Cell Cycle Regulatory Cyclin D1 Research Articles

DROSHA Regulates Mesenchymal Gene Expression in Wilms Tumor.

Wilms tumor, the most common pediatric kidney cancer, resembles embryonic renal progenitors. Currently, there are no ways to therapeutically target Wilms tumor driver mutations, such as in the microRNA processing gene DROSHA. In this study, we used a "multiomics" approach to define the effects of DROSHA mutation in Wilms tumor. We categorized Wilms tumor mutations into four mutational subclasses with unique transcriptional effects: microRNA processing, MYCN activation, chromatin remodeling, and kidney developmental factors. In particular, we find that DROSHA mutations are correlated with de-repressing microRNA target genes that regulate differentiation and proliferation and a self-renewing, mesenchymal state. We model these findings by inhibiting DROSHA expression in a Wilms tumor cell line, which led to upregulation of the cell cycle regulator cyclin D2 (CCND2). Furthermore, we observed that DROSHA mutations in Wilms tumor and DROSHA silencing in vitro were associated with a mesenchymal state with aberrations in redox metabolism. Accordingly, we demonstrate that Wilms tumor cells lacking microRNAs are sensitized to ferroptotic cell death through inhibition of glutathione peroxidase 4, the enzyme that detoxifies lipid peroxides. Implications: This study reveals genotype-transcriptome relationships in Wilms tumor and points to ferroptosis as a potentially therapeutic vulnerability in one subset of Wilms tumor.

The Role of Zinc Finger Proteins in the Fate Determination of Mesenchymal Stem Cells during Osteogenic and Adipogenic Differentiation

Zinc finger proteins (ZFPs) constitute a crucial group of transcription factors widely present in various organisms. They act as transcription factors, nucleases, and RNA-binding proteins, playing significant roles in cell differentiation, growth, and development. With extensive research on ZFPs, their roles in the determination of mesenchymal stem cells (MSCs) fate during osteogenic and adipogenic differentiation processes have become increasingly clear. ZFP521, for instance, is identified as an inhibitor of the Wnt signaling pathway and RUNX2's transcriptional activity, effectively suppressing osteogenic differentiation. Moreover, ZFP217 contributes to the inhibition of adipogenic differentiation by reducing the M6A level of the cell cycle regulator cyclin D1 (CCND1). In addition, other ZFPs can also influence the fate of mesenchymal stem cells (MSCs) during osteogenic and adipogenic differentiation through various signaling pathways, transcription factors, and epigenetic controls, participating in the subsequent differentiation and maturation of precursor cells. Given the prevalent occurrence of osteoporosis, obesity, and related metabolic disorders, a comprehensive understanding of the regulatory mechanisms balancing bone and fat metabolism is essential, with a particular focus on the fate determination of MSCs in osteogenic and adipogenic differentiation. In this review, we provide a detailed summary of how zinc finger proteins influence the osteogenic and adipogenic differentiation of MSCs through different signaling pathways, transcription factors, and epigenetic mechanisms. Additionally, we outline the regulatory mechanisms of ZFPs in controlling osteogenic and adipogenic differentiation based on various stages of MSC differentiation.

Loss of microRNA-21 protects against acetaminophen-induced hepatotoxicity in mice.

