p27 specifically decreases in squamous carcinoma, and mediates NNK-induced transformation of human bronchial epithelial cells.

Lung cancer remains the leading cause of cancer-related deaths, with cigarette smoking being the most critical factor, linked to nearly 90% of lung cancer cases. NNK, a highly carcinogenic nitrosamine found in tobacco, is implicated in the lung cancer-causing effects of cigarette smoke. Although NNK is known to mutate or activate certain oncogenes, its potential interaction with p27 in modulating these carcinogenic effects is currently unexplored. Recent studies have identified specific downregulation of p27 in human squamous cell carcinoma, in contrast to adenocarcinoma. Additionally, exposure to NNK significantly suppresses p27 expression in human bronchial epithelial cells. Subsequent studies indicates that the downregulation of p27 is pivotal in NNK-induced cell transformation. Mechanistic investigations have shown that reduced p27 expression leads to increased level of ITCH, which facilitates the degradation of Jun B protein. This degradation in turn, augments miR-494 expression and its direct regulation of JAK1 mRNA stability and protein expression, ultimately activating STAT3 and driving cell transformation. In summary, our findings reveal that: (1) the downregulation of p27 increases Jun B expression by upregulating Jun B E3 ligase ITCH, which then boosts miR-494 transcription; (2) Elevated miR-494 directly binds to 3'-UTR of JAK1 mRNA, enhancing its stability and protein expression; and (3) The JAK1/STAT3 pathway is a downstream effector of p27, mediating the oncogenic effect of NNK in lung cancer. These findings provide significant insight into understanding the participation of mechanisms underlying p27 inhibition of NNK induced lung squamous cell carcinogenic effect.

Upregulation of LncRNA UCA1 promotes cardiomyocyte proliferation by inhibiting the miR-128/SUZ12/P27 pathway.

Enhancing cardiomyocyte proliferation is essential to reverse or slow down the heart failure progression in many cardiovascular diseases such as myocardial infarction (MI). Long non-coding RNAs (lncRNAs) have been reported to regulate cardiomyocyte proliferation. In particular, lncRNA urothelial carcinoma-associated 1 (lncUCA1) played multiple roles in regulating cell cycle progression and cardiovascular diseases, making lncUCA1 a potential target for promoting cardiomyocyte proliferation. However, the role of lncUCA1 in cardiomyocyte proliferation remains unknown. This study aimed at exploring the function and underlying molecular mechanism of lncUCA1 in cardiomyocyte proliferation. Quantitative RT-PCR showed that lncUCA1 expression decreased in postnatal hearts. Gain-and-loss-of-function experiments showed that lncUCA1 positively regulated cardiomyocyte proliferation in vitro and in vivo. The bioinformatics program identified miR-128 as a potential target of lncUCA1, and loss of miR-128 was reported to promote cardiomyocyte proliferation by inhibiting the SUZ12/P27 pathway. Luciferase reporter assay, qRT-PCR, western blotting, and immunostaining experiments further revealed that lncUCA1 acted as a ceRNA of miR-128 to upregulate its target SUZ12 and downregulate P27, thereby increasing cyclin B1, cyclin E, CDK1 and CDK2 expression to promote cardiomyocyte proliferation. In conclusion, upregulation of lncRNA UCA1 promoted cardiomyocyte proliferation by inhibiting the miR-128/SUZ12/P27 pathway. Our results indicated that lncUCA1 might be a new therapeutic target for stimulating cardiomyocyte proliferation.

Fatty Acid Synthase Promotes Hepatocellular Carcinoma Growth via S-Phase Kinase-Associated Protein 2/p27KIP1 Regulation.

