Retinoblastoma Phosphorylation Research Articles

Stabilization of GTSE1 by cyclin D1-CDK4/6 promotes cell proliferation: relevance in cancer prognosis.

Cyclin D1 is the activating subunit of the cell cycle kinases CDK4 and CDK6, and its dysregulation is a well-known oncogenic driver in many human cancers. The biological function of cyclin D1 has been primarily studied by focusing on the phosphorylation of the retinoblastoma (RB) gene product. Here, using an integrative approach combining bioinformatic analyses and biochemical experiments, we show that GTSE1 (G2 and S phases expressed protein 1), a protein positively regulating cell cycle progression, is a previously unknown substrate of cyclin D1-CDK4/6. The phosphorylation of GTSE1 mediated by cyclin D1-CDK4/6 inhibits GTSE1 degradation, leading to high levels of GTSE1 also during the G1 phase of the cell cycle. Functionally, the phosphorylation of GTSE1 promotes cellular proliferation and is associated with poor prognosis within a pan-cancer cohort. Our findings provide insights into cyclin D1's role in cell cycle control and oncogenesis beyond RB phosphorylation.

Abstract 2849: Palbociclib as a novel therapeutic for desmoplastic small round cell tumor

Abstract Desmoplastic small round cell tumor (DSRCT) is an aggressive, pediatric cancer caused by the EWSR1-WT1 fusion oncoprotein. Targeted therapies are lacking and the current standard of care, multimodal therapy, is insufficient, leading to a 5-year survival rate of less than 25%. While the EWSR1-WT1 oncoprotein is critical to growth in vitro, its importance has yet to be examined in vivo. Further, the set of EWSR1-WT1 downstream targets critical to oncogenesis and the mechanism by which the two EWSR1-WT1 isoforms regulate them remain underinvestigated. Here we generate a toolkit of DSRCT cell lines that deplete EWSR1-WT1 in a doxycycline (dox)-inducible manner. This toolkit enabled us to establish the essentiality of EWSR1-WT1 not only in vitro but also for the first time in vivo. Using this toolkit, we performed RNA sequencing on four DSRCT cell lines and established the most comprehensive analysis of EWSR1-WT1 downstream targets to date. We discovered novel mechanistic insights into EWSR1-WT1 functionality including the uniqueness of its transcriptional alterations as compared to native WT1, the direct role of EWSR1-WT1 binding in gene upregulation, and the dominant role of the E-KTS isoform in transcription. Given the rarity of DSRCT, we focused on downstream targets with existing inhibitors and perform a drug screen that identified the CDK4/6 inhibitor palbociclib as a novel DSRCT therapeutic. We found that the E-KTS isoform of EWSR1-WT1 binds to the CCND1 promoter, leads to CCND1 expression, and stimulates DSRCT growth through the CCND-CDK4/6-RB axis. Palbociclib treatment conversely reduced DSRCT growth by preventing retinoblastoma phosphorylation and blocking the transition from G1 to S phase. Palbociclib treatment was effective not only in vitro but also in vivo where it significantly reduced tumor growth in two xenograft models of DSRCT. Given these novel findings and palbociclib’s previous approval by the FDA for the treatment of breast cancer, we advance palbociclib as an exciting DSRCT therapy that warrants urgent clinical investigation. Citation Format: Justin W. Magrath, Shruthi Sanjitha Sampath, Dane A. Flinchum, Alifiani B. Hartono, Ilon N. Goldberg, Julia R. Boehling, Suzana D. Savkovic, Sean B. Lee. Palbociclib as a novel therapeutic for desmoplastic small round cell tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2849.

UHRF2 accumulates in early G1-phase after serum stimulation or mitotic exit to extend G1 and total cell cycle length

ABSTRACT Ubiquitin like with PHD and ring finger domains 2 (UHRF2) regulates the cell cycle and epigenetics as a multi-domain protein sharing homology with UHRF1. UHRF1 functions with DNMT1 to coordinate daughter strand methylation during DNA replication, but UHRF2 can’t perform this function, and its roles during cell cycle progression are not well defined. UHRF2 role as an oncogene vs. tumor suppressor differs in distinct cell types. UHRF2 interacts with E2F1 to control Cyclin E1 (CCNE1) transcription. UHRF2 also functions in a reciprocal loop with Cyclin E/CDK2 during G1, first as a direct target of CDK2 phosphorylation, but also as an E3-ligase with direct activity toward both Cyclin E and Cyclin D. In this study, we demonstrate that UHRF2 is expressed in early G1 following either serum stimulation out of quiescence or in cells transiting directly out of M-phase, where UHRF2 protein is lost. Further, UHRF2 depletion in G2/M is reversed with a CDK1 specific inhibitor. UHRF2 controls expression levels of cyclins and CDK inhibitors and controls its own transcription in a negative-feedback loop. Deletion of UHRF2 using CRISPR/Cas9 caused a delay in passage through each cell cycle phase. UHRF2 loss culminated in elevated levels of cyclins but also the CDK inhibitor p27KIP1, which regulates G1 passage, to reduce retinoblastoma phosphorylation and increase the amount of time required to reach G1/S passage. Our data indicate that UHRF2 is a central regulator of cell-cycle pacing through its complex regulation of cell cycle gene expression and protein stability.

