PTEN-mediated dephosphorylation of 53BP1 confers cellular resistance to DNA damage in cancer cells.

Homologous recombination (HR) repair for DNA double-strand breaks (DSBs) is critical for maintaining genome stability and conferring the resistance of tumor cells to chemotherapy. Nuclear PTEN which contains both phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and protein phosphatase plays a key role in HR repair, but the underlying mechanism remains largely elusive. We find that SUMOylated PTEN promotes HR repair but represses nonhomologous end joining (NHEJ) repair by directly dephosphorylating TP53-binding protein 1 (53BP1). During DNA damage responses (DDR), tumor suppressor ARF (p14ARF) was phosphorylated and then interacted efficiently with PTEN, thus promoting PTEN SUMOylation as an atypical SUMO E3 ligase. Interestingly, SUMOylated PTEN was subsequently recruited to the chromatin at DSB sites. This was because SUMO1 that was conjugated to PTEN was recognized and bound by the SUMO-interacting motif (SIM) of breast cancer type 1 susceptibility protein (BRCA1), which has been located to the core of 53BP1 foci on chromatin during S/G2 stage. Furthermore, these chromatin-loaded PTEN directly and specifically dephosphorylated phosphothreonine-543 (pT543) of 53BP1, resulting in the dissociation of the 53BP1 complex, which facilitated DNA end resection and ongoing HR repair. SUMOylation-site-mutated PTENK254R mice also showed decreased DNA damage repair in vivo. Blocking the PTEN SUMOylation pathway with either a SUMOylation inhibitor or a p14ARF(2-13) peptide sensitized tumor cells to chemotherapy. Our study therefore provides a new mechanistic understanding of PTEN in HR repair and clinical intervention of chemoresistant tumors.

MDM2-Targeting Reassembly Peptide (TRAP) Nanoparticles for p53-Based Cancer Therapy.

Gene mutations and functional inhibition are the major obstacles for p53-mediated oncotherapy. For p53-wild-type tumors, the underlying mechanisms of functional inhibition of p53 during oncogenesis are unknown. The results reveal that the expression of the MDM2 inhibitor ARF is inhibited in p53-wild-type tumors, indicating that the restoration of ARF could be a potential oncotherapy strategy for p53-wild-type tumors. Therefore, ARF-mimetic MDM2-targeting reassembly peptide nanoparticles (MtrapNPs) for p53-based tumor therapy is developed. The results elucidated that the MtrapNPs respond to and form a nanofiber structure with MDM2. By trapping MDM2, the MtrapNPs stabilize and activate p53 for the inhibition of p53-wild-type tumors. In most cases, reactivated mutant p53 is inhibited and degraded by MDM2. In the present study, MtrapNPs are used to load and deliver arsenic trioxide, a p53 mutation rescuer, for p53-mutated tumor treatment in both orthotopic and metastatic models, and they exhibit significant therapeutic effects. Therefore, the study provides evidence supporting a link between decreased ARF expression and tumor development in patients with p53-wild-type tumors. Thus, the MDM2-trap strategy, which addresses both the inhibition and mutations of p53, is an efficient strategy for the treatment of p53-mutated tumors.

MLL3 regulates the CDKN2A tumor suppressor locus in liver cancer.

Mutations in genes encoding components of chromatin modifying and remodeling complexes are among the most frequently observed somatic events in human cancers. For example, missense and nonsense mutations targeting the mixed lineage leukemia family member 3 (MLL3, encoded by KMT2C) histone methyltransferase occur in a range of solid tumors, and heterozygous deletions encompassing KMT2C occur in a subset of aggressive leukemias. Although MLL3 loss can promote tumorigenesis in mice, the molecular targets and biological processes by which MLL3 suppresses tumorigenesis remain poorly characterized. Here, we combined genetic, epigenomic, and animal modeling approaches to demonstrate that one of the mechanisms by which MLL3 links chromatin remodeling to tumor suppression is by co-activating the Cdkn2a tumor suppressor locus. Disruption of Kmt2c cooperates with Myc overexpression in the development of murine hepatocellular carcinoma (HCC), in which MLL3 binding to the Cdkn2a locus is blunted, resulting in reduced H3K4 methylation and low expression levels of the locus-encoded tumor suppressors p16/Ink4a and p19/Arf. Conversely, elevated KMT2C expression increases its binding to the CDKN2A locus and co-activates gene transcription. Endogenous Kmt2c restoration reverses these chromatin and transcriptional effects and triggers Ink4a/Arf-dependent apoptosis. Underscoring the human relevance of this epistasis, we found that genomic alterations in KMT2C and CDKN2A were associated with similar transcriptional profiles in human HCC samples. These results collectively point to a new mechanism for disrupting CDKN2A activity during cancer development and, in doing so, link MLL3 to an established tumor suppressor network.