Background and Objectives: Aberrant upregulation of fatty acid synthase (FASN), catalyzing de novo synthesis of fatty acids, occurs in various tumor types, including human hepatocellular carcinoma (HCC). Although FASN oncogenic activity seems to reside in its pro-lipogenic function, cumulating evidence suggests that FASN's tumor-supporting role might also be metabolic-independent. Materials and Methods: In the present study, we show that FASN inactivation by specific small interfering RNA (siRNA) promoted the downregulation of the S-phase kinase associated-protein kinase 2 (SKP2) and the consequent induction of p27KIP1 in HCC cell lines. Results: Expression levels of FASN and SKP2 directly correlated in human HCC specimens and predicted a dismal outcome. In addition, forced overexpression of SKP2 rendered HCC cells resistant to the treatment with the FASN inhibitor C75. Furthermore, FASN deletion was paralleled by SKP2 downregulation and p27KIP1 induction in the AKT-driven HCC preclinical mouse model. Moreover, forced overexpression of an SKP2 dominant negative form or a p27KIP1 non-phosphorylatable (p27KIP1-T187A) construct completely abolished AKT-dependent hepatocarcinogenesis in vitro and in vivo. Conclusions: In conclusion, the present data indicate that SKP2 is a critical downstream effector of FASN and AKT-dependent hepatocarcinogenesis in liver cancer, envisaging the possibility of effectively targeting FASN-positive liver tumors with SKP2 inhibitors or p27KIP1 activators.

Targeting calciumopathy for neuroprotection: focus on calcium channels Cav1, Orai1 and P2X7.

As the uncontrolled entry of calcium ions (Ca2+) through plasmalemmal calcium channels is a cell death trigger, the conjecture is here raised that mitigating such an excess of Ca2+ entry should rescue from death the vulnerable neurons in neurodegenerative diseases (NDDs). However, this supposition has failed in some clinical trials (CTs). Thus, a recent CT tested whether isradipine, a blocker of the Cav1 subtype of voltage-operated calcium channels (VOCCs), exerted a benefit in patients with Parkinson's disease (PD); however, outcomes were negative. This is one more of the hundreds of CTs done under the principle of one-drug-one-target, that have failed in Alzheimer's disease (AD) and other NDDs during the last three decades. As there are myriad calcium channels to let Ca2+ ions gain the cell cytosol, it seems reasonable to predict that blockade of Ca2+ entry through a single channel may not be capable of preventing the Ca2+ flood of cells by the uncontrolled Ca2+ entry. Furthermore, as Ca2+ signaling is involved in the regulation of myriad functions in different cell types, it seems also reasonable to guess that a therapy should be more efficient by targeting different cells with various drugs. Here, we propose to mitigate Ca2+ entry by the simultaneous partial blockade of three quite different subtypes of plasmalemmal calcium channels that is, the Cav1 subtype of VOCCs, the Orai1 store-operated calcium channel (SOCC), and the purinergic P2X7 calcium channel. All three channels are expressed in both microglia and neurons. Thus, by targeting the three channels with a combination of three drug blockers we expect favorable changes in some of the pathogenic features of NDDs, namely (i) to mitigate Ca2+ entry into microglia; (ii) to decrease the Ca2+-dependent microglia activation; (iii) to decrease the sustained neuroinflammation; (iv) to decrease the uncontrolled Ca2+ entry into neurons; (v) to rescue vulnerable neurons from death; and (vi) to delay disease progression. In this review we discuss the arguments underlying our triad hypothesis in the sense that the combination of three repositioned medicines targeting Cav1, Orai1, and P2X7 calcium channels could boost neuroprotection and delay the progression of AD and other NDDs.

Seliciclib: A New Treatment for Cushing's Disease?

Previous studies have suggested that corticotroph tumours are associated with the overexpression of cyclin E and that the inactivation of cyclin-dependent kinases, which activate cyclin E, may have antisecretory and antiproliferative effects. Seliciclib, also known as R-roscovitine, is a pituitary-targeting agent shown to inhibit the growth of corticotroph tumour cells via cyclin E and retinoblastoma protein-mediated pathways. A recent study investigated the role of seliciclib in regulating biochemical parameters in a small number of patients with Cushing's disease, providing preliminary data on its possible therapeutic effectiveness in treating this disorder.