Inhibition of Autophagy Aggravates Arachis hypogaea L. Skin Extracts-Induced Apoptosis in Cancer Cells.

The skin of Arachis hypogaea L. (peanut or groundnut) is a rich source of polyphenols, which have been shown to exhibit a wider spectrum of noteworthy biological activities, including anticancer effects. However, the anticancer activity of peanut skin extracts against melanoma and colorectal cancer (CRC) cells remains elusive. In this study, we systematically investigated the cytotoxic, antiproliferative, pro-apoptotic, and anti-migration effects of peanut skin ethanolic extract and its fractions on melanoma and CRC cells. Cell viability results showed that the ethyl acetate fraction (AHE) of peanut skin ethanolic crude extract and one of the methanolic fractions (AHE-2) from ethyl acetate extraction exhibited the highest cytotoxicity against melanoma and CRC cells but not in nonmalignant human skin fibroblasts. AHE and AHE-2 effectively modulated the cell cycle-related proteins, including the suppression of cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 6 (CDK6), phosphorylation of Retinoblastoma (p-Rb), E2F1, Cyclin A, and activation of tumor suppressor p53, which was associated with cell cycle arrest and paralleled their antiproliferative efficacies. AHE and AHE-2 could also induce caspase-dependent apoptosis and inhibit migration activities in melanoma and CRC cells. Moreover, it is noteworthy that autophagy, manifested by microtubule-associated protein light chain 3B (LC3B) conversion and the aggregation of GFP-LC3, was detected after AHE and AHE-2 treatment and provided protective responses in cancer cells. Significantly, inhibition of autophagy enhanced AHE- and AHE-2-induced cytotoxicity and apoptosis. Together, these findings not only elucidate the anticancer potential of peanut skin extracts against melanoma and CRC cells but also provide a new insight into autophagy implicated in peanut skin extracts-induced cancer cell death.

THU543 Acquired Resistance To CDK4/6 Inhibitors Is Associated With Upregulated IGF1R And IR Signaling

Abstract Disclosure: S.L. Gerhardt: None. C. Baar: None. I. Johnson: None. D. Sachdev: None. The most common subtype of breast cancer is hormone receptor-positive (HR+). While these patients are successfully treated with endocrine therapies (ET), over 30% of patients develop resistance and require other therapies. Cyclin-dependent kinases (CDKs) 4 and 6 are required for cell cycle progression. Three CDK4/6 inhibitors (CDK4/6i), palbociclib, abemaciclib, and ribociclib, have recently been approved in combination with ET. CDK4/6i block phosphorylation of retinoblastoma (Rb) and cause G1/G0 arrest and are standard of care for treating metastatic HR+ Her2- advanced breast cancer with ET. Patients who respond to the CDK4/6i eventually develop resistance to the drugs. Thus, understanding the mechanisms of resistance to CDK4/6i and the role of growth-factor signaling will allow the identification of other pathways that can be targeted. To understand the mechanisms of resistance, we generated HR+ breast cancer cells (MCF-7) with acquired resistance to palbociclib (MCF-7/PalboR) or abemaciclib (MCF-7/AbemaR) by culturing them in increasing concentrations of the drugs. As shown by others, we found that acquired resistance to CDK4/6i results in loss of Rb and thus Rb phosphorylation. We analyzed signaling pathways and in vitro proliferation in the MCF-7/PalboR and MCF-7/AbemaR compared to the CDK4/6i sensitive matched parent cells (MCF-7L MP). MCF-7/PalboR and MCF-7/AbemaR had increased levels of CDK2 and cyclin E2. Both MCF-7/PalboR and MCF-7/AbemaR cells had upregulated type I IGF receptor (IGF1R) and insulin receptor (IR) protein levels compared MCF-7L MP as assayed by western blotting and flow cytometry. MCF-7/PalboR and MCF-7/AbemaR showed higher transcript levels of the fetal form of IR (IR-A) which is not expressed in normal adult tissues. IR-A plays a role in cancer progression and has been implicated in resistance to ET. MCF-7/PalboR and MCF-7/AbemaR had increased sensitivity to signaling by insulin-like growth factor I (IGF-I) and insulin compared to MCF-7 MP, with much lower concentrations of IGF-I and insulin phosphorylating the receptors and activating downstream MAPK and PI3K/Akt/mTOR pathways. Further, IGF-I and insulin mediated growth was enhanced to a statistically greater extent in MCF-7/PalboR and MCF-7/AbemaR compared to MCF-7 MP, and this enhanced growth was not blocked by 1200 nM of palbociclib or abemaciclib. MCF-7/PalboR cells were cross resistant to abemaciclib and MCF-7/AbemaR to palbociclib. Combined treatment with CDK4/6i and rapamycin, an mTOR pathway inhibitor, was not sufficient to overcome growth-factor mediated proliferation in MCF-7/PalboR and MCF-7/AbemaR but was in MCF-7 MP. These data indicate that acquired resistance to CDK4/6i is mediated through upregulated IGF1R and IR signaling. Our data suggest that combining CDK4/6i with drugs targeting IGF1R, IR-A, and mTOR signaling should be tested to delay resistance to CDK4/6i. Presentation: Thursday, June 15, 2023

