AbstractAll-trans Retinoic Acid Research Articles

Abstract 1836: Combining a novel retinoic acid receptor-γ agonist with immune checkpoint blockade represses lung cancer growth in vivo

Abstract All-trans-retinoic acid (ATRA), a pan-agonist for retinoic acid receptors (RARs), regulates diverse cellular functions including growth, differentiation and immune function. We report here that ATRA-treatment represses tumor growth in syngeneic, immunocompetent but not in immunodeficient mice. Tumor immune microenvironment was implicated since depletion of cytotoxic T lymphocytes antagonized these effects in syngeneic mice. Combining ATRA with immune checkpoint blockade did not inhibit lung cancer growth in mice. We sought to augment retinoid anti-tumor effects without affecting its pro-tumorigenicity. We previously reported that CD38 mediated resistance to checkpoint blockade in murine 344SQ lung cancer cells via RARα transcriptional activation of CD38 expression. Yet, combining the RARα antagonist (IRX6696) with anti-PD-L1 did not augment anti-tumorigenicity in transplanted 344SQ cells in syngeneic mice. Prior work implicated RARγ in regulating T cell response. Combining the novel RARγ agonist (IRX4647) with anti-PD-L1 statistically-significantly repressed 344SQ lung cancer cell growth in syngeneic mice. This line is relatively resistant to checkpoint blockade. Immunofluorescent analysis of these treated tumors revealed that combined IRX4647 and anti-PD-L1 treatments reduced CD38 expression in the tumor stroma relative to IRX4647 or anti-PD-L1 treatment alone. Statistically-significantly elevated helper (CD4+) T cells were detected in treated tumors along with increased IL-5 and IL-13 expression observed in plasma and tumors. These cytokines can activate helper T cells, altering lung cancer growth. These microenvironment effects were associated with in vivo anti-tumorigenicity. IRX4647-treatment did not appreciably alter in vitro growth of lung cancer cells although retinoid receptors expression profiles were affected. Pharmacokinetic study of IRX4647 found its plasma half-life was 6 hours. Combining an RARγ agonist with immune checkpoint blockade exerted superior anti-neoplastic efficacy against lung cancer versus an ATRA-based regimen. Given these findings we propose exploring activity of this RARγ agonist with an optimal checkpoint inhibitor in a lung cancer clinical trial. Citation Format: Cheng-Hsin Wei, Lu Huang, Blair Kreh, Xiuxia Liu, Liliya Tyutyunyk-Massey, Zibo Chen, Mi Shi, Vidyasagar Vuligonda, Martin Sanders, Serguei Kozlov, King Chan, Amir Horowitz, Mary Carrington, Patrick Hwu, Weiyi Peng, Ethan Dmitrovsky, Xi Liu. Combining a novel retinoic acid receptor-γ agonist with immune checkpoint blockade represses lung cancer growth in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1836.

All-trans retinoic acid changes muscle fiber type via increasing GADD34 dependent on MAPK signal.

All-trans retinoic acid (ATRA) increases the sensitivity to unfolded protein response in differentiating leukemic blasts. The downstream transcriptional factor of PERK, a major arm of unfolded protein response, regulates muscle differentiation. However, the role of growth arrest and DNA damage-inducible protein 34 (GADD34), one of the downstream factors of PERK, and the effects of ATRA on GADD34 expression in muscle remain unclear. In this study, we identified ATRA increased the GADD34 expression independent of the PERK signal in the gastrocnemius muscle of mice. ATRA up-regulated GADD34 expression through the transcriptional activation of GADD34 gene via inhibiting the interaction of homeobox Six1 and transcription co-repressor TLE3 with the MEF3-binding site on the GADD34 gene promoter in skeletal muscle. ATRA also inhibited the interaction of TTP, which induces mRNA degradation, with AU-rich element on GADD34 mRNA via p-38 MAPK, resulting in the instability of GADD34 mRNA. Overexpressed GADD34 in C2C12 cells changes the type of myosin heavy chain in myotubes. These results suggest ATRA increases GADD34 expression via transcriptional and post-transcriptional regulation, which changes muscle fiber type.