Acetaminophen (APAP)-induced Acute Liver Failure (ALF) is recognized as the most common cause of ALF in Western societies. APAP-induced ALF is characterized by coagulopathy, hepatic encephalopathy, multi-organ failure, and death. MicroRNAs are small, non-coding RNAs that regulate gene expression at the post-transcriptional level. MicroRNA-21 (miR-21) is dynamically expressed in the liver and is involved in the pathophysiology of both acute and chronic liver injury models. We hypothesize that miR-21genetic ablation attenuates hepatotoxicity following acetaminophen intoxication. Eight-week old miR-21knockout (miR21KO) or wild-type (WT) C57BL/6N male mice were injected with acetaminophen (APAP, 300mg/kg BW) or saline. Mice were sacrificed 6 or 24h post-injection. MiR21KO mice presented attenuation of liver enzymes ALT, AST, LDH compared with WT mice 24h post-APAP treatment. Moreover, miR21KO mice had decreased hepatic DNA fragmentation and necrosis than WT mice after 24h of APAP treatment. APAP-treated miR21KO mice showed increased levels of cell cycle regulators CYCLIN D1 and PCNA, increased autophagy markers expression (Map1LC3a, Sqstm1) and protein (LC3AB II/I, p62), and an attenuation of the APAP-induced hypofibrinolytic state via (PAI-1) compared with WT mice 24 post-APAP treatment. MiR-21 inhibition could be a novel therapeutic approach to mitigate APAP-induced hepatotoxicity and enhance survival during the regenerative phase, particularly to alter regeneration, autophagy, and fibrinolysis. Specifically, miR-21 inhibition could be particularly useful when APAP intoxication is detected at its late stages and the only available therapy is minimally effective.

Chronic PFOA exposure in vitro causes acquisition of multiple tumor cell characteristics in rat liver cells

Perfluorooctanoic acid (PFOA) is tumorigenic in rats and mice and potentially tumorigenic in humans. Here, we studied long-term PFOA exposure with an in vitro transformation model using the rat liver epithelial cell, TRL 1215. Cells were cultured in 10 μM (T10), 50 μM (T50) and 100 μM (T100) PFOA for 38 weeks and compared to passage-matched control cells. T100 cells showed morphological changes, loss of cell contact inhibition, formation of multinucleated giant and spindle-shaped cells. T10, T50, and T100 cells showed increased LC50 values 20%, 29% to 35% above control with acute PFOA treatment, indicating a resistance to PFOA toxicity. PFOA-treated cells showed increases in Matrix metalloproteinase-9 secretion, cell migration, and developed more and larger colonies in soft agar. Microarray data showed Myc pathway activation at T50 and T100, associating Myc upregulation with PFOA-induced morphological transformation. Western blot confirmed that PFOA produced significant increases in c-MYC protein expression in a time- and concentration-related manner. Tumor invasion indicators MMP-2 and MMP-9, cell cycle regulator cyclin D1, and oxidative stress protein GST were all significantly overexpressed in T100 cells. Taken together, chronic in vitro PFOA exposure produced multiple cell characteristics of malignant progression and differential gene expression changes suggestive of rat liver cell transformation.

Inhibition of the JAK2/STAT3 pathway and cell cycle re-entry contribute to the protective effect of remote ischemic pre-conditioning of rat hindlimbs on cerebral ischemia/reperfusion injury.

Remote ischemic pre-conditioning (RIPC) protects against ischemia/reperfusion (I/R) injury. However, the mechanisms underlying this protection remain unclear. In the present study, we investigated the role of Janus-activated kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway and cell cycle arrest, and their relationship with neuronal apoptosis following RIPC. A rat cerebral I/R injury model was induced by middle cerebral artery occlusion (MCAO), and AG490 was used to investigate the mechanisms of RIPC. p-JAK2-, p-STAT3-, cyclin D1-, and cyclin-dependent kinase 6 (CDK6) expression was assessed by Western blotting and immunofluorescence staining. RIPC reduced the infarct volume, improved neurological function, and increased neuronal survival. Furthermore, p-JAK2 and p-STAT3 were detected during the initial phase of reperfusion; the expression levels were significantly increased at 3 and 24 h after reperfusion and were suppressed by RIPC. Additionally, the MCAO-induced upregulation of the cell cycle regulators cyclin D1 and CDK6 was ameliorated by RIPC. Meanwhile, cyclin D1 and CDK6 were colocalized with p-STAT3 in the ischemic brain. RIPC ameliorates the induction of the JAK2/STAT3 pathway and cell cycle regulators cyclin D1 and CDK6 by MCAO, and this net inhibition of cell cycle re-entry by RIPC is associated with downregulation of STAT3 phosphorylation.

