Expression Of Cyclin Research Articles

Fluorinated chalcone arrests cell cycle progression in Triple-Negative Breast Cancer Cells by decreasing the expression of cyclin A, cyclin B1 and Cdc 2 protein

Introducción. El cáncer de mama triple negativo (TNBC) representa alrededor del 20 % de todos los casos de cáncer de mama a nivel mundial, siendo uno de los cánceres más agresivos, ya que se asocian con grados tumorales avanzados, así como con alteraciones en la expresión de genes implicados en la regulación de la proliferación y supervivencia celular. Las chalconas [(1,3-diaril)-2-propen-1-ona] son compuestos orgánicos valiosos para el desarrollo de fármacos, los cuales han mostrado actividad anticancerígena frente a diversas células tumorales, incluido el carcinoma mamario, debido a que modulan la expresión génica en diversos mecanismos asociados al crecimiento y proliferación celular. Objetivo. Identificar genes que pudieran estar involucrados con el efecto anticancerígeno de la chalcona fluorada, utilizando la línea celular MDA-MB-231 como modelo de esta condición. Metodología. Sintetizamos el derivado de chalcona (E)-3-(4-fluorofenil)-1-(2-pirazinil)-prop-2-en-1-ona, por el método de condensación de Claisen-Schmidt. Luego, utilizando un enfoque de secuenciación de próxima generación (NGS), comparamos los transcriptomas de las células MDA-MB-231 tratadas frente a las no tratadas, para identificar los genes involucrados en el efecto anticancerígeno de la chalcona fluorada. Resultados. Encontramos que este compuesto regula a la baja varios genes involucrados en la progresión del ciclo celular, entre ellos: ciclina A, ciclina B1 y Cdc2, además de aumentar la expresión de genes que regulan negativamente este mecanismo, incluyendo: p21 y p27. Conclusión. Esta investigación indica que la chalcona fluorada detiene la progresión del ciclo celular mediante la regulación de genes importantes implicados en este mecanismo.

Crystalline silica-exposed human lung epithelial cells presented enhanced anchorage-independent growth with upregulated expression of BRD4 and EZH2 in autocrine and paracrine manners.

Crystalline silica-induced inflammation possibly facilitates carcinogenesis. Here, we investigated its effect on lung epithelium damage. We prepared conditioned media of immortalized human bronchial epithelial cell lines (hereinafter bronchial cell lines) NL20, BEAS-2B, and 16HBE14o- pre-exposed to crystalline silica (autocrine crystalline silica conditioned medium), a phorbol myristate acetate-differentiated THP-1 macrophage line, and VA13 fibroblast line pre-exposed to crystalline silica (paracrine crystalline silica conditioned medium). As cigarette smoking imposes a combined effect on crystalline silica-induced carcinogenesis, a conditioned medium was also prepared using the tobacco carcinogen benzo[a]pyrene diol epoxide. Crystalline silica-exposed and growth-suppressed bronchial cell lines exhibited enhanced anchorage-independent growth in autocrine crystalline silica and benzo[a]pyrene diol epoxide conditioned medium compared with that in unexposed control conditioned medium. Crystalline silica-exposed nonadherent bronchial cell lines in autocrine crystalline silica and benzo[a]pyrene diol epoxide conditioned medium showed increased expression of cyclin A2, cdc2, and c-Myc, and of epigenetic regulators and enhancers, BRD4 and EZH2. Paracrine crystalline silica and benzo[a]pyrene diol epoxide conditioned medium also accelerated the growth of crystalline silica-exposed nonadherent bronchial cell lines. Culture supernatants of nonadherent NL20 and BEAS-2B in crystalline silica and benzo[a]pyrene diol epoxide conditioned medium had higher EGF concentrations, whereas those of nonadherent 16HBE14o- had higher TNF-α levels. Recombinant human EGF and TNF-α promoted anchorage-independent growth in all lines. Treatment with EGF and TNF-α neutralizing antibodies inhibited cell growth in crystalline silica conditioned medium. Recombinant human TNF-α induced BRD4 and EZH2 expression in nonadherent 16HBE14o-. The expression of γH2AX occasionally increased despite PARP1 upregulation in crystalline silica-exposed nonadherent lines with crystalline silica and benzo[a]pyrene diol epoxide conditioned medium. Collectively, crystalline silica- and benzo[a]pyrene diol epoxide-induced inflammatory microenvironments comprising upregulated EGF or TNF-α expression may promote crystalline silica-damaged nonadherent bronchial cell proliferation and oncogenic protein expression despite occasional γH2AX upregulation. Thus, carcinogenesis may be cooperatively aggravated by crystalline silica-induced inflammation and genotoxicity.