Immortalized murine tenocyte cells: a novel and innovative tool for tendon research.

Primary tenocytes rapidly undergo senescence and a phenotypic drift upon in vitro monolayer culture, which limits tendon research. The Ink4a/Arf locus encodes the proteins p16Ink4a/Arf and p14ARF (p19ARF in mice) that regulate cell cycle progression and senescence. We here established an immortalized cell line using tenocytes isolated from Ink4a/Arf deficient mice (Ink4a/Arf-/-). These cells were investigated at three distinct time points, at low (2-5), intermediate (14-17) and high (35-44) passages. Wild-type cells at low passage (2-5) served as controls. Ink4a/Arf-/- tenocytes at all stages were comparable to wild-type cells regarding morphology, expression of tenogeneic genes (collagen type 1, 3 and 5, Scleraxis, Tenomodulin and Tenascin-C), and surface markers (CD29, CD44 and CD105) and form 3D tendon-like structures. Importantly, Ink4a/Arf-/- tenocytes maintained their phenotypic features and proliferation potential in culture for more than 40 passages and also following freeze-thaw cycles. In contrast, wild-type tenocytes underwent senescence starting in passage 6. These data define Ink4a/Arf-/- tenocytes as novel tool for in vitro tendon research and as valuable in vitro alternative to animal experiments.

Computational screening and analysis of deleterious nsSNPs in human p14ARF (CDKN2A gene) protein using molecular dynamic simulation approach.

Cyclin-dependent kinase inhibitor 2 A (CDKN2A) gene belongs to the cyclin-dependent kinase family that code for two transcripts (p16INK4A and p14ARF), both work as tumor suppressors proteins. The mutation that occurs in the p14ARF protein can lead to different types of cancers. Single nucleotide polymorphisms (SNPs) are an important type of genetic alteration that can lead to different types of diseases. In this study, we applied the computational strategy on human p14ARF protein to identify the potential deleterious nsSNPs and check their impact on the structure, function, and protein stability. We applied more than ten prediction tools to screen the retrieved 288 nsSNPs, consequently extracting four deleterious nsSNPs i.e., rs139725688 (R10G), rs139725688 (R21W), rs374360796 (F23L) and rs747717236 (L124R). Homology modeling, conservation and conformational analysis of mutant models were performed to examine the divergence of these variants from the native p14ARF structure. All-atom molecular dynamics simulation revealed a significant impact of these mutations on protein stability, compactness, globularity, solvent accessibility and secondary structure elements. Protein-protein interactions indicated that p14ARF operates as a hub linking clusters of different proteins and that changes in p14ARF may result in the disassociation of numerous signal cascades. Our current study is the first survey of computational analysis on p14ARF protein that determines the association of these nsSNPs with the altered function of p14ARF protein and leads to the development of various types of cancers. This research proposes the described functional SNPs as possible targets for proteomic investigations, diagnostic procedures, and treatments.Communicated by Ramaswamy H. Sarma.

Induction of Immune-Stimulating Factors and Oncolysis Upon p14ARF Gene Transfer in Melanoma Cell Lines.

Together with an anti-tumor immune response, oncolysis using a recombinant viral vector promises to eliminate cancer cells by both gene transfer and host-mediated functions. In this study we explore oncolysis induced by nonreplicating adenoviral vectors used for p14ARF and interferon-ß (hIFNß) gene transfer in human melanoma cell lines, revealing an unexpected role for p14ARF in promoting cellular responses predictive of immune stimulation. Oncolysis was confirmed when UACC-62 (p53 wild-type) cells succumbed upon p14ARF gene transfer in vitro, whereas SK-Mel-29 (p53-mutant) benefitted from its combination with hIFNß. In the case of UACC-62, in situ gene therapy in nude mice yielded reduced tumor progression in response to the p14ARF and hIFNß combination. Potential for immune stimulation was revealed where p14ARF gene transfer in vitro was sufficient to induce emission of immunogenic cell death factors in UACC-62 and upregulate pro-immune genes, including IRF1, IRF7, IRF9, ISG15, TAP-1, and B2M. In SK-Mel-29, p14ARF gene transfer induced a subset of these factors. hIFNß was, as expected, sufficient to induce these immune-stimulating genes in both cell lines. This work is a significant advancement for our melanoma gene therapy strategy because we revealed not only the induction of oncolysis, but also the potential contribution of p14ARF to immune stimulation.

