Overexpression of cyclin F/CCNF as an independent prognostic factor for poor survival in clear cell renal cell carcinoma.

Cyclin F (encoded by CCNF gene) has been reported to be implicated in the pathobiology of several human cancers. However, its potential clinical significance in clear cell renal cell carcinoma (ccRCC) remains unknown. The present study aimed to evaluate the potential significance of cyclin F, assessed by immunohistochemical (IHC) staining and molecular (bioinformatics) techniques, as a prognostic marker in ccRCC in relation to clinicopathological features and outcomes. IHC staining was performed using two independent ccRCC tissue array cohorts, herein called tissue macroarray (TMA)_1 and tissue microarray (TMA)_2, composed of 108 ccRCCs and 37 histologically normal tissues adjacent to the tumor (NAT) and 192 ccRCCs and 16 normal kidney samples, respectively. The mRNA expression data were obtained from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) public datasets, followed by bioinformatics analysis of biological mechanisms underlying prognosis. The relationship between immune cell infiltration level and CCNF expression in ccRCC was investigated using the Tumor Immune Estimation Resource 2.0 (TIMER2) and Gene Expression Profiling Interactive Analysis 2 (GEPIA2). Cyclin F expression was significantly elevated in ccRCC lesions compared to both NAT and normal renal tissues. Likewise, CCNF mRNA was markedly increased in ccRCCs relative to non-cancerous tissues. In all analyzed cohorts, tumors with features of more aggressive behavior were more likely to display cyclin F/CCNF-high expression than low. Furthermore, patients with high cyclin F/CCNF expression had shorter overall survival (OS) times than those with low expression. In addition, multivariable analysis revealed that cyclin F/CCNF-high expression was an independent prognostic factor for poor OS in ccRCC. Enrichment analysis for mechanistically relevant processes showed that CCNF and its highly correlated genes initiate the signaling pathways that eventually result in uncontrolled cell proliferation. CCNF expression was also correlated with immune cell infiltration and caused poor outcomes depending on the abundance of tumor-infiltrating immune cells in ccRCC. Our findings suggest that cyclin F/CCNF expression is likely to have an essential role in ccRCC pathobiology through regulating multiple oncogenic signaling pathways and affecting the tumor immune microenvironment and may serve as prognostic biomarker and promising therapeutic target in ccRCC.

Fascin in migration and metastasis of breast cancer cells - A review.

Cancer cell migration and metastasis are the biggest problems in the treatment of cancer patients. The most aggressive breast cancer (BC) is the triple-negative type. Therefore, effective therapeutic targets that limit cell migration are sought. One such target may be fascin, as its overexpression is characteristic to triple-negative breast cancer. The high level of fascin enables the formation of protrusion and thus promotes the invasion of cancer cells. Fascin also shows co-localization or functional relationships with other proteins. These are proteins involved in the epithelial-mesenchymal transition process, vimentin, cadherins, ß-catenin, and matrix metalloproteinases 2/9 (MMP-2/9). Fascin is also involved in many signaling pathways protein kinase C-δ (PKCδ), Wnt/ß-catenin, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and phosphatidylinositol 3-kinase (PI3K)-Akt. Therefore, in this article, we review currently available in vitro studies and compare them with The Cancer Genome Atlas (TCGA) data analysis of BC patients to demonstrate the role of fascin in the migration and invasion of cancer cells.

The role of cyclin Y in normal and pathological cells.

The family protein of cyclins, as well as cyclin-dependent kinases (CDKs) cooperating with them, are broadly researched, as a matter of their dysfunction may lead to tumor transformation. Cyclins are defined as key regulators that have a controlling function of the mammalian nuclear cell divides. Cyclin Y (CCNY) is a recently characterized member of the cyclin family and was first identified from the human testis cDNA library. It is an actin-binding protein acting through decreased actin dynamics at a steady state and during glycine-induced long-term potentiation (LTP) and involves the inhibition of cofilin activation. What is more, CCNY is a positive regulatory subunit of the CDK14/PFTK1 complexes affected by the activation of the Wnt signaling pathway in the G2/M phase by recruiting CDK14/PFTK1 to the plasma membrane and promoting phosphorylation of LRP6. The expression of CCNY has been significantly mentioned within the cell migration and invasion activity both in vivo and in vitro. The aim of this review is evaluation of the expression of CCNY in the physiology processes and compare the expression of this protein in cancer cells, taking into account the impact of the level of expression on tumor progression.

