Cell Cycle Phase-dependent Manner Research Articles

Abstract PO3-28-12: Targeting the KCa3.1 potassium channel arrests ER+ breast cancer growth and re-sensitizes to drug resistance

Abstract Standard of care for breast cancer (BC) treatment including Selective-Estrogen Receptor (ER)-modulators and/or degraders (SERM; tamoxifen, SERD;Fulvestrant) or cyclin-dependent kinases CDK4 and CDK6 (palbociclib) can be effective therapeutic strategies. However, drug toxicity and/or drug resistance severely limit these approaches and patients succumb to the disease. Therefore, there is an urge to develop new therapeutic strategies against ER+-BC. Ion channels are general pharmacological targets however, little is known about the role of this class of proteins in cancer biology. We discovered that expression of the KCa3.1 potassium ion channel is downregulated in ER+-BC. Also, patients expressing high level of KCa3.1 present a better overall survival (OS) and relapse free survival when compared with patients with low expression. Thus, these data indicate that KCa3.1 is a prognostic factor in ER+-BC and that high activity of this channel plays a fundamental role in an oncosuppressor signature. Our central hypothesis is that pharmacological targeting KCa3.1 with activator molecules arrests growth of ER+-BC. We discovered that ER+-BC patient-derived organoids (PDO) and cell lines treated with KCa3.1 agonists including the FDA approved Chlorzoxazone, (CZX) arrests significantly inhibit tumor growth. Activation of KCa3.1 arrested the cell cycle in the G0/G1 phase by activating a senescent-like phenotype. Furthermore, our studies on the biochemical signaling that underlines this event demonstrated that increased expression level of KCa3.1 protein associated with lower expression level of ERa in a cell cycle phase-dependent manner. Furthermore, use of KCa3.1activator produced a proteasomal-dependent ERa degradation. To better characterize the therapeutic benefit of targeting the KCa3.1, we assessed the effects of CZX on developed several tamoxifen and/or palbociclib resistant cell lines and PDO models. We found that the combination of CZX strongly potentiated the lethal effects of both tamoxifen and palbociclib in the relative resistant models. We conclude that pharmacological targeting KCa3.1 potassium channel in ER+-BC can be considered a novel, safe and efficacious therapeutic strategy. Citation Format: Davide Delisi, Metin Cetin, Ozge Saatci, Ozgur Sahin, Saverio Gentile. Targeting the KCa3.1 potassium channel arrests ER+ breast cancer growth and re-sensitizes to drug resistance [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-28-12.

RNA-Binding RING E3-Ligase DZIP3/hRUL138 Stabilizes Cyclin D1 to Drive Cell-Cycle and Cancer Progression.

DZIP3/hRUL138 is a poorly characterized RNA-binding RING E3-ubiquitin ligase with functions in embryonic development. Here we demonstrate that DZIP3 is a crucial driver of cancer cell growth, migration, and invasion. In mice and zebrafish cancer models, DZIP3 promoted tumor growth and metastasis. In line with these results, DZIP3 was frequently overexpressed in several cancer types. Depletion of DZIP3 from cells resulted in reduced expression of Cyclin D1 and a subsequent G1 arrest and defect in cell growth. Mechanistically, DZIP3 utilized its two different domains to interact and stabilize Cyclin D1 both at mRNA and protein levels. Using an RNA-binding lysine-rich region, DZIP3 interacted with the AU-rich region in 3' untranslated region of Cyclin D1 mRNA and stabilized it. Using a RING E3-ligase domain, DZIP3 interacted and increased K63-linked ubiquitination of Cyclin D1 protein to stabilize it. Remarkably, DZIP3 interacted with, ubiquitinated, and stabilized Cyclin D1 predominantly in the G1 phase of the cell cycle, where it is needed for cell-cycle progression. In agreement with this, a strong positive correlation of mRNA expression between DZIP3 and Cyclin D1 in different cancer types was observed. Additionally, DZIP3 regulated several cell cycle proteins by modulating the Cyclin D1-E2F axes. Taken together, this study demonstrates for the first time that DZIP3 uses a unique two-pronged mechanism in its stabilization of Cyclin D1 to drive cell-cycle and cancer progression. SIGNIFICANCE: These findings show that DZIP3 is a novel driver of cell-cycle and cancer progression via its control of Cyclin D1 mRNA and protein stability in a cell-cycle phase-dependent manner. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/2/315/F1.large.jpg.

Ubiquitin System

Ever since the discovery of ubiquitin in 1975[...].

IRES mediated translational regulation of p53 isoforms

p53 is a well known tumor suppressor protein that plays a critical role in cell cycle arrest and apoptosis. It has several isoforms which are produced by transcriptional and posttranscriptional regulatory mechanisms. p53 mRNA has been demonstrated to be translated into two isoforms, full-length p53 (FL-p53) and a truncated isoform ΔN-p53 by the use of alternative translation initiation sites. The mechanism of translation regulation of these two isoforms was further elucidated by the discovery of IRES elements in the p53 mRNA. These two IRESs were shown to regulate the translation of p53 and ΔN-p53 in a distinct cell-cycle phase-dependent manner. This review focuses on the current understanding of the regulation of p53 IRES mediated translation and the role of cis and trans acting factors that influence expression of p53 isoforms.