Synthesis and Characterization of Hydroxyl-Pinacolone Retinoate

All-trans retinoic acid (10) was synthesized by β-ionone (5) through knoevenagel condensation, reduction and hydrolysis reaction. Then, hydroxyl-pinacolone retinoate (11) was prepared by the esterification with 1-hydroxy- 3, 3-dimethylbutan-2-ketone. This route not only reduces the synthesis steps but also helps to increase the yield. The structure of the product was characterized by 1H NMR and 13C NMR.

Retinoic acid induces hyaluronic acid production through the klotho-mediated EGFR signaling pathway in human epidermal keratinocytes

All-trans retinoic acid (RA) is an effective anti-aging chemical substance widely used in skin-care products. RA compromises epidermal differentiation and induces keratinocyte proliferation, causing hyaluronic acid production through mechanisms that are not completely understood. Klotho protein causes the differentiation of human epidermal keratinocytes. Klotho gene expression is mediated by epidermal growth factor (EGF), which inhibits cell apoptosis in aging-related diseases. The klotho gene causes human aging syndrome, including short lifespan, skin atrophy, and osteoporosis. We investigated the relationship between RA and klotho in epidermal keratinocytes for the first time. In human epidermal keratinocytes, RA induced klotho gene expression. Treatment with both RA and recombinant klotho induced hyaluronic acid production in human epidermal keratinocytes. However, in klotho small interfering RNA (siRNA)-transfected keratinocytes, RA produced less hyaluronic acid than in the control group, indicating that RA may partially regulate hyaluronic acid production through a klotho-dependent pathway. Knockdown of klotho gene expression inactivated the EGFR-extracellular signal-regulated kinase (ERK) signaling pathway, which is involved in hyaluronic acid production. We concluded that the effect of RA on hyaluronic acid production is partly regulated through the klotho-mediated EGFR signaling pathway in human epidermal keratinocytes.

Inhibition of pyrimidine biosynthesis by strobilurin derivatives induces differentiation of acute myeloid leukemia cells

All-trans retinoic acid–based differentiation therapies have succeeded in the treatment of acute promyelocytic leukemia, which is a rare subtype of acute myeloid leukemia (AML). Their clinical efficacy is negligible, however, for other subtypes of AML. Here, we showed that strobilurin derivatives, a well-established class of inhibitors of mitochondrial electron transport chain (ETC) complex III, possessed differentiation-inducing activity in AML cells. Impairment of mitochondrial ETC activity was involved in the differentiation effects of strobilurin derivatives, where reactive oxygen species generation appeared unnecessary. Conversely, strobilurin derivative-mediated differentiation was triggered by pyrimidine deficiency, which resulted from the inhibition of the mitochondrial-coupled dihydroorotate dehydrogenase enzyme. Moreover, strobilurin derivative-mediated pyrimidine depletion led to the activation of the Akt/mTOR cascade, which was required for the differentiation. Our study provided evidence that strobilurin derivatives may represent a novel class of differentiation-inducing agents for the treatment of AML.

Trametinib enhances ATRA-induced differentiation in AML cells

All-trans retinoic acid (ATRA) is only clinically useful in acute promyelocytic leukemia (APL), but not other subtypes of acute myeloid leukemia (AML). In the present study, a clinically achievable concentration of trametinib, a highly selective inhibitor of MEK, enhanced ATRA-induced differentiation in AML cell lines, HL-60 and U937 as well as AML primary cells. Moreover, trametinib–ATRA (tra–ATRA) co-treatment restored ATRA sensitivity in ATRA-resistant AML cell line, HL-60Res. The protein level of STAT3 and the phosphorylation of Akt or JNK were enhanced with tra–ATRA treatment in HL-60, U937, and HL-60Res cells, respectively. Furthermore, tra–ATRA-induced differentiation in HL-60, U937, and HL-60Res cells was inhibited by STAT3, PI3K, and JNK inhibitors, respectively. Therefore, STAT3, Akt, and JNK signaling pathways were involved in tra–ATRA-induced differentiation in HL-60, U937, and HL-60Res cells, respectively. Taken together, our findings may provide novel therapeutic strategies for AML patients.

