly transformed immature myeloid precursors. A very attractive way to treat myeloid leukemia, which is now called ‘differentiation therapy’, was proposed as in vitro studies have shown that a variety of agents stimulate differentiation of the cell lines isolated from leukemic patients. One of the differentiation-inducing agents, all-trans retinoic acid (ATRA), which can induce granulocytic differentiation in myeloid leukemic cell lines, has been introduced into clinics to treat patients with acute promyelocytic leukemia (APL) in which a PML-RARA fusion protein is generated by a t(15;17)(q22;q12) chromosomal translocation. Because differentiation therapy using ATRA has significantly improved prognosis for patients with APL, many efforts have been made to find alternative differentiating agents. Since 1,25-dihydroxyvitamin D3 (1,25D) is capable of inducing in vitro monocyte/macrophage differentiation of myeloid leukemic cells, clinical trials have been performed to estimate its potential to treat patients with AML or myelodysplastic syndrome (MDS). Unfortunately therapeutic concentrations of 1,25D can induce potentially fatal systemic hypercalcemia, thus limiting clinical utility of that compound. Attempts to overcome this problem have focused on the synthesis of 1,25D analogs (VDAs) which retain differentiation inducing potential, but lack its hypercalcemic effects. This review aims to discuss current problems and potential solutions in differentiation therapy of AML.

Acute myelogenous leukemia (AML) is a disease characterized by dysregulated cell proliferation associated with impaired cell differentiation, and current treatment regimens rarely save the patient. Thus, new mechanism-based approaches are needed to improve prognosis of this disease. We have investigated in preclinical studies the potential anti-leukemia use of the plant-derived polyphenol Silibinin (SIL) in combination with 1,25-dihydroxyvitamin D3 (1,25D). Although most of the leukemic blasts ex vivo responded by differentiation to treatment with this combination, the reasons for the absence of SIL-1,25D synergy in some cases were unclear. Here we report that failure of SIL to enhance the action of 1,25D is likely due to the SIL-induced increase in the activity of differentiation-antagonizing cell components, such as ERK5. This kinase is under the control of Cot1/Tlp2, and inhibition of Cot1 activity by a specific pharmacological inhibitor 4-(3-chloro-4-fluorophenylamino)-6-(pyridin-3-yl-methylamino-3-cyano-[1-7]-naphthyridine, or by Cot1 siRNA, increases the differentiation by SIL/1,25D combinations. Conversely, over-expression of a Cot1 construct increases the cellular levels of P-ERK5, and SIL/1,25D-induced differentiation and cell cycle arrest are diminished. It appears that reduction in ERK5 activity by inhibition of Cot1 allows SIL to augment the expression of 1,25D-induced differentiation promoting factors and cell cycle regulators such as p27 (Kip1) , which leads to cell cycle arrest. This study shows that in some cell contexts SIL/1,25D can promote expression of both differentiation-promoting and differentiation-inhibiting genes, and that the latter can be neutralized by a highly specific pharmacological inhibitor, suggesting a potential for supplementing treatment of AML with this combination of agents.

Phosphorylation of proteins is a post-translational modification that alters their structures and leads to the changes in their activities, functions and biological responses. Enzymes that transfer phosphate groups to tyrosine residues on protein substrates are named protein tyrosine kinases (PTKs). Two main classes of PTKs have been distinguished: the transmembrane receptor PTKs (RPTKs) and the cytoplasmic non-receptor PTKs (NRPTKs). Both classes of enzymes are involved in multiple signaling pathways regulating several aspects of cells growth, proliferation, differentiation and apoptosis. Deregulation of PTKs, mainly overexpression and mutations resulting in constitutive activation, is tightly connected with malignant transformation and carcinogenesis. In this chapter, we will discuss signaling pathways engaging PTKs and their implications in the initiation and progression of several human malignancies. We will point out the principal cancer cell types known to be candidates for tyrosine kinases inhibitors (TKIs)-or monoclonal antibodies-based targeted therapies.

1,25-Dihydroxyvitamin D3 (1,25D) regulates gene transcription through the nuclear vitamin D receptor (VDR) and initiates rapid cellular responses via an unknown mechanism. Here we report that 1,25D induces a rapid increase in synthesis of VDR protein and its transport to the nucleus. These results are similarly obtained in myeloid leukemia cell lines, and in blast cells from blood of patients diagnosed with acute myeloid leukemia, subtypes M2 and M4. Our results suggest that stability of unliganded VDR is LY294002- and PD98059-dependent, and that ligation of VDR leads to its increased translation and nuclear translocation. The receptor localized in the cell nucleus is not exported back to the cytosol by exportin 1. We also show that the cytosolic portion of VDR in leukemia cells is localized in the vicinity of the plasma membrane, close to the F-actin cytoskeleton.