Propranolol blocks osteosarcoma cell cycle progression, inhibits angiogenesis and slows xenograft growth in combination with cisplatin-based chemotherapy

Osteosarcoma is still associated with limited response to standard-of-care therapy and alarmingly elevated mortality rates, especially in low- and middle-income countries. Despite multiple efforts to repurpose β-blocker propranolol in oncology, its potential application in osteosarcoma management remains largely unexplored. Considering the unsatisfied clinical needs of this aggressive disease, we evaluated the antitumoral activity of propranolol using different in vitro and in vivo osteosarcoma preclinical models, alone or in addition to chemotherapy. Propranolol significantly impaired cellular growth in β2-adrenergic receptor-expressing MG-63 and U-2OS cells, and was capable of blocking growth-stimulating effects triggered by catecholamines. siRNA-mediated ADRB2 knockdown in MG-63 cells was associated with decreased cell survival and a significant attenuation of PPN anti-osteosarcoma activity. Direct cytostatic effects of propranolol were independent of apoptosis induction and were associated with reduced mitosis, G0/G1 cell cycle arrest and a significant down-regulation of cell cycle regulator Cyclin D1. Moreover, colony formation, 3D spheroid growth, cell chemotaxis and capillary-like tube formation were drastically impaired after propranolol treatment. Interestingly, anti-migratory activity of β-blocker was associated with altered actin cytoskeleton dynamics. In vivo, propranolol treatment (10 mg/kg/day i.p.) reduced the early angiogenic response triggered by MG-63 cells in nude mice. Synergistic effects were observed in vitro after combining propranolol with chemotherapeutic agent cisplatin. Sustained administration of propranolol (10 mg/kg/day i.p., five days a week), alone and especially in addition to low-dose metronomic cisplatin (2 mg/kg/day i.p., three times a week), markedly reduced xenograft progression. After histological analysis, propranolol and cisplatin combination resulted in low tumor mitotic index and increased tumor necrosis. β-blockade using propranolol seems to be an achievable and cost-effective therapeutic approach to modulate osteosarcoma aggressiveness. Further translational studies of propranolol repurposing in osteosarcoma are warranted.

Type 2 Diabetes, Independent of Obesity and Age, Is Characterized by Senescent and Dysfunctional Mature Human Adipose Cells.

Obesity with dysfunctional adipose cells is the major cause of the current epidemic of type 2 diabetes (T2D). We examined senescence in human adipose tissue cells from age- and BMI-matched individuals who were lean, obese, and obese with T2D. In obese individuals and, more pronounced, those with T2D, we found mature and fully differentiated adipose cells to exhibit increased senescence similar to what we previously have shown in the progenitor cells. The degree of adipose cell senescence was positively correlated with whole-body insulin resistance and adipose cell size. Adipose cell protein analysis revealed dysfunctional cells in T2D with increased senescence markers reduced PPAR-γ, GLUT4, and pS473AKT. Consistent with a recent study, we found the cell cycle regulator cyclin D1 to be increased in obese cells and further elevated in T2D cells, closely correlating with senescence markers, ambient donor glucose, and, more inconsistently, plasma insulin levels. Furthermore, fully differentiated adipose cells were susceptible to experimentally induced senescence and to conditioned medium increasing cyclin D1 and responsive to senolytic agents. Thus, fully mature human adipose cells from obese individuals, particularly those with T2D become senescent, and SASP secretion by senescent progenitor cells can play an important role in addition to donor hyperinsulinemia.

Fisetin Inhibits Trypsin Activity and Suppresses the Growth of Colorectal Cancer in Vitro and in Vivo

Colorectal cancer (CRC) is a malignant tumor with high incidence and bad prognosis. Therapies, which are more safe and effective, are urgently needed. Trypsin is proved to be crucial to cancer proliferation and migration, therefore, it is possible to control cancers by modulating its activity. Fisetin is a flavone with trypsin inhibition properties that was screened from more than 45 compounds derived from traditional Chinese medicine (TCM). However, the effects and mechanisms of fisetin on CRC have not been well investigated. In this study, we evaluated the effects of fisetin on 2 different CRC cell lines. Fisetin remarkably inhibited CRC cell proliferation and migration, as well as induced cell apoptosis and Go/G1 phase arrest in a dose-dependent manner. Mechanistic studies revealed that these effects were mediated partially through signaling pathways involving cell cycle regulators p21, p27, cyclinD1, and NF kappa B (NF-κB) p65. Administration of fisetin also significantly suppressed the tumor growth in tumor-bearing NOD/Shi-scid-IL2R gamma (null) (NOG) mice that had been inoculated with human HCT116 cells. Fisetin at the given dosage did not induce significant acute or chronic toxicity in rats. These data provide a potential therapeutic strategy for CRC.