Therapeutic Potential of Targeting p27kip1 in Plaque Vulnerability.

Atherosclerosis, a critical contributor to coronary artery diseases, involves the accumulation of cholesterol, fibrin, and lipids within arterial walls, inciting inflammatory reactions culminating in plaque formation. This multifaceted interplay encompasses excessive fibrosis, fatty plaque development, vascular smooth muscle cell (VSMC) proliferation, and leukocyte migration in response to inflammatory pathways. While stable plaques demonstrate resilience against complications, vulnerable ones, with lipid-rich cores, necrosis, and thin fibrous caps, lead to thrombosis, myocardial infarction, stroke, and acute cerebrovascular accidents. The nuanced phenotypes of VSMCs, modulated by gene regulation and environmental cues, remain pivotal. Essential markers like alpha-SMA, myosin heavy chain, and calponin regulate VSMC migration and contraction, exhibiting diminished expression during VSMC de-differentiation and proliferation. p27kip, a CDK inhibitor, shows promise in regulating VSMC proliferation and appears associated with TNF-α-induced pathways impacting unstable plaques. Oncostatin M (OSM), an IL-6 family cytokine, correlates with MMP upregulation and foam cell formation, influencing plaque development. Efforts targeting mammalian target of rapamycin (mTOR) inhibition, notably using rapamycin and its analogs, demonstrate potential but pose challenges due to associated adverse effects. Exploration of the impact of p27kip impact on plaque macrophages presents promising avenues, yet its complete therapeutic potential remains untapped. Similarly, while OSM has exhibited potential in inducing cell cycle arrest via p27kip, direct links necessitate further investigation. This critical review discusses the role of mTOR, p27kip, and OSM in VSMC proliferation and differentiation followed by the therapeutic potential of targeting these mediators in atherosclerosis to attenuate plaque vulnerability.

PD-L1 intrinsically promotes the proliferation of breast cancer cells through the SKP2-p27/p21 axis.

BACKGROUND: PD-L1 intrinsically promotes tumor progression through multiple mechanisms, which potentially leads to resistance to anti-PD-1/PD-L1 therapies. The intrinsic effect of PD-L1 on breast cancer (BC) cell proliferation has not been fully elucidated. METHODS: we used proteomics, gene expression knockdown (KD), quantitative immunofluorescence (qIF), western blots, functional assays including colony-forming assay (CFA) and real-time cell analyzer (RTCA), and in vivo data using immunohistochemistry in breast cancer patients. RESULTS: PD-L1 promoted BC cell proliferation by accelerating cell cycle entry at the G1-to-S phase transition. Global proteomic analysis of the differentially expressed nuclear proteins indicated the involvement of several proliferation-related molecules, including p21CIP1/WAF1. Western blotting and qIF demonstrated the higher expression of SKP2 and the lower expression of p21CIP1/WAF1 and p27Kip1 in PD-L1 expressing (PD-L1pos) cells as compared to PD-L1 KD (PD-L1KD) cells. Xenograft-derived cells and the TCGA BC dataset confirmed this relationship in vivo. Functionally, CFA and RTCA demonstrated the central role of SKP2 in promoting PD-L1-mediated proliferation. Finally, immunohistochemistry in 74 breast cancer patients confirmed PD-L1 and SKP-p21/p27 axis relationship, as it showed a highly statistically significant correlation between SKP2 and PD-L1 expression (p < 0.001), and both correlated significantly with the proliferation marker Ki-67 (p < 0.001). On the other hand, there was a statistically significant inverse relationship between PD-L1 and p21CIP1/WAF1 expression (p = 0.005). Importantly, double negativity for p21CIP1/WAF1 and p27Kip1 correlated significantly with PD-L1 (p < 0.001), SKP2 (p = 0.002), and Ki-67 (p = 0.002). CONCLUSIONS: we have demonstrated the role of the SKP2-p27/p21 axis in intrinsic PD-L1-enhanced cell cycle progression. Inhibitors of SKP2 expression can alleviate resistance to ICPIs.