Impact of Transcript (p16/p14ARF) Alteration on Cancer Risk in CDKN2A Germline Pathogenic Variant Carriers.

BACKGROUND: Few studies have evaluated the relationship between CDKN2A germline pathogenic variants (GPV), transcript (p16/p14ARF) alteration, and cancer risk. METHODS: Standardized incidence ratios (SIRs) comparing cancer risk with the general population were calculated for 385 CDKN2A GPV carriers from 2 large cohorts (259 United States and 126 Swedish individuals) using Poisson regression; statistical significance was defined as P less than .002 (Bonferroni correction). Cumulative incidence is reported for melanoma and nonmelanoma cancer. RESULTS: Incidence was increased for melanoma (SIR = 159.8, 95% confidence interval [CI] = 132.1 to 193.2), pancreatic cancer (SIR = 24.1, 95% CI = 14.7 to 39.4), head and neck squamous cell carcinoma (SIR = 16.2, 95% CI = 9.5 to 27.6), and lung cancer (SIR = 5.6, 95% CI = 3.4 to 9.1) in GPV carriers. Similar associations were observed with p16 alteration. Combined p16 and p14ARF alteration was associated with increased incidence of esophageal cancer (SIR = 16.7, 95% CI = 5.7 to 48.9) and malignant peripheral nerve sheath tumor (SIR = 113.0, 95% CI = 16.4 to 780.9), although cancer events were limited (n < 5 for each malignancy). Cumulative incidence at age 70 years for melanoma and nonmelanoma cancer was 68.3% (95% CI = 68.0% to 68.6%) and 35.2% (95% CI = 34.9% to 35.6%), respectively. A total 89% of smoking-related cancers (lung, head and neck squamous cell carcinoma, pancreatic, esophageal) occurred in ever smokers. CONCLUSION: These findings highlight the impact of p16 and p14ARF alteration on cancer risk. Smoking was an important risk factor for smoking-related cancers in our study.

CDKN2A exon 1B deletion predisposing to melanoma and neural system tumour syndrome.

CDKN2A at chromosome positon 9p21 is a tumour suppressor gene encoding the cell cycle regulators p16 and p14ARF. While melanoma is associated with variants affecting both transcripts, families with mutations involving the p14ARF-specific exon 1B may be predisposed to central nervous system tumours. We describe a family with a deletion of exon 1B in CDKN2A, who had multiple cutaneous melanomas, neural tumours and various malignancies.

Effect of Sodium Butyrate on p16INK4a, p14ARF, p15INK4b, Class I HDACs (HDACs 1, 2, 3) Class II HDACs (HDACs 4, 5, 6), Cell Growth Inhibition and Apoptosis Induction in Pancreatic Cancer AsPC-1 and Colon Cancer HCT-116 Cell Lines.

