Multiple Cellular Programs Elicited by ATRA: Apoptosis, Cell Cycle Arrest, Growth Inhibition and Immune Activation in APL and non-APL Contexts

Leukemia is the most common malignant disease in children with high incidence and mortality rates, and a poor treatment effect. The aim of the present study was to examine the changes in the expression of homeobox (Hox) A5 gene and its relationship with cell cycle and apoptosis through the intervention of human K562 myeloid leukemia cell line by all-trans retinoic acid (ATRA), to analyze the role of HOXA5 in the pathogenesis and development process of myeloid leukemia. The optimal concentration of ATRA to be used with K562 cells was determined using a cell counting kit-8 (CCK-8). After 24, 72 and 48 h following treatment of K562 cells with 10 µmol/l ATRA, cell cycle events and apoptosis were measured using flow cytometry. HOXA5 mRNA and protein expression in K562 cells was assessed by RT-PCR and western blot analysis, and the relationship between HOXA5 expression and cell cycle and apoptosis was analyzed. The HOXA5 mRNA and protein expression levels were increased following treatment with ATRA in K562 cells. Apoptosis was increased significantly. The cell cycle was inhibited in G0/G1 phase. Cell proliferation was also inhibited. HOXA5 mRNA and protein expression rates positively correlated with cell apoptosis and the increased percentage and cell cycle of the G0/G1 phase. However, HOXA5 negatively correlated with the reduced percentage of S stage. In conclusion, the expression of HOXA5 in cells was increased following treatment with ATRA in K562 cells, in a time-dependent manner. Additionally, ATRA may inhibit the proliferation of K562 cells and promote apoptosis by upregulating the HOXA5 mRNA and protein expression.

… acid (ATRA) on cell cycle and apoptosis of … cell cycle-related proteins as markers of proliferation potential, we demonstrated that ATRA exerts its antiproliferative effecton MCF-7 cells in …

… model, although Nur77 does play an intrinsic part in the course of apoptosis- and cell cycle arrest-induction, the way by which it operates in these processes should be other than its …

… mechanism of ATRA-induced differentiation of APL cells, our … genes in HL-60 cells upon treatment with ATRA by use of PCR-… with ATRA-induced cell cycle arrest and cell differentiation …

Background/Aims: All-trans retinoic acid (ATRA), the active form of vitamin A, plays an important role in the growth arrest of numerous types of cancer cells. It has been indicated that cyclin-dependent kinase 5 (Cdk5) activity can be affected by ATRA treatment. Our previous results demonstrate the involvement of Cdk5 in the fate of prostate cancer cells. The purpose of this study is to examine whether Cdk5 is involved in ATRA-induced growth arrest of the castration-resistant cancer cell line DU145 through up-regulating Cdk inhibitor protein, p27. Methods: DU145 cells were treated with ATRA, and cell proliferation, protein expression, and protein localization of Cdk5/p27 were examined. Cell proliferation and cell cycle distribution were also determined under Cdk5 inhibition induced by inhibitor or knockdown. Results: ATRA treatment inhibited DU145 cell proliferation and significantly increased p27 expression through Cdk5 up-regulation. Immunocytochemical data showed that a Cdk5 inhibitor reduced ATRA-triggered nuclear distribution of p27 in DU145 cells. The proliferation inhibition and G1 phase accumulation of DU145 cells were significantly increased by ATRA treatment, whereas Cdk5 inhibitor and siRNA could reverse these effects. Conclusions: Our results demonstrate that ATRA induced growth inhibition in castration-resistant prostate cancer cells through activating Cdk5 and p27. We hope this finding will increase the knowledge of prostate cancer treatment and can be applied in patients' nutritional control in the future.

… is accompanied by a G0/G1 arrest, yet how aTRa couples cell cycle arrest to differentiation … The proteasome inhibitor PS-341 inhibits growth, induces apoptosis and overcomes drug …

All-trans retinoic acid (ATRA) is a potential therapeutic agent for the treatment of hematopoietic malignancies, because of its function as an inducer of terminal differentiation of leukemic blasts. Although the efficacy of ATRA as an anticancer drug has been demonstrated by the successful treatment of acute promyelocytic leukemia (APL), the molecular mechanisms of ATRA-induced cell cycle arrest of myeloid cells have not been fully investigated. In this study, we show that the onset of ATRA-induced G0/G1 arrest of human monoblastic U-937 cells is linked to a sharp down-regulation of c-Myc and cyclin E levels and an increase in p21WAF1/CIP1 expression. This is followed by an increase in p27Kip1 protein expression due to enhanced protein stability. The importance of an early decrease in Myc expression for these events was demonstrated by the failure of a U-937 subline with constitutive exogenous expression of v-Myc to cell cycle arrest and regulate cyclin E and p27Kip1 in response to ATRA. Preceding the initiation of G1 arrest, a transient rise in retinoblastoma protein (pRb), p107, and cyclin A levels was detected. Later, a rapid fall in the levels of cyclins A and B and a coordinate dephosphorylation of pRb at Ser780, Ser795, and Ser807/811 coincided with the accumulation of cells in G1. These results thus identify a decrease in c-Myc and cyclin E levels and a posttranscriptional up-regulation of p27Kip1 as important early changes, and position them in the complex chain of events regulating ATRA-induced cell cycle arrest of myeloid cells.

… apoptosis in a dose-dependent manner. atRA also caused a G1 block in the cell cycle with … the effect of atRA on cell growth and cell cycle distribution in MEPM cells, and clarified the …

… atRA induced apoptosis and cell cycle arrest (2). However, the mechanisms behind the atRA-induced apoptosis and cell cycle arrest in … the atRA-induced apoptosis and cell cycle arrest, …

… ATRA-ND induced generation of ROS, cell cycle arrest, and apoptosis in cultured MCL cells. … ATRA-ND induced generation of ROS was inhibited by co-incubation with Ro 41-5253 (Fig …

Of all neoplasms found in women, cervical cancer has the third highest incidence and causes the fourth most deaths. All-trans retinoic acid (ATRA) may be with chemopreventive potential on cervical cancer, but the mechanisms underlying is not clear. To investigate the mechanisms, human cervical cancer HeLa cells were treated with ATRA for 1, 2, 3, or 4 days in vitro. We found that ATRA inhibited the growth of HeLa cells in a dose-dependent manner at the concentrations from 0.3 to 9.6 μmol/L. Flow cytometric analysis showed that HeLa cells were arrested at G0/G1 phase by ATRA, and the aneuploidy was found when cells were treated for 4 days, which is the first report that ATRA causes aneuploid cycle in HeLa cells. The expression of human telomerase catalytic subunit messenger RNA was decreased remarkably by ATRA. These findings suggested that the inhibition of telomerase activity and arrest of cells at G0/G1 phase might be the key steps through which ATRA inhibits the proliferation of HeLa cells. Our results provide a possible mechanistic explanation for the growth inhibitory effect of ATRA on HeLa cells. Therefore, retinoids may have therapeutic potential to complement current treatments of cervical cancers.

Abnormal dendritic cell differentiation and accumulation of immature myeloid suppressor cells (ImC) is one of the major mechanisms of tumor escape. We tested the possibility of pharmacologic regulation of myeloid cell differentiation using all-trans-retinoic acid (ATRA). Eighteen patients with metastatic renal cell carcinoma were treated with ATRA followed by s.c. interleukin 2 (IL-2). Eight healthy individuals comprised a control group. As expected, the cancer patients had substantially elevated levels of ImC. We observed that ATRA dramatically reduced the number of ImC. This effect was observed only in patients with high plasma concentration of ATRA (>150 ng/mL), but not in patients with lower ATRA concentrations (<135 ng/mL). Effects of ATRA on the proportions of different dendritic cell populations were minor. However, ATRA significantly improved myeloid/lymphoid dendritic cell ratio and the ability of patients' mononuclear cells to stimulate allogeneic T cells. This effect was associated with significant improvement of tetanus-toxoid–specific T-cell response. During the IL-2 treatment, the ATRA effect was completely eliminated. To assess the role of IL-2, specimens from 15 patients with metastatic renal cell carcinoma who had been treated with i.v. IL-2 alone were analyzed. In this group also, IL-2 significantly reduced the number and function of dendritic cells as well as T-cell function. These data indicate that ATRA at effective concentrations eliminated ImC, improved myeloid/lymphoid dendritic cell ratio, dendritic cell function, and antigen-specific T-cell response. ATRA treatment did not result in significant toxicity and it could be tested in therapeutic combination with cancer vaccines. (Cancer Res 2006; 66(18): 9299-307)

The vitamin A metabolite all-trans retinoic acid (ATRA) plays a key role in immune response, but effects of ATRA on cancer-associated immunity remains unclear. Previously, we have shown that ATRA regulates the expression of PD-L1 in gastric cancer (GC) cells. We herein reported the mechanism underlying ATRA-induced PD-L1 expression in GC cells and the effects of ATRA on cancer-associated immunosuppression in vitro and in vivo. ATRA enhanced PD-L1 expression through increasing its protein stability and protein synthesis, which was suppressed by JAK pan-inhibitor ruxolitinib (RUX), but was enhanced by the combination with IFN-γ. In T-cell-mediated killing assay, the upregulation of PD-L1-induced by ATRA rendered GC cells strongly resistant to activated T-cell killing, which was reversed by RUX. In vivo, PD-L1 antibody restricted tumor growth, however, ATRA antagonized PD-L1 antibody efficacy. Importantly, RUX not only inhibited the expression of PD-L1 induced by ATRA, but also re-sensitive GC cells to PD-L1 antibody. In conclusion, our study illustrated that ATRA attenuated the effect of PD-L1 blockade through upregulating PD-L1 and blocking PD-L1 expression is an important role for the generation of effective anti-tumor immune response in the combination of immunotherapy and chemotherapy or targeted therapy.