The novel polyfluoroalkyl benzenesulfonate OBS exposure induces cell cycle arrest and senescence of rat pituitary cell GH3 via the p53/p21/RB pathway

Sodium p-perfluorous nonenoxybenzene sulfonate (OBS), an economical alternative to perfluorooctane sulfonate (PFOS) in multiple industrial fields, is widely detected in the environment. The toxicity of OBS has received increasing attention. Pituitary cells are components of the endocrine system and act as vital regulators of homeostatic endocrine balance. However, the effects of OBS on pituitary cells remain unknown. The present study explores the effects of OBS (0.5, 5, and 50 μM) on GH3 rat pituitary cells after treatment for 24, 48, and 72 h. We found that OBS significantly inhibited cell proliferation in GH3 cells with remarkable senescent phenotypes, including enhanced SA-β-gal activity and expression of senescence-associated secretory phenotype (SASP)-related genes, cell cycle arrest, and upregulation of the senescence-related proteins γ-H2A.X and Bcl-2. OBS caused significant cell cycle arrest of GH3 cells at the G1-phase and concomitantly downregulated the expression of some key proteins for the G1/S transition, including cyclin D1 and cyclin E1. Consistently, the phosphorylation of retinoblastoma (RB), which plays a central role in regulating the cell cycle, was prominently reduced after OBS exposure. Furthermore, OBS notably activated the p53-p21 signalling pathway in GH3 cells, as evidenced by increased p53 and p21 expressions, enhanced p53 phosphorylation, and augmented p53 nuclear import. To our knowledge, this study is the first to reveal that OBS triggers senescence in pituitary cells via the p53-p21-RB signalling pathway. Our study demonstrates a novel toxic effect of OBS in vitro, and provides new perspectives for understanding the potential toxicity of OBS.

Abstract 5989: Targeting RRM2 & cell cycle for breast cancer treatment

Abstract Endocrine treatment is an effective first-line therapy for targeting ER positive, HER2 negative breast cancer. But success is limited by development of acquired resistance due to long-term therapy. Cyclin D1 and cyclin dependent kinase 4/6 (CDK4/6) complex causes phosphorylation and subsequent inactivation of retinoblastoma (Rb) tumor suppressor protein which promotes progression of the cell cycle from G1 to S phase. This observation led the development of the first CDK4/6 inhibitor palbociclib (Ibrance; Pfizer) which induces cell cycle arrest at G1 phase in cancer cells. Owing to intrinsic and acquired drug resistance development, success is limited despite promising clinical outcomes. This situation necessitates the development of potential combination strategies to overcome drug resistance. Ribonucleotide reductase (RR) is a rate limiting enzyme in DNA synthesis consisting of two subunits RRM1 and RRM2. Didox inhibits ribonucleotide reductase subunit 2 (RRM2) which ultimately blocks DNA synthesis. Combination of didox with palbociclib is a potential strategy to target ER positive and ER negative/triple negative breast cancer. Previously, we have shown that didox (DDX) can significantly halt malignant breast cancer cell division in combination with the anthracycline drug doxorubicin by targeting RRM2, mutant p53 and NFkB regulatory proteins. Recently, we confirmed that didox in combination with palbociclib significantly lowers ER positive and ER negative breast cancer and their palbociclib resistant counterparts’ growth compared to no treatment or palbociclib treatment. We also confirmed that ER positive MCF7 and ER negative MDA-MB-468 parental breast cancer cells IC50 of palbociclib drug is lower than their palbociclib resistant counterparts. Here, we are reporting that didox decreases cell cycle proteins alone or in combination with palbociclib in ER positive MCF7 and ER negative MDA-MB-468 parental and palbociclib resistant breast cancer cells. This finding opens a novel approach for targeting both ER positive as well as ER negative breast cancer treatment. We are also reporting that didox treatment modulates cyclin D1 (CCND1) and RRM2 expression in MCF7 and MDA-MB-468 breast cancer along with their palbociclib resistant counterparts. Additionally, we observed that didox alone or in combination with palbociclib alters the cell cycle of MCF7 and MDA-MB-468 parental and palbociclib resistant breast cancer cells. Our data presents a novel and promising approach for the treatment of ER positive and ER negative breast cancer that involves inhibition of RRM2 and cell cycle that merits further clinical investigation in animal and human models. Citation Format: Nahid Sultana, Howard L. Elford, Jesika S. Faridi. Targeting RRM2 & cell cycle for breast cancer treatment. [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 5989.