Epigenetically silenced apoptosis-associated tyrosine kinase (AATK) facilitates a decreased expression of Cyclin D1 and WEE1, phosphorylates TP53 and reduces cell proliferation in a kinase-dependent manner

Silencing of the Apoptosis associated Tyrosine Kinase gene (AATK) has been described in cancer. In our study, we specifically investigated the epigenetic inactivation of AATK in pancreatic adenocarcinoma, lower grade glioma, lung, breast, head, and neck cancer. The resulting loss of AATK correlates with impaired patient survival. Inhibition of DNA methyltransferases (DNMTs) reactivated AATK in glioblastoma and pancreatic cancer. In contrast, epigenetic targeting via the CRISPR/dCas9 system with either EZH2 or DNMT3A inhibited the expression of AATK. Via large-scale kinomic profiling and kinase assays, we demonstrate that AATK acts a Ser/Thr kinase that phosphorylates TP53 at Ser366. Furthermore, whole transcriptome analyses and mass spectrometry associate AATK expression with the GO term ‘regulation of cell proliferation’. The kinase activity of AATK in comparison to the kinase-dead mutant mediates a decreased expression of the key cell cycle regulators Cyclin D1 and WEE1. Moreover, growth suppression through AATK relies on its kinase activity. In conclusion, the Ser/Thr kinase AATK represses growth and phosphorylates TP53. Furthermore, expression of AATK was correlated with a better patient survival for different cancer entities. This data suggests that AATK acts as an epigenetically inactivated tumor suppressor gene.

Deferasirox shows inhibition activity against cervical cancer in vitro and in vivo

ObjectiveIron depletion may be a novel therapeutic strategy for cancer. This study aimed to assess the inhibition effects of deferasirox (DFX), an oral iron chelator, on cervical cancer. MethodsIn this study, we performed immunohistochemical analysis, enzyme-linked immunoassay, cell viability and invasive ability assay, cell cycle and apoptosis analysis, protein expression investigation, molecular mechanism investigation, and in vivo murine xenograft model to evaluate the impact of DFX on cervical cancer. ResultsThe cervical cancer cell lines viability decreased and cell apoptosis was induced after DFX incubation. Additionally, DFX promoted cell cycle arrest by regulating the expression of cell cycle regulators cyclin D1, cyclin E and proliferating cell nuclear antigen (PCNA) in cervical cancer cell lines. DFX also decreased cell invasion by upregulating the expression of NDRG1 and downregulating c-Myc. The activation of Akt and the MEK/ERK signaling pathway was inhibited by DFX. DFX also significantly suppressed xenograft tumor growth, decreased the levels of ferritin in serum and tumor tissue, reduced iron deposits and reactive oxygen species (ROS) levels in xenografts of DFX-treated group compared with the control group, with no serious side effects. ConclusionPresent study demonstrated the inhibitory effect of DFX against cervical cancer, and provided a potential therapeutic agent for cervical cancer.

In Vivo Screen Identifies Liver X Receptor Alpha Agonism Potentiates Sorafenib Killing of Hepatocellular Carcinoma.

In Vivo Screen Identifies Liver X Receptor Alpha Agonism Potentiates Sorafenib Killing of Hepatocellular Carcinoma.