The active form of vitamin D, 1alpha,25-dihydroxyvitamin D3 (1,25D), has a broad range of effects which are mediated by nuclear vitamin D receptor (VDR). Many experiments that investigate the role of VDR can be done in human acute myeloid leukemia (AML) cells, since these cells are responsive to 1,25D and express VDR in a 1,25D-regulated manner. In this paper we show that in HL60 and in THP-1 cells VDR protein interacts with heat shock protein 90 (Hsp90) and that Hsp90 is important for differentiation of AML cells. Geldanamycin (GA), an Hsp90 inhibitor, is able to suppress 1,25-induced differentiation of HL60 cells.

Protein tyrosine kinases (PTKs) are enzymes that transfer phosphate groups to tyrosine residues on protein substrates. Phosphorylation of proteins causes changes in their function and/or enzymatic activity resulting in specific biological responses. There are two classes of PTKs: the transmembrane receptor PTKs and the cytoplasmic non-receptor PTKs (NRTKs). NRTKs are involved in transduction of signals originating from extracellular clues, which often interact with transmembrane receptors. Thus, they are important components of signaling pathways which regulate fundamental cellular functions such as cell differentiation, apoptosis, survival, and proliferation. The activity of NRTKs is tightly regulated, and de-regulation and/or overexpression of NRTKs has been implicated in malignant transformation and carcinogenesis. Research on NRTKs has shed light on the mechanisms of a number of cellular processes including those involved in carcinogenesis. Not surprisingly, several tyrosine kinase inhibitors are in use as treatment for a number of malignancies, and more are under investigation. This review deals with the structure, function, and signaling pathways of nine main families of NRTKs in normal and cancer cells.

Acute myeloid leukemia (AML) cell lines can be driven to differentiate to monocyte-like cells by 1,25- dihydroxyvitamin D3 (1,25D) and to granulocyte-like cells by all-trans-retinoic acid (ATRA). Both compounds activate their specific intracellular receptors, vitamin D receptor (VDR) and retinoic acid receptors (RARs) respectively. Inside the cells 1,25D is degraded to calcitrioic acid by a mitochondrial enzyme CYP24A1, while ATRA is degraded to several polar metabolites by CYP26. NADPH-cytochrome P450 oxidoreductase (POR) is a membrane-bound enzyme required for electron transfer to cytochrome P450 (CYP), vital in the processes of the metabolism of drugs and steroid production in humans. In this paper we report that POR in AML cells, from both cell lines and patients, is upregulated by ATRA and by 1,25D at the level of mRNA and protein. Partial silencing of POR in HL60 cells resulted in augmented differentiation response to 1,25D.

Vitamin D derivatives, including its physiological form 1α,25(OH)2 vitamin D3 (1,25D), have anti-tumor actions demonstrated in cell culture and confirmatory epidemiological associations are frequently reported. However, their promise for use in the cancer clinic is still incompletely fulfilled, suggesting that a better understanding of the molecular events initiated by these compounds is needed for therapeutic advances. While ERK1/2 has been intensely investigated and is known to transmit signals for cell survival, growth, and differentiation, the role of other MAPK pathways has been studied sporadically. Therefore, we utilized acute myeloid leukemia (AML) cells in culture (HL60 and U937), to determine if ERK5 has a role in 1,25D-induced terminal differentiation which is distinct from the previously shown involvement of ERK1/2. We previously found that inhibition of kinase activity of ERK5 by specific pharmacological inhibitors BIX02189 or XMD8-92 results in higher expression of general myeloid marker CD11b, but a lower expression of the monocytic marker CD14. In contrast, the inhibition of the ERK1/2 pathway by PD98059 or U0126 reduced the expression of all differentiation markers studied. We report here for the first time that the differentiation changes induced by ERK5 inhibitors are accompanied by the inhibition of cell proliferation, and this occurs in the both G1 and G2 phases of the cell cycle. Of note, inhibition of ERK5 auto-phosphorylation by XMD8-92 results in a particularly robust cell cycle arrest in G2 phase in AML cells. This study provides a link between the 1,25D-elevated ERK5 pathway and changes in the cell cycle phase transitions in AML cells. Thus, combinations of vitamin D derivatives and ERK5 inhibitors may be more successful in cancer clinics than 1,25D or analogs alone. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Some leukemic cell lines can be driven to differentiate to monocyte-like cells by 1,25-dihydroxyvitamin D(3) (1,25D) and to granulocyte-like cells by all-trans retinoic acid (ATRA). Acute myloid leukemias (AMLs) are heterogeneous blood malignancies characterized by a block at various stages of hematopoietic differentiation and there are more than 200 known chromosome translocations and mutations in leukemic cells of patients diagnosed with AML. Because of the multiplicity in the genetic lesions causing the disease, AMLs are particularly difficult to treat successfully. In particular, various AML cells to a variable degree respond to 1,25D-based differentiation and only one type of AML undergoes successfully ATRA-based differentiation therapy. In this paper we describe that AML cell line KG-1 is resistant to 1,25D-induced monocytic differentiation, while sensitive to ATRA-induced granulocytic differentiation. We show that KG-1 cells have very low level of VDR protein and that expression of VDR mRNA is upregulated by ATRA. We show for the first time that this regulation is cell context-specific, because in another AML cell line, HL60, VDR mRNA is downregulated by ATRA. ATRA-induced VDR protein in cytosol of KG-1 cells can be further activated by 1,25D to induce monocytic differentiation of these cells.