Discovery of N,4-Di(1H-pyrazol-4-yl)pyrimidin-2-amine-Derived CDK2 Inhibitors as Potential Anticancer Agents: Design, Synthesis, and Evaluation.

Cyclin-dependent kinase 2 (CDK2) has been garnering considerable interest as a target to develop new cancer treatments and to ameliorate resistance to CDK4/6 inhibitors. However, a selective CDK2 inhibitor has yet to be clinically approved. With the desire to discover novel, potent, and selective CDK2 inhibitors, the phenylsulfonamide moiety of our previous lead compound 1 was bioisosterically replaced with pyrazole derivatives, affording a novel series of N,4-di(1H-pyrazol-4-yl)pyrimidin-2-amines that exhibited potent CDK2 inhibitory activity. Among them, 15 was the most potent CDK2 inhibitor (Ki = 0.005 µM) with a degree of selectivity over other CDKs tested. Meanwhile, this compound displayed sub-micromolar antiproliferative activity against a panel of 13 cancer cell lines (GI50 = 0.127-0.560 μM). Mechanistic studies in ovarian cancer cells revealed that 15 reduced the phosphorylation of retinoblastoma at Thr821, arrested cells at the S and G2/M phases, and induced apoptosis. These results accentuate the potential of the N,4-di(1H-pyrazol-4-yl)pyrimidin-2-amine scaffold to be developed into potent and selective CDK2 inhibitors for the treatment of cancer.

Abstract OT3-23-01: VELA: A first-in-human phase 1/2 study of BLU-222, a potent, selective cyclin-dependent kinase (CDK) 2 inhibitor in patients with cyclin E1 gene (CCNE1)-amplified or CDK4/6 inhibitor-resistant advanced solid tumors