TG-interacting factor 1 regulates mitotic clonal expansion during adipocyte differentiation.

Obesity is one of the significant health challenges in the world and is highly associated with abnormal adipogenesis. TG-interacting factor 1 (TGIF1) is essential for differentiating murine adipocytes and human adipose tissue-derived stem cells. However, the mode of action needs to be better elucidated. To investigate the roles of TGIF1 in differentiation in-depth, CRISPR/Cas9 knockout technology was performed to generate TGIF1-silenced preadipocytes. The absence of TGIF1 in 3 T3-F442A preadipocytes abolished lipid accumulation throughout the differentiation using Oil Red O staining. Conversely, we established 3 T3-F442A preadipocytes stably expressing TGIF1 and doxycycline-inducible TGIF1 in TGIF1-silenced 3 T3-F442A preadipocytes. Remarkably, the induction of TGIF1 by doxycycline during the initial differentiation phase successfully promoted lipid accumulation in TGIF1-silenced 3 T3-F442A cells. We further explored the mechanisms of TGIF1 in early differentiation. We demonstrated that TGIF1 promoted the mitotic clonal expansion via upregulation of CCAAT/enhancer-binding proteins ß expression, interruption with peroxisome proliferators activated receptor γ downstream regulation, and inhibition of p27kip1 expression. In conclusion, we strengthen the pivotal roles of TGIF1 in early differentiation, which might contribute to resolving obesity-associated metabolic syndromes.

Albiflorin ameliorates thioacetamide-induced hepatic fibrosis: The involvement of NURR1-mediated inflammatory signaling cascades in hepatic stellate cells activation.

Thioacetamide (TAA) within the liver generates hepatotoxic metabolites that can be induce hepatic fibrosis, similar to the clinical pathological features of chronic human liver disease. The potential protective effect of Albiflorin (ALB), a monoterpenoid glycoside found in Paeonia lactiflora Pall, against hepatic fibrosis was investigated. The mouse hepatic fibrosis model was induced with an intraperitoneal injection of TAA. Hepatic stellate cells (HSCs) were subjected to treatment with transforming growth factor-beta (TGF-ß), while lipopolysaccharide/adenosine triphosphate (LPS/ATP) was added to stimulate mouse peritoneal macrophages (MPMs), leading to the acquisition of conditioned medium. For TAA-treated mice, ALB reduced ALT, AST, HYP levels in serum or liver. The administration of ALB reduced histopathological abnormalities, and significantly regulated the expressions of nuclear receptor-related 1 protein (NURR1) and the P2X purinoceptor 7 receptor (P2×7r) in liver. ALB could suppress HSCs epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) deposition, and pro-inflammatory factor level. ALB also remarkably up-regulated NURR1, inhibited P2×7r signaling pathway, and worked as working as C-DIM12, a NURR1 agonist. Moreover, deficiency of NURR1 in activated HSCs and Kupffer cells weakened the regulatory effect of ALB on P2×7r inhibition. NURR1-mediated inhibition of inflammatory contributed to the regulation of ALB ameliorates TAA-induced hepatic fibrosis, especially based on involving in the crosstalk of HSCs-macrophage. Therefore, ALB plays a significant part in the mitigation of TAA-induced hepatotoxicity this highlights the potential of ALB as a protective intervention for hepatic fibrosis.

Glyphosate-based herbicide worsens alterations induced by cafeteria diet on rat uterus.