1α,25-Dihydroxyvitamin D3(1,25(OH)2D) exerts its biological activities through vitamin D receptor (VDR), which is a member of the superfamily of steroid receptors, that act as ligand-dependent transcription factors. Ligated VDR in complex with retinoid X receptor (RXR) binds to regulatory regions of 1,25(OH)2D-target genes. 1,25(OH)2D is able to induce differentiation of leukemic blasts towards macrophage-like cells. Many different acute myeloid leukemia (AML) cell lines respond to 1,25(OH)2D by increasing CD14 cell surface receptor, some additionally upregulate CD11b and CD11c integrins. In untreated AML cells VDR protein is present in cytosol at a very low level, even though its mRNA is continuously expressed. Ligation of VDR causes protein stabilization and translocation to the cell nuclei, where it regulates transcription of target genes. Several important groups of genes are regulated by 1,25(OH)2D in HL60 cells. These genes include differentiation-related genes involved in macrophage function, as well as a gene regulating degradation of 1,25(OH)2D, namely CYP24A1. We summarize here the data which demonstrate that though some cellular responses to 1,25(OH)2D in AML cells are transcription-dependent, there are many others which depend on intracellular signal transduction, protein trafficking and stabilization. The final effect of 1,25(OH)2D action in leukemic cells requires all these acting together.

Publisher Summary Differentiation can be considered to be, in essence, a persistent pattern of expression of previously dormant genes, which results in new functional capabilities of the differentiated cell. The new functions require cellular resources that compete with and finally titrate out the resources required for proliferation, and allow an accumulation of negative regulators of the cell cycle, which then predominate over the positive regulators. Thus, there is a reciprocal relationship between cellular differentiation and cell cycle progression/proliferation, though there is also evidence that differentiation and cycle arrest need not be strictly coupled. Cell cycle changes in differentiating cells need not take place immediately—in some cells there is at first a boost of proliferation—as in normal hematopoiesis, or in HL60 and U937 cells differentiating in response to derivatives of vitamin D 3 . However, even in these cases, there is an eventual slowdown of the cell cycle traverse and cessation of proliferation of differentiated cells. Consequently, numerous attempts are being made to exploit the differentiating actions of vitamin D and its analogs to induce proliferative quiescence of neoplastic cells, and thus increase the range of options for optimal therapy of human cancer.

Current understanding of the vitamin D-induced differentiation of neoplastic cells, which results in the generation of cells that acquire near-normal, mature phenotype is summarized here. The criteria by which differentiation is recognized in each cell type are provided, and only those effects of 1α,25-dihydroxyvitamin D3 (1,25D) on cell proliferation and survival which are associated with the differentiation process are emphasized. The existing knowledge of the signaling pathways that lead to vitamin-D-induced differentiation of colon, breast, prostate, squamous cell carcinoma (SCC), osteosarcoma, and myeloid leukemia cancer cells is outlined. Where known, the distinctions between the different mechanisms of 1,25D-induced differentiation which are cell-type-specific and cell-context-specific are pointed out. A considerable body of evidence suggests that several types of human cancer cells can be suitable candidates for chemoprevention or differentiation therapy with vitamin D. However, further studies of the underlying mechanisms are needed to gain further insights on how to improve the therapeutic approaches that incorporate vitamin D derivatives.