Abstract Background The regulation of cell growth and proliferation is dependent on cyclins and CDKs. The formation of the cyclin D-CDK4/6 complex increases the expression of cyclin E1 and E2. Cyclin E1 and E2 bind to and activate CDK2; this results in a cyclin E/CDK2 complex that assists with downstream expression of DNA synthesis machinery. The use of CDK4/6 inhibitors such as palbociclib or ribociclib is an effective treatment in patients with hormone receptor-positive (HR+), human epithelial growth factor receptor-2 negative (HER2-) breast cancer; however, resistance to treatment eventually occurs. Aberrant cyclin E/CDK2 activity has been identified as a potential resistance mechanism by which tumors can evade CDK4/6 inhibitors. BLU-222 is an oral, investigational, potent, and selective CDK2 inhibitor. In preclinical studies, BLU-222 treatment in combination with ribociclib led to durable tumor regression in both CDK4/6-resistant and sensitive models of HR+HER2- breast cancer. Trial design VELA (NCT05252416) is an international, open-label, first-in-human phase 1/2 study evaluating the safety, tolerability, pharmacokinetics, pharmacodynamics, and efficacy of BLU-222 in adult patients with CCNE1-amplified tumors or with HR+HER2- breast cancer with disease progression on CDK4/6 inhibitors. In phase 1 and 2, patients aged ≥18 years with an Eastern Cooperative Oncology Group performance status 0–2 are eligible. In phase 2, all patients must have ≥1 measurable target lesion per Response Evaluation Criteria in Solid Tumors version 1.1. Primary endpoints include assessing the safety of BLU-222 as a single agent or BLU-222 in combination with either carboplatin or ribociclib and/or fulvestrant (phase 1 and 2), identifying the maximum tolerated dose and/or recommended phase 2 dose (phase 1), and determining the objective response rate (phase 2). In the phase 1 dose-escalation part, patients with any advanced solid tumor with progression on standard of care (SOC) will receive BLU-222; patients with gastric or endometrial cancer (EC) with progression on ≥2 prior therapies (including ≥1 platinum-based therapy) or with CCNE1-amplified platinum-resistant/refractory ovarian cancer (OC) will receive BLU-222 and carboplatin; patients with HR+HER- breast cancer with progression on CDK4/6 inhibitors will receive BLU-222, ribociclib, and fulvestrant. In the phase 2 dose-expansion part, patients with CCNE1-amplified tumors including EC (progression on ≥2 prior therapies), platinum-resistant/refractory OC, or other advanced solid tumors (progression after SOC) will receive BLU-222 monotherapy; patients with CCNE1-amplified platinum-resistant/refractory OC will receive BLU-222 and carboplatin; and patients with CDK4/6 inhibitor-resistant HR+HER2- breast cancer will receive BLU-222 and fulvestrant with/without ribociclib. Pharmacokinetic parameters will be calculated using standard non-compartmental methods from the plasma concentration–time data. Tissue biopsies will be collected during cycle 1 to assess the phosphorylation of retinoblastoma 1 (Rb1) protein which will be used as a pharmacodynamic marker to assess target inhibition. Dose escalation is ongoing and approximately 50 sites are anticipated to enroll patients across North America, Europe, and the Asia/Pacific region. Citation Format: Manish R Patel, Dejan Juric, Brian S Henick, Kathleen N Moore, Doreen Do, Joshua Chapman, Hui Zhang, Maria Roche, Kate J Newberry, Mikael Rinne, Timothy A Yap. VELA: A first-in-human phase 1/2 study of BLU-222, a potent, selective cyclin-dependent kinase (CDK) 2 inhibitor in patients with cyclin E1 gene (CCNE1)-amplified or CDK4/6 inhibitor-resistant advanced solid tumors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-23-01.

2-Anilino-4-(1-methyl-1H-pyrazol-4-yl)pyrimidine-derived CDK2 inhibitors as anticancer agents: Design, synthesis & evaluation

Deregulation of cyclin-dependent kinase 2 (CDK2) and its activating partners, cyclins A and E, is associated with the pathogenesis of a myriad of human cancers and with resistance to anticancer drugs including CDK4/6 inhibitors. Thus, CDK2 has become an attractive target for the development of new anticancer therapies and for the amelioration of the resistance to CDK4/6 inhibitors. Bioisosteric replacement of the thiazole moiety of CDKI-73, a clinically trialled CDK inhibitor, by a pyrazole group afforded 9 and 19 that displayed potent CDK2-cyclin E inhibition (Ki = 0.023 and 0.001 μM, respectively) with submicromolar antiproliferative activity against a panel of cancer cell lines (GI50 = 0.025–0.780 μM). Mechanistic studies on 19 with HCT-116 colorectal cancer cells revealed that the compound reduced the phosphorylation of retinoblastoma at Ser807/811, arrested the cells at the G2/M phase, and induced apoptosis. These results highlight the potential of the 2-anilino-4-(1-methyl-1H-pyrazol-4-yl)pyrimidine series in developing potent and selective CDK2 inhibitors to combat cancer.

Plakophilin 3 facilitates G1/S phase transition and enhances proliferation by capturing RB protein in the cytoplasm and promoting EGFR signaling

Plakophilin 3 (PKP3) is a component of desmosomes and is frequently overexpressed in cancer. Using keratinocytes either lacking or overexpressing PKP3, we identify a signaling axis from ERK to the retinoblastoma (RB) protein and the E2F1 transcription factor that is controlled by PKP3. RB and E2F1 are key components controlling G1/S transition in the cell cycle. We show that PKP3 stimulates the activity of ERK and its target RSK1. This inhibits expression of the transcription factor RUNX3, a positive regulator of the CDK inhibitor CDKN1A/p21, which is also downregulated by PKP3. Elevated CDKN1A prevents RB phosphorylation and E2F1 target gene expression, leading to delayed S phase entry and reduced proliferation in PKP3-depleted cells. Elevated PKP3 expression not only increases ERK activity but also captures phosphorylated RB (phospho-RB) in the cytoplasm to promote E2F1 activity and cell-cycle progression. These data identify a mechanism by which PKP3 promotes proliferation and acts as an oncogene.