Annexin A2 and PSF proteins interact with p53 IRES and regulate translation of p53 mRNA

p53 mRNA has been shown to be translated into two isoforms, full-length p53 (FL-p53) and a truncated isoform ΔN-p53, which modulates the functions of FL-p53 and also has independent functions. Previously, we have shown that translation of p53 and ΔN-p53 can be initiated at Internal Ribosome Entry Sites (IRES). These two IRESs were shown to regulate the translation of p53 and ΔN-p53 in a distinct cell-cycle phase-dependent manner. Earlier observations from our laboratory also suggest that the structural integrity of the p53 RNA is critical for IRES function and is compromised by mutations that affect the structure as well as RNA protein interactions. In the current study, using RNA affinity approach we have identified Annexin A2 and PTB associated Splicing Factor (PSF/SFPQ) as novel ITAFs for p53 IRESs. We have showed that the purified Annexin A2 and PSF proteins specifically bind to p53 IRES elements. Interestingly, in the presence of calcium ions Annexin A2 showed increased binding with p53 IRES. Immunopulldown experiments suggest that these two proteins associate with p53 mRNA ex vivo as well. Partial knockdown of Annexin A2 and PSF showed decrease in p53 IRES activity and reduced levels of both the p53 isoforms. More importantly the interplay between Annexin A2, PSF and PTB proteins for binding to p53mRNA appears to play a crucial role in IRES function. Taken together, our observations suggest pivotal role of two new trans-acting factors in regulating the p53-IRES function, which in turn influences the synthesis of p53 isoforms.

Polypyrimidine tract binding protein regulates IRES-mediated translation of p53 isoforms

The p53 tumor suppressor protein plays a key role in maintaining genomic integrity. Enhanced expression of p53 during genotoxic stress is due to both increased protein stability and translational up regulation. Previous reports have shown that p53 mRNA is translated from an alternative initiation codon to produce N-terminal truncated isoform (ΔN-p53) besides full-length p53. We have demonstrated that two internal ribosome entry sites (IRESs) regulate the translation of p53 and ΔN-p53 in a distinct cell-cycle phase-dependent manner. Here, we report that polypyrimidine tract-binding protein (PTB) is a p53 IRES interacting trans-acting factor. PTB protein binds specifically to both the p53 IRESs but with differential affinity. siRNA-mediated knockdown of PTB protein results in reduction of activity of both IRESs and also the levels of both the isoforms. It is well known that DNA-damaging agents such as doxorubicin enhance the expression of p53. Our results indicate that during doxorubicin treatment, PTB protein translocates from nucleus to the cytoplasm, probably to facilitate IRES mediated p53 translation. These observations suggest that the relative cytoplasmic abundance of PTB protein, under DNA-damaging conditions, might contribute to regulating the coordinated expression of the p53 isoforms, owing to the differential affinity of PTB binding to the two p53 IRESs.

Loss of the VHR dual-specific phosphatase causescell-cycle arrest and senescence

Protein tyrosine phosphatases regulate important processes in eukaryotic cells and have critical functions in many human diseases including diabetes to cancer. Here, we report that the human Vaccinia H1-related (VHR) dual-specific protein tyrosine phosphatase regulates cell-cycle progression and is itself modulated during the cell cycle. Using RNA interference (RNAi), we demonstrate that cells lacking VHR arrest at the G1-S and G2-M transitions of the cell cycle and show the initial signs of senescence, such as flattening, spreading, appearance of autophagosomes, beta-galactosidase staining and decreased telomerase activity. In agreement with this notion, cells lacking VHR were found to upregulate p21(Cip-Waf1), whereas they downregulated the expression of genes for cell-cycle regulators, DNA replication, transcription and mRNA processing. Loss of VHR also caused a several-fold increase in serum-induced activation of its substrates, the mitogen-activated protein (MAP) kinases Jnk and Erk. VHR-induced cell-cycle arrest was dependent on this hyperactivation of Jnk and Erk, and was reversed by Jnk and Erk inhibition or knock-down. We conclude that VHR is required for cell-cycle progression as it modulates MAP kinase activation in a cell-cycle phase-dependent manner.

Heterogeneous nuclear ribonucleoprotein C modulates translation of c-myc mRNA in a cell cycle phase-dependent manner.