Influence of postnatal overfeeding on postnatal heart development in juvenile mice

Shortly after birth, cardiomyocytes progressively lose their ability to proliferate to switch towards a hypertrophic growth and this ability is known to be influenced by nutritional factors. Postnatal overfeeding (PNOF) induced by litter size reduction in rodents reproduces the impact of childhood overnutrition and was shown to alter levels of circulating metabolites (lipids and glucose). Moreover, PNOF is known to have a major impact on metabolic status and cardiac function in adulthood. The aim of this work was to study the impact of PNOF on postnatal cardiac proliferation and apoptosis in juvenile mice. C57BL/6 male pups were raised in litter adjusted either to 9 or 3 pups for control (NF) and PNOF group respectively. At 7, 10 or 24 days after birth, hearts were harvested for immunostaining of proliferation marker Ki67 or apoptosis marker caspase 3. Cardiomyocyte number was also counted in hearts of 24 days-old mice and RT-qPCR were performed to study different cyclin mRNA expressions. At 7, 10 and 24 days, body weight and heart mass of PNOF mice was significantly higher than control mice. The proliferation rate of cardiomyocytes in PNOF hearts was decreased at 7 and 10 days with no signs of apoptosis at 7 days. At 24 days-old, even though the proliferation rate was not different between the two groups, the number of cardiomyocytes per mg of tissue was smaller in PNOF hearts and associated to a decreased expression of cyclins D1 and A2. In this study we observed that PNOF does impact the postnatal proliferative window of cardiomyocytes by reducing their proliferation rate 7 and 10 days after birth. This leads to a diminution of cardiomyocytes number in PNOF hearts at weaning and is associated to a decreased expression of specific cyclins essential for the cell cycle progression. Such a decrease could later be involved in the long-term cardiac alterations observed in PNOF mice.

Regulation of the cell cycle and P13K/AKT/mTOR signaling pathway by phthalates in normal human breast cells

ObjectiveTo investigate the impact of phthalates, including Butyl benzyl phthalate (BBP), di(n-butyl) phthalate (DBP), and di(2-ethylhexyl) phthalate (DEHP), in breast carcinogenesis. Materials and methodsMCF-10A normal breast cells were treated with phthalates (100 nM) and 17β-estradiol (E2, 10 nM), which were co-cultured with fibroblasts from normal mammary tissue adjacent to estrogen receptor positive primary breast cancers. Cell viability was determined using a 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cell cycles were analyzed using flow cytometry. The proteins involving cell cycles and P13K/AKT/mTOR signaling pathway were then evaluated by Western blot analysis. ResultsMCF-10A co-cultured cells treated with E2, BBP, DBP, and DEHP exhibited a significant increase in cell viability using MTT assay. The expressions of P13K, p-AKT, and p-mTOR, as well as PDK1 expression, were significantly higher in MCF-10A cells treated with E2 and phthalates. E2, BBP, DBP, and DEHP significantly increased cell percentages in the S and G2/M phases. The significantly higher expression of cyclin D/CDK4, cyclin E/CDK2, cyclin A/CDK2, cyclin A/CDK1, and cyclin B/CDK1 in MCF-10A co-cultured cells were induced by E2 and these three phthalates. ConclusionThese results provide consistent data regarding the potential role of phthalates exposure in the stimulating proliferation of normal breast cells, enhancing cell viability, and driving P13K/AKT/mTOR signaling pathway and cell cycle progression. These findings strongly support the hypothesis that phthalates may play a crucial role in breast tumorigenesis.

Daidzein and Chicory Extract Arrest the Cell Cycle via Inhibition of Cyclin D/CDK4 and Cyclin A/CDK2 Gene Expression in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers, associated with a high rate of mortality. A disturbance between cell proliferation and cell death is one of the cancer hallmarks including HCC. Cell proliferation is mainly controlled by the cell cycle. The arrest of the cell cycle is one of the important targets of anticancer agents. The present study tries to clarify the exact role of some natural products such as daidzein (DAZ) and alcoholic chicory leaf extract (CE), as possible regulators of cell cycle and apoptosis. HCC in rats was induced using diethylnitrosamine (DENA). Ninety rats were allocated and divided equally into nine groups, treated with CE, DAZ, a combination of both, and sorafenib with non-treated control groups. Treatment with CE, DAZ, and their combination significantly downregulated hepatic tissue expression of cyclin D1/CDK4 axis as well as cyclin A/CDK2 axis. The suggested therapeutic protocol inhibited the proliferation and dampened Bcl-2 expression. Furthermore, the efficiency of combining CE and DAZ demonstrated a potency comparable to sorafenib in terms of cyclin D/CDK4 axis expression, as well as; this combination protocol was more potent in revealing a potentiated inhibitory effect on cyclin A and Ki-67 expression. Treatment with DAZ or CE alone, or in combination, could possess an inhibitory effect on hepatocarcinogenesis via cell cycle arrest, inhibition of proliferation through suppression of Ki-67 expression, and apoptosis induction, mediated by downregulation of Bcl-2.