Summary Although all-trans retinoic acid (atRA) is a key regulator of intestinal immunity, its role in colorectal cancer (CRC) is unknown. We found that mice with colitis-associated CRC had a marked deficiency in colonic atRA due to alterations in atRA metabolism mediated by microbiota-induced intestinal inflammation. Human ulcerative colitis (UC), UC-associated CRC, and sporadic CRC specimens have similar alterations in atRA metabolic enzymes, consistent with reduced colonic atRA. Inhibition of atRA signaling promoted tumorigenesis whereas atRA supplementation reduced tumor burden. The benefit of atRA treatment was mediated by cytotoxic CD8+ T cells, activated due to MHCI upregulation on tumor cells. Consistent with these findings, increased colonic expression of the atRA-catabolizing enzyme, CYP26A1, correlated with reduced frequencies of tumoral cytotoxic CD8+ T cells and with worse disease prognosis in human CRC. These results reveal a mechanism by which microbiota drive colon carcinogenesis and highlight atRA metabolism as a therapeutic target for CRC.

Vitamin A plays an essential role in the maintenance of gut homeostasis but its interplay with chemokines has not been explored so far. Using an in vitro model system we studied the effects of human colonic epithelial cells (Caco2, HT-29, and HCT116) derived inflammatory stimuli on monocyte-derived dendritic cells and macrophages. Unstimulated Caco2 and HT-29 cells secreted CCL19, CCL21, and CCL22 chemokines, which could attract dendritic cells and macrophages and induced CCR7 receptor up-regulation by retinoic-acid resulting in dendritic cell migration. The chemokines Mk, CXCL16, and CXCL7 were secreted by all the 3 cell lines tested, and upon stimulation by IL-1β or TNF-α this effect was inhibited by ATRA but had no impact on CXCL1, CXCL8, and CCL20 secretion in response to IL-1β. In the presence of ATRA the supernatants of these cells induced CD103 expression on monocyte-derived dendritic cells and when conditioned by ATRA and cocultured with CD4+ T-lymphocytes they reduced the proportion of Th17 T-cells. However, in the macrophage-T-cell cocultures the number of these effector T-cells was increased. Thus cytokine-activated colonic epithelial cells trigger the secretion of distinct combinations of chemokines depending on the proinflammatory stimulus and are controlled by retinoic acid, which also governs dendritic cell and macrophage responses.

… pattern of ATRA, we discuss the current knowledge of ATRA’s … which ATRA exerts its function and the application of ATRA, both as a single agent and in combination with other cancer …

NK cell is an innate immune system lymphocyte lineage with natural cytotoxicity. Its optimal use in the clinic requires in vitro expansion and activation. Cytokines and encounter with target cells activate NK cells and induce proliferation, and this could depend on the presence of other immune cells. Here we activated PBMCs during 5 days with IL-2, with IL-2 plus the tumor cell line K562 and with the lymphoblastoid cell line R69 and perform integrated analyses of microRNA and mRNA expression profiles of purified NK cells. The samples cluster depending on the stimuli and not on the donor, indicating that the pattern of NK cell stimulation is acutely well conserved between individuals. Regulation of mRNA expression is tighter than that of miRNA expression. All stimuli induce a common preserved genetic remodeling. In addition, encounter with target cells mainly activates pathways related to metabolism. Different target cells induce different NK cell remodeling which affects cytokine response and cytotoxicity, supporting the notion that encounter with different target cells significantly changing the activation pattern. We validate our analysis by showing that activation down regulates miR-23a, which is a negative regulator of cathepsin C (CTSC) mRNA, a gene up regulated by all stimuli. The peptidase CTSC activates the granzymes, the main effector proteases involved in NK cell cytotoxicity. All-trans retinoic acid (ATRA), which induces miR-23a expression, decreases CTSC expression and granzyme B activity leading to impaired NK cell cytotoxicity in an in vivo mouse model.

Background. Vitamin A derivative all-trans retinoic acid (ATRA) is considered as a potent chemotherapeutic drug for its capability of regulating cell growth and differentiation. We studied the effect of ATRA on MMP-2 in MCF-7, human breast cancer cells, and the probable signaling pathways which are affected by ATRA on regulating pro-MMP-2 activity and expression. Methods. Gelatin zymography, RT-PCR, ELISA, Western blot, Immunoprecipitation, and Cell adhesion assay are used. Results. Gelatin zymography showed that ATRA caused a dose-dependent inhibition of pro-MMP-2 activity. ATRA treatment downregulates the expression of MT1-MMP, EMMPRIN, FAK, NF-kB, and p-ERK. However, expression of E-cadherin, RAR, and CRABP increased upon ATRA treatment. Binding of cells to extra cellular matrix (ECM) protein fibronectin reduced significantly after ATRA treatment. Conclusions. The experimental findings clearly showed the inhibition of MMP-2 activity upon ATRA treatment. This inhibitory effect of ATRA on MMP-2 activity in human breast cancer cells (MCF-7) may result due to its inhibitory effect on MT1-MMP, EMMPRIN, and upregulation of TIMP-2. This study is focused on the effect of ATRA on MMP, MMP-integrin-E-cadherin interrelationship, and also the effect of the drug on different signaling molecules which may involve in the progression of malignant tumor development.

All-trans retinoic acid (ATRA) is the most relevant and functionally active metabolite of Vitamin-A. From a therapeutic standpoint, ATRA is the first example of pharmacological agent exerting its anti-tumor activity via a cell differentiating action. In the clinics, ATRA is used in the treatment of Acute Promyelocytic Leukemia, a rare form of myeloid leukemia with unprecedented therapeutic results. The extraordinary effectiveness of ATRA in the treatment of Acute Promyelocytic Leukemia patients has raised interest in evaluating the potential of this natural retinoid in the treatment of other types of neoplasias, with particular reference to solid tumors. The present article provides an overview of the available pre-clinical and clinical studies focussing on ATRA as a therapeutic agent in the context of breast cancer from a holistic point of view. In detail, we focus on the direct effects of ATRA in breast cancer cells as well as the underlying molecular mechanisms of action. In addition, we summarize the available information on the action exerted by ATRA on the breast cancer micro-environment, an emerging determinant of the progression and invasive behaviour of solid tumors. In particular we discuss the recent evidences of ATRA activity on the immune system. Finally, we analyse and discuss the results obtained with the few ATRA-based clinical trials conducted in the context of breast cancer.

PD-1/PD-L1 inhibitor treatments are relatively inefficacious in advanced cervical cancer patients. The presence of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment may be one significant barrier to efficacy. It has been shown that all-trans retinoic acid (ATRA) can differentiate MDSCs into mature myeloid cells. However, whether ATRA suppression of MDSCs function could enhance PD-L1 blockade-mediated tumor immunotherapy remains unknown. Here, the frequency of tumor-infiltrating MDSCs in cervical cancer patients was measured. ATRA was used to target MDSCs both in vitro and in tumor-bearing mice. The impact of ATRA on the human cell line HeLa was also investigated. The frequency of MDSCs and T cells was determined by flow cytometry. The expression of immunosuppressive genes was measured with quantitative real time-PCR and infiltration of immune cells was assessed by immunohistochemical examination. We found that tumor-infiltrating PD-L1+ MDSCs were more prevalent in cervical cancer patients. Blockade of PD-L1 expression in MDSCs with anti-PD-L1 antibody cannot relieve the suppressive activity of MDSCs induced by HeLa cells, while ATRA efficiently abrogated the suppressive activity of MDSCs. Furthermore, ATRA had no effect on PD-L1 expression in HeLa cells in vitro. In in vivo treatment, ATRA decreased MDSCs accumulation and increased the frequency of CD8+ T cells in BALB/C mice with U14 cervical tumors. Importantly, a combination treatment of ATRA and anti-PD-L1 antibody further delayed U14 tumor growth and increased the proportion of CD62L−CD8+ T cells, CD62L−CD4+ T cells, CD107a+CD8+ T cells as well as IFN-γ and TNF-α levels in tumors. Our results provide a rationale for the use of ATRA to suppress MDSCs and enhance anti-PD-L1 cancer immunotherapy in cervical cancer.