Primitive myeloid leukemic cell lines can be driven to differentiate to monocyte-like cells by 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and, therefore, 1,25(OH)(2)D(3) may be useful in differentiation therapy of myeloid leukemia and myelodysplastic syndromes (MDS). Recent studies have provided important insights into the mechanism of 1,25(OH)(2)D(3)-stimulated differentiation. For myeloid progenitors to complete monocytic differentiation a complex network of intracellular signals has to be activated and/or inactivated in a precise temporal and spatial pattern. 1,25(OH)(2)D(3) achieves this change to the 'signaling landscape' by (i) direct genomic modulation of the level of expression of key regulators of cell signaling and differentiation pathways, and (ii) activation of intracellular signaling pathways. An improved understanding of the mode of action of 1,25(OH)(2)D(3) is facilitating the development of new therapeutic regimens.

This study was designed to compare the differentiation-inducing potential of 1,25-dihydroxyvitamin D 3 (1,25D) with some analogs (VDAs) in a panel of acute myeloid leukemia (AML) cell lines and in blast cells isolated from patients with AML. Of the cell lines studied, HL60 proved to be the most sensitive to each of the differentiation-inducing agents when compared to THP-1, NB-4 and U-937 cell lines. Three of the VDAs tested (PRI-1906, PRI-2191 and PRI-2201) were similarly effective as 1,25D in all the cell lines tested. However, blast cells from AML showed a varying sensitivity towards 1,25D. For example, blast cells isolated from patients in which the whole or part of chromosome 7 was deleted were extremely sensitive to 1,25D and its analogs. In contrast, 1,25D failed to increase the expression of differentiation markers in blast cells isolated from patients carrying activating mutations in Flt3 gene. Since, the expression of vitamin D receptor (VDR) in cells with Flt3 mutations was increased to the same extent as in other AML cells this suggests that failure of these cells to differentiate lies downstream of the receptor. That blast cells with different cytogenetic abnormalities have dissimilar responses to 1,25D and its analogs, may have implications in the use of 1,25D as a ‘differentiation therapy’ for myeloid leukemias. The analog PRI-2191 (tacalcitol) was found to be the most potent in inducing patient's cells differentiation.

Human myeloid leukemia cells exposed to 1,25-dihydroxyvitamin D(3) (1,25D), a major cancer chemopreventive agent, acquire features of normal monocytes and arrest in the G(1) phase of the cell cycle, due to the upregulation of p27(Kip1) and p21(Cip1), but the mechanism is not clear. Here evidence is provided that an exposure of HL60 and U937 cells to low (1-10 nM) concentrations of 1,25D decreases the expression of miR181a and miR181b in a concentration and time-dependent manner. Since the predicted miR181 targets include the 3'-UTR of p27(Kip1), we expressed pre-miR181a in these cells, and found that the elevation of cellular miR181a levels abrogates the 1,25D-induced increase in p27(Kip1) at both mRNA and protein levels. In contrast, transfection of pre-miR181a resulted in a slight elevation of p21(Cip1) expression. Importantly, transfection of pre-miR181a blunted the effect of 1,25D on the expression of monocytic differentiation markers, and reduced the G(1) block in 1,25D-treated cells, while transfection of anti-miR181a increased 1,25D-induced differentiation. Together, these data show that miR181a participates in 1,25D-induced differentiation of HL60 and U937 cells, and suggest that a high constitutive expression of members of miR181 family may contribute to the malignant phenotype in the myeloid lineage.

This paper reviews the current understanding of the vitamin D-induced differentiation of neoplastic cells, which results in the generation of cells that acquire near-normal, mature phenotype. Examples of the criteria by which differentiation is recognized in each cell type are provided, and only those effects of 1alpha,25-dihydroxyvitamin D(3) (1,25D) on cell proliferation and survival that are associated with the differentiation process are emphasized. The existing knowledge, often fragmentary, of the signaling pathways that lead to vitamin D-induced differentiation of colon, breast, prostate, squamous cell carcinoma, osteosarcoma, and myeloid leukemia cancer cells is outlined. The important distinctions between the different mechanisms of 1,25D-induced differentiation that are cell-type and cell-context specific are pointed out where known. There is a considerable body of evidence that the principal human cancer cells can be suitable candidates for chemoprevention or differentiation therapy with vitamin D. However, further studies are needed to fully understand the underlying mechanisms in order to improve the therapeutic approaches.