BACKGROUND: In higher eukaryotes, cell-cycle transitions are regulated by different cyclin-dependent kinases (Cdks) and Cdk inhibitors (CKIs). CKIs include two groups, the Ink4 (p16INK4a, p15INK4b, p18INK4c, and p19INK4d) and the Cip/Kip (p21Cip1, p27Kip1, and p57Kip2) families. The hyperactivity of histone deacetylases (HDACs) is associated with cancer induction. Histone deacetylase inhibitors (HDACIs) such as sodium butyrate (NaBT) can inhibit HDAC activity resulting in apoptosis induction. The present study was designed to investigate the effect of sodium butyrate on p16INK4a, p14ARF, p15INK4b, class I HDACs (HDACs 1, 2, 3), and class II HDACs (HDACs 4, 5, 6), cell growth inhibition, and apoptosis induction in pancreatic cancer AsPC-1 and colon cancer HCT-116 cell lines. In fact, we want to know whether sodium butyrate can reactivate Ink4 and Cip/Kip families by HDACs inhibition. MATERIALS AND METHODS: The AsPC-1 and HCT-116 cells were treated with sodium butyrate at different periods. Then, the MTT assay, cell apoptosis assay, and qRT-PCR were done to determine viability, apoptosis, and the relative expression level of the genes respectively. RESULTS: The sodium butyrate increased p16INK4a, p14ARF, and p15INK4b and decreased class I and II HDACs significantly. Besides, HCT-116 cell was more sensitive to sodium butyrate in comparison to AsPC-1 cell. CONCLUSION: The sodium butyrate can reactivate the p16INK4a, p14ARF, and p15INK4b through inhibition of HDACs in AsPC-1 and HCT-116 cell lines.

Farnesoid X receptor functions in cervical cancer via the p14ARF-mouse double minute 2-p53 pathway.

BACKGROUND: Cervical cancer is the second most common cancer among women living in developing countries. Farnesoid X receptor (FXR) is a member of the nuclear receptor family, which regulates the development and proliferation of cancer. However, the role of and molecular mechanism by which FXR acts in cervical cancer are still unknown. METHODS AND RESULTS: The relationship between FXR and the proliferation of cervical cancer cell lines was detected by MTT and colony formation assays. Immunohistochemistry was used to detect the expression of FXR in cervical cancer tissue slides. Western blotting was used to detect the expression of p14ARF, mouse double minute 2 (MDM2) and p53 when FXR was overexpressed or siRNA was applied. Western blotting was used to detect the expression of MDM2 and p53 when pifithrin-α (PFT-α) was applied. FXR activation inhibited the proliferation of cervical cancer cell lines. FXR was significantly decreased in cervical squamous cell carcinoma, which was correlated with TNM stage, but not with metastasis. Overexpression of FXR activated the p14ARF-MDM2-p53 pathway. As a p53 inhibitor, PFT-α increased MDM2 in Lenti-vector cells, but had no effect on MDM2 in Lenti-FXR cells. CONCLUSIONS: FXR inhibits cervical cancer by upregulating the p14ARF-MDM2-p53 pathway. Activation of FXR may be a potential strategy for the treatment of cervical cancer.

Anticancer and Antimicrobial Evaluations on Alternative Reading Frame (ARF) Peptides and their Derivatives.

BACKGROUND: Alternative reading frame (ARF) protein up-regulates the intracellular level of a tumour suppressor protein, p53, by blocking MDM2 mediated p53 ubiquitination. The two homologous forms of ARF proteins are p19ARF in mice and p14ARF in humans. In our study, p19ARF-derived peptide ARF (26-44) and its cell-penetrating peptide conjugate Tat-ARF (26-44), p14ARF-derived peptide ARF (1-22), and its NrLS conjugate ARF (1-22)-NrLS were designed, and their anticancer properties were investigated. OBJECTIVE: Our objective is to study the anticancer and antimicrobial properties of ARF-derived peptides and their cell-penetrating and NrLS conjugates. METHODS: Peptides synthesized using solid-phase peptide synthesis (SPPS) were purified using RPHPLC and characterized using Bruker MALDI-TOF mass spectrometry. Cytotoxicity was evaluated on HeLa and BE(2)-C cells by cell viability IC50 determination. Minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. Morphological studies were carried out using SEM and TEM techniques, live/dead staining, ROS and Hoest staining. RESULTS: Peptides Tat-ARF (1-22) and ARF (1-22)-NrLS exhibited potent cytotoxic effects, comparable to the known standard cisplatin. Cellular morphological studies showed signs of apoptosis which were confirmed by reactive oxygen species (ROS) generation and Hoechst nuclear staining. ARF peptides showed potent antimicrobial activities at low micromolar concentrations without haemolysis. CONCLUSION: Tat modification improved the activity of ARF (26-44) by 9 folds against HeLa and 5 folds against BE(2)-C cells. NrLS modification of ARF (1-22) imparted 12 fold potency against HeLa and 2-fold potency against BE(2)-C cells. This study helps to further understand the effect of these peptides on MDM2 proteins and their role in the apoptosis signalling pathway.