Simple Summary Vast improvements in our understanding of the host’s immune response to tumor cells has dramatically improved cancer treatment in the last 20 years. Naturally occurring cancers in domesticated dogs closely model the immune response to cancers in humans. Therefore, understanding similarities and differences between the immune systems of domesticated dogs and people is critical to translating results relating to immune-targeting cancer therapies between species. To this end, this study evaluated the ability of all-trans retinoic acid, a vitamin A derivative, to decrease the production of pro-cancer substances from a particular immune cell (macrophages). These macrophages were developed in vitro from the blood of dogs with naturally occurring cancers. Our data revealed the ability of all-trans retinoic acid (ATRA) to decrease macrophage production of substances that have been previously reported to increase tumor cell invasion and spread as well as inhibit an effective anti-tumor immune response. We also confirmed the inhibitory activity of one of these substances on critical anti-cancer immune cells, T-lymphocytes. While additional studies are needed to show a direct link between ATRA treatment, macrophage inhibition, and subsequent T-lymphocyte stimulation, these preliminary findings suggest a potential role for ATRA in modulating cancer immunity in dogs. Abstract Immunosuppressive myeloid cells in the tumor microenvironment play a major role in suppressing tumor immunity via the production of arginase, IL-10, and others. The objectives of this study were to determine the ability of all-trans retinoic acid (ATRA) to decrease the expression of arginase and other soluble mediators by canine monocyte-derived macrophages (MDMs) and to determine the inhibitory activity of arginase on canine T-lymphocytes. The immunomodulatory ability of ATRA (2 µM) on canine MDMs was evaluated via reverse transcription quantitative PCR (RT-qPCR), flow cytometry, arginase activity assay, and enzyme-linked immunoassay (ELISA). Arginase effects on T-lymphocyte phenotype and proliferation were then evaluated by flow cytometry. ATRA consistently decreased MDM expression of IL6, TGFB1, NOS2, ARG1, and CIITA transcripts, by approximately 2–4-fold, although this did not reach statistical significance for ARG1 or CIITA. Furthermore, arginase activity was decreased in ATRA-treated MDMs while the MDM phenotype remained unchanged. Arginase decreased the expression of granzyme B on CD8+ T-lymphocytes and inhibited CD4+ and CD8+ T-lymphocyte proliferation. These findings suggested that ATRA could inhibit canine MDM production of soluble inflammatory/immunosuppressive mediators. These data also revealed that arginase decreased canine T-lymphocyte proliferation and granzyme B expression. Further studies are needed to determine whether ATRA could reverse the immunosuppressive effects of myeloid cells on canine T-lymphocytes in vivo.

All-trans retinoic acid (ATRA), a recognized differentiating agent, has significant potential in the personalized/stratified treatment of breast cancer. The present study reports on the molecular mechanisms underlying the anti-tumor activity of ATRA in breast cancer. The work is based on transcriptomic experiments performed on ATRA-treated breast cancer cell-lines, short-term tissue cultures of patient-derived mammary-tumors and a xenograft model. ATRA upregulates gene networks involved in interferon-responses, immune-modulation and antigen-presentation in retinoid-sensitive cells and tumors characterized by poor immunogenicity. ATRA-dependent upregulation of these gene networks is caused by a viral mimicry process, involving the activation of endogenous retroviruses. ATRA induces a non-canonical type of viral mimicry, which results in increased expression of the IRF1 (Interferon Responsive Factor 1) transcription factor and the DTX3L (Deltex-E3-Ubiquitin-Ligase-3L) downstream effector. Functional knockdown studies indicate that IRF1 and DTX3L are part of a negative feedback loop controlling ATRA-dependent growth inhibition of breast cancer cells. The study is of relevance from a clinical/therapeutic perspective. In fact, ATRA stimulates processes controlling the sensitivity to immuno-modulatory drugs, such as immune-checkpoint-inhibitors. This suggests that ATRA and immunotherapeutic agents represent rational combinations for the personalized treatment of breast cancer. Remarkably, ATRA-sensitivity seems to be relatively high in immune-cold mammary tumors, which are generally resistant to immunotherapy.

Background All-trans retinoic acid (ATRA), a derivative of vitamin A, potently induces cell maturation in acute promyelocytic leukemia (APL). However, this therapeutic effect is attenuated or absent in non-APL acute myeloid leukemia (AML) and solid tumors. This study aims to elucidate the mechanisms underlying ATRA resistance and identify strategies to overcome it. Methods Integrated high-throughput sequencing—including single-cell multiomics, digital RNA with pertUrbation of genes (DRUG)-seq, bulk RNA-seq, ATAC-seq and CUT&Tag—was performed on AML and solid tumor cell lines treated with ATRA and the mTOR inhibitor PP242. Functional validation was conducted using flow cytometry, siRNA transfection, morphological assessment, Western Blotting and cell line derived xenografts (CDX). Pan-cancer analysis leveraged the Tahoe-100 M pharmacological perturbation dataset to assess metabolic responses across 50 cancer cell lines. Results ATRA simultaneously activates myeloid differentiation programs and a hyperactive metabolic state that impedes terminal maturation in non-APL AML cells. Pharmacological inhibition of the mechanistic target of the mTOR pathway with PP242 suppresses this metabolic surge and redirects transcriptional output toward effective differentiation. The Tahoe-100 M analysis reveals that cancers harboring oncogenic RAS mutations exhibit heightened metabolic responsiveness to ATRA, a vulnerability confirmed in RAS-mutant lung and colon carcinoma cell lines. In these models, ATRA and PP242 synergistically promotes cell maturation and reduces viability. Conclusions These findings uncover a conserved metabolic barrier that limits ATRA-induced differentiation beyond APL. mTOR acts as a critical node governing the balance between pro-differentiation and pro-metabolic signals. Co-targeting mTOR overcomes this barrier, particularly in RAS-mutant malignancies, providing a genotype-informed rationale for extending differentiation therapy to broader cancer contexts. Supplementary information The online version contains supplementary material available at 10.1186/s12967-026-08218-7.

… significantly increased as a result of giving ATRA treatment to tumor cell-injected mice. These hosts … (Citation1) Immune response involved here may be by T H 1 response. Delayed-type …

ABSTRACT Tumor immune escape is a critical problem which frequently accounts for the failure of therapeutic tumor vaccines. Among the most potent suppressors of tumor immunity are myeloid derived suppressor cells (MDSCs). MDSCs can be targeted by all-trans-retinoic-acid (atRA), which reduced their numbers and increased response rates in several vaccination studies. However, not much is known about the optimal administration interval between atRA and the vaccine as well as about its mode of action. Here we demonstrate in 2 different murine tumor models that mice unresponsive to a therapeutic vaccine harbored higher MDSC numbers than did responders. Application of atRA overcame MDSC-mediated immunosuppression and restored tumor control. Importantly, atRA was protective only when administered 3 d after vaccination (delayed treatment), whereas simultaneous administration even decreased the anti-tumor immune response and reduced survival. When analyzing the underlying mechanisms, we found that delayed, but not simultaneous atRA treatment with vaccination abrogated the suppressive capacity in monocytic MDSCs and instead caused them to upregulate MHC-class-II. Consistently, MDSCs from patients with colorectal carcinoma also failed to upregulate HLA-DR after ex vivo treatment with TLR-ligation. Overall, we demonstrate that atRA can convert non-responders to responders to vaccination by suppressing MDSCs function and not only by reducing their number. Moreover, we identify a novel, strictly time-dependent mode of action of atRA to be considered during immunotherapeutic protocols in the future.

… respond to ATRA and clinical trials that tested ATRA response in non-APL AML patients have … epigenetic aspects of susceptibility to RA-induced differentiation in APL and non-APL AML. …

Many cancers arise in a tissue stem cell, and cell differentiation is impaired resulting in an accumulation of immature cells. The introduction of all-trans retinoic acid (ATRA) in 1987 to treat acute promyelocytic leukemia (APL), a rare subtype of acute myeloid leukemia (AML), pioneered a new approach to obtain remission in malignancies by restoring the terminal maturation of leukemia cells resulting in these cells having a limited lifespan. Differentiation therapy also offers the prospect of a less aggressive treatment by virtue of attenuated growth of leukemia cells coupled to limited damage to normal cells. The success of ATRA in differentiation therapy of APL is well known. However, ATRA does not work in non-APL AML. Here we examine some of the molecular pathways towards new retinoid-based differentiation therapy of non-APL AML. Prospects include modulation of the epigenetic status of ATRA-insensitive AML cells, agents that influence intracellular signalling events that are provoked by ATRA, and the use of novel synthetic retinoids.

ABSTRACT Acute myeloid leukemia (AML) is an aggressive, often fatal hematopoietic malignancy. All-trans retinoic acid (atRA), one of the first molecularly targeted drugs in oncology, has greatly improved the outcome of a subtype of AML, acute promyelocytic leukemia (APL). In contrast, atRA has so far provided little therapeutic benefit in the much larger group of patients with non-APL AML. Attempts to identify genetically or molecularly defined subgroups of patients that may respond to atRA have not yielded consistent results. Since AML is a stem cell-driven disease, understanding the effectiveness of atRA may require an appreciation of its impact on AML stem cells. Recent studies reported that atRA decreased stemness of AML with an FLT3-ITD mutation, yet increased it in AML1-ETO driven or EVI1-overexpressing AML. This review summarizes the role of atRA in normal hematopoiesis and in AML, focusing on its impact on AML stem cells.