From mice to men.

All-trans retinoic acid induces functional and structural plasticity of synapses in human cortical circuits through the engagement of the spine apparatus.

The Effects of Voriconazole on Metabolism of All-Trans Retinoic Acid in the Treatment of Acute Promyelocytic Leukemia: A Case Report

All-trans retinoic acid (ATRA) is a derivative of vitamin A and is the mainstay treatment of acute promyelocytic leukemia (APL). Hypercalcemia is a rare yet important side-effect of ATRA, especially when it is used concomitantly with a medication that impedes its metabolism by inhibiting cytochrome P-450 in the liver and thus increasing the duration of exposure to ATRA. Azole antifungal drugs such as voriconazole are frequently used in patients undergoing chemotherapy due to a high incidence of fungal infections. These medications inhibit two vital enzymes of cytochrome P-450, CYP2C9 and CYP3A4, potentiating the effects of ATRA on calcium metabolism. We present a case of a nine-year-old girl who underwent chemotherapy with all-trans retinoic acid for acute promyelocytic leukemia. The patient was given an anti-fungal cover with voriconazole for extensive fungal chest infection simultaneously. She was found to have asymptomatic hypercalcemia on routine follow-up during the consolidation phase. Both medications were stopped. Subsequently, she was admitted to the ward and managed conservatively with hydration. Serum calcium levels were returned to normal within six days after stopping the combination of ATRA and voriconazole. We underscore that the use of anti-fungal medications should be limited while using ATRA. However, strict monitoring must be done when a combination of these drugs is started, if necessary.

All-trans retinoic acid has renoprotective effects on Cisplatin-induced acute kidney injury in rats.

All-trans retinoic acid (ATRA) is the active metabolites of vitamin A; it has antioxidant effects, and can regulate apoptosis. This study aims to observe the effects of ATRA on cisplatin-induced acute kidney injury, where a total of 96 Sprague-Dawley rats (180–220 g) were divided into four groups; (24 rats in each); normal group, DMSO group, cisplatin group, and cisplatin with retinoic acid group. Eight rats were sacrificed on days 3, 7, and 11 in each group. Serum creatinine, blood urea nitrogen and MAU levels were assayed, the activities of Catalase (CAT), glutathione reductase (GSH) and malondialdehyde (MDA) were tested in kidney tissue, expressions of nuclear factor- kB (NF- kB), Caspase-3, and TGFβ1 were determined by real time PCR, also Histopathological changes in kidney tissue were determined. Our study found that the levels of serum creatinine, BUN, MAU, and MDA in cisplatin group were significantly increased, while the activities of CAT and GSH were significantly decreased, as well as, the expression levels of caspase-3, NF-kB, and TGFβ1 were significantly increased in cisplatin group. In contrast, ATRA group showed lower levels of serum creatinine, BUN, MAU and MDA (p < 0.05), and increased levels of CAT and GSH (p < 0.05), which reached the highest levels at day 11, also the expression levels of caspase-3, NF-kB and TGFβ1 decreased significantly in ATRA group (P< 0.05). Our study concluded that, ATRA has a protective effect on cisplatin-induced acute kidney injury in rats, and it is associated with inhibition of lipid peroxidation, lower inflammation and lower apoptosis.

Vav1 Sustains the In Vitro Differentiation of Normal and Tumor Precursors to Insulin Producing Cells Induced by all-Trans Retinoic Acid (ATRA)

All-trans retinoic acid (ATRA) promotes the development and the function of insulin producing cells and induces partial differentiation of pancreatic tumor cells. A number of evidences clearly indicate that the ATRA mediated signaling may have a substantial role in therapeutic approaches based on restoration of functional β-cells. Among the proteins up-regulated by ATRA, Vav1 is involved in maturation and function of haematopoietic cells and is essential for retinoids induced differentiation of tumor promyelocytes. The presence of Vav1 in solid tissues, including pancreas, is considered ectopic and no role in the differentiation of human epithelial cells has so far been described. We demonstrated here that Vav1 sustains the maturation to β-cells of the normal precursors human Biliary Tree Stem/progenitor Cells (hBTSCs) induced by a differentiation medium containing ATRA and that, in the mature normal pancreas, insulin-producing cells express variable levels of Vav1. Using pancreatic ductal adenocarcinoma (PDAC)-derived cells, we also revealed that the ATRA induced up-modulation of Vav1 is essential for the retinoid-induced trans-differentiation of neoplastic cells into insulin producing cells. The results of this study identify Vav1 as crucial molecule in ATRA induced maturation of insulin producing cells and suggest this protein as a marker for new strategies ended to restore functional β-cells.Graphical abstract