Retinoblastoma protein as an intrinsic BRD4 inhibitor modulates small molecule BET inhibitor sensitivity in cancer

Bromodomain and extraterminal (BET) proteins including BRD4 play important roles in oncogenesis and immune inflammation. Here we demonstrate that cancer cells with loss of the retinoblastoma (RB) tumor suppressor became resistant to small molecule bromodomain inhibitors of BET proteins. We find that RB binds to bromodomain-1 (BD1) of BRD4, but binding is impeded by CDK4/6-mediated RB phosphorylation at serine-249/threonine-252 (S249/T252). ChIP-seq analysis shows RB knockdown increases BRD4 occupancy at genomic loci of genes enriched in cancer-related pathways including the GPCR-GNBIL-CREB axis. S249/T252-phosphorylated RB positively correlates with GNBIL protein level in prostate cancer patient samples. BET inhibitor resistance in RB-deficient cells is abolished by co-administration of CREB inhibitor. Our study identifies RB protein as a bona fide intrinsic inhibitor of BRD4 and demonstrates that RB inactivation confers resistance to small molecule BET inhibitors, thereby revealing a regulatory hub that converges RB upstream signaling onto BRD4 functions in diseases such as cancer.

PP2A-B55 and its adapter proteins IER2 and IER5 regulate the activity of RB family proteins and the expression of cell cycle-related genes.

The retinoblastoma (RB) tumour suppressor protein regulates cell proliferation, motility, differentiation and apoptosis. The phosphorylation state of RB is modulated by kinases and phosphatases, and RB exhibits phosphorylation-sensitive interactions with E2F family transcription factors. Here, we characterize RB dephosphorylation by protein phosphatase 2A (PP2A). The growth factor-inducible immediate early response (IER) proteins IER2 and IER5 possess an adapter-like function in which IER proteins bind to both PP2A and its target proteins and enhance PP2A activity towards the proteins. IER2 interacts with RB and facilitates dephosphorylation of RB at T821/T826 by PP2A. In IER2 knockdown cells, elevated phosphorylation of RB resulted in reduced binding of RB to the promoters and derepression of cyclin D1 and p21. IER5 binds to both RB and RB-like 1 (p107/RBL1), enhances dephosphorylation of these proteins by PP2A and represses the expression of various cell cycle-related genes. However, IER2-regulated dephosphorylation at T821/T826 is not necessary for the repression function of RB in cell mobility-related gene expression. Our data identify PP2A adapter proteins as critical regulators of RB family proteins and suggest that the phosphorylation status of RB differentially affects gene expression.

Farrerol exhibits inhibitory effects on lung adenocarcinoma cells by activating the mitochondrial apoptotic pathway.

Farrerol is an herbal compound extracted from rhododendron. Here, our study is to investigate biological effects of farrerol on lung adenocarcinoma (LAC) cells. Human LAC cell lines and xenograft mouse model were utilized to define the effects of farrerol on tumor growth. Our findings indicated that farrerol significantly reduced LAC cell viability as well as the colony-forming capacity. Flow cytometry analysis demonstrated that farrerol contributed to cell apoptosis and G0/G1 phase cell cycle arrest. Mechanistically, farrerol treatment upregulated proapoptotic molecules (Bak, Bid, cleaved caspase-3 and cleaved caspase-9) and senescence markers (p16 and p2), but downregulated antiapoptosis genes (Bcl-2 and Bcl-XL) and cell cycle-associated genes (CyclinD1 and CDK4); meanwhile, the phosphorylation of retinoblastoma (Rb) protein was attenuated upon pretreatment of LAC cells with farrerol in comparison to untreated control. Further studies indicated that farrerol elevated reactive oxygen species levels, activating mitochondrial apoptotic pathway and causing cell apoptosis. However, exposure to farrerol did not result in significant apoptosis in normal lung epithelial cells, suggesting a tumor-specific effect of farrerol on LAC cells. In animal model, farrerol showed a significant inhibitory effect on LAC xenograft tumor growth. And gene expressions in tumor tissues, as mentioned above, were in line with the in vitro results. Taken together, these results suggested that farrerol caused LAC cell apoptosis by activating mitochondrial apoptotic pathway, whereas farrerol treatment had no notable effect on normal lung epithelial cells. Farrerol might be an effective therapeutic drug for LAC.

Retinoblastoma-E2F Transcription Factor Interplay Is Essential for Testicular Development and Male Fertility.