Downregulation of MMP-9 Enhances the Anti-Migratory Effect of Cyclophosphamide in MDA-MB-231 and MCF-7 Breast Cancer Cell Lines.

Metastasis is one of the most urgent issues in breast cancer patients. One of the factors necessary in the migration process is the remodeling of the extracellular matrix (ECM). Metalloproteinases (MMPs) can break down the elements of the ECM, which facilitates cell movement. Many highly aggressive tumors are characterized by high levels of MMPs. In the case of breast cancer, the association between MMP-9 and the migration potential and invasiveness of cells has been demonstrated. In addition, reports indicating increased migration of breast cancer cells after the administration of the commonly used cytostatic cyclophosphamide (CP) are particularly disturbing. Hence, our research aimed to assess the effect of CP treatment on MDA-MB-231 and MCF-7 cells and how this response is influenced by the downregulation of the MMP-9 level. The obtained results suggest that CP causes a decrease in the survival of breast cancer cells of various invasiveness, and the downregulation of MMP-9 enhances this effect, mainly by inducing apoptosis. Moreover, in the group of MMP-9 siRNA-transfected CP-treated cells, a more severe reduction in invasion and migration of cells of both lines was observed, as indicated by the migration and invasion transwell assays and Wound healing assay. Hence, we suggest that CP alone may not result in satisfactory therapeutic effects. On the other hand, the use of combination therapy targeting MMP-9, together with the CP, could improve the effectiveness of the treatment. Additionally, we confirmed a relationship between the levels of MMP-9 and cytokeratin 19 (CK19).

CRISPR-Based Activation of Endogenous Expression of TPM1 Inhibits Inflammatory Response of Primary Human Coronary Artery Endothelial and Smooth Muscle Cells Induced by Recombinant Human Tumor Necrosis Factor α.

Tumor necrosis factor α (TNFα) is one of the most important proinflammatory cytokines, which affects many processes associated with the growth and characteristics of endothelial, smooth muscle, and immune system cells. However, there is no correlation between most in vivo and in vitro studies on its role in endothelial cell proliferation and migration. In this study, we examined the effect of recombinant human (rh) TNFα produced in HEK293 cells on primary human coronary artery endothelial cells (pHCAECs) in the context of F-actin organization and such processes as migration and adhesion. Furthermore, we evaluated the possibility of the inhibition of the endothelial inflammatory response by the CRISPR-based regulation of TPM1 gene expression. We showed that TNFα-induced activation of pHCAECs was related to the reorganization of the actin cytoskeleton into parallel-arranged stress fibers running along the longer axis of pHCAECs. It allowed for the directed and parallel motion of the cells during coordinated migration. This change in F-actin organization promoted strong but discontinuous cell-cell contacts involved in signalization between migrating cells. Moreover, this form of intercellular connections together with locally increased adhesion was related to the formation of migrasomes and further migracytosis. Stabilization of the actin cytoskeleton through the CRISPR-based activation of endogenous expression of TPM1 resulted in the inhibition of the inflammatory response of pHCAECs following treatment with rh TNFα and stabilization of cell-cell junctions through reduced cleavage of vascular endothelial cadherin (VE-cadherin) and maintenance of the stable levels of α- and ß-catenins. We also showed that CRISPR-based activation of TPM1 reduced inflammatory activation, proliferation, and migration of primary human coronary artery smooth muscle cells. Therefore, products of the TPM1 gene may be a potential therapeutic target for the treatment of proinflammatory vascular disorders.

Cyclin F Downregulation Affects Epithelial-Mesenchymal Transition Increasing Proliferation and Migration of the A-375 Melanoma Cell Line.