C/EBPbeta is known to be important for monocytic differentiation and macrophage function. Here, we found that expression of all three C/EBPbeta isoforms induced in HL60 cells by 1,25-dihydroxyvitamin D3 (1,25D) was upregulated in a sustained manner that correlates with the appearance of monocytic phenotype and with the G1 phase cell cycle arrest. In 1,25D-resistant HL60-40AF cells, isoforms beta-1 and beta-3 were expressed at levels comparable to 1,25D-sensitive HL60-G cells, but isoform beta-2 was difficult to detect. Treatment of sensitive HL60 cells with 1,25D resulted in predominantly nuclear localization of C/EBP isoforms beta-2 and beta-3, while a large proportion of C/EBPbeta-1 remained in the cytoplasm. Attenuation of the MEK-ERK MAPK pathway by the inhibitor PD98059 markedly reduced the expression, 1,25D-induced phosphorylation and nuclear localization of C/EBPbeta-2 and C/EBPbeta-3. Interestingly, only the lower molecular mass isoforms of C/EBPbeta phosphorylated on Thr235 were found in the nuclei, while C/EBPbeta-1 was constitutively phosphorylated and was detected principally in the cytoplasmic fraction. Although the role of C/EBPbeta isoforms in 1,25D-induced differentiation is complex, our results taken together strongly suggest that the phosphorylation of C/EBPbeta isoforms on Thr235 takes place mainly via the MEK-ERK pathway and that C/EBPbeta-2 is the principal transcription factor in this cell system.

Induced differentiation of malignant cells presents a treatment alternative less toxic than the conventional therapeutic approaches. In particular, Acute Myeloid Leukemia (AML) cells have been shown to differentiate along the monocytic lineage when treated with the active form of vitamin D3 (1,25D), and along the granulocytic lineage after exposure to all-trans retinoic acid (ATRA). While induced differentiation of human AML cells is associated with cell cycle blocks and cessation of cell proliferation, whether treated with 1,25D or ATRA, 1,25D-induced monocytic differentiation is accompanied by somewhat increased cell survival during drug-elicited cellular stress, which counters the therapeutic objective of malignant cell elimination. In contrast, granulocytic differentiation leads to cell death, suggesting that this is the preferred mode of differentiation in a therapeutic setting. In the current study we have observed that inhibition of the MEK5/ERK5 MAPK pathway in 1,25D-treated human AML cells switches the phenotype from monocytic to granulocytic, as evidenced by decreased expression of monocytic markers CD14 and Monocyte Specific Esterase (MSE), concurrent with an increase in general myeloid/granulocytic markers CD11b and CD11c, as well as NBT positivity. On the molecular level, the switch from the monocytic to granulocytic phenotype was accompanied by reduced levels of phosphorylation of transcription factor (TF) C/EBPb but increased phosphorylation of C/EBPa. The requirement for activated ERK5 in monocytic differentiation was also shown by the use of Bix 02189, a specific inhibitor of MEK5 kinase activity, and by the transfection of siRNA to ERK5. Inhibition of ERK1/2 activity by PD098059 reduced monocytic differentiation, but did not alter the 1,25D-induced monocytic phenotype, showing that ERK1/2 and ERK5 serve different functions in differentiation signaling. Importantly, ERK5 appears to control the activation of the TF c-jun/AP1, previously shown to be required for 1, 25D- induced monocytic differentiation, and of the TF MEF2C, not previously linked to 1,25D action or to monocytic differentiation. Thus, our results suggest that ERK5 inhibitors should be evaluated as potentiators of the therapeutic effects of vitamin D analogs in the proposed clinical trials of AML therapy, and raise the possibility that ERK5 inhibitors may also augment the granulocytic differentiation induced by ATRA in the treatment of APL. (Supported by the NIH grant R01-CA-077422-22 from the National Cancer Institute). Citation Format: Xuening Wang, Ajay Menon, Elzbieta Gocek, Michael Danilenko, Jonathan S. Harrison, George P. Studzinski. Inhibition of the MEK5/ERK5 pathway can redirect 1,25-dihydroxyvitamin D3-treated human AML cells from monocytic to granulocytic lineage of differentiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5451. doi:10.1158/1538-7445.AM2013-5451