Potentiation of ATRA Activity in HL-60 Cells by Targeting Methylation Enzymes

All Trans Retinoic Acid (ATRA) is a Differentiation Inducer (DI) of Acute Promyelocytic Leukemia (APL) with proven clinical utility. Its benefits for other types of Acute Myelocytic Leukemia (AML) have been limited. In APL, ATRA targets the PML-RARA (promyelocytic leukemia/retinoic acid receptor-alpha)/DNA methyltransferase (DNMT)/Histone Deacetylase (HDAC) complex, facilitating its

All-trans retinoic acid regulated prohibitin by retinoic acid receptor α in hypoxia-induced renal tubular epithelial cell injury

All-trans retinoic acid (ATRA) is a critical component in cell processes such as cell growth, differentiation and apoptosis, and it is also crucial in the regulation of extracellular matrix (ECM) deposition. Prohibitin (PHB) can regulate cell proliferation, apoptosis and differentiation. The current study investigated whether ATRA regulated PHB is induced by hypoxia/reoxygenation injury in renal tubular epithelial cells (RTEC), using gene interference treatments (knockdown or overexpression of RARα). Our results indicate that ATRA can augment the expression of RARα and PHB proteins and reduce the expression of TGF-β1, FN and Col-IV proteins. PHB expression was reduced in an ATRA treated RARα- group, and TGF-β1, FN and Col-IV were up-regulated compared to the ATRA treated RARα+ group. We postulate that ATRA can induce the PHB expression by RARα in hypoxia/reperfusion related RTEC injury.

Abstract 3279: All-trans-retinoic acid (ATRA) markedly augments anti-tumor immunity

Abstract All-trans-retinoic acid (ATRA) engages diverse actions on populations of immune cells. These include effects on regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), Th17 and other immune cells. Prior work found that ATRA can induce anti-tumor immunity through MDSCs and CD8+ T cells and pro-tumor immunity by increasing Treg cells. In this study, we explored how ATRA modulated anti-tumor immunity in solid tumors. To do this, syngeneic lung cancer and colon cancer models were independently developed by optimizing subcutaneous injections of ED1SQ4 lung cancer and MC38 colon cancer cells into immune-competent mice. ATRA-treatment markedly repressed both syngeneic lung cancer and colon cancer growth as compared to vehicle-treated mice. To exclude the possibility that ATRA directly suppressed growth of cancer cells, ED1SQ4 and MC38 cells were independently implanted subcutaneously into athymic mice in order to allow these lung and colon cancer xenografts to grow. Notably, ATRA-treatment did not reduce the growth of these respective xenograft tumors. Intriguingly, ATRA did not suppress in vitro proliferation of either ED1SQ4 or MC38 cells. This implicated ATRA as engaging mechanisms that affect the tumor microenvironment. To learn if the observed anti-tumorigenic activity was mediated by regulation of T cell immunity, CD4+ or CD8+ T cell populations were depleted in syngeneic mice before in vivo ATRA-treatment. CD4+ and CD8+ T cell depletion from syngeneic mice at least partially reversed the tumor suppressive activity of ATRA. Flow cytometry assays conducted on intratumoral immune cells revealed that ATRA-treatment decreased the CD8+ T to Treg cellular ratios while increasing the ratios of CD8+ T to Treg cells. Cytokine and chemokine assays were performed on these syngeneic cancers as well as on peripheral blood harvested from ATRA or vehicle-treated syngeneic mice in order to identify mediators of the observed anti-tumorigenic effects. Findings will be presented. Taken together, this study uncovered a previously unrecognized role for ATRA in augmenting immunotherapy. These preclinical immunotherapy findings can be translated into the cancer clinic. Citation Format: Lu Huang, Zibo Chen, Leila Williams, Yulong Chen, Yunfei Wang, Masanori Kawakami, Jason Roszik, Patrick Hwu, Ethan Dmitrovsky, Weiyi Peng, Xi Liu. All-trans-retinoic acid (ATRA) markedly augments anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3279.