Transglutaminase 2 regulates terminal erythroid differentiation via cross-linking activity.

Transglutaminase 2 (TGM2) is a versatile enzyme that modulates cell survival and differentiation. However, its role in terminal erythroid differentiation is poorly understood. In this study, we investigated the function of TGM2 in primary fetal liver erythroid differentiation. We predicted TGM2 as an upstream regulator via ingenuity pathway analysis (IPA), and found that its expression was increased at both RNA and protein level during terminal erythroid differentiation. TGM2 cross-linking activity inhibitors GK921 and Z-DON suppressed erythroid maturation and enucleation, while its GTPase inhibitor LDN27219 had no such effect. Z-DON treatment arrested differentiation at basophilic erythroblast stage, and interfered with cell cycle progression. RT-PCR demonstrated decreased GATA-1 and KLF1, and disarranged cyclin, CDKI and E2F family genes expression after Z-DON treatment. In conclusion, TGM2 regulates terminal erythroid differentiation through its cross-linking enzyme activity.

Hydroxylated polymethoxyflavones reduce the activity of pancreatic lipase, inhibit adipogenesis and enhance lipolysis in 3T3-L1 mouse embryonic fibroblast cells

Hydroxylated polymethoxyflavones (HPMFs) have been shown to possess various anti-disease effects, including against obesity. This study investigates the anti-obesity effects of HPMFs in further detail, aiming to gain understanding of their mechanism of action in this context. The current study demonstrates that two HPMFs; 3′-hydroxy-5,7,4′,5′-tetramethoxyflavone (3′OH-TetMF) and 4′-hydroxy-5,7,3′,5′-tetramethoxyflavone (4′OH-TetMF) possess anti-obesity effects. They both significantly reduced pancreatic lipase activity in a competitive manner as demonstrated by molecular docking and kinetic studies. In cell studies, it was revealed that both of the HPMFs suppress differentiation of 3T3-L1 mouse embryonic fibroblast cells during the early stages of adipogenesis. They also reduced expression of key adipogenic and lipogenic marker genes, namely peroxisome proliferator-activated receptor-gamma (PPARγ), CCAAT/enhancer-binding protein α and β (C/EBP α and β), adipocyte binding protein 2 (aP2), fatty acid synthase (FASN), and sterol regulatory element-binding protein 1 (SREBF 1). They also enhanced the expression of cell cycle genes, i.e., cyclin D1 (CCND1) and C-Myc, and reduced cyclin A2 expression. When further investigated, it was also observed that these HPMFs accelerate lipid breakdown (lipolysis) and enhance lipolytic genes expression. Moreover, they also reduced the secretion of proteins (adipokines), including pro-inflammatory cytokines, from mature adipocytes. Taken together, this study concludes that these HPMFs have anti-obesity effects, which are worthy of further investigation.

LncRNA TUSC7 sponges miR-10a-5p and inhibits BDNF/ERK pathway to suppress glioma cell proliferation and migration.

Gliomas as primary cerebral malignancies frequently occurring in adults have relatively high morbidity and mortality. The underlying role of long non-coding ribonucleic acids (lncRNAs) in malignancies has attracted much attention, among which tumor suppressor candidate 7 (TUSC7) is a novel tumor suppressor gene whose regulatory mechanism in human cerebral gliomas remains inconclusive. In this study, bioinformatics analysis indicated that TUSC7 could specifically bind to microRNA (miR)-10a-5p, and according to quantitative polymerase chain reaction (q-PCR), miR-10a-5p was up-regulated in human glioma cells and negatively correlated with TUSC7 expression. Dual-luciferase reporter gene assay showed the ability of TUSC7 to bind to miR-10a-5p, and overexpression of TUSC7 notably inhibited miR-10a-5p expression, restrained human glioma cell proliferation and migration, and regulated cell cycle and cyclin expression via the brain-derived neurotrophic factor/extracellular signal-regulated kinase (BDNF/ERK) pathway. The inhibitory effect of TUSC7 on miR-10a-5p was also verified by designing miR-10a-5p overexpression and knockdown panels for wound healing, Transwell and Western blotting assays. TUSC7 suppresses human glioma cell proliferation and migration by negatively modulating miR-10a-5p and inhibiting the BDNF/ERK pathway, thus acting as a tumor suppressor gene in human gliomas.