BACKGROUND: Cyclins are well-known cell cycle regulators. The activation of cyclin-dependent kinases by cyclins allows orchestration of the complicated cell cycle machinery and drives the cell from the G1 phase to the end of the mitotic phase. In recent years, it has become evident that cyclins are involved in processes beyond the cell cycle. Cyclin F does not activate CDKs but forms part of the Skp1-Cul1-F-box (SCF) complex where it is responsible for protein target recognition and subsequent degradation in a proteasome-dependent manner. RESULTS: Here, we report that the downregulation of cyclin F in the A-375 melanoma cell line increases cell viability and colony formation in a cell cycle independent manner. Lower levels of cyclin F do not appear to affect the cell cycle, based on flow cytometry measuring BrdU incorporation and propidium iodide staining. By means of immunofluorescence staining and Western blot analysis, we observed changes in cell morphology-related markers which suggested ongoing epithelial-mesenchymal transition (EMT) in response to cyclin F downregulation. Increases in vimentin and N-cadherin protein levels, decreases in levels of epithelial markers such as ZO-1, along with changes in morphology to a spindle-like shape with the appearance of actin stress fibers, are all hallmarks of EMT. These changes are associated with increased invasive and migratory potential, based on 2D migration assays. Moreover, we observe an increase in RhoABC, talin and paxillin levels, the proteins involved in controlling cell signaling and motility. Lastly, upon knocking down cyclin F expression, we observed a decrease in thrombospondin-1 expression, suggesting a role of cyclin F in angiogenesis. CONCLUSION: Cyclin F depletion induces proliferation and EMT processes in the A-375 melanoma model.

Cyclin F is involved in response to cisplatin treatment in melanoma cell lines.

Cyclin F is a non­canonical cyclin which is a part of the SKP1­CUL1­F­box protein (SCF) E3 ubiquitin­protein ligase complex. Cyclin F is responsible for target recognition, ubiquitination, and degradation of various molecular targets. This protein also controls genome stability through the degradation of ribonucleotide reductase subunit M2 (RRM2). In the present study, the difference between cyclin F expression in cell lines derived from primary and metastatic melanoma, A375 and RPMI­7951, respectively, were investigated using a western blot analysis and flow cytometry assays. A decrease in cyclin F expression in the A375 cells and an increase in RPMI­7951 cells after cisplatin treatment were observed. These changes may be related to a mutation in p53 in the RPMI­7951 cell line. Flow cytometry was conducted to observe that the RPMI­7951 cell line exhibited greater susceptibility to cisplatin, associated with lack of proper cell cycle control. Therefore, it is possible that cyclin F may modulate drug response in melanoma. The presented data describe cyclin F as a new potential factor that contributes to drug resistance in melanoma patients.

Expression of cyclin B1, D1 and K in non­small cell lung cancer H1299 cells following treatment with sulforaphane.

Sulforaphane (SFN) was first isolated from broccoli sprout and it is present at high concentrations in plants belonging to the Cruciferae family. The chemotherapeutic and anti­cancerogenic capacities of SFN have been demonstrated by inhibition of cancer cell proliferation in several cancer cell lines. The aim of the present study was to evaluate the effect of SFN on apoptosis, cell cycle and expression of selected cell cycle­associated proteins: Cyclin B1, cyclin D1 and cyclin K in the H1299 cell line. The non­small cell lung cancer cell line H1299 was treated with increasing concentrations of SFN (5, 10 and 15 µM) for two days. After incubation, the percentage of cells in the individual cell cycle phases, as well as the percentage of necrotic and apoptotic cells, were estimated using flow cytometry. The expression of cyclins was examined by immunofluorescence staining, flow cytometry, western blot analysis and qRT­PCR. Cyclin K was characterized by nuclear localization and increased expression after treatment with SFN. The expression data were confirmed by qRT­PCR. SFN­induced cell cycle arrest was associated with a decrease in cyclin B1 expression. Cells treated with SFN were also characterized by higher cyclin D1 and cyclin K expression. These data suggest the involvement of cyclin K in response to SFN. Moreover, we investigated the prognostic value of cyclin K, CDK12 and CDK13 in adenocarcinoma patients using 'The Kaplan­Meier plotter' (KM plotter) database. It was shown that high expression of CDK12 and CDK13 but no cyclin K proteins is associated with worse overall survival among adenocarcinoma patients.