Linc01133 contributes to gastric cancer growth by enhancing YES1-dependent YAP1 nuclear translocation via sponging miR-145-5p

The long intergenic non-coding RNA linc01133 is reported to be oncogenic in various malignancies. However, the role and mechanism of linc01133 in regulating gastric cancer growth is still not clear. In the present study, we found that linc01133 was significantly upregulated in gastric cancer tissues compared to non-tumorous gastric tissues. Linc01133 over-expression significantly correlated with tumor size and tumor differentiation in gastric cancer patients. The expression of linc01133 was regulated by c-Jun and c-Fos collaboratively. In both in vitro and in vivo studies, linc01133 was shown to promote gastric cancer cell growth. Linc01133 localized in the cytoplasm and functioned as an endogenous competing RNA of miR-145-5p to upregulate the expression of YES1, which was proved to be the target gene of miR-145-5p. By promoting YES1-dependent YAP1 nuclear translocation, linc01133 upregulated the expression of the key cell cycle regulators CDK4, CDK6 and cyclin D1 to promote G1-S phase transition. Thus, our study unveiled the function and mechanism of linc01133 regulating cell cycle progression in gastric cancer.

PDK1 Regulates the Lengthening of G1 Phase to Balance RGC Proliferation and Differentiation during Cortical Neurogenesis.

During cortical development, the balance between progenitor self-renewal and neurogenesis is critical for determining the size/morphology of the cortex. A fundamental feature of the developing cortex is an increase in the length of G1 phase in RGCs over the course of neurogenesis, which is a key determinant of progenitor fate choice. How the G1 length is temporally regulated remains unclear. Here, Pdk1, a member of the AGC kinase family, was conditionally disrupted by crossing an Emx1-Cre mouse line with a Pdk1fl/fl line. The loss of Pdk1 led to a shorter cell cycle accompanied by increased RGC proliferation specifically at late rather than early/middle neurogenic stages, which was attributed to impaired lengthening of G1 phase. Coincidently, apical-to-basal interkinetic nuclear migration was accelerated in Pdk1 cKO cortices. Consequently, we detected an increased neuronal output at P0. We further showed the significant upregulation of the cell cycle regulator cyclin D1 and its activator Myc in the cKO cortices relative to those of control animals. Overall, we have identified a novel role for PDK1 in cortical neurogenesis. PDK1 functions as an upstream regulator of the Myc-cyclin D1 pathway to control the lengthening of G1 phase and the balance between RGC proliferation and differentiation.

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha protects a fibrotic liver from partial hepatectomy-induced advanced liver injury through regulating cell cycle arrest.

BackgroundA fibrotic liver may have an impaired regenerative capacity. Because liver transplantation is donor limited, understanding the regenerative ability of a fibrotic liver is important.MethodsA two‐thirds partial hepatectomy (PH) was performed in C57Bl/6 mice with or without carbon tetrachloride (CCl4) treatment. Liver regeneration in the fibrotic liver after PH was assessed by the intrahepatic expression of the cell cycle regulators p53, p21, cyclin D1, c‐Fos and CDK2 using Western blot analysis. In addition, the expression of PGC‐1α and the cell proliferation‐related proteins PCNA and phosphate histone H3 was determined by Western blot and immunohistochemical staining analyses. Histone epigenetic modification of the PGC‐1α promoter was investigated through chromatin immunoprecipitation (ChIP) and reverse transcription–quantitative polymerase chain reaction (RT‐qPCR) assays. The impact of PGC‐1α on liver regeneration after PH was further evaluated in PGC‐1α‐knockout mice.ResultsA decreased expression of PGC‐1α and liver regeneration‐related genes in the fibrotic liver was detected after a PH. Histone acetylation at the PGC‐1α promoter led to increases in PGC‐1α expression and the survival rate in the fibrotic group after a PH. PGC‐1α‐mediated liver regeneration was further demonstrated in PGC‐1αf/falbcre+/0 mice.ConclusionTargeting PGC‐1α may represent a strategy to improve the treatment of PH in patients with liver fibrosis.

Cold-pressed oil from Citrus aurantifolia inhibits the proliferation of vascular smooth muscle cells via regulation of PI3K/MAPK signaling pathways.