Activation of p70S6 Kinase-1 in Mesenchymal Stem Cells Is Essential to Lung Tissue Repair.

All‐trans retinoic acid (ATRA) or mesenchymal stem cells (MSCs) have been shown to promote lung tissue regeneration in animal models of emphysema. However, the reparative effects of the combination of the two and the role of p70S6 kinase‐1 (p70S6k1) activation in the repair process have not been defined. Twenty‐one days after intratracheal instillation of porcine pancreatic elastase (PPE), MSC and/or 10 days of ATRA treatment was initiated. Thirty‐two days later, static lung compliance (Cst), mean linear intercepts (MLIs), and alveolar surface area (S) were measured. After PPE, mice demonstrated increased values of Cst and MLI, and decreased S values. Both ATRA and MSC transfer were individually effective in improving these outcomes while the combination of ATRA and MSCs was even more effective. The combination of p70S6k1−/− MSCs transfer followed by ATRA demonstrated only modest effects, and rapamycin treatment of recipients with wild‐type (WT) MSCs and ATRA failed to show any effect. However, transfer of p70S6k1 over‐expressing‐MSCs together with ATRA resulted in further improvements over those seen following WT MSCs together with ATRA. ATRA activated p70S6k1 in MSCs in vitro, which was completely inhibited by rapamycin. Tracking of transferred MSCs following ATRA revealed enhanced accumulation and extended survival of MSCs in recipient lungs following PPE but not vehicle instillation. These data suggest that in MSCs, p70S6k1 activation plays a critical role in ATRA‐enhanced lung tissue repair, mediated in part by prolonged survival of transferred MSCs. p70S6k1‐activated MSCs may represent a novel therapeutic approach to reverse the lung damage seen in emphysema. stem cells translational medicine 2018;7:551–558

All-trans Retinoic Acid-induced Pseudotumor Cerebri during Treatment of Acute Promyelocytic Leukemia: A Case Report

All-trans retinoic acid (ATRA), is a first line treatment of acute promyelocytic leukemia (APML). It is a derivative of vitamin A, and a relatively safe drug, but use of ATRA can lead to side effects including some life-threatening conditions such as differentiation syndrome and pseudotumor cerebri (PC). PC is a rare disorder characterized by headache, papilledema, and/or vision loss despite normal composition of cerebrospinal fluid and normal radiological imaging of brain. It may arise as a complication of ATRA during treatment of APML. ATRA induced PC occurs predominantly in the pediatric age group. We report a rare case of ATRA-induced PC in an adult, 25 year old female undergoing consolidation treatment for APML. On discontinuation of ATRA and treatment with acetazolamide and corticosteroids symptoms improved within 5 days. ATRA was reinstituted within 2 weeks at reduced dose with prophylactic acetazolamide. Later ATRA was tolerated well by the patient and consolidation therapy was completed.

Dysregulatory effects of retinoic acid isomers in late zebrafish embryos.

All-trans retinoic acid (atRA) and 9-cis retinoic acid (9cRA) are two natural derivatives of vitamin A that contribute to the normal vertebrate development by affecting gene expression through the retinoic acid signalling pathway. We show transcriptomic effects of the ectopic addition of atRA or 9cRA to zebrafish embryos at the posthatching embryonic stage. Exposure for 24 or 72h to sublethal concentrations of both isomers resulted in characteristic transcriptome changes, in which many proliferation and development-related genes became underexpressed, whereas genes related to retinoid metabolism and some metabolic functions became overrepresented. While short and long exposures elicit essentially the same set of genes, atRA specifically induced expression of a specific subset of proteases, likely acting at the extracellular level, and of elements of the response to xenobiotics. These results reflect the well-known antiproliferative activity of retinoids, and they suggest a dysregulation of the developmental process at final stages of embryogenesis. They also indicate a potential role of endopeptidases as markers of developmental alterations, as well as their possible control by the retinoic signalling pathway. We propose to monitor mRNA levels of cyp16a, cyp16b, and cyp16c in zebrafish embryos as a bioassay for retinoid disruption.