… granulocytic differentiation of non‐APL myeloid cell lines independent of addition of ATRA or … is required during ATRA‐induced differentiation of APL cells. Taken together, C/EBPβ is an …

Abstract 3305 The differentiating agent ATRA has been used successfully in the treatment of acute promyelocytic leukemia (APL). By comparison, non-APL AML has not shown similar sensitivity to ATRA induced differentiation. Recent data has suggested that a subset of de novo AML patients with nucleophosmin (NPM1) mutations may benefit from addition of ATRA to conventional therapy. The NPM1 gene has several functions affecting cell cycle proliferation including regulation of ribosome biogenesis and centrosome duplication and it acts as a histone chaperone. Mutation of the NPM1 gene leads to differentiation arrest contributing to AML pathogenesis. We hypothesized that leukemia cells with NPM1 mutations could be induced to undergo differentiation. We tested this hypothesis with the NPM1 mutant AML cell line OCI-AML3 and compared the results to identical assays using the AML cell line HL-60 which has been previously well documented to differentiate in response to ATRA therapy. OCI-AML3 and HL-60 cell lines were treated for 5 days with control media and four ATRA doses including 0.2 μM, 1 μM, 5 μM, and 25 μM. Cell viability was assessed by flow cytometry. Compared to the control condition, OCI-AML3 cells treated with the lowest dose of ATRA (0.2 μM) had a live cell count 21.6% of the control. HL-60 cells treated at even the highest ATRA dose (25 uM) had a live cell count 79.3% of the control. Due to the sensitivity of OCI-AML3 cells to the toxic effects of ATRA, the experiment was repeated with lower doses of ATRA including 0.001 μM, 0.01 μM and 0.1 μM. At the lowest dose of ATRA (0.001 μM), OCI-AML3 cells demonstrated a cell viability of 49% with further decrease to 26% at 0.1 μM dose of ATRA. At similar ATRA doses, cell viability for HL-60 cells was 91% and 85%, respectively (see table 1). Table 1: Cell viability as a percent of control cells after 5 days of treatment at three different doses of ATRA in OCI-AML3 and HL-60 cell lines. Cell Line: ATRA 0.001 μM ATRA 0.01 μM ATRA 0.1 μM OCI-AML3 49% 33% 26% HL-60 91% 91% 85% We subsequently determined the time course of changes in cell growth and the extent of differentiation at each point was determined by morphologic assessment. Both cell lines were treated with ATRA at doses of 0.001 μM, 0.01 μM, 0.1 μM, and 1 μM for a total of 4 days. Each day viable cell number was determined. In contrast to the HL-60 cells which had continued growth in lower ATRA doses, OCI-AML3 cells demonstrated exquisite sensitivity to growth arrest at the lowest doses of ATRA. Cell morphology was assessed daily with modified Wright-Giemsa staining of cells. Cells were examined for signs of myeloid differentiation including decrease in nuclear to cytoplasmic (N/C) ratio, nuclear segmentation, and cytoplasmic granules and vacuoles. At the lowest dose of ATRA (0.001 μM), after 4 days of exposure, significant number of OCI-AML3 cells demonstrated morphologic evidence of differentiation. At this ATRA dose and exposure interval, HL-60 cells showed no evidence of differentiation. At an ATRA dose of 1 μM (considered a standard dose used for differentiation of HL-60 cells), the OCI-AML3 cells showed differentiation changes as early as day 2 with nuclear segmentation and decreased N/C ratio while HL-60 cells did not show any change at this time point. After 4 days of ATRA exposure, most OCI-AML3 cells showed segmented nuclei and vacuolated cytoplasm, whereas HL-60 cells showed less distinct signs of differentiation with some cytoplasm granules and cup shaped nuclei. This data suggests that leukemic cells with NPM mutations may be susceptible to the pro-differentiating properties of ATRA. Further substantiation of this data with primary human specimens may ultimately provide the rationale for a novel therapeutic option using ATRA-based differentiation therapy for subsets of non-APL AML. Disclosures: No relevant conflicts of interest to declare.

Summary All-trans retinoid acid (ATRA) can induce terminal differentiation of acute promyelocytic leukemia (APL), also known as the M3 subtype of acute myeloid leukemia (AML). However, non-APL types of AML respond poorly to ATRA-induced differentiation, and the mechanism underlying cell-type-specific resistance against ATRA remains unclear. Here, we use single-cell transcriptome analysis to compare the differentiation trajectories of two AML cell types during ATRA treatment. We show that in NB4 (APL/AML-M3) cells, ATRA activates canonical myeloid lineage factors—including SPI1, CEBPE, and STAT1—to direct near-normal differentiation toward mature granulocytes. By contrast, in HL60 (AML-M2) cells, ATRA-induced differentiation is incomplete and promiscuous, which is characterized by coinduction of both myelopoiesis and lymphopoiesis gene expression programs, as well as transient activation of cis-regulatory elements associated with myeloid differentiation. Our study suggests that the differentiation inducing capacity of ATRA in certain subtypes of AML may be compromised by therapy-induced lineage promiscuity.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by diverse genetic abnormalities. The standard of care remains to be chemotherapy and stem cell transplantation. In acute promyelocytic leukaemia (APL), differentiation therapy with all‐trans retinoic acid (ATRA) has significantly improved outcomes. Despite this, the success of ATRA has yet to be transferred to non‐APL AML. Exploring combinations to enhance the efficacy of ATRA in non‐APL AML remains a key focus. To investigate the therapeutic effect of ATRA in combination with cyclin‐dependent kinase 4/6 (CDK4/6) inhibitors in non‐APL AML. Non‐APL AML cell lines and primary patient samples were treated with ATRA and CDK4/6 inhibitors. Key outcomes included differentiation, proliferation, cell viability and colony‐forming capacity. Combination synergy was evaluated, and gene expression analysis identified pathways associated with therapeutic effects. The combination demonstrated dose‐dependent effects, enhancing differentiation and reducing proliferation, cell viability and colony‐forming capacity. A synergistic effect was observed across AML cell lines. Gene expression profiling revealed the co‐regulation of differentiation‐associated genes, unveiling the mechanisms driving therapeutic synergy. Combination of CDK4/6 inhibitors with ATRA shows potential for differentiation‐based AML treatment. This approach offers a promising avenue for improved outcomes in non‐APL AML.

… resistance to ATRA. Using ATRA sensitive NB4 and ATRA-resistant derivative MR2 cell lines, … /CDDO combination may be useful transcriptional/differentiation therapy in non-APL AML. …

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.

… APL is the first example of a human cancer that can be effectively treated with the differentiation inducer all-trans retinoic acid (ATRA… also affects PML in non-APL cells, suggesting that …

<div>Abstract<p><b>Purpose:</b> All trans-retinoic acid (ATRA) is successful in treating acute promyelocytic leukemia (APL) by inducing terminal differentiation-mediated cell death, but it has limited activity in non-APL acute myeloid leukemia (AML). We aim to improve ATRA therapy of AML by enhancing apoptosis through repression of the antiapoptotic proteins Bcl-2 and Mcl-1.</p><p><b>Experimental Design:</b> APL and AML cell lines, as well as primary AML samples, were used to explore the mechanisms regulating differentiation and apoptosis during ATRA treatment. Stable transfection and gene silencing with siRNA were used to identify the key factors that inhibit apoptosis during induction of differentiation and drugs that accelerate apoptosis.</p><p><b>Results:</b> In differentiation-responsive AML cells, ATRA treatment induces long-lasting repression of Bcl-2 while first upmodulating and then reducing the Mcl-1 level. The Mcl-1 level appears to serve as a gatekeeper between differentiation and apoptosis. During differentiation induction, activation of MEK/ERK and PI3K/Akt pathways by ATRA leads to activation of p90RSK and inactivation of glycogen synthase kinase 3β (GSK3β), which increase Mcl-1 levels by increasing its translation and stability. Sorafenib blocks ATRA-induced Mcl-1 increase by reversing p90RSK activation and GSK3β inactivation, maintains the repressed Bcl-2 level, and enhances ATRA induced apoptosis in non-APL AML cell lines and in primary AML cells.</p><p><b>Conclusions:</b> Inhibition of Mcl-1 is required for apoptosis induction in ATRA differentiation-responsive AML cells. ATRA and sorafenib can be developed as a novel drug combination therapy for AML patients because this drug combination augments apoptosis by inhibiting Bcl-2 and Mcl-1. <i>Clin Cancer Res; 22(5); 1211–21. ©2015 AACR</i>.</p></div>

… To investigate whether dasatinib could be used to restore ATRA-induced differentiation in primary AML cells representing a non-APL subtype, we treated leukemic blasts from a patient …

Although the mixed lineage leukemia 5 (MLL5) gene has prognostic implications in acute promyelocyte leukemia (APL), the underlying mechanism remains to be elucidated. Here, we demonstrate the critical role exerted by MLL5 in APL regarding cell proliferation and resistance to drug-induced apoptosis, through mtROS regulation. Additionally, MLL5 overexpression increased the responsiveness of APL leukemic cells to all-trans retinoic acid (ATRA)-induced differentiation, via regulation of the epigenetic modifiers SETD7 and LSD1. In silico analysis indicated that APL blasts with MLL5high transcript levels were associated with retinoic acid binding and downstream signaling, while MLL5low blasts displayed decreased expression of epigenetic modifiers (such as KMT2C, PHF8 and ARID4A). Finally, APL xenograft transplants demonstrated improved engraftment of MLL5-expressing cells and increased myeloid differentiation over time. Concordantly, evaluation of engrafted blasts revealed increased responsiveness of MLL5-expressing cells to ATRA-induced granulocytic differentiation. Together, we describe the epigenetic changes triggered by the interaction of MLL5 and ATRA resulting in enhanced granulocytic differentiation.