Vascular occlusive disease is a chronic disease with significant morbidity and mortality. Although a variety of therapies and medications have been developed, the likelihood of disease re-emergence is high and this can be life-threatening. Based on a previous screening experiment related to vascular obstructive diseases using 34 types of essential oils, cold-pressed oil (CpO) from Citrus aurantifolia (lime) has been demonstrated to have the best effect for the inhibition of vascular smooth muscle cells (VSMCs) proliferation. The aim of the present study was to evaluate the effect of lime CpO on the pathological changes of VSMCs. To determine this, the effect of lime CpO on VSMC proliferation, a major cause of vascular disease, was investigated. To determine the safe concentration interval for toxicity of CpO during VSMC culture, a dilution of 1x10-5 was determined using Cell Counting Kit-8 assay, which was confirmed to be non-toxic using a lactate dehydrogenase assay. To examine the effect of lime CpO in cellular signaling pathways, changes in phosphorylation of both the PI3K/AKT/mTOR and extracellular signal-regulated MEK/ERK signaling pathways with serum were investigated. Furthermore, lime CpO with FBS also significantly decreased the expression levels of the cell cycle regulators cyclin D1 and proliferating cell nuclear antigen. Additionally, lime CpO with FBS significantly inhibited the sprouting of VSMCs in an ex vivo culture system. These results suggested that lime CpO inhibited the abnormal proliferation of VSMCs and can be developed as a nature-based therapeutic agent for obstructive vascular disease.

Cyclin D2 overexpression drives B1a-derived MCL-like lymphoma in mice.

Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma with poor long-term overall survival. Currently, MCL research and development of potential cures is hampered by the lack of good in vivo models. MCL is characterized by recurrent translocations of CCND1 or CCND2, resulting in overexpression of the cell cycle regulators cyclin D1 or D2, respectively. Here, we show, for the first time, that hematopoiesis-specific activation of cyclin D2 is sufficient to drive murine MCL-like lymphoma development. Furthermore, we demonstrate that cyclin D2 overexpression can synergize with loss of p53 to form aggressive and transplantable MCL-like lymphomas. Strikingly, cyclin D2-driven lymphomas display transcriptional, immunophenotypic, and functional similarities with B1a B cells. These MCL-like lymphomas have B1a-specific B cell receptors (BCRs), show elevated BCR and NF-κB pathway activation, and display increased MALT1 protease activity. Finally, we provide preclinical evidence that inhibition of MALT1 protease activity, which is essential for the development of early life-derived B1a cells, can be an effective therapeutic strategy to treat MCL.

KDM4A regulates myogenesis by demethylating H3K9me3 of myogenic regulatory factors

Histone lysine demethylase 4A (KDM4A) plays a crucial role in regulating cell proliferation, cell differentiation, development and tumorigenesis. However, little is known about the function of KDM4A in muscle development and regeneration. Here, we found that the conditional ablation of KDM4A in skeletal muscle caused impairment of embryonic and postnatal muscle formation. The loss of KDM4A in satellite cells led to defective muscle regeneration and blocked the proliferation and differentiation of satellite cells. Myogenic differentiation and myotube formation in KDM4A-deficient myoblasts were inhibited. Chromatin immunoprecipitation assay revealed that KDM4A promoted myogenesis by removing the histone methylation mark H3K9me3 at MyoD, MyoG and Myf5 locus. Furthermore, inactivation of KDM4A in myoblasts suppressed myoblast differentiation and accelerated H3K9me3 level. Knockdown of KDM4A in vitro reduced myoblast proliferation through enhancing the expression of the cyclin-dependent kinase inhibitor P21 and decreasing the expression of cell cycle regulator Cyclin D1. Together, our findings identify KDM4A as an important regulator for skeletal muscle development and regeneration, orchestrating myogenic cell proliferation and differentiation.

Phyllanthin inhibits MOLT-4 leukemic cancer cell growth and induces apoptosis through the inhibition of AKT and JNK signaling pathway.