Effect of Ethanolic Extract of Moringa oleifera, Lam. Roots on Expression of Cyclin D1 in Testicular Tissue of Metabolic Syndrome Induced Wistar Rats

Background: Metabolic syndrome (MS) impacts on inhibition process of spermatogenesis by damaging various components of cell cycle proteins. Cyclin D1 protein which plays role in regulating the cell cycle of spermatogenic cells is depleted due to the metabolic syndrome. Variation results of M. oleifera studies on fertility are interesting for further research. This study aimed to determine the effect of ethanolic extract of M. oleifera roots towards expression of testicular cyclin D1 of metabolic syndrome induced Wistar rats.Subjects and Method: This study was an experimental laboratory. Thirty subjects of male Wistar rats were randomly divided into 5 groups consisted of 6 rats, control group (K1), MS group (K2), and treatment groups (K3, K4, and K5) which were given doses of 150, 250, and 350 mg/kgBW, respectively for 28 days. Induction of MS was achieved by high-fat diet for 28 days and injection of streptozotocin-nicotinamide (STZ-NA) on day 25. Cyclin D1 data was calculated using Intensity Distribution Score (IDS) and analyzed using one-way ANOVA test followed by post hoc multiple comparisons with Tukey HSD. Simple liniar regression test conducted on cyclin D1 data of K2, K3, K4, and K5.Results: One-way ANOVA test showed that the administration of ethanolic extract M. oleifera Lam. roots with doses of 150, 250, and 350 mg/kgBW in 28 days increased cyclin D1 significantly (p= 0.000). There were significant differences between K1 and K2 (p= 0.000), K2 and K3 (p= 0.000), K2 and K4 (p= 0.000), K2 and K5 (P= 0.000). Liniar regression test showed significant (p= 0.000) and moderate effect (R2= 0.65) of doses of ethanolic extract of M. oleifera roots on the expression of cyclin D1.Conclusion: The administration of ethanolic extract Moringa oleifera Lam. roots with doses of 150, 250, and 350 mg/kgBW for 28 days increased cyclin D1 in testicular rats.

1,6-Hexanediol regulates angiogenesis via suppression of cyclin A1-mediated endothelial function

BackgroundAngiogenesis plays important roles in physiological and pathologic conditions, but the mechanisms underlying this complex process often remain to be elucidated. In recent years, liquid–liquid phase separation (LLPS) has emerged as a new concept to explain many cellular functions and diseases. However, whether LLPS is involved in angiogenesis has not been studied until now. Here, we investigated the potential role of LLPS in angiogenesis and endothelial function.ResultsWe found 1,6-hexanediol (1,6-HD), an inhibitor of LLPS, but not 2,5-hexanediol (2,5-HD) dramatically decreases neovascularization of Matrigel plug and angiogenesis response of murine corneal in vivo. Moreover, 1,6-HD but not 2,5-HD inhibits microvessel outgrowth of aortic ring and endothelial network formation. The endothelial function of migration, proliferation, and cell growth is suppressed by 1,6-HD. Global transcriptional analysis by RNA-sequencing reveals that 1,6-HD specifically blocks cell cycle and downregulates cell cycle-related genes including cyclin A1. Further experimental data show that 1,6-HD treatment greatly reduces the expression of cyclin A1 but with minimal effect on cyclin D1, cyclin E1, CDK2, and CDK4. The inhibitory effect of 1,6-HD on cyclin A1 is mainly through transcriptional regulation because proteasome inhibitors fail to rescue its expression. Furthermore, overexpression of cyclin A1 in HUVECs largely rescues the dysregulated tube formation upon 1,6-HD treatment.ConclusionsOur data reveal a critical role of LLPS inhibitor 1,6-HD in angiogenesis and endothelial function, which specifically affects endothelial G1/S transition through transcriptional suppression of CCNA1, implying LLPS as a possible novel player to modulate angiogenesis, and thus, it might represent an interesting therapeutic target to be investigated in clinic angiogenesis-related diseases in future.

Abstract 2132: Tumor heterogeneity of CCNE1 copy number assessed by fluorescence in situ hybridization (FISH) in ovarian and uterine cancers and correlation with cyclin E protein expression