… assays were performed to show that all-trans retinoic acid (atRA) antagonized platelet-… growth factor-BB and serum-stimulated SMC growth. Growth inhibition was distal to early growth-…

Interest has been increasingly focused on all-trans-retinoic acid (tRA) and 13-cis-retinoic acid (13cRA) in cancer chemoprevention and treatment. We have examined the in vitro effects of these 2 retinoic acids (RAs) on human breast-cancer cell lines MCF-7 and ZR-75.1 (both estrogen-receptor-positive, ER+) and MDA-MB-231 (estrogen-receptor-negative, ER-), in terms of inhibition of proliferation and induction of apoptosis. Both retinoic acids exerted an evident dose-dependent growth inhibition, although in the ER- cell line the anti-proliferative effect was obtained only with the highest concentration used; the anti-proliferative activity of tRA was more evident than 13cRA on all 3 tested cell lines. tRA and 13cRA induced apoptosis in MCF-7 and MDA-MB-231 cell lines, but not in ZR-75.1. The apoptotic phenomenon was clearly time-dependent, and in our experience it was not related to the arrest in a specific phase of cell cycle. After treatment with RAs the levels of bcl-2 were reduced in MCF-7, while in ZR-75.1 and in MDA-MB-231 no treatment-related modifications were observed. An analysis of estrogen-receptor status, used as a marker of differentiation, demonstrated that after treatment with RAs the levels of estrogen receptor (ER) decreased in ZR-75.1 only. Our study indicates that the anti-proliferative effects of RAs are sustained by induction of apoptosis in MCF-7 and MDA-MB-231 cells, while in ZR-75.1 cells an induction of differentiation without apoptosis was the prevalent mechanism of growth inhibition. Our results encourage further studies on in vivo effects of these retinoids in breast cancer.

… by inhibiting cell proliferation and inducing cell differentiation, apoptosis, and growth arrest, and … However, there is no conclusive evidence of retinoids inducing cell growth arrest in MBs. …

… all-trans retinoic acid (ATRA) and TGF-β1 led to the enhancement of ATRA-induced suppression of cell proliferation, which is accompanied by inhibition … level of p27, inhibitors of cyclin-…

Besides their growth-inhibiting and differentiation-inducing properties, retinoids have been shown to exert immunomodulatory and anti-inflammatory functions by mechanisms that are not well understood. Tumor necrosis factor-α (TNF), a cytokine produced by mononuclear phagocytes, has been shown to be an important mediator of endotoxin-induced septic shock, cachexia, bone resorption, and inflammation. Nitric oxide may also have a role in septic shock, hypotension, and vasodilatation. In this study, we examined the effects of retinoids on the production of TNF and nitric oxide by murine peritoneal macrophages. Of the various retinoids studied, all-trans-retinoic acid (RA) was most potent; it almost completely inhibited the production of TNF by macrophages activated with endotoxin and interferon-γ. The inhibitory effect was dependent on the dose and duration of RA exposure to macrophages. RA also blocked phorbol ester-induced TNF production in a macrophage cell line (RAW 264.7). Besides TNF, the retinoid suppressed the production of nitric oxide from activated peritoneal macrophages. The importance of these results in relation to controlling various harmful effects of cytokines released by activated macrophages is discussed. J. Leukoc. Biol. 55: 336–342; 1994.

Retinoic acid (RA) metabolites of vitamin A are key regulators of gene expression involved in embryonic development and maintenance of epithelial tissues. The cellular effects of RA are dependent upon the complement of nuclear receptors expressed (RARs and RXRs), which transduce retinoid signals into transcriptional regulation, the presence of cellular retinoid-binding proteins (CRABP and CRBP), which may be involved in RA metabolism, and the activity of RA metabolizing enzymes. We have been using the zebrafish as a model to study these processes. To identify genes regulated by RA during exogenous RA exposure, we utilized mRNA differential display. We describe the isolation and characterization of a cDNA, P450RAI, encoding a novel member of the cytochrome P450 family. mRNA transcripts for P450RAI are expressed normally during gastrulation, and in a defined pattern in epithelial cells of the regenerating caudal fin in response to exogenous RA. In COS-1 cells transfected with the P450RAI cDNA, all-trans-RA is rapidly metabolized to more polar metabolites. We have identified 4-oxo-RA and 4-OH-RA as major metabolic products of this enzyme. P450RAI represents the first enzymatic component of RA metabolism to be isolated and characterized at the molecular level and provides key insight into regulation of retinoid homeostasis.

… ATRA caused a decrease in tumor DNA, RNA, and protein content of the BON cell tumors paralleling the growthinhibitory effects that we observed (Fig. 5). The ratio of protein to DNA is …

Retinoic acid (RA) stimulates the clonal proliferation of mature bone marrow progenitor cells and inhibits the growth of leukemic progenitors, whereas its effects on normal primitive hematopoietic progenitors have not yet been investigated. This study investigated the ability of all-trans- and 9-cis-RA to modulate the proliferation and differentiation of murine Lin-Sca-1+ bone marrow progenitor cells. Both RA isoforms inhibited in a reversible and dose-dependent fashion, the proliferation of multi- but not single-factor responsive Lin-Sca-1+ progenitor cells. The 50% effective dose was 10 nM for both all-trans- and 9-cis-RA. Maximum inhibition was observed at 100-1,000 nM RA, resulting in a 50-75% reduction in the number of proliferative clones. Lin-Sca-1+ cells with high proliferative potential were preferentially inhibited by RA, resulting in a 80-100% inhibition depending on the hematopoietic growth factors stimulating their growth. The inhibitory effects of RA were directly mediated on the target cell, since the effects were observed at the single cell level. Furthermore, autocrine transforming growth factor beta (TGF-beta) production can probably not account for the observed inhibitory effects of RA, since a TGF-beta neutralizing antibody did not block RA-induced inhibition. Whereas RA, in general, is a differentiation-inducing agent, treatment of Lin-Sca- 1+ progenitors resulted in the accumulation of an increased fraction of blasts and immature myeloid cells. Thus, RA inhibits the proliferation as well as differentiation of normal primitive hematopoietic progenitor cells.

… cell cancer (HNSCC), retinoids are especially active in … the growth inhibiting activity of all-trans retinoic acid (all-trans RA… one strong growth inhibition after 72 h exposure to all-trans RA. …

Peripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with generally poor outcomes following standard therapy. Few candidate therapeutic targets have been identified to date. Retinoic acid receptor alpha (RARA) is a transcription factor that modulates cell growth and differentiation in response to retinoids. While retinoids have been used to treat some cutaneous T-cell lymphomas (CTCLs), their mechanism of action and the role of RARA in CTCL and other mature T-cell lymphomas remain poorly understood. After identifying a PTCL with a RARAR394Q mutation, we sought to characterize the role of RARA in T-cell lymphoma cells. Overexpressing wild-type RARA or RARAR394Q significantly increased cell growth in RARAlow cell lines, while RARA knockdown induced G1 arrest and decreased expression of cyclin-dependent kinases CDK2/4/6 in RARAhigh cells. The retinoids, AM80 (tamibarotene) and all-trans retinoic acid, caused dose-dependent growth inhibition, G1 arrest, and CDK2/4/6 down-regulation. Genes down-regulated in transcriptome data were enriched for cell cycle and G1-S transition. Finally, RARA overexpression augmented chemosensitivity to retinoids. In conclusion, RARA drives cyclin-dependent kinase expression, G1-S transition, and cell growth in T-cell lymphoma. Synthetic retinoids inhibit these functions in a dose-dependent fashion and are most effective in cells with high RARA expression, indicating RARA may represent a therapeutic target in some PTCLs.