The c-myc proto-oncogene plays a key role in the proliferation, differentiation, apoptosis, and regulation of the cell cycle. Recently, it was demonstrated that the 5' nontranslated region (5' NTR) of human c-myc mRNA contains an internal ribosomal entry site (IRES). In this study, we investigated cellular proteins interacting with the IRES element of c-myc mRNA. Heterogeneous nuclear ribonucleoprotein C (hnRNP C) was identified as a cellular protein that interacts specifically with a heptameric U sequence in the c-myc IRES located between two alternative translation initiation codons CUG and AUG. Moreover, the addition of hnRNP C1 in an in vitro translation system enhanced translation of c-myc mRNA. Interestingly, hnRNP C was partially relocalized from the nucleus, where most of the hnRNP C resides at interphase, to the cytoplasm at the G(2)/M phase of the cell cycle. Coincidently, translation mediated through the c-myc IRES was increased at the G(2)/M phase when cap-dependent translation was partially inhibited. On the other hand, a mutant c-myc mRNA lacking the hnRNP C-binding site, showed a decreased level of translation at the G(2)/M phase compared to that of the wild-type message. Taken together, these findings suggest that hnRNP C, via IRES binding, modulates translation of c-myc mRNA in a cell cycle phase-dependent manner.

Cell-cycle modulation of CK2 activity in tobacco BY-2 cells.

Protein kinase CK2 is an ubiquitous Ser/Thr kinase essential for cell growth. We have used the highly synchronizable tobacco BY-2 cell line to investigate whether CK2 activity and expression are regulated in a cell cycle phase-dependent manner in higher plants. Specific cDNA probes for tobacco CK2alpha and beta subunits, respectively, and polyclonal antibodies recognising alpha and beta subunits separately, were obtained to determine mRNA and protein levels of both subunits. Our results show that CK2 activity oscillates throughout the cell cycle, peaking at G1/S and M phases, due to a post-translational regulation of the tetrameric enzyme. Additional levels of control of CK2 expression operate in relation to the proliferative state of the cells, including differential accumulation of alpha and beta transcripts and post-transcriptional regulation of protein levels (beta subunit). Moreover, in vivo inhibition of CK2 activity corroborates the requirement of the functional CK2 to progress through the cell division cycle, and suggests that CK2 might play an important role at the G2/M checkpoint.

Human topoisomerase II alpha is phosphorylated in a cell-cycle phase-dependent manner by a proline-directed kinase.

Topoisomerase II is essential for chromosome condensation and segregation at mitosis in eukaryotic cells, but the mechanism of its regulation is not clearly understood. We have investigated whether or not the alpha isozyme of human topoisomerase II is phosphorylated in a cell-cycle phase-dependent manner. Two-dimensional tryptic phosphopeptide mapping revealed that several sites on HeLa topoisomerase II alpha protein were phosphorylated predominantly or exclusively during the G2 and M phases. To identify the protein kinases involved in this cell-cycle phase-specific phosphorylation, oligohistidine-tagged recombinant domains of the topoisomerase II alpha protein were expressed in Escherichia coli, purified by affinity chromatography and phosphorylated in vitro by different protein kinases. Phosphorylation of the C-terminal domain of the topoisomerase II alpha protein by the universal mitotic controller, p34cdc2, generated multiple tryptic phosphopeptides, many of which corresponded to the G2/M-phase-specific phosphorylation sites observed in vivo. The same phosphopeptides were obtained following phosphorylation of the C-terminal domain in vitro by the mitogen-activated protein kinase. Site-directed mutagenesis studies identified five of these sites of phosphorylation, each of which comprised a serine-proline motif. Our data implicate one or more proline-directed kinases in the cell-cycle-dependent regulation of topoisomerase II alpha enzyme activity in human cells.

Wound-factor-induced and cell cycle phase-dependent expression of 9E3/CEF4, the avian gro gene.

The gro genes encode for secreted proteins with sequence homologies to inflammatory mediators. Little is known about the function of these proteins or their regulation. The chicken gro (9E3/CEF4) is expressed abundantly in the cells of proliferating cultures but at very low levels in confluent cultures. In vivo, this gene is expressed in connective tissue and overexpressed at sites of injury, especially in areas of neovascularization. Here we provide a bridge between these observations by examining in culture the effect on 9E3 expression and DNA synthesis induced by cell damage and by addition of factors known to be released on wounding. We mimicked wounding by scraping swaths across confluent cultures of embryonic fibroblasts and determined the time dependence of expression of 9E3 mRNA and incorporation of 3H-thymidine. We find that 9E3 is (1) transiently expressed after "wounding" or serum-stimulation; (2) expressed in a cell cycle phase-dependent manner; it is triggered during the G0-G1 transition or early in G1 and subsides during S-phase; and (3) stimulated to high levels by a-fibroblast growth factor (aFGF), bFGF, transforming growth factor alpha (TGF alpha), and TGF beta, to intermediate levels by platelet-derived growth factor and not stimulated by epidermal growth factor. We also find that cells that are constantly cycling do not express 9E3, indicating that they skip either the portion of the cell cycle where 9E3 is induced or that they constitutively express a repressor of transcription or an RNA-degrading enzyme. Taken together, these observations suggest that the product of this gene could play more than one role in vivo. For example, in normal tissues the 9E3 protein could be involved in the exit of cells from the resting stage, whereas during wound healing the secreted protein or its cleavage products also could play a role in angiogenesis.