Abstract Amplification of CCNE1 is a recurrent genetic alteration that has been associated with chemoresistance and poor prognosis in gynecological malignancies. We identified Protein Kinase, Membrane Associated Tyrosine/Threonine 1 (PKMYT1) as a novel synthetic lethal target in CCNE1 amplified cancers. In this genomic setting, PKMYT1 inhibition causes premature mitotic entry, mitotic catastrophe and, in turn, cell death. In pre-clinical models, correlation between CCNE1 copy number, cyclin E1 expression and sensitivity to PKMYT1 inhibition is imperfect. Thus, a deep understanding of CCNE1 amplification as a predictive biomarker for PKMYT1 inhibition is critical for refining patient selection strategies and strengthening our understanding of the mechanism of action. These learnings will be applied to correlative biomarker analyses in clinical trials evaluating RP-6306, a first-in-class, potent, and highly selective PKMYT1 inhibitor, in tumors with CCNE1 amplifications (NCT04855656, NCT05147272, NCT05147350). In this study, multiple features of CCNE1 were examined using a combination of whole genome and targeted next generation sequencing (NGS), FISH, and immunohistochemistry (IHC). Cyclin E1 levels were evaluated across tumor tissue microarrays spanning 5 tumor indications: triple-negative breast (n=100), colorectal (n=100), gastric (n=100), uterine (n=49), ovarian (n=100) cancers. The highest cyclin E1 protein levels were found in ovarian and uterine malignancies, consistent with indications with high prevalence of CCNE1 amplification (median H-score ovarian = 125; uterine = 100; gastric = 45; TNBC = 48 and CRC = 24). To explore CCNE1 amplification on a single cell level, we established a novel FISH assay using a subtelomeric q-arm control probe. The subtelomeric q-arm band is more frequently diploid than other regions on chromosome 19. Analytical validation of the FISH assay demonstrated sensitivity of 95%, specificity of 95.2%, and overall concordance of 95.1% when compared to a targeted sequencing panel (SNIPDx®). Within the uterine and ovarian cohorts, cancers harboring CCNE1 amplification were found to display higher cyclin E1 protein levels than non-amplified tumors (p-val=0.0002) and cyclin E1 expression was positively correlated with CCNE1 copy number (Spearman ρ=0.67; p-value=4.1e-05). Substantial intratumoral spatial heterogeneity of CCNE1 copy number when measured by FISH was observed across ovarian and uterine tumors. The potential impact of CCNE1 amplification heterogeneity, cyclin E1 protein levels and CCNE1 amplification fragment size on response to RP-6306 in preclinical models are currently being investigated and will be reported. Citation Format: Adam Petrone, Elia Aguado-Fraile, Sunantha Sethuraman, Adrienne Johnson, Ian Silverman, Gary Marshall, Jorge S. Reis-Filho, John Iafrate, Artur Veloso, Victoria Rimkunas. Tumor heterogeneity of CCNE1 copy number assessed by fluorescence in situ hybridization (FISH) in ovarian and uterine cancers and correlation with cyclin E protein expression [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 2132.

Abstract 2153: Cyclin E1 protein overexpression sensitizes ovarian cancer cells to ZN-c3, a novel, selective and oral bioavailable inhibitor of Wee1

Abstract CCNE1 gene amplification is associated with overexpression of Cyclin E1 protein and is an important oncogenic driver. CCNE1 amplification is common in high grade serous ovarian carcinomas (HGSOC) and is associated with platinum resistance and poor patient outcomes. Importantly, Cyclin E1 overexpression can also occur in the absence of gene amplification in ovarian cancer. Overexpression of Cyclin E1 increases CDK2 activity and accelerates entry into S phase of cell cycle, resulting in replication stress and rendering cells more dependent on DNA repair. WEE1 participates in the DNA damage response by controlling essential checkpoints of the cell cycle, preventing cells from entering mitosis and allowing for DNA repair before cell cycle progression. Thus, we hypothesize that ovarian cancers overexpressing Cyclin E1, either via CCNE1 gene amplification or through other independent mechanisms, are more sensitive to WEE1 inhibition. Here, we tested this hypothesis in a range of preclinical cancer models using azenosertib, a novel, selective, and orally bioavailable WEE1 inhibitor currently in clinical development. Ovarian cancer cell lines overexpressing Cyclin E1 protein (Cyclin E1high) were more sensitive to azenosertib than ovarian cancer cell lines with lower levels of Cyclin E1 protein (Cyclin E1low) inducing significant cytotoxic effects (GRmax < -0.5). In Cyclin E1low ovarian cancer cell lines, overexpression of Cyclin E1 increased sensitivity to azenosertib. In contrast, silencing of CDK2 in Cyclin E1high cells reduced sensitivity to azenosertib. In vivo, higher levels of baseline expression of Cyclin E1 in tumor cells were associated with better response to azenosertib, with tumor growth inhibition (TGI) ranging from 51.5% in a Cyclin E1low SKOV3 model to 88% in a Cyclin E1high OVCAR3 model. Azenosertib synergized with chemotherapy agents in multiple ovarian cancer models. In vitro, we report that Cyclin E1high cancer cells were more sensitive to the combination of azenosertib and chemotherapeutics than Cyclin E1low cancer cells. In vivo, the synergy between azenosertib and paclitaxel was stronger in Cyclin E1high OVCAR3 model (46% reduction of initial tumor volume/104% TGI) than Cyclin E1low A2780 model (85% TGI). Taken together, these data suggest that Cyclin E1 overexpression via gene amplification or independent mechanisms sensitize ovarian cancer cells to azenosertib alone or in combination with chemotherapy. This observation supports the use of Cyclin E1 expression to enrich for azenosertib responders in ovarian cancer and possibly other tumor types. Two subsets of Cyclin E1-driven platinum resistant ovarian cancer patients, as defined by gene amplification or protein overexpression, are being enrolled in an azenosertib monotherapy trial. Citation Format: Jianhui Ma, Wen Liu, Heekyung Chung, Hooman Izadi, Petrus DeJong, Olivier Harismendy, Jia li, Fernando Doñate, Ahmed Samatar, Mark Lackner, Laure Escoubet, Kevin Bunker, Daehwan Kim, Nathan Jameson, Tugba Yildiran Ozmen, Kangjin Jeong, Dong Zhang, Wen-An Pan, Gordon Mills. Cyclin E1 protein overexpression sensitizes ovarian cancer cells to ZN-c3, a novel, selective and oral bioavailable inhibitor of Wee1 [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 2153.