The anticarcinogenic activities of retinoic acid (RA) are believed to be mediated by the nuclear RA receptor (RAR) and by the RA-binding protein cellular RA-binding protein-II (CRABP-II). In MCF-7 mammary carcinoma cells, growth inhibition by RA entails an early cell cycle arrest followed by induction of apoptosis. Here, we aimed to obtain insights into the initial cell cycle response. We show that a 3- to 5-h RA pulse is sufficient for inducing a robust growth arrest 2 to 4 days later, demonstrating inhibition of the G1-S transition by RA is triggered by immediate-early RAR targets and does not require the continuous presence of the hormone throughout the arrest program. Expression array analyses revealed that RA induces the expression of several genes involved in cell cycle regulation, including the p53-controlled antiproliferative gene B-cell translocation gene, member 2 (Btg2) and the BTG family member Tob1. We show that induction of Btg2 by RA does not require de novo protein synthesis and is augmented by overexpression of CRABP-II. Additionally, we identify a RA response element in the Btg2 promoter and show that the element binds retinoid X receptor/RAR heterodimers in vitro, is occupied by the heterodimers in cells, and can drive RA-induced activation of a reporter gene. Hence, Btg2 is a novel direct target for RA signaling. In concert with the reports that Btg2 inhibits cell cycle progression by down-regulating cyclin D1, induction of Btg2 by RA was accompanied by a marked decrease in cyclin D1 expression. The observations thus show that the antiproliferative activity of RA in MCF-7 cells is mediated, at least in part, by Btg2. [Cancer Res 2007;67(2):609–15]

We described recently the growth inhibitory effects of the novel compound acyclic retinoid (ACR) in human hepatoma cell lines (M. Suzui et al., Cancer Res., 62: 3997–4006, 2002). In this study we examined the cellular and molecular effects of ACR on human squamous cell carcinoma (SCC) cells. ACR inhibited growth of the esophageal SCC cell line HCE7, and the head and neck SCC cell lines YCU-N861 and YCU-H891, with IC50 values of ∼10, 25, and 40 μm, respectively. Detailed studies were then done with HCE7 cells. Treatment of these cells with 10 μm ACR caused an increase of cells in G0-G1 and induced apoptosis. This was associated with two phases of molecular events. During phase 1, which occurred within 6–12 h, there was an increase in the retinoic acid receptor β (RARβ) and p21CIP1 proteins, and their corresponding mRNAs, and a decrease in the hyperphosphorylated form of the retinoblastoma protein. During phase 2, which occurred at ∼24 h, there was a decrease in the cellular level of transforming growth factor α, and the phosphorylated (i.e., activated) forms of the epidermal growth factor receptor, Stat3, and extracellular signal-regulated kinase proteins, and a decrease in both cyclin D1 protein and mRNA. Reporter assays indicated that ACR inhibited the transcriptional activity of the cyclin D1, c-fos, and activator protein promoters. On the other hand, ACR markedly stimulated the activity of a retinoic acid response element-CAT reporter when the cells were cotransfected with a RARβ expression vector. A hypothetical model explaining these two phases is presented. The diverse effects that we obtained with ACR suggest that this agent might be useful in the chemoprevention and/or therapy of human SCCs.

Background Our study and several studies have reported that in some cancers, including pancreatic ductal adenocarcinoma (PDAC), the expression of squamous lineage markers, such as esophagus-tissue-specific genes, correlated with a poor prognosis. However, the mechanism by which the acquisition of squamous lineage phenotypes leads to a poor prognosis remains unclear. We previously reported that retinoic acid signaling via retinoic acid receptor γ (RARγ signaling) determines the differentiation lineage into the esophageal squamous epithelium. These findings hypothesized that the activation of RARγ signaling contributed to acquiring squamous lineage phenotypes and malignant behavior in PDAC. Methods This study utilized public databases and immunostaining of surgical specimens to examine RARγ expression in PDAC. We evaluated the function of RARγ signaling by inhibitors and siRNA knockdown using a PDAC cell line and patient-derived PDAC organoids. The mechanism of the tumor-suppressive effects by blocking RARγ signaling was examined by a cell cycle analysis, apoptosis assays, RNA sequencing and Western blotting. Results RARγ expression in pancreatic intraepithelial neoplasia (PanIN) and PDAC was higher than that in the normal pancreatic duct. Its expression correlated with a poor patient prognosis in PDAC. In PDAC cell lines, blockade of RARγ signaling suppressed cell proliferation by inducing cell cycle arrest in the G1 phase without causing apoptosis. We demonstrated that blocking RARγ signaling upregulated p21 and p27 and downregulated many cell cycle genes, including cyclin-dependent kinase 2 (CDK2), CDK4 and CDK6. Furthermore, using patient-derived PDAC organoids, we confirmed the tumor-suppressive effect of RARγ inhibition and indicated the synergistic effects of RARγ inhibition with gemcitabine. Conclusions This study clarified the function of RARγ signaling in PDAC progression and demonstrated the tumor-suppressive effect of selective blockade of RARγ signaling against PDAC. These results suggest that RARγ signaling might be a new therapeutic target for PDAC.

Despite the expectation that retinoic acid receptor could be the potential therapeutic targets for pancreatic cancers, there has been the lack of information about the role and the impact of retinoic acid receptor gamma (RARγ, RARG) on pancreatic cancer, unlike other two RARs. Herein, we applied TCGA and GEO database to show that the expression and prognosis of RARG is closely related to pancreatic cancer, which demonstrates that RARG is commonly overexpressed in human pancreatic cancer and is an independent diagnostic marker predicting the poor prognosis of pancreatic cancer patients. In addition, we demonstrated that the reduction in the expression of RARG in human pancreatic cancer cells dramatically suppress their proliferation and tumor growth in vivo, partially attributable to the downregulation of tumor‐supporting biological processes such as cell proliferation, antiapoptosis and metabolism and the decreased expression of various oncogenes like MYC and STAT3. Mechanistically, RARG binds on the promoters of MYC, STAT3, and SLC2A1 which is distinguished from well‐known conventional Retinotic acid response elements (RAREs) and that the binding is likely to be responsible for the epigenetic activation in the level of chromatin, assessed by the measurement of deposition of the gene activation marker histone H3 K27 acetylation (H3K27ac) using ChIP‐qPCR. In this study, we reveal that RARG plays important role in the tumorigenesis of pancreatic cancer and represents new therapeutic targets for human pancreatic cancer.

… signaling on APL cell differentiation, in which ATRA coordinates G1 arrest and transition into differentiation by inducing MAT1 degradation and PML/RAR … We determined a cell cycle …

… tumor cells is regulated through signaling involving cell … the cell cycle do not mediate the observed cell cycle changes… of cells in different phases of the cell cycle in T-47D cells …

… RAR and RXR modulators. This has been aided by recent advances in the understanding of the biological role of RARs … based on RXR and RAR modulators, with a focus on cancer and …

Summary Cellular senescence can exert dual effects in tumors, either suppressing or promoting tumor progression. The senescence-associated secretory phenotype (SASP), released by senescent cells, plays a crucial role in this dichotomy. Consequently, the clinical challenge lies in developing therapies that safely enhance senescence in cancer, favoring tumor-suppressive SASP factors over tumor-promoting ones. Here, we identify the retinoic-acid-receptor (RAR) agonist adapalene as an effective pro-senescence compound in prostate cancer (PCa). Reactivation of RARs triggers a robust senescence response and a tumor-suppressive SASP. In preclinical mouse models of PCa, the combination of adapalene and docetaxel promotes a tumor-suppressive SASP that enhances natural killer (NK) cell-mediated tumor clearance more effectively than either agent alone. This approach increases the efficacy of the allogenic infusion of human NK cells in mice injected with human PCa cells, suggesting an alternative therapeutic strategy to stimulate the anti-tumor immune response in "immunologically cold" tumors.

… RARs form heterodimers with RXRs that bind preferentially to retinoic acid response … targets of the various retinoid signaling pathways that regulate cell cycle progression and apoptosis. …

Retinoids are potent inducers of cell cycle arrest and differentiation of numerous cell types, notably granulocytes. However the mechanisms by which retinoids mediate cell cycle arrest during differentiation remain unclear. We have used myeloid differentiation to characterize the molecular pathways that couple cell cycle withdrawal to terminal differentiation. Using primary cells from mice deficient for either the cyclin-dependent kinase inhibitor (CDKi) p27Kip1, the Myc antagonist Mad1, or both Mad1 and p27Kip1, we observed that signals mediated through retinoic acid receptor α (RARα), but not RARβ or γ, required both Mad1 and p27Kip1 to induce cell cycle arrest and to accelerate terminal differentiation of granulocytes. Although RARα did not directly regulate Mad1 or p27Kip1, the RARα target gene C/EBPϵ directly regulated transcription of Mad1. Induction of C/EBPϵ activity in granulocytic cells led to rapid induction of Mad1 protein and transcript, with direct binding of C/EBPϵ to the Mad1 promoter demonstrated through chromatin immunoprecipitation assay. These data demonstrate that cell cycle arrest in response to RARα specifically requires Mad1 and p27Kip1 and that Mad1 is transcriptionally activated by CCAAT/enhancer-binding protein ϵ (C/EBPϵ). Moreover, these data demonstrate selectivity among the RARs for cell cycle arrest pathways and provide a direct mechanism to link differentiation induction and regulation of the Myc antagonist Mad1.