Exposure to glyphosate-based herbicides (GBH) and consumption of cafeteria (CAF) diet, which are widespread in Western society, seem to be associated with endometrial hyperplasia (EH). Here, we aimed to evaluate the effects of a subchronic low dose of GBH added to the CAF diet on the rat uterus. Female Wistar rats were fed from postnatal day (PND)21 until PND240 with chow (control) or CAF diet. Since PND140, rats also received GBH (2 mg of glyphosate/kg/day) or water through food, yielding four experimental groups: control, CAF, GBH, and CAF+GBH. On PND240, CAF and CAF+GBH animals showed an increased adiposity index. With respect to the control group, no changes in the serum levels of 17ß-estradiol and progesterone were found. However, progesterone levels were higher in the CAF+GBH group than in the CAF and GBH groups. In the uterus, both studied factors alone and in combination induced morphological and molecular changes associated with EH. Furthermore, the addition of GBH provoked an increased thickness of subepithelial stroma in rats fed with the CAF diet. As a consequence of GBH exposure, CAF+GBH rats exhibited an increased density of abnormal gland area, considered preneoplastic lesions, as well as a reduced PTEN and p27 expression, both tumor suppressor molecules that inhibit cell proliferation, with respect to control rats. These results indicate that the addition of GBH exacerbates the CAF effects on uterine lesions and that the PTEN/p27 signaling pathway seems to be involved. Further studies focusing on the interaction between unhealthy diets and environmental chemicals should be encouraged to better understand uterine pathologies.

Phosphorylation of TG-interacting factor 1 at carboxyl-terminal sites in response to insulin regulates adipocyte differentiation.

TG-interacting factor 1 (TGIF1) contributes to the differentiation of murine white preadipocyte and human adipose tissue-derived stem cells; however, its regulation is not well elucidated. Insulin is a component of the adipogenic cocktail that induces ERK signaling. TGIF1 phosphorylation and sustained stability in response to insulin were reduced through the use of specific MEK inhibitor U0126. Mutagenesis at T235 or T239 residue of TGIF1 in preadipocytes led to dephosphorylation of TGIF1. The reduced TGIF1 stability resulted in an increase in p27kip1 expression, a decrease in phosphorylated Rb expression and cellular proliferation, and a reduced accumulation of lipids compared to the TGIF1-overexpressed cells. These findings highlight that insulin/ERK-driven phosphorylation of the T235 or T239 residue at TGIF1 is crucial for adipocyte differentiation.

The E3 ligase SMURF1 stabilizes p27 via UbcH7 catalyzed K29-linked ubiquitin chains to promote cell migration SMURF1-UbcH7 K29 ubiquitination of p27 and cell migration.

Ubiquitination is a key regulator of protein stability and function. The multifunctional protein p27 is known to be degraded by the proteasome following K48-linked ubiquitination. However, we recently reported that when the ubiquitin-conjugating enzyme UbcH7 (UBE2L3) is overexpressed, p27 is stabilized, and cell cycle is arrested in multiple diverse cell types including eye lens, retina, HEK-293, and HELA cells. However, the ubiquitin ligase associated with this stabilization of p27 remained a mystery. Starting with an in vitro ubiquitination screen, we identified RSP5 as the yeast E3 ligase partner of UbcH7 in the ubiquitination of p27. Screening of the homologous human NEDD4 family of E3 ligases revealed that SMURF1 but not its close homolog SMURF2, stabilizes p27 in cells. We found that SMURF1 ubiquitinates p27 with K29O but not K29R or K63O ubiquitin in vitro, demonstrating a strong preference for K29 chain formation. Consistent with SMURF1/UbcH7 stabilization of p27, we also found that SMURF1, UbcH7, and p27 promote cell migration, whereas knockdown of SMURF1 or UbcH7 reduces cell migration. We further demonstrated the colocalization of SMURF1/p27 and UbcH7/p27 at the leading edge of migrating cells. In sum, these results indicate that SMURF1 and UbcH7 work together to produce K29-linked ubiquitin chains on p27, resulting in the stabilization of p27 and promoting its cell-cycle independent function of regulating cell migration.

UBE4B regulates p27 expression in A549 NSCLC cells through regulating the interaction of HuR and the p27 5' UTR.