The retinoblastoma (RB) protein family members (pRB, p107 and p130) are key regulators of cell cycle progression, but also play crucial roles in apoptosis, and stem cell self-renewal and differentiation. RB proteins exert their effects through binding to E2F transcription factors, which are essential developmental and physiological regulators of tissue and organ homeostasis. According to the canonical view, phosphorylation of RB results in release of E2Fs and induction of genes needed for progress of the cell cycle. However, there are eight members in the E2F transcription factor family with both activator (E2F1-3a) and repressor (E2F3b–E2F8) roles, highlighting the functional diversity of RB-E2F pathway. In this review article we summarize the data showing that RB-E2F interaction is a key cell-autonomous mechanism responsible for establishment and maintenance of lifelong male fertility. We also review the expression pattern of RB proteins and E2F transcription factors in the testis and male germ cells. The available evidence supports that RB and E2F family members are widely and dynamically expressed in the testis, and they are known to have versatile roles during spermatogenesis. Knowledge of the function and significance of RB-E2F interplay for testicular development and spermatogenesis comes primarily from gene knock-out (KO) studies. Several studies conducted in Sertoli cell-specific pRB-KO mice have demonstrated that pRB-mediated inhibition of E2F3 is essential for Sertoli cell functional maturation and cell cycle exit, highlighting that RB-E2F interaction in Sertoli cells is paramount to male fertility. Similarly, ablation of either pRB or E2F1 in the germline results in progressive testicular atrophy due to germline stem cell (GSC) depletion, emphasizing the importance of proper RB-E2F interplay for germline maintenance and lifelong sperm production. In summary, while balanced RB-E2F interplay is essential for cell-autonomous maintenance of GSCs and, the pRB-E2F3 system in Sertoli cells is critical for providing GSC niche thus laying the basis for spermatogenesis.

FGF19 Is Coamplified With CCND1 to Promote Proliferation in Lung Squamous Cell Carcinoma and Their Combined Inhibition Shows Improved Efficacy.

Lung squamous cell carcinoma (LUSC) remains as a major cause of cancer-associated mortality with few therapeutic options. Continued research on new driver genes is particularly important. FGF19, a fibroblast growth factor, is frequently observed as amplified in human LUSC, which is also associated with multiple genomic gains and losses. However, the importance of these associated changes is largely unknown. In this study, we aimed to clarify a novel mechanism that link neighboring oncogene co-amplification in the development of LUSC. We found that FGF19 was co-amplified and co-expressed with its neighboring gene CCND1 in a subset of LUSC patients and associated with poor prognosis. Moreover, FGF19 combined with CCND1 promoted the cell cycle progression of LUSC cells. Mechanistically, FGF19 also enhanced CCND1 expression by activating FGFR4-ERK1/2 signaling and strengthening CCND1-induced phosphorylation and inactivation of retinoblastoma (RB). In a murine model of lung orthotopic cancer, knockdown of CCND1 was found to prolong survival by attenuating FGF19-induced cell proliferation. Furthermore, the combination treatment of the FGFR4 inhibitor BLU9931 and the CDK4/6 inhibitor palbociclib potentiated the growth inhibition and arrested cells in G1 phase. In vivo, co-targeting FGFR4 and CDK4/6 also showed marked inhibition of tumor growth than single agent treatment. These findings further elucidate the oncogenic role of FGF19 in LUSC and provide insights into how the co-amplification of neighboring genes synergistically function to promote cancer growth, and combined inhibition against both FGF19 and CCND1 is more effective.

The pro-tumorigenic activity of p38\u03b3 overexpression in nasopharyngeal carcinoma

It is urgent to identify and validate biomarkers for early diagnosis and efficient treatment of nasopharyngeal carcinoma (NPC). Recent studies have proposed p38 gamma (p38γ) as a cyclin-dependent kinase (CDK)-like kinase that phosphorylates retinoblastoma (Rb) to promote cyclins expression and tumorigenesis. Here the Gene Expression Profiling Interactive Analysis (GEPIA) database and results from the local NPC tissues demonstrate that p38γ is significantly upregulated in NPC tissues, correlating with poor overall survival. Furthermore, p38γ mRNA and protein expression is elevated in established NPC cell lines (CNE-1 HONE-1 and CNE-2) and primary human NPC cells, but low expression detected in human nasal epithelial cells. In established and primary NPC cells, p38γ depletion, using the shRNA strategy or the CRISPR/Cas9 gene-editing method, largely inhibited cell growth, proliferation and migration, and induced significant apoptosis activation. Contrarily, ectopic p38γ overexpression exerted opposite activity and promoted NPC cell proliferation and migration. Retinoblastoma (Rb) phosphorylation and cyclin E1/A expression were decreased in NPC cells with p38γ silencing or knockout, but increased after p38γ overexpression. Moreover, mitochondrial subcellular p38γ localization was detected in NPC cells. Significantly, p38γ depletion disrupted mitochondrial functions, causing mitochondrial depolarization, reactive oxygen species production, oxidative injury and ATP depletion in NPC cells. In vivo, intratumoral injection of adeno-associated virus-packed p38γ shRNA potently inhibited primary human NPC xenograft growth in nude mice. In p38γ shRNA virus-injected NPC xenograft tissues, p38γ expression, Rb phosphorylation, cyclin E1/A expression and ATP levels were dramatically decreased. Taken together, we conclude that p38γ overexpression is required for NPC cell growth, acting as a promising therapeutic target of NPC.