Due to the anti-proliferative and anti-apoptotic effects of retinoic acid (RA), this hormone has emerged as a target for several diseases, including cancer. However, development of retinoid resistance is a critical issue and efforts to understand the retinoid signaling pathway may identify useful biomarkers for future clinical trials. Apoptotic responses of RA are exhibited through the cellular RA-binding protein II (CRABPII)/retinoic acid receptor (RAR) signaling cascade. Delivery of RA to RAR by CRABPII enhances the transcriptional activity of genes involved in cell death and cell cycle arrest. The purpose of this study was to investigate the role of curcumin in sensitizing RA-resistant triple-negative breast cancer (TNBC) cells to RA-mediated apoptosis. We provide evidence that curcumin upregulates the expression of CRABPII, RARβ and RARγ in two different TNBC cell lines. Co-treatment of the cells with curcumin and RA results in increased apoptosis as demonstrated by elevated cleavage of poly(ADP-ribose) polymerase and cleaved caspase-9. Additionally, silencing CRABPII reverses curcumin sensitization of TNBC cells to the apoptotic inducing effects of RA. These findings provide mechanistic insights into sensitizing TNBC cells to RA-mediated cell death by curcumin-induced upregulation of the CRABPII/RAR pathway.

The mechanisms whereby vitamin A stimulates the immune system are poorly understood. In the current study, we attempted to elucidate the potential mechanisms of action of all-trans retinoic acid (atRA) on proliferation of human T lymphocytes. We found that physiological levels of atRA potently augmented T cell proliferation when added in combination with common T cell-stimulating agents. This was reflected in a time- and concentration-dependent stimulation of the cell cycle machinery. The presence of atRA led to elevated levels of cyclin D3, -E, and -A, decreased levels of p27Kip1, increased activity of cyclin-dependent kinase 2, and enhanced phosphorylation of the retinoblastoma protein (pRB). The atRA-mediated changes in the cell cycle machinery were late events, appearing after 20 h of stimulation, indicating that the effects of atRA were indirect. atRA did not alter the expression of the high-affinity IL-2R. However, the level of IL-2 secreted by T cells was strongly enhanced by atRA. rIL-2 was able to substitute for the effects of atRA on the cell cycle machinery and on DNA synthesis, and blocking the IL-2R markedly inhibited atRA-induced cell proliferation and pRB phosphorylation. A retinoic acid receptor (RAR)-selective agonist and 9-cis-RA had the same potency as atRA on T cell proliferation and IL-2 secretion, whereas a retinoid X receptor-selective agonist had only marginal effects. Furthermore, a RAR-selective antagonist completely suppressed T cell proliferation and pRB phosphorylation induced by atRA. Taken together, these results suggest that atRA stimulates the cell cycle machinery and proliferation of normal human T cells by increasing IL-2 secretion through mechanisms involving RARs.

… cell cycle progression from G1 to S phase. In summary, acyclo-… acid inhibited cancer cell growth and interacted with RAR. … signaling, AP-1 transcription factor activation, and cell cycle …

… cellular mechanisms of retinoid action during the past decade. One of the cellular mechanisms retinoids have been implicated in is the initiation and modulation of apoptosis in normal …

… of new active retinoids and the identification of two distinct families of retinoid receptors has … these receptors and of mechanisms involved in the retinoid-induced apoptosis. In this review …

… Certain synthetic retinoid-related molecules induce apoptosis … retinoid targets that mediate their apoptotic activity will enhance our understanding of the mechanism of this novel retinoid …

… , however, the process of apoptosis and the underlying mechanisms have been the object of … activation in the process of retinoid-induced apoptosis, we evaluated whether ATRA, 9-cis …

… This review focuses on the involvement of RA in regulating apoptotic … apoptotic pathways, and then addresses available information on the mechanisms by which RA induces apoptosis …

… two kinds of nucIear receptors,retinoic acid receptor ( RAR ) and retinoic acid X receptor (RXR… diverse retinoids and receptor agonists in inhibiting proIiferation and inducing apoptosis of …

… acid via unknown mechanisms. It was previously shown that … cis retinoic acid induce apoptosis of mouse thymocytes and purified CD4 + CD8 + cells in ex vivo cultures, with 9-cis retinoic …

… , its mechanisms of action involve … , induces apoptosis dependent on caspase-3 activation, and activates the transcription factor AP-1 in cultured Sertoli cells. Moreover, RA induced a …

… The synthetic retinoid CD437/AHPN selectively induces apoptosis in human lung cancer cells … Implication of multipie mechanisms in apoptosis induced by the synthetic retinoid CD437/…

… Retinoids have been linked to the induction of apoptosis in … mechanisms involved in the retinoid-mediated induction of … role of retinoid receptors in the induction of apoptosis in myeloid …

All-trans-retinoic acid and 9-cis-retinoic acid have been reported to have inhibitory effects on pancreatic adenocarcinoma cells and we have shown that this is partly due to induction of apoptosis. In this study, the mechanisms whereby 9-cis-retinoic acid induces apoptosis in these cells were investigated. An involvement of the Bcl-2 family of proteins was shown, such that 9-cis-retinoic acid causes a decrease in the Bcl-2/Bax ratio. Overexpression of Bcl-2 also resulted in inhibition of apoptosis induced by 9-cis-retinoic acid. Furthermore, two broad-range caspase inhibitors blocked DNA fragmentation induced by 9-cis-retinoic acid, but had no effect on viability defined by mitochondrial activity. Using synthetic retinoids, which bind selectively to specific retinoic acid receptor subtypes, we further established that activation of retinoic acid receptor-γ is essential for induction of apoptosis. Only pan-retinoic acid receptor and retinoic acid receptor-γ selective agonists reduced viability and a cell line expressing very low levels of retinoic acid receptor-γ is resistant to the effects of 9-cis-retinoic acid. A retinoic acid receptor-β/γ selective antagonist also suppressed the cytotoxic effects of 9-cis-retinoic acid in a dose-dependent manner. This study provides important insight into the mechanisms involved in suppression of pancreatic tumour cell growth by retinoids. Our results encourage further work evaluating the clinical use of receptor subtype selective retinoids in pancreatic carcinoma.

All-trans-retinoic acid (ATRA) is the drug of choice in the treatment of acute promyelocytic leukemia (APL). ATRA induces both in vitro and in vivo differentiation of APL cells into mature granulocytes. However, the molecular mechanisms involved in ATRA-dependent growth inhibition and cellular differentiation are not presently understood. The NB4 cell line, which is derived from the bone marrow of a patient with APL during relapse, can be used as a model system to study the growth and differentiation of APL cells. Because interferon (IFN) regulatory factors (IRF-1 and IRF-2) and other IFN-inducible gene products regulate cell growth, we analyzed the effects of ATRA on the expression of these genes. We show that ATRA directly activates IRF-1 gene expression, followed by activation of IRF-2 and 2′–5′ oligoadenylate synthetase (OAS) gene expression with slower kinetics. In addition to NB4 cells, ATRA also activated IRF-1 gene expression in HL-60, U937, and THP-1 cells, which all respond to ATRA by growth inhibition. A more than additive increase in IRF-1 gene expression was seen with ATRA and IFN-gamma in NB4 cells. ATRA did not activate nuclear factor kappa B or signal transducer and activator of transcription (STAT) activation pathways, suggesting that an alternate mechanism is involved in IRF-1 gene activation. The ATRA-induced expression of IRF-1, an activator of transcription and repressor of transformation, may be one of the molecular mechanisms of ATRA-induced growth inhibition, and the basis for the synergistic actions of ATRA and IFNs in myeloid leukemia cells.

Vitamin D receptor (VDR) is present in multiple blood cells, and the hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is essential for the proper functioning of the immune system. The role of retinoic acid receptor α (RARα) in hematopoiesis is very important, as the fusion of RARα gene with PML gene initiates acute promyelocytic leukemia where differentiation of the myeloid lineage is blocked, followed by an uncontrolled proliferation of leukemic blasts. RARα takes part in regulation of VDR transcription, and unliganded RARα acts as a transcriptional repressor to VDR gene in acute myeloid leukemia (AML) cells. This is why we decided to examine the effects of the combination of 1,25D and all-trans-retinoic acid (ATRA) on VDR gene expression in normal human and murine blood cells at various steps of their development. We tested the expression of VDR and regulation of this gene in response to 1,25D or ATRA, as well as transcriptional activities of nuclear receptors VDR and RARs in human and murine blood cells. We discovered that regulation of VDR expression in humans is different from in mice. In human blood cells at early stages of their differentiation ATRA, but not 1,25D, upregulates the expression of VDR. In contrast, in murine blood cells 1,25D, but not ATRA, upregulates the expression of VDR. VDR and RAR receptors are present and transcriptionally active in blood cells of both species, especially at early steps of blood development.