Mutation of the Conserved Threonine 8 within the Human ARF Tumour Suppressor Protein Regulates Autophagy.

BACKGROUND: The ARF tumour suppressor plays a well-established role as a tumour suppressor, halting cell growth by both p53-dependent and independent pathways in several cellular stress response circuits. However, data collected in recent years challenged the traditional role of this protein as a tumour suppressor. Cancer cells expressing high ARF levels showed that its expression, far from being dispensable, is required to guarantee tumour cell survival. In particular, ARF can promote autophagy, a self-digestion pathway that helps cells cope with stressful growth conditions arising during both physiological and pathological processes. METHODS: We previously showed that ARF is regulated through the activation of the protein kinase C (PKC)-dependent pathway and that an ARF phospho-mimetic mutant on the threonine residue 8, ARF-T8D, sustains cell proliferation in HeLa cells. We now explored the role of ARF phosphorylation in both basal and starvation-induced autophagy by analysing autophagic flux in cells transfected with either WT and ARF phosphorylation mutants by immunoblot and immunofluorescence. RESULTS: Here, we show that endogenous ARF expression in HeLa cells is required for starvation-induced autophagy. Further, we provide evidence that the hyper-expression of ARF-T8D appears to inhibit autophagy in both HeLa and lung cancer cells H1299. This effect is due to the cells' inability to elicit autophagosomes formation upon T8D expression. CONCLUSIONS: Our results lead to the hypothesis that ARF phosphorylation could be a mechanism through which the protein promotes or counteracts autophagy. Several observations underline how autophagy could serve a dual role in cancer progression, either protecting healthy cells from damage or aiding cancerous cells to survive. Our results indicate that ARF phosphorylation controls protein's ability to promote or counteract autophagy, providing evidence of the dual role played by ARF in cancer progression.

Differential regulation of p27Kip1 depending on culture conditions and its correlation with status of p14ARF and p53.

p27Kip1 is known as a major cyclin-dependent kinase inhibitor and a tumor suppressor, and often functionally hampered at protein level. p27 protein expression levels are frequently low in various cancers and negatively correlated with malignancy of cancer. However, in our previous study, we discovered that p27 overexpression does not inhibit the proliferation of two cancer cell lines due to a functional suppression of p27 by nucleophosmin isoform 1 (NPM1); that is, a qualitative, not quantitative, suppression of p27 function occurs in these cancer cell lines. To clarify the regulation of p27 in several types of cancer, we investigated p27 function in other cancer cell lines, based on proliferation assays in those cell lines carrying doxycycline-inducible p27, and found that MDAH041 cells which express p14ARF, an antagonist of NPM1, show growth inhibition depending on p27 induction. Moreover, to investigate p27 function under anchorage-independent culture conditions, we performed soft agar colony formation assay and observed that the colony formation of some cell lines carrying wild-type p53, a major tumor suppressor, was inhibited depending on p27 induction. These results suggest that p27 function is regulated differentially among cancer cell types under anchorage-dependent and anchorage-independent culture conditions.

Exercise effects on DNA methylation in EVL, CDKN2A (p14, ARF), and ESR1 in colon tissue from healthy men and women.

Physical activity reduces risk of colon cancer by 20-30%. Aberrant methylation patterns are common epigenetic alterations in colorectal adenomas, and cancers and play a role in cancer initiation and progression. Alterations identified in normal colon tissue represent apotential 'field cancerization' process, where normal colon is primed for carcinogenesis. Here, we investigate methylation patterns in three genes -Ena/VASP-like (EVL), (CDKN2A (p14, ARF)), and Oestrogen Receptor-1 (ESR1)- in normal colon tissue collected at baseline and 12 months from 202 sedentary men and women, 40-75 years, enrolled in a randomized controlled trial testing an exercise intervention vs. control (http://clinicaltrials.gov/show/NCT00668161). Participants were randomized to moderate-to-vigorous intensity exercise, 60 minutes/day, 6 days/week for 12 months, or usual lifestyle. Sigmoid colon biopsies were obtained at baseline and 12-months, DNA extracted, and bisulphite converted. Droplet digital methylation-specific PCR was performed for EVL, p14ARF, and ESR1. Generalized estimating equations modification of linear regression was used to model relationships between intervention effects and gene methylation levels, adjusting for possible confounders.There were no statistically significant differences between methylation patterns at 12-months between exercisers and controls. ESR1 methylation patterns differed by sex: women -10.58% (exercisers) +11.10% (controls); men +5.54% (exercisers), -8.16% (controls) (P=0.05), adjusting for BMI and age. There were no statistically significant changes in methylation patterns in any gene stratified by change in VO2max or minutes/week of exercise.While no statistically significant differences were found in gene methylation patterns comparing exercises vs. controls, 12-month exercise effects on ESR1 methylation differed by sex, warranting further study.