Ubiquitination factor E4B (UBE4B) has a tumor-promoting effect, demonstrated by its aberrant expression in various types of cancers, and in vitro studies have shown that the retardation of cancer cell proliferation can be induced by targeting UBE4B. However, the molecular pathways through which UBE4B exerts its oncogenic activities have not yet been clearly identified and existing knowledge is limited to p53 and its subsequent downstream targets. In this study, we demonstrated that UBE4B regulates p27 expression in A549 cells via the cap-independent translation pathway following treatment with rapamycin and cycloheximide (CHX). Subsequently, we identified that UBE4B regulates p27 translation by regulating the interaction between human antigen R (HuR) and the p27 internal ribosomal entry site (IRES). First, UBE4B interacts with HuR, which inhibits p27 translation through the IRES. Secondly, the interaction between HuR and the p27 IRES was diminished by UBE4B depletion and enhanced by UBE4B overexpression. Finally, HuR depletion-induced growth retardation, accompanied by p27 accumulation, was restored by UBE4B overexpression. Collectively, these results suggest that the oncogenic properties of UBE4B in A549 cells are mediated by HuR, suggesting the potential of targeting the UBE4B-HuR-p27 axis as a therapeutic strategy for lung cancer.

circUBE3C modulates myoblast development by binding to miR-191 and upregulating the expression of p27.

Noncoding RNAs, including miRNAs (microRNAs) and circRNAs (circular RNA), are crucial regulators of myoblast proliferation and differentiation during muscle development. However, the specific roles and molecular mechanisms of circRNAs in muscle development remain poorly understood. Based on the existing circRNA-miRNA-mRNA network, our study focuses on circUBE3C, exploring its differential expression in fetal and adult muscle tissue of the cattle and investigating its impact on myoblast proliferation, apoptosis, and differentiation. The functional analysis of overexpression plasmids and siRNAs (small interfering RNAs) targeting circUBE3C was comprehensively evaluated by employing an array of advanced assays, encompassing CCK-8 (cell counting kit-8), EdU (5-ethynyl-20-deoxyuridine), flow cytometry, western blot analysis, and RT-qPCR. In vivo investigations indicated that overexpression of circUBE3C impedes the process of skeletal muscle regeneration. Mechanistically, we demonstrated that circUBE3C interacts with miR-191 and alleviates the suppression of p27 through cytoplasmic separation, bioinformatics prediction, dual-luciferase reporter assay, and RIP (RNA immunoprecipitation). Our findings indicate that the novel circRNA circUBE3C competitively binds to miR-191, thereby inhibiting proliferation and promoting apoptosis in bovine primary myoblasts and unveiling a regulatory pathway in bovine skeletal muscle development. These findings expand our understanding of circRNA functions in mammals and provide a basis for further exploration of their role in myogenesis and muscle diseases.

Up and away with cervical cancer: IL-29 is a promising cytokine for immunotherapy of cervical cancer due to its powerful upregulation of p18, p27, and TRAILR1.

Cervical cancer is one of the most common types of female cancers worldwide. IL-29 is an interesting cytokine in the IFNλ family. Its role in the pathogenesis of neoplasia is complicated and has been studied in other cancers, such as lung cancer, gastric cancer, and colorectal cancer. IL-29 has been previously reported to promote the growth of pancreatic cancer. However, the direct role of IL-29 in cervical cancer has not been studied yet. This study was performed to investigate the direct effect on cervical cancer cell growth. Clonogenic survival assay, cell proliferation, and caspase-3 activity kits were used to evaluate the effects of IL-29 on cell survival, proliferation, and apoptosis of a well-studied cervical cancer cell line, SiHa. We further investigated the potential molecular mechanisms by using RT-PCR and IHC. We found that the percentage of colonies of SiHa cells was decreased in the presence of IL-29. This was consistent with a decreased OD value of cancer cells. Furthermore, the relative caspase-3 activity in cancer cells increased in the presence of IL-29. The anti-proliferative effect of IL-29 on cancer cells correlated with increased expression of the anti-proliferative molecules p18 and p27. The pro-apoptotic effect of IL-29 on cancer cells correlated with increased expression of the pro-apoptotic molecule TRAILR1. IL-29 inhibits cervical cancer cell growth by inhibiting cell proliferation and promoting cell apoptosis. Thus, IL-29 might be a promising cytokine for immunotherapy of cervical cancer.