Abstract 5988: A non-canonical CDK9 complex mediates endocycling in polyaneuploid cancer cell (PACC) state

Abstract Once prostate cancer has spread from its primary site, treatment is limited to systemically administered therapy. While tumors initially respond to these therapies, eventually, all therapies fail, and the tumor recurs. Ours and others' recent studies have shown the presence of a universal mechanism of resistance: accession of the polyaneuploid cancer cell (PACC) state. The PolyAneuploid Transition (PAT) is initiated when a cancer cell is subjected to external stress, including therapy (we have tested three classes of chemotherapy and radiation), and accesses an alternative non-mitotic cell cycle known as endocycling, resulting in a greater-than-G2 polyploidization of the aneuploid genome (polyaneuploidy). While in this pro-survival PACC state, we have shown that cells are resistant to all forms of cytotoxic therapy. Initial RNA sequencing of cells that have accessed the PACC state in response to multiple classes of chemotherapy, including docetaxel and cisplatin, demonstrate a remodeled transcriptional profile that is distinct from control cultures. We hypothesize that accession of the resistance-mediating PACC state requires transcriptional remodeling. Our data show that cells in the PACC state had increased overall transcriptional activity. The multi protein complex pTEFB (positive transcription elongation factor) is a critical driver of RNA Polymerase II function and is composed of multiple subunits, including CDK9, a cyclin regulatory subunit, and a transcription factor. CDK9 phosphorylation was significantly elevated during accession and persistence in the PACC state. Genetic inhibitionof CDK9 by siRNA led to decreased expression of cyclin A which plays a role in S phase, both in the mitoticcycle and in the PACC endocycle. Interestingly, despite increased CDK9 levels, known CDK9 pTEFb complex members cyclin T1 and BRD4 were both decreased in cells in the PACC state. We treated cancer cells induced to enter the PACC state upon exposure to cisplatin with pharmaceutical inhibitors of CDK9, Flavopiridol and Fadraciclib. Combination of CDK9 inhibition with chemotherapy showed a suppressive effect on the PACC population. Our findings therefore suggest that a non-canonical CDK9 complex could be critical role for the endocycling to access and maintain the PACC state and represents a therapeutic candidate to eliminate the resistance-mediating PACC state. Citation Format: Cheng-fan Lee, Michael Loycano, Luke LOFTUS, Laurie Kostecka, Melvin Li, Sarah Amend, Kenneth Pienta. A non-canonical CDK9 complex mediates endocycling in polyaneuploid cancer cell (PACC) state. [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 5988.

Activation of skeletal muscle FAPs by LPA requires the Hippo signaling via the FAK pathway

Lysophosphatidic acid (LPA) is a lysophospholipid that signals through six G-protein coupled receptors (LPARs), LPA1 to LPA6. LPA has been described as a potent modulator of fibrosis in different pathologies. In skeletal muscle, LPA increases fibrosis-related proteins and the number of fibro/adipogenic progenitors (FAPs). FAPs are the primary source of ECM-secreting myofibroblasts in acute and chronic damage. However, the effect of LPA on FAPs activation in vitro has not been explored. This study aimed to investigate FAPs’ response to LPA and the downstream signaling mediators involved. Here, we demonstrated that LPA mediates FAPs activation by increasing their proliferation, expression of myofibroblasts markers, and upregulation of fibrosis-related proteins. Pretreatment with the LPA1/LPA3 antagonist Ki16425 or genetic deletion of LPA1 attenuated the LPA-induced FAPs activation, resulting in decreased expression of cyclin e1, α-SMA, and fibronectin. We also evaluated the activation of the focal adhesion kinase (FAK) in response to LPA. Our results showed that LPA induces FAK phosphorylation in FAPs. Treatment with the P-FAK inhibitor PF-228 partially prevented the induction of cell responses involved in FAPs activation, suggesting that this pathway mediates LPA signaling. FAK activation controls downstream cell signaling within the cytoplasm, such as the Hippo pathway. LPA induced the dephosphorylation of the transcriptional coactivator YAP (Yes-associated protein) and promoted direct expression of target pathway genes such as Ctgf/Ccn2 and Ccn1. The blockage of YAP transcriptional activity with Super-TDU further confirmed the role of YAP in LPA-induced FAPs activation. Finally, we demonstrated that FAK is required for LPA-dependent YAP dephosphorylation and the induction of Hippo pathway target genes. In conclusion, LPA signals through LPA1 to regulate FAPs activation by activating FAK to control the Hippo pathway.