The functional role of inherited CDKN2A variants in childhood acute lymphoblastic leukemia.

OBJECTIVE: Genetic alterations in CDKN2A tumor suppressor gene on chromosome 9p21 confer a predisposition to childhood acute lymphoblastic leukemia (ALL). Genome-wide association studies have identified missense variants in CDKN2A associated with the development of ALL. This study systematically evaluated the effects of CDKN2A coding variants on ALL risk. METHODS: We genotyped the CDKN2A coding region in 308 childhood ALL cases enrolled in CCCG-ALL-2015 clinical trials by Sanger Sequencing. Cell growth assay, cell cycle assay, MTT-based cell toxicity assay, and western blot were performed to assess the CDKN2A coding variants on ALL predisposition. RESULTS: We identified 10 novel exonic germline variants, including 6 missense mutations (p.A21V, p.G45A and p.V115L of p16INK4A; p.T31R, p.R90G, and p.R129L of p14ARF) and 1 nonsense mutation and 1 heterozygous termination codon mutation in exon 2 (p16INK4A p.S129X). Functional studies indicate that five novel variants resulted in reduced tumor suppressor activity of p16INK4A, and increased the susceptibility to the leukemic transformation of hematopoietic progenitor cells. Compared to other variants, p.H142R contributes higher sensitivity to CDK4/6 inhibitors. CONCLUSION: These findings provide direct insight into the influence of inherited genetic variants at the CDKN2A coding region on the development of ALL and the precise clinical application of CDK4/6 inhibitors.

Genetic and methylation status of CDKN2A (p14ARF/p16INK4A) and TP53 genes in recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) is a rare and chronic disease affecting the upper airway with papillomatous lesions caused by the human papillomavirus (HPV) infection, especially HPV-6 and/or HPV-11 types. Little is known about the genetic and epigenetic drivers in RRP pathophysiology. For this purpose, we analyzed 27 papillomatous lesions from patients with RRP to evaluate somatic mutations and methylation status in CDKN2A (p14ARF/p16INK4A) and TP53, which are key tumor suppressor genes for the cell cycle control. Sanger sequencing analysis revealed one somatic mutation in TP53 (c.733_734insA) and four mutations in CDKN2A (c.-30G > T, c.29_30insA, c.69delT, and c.300C > A). These mutations were observed in 10 patients, 6 of which carried double mutation. Furthermore, 50% (5/10) of these patients carrying somatic mutations had RRP severity, representing 62.5% (5/8) of the severity cases in this study, albeit no significant association was found between somatic mutations and disease severity. Methylation-specific polymerase chain reaction assays revealed p14ARF promoter hypermethylation in 100% of cases, followed by TP53 (96.3%) and p16INK4A (55.6%), suggesting the influence of HPV in the DNA methylation machinery. In conclusion, somatic mutations were not common events identified in patients with RRP. However, epigenetic modulation by high methylation rates, particularly for the p14ARF/TP53 pathway, seems to be in the course of RRP development.

A murine mesenchymal stem cell model for initiating events in osteosarcomagenesis points to CDK4/CDK6 inhibition as a therapeutic target.