Syndromic MEN1 parathyroid adenomas consist of both subclonal nodules and clonally independent tumors.

Primary hyperparathyroidism with parathyroid tumors is a typical manifestation of Multiple Endocrine Neoplasia Type 1 (MEN1) and is historically termed "primary hyperplasia". Whether these tumors represent a multi-glandular clonal disease or hyperplasia has not been robustly proven so far. Loss of Menin protein expression is associated with inactivation of both alleles and a good surrogate for a MEN1 gene mutation. The cyclin-dependent kinase inhibitor 1B (CDKN1B) gene is mutated in MEN4 and encodes for protein p27 whose expression is poorly studied in the syndromic MEN1 setting.Here, we analyzed histomorphology and protein expression of Menin and p27 in parathyroid adenomas of 25 patients of two independent, well-characterized MEN1 cohorts. The pattern of loss of heterozygosity (LOH) was assessed by fluorescence in situ hybridization (FISH) in one MEN1-associated parathyroid adenoma. Further, next-generation sequencing (NGS) was performed on eleven nodules of four MEN1 patients.Morphologically, the majority of MEN1 adenomas consisted of multiple distinct nodules, in which Menin expression was mostly lost and p27 protein expression reduced. FISH analysis revealed that most nodules exhibited MEN1 loss, with or without the loss of centromere 11. NGS demonstrated both subclonal evolution and the existence of clonally unrelated tumors.Syndromic MEN1 parathyroid adenomas therefore consist of multiple clones with subclones, which supports the current concept of the novel WHO classification of parathyroid tumors (2022). p27 expression was lost in a large fraction of MEN1 parathyroids and must therefore be used with caution in suggesting MEN4.

Endothelial cell flow-mediated quiescence is temporally regulated and utilizes the cell cycle inhibitor p27.

Background: Endothelial cells regulate their cell cycle as blood vessels remodel and transition to quiescence downstream of blood flow-induced mechanotransduction. Laminar blood flow leads to quiescence, but how flow-mediated quiescence is established and maintained is poorly understood. Methods: Primary human endothelial cells were exposed to laminar flow regimens and gene expression manipulations, and quiescence depth was analyzed via time to cell cycle re-entry after flow cessation. Mouse and zebrafish endothelial expression patterns were examined via scRNA seq analysis, and mutant or morphant fish lacking p27 were analyzed for endothelial cell cycle regulation and in vivo cellular behaviors. Results: Arterial flow-exposed endothelial cells had a distinct transcriptome, and they first entered a deep quiescence, then transitioned to shallow quiescence under homeostatic maintenance conditions. In contrast, venous-flow exposed endothelial cells entered deep quiescence early that did not change with homeostasis. The cell cycle inhibitor p27 (CDKN1B) was required to establish endothelial flow-mediated quiescence, and expression levels positively correlated with quiescence depth. p27 loss in vivo led to endothelial cell cycle upregulation and ectopic sprouting, consistent with loss of quiescence. HES1 and ID3, transcriptional repressors of p27 upregulated by arterial flow, were required for quiescence depth changes and the reduced p27 levels associated with shallow quiescence. Conclusions: Endothelial cell flow-mediated quiescence has unique properties and temporal regulation of quiescence depth that depends on the flow stimulus. These findings are consistent with a model whereby flow-mediated endothelial cell quiescence depth is temporally regulated downstream of p27 transcriptional regulation by HES1 and ID3. The findings are important in understanding endothelial cell quiescence mis-regulation that leads to vascular dysfunction and disease.

NNMT/1-MNA Promote Cell-Cycle Progression of Breast Cancer by Targeting UBC12/Cullin-1-Mediated Degradation of P27 Proteins.