Among cancers, leukemia is a multistepprogression that involves genetic modifications of normal hematopoietic progenitor cells to cancerous cells. In recent times, leukemia cases and their mortality rate have increased rapidly. Therefore, the immense need for a therapeutic approach is crucial that can control this type of cancer. Phyllanthin is a lignan compound constituent from the Phyllanthus species and has numerous beneficial effects as a dietary component. The present study aims to determine the impact of phyllanthin on the MOLT-4 cytotoxic effect. MOLT-4 cells and MS-5 cells were cultured at different concentrations of phyllanthin (5, 10, 25, 50, 75, and 100 μM/ml), and the viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The level of reactive oxygen species, the membrane potential of mitochondria, apoptosis by 2',7'-dichlorofluorescin-diacetate (DCF-DA), rhodamine, acridine orange (AO)/ethidium bromide (EB), 4',6-diamidino-2-phenylindole (DAPI)/propidium iodide (PI) staining, gene expression of signaling molecules, and protein levels were assessed by reverse-transcription polymerase chain reactionand western blot analysis. Phyllanthin did not show toxicity toward MS-5 cells and significantly decreased the cell viability of MOLT-4 cells with an IC50 value of 25 µM/ml. Also, phyllanthin induced the production of reactive oxygen species and led to the loss of mitochondrial membrane potential. AO/EB and DAPI/PI staining fluorescent image confirmed the induction of apoptosis by phyllanthin treatment. The messenger RNA (mRNA) expression of cell cycle regulator cyclin D1, antiapoptotic gene Bcl-2, NF-κB, and TNF-αdecreased, but the proapoptotic Bax mRNA expression was increased. The phosphorylated protein levels of p-PI3K1/2, p-ERK1/2, and p-AKT were decreased, whereas the levels of p-p38 andp-JNKT1/2 increased. Our results confirmed that phyllanthin inhibits the MOLT-4 cells, increases apoptosis, and inhibits MOLT-4 migration and cell invasion. Therefore, phyllanthin can be used as a potential target for leukemia treatment.

Abstract PD8-04: Antitumor activity of Z-endoxifen (ENDX) is mediated via PKCβ1-dependent ERα loss and cell cycle arrest in ERα-positive breast cancer