Activation of G0S2 is coordinated by recruitment of PML/RARα and C/EBPɛ to its promoter during ATRA-induced APL differentiation

All-trans retinoic acid (ATRA) binds the promyelocytic leukemia/retinoic acid receptor α (PML/RARα) fusion protein and is an effective oncogene-targeted therapy for acute promyelocytic leukemia (APL). However, the molecular basis of PML/RARα-mediated transcriptional control during ATRA-induced differentiation is unclear. Previous studies have shown that the PML/RARα fusion protein behaves as a type II nuclear receptor, binding to DNA regardless of ligand status. Here, we performed a series of chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) experiments, demonstrating that there is an additional mode of action of PML/RARα, wherein PML/RARα does not bind DNA in the absence of ATRA but binds DNA and activates adjacent genes in the presence of ATRA. This mode of action is similar to that of a type I nuclear receptor and is highlighted by activation of G0/G1 switch gene 2 (G0S2) during ATRA-induced neutrophil differentiation of leukemia cell lines (NB4 and PR9) and primary human APL cells. C/EBPε occupancy of the G0S2 promoter was elevated in parallel with recruitment of PML/RARα in ATRA-treated NB4, PR9, and primary APL cells. Furthermore, we verified that the p30 isoform of C/EBPε is crucial for activation of G0S2 and that PML/RARα interacts physically and cooperates functionally with C/EBPε to up-regulate G0S2 Our data not only demonstrate a new mode of action of PML/RARα but also suggest a novel model in which PML/RARα synergizes with C/EBPε to reactivate the C/EBPε target G0S2, thereby contributing to ATRA-mediated APL differentiation and potentially, clinical remission.

Abstract 4532: Deregulation of retinoic acid (RA) epigenetic control of sphingolipid signaling

Abstract All-trans retinoic acid (RA), the bioactive derivative of vitamin A, can paradoxically exert both anti-cancer and cancer-promoting actions. According to our studies, RA promotes breast cancer cell growth and invasion whenever it fails to exert its genome-wide epigenetic control of transcription via the RA receptor alpha (RARA). Factors that negatively affect RARA genomic function lead to genome-wide deregulation of the transcriptome epigenetically regulated by RA, including RARA-target genes controlling critical sphingolipid functions. We show here that functional inhibition of genomic RARA concomitantly leads to epigenetic repression of neutral sphingomyelinase 2 (nSMase2/SMDP3), a critical enzyme involved in the synthesis of pro-apoptotic ceramide, and upregulation of S1PR1, a key receptor of sphingosine-1-phosphate (S1P), a sphingolipid with pro-proliferative and pro-invasive activity. Selective activation of S1PR1 recapitulates RA pro-proliferative/pro-invasive action, while selective inhibition of S1PR1 counteracts it. Apparently, activation of the S1P-S1PR1 axis, combined with lack of RARA-mediated epigenetic control of SMPD3-ceramide axis, contributes to determine RA tumorigenic action. This study was supported by the NCI R01 CA127614 grant (NS). Citation Format: Stefano Rossetti, Vincenzo Gagliostro, Nicoletta Sacchi. Deregulation of retinoic acid (RA) epigenetic control of sphingolipid signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4532.