Potential role of cyclin F mRNA expression in the survival of skin melanoma patients: Comprehensive analysis of the pathways altered due to cyclin F upregulation.

Cyclin F is a part of the Skp, Cullin, F-box containing ligase complex. The activity of cyclin F includes cell cycle control, centrosome duplication and response to DNA damage. The cyclin F expression pattern is very similar to cyclin A, but cyclin F is an orphan cyclin without its cyclin-dependent kinase partner. There is little evidence concerning the role of cyclin F in cancer. In the present study, for the first time, we present analysis from The Cancer Genome Atlas (TCGA) data in the context of expression of cyclin F mRNA in melanoma patients. Our original in silico analysis, not published elsewhere before, revealed that high expression of cyclin F in melanoma patients is associated with worse overall survival. Cyclin F and ribonucleotide reductase family member 2 (RRM2) compose a functional axis responsible for nucleotide metabolism. Impairment in this pathway may contribute to increased DNA damage repair and drug resistance. Additionally, we analyzed the expression of RRM2 mRNA and discovered that high expression of RRM2 is associated with worse overall survival. To shed more light on cyclin F overexpression in melanoma, we analyzed all protein data available in the TCGA melanoma dataset. It was found that in patients with upregulated cyclin F mRNA, we noted increased activity of pathways related to cell cycle and DNA damage repair. These data will support further in vitro and in vivo studies on the involvement of cyclin F in skin cutaneous melanoma.

Combretastatins: In vitro structure-activity relationship, mode of action and current clinical status.

For the first time combretastatins were isolated from African willow tree Combretum Caffrum. Subsequent studies have shown the impact of combretastatin A4 phosphate, a water-soluble prodrug, on endothelial cells in tumor vascular system. The same effect was not observed in the vascular system. This selectivity is associated with combretastatins mechanism of action: binding to colchicine domain of microtubules, which affects the cytoskeleton functionality of immature endothelial cells. At the same time, combretastatins directly induce cell death via apoptosis and/or mitotic catastrophe pathways. The combination of both elements makes combretastatin an anticancer compound of high efficiency. The cis-configuration is crucial for its biological activity. To date, many derivatives were synthesized. The attempts to resolve spontaneous isomerization to less active trans-stilbene derivative are still in progress. This issue seems to be overcome by incorporation of the ethene bridge with heterocyclic moiety in combretastatins structure. This modification retains the cis-configuration and prevents isomerization. Nevertheless, combretastatin A4 phosphate disodium is still the most potent compound of this group. The combination therapy, which is the most effective treatment, includes combretastatin A4 phosphate (CA4P) and conventional chemotherapeutics and/or radiotherapy. CA4P is relatively well tolerated giving adverse events of moderate severity, which includes: nausea, vomiting, headache, and tumor pain. The aforementioned effects subside on the day of drug administration or on the following day.

Activity of cyclin B1 in HL-60 cells treated with etoposide.

Cyclin B1 triggers G2/M phase transition phosphorylating with its catalytical partner - Cdc2 many of the molecular targets essential for cell cycle progression. Human leukemia cell line HL-60 were treated with increasing doses of etoposide (ETP) (0.5; 0.75; 1µM) to investigate how the drug affects cell morphology, viability, cell cycle distribution and expression of cyclin B1. To achieve this aim we applied light and transmission electron microscopy to observe morphological and ultra structural changes, image-based cytometry for apoptosis evaluation and cell cycle analysis, and then we conducted immunohistochemical and immunofluorescence staining to visualize cyclin localization and expression. Quantitive data about cyclin B1 expression were obtained from flow cytometry. Etoposide caused decrease in cell viability, induced apoptosis and G2/M arrest accompanied by enhanced expression of cyclin B1. Changes in expression and localization of cyclin B1 may constitute a part of the mechanism responsible for resistance of HL-60 cells to etoposide. Our results may reflect involvement of cyclin B1 in opposite processes - apoptosis induction and maintenance of cell viability in leukemia cells. We hypothesized possible roles and pathways by which cyclin B1 takes part in drug treatment response and chemosensitivity.