P16ink4a overexpression ameliorates cardiac remodeling of mouse following myocardial infarction via CDK4/pRb pathway

BackgroundP16ink4a can accumulate in senescent cells and can be induced by different oncogenic stimulations. These functions make p16ink4a a biomarker of senescence and cancer. However, the exact role of p16ink4a remains unclear in cardiovascular disease. This study was aimed to investigate the role of p16ink4a in cardiac remodeling after myocardial infarction (MI). MethodsIn vivo, gain and loss of function experiments using p16ink4a overexpression and knockdown adenovirus were induced to determine the effect of p16ink4a on cardiac structure and function after MI. The in vitro effects of p16ink4a were evaluated by overexpression and knockdown adenovirus of p16ink4a on isolated neonatal mouse cardiac myocytes (NMCMs) and neonatal mouse cardiac fibroblasts (NMCFs). ResultsExpression level of p16ink4a was increased after MI and enriched in the infarction area. In vivo, overexpression of p16ink4a protected, while knockdown of p16ink4a worsened cardiac function. In vitro, p16ink4a did not influence the hypertrophy of NMCMs. Overexpression of p16ink4a inhibited the proliferation and migration of NMCFs and reduced the level of collagen I and α-SMA. Consistently, knockdown of p16ink4a in vitro displayed the opposite effects. Further mechanism studies revealed that p16ink4a affected the expression level of cyclin-dependent kinase 4 (CDK4) and phosphorylation of retinoblastoma (pRb), which could be a potential pathway in regulating cardiac remodeling after MI. ConclusionOverexpression of 16ink4a in cardiac fibroblasts can ameliorate cardiac dysfunction and attenuate pathological cardiac remodeling in mice after MI by regulating the p16ink4a/CDK4/pRb pathway.

Tumor suppressor p53 promotes ferroptosis in oxidative stress conditions independent of modulation of ferroptosis by p21, CDKs, RB, and E2F

p53 is a well-established critical cell cycle regulator. By inducing transcription of the gene encoding p21, p53 inhibits cyclin-dependent kinase (CDK)-mediated phosphorylation of cell cycle inhibitor retinoblastoma (RB) proteins. Phosphorylation of RB releases E2F transcription factor proteins that transactivate cell cycle-promoting genes. Here, we sought to uncover the contribution of p53, p21, CDK, RB, and E2F to the regulation of ferroptosis, an oxidative form of cell death. Our studies have uncovered unexpected complexity in this regulation. First, we showed that elevated levels of p53 enhance ferroptosis in multiple inducible and isogenic systems. On the other hand, we found that p21 suppresses ferroptosis. Elevation of CDK activity also suppressed ferroptosis under conditions where p21 suppressed ferroptosis, suggesting that the impact of p21 must extend beyond CDK inhibition. Furthermore, we showed that overexpression of E2F suppresses ferroptosis in part via a p21-dependent mechanism, consistent with reports that this transcription factor can induce transcription of p21. Finally, deletion of RB genes enhanced ferroptosis. Taken together, these results show that signals affecting ferroptotic sensitivity emanate from multiple points within the p53 tumor suppressor pathway.

In Silico and Cellular Differences Related to the Cell Division Process between the A and B Races of the Colonial Microalga Botryococcus braunii.

Botryococcus braunii produce liquid hydrocarbons able to be processed into combustion engine fuels. Depending on the growing conditions, the cell doubling time can be up to 6 days or more, which is a slow growth rate in comparison with other microalgae. Few studies have analyzed the cell cycle of B. braunii. We did a bioinformatic comparison between the protein sequences for retinoblastoma and cyclin-dependent kinases from the A (Yamanaka) and B (Showa) races, with those sequences from other algae and Arabidopsis thaliana. Differences in the number of cyclin-dependent kinases and potential retinoblastoma phosphorylation sites between the A and B races were found. Some cyclin-dependent kinases from both races seemed to be phylogenetically more similar to A. thaliana than to other microalgae. Microscopic observations were done using several staining procedures. Race A colonies, but not race B, showed some multinucleated cells without chlorophyll. An active mitochondrial net was detected in those multinucleated cells, as well as being defined in polyphosphate bodies. These observations suggest differences in the cell division processes between the A and B races of B. braunii.