Cell cycle dysregulation is a defining feature of breast cancer. Here, 1-methyl-nicotinamide (1-MNA), metabolite of nicotinamide N-methyltransferase(NNMT) is identified, as a novel driver of cell-cycle progression in breast cancer. NNMT, highly expressed in breast cancer tissues, positively correlates with tumor grade, TNM stage, Ki-67 index, and tumor size. Ablation of NNMT expression dramatically suppresses cell proliferation and causes cell-cycle arrest in G0/G1 phase. This phenomenon predominantly stems from the targeted action of 1-MNA, resulting in a specific down-regulation of p27 protein expression. Mechanistically, 1-MNA expedites the degradation of p27 proteins by enhancing cullin-1 neddylation, crucial for the activation of Cullin-1-RING E3 ubiquitin ligase(CRL1)-an E3 ubiquitin ligase targeting p27 proteins.  NNMT/1-MNA specifically up-regulates the expression of UBC12, an E2 NEDD8-conjugating enzyme required for cullin-1 neddylation. 1-MNA showes high binding affinity to UBC12, extending the half-life of UBC12 proteins via preventing their localization to lysosome for degradation. Therefore, 1-MNA is a bioactive metabolite that promotes breast cancer progression by reinforcing neddylation pathway-mediated p27 degradation. The study unveils the link between NNMT enzymatic activity with cell-cycle progression, indicating that 1-MNA may be involved in the remodeling of tumor microenvironment.

Cells use multiple mechanisms for cell-cycle arrest upon withdrawal of individual amino acids.

Amino acids are required for cell growth and proliferation, but it remains unclear when and how amino acid availability impinges on the proliferation-quiescence decision. Here, we used time-lapse microscopy and single-cell tracking of cyclin-dependent kinase 2 (CDK2) activity to assess the response of individual cells to withdrawal of single amino acids and found strikingly different cell-cycle effects depending on the amino acid. For example, upon leucine withdrawal, MCF10A cells complete two cell cycles and then enter a CDK2-low quiescence, whereas lysine withdrawal causes immediate cell-cycle stalling. Methionine withdrawal triggers a restriction point phenotype similar to serum starvation or Mek inhibition: upon methionine withdrawal, cells complete their current cell cycle and enter a CDK2-low quiescence after mitosis. Modulation of restriction point regulators p21/p27 or cyclin D1 enables short-term rescue of proliferation under methionine and leucine withdrawal, and to a lesser extent lysine withdrawal, revealing a checkpoint connecting nutrient signaling to cell-cycle entry.

Genistein Induces Antiproliferative Activity and Apoptosis in Human Osteosarcoma Saos-2 Cells.

BACKGROUND/AIM: Genistein (4', 5, 7-trihydroxyisoflavone) and daidzein (4', 7-dihydroxyisoflavone) are isoflavones derived from soybean and have anti-cancer effects in various cells. However, the effects of genistein and daidzein on the human osteosarcoma cell line Saos-2 has not been investigated before. MATERIALS AND METHODS: Human osteosarcoma Saos-2 cells were treated with genistein for 24 and 48 hours. Cytotoxicity and apoptosis were measured. RESULTS: Genistein significantly inhibited proliferation of Saos-2 cells stronger than daidzein in a dose-dependent manner (0 to 80 µM). Genistein also significantly suppressed Saos-2 cell viability in a dose-dependent manner (0 to 100 µM). In contrast, daidzein did not affect Saos-2 cell viability. Real-time PCR revealed that genistein caused G1-arrest by increasing the expression of p21 and p27 mRNAs in Saos-2 cells. In addition, genistein induced apoptosis through the up-regulation of effector caspase-3/7 activity in Saos-2 cells. Genistein also enhanced initiator caspase-9 and TNF-α mRNA expression in cells. CONCLUSION: Genistein may inhibit proliferation through the up-regulation of p21 and p27 and viability by inducing apoptosis in Saos-2 cells.