Cell cycle dysregulation on prenatal and postnatal arsenic exposure in skin of Wistar rat neonates

This study explores the effects of prenatal and postnatal (until weaning period) arsenic exposure given via pregnant females on Wistar rat neonates. Pregnant female rats were divided in four groups – control, low dose, moderate dose and high dose groups of sodium arsenite exposure during gestation and weaning period. Half of the neonates were sacrificed at day 1 of birth and other half at day 21 of birth. Cell cycle analysis in epidermal keratinocytes using flowcytometer revealed that there was a consistent increase in number of cells in G2/M phase from 0.04% in control group to 0.88%, 1.59% and 2.77% in low, moderate and high dose groups respectively for neonates sacrificed at day-1. Whereas, the increase in number of cells with increasing doses in G2/M phase of neonates sacrificed at day-21 was from 3.44% to 5.1%, 6.82%, and 9.17%. At postnatal day 21, mRNA expression of Cyclin A and B1, p53, Caspases 3, 7 and 9, and Bax were found to be up-regulated. Whereas that of Cyclin E, CDK 1 and 2 and Bcl2 were down regulated consistently in skin tissues of arsenic exposed groups.

SUZ12 promotes the malignant behavior of gastric cancer cells by CDKs.

Zeste 12 Homolog (SUZ12), as a transcription factor, has been found to be highly expressed in a variety of tumors and promote tumor progression. We focus on revealing its role and mechanism in gastric cancer. Cellular level studies were conducted in mouse gastric cancer MFC cells by performing overexpression of SUZ12, overexpression of CDK6, and treatment with CDK6 inhibitor, respectively. Changes in cell viability, invasion, metastasis and colony formation were detected, and the expression variations of cell cycle regulatory proteins CDK6, P21, and Cyclin D were determined. During the animal experimentation, a mouse xenograft model was established. After transplantation of SUZ12-overexpressing MFC-SUZ12, the tumor growth was compared with that in MFC, and the tissue expressions of CDK-6, SUZ12, and Cyclin D were examined. Overexpression of SUZ12 could enhance the viability of MFC cells while upregulating their migration, invasion and colony formation abilities, which promoted the expression of CDK6, P21, and Cyclin D. CDK6 inhibitor, on the other hand, could suppress the effects of SUZ12 overexpression and weaken the cell viability and malignant behavior. Overexpression of CDK6 also promoted the MFC viability and malignant behavior. We found that SUZ12 exerted its effects by promoting the expression of downstream cyclin CDK6. At the animal level, the mice xenografted with SUZ12-overexpressing MFC cells exhibited larger tumor volumes, as well as elevated cyclin expression.SUZ12 promotes the proliferation and malignant behavior of gastric cancer cells by regulating the expression of downstream CDK6.

Copy number variations and protein expression of Cyclin E1 and Epithelial cell transforming sequence 2 genes predict the chemotherapeutic response in patients with serous ovarian carcinoma.

Serous ovarian carcinoma (SOC) is characterized by extreme genomic instability, chromosomal rearrangements and copy number variations (CNVs) leading to the development of early metastasis and chemo-resistance. The present study was designed to observe the role of CNVs of Cyclin E1 (CCNE1) and Epithelial cell transforming sequence- 2 (ECT2) genes and their encoded proteins in predicting the chemotherapeutic response in SOC patients. This observational analytical study was conducted at University of Health Sciences, Lahore, Pakistan from December 2019 till June 2022.The study included twenty-five SOC patients with resectable ovarian tumors and twenty-five control subjects. The patients were followed-up for six months for their response to chemotherapy. The CNVs in CCNE1 and ECT-2 genes were determined by real time PCR while serum levels of encoded proteins were determined in controls and cases, before and after six months of treatment, through ELISA. The response to chemotherapy was categorized as sensitive or resistant based on serum CA-125 levels and radiological scans. The copy number variations in CCNE1 and ECT2 genes showed association with the clinic-pathological characteristics and chemotherapy response. Statistically significant difference was found between the mean pre-chemotherapy protein levels of CCNE1 in cases than controls (p-value <0.001) and between the mean pre and post-chemotherapy protein levels of CCNE1 and ECT2 (p-value <0.001) in SOC patients. The copy number variations of CCNE1 and ECT2 genes and their protein expression are positively associated with chemotherapeutic response in SOC patients.

Loss of IRF8 inhibits the growth of acute myeloid leukemia cells.