The effect of sulforaphane on the cell cycle, apoptosis and expression of cyclin D1 and p21 in the A549 non-small cell lung cancer cell line.

Sulforaphane (SFN) is present in plants belonging to Cruciferae family and was first isolated from broccoli sprouts. Chemotherapeutic and anticarcinogenic properties of sulforaphane were demonstrated, however, the underlying mechanisms are not fully understood. In this study we evaluated the expression of cyclin D1 and p21 protein in SFN-treated A549 cells and correlated these results with the extent of cell death and/or cell cycle alterations, as well as determined a potential contribution of cyclin D1 to cell death. A549 cells were treated with increasing concentrations of SFN (30, 60 and 90 µM) for 24 h. Morphological and ultrastructural changes were observed using light, transmission electron microscope and videomicroscopy. Image-based cytometry was applied to evaluate the effect of SFN on apoptosis and the cell cycle. Cyclin D1 and p21 expression was determined by flow cytometry, RT-qPCR and immunofluorescence. siRNA was used to evaluate the role of cyclin D1 in the process of suforaphane-induced cell death. We found that the percentage of cyclin D1-positive cells decreased after the treatment with SFN, but at the same time mean fluorescence intensity reflecting cyclin D1 content was increased at 30 µM SFN and decreased at 60 and 90 µM SFN. Percentage of p21-positive cells increased following the treatment, with the highest increase at 60 µM SFN, at which concentration mean fluorescence intensity of this protein was also significantly increased. The 30-µM dose of SFN induced an increased G2/M phase population along with a decreased polyploid fraction of cells, which implies a functional G2/M arrest. The major mode of cell death induced by SFN was necrosis and, to a lower degree apoptosis. Transfection with cyclin D1-siRNA resulted in significantly compromised fraction of apoptotic and necrotic cells, which suggests that cyclin D1 is an important determinant of the therapeutic efficiency of SFN in the A549 cells.

Expression of cyclin A in A549 cell line after treatment with arsenic trioxide.

BACKGROUND: Arsenic trioxide (ATO) is an effective drug used in acute promyelocytic leukemia (AML). Many reports suggest that ATO can also be applied as an anticancer agent for solid tumors in the future. The influence of arsenic trioxide on the expression of different cell cycle regulators is poorly recognized. The purpose of the current study is to investigate how arsenic trioxide affects cyclin A expression and localization in the A549 cell line. MATERIALS AND METHODS: Morphological and ultrastructural changes in A549 cells were observed using light and transmission electron microscopes. Cyclin A localization was determined by immunofluorescence. Image-based cytometry was applied to evaluate the effect of arsenic trioxide on apoptosis and the cell cycle. Expression of cyclin A mRNA was quantified by real-time PCR. RESULTS: After treatment with arsenic trioxide, increased numbers of cells with cytoplasmic localization of cyclin A were observed. The doses of 10 and 15 µM ATO slightly reduced expression of cyclin A mRNA. The apoptotic phenotype of cells was poorly represented, and the Tali imagebased cytometry analysis showed low percentages of apoptotic cells. The A549 population displayed an enriched fraction of cells in G0/G1 phase in the presence of 5µM ATO, whereas starting from the higher concentrations of the drug, i.e. 10 and 15 µM ATO, the G2/M fraction was on the increase. DISCUSSION: Low expression of cyclin A in the A549 cell line may constitute a potential factor determining arsenic trioxide resistance. It could be hypothesized that the observed alterations in cyclin A expression/distribution may correlate well with changes in cell cycle regulation in our model, which in turn determines the outcome of the treatment.