… We have shown that ATRA activates IRF-1 gene expression in … respond to ATRA by growth inhibition. In addition, during … is expressed primarily in the cells of the immune system.[221 …

… response to pathogens are controversial. This study investigated the mechanisms by which all-trans retinoic acid (atRA) modulates the host immune response in … /kg atRA increased the …

BackgroundTh2 cytokine responses are enhanced by all trans retinoic acid (ATRA), the bioavailable form of vitamin A. Retinoic acid receptor alpha (RARα) is the high affinity receptor for ATRA that mediates these pro-Th2 effects. We have previously characterized two major human Th2 subpopulations: IL-5- Th2 (IL-5-, IL-4+, IL-13+) and IL-5+ Th2 cells (IL-5+, IL-4+, IL-13+), which represent less and more highly differentiated Th2 cells, respectively. We hypothesized that the pro-Th2 effects of ATRA may differentially affect these Th2 subpopulations.MethodsSpecific cytokine producing Th2 subpopulations were identified using intracellular cytokine staining. Proliferation was measured using the Cell Trace Violet proliferation tracking dye. Apoptotic cells were identified using either annexin-V or active caspase 3 staining. Th2 gene expression was measured using quantitative polymerase chain reaction.ResultsATRA increased the output of Th2 cells from house dust mite allergen (HDM) specific short-term cell lines, and this enhancement was limited to the IL-5+ Th2 subpopulation. Conversely, the RARα antagonist Ro415253 decreased Th2 cell output from these cultures, and this effect was again limited to the IL-5+ Th2 subpopulation. ATRA and Ro415253 respectively augmented and inhibited Th2 cell proliferation, and this affect was more pronounced for the IL-5+ vs. IL-5- Th2 subpopulation. ATRA and Ro415253 respectively augmented and inhibited the expression of IL5 in a significant manner, which was not found for IL4 or IL13.ConclusionsWe report that the reciprocal regulation of Th2 cytokine expression and proliferation by RARα modulators are largely limited to modulation of IL-5 gene expression and to proliferation of the highly differentiated IL-5+ Th2 subpopulation. These results suggest that RARα antagonism is a potential means to therapeutically target allergic inflammation.Trial registrationClinicaltrials.gov identifier: NCT01212016

ABSTRACT Pigs infected with Ascaris suum or controls were given 100 μg (low-dose) or 1,000 μg (high-dose) all-trans retinoic acid (ATRA)/kg body weight in corn oil or corn oil alone per os on days after inoculation (DAI) −1, +1, and +3 with infective eggs. Treatment with ATRA increased interleukin 4 (IL4) and IL12p70 in plasma of infected pigs at 7 DAI and augmented bronchoalveolar lavage (BAL) eosinophilia observed at 7 and 14 DAI. To explore potential molecular mechanisms underlying these observations, a quantitative real-time reverse transcription (RT)-PCR array was used to examine mRNA expression in tissue. Ascaris-infected pigs had increased levels of liver mRNA for T-helper-2 (Th2)-associated cytokines, mast cell markers, and T regulatory (Treg) cells, while infected pigs given ATRA had higher IL4, IL13, CCL11, CCL26, CCL17, CCL22, and TPSB1 expression. Gene expression for Th1-associated markers (IFNG, IL12B, and TBX21), the CXCR3 ligand (CXCL9), IL1B, and the putative Treg marker TNFRSF18 was also increased. Expression of IL4, IL13, IL1B, IL6, CCL11, and CCL26 was increased in the lungs of infected pigs treated with ATRA. To determine a putative cellular source of eosinophil chemoattractants, alveolar macrophages were treated with IL4 and/or ATRA in vitro. IL4 induced CCL11, CCL17, CCL22, and CCL26 mRNA, and ATRA increased the basal and IL4-stimulated expression of CCL17 and CCL22. Thus, ATRA augments a diverse Th1-, Th2-, Treg-, and inflammation-associated response in swine infected with A. suum, and the increased BAL eosinophilia may be related to enhanced induction of eosinophil chemokine activity by alveolar macrophages.

All-trans retinoic acid (ATRA) has been proved to protect liver from ischemia/reperfusion (IR) injury, however, its mechanism is still unclear. This study is to investigate the mechanism of effect of ATRA on innate immunity in mice liver IR injury. Before operation, mice were gavaged by ATRA at 15 mg/kg/d for two weeks, and then the liver was underwent 70% ischemia (90 min) and reperfusion (6 h). Liver function was assessed by serum alanine aminotransferase (sALT), serum aspartate aminotransferase (sAST). Real-time PCR and Western blot were to detect the level of mRNA and protein. In vitro, RAW264.7 macrophages were treatment with ATRA (1 µM) or LE540 (5 µM, a retinoic acid receptor α (RARα) receptor antagonist) before lipopolysaccharide (100 ng/mL) stimulation. In vivo, ATRA protected the liver from IR injury by improving hepatocellular function (sALT and sAST), decreasing cell apoptosis and inhibiting inflammatory response (i.e., the level of toll-like receptor 4, transcription factor nuclear factor-κBp65, interleukin (IL)-1β, IL-6, and tumor necrosis factor-α). When RARα was blocked by LE540 in RAW264.7 macrophages, the inflammatory cytokines were enhancing, along with a decline of Akt phosphorylation but Forkhead box o (Foxo) 1, compared with the ATRA group. In summary, ATRA regulates in part the innate immunity to protect liver from IR injury by RARα/Akt/Foxo1 pathway.

ABSTRACT Objectives Multiple sclerosis (MS) is a potentially disabling autoimmune disease of the central nervous system. Neither the pathogenesis nor the effectiveness of treatment of MS has been fully understood. This in vitro trial evaluated the beneficial immunomodulatory effects of single and combined treatments of all-trans retinoic acid (ATRA) and docosahexaenoic acid (DHA) on the peripheral blood mononuclear cells (PBMCs) of relapsing-remitting MS (RRMS) patients who were receiving interferon beta (IFN-β). Methods The PBMCs of 15 RRMS patients were isolated, cultured, and treated with single and combined treatments of ATRA and DHA. The expressions of IL-2, IL-4, T-bet, and GATA3 genes were evaluated using real-time PCR. Results The results showed that a single treatment of ATRA could significantly suppress the gene expression of the pro-inflammatory cytokine, IL-2 (P < 0.05), and related transcription factor, T-bet (P < 0.001). The gene expression level of the anti-inflammatory cytokine, IL-4, and its transcription factor, GATA3, were not significantly changed. The expression of IL-2 and T-bet genes was significantly decreased in combination treatments of ATRA and DHA (P < 0.001). Significant suppression of IL-2 and T-bet (P < 0.001) was observed in ATRA and DHA combination therapy with half doses of their single treatment, which suggested a synergistic effect of these components. Discussion Co-administration of vitamin A and DHA, an omega-3 fatty acid derivative, may exert a synergistic effect in modulating the immune system in MS patients; however, more studies are needed to evaluate the exact effects and mechanism of their actions on the immune cells.

… coordinating the process of differentiation with arrest in the G/G… associated with retinoic acid induced-growth arrest in human … inhibition of proliferation of the immature leukemic blasts [6]. …

Retinoic acid (RA) induces cell cycle arrest of hormone-dependent human breast cancer (HBC) cells. Previously, we demonstrated that RA-induced growth arrest of T-47D HBC cells …

… retinoic acid receptor β 2 (RARβ 2 ) knockout cell line exhibits no growth arrest in response to all-trans-retinoic acid … RARγ −/− cell lines do growth arrest in response to RA. To examine …

… We report that All-Tans Retinoic Acid (ATRA) slowed RMS cell proliferation and cell … preclude continued cell proliferation and eventual relapse. When ATRA and Cis-Retinoic Acid (CRA) …

MAPK signaling is required for retinoic acid (RA)-triggered G0 cell cycle arrest and cell differentiation, but the mechanism is not well defined. In this study, RA is found to cause MAPK activation with sustained association of RAF to MEK or ERK, leading to a MAPK-dependent accumulation of p21Waf1/Cip1 and binding to CDK2 blocking G1/S transition. BLR1, a chemokine receptor, was found to function as a critical component of RA-triggered MAPK signaling. Unlike wild-type parental cells, RA-treated BLR1 knock-out cells failed to show RAF and consequential MEK and ERK phosphorylation, failed to accumulate CDK inhibitors that control G1/S transition, and failed to differentiate and arrest in response to RA, whereas ectopically overexpressing BLR1 enhanced MAPK signaling and caused accelerated RA-induced differentiation and arrest. Ectopic overexpression of RAF enhanced BLR1 expression in response to RA, whereas inhibition of RAF or MEK by inhibitors or knockdown of RAF by short interfering RNA diminished RA-induced BLR1 expression and attenuated differentiation and growth arrest. Ectopic expression of the RAF CR3, the catalytically active domain, in the BLR1 knock-out restored RA-induced MAPK activation and the ability to differentiate and arrest, indicating that RAF effects MAPK signaling by BLR1 to propel differentiation/arrest. Taken together, RA induces cell differentiation and growth arrest through activation of a novel MAPK pathway with BLR1 as a critical component in a positive feedback mechanism that may contribute to the prolonged MAPK signaling propelling RA-induced cell cycle arrest and differentiation.

… We are interested in examining the influence of retinoic acid (RA) on proliferation, apoptosis, cytotoxicity, migration, and invasion in dependence of the differentiation of neuroblastoma …

… arrest and full expression of the differentiated phenotype required about 3 days, c-myc mRNA declined abruptly between 8 and 16 h after the addition of retinoic acid … limit proliferation in …

… and increased p21 leading to cell arrest. Overall, data show … and proliferative/anti-proliferative potential of different retinoid … intracellular retinoids, ROH and RA, on cell proliferation and …

… We have previously shown that 9-cis-, 13-cis- and all-trans-retinoic acid (RA) powerfully inhibit LCL proliferation at concentrations corresponding to therapeutically achievable plasma …