The transcription factor interferon regulatory factor 8 (IRF8), as a member of the IRF family, is essential for myeloid cell differentiation. However, the precise role of IRF8 in the pathogenesis of acute myeloid leukemia (AML) remains unknown. By using multivariate analysis, we discovered that high IRF8 expression was an independent poor predictor of overall survival (OS) in AML patients from our clinical follow-up study. The proliferation of three AML cell lines was significantly inhibited by shRNA-mediated knockdown of IRF8, owing to cell cycle S-phase arrest. Furthermore, we demonstrated that knocking down IRF8 could suppress the expression of CyclinA and CyclinB1, resulting in a shift in cell cycle distribution. Loss of IRF8 in AML cells decreased the expression of STAT3 and phosphor-STAT3 (pSTAT3), which are key factors in JAK/STAT signal pathway and are important for AML progression. Using a xenograft mouse model, we discovered the antiproliferative effect of losing IRF8 in vivo. In conclusion, this study found that IRF8 may play a prognostic factor and therapeutic target in AML.

Adipose Mesenchymal Stromal Cell-Derived Exosomes Carrying MiR-122-5p Antagonize the Inhibitory Effect of Dihydrotestosterone on Hair Follicles by Targeting the TGF-β1/SMAD3 Signaling Pathway.

Androgenic alopecia (AGA) is the most common type of hair loss, where local high concentrations of dihydrotestosterone (DHT) in the scalp cause progressive shrinkage of the hair follicles, eventually contributing to hair loss. Due to the limitations of existing methods to treat AGA, the use of multi-origin mesenchymal stromal cell-derived exosomes has been proposed. However, the functions and mechanisms of action of exosomes secreted by adipose mesenchymal stromal cells (ADSCs-Exos) in AGA are still unclear. Using Cell Counting Kit-8 (CCK8) analysis, immunofluorescence staining, scratch assays, and Western blotting, it was found that ADSC-Exos contributed to the proliferation, migration, and differentiation of dermal papilla cells (DPCs) and up-regulated the expression of cyclin, β-catenin, versican, and BMP2. ADSC-Exos also mitigated the inhibitory effects of DHT on DPCs and down-regulated transforming growth factor-beta1 (TGF-β1) and its downstream genes. Moreover, high-throughput miRNA sequencing and bioinformatics analysis identified 225 genes that were co-expressed in ADSC-Exos; of these, miR-122-5p was highly enriched and was found by luciferase assays to target SMAD3. ADSC-Exos carrying miR-122-5p antagonized DHT inhibition of hair follicles, up-regulated the expression of β-catenin and versican in vivo and in vitro, restored hair bulb size and dermal thickness, and promoted the normal growth of hair follicles. So, ADSC-Exos enhanced the regeneration of hair follicles in AGA through the action of miR-122-5p and the inhibition of the TGF-β/SMAD3 axis. These results suggest a novel treatment option for the treatment of AGA.

Lactobacillus rhamnosus GG cell-free supernatant as a novel anti-cancer adjuvant

BackgroundGut microbiota modulation has been demonstrated to be effective in protecting patients against detrimental effects of anti-cancer therapies, as well as to improve the efficacy of certain anti-cancer treatments. Among the most characterized probiotics, Lactobacillus rhamnosus GG (LGG) is currently utilized in clinics to alleviate diarrhea, mucositis or intestinal damage which might be associated with several triggers, including Clostridium difficile infections, inflammatory gut diseases, antibiotic consumption, chemotherapy or radiation therapy. Here, we investigate whether LGG cell-free supernatant (LGG-SN) might exert anti-proliferative activity toward colon cancer and metastatic melanoma cells. Moreover, we assess the potential adjuvant effect of LGG-SN in combination with anti-cancer drugs.MethodsLGG-SN alone or in combination with either 5-Fuorouracil and Irinotecan was used to treat human colon and human melanoma cancer cell lines. Dimethylimidazol-diphenyl tetrazolium bromide assay was employed to detect cellular viability. Trypan blue staining, anti-cleaved caspase-3 and anti-total versus anti-cleaved PARP western blots, and annexin V/propidium iodide flow cytometry analyses were used to assess cell death. Flow cytometry measurement of cellular DNA content (with propidium iodide staining) together with qPCR analysis of cyclins expression were used to assess cell cycle.ResultsWe demonstrate that LGG-SN is able to selectively reduce the viability of cancer cells in a concentration-dependent way. While LGG-SN does not exert any anti-proliferative activity on control fibroblasts. In cancer cells, the reduction in viability is not associated with apoptosis induction, but with a mitotic arrest in the G2/M phase of cell cycle. Additionally, LGG-SN sensitizes cancer cells to both 5-Fluorouracil and Irinotecan, thereby showing a positive synergistic action.ConclusionOverall, our results suggest that LGG-SN may contain one or more bioactive molecules with anti-cancer activity which sensitize cancer cells to chemotherapeutic drugs. Thus, LGG could be proposed as an ideal candidate for ground-breaking integrated approaches to be employed in oncology, to reduce chemotherapy-related side effects and overcome resistance or relapse issues, thus ameliorating the therapeutic response in cancer patients.