Abstract 2101: A gene-expression fingerprint predicting sensitivity to all-trans-retinoic acid in breast cancer cells is tumor-context independent

Abstract All-trans retinoic acid (ATRA) and derived natural as well as synthetic retinoids are promising agents in the treatment and chemoprevention of various types of neoplasia, including mammary tumors. ATRA is an important component of the therapeutic schemes used for the treatment of a rare form of Acute Myelogenous Leukemia known as Acute Promyelocytic Leukemia (APL). A rational use of the paradigmatic retinoid, ATRA, in a heterogeneous disease, like breast cancer, requires the definition of the cellular and molecular determinants of sensitivity to the agent. The major aim of the study was the definition of a predictive gene expression fingerprint that can be used for the selection of patients who may benefit from treatment protocols containing ATRA. To this purpose, we selected 53 breast cancer cell lines characterized for the constitutive whole genome gene expression profiles. The sensitivity of 30 cell lines (training set) to the anti-proliferative action of ATRA was defined after challenge with increasing concentrations of the retinoid for 3, 6 and 9 days. This analysis established that Luminal and ER+ cell lines are enriched within the ATRA sensitive group. In contrast, cell lines characterized by a Basal-like phenotype, according to the PAM50 gene expression signature, are generally refractory to the growth inhibitory action of ATRA. The sensitivity of Luminal-A and Luminal-B and the general refractoriness of Basal-like tumors to ATRA was validated in short-term tissue slice cultures of surgical breast cancer specimens. The training set was used to define a gene-expression fingerprint consisting of morethan 100 genes significantly associated with ATRA sensitivity. The fingerprint was generated by reprocessing the RNA sequencing data contained in the CCLE (Cancer Cell line Encyclopedia) of the Broad Institute and it was built from approximately 60,000 coding and non-coding loci. The approach involved the use of general linear models (machine learning algorithm). The identified gene-expression fingerprint was subsequently used to successfully predict ATRA sensitivity in a test set consisting of the remaining 23 cell lines. As a first step towards the use of the fingerprint for the stratification of patients, we evaluated the proportion of predicted ATRA sensitive breast tumors in the TCGA dataset. In accordance with the cell line and primary tumor data, approximately 30% of the Luminal tumors present with a high similarity score to the identified gene expression fingerprint associated with ATRA sensitivity. In contrast, only 5% of the Basal-like or Triple-negative mammary tumors are characterized by the same high similarity score. Interestingly, the ATRA sensitivity signature seems to be tumor context independent, as it correctly identifies the Acute Promyelocytic Leukemia patients present in the Acute Myelogenous Leukemia patients present in two publicly available datasets. Citation Format: Enrico Garattini, Maurizio Gianni’, Marco Bolis, Maddalena Fratelli, Gabriela Paroni, Mineko Terao. A gene-expression fingerprint predicting sensitivity to all-trans-retinoic acid in breast cancer cells is tumor-context independent. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2101.

Effect of NADPH–cytochrome P450 reductase on all-trans-retinoic acid efficacy and cytochrome P450 26A1 expression in human myeloid leukaemia HL-60 cells

All-trans-retinoic acid (ATRA), a naturally occurring metabolite of vitamin A, has been shown to have great potential as an antitumorigenic drug to treat acute leukaemia by promoting cancer cell differentiation. Cytochrome P450 oxidoreductase (POR) is the only obligate electron donor for all of the microsomal cytochrome P450 enzymes including CYP26A1 which is highly specific for ATRA metabolism and efficacy in human myeloid leukaemia cells. In this study, we aimed to investigate the effect of POR on ATRA efficacy and CYP26A1 expression in human myeloid leukaemia HL-60 cells. Stably expressed POR and POR-RNAi HL-60 cell lines were established by transfecting POR overexpression or RNAi (RNA interference) vectors mediated by lentivirus. The protein expression of POR and CYP26A1 was examined by Western blot. The potential roles of POR on ATRA efficacy in HL-60 cells were explored by cell viability assay, cell cycle distribution, cellular differentiation and apoptosis analysis. All-trans-retinoic acid treatment caused the expression of POR upregulation and CYP26A1 downregulation in dose- and time-dependent manners. POR overexpression decreased CYP26A1 expression in HL-60 cells. When POR gene was interfered, the downregulation of CYP26A1 expression by ATRA was abolished. In addition, POR overexpression in HL-60 cells significantly compromised ATRA-induced cell proliferation inhibition, cell cycle arrest, differentiation and apoptosis, whereas downregulation of POR significantly potentiated ATRA effects. Our study therefore suggested that POR played an important role in regulating ATRA efficacy and CYP26A1 expression in HL-60 cells.