Abstract Background: The tamoxifen (TAM) metabolite, ENDX, demonstrated promising antitumor activity in endocrine resistant breast cancer (BC) in both phase I and phase II settings. Furthermore, ENDX resulted in superior in vivo antitumor activity compared to TAM and letrozole in aromatase-expressing aromatase inhibitor-sensitive and resistant MCF7AC1 models. Recently, we identified protein kinase C beta 1 (PKCβ1), which regulates cell proliferation and tumorigenic transformation, as a novel target of ENDX. ENDX-bound PKCβ1 at concentrations achieved in phase I/II ENDX studies (100-300 nM). In contrast, TAM binding to PKCβ1 occurred at concentrations 7-10 folds higher (2 μM) than achievable with TAM 20 mg/day dosing. However, the clinical relevance of targeting PKCβ1 kinase activity is unclear, since drugs that target PKCβ1 (enzastaurin) have been ineffective in BC and other solid tumors. Therefore, we sought to understand how ENDX altered PKCβ1 and to further compare and contrast ENDXs effects to that of PKCβ1 kinase inhibition in ERα+ BC. Methods: The effects of PKCβ1 silencing and ENDX treatment on gene expression was analyzed by RNAseq in MCF7AC1 cells. The impact of PKCβ1-silencing on cell cycle was evaluated by flow cytometry. Protein expression of cell cycle regulators in PKCβ1 and ENDX-treated MCF7AC1 and T47D cells were compared to TAM and enzastaurin in vitro and to letrozole, TAM or control in vivo. The effects of PKCβ1 and drugs on growth were analyzed by cell proliferation assays. PRKCB gene amplification was assessed in primary tumors using TCGA data and in metastatic tumors using whole-exome sequencing data from patients enrolled in the PROMISE study (NCT 03281902). Results: RNAseq analysis revealed E2F targets and G2M checkpoints as the top hallmark genesets significantly downregulated in both PKCβ1-silenced and ENDX-treated MCF7AC1 cells. Flow cytometry demonstrated that PKCβ1 silencing increased G1 and reduced S phases of the cell cycle. Western blot analyses of PKCβ1-silenced MCF7AC1 and T47D cells displayed reduced protein levels of the cell cycle regulators Cyclin D1, Retinoblastoma (Rb), phospho-RbS807/811, CDK4, Chk1 and E2F1 that regulate G1/S transition. While short term ENDX (48 hours) treatment did not alter PKCβ1 levels, prolonged in vitro ENDX treatment profoundly reduced PKCβ1 protein levels and the aforementioned cell cycle regulators, faithfully replicating PKCβ1 silencing effects. In contrast, enzastaurin had no impact on proliferation or cell cycle proteins in either model. Consistent with this finding, ENDX, but not TAM or letrozole, reduced protein levels of ERα and cell cycle regulators in vivo. Overexpression of PKCβ1 induced TAM, but not ENDX, resistance and had little impact on responsiveness to enzastaurin. While PRKCB gene amplification was uncommon in newly diagnosed ERα+/HER2- BC (5%, TCGA), PRKCB was amplified in 40% of metastatic ERα+/HER2- BC (PROMISE study). Conclusion: We have confirmed the relevance of a new ENDX target, PKCβ1, in ERα+/HER2- BC. While targeting PKCβ1 kinase activity elicited no anticancer effects in ERα+ cells, PKCβ1 downregulation, either by siRNA or ENDX, resulted in profound ERα turnover, reduced protein levels of essential cell cycle mediators and profoundly inhibited cell proliferation. Furthermore, PKCβ1 protein expression is associated with TAM, but not ENDX, resistance, a finding whose clinical relevance is further magnified by identification of PRKCB amplification in metastatic ERα+ BC, confirming its potential importance in progression. Efforts are currently underway to elucidate the mechanistic basis for ENDX-induced PKCβ1 and ERα degradation and the contribution of these effects to the superior antitumor activity of ENDX in ERα+ BC. Citation Format: Swaathi Jayaraman, Mary J Kuffel, Krishna R Kalari, Kevin J Thompson, Xiaojia Tang, Vera J Suman, Elizabeth S Bruinsma, Ciara C O'Sullivan, Liewei Wang, Richard Weinshilboum, James N Ingle, John R Hawse, Matthew P Goetz. Antitumor activity of Z-endoxifen (ENDX) is mediated via PKCβ1-dependent ERα loss and cell cycle arrest in ERα-positive breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD8-04.

IRF8 Is an AML-Specific Susceptibility Factor That Regulates Signaling Pathways and Proliferation of AML Cells.

Simple SummaryDespite progress, acute myeloid leukemia (AML) remains one of the deadliest cancer diseases. The identification of novel molecular targets may allow developing innovative and alternative treatment options for AML. Using public data from genome-edited cancer cells, we identified factors that are specifically essential for AML cell growth. We validated the critical role of the transcription factor IRF8 and demonstrated that it modulates the function of the cells by regulating important signaling molecules. These results support that IRF8 may be a suitable molecular target for the treatment of AML.Personalized treatment of acute myeloid leukemia (AML) that target individual aberrations strongly improved the survival of AML patients. However, AML is still one of the most lethal cancer diseases of the 21st century, demonstrating the need to find novel drug targets and to explore alternative treatment strategies. Upon investigation of public perturbation data, we identified the transcription factor IRF8 as a novel AML-specific susceptibility gene in humans. IRF8 is upregulated in a subset of AML cells and its deletion leads to impaired proliferation in those cells. Consistently, high IRF8 expression is associated with poorer patients’ prognoses. Combining gene expression changes upon IRF8 deletion and the genome-wide localization of IRF8 in the AML cell line MV4-11, we demonstrate that IRF8 directly regulates key signaling molecules, such as the kinases SRC and FAK, the transcription factors RUNX1 and IRF5, and the cell cycle regulator Cyclin D1. IRF8 loss impairs AML-driving signaling pathways, including the WNT, Chemokine, and VEGF signaling pathways. Additionally, many members of the focal adhesion pathway showed reduced expression, providing a putative link between high IRF8 expression and poor prognosis. Thus, this study suggests that IRF8 could serve as a biomarker and potential molecular target in a subset of human AMLs.