Osteosarcoma is a high-grade bone-forming neoplasm, with a complex genome. Tumours frequently show chromothripsis, many deletions, translocations and copy number alterations. Alterations in the p53 or Rb pathway are the most common genetic alterations identified in osteosarcoma. Using spontaneously transformed murine mesenchymal stem cells (MSCs) which formed sarcoma after subcutaneous injection into mice, it was previously demonstrated that p53 is most often involved in the transformation towards sarcomas with complex genomics, including osteosarcoma. In the current study, not only loss of p53 but also loss of p16Ink4a is shown to be a driver of osteosarcomagenesis: murine MSCs with deficient p15Ink4b, p16Ink4a, or p19Arf transform earlier compared to wild-type murine MSCs. Furthermore, in a panel of nine spontaneously transformed murine MSCs, alterations in p15Ink4b, p16Ink4a, or p19Arf were observed in eight out of nine cases. Alterations in the Rb/p16 pathway could indicate that osteosarcoma cells are vulnerable to CDK4/CDK6 inhibitor treatment. Indeed, using two-dimensional (n = 7) and three-dimensional (n = 3) cultures of human osteosarcoma cell lines, it was shown that osteosarcoma cells with defective p16INK4A are sensitive to the CDK4/CDK6 inhibitor palbociclib after 72-hour treatment. A tissue microarray analysis of 109 primary tumour biopsies revealed a subset of patients (20-23%) with intact Rb, but defective p16 or overexpression of CDK4 and/or CDK6. These patients might benefit from CDK4/CDK6 inhibition, therefore our results are promising and might be translated to the clinic.

MiR-6838-5p facilitates the proliferation and invasion of renal cell carcinoma cells through inhibiting the DMTF1/ARF-p53 axis.

Renal cell carcinoma (RCC) is one of the most common renal malignancies in the urinary system. Numerous studies have demonstrated that miRNAs can regulate tumorigenesis and progression. This study aims to investigate the role and regulatory mechanism of miR-6838-5p in RCC. Our study confirmed that miR-6838-5p was upregulated in human RCC tissues (30/42, 77.43%, P < 0.01) and RCC cell lines (P < 0.05) compared to adjacent non-neoplastic tissues and normal renal epithelial cells. In vitro, overexpression of miR-6838-5p enhanced cell proliferation and invasion in human RCC cell lines (ACHN and 786-O), which were detected by CCK-8, Transwell and Colony formation assays (P < 0.05), and knockdown of miR-6838-5p suppressed cell proliferation and invasion (P < 0.05). Results of Bioinformatics analysis combined with Dual-luciferase reporter gene assay demonstrated that miR-6838-5p could bind to Cyclin D binding myb-like transcription factor 1 (DMTF1). In addition, RT-qPCR and Western blotting confirmed that DMTF1 was downregulated in RCC tissues and cell lines. Meanwhile, it was demonstrated that overexpression of miR-6838-5p inhibited DMTF1 level in ACHN cells. Next, we confirmed that DMTF1 overexpression reversed the inhibitory effects of overexpression of miR-6838-5p on phosphatase and tensin homolog (PTEN), tumor protein 53(p53), murine double minute 2 (MDM2) and alternative reading frame (ARF) protein levels in the ARF-p53 signaling pathway. In conclusion, our research showed that miR-6838-5p enhanced the proliferation and invasion of RCC cells by inhibiting the DMTF1/ARF-p53 axis.

Tumor-associated antigen Prame targets tumor suppressor p14/ARF for degradation as the receptor protein of CRL2Prame complex.

Protein Preferentially Expressed Antigen in Melanoma (Prame), a tumor-associated antigen, has been found to frequently overexpress in various cancers, which indicates advanced cancer stages and poor clinical prognosis. Moreover, previous reports noted that Prame functions as a substrate recognizing receptor protein of Cullin RING E3 ligases (CRLs) to mediate potential substrates degradation through Ubiquitin Proteasome System (UPS). However, none of the Prame specific substrate has been identified so far. In this study, proteomic analysis of RBX1-interacting proteins revealed p14/ARF, a well-known tumor suppressor, as a novel ubiquitin target of RBX1. Subsequently, immunoprecipitation and in vivo ubiquitination assay determined Cullin2-RBX1-Transcription Elongation Factor B Subunit 2 (EloB) assembled CRL2 E3 ligase complex to regulate the ubiquitination and subsequent degradation of p14/ARF. Finally, through siRNA screening, Prame was identified as the specific receptor protein responsible for recognizing p14/ARF to be degraded. Additionally, via bioinformatics analysis of TCGA database and clinical samples, Prame was determined to overexpress in tumor tissues vs. paired adjacent tissues and associated with poor prognosis of cancer patients. As such, downregulation of Prame expression significantly restrained cancer cell growth by inducing G2/M phase cell cycle arrest, which could be rescued by simultaneously knocking down of p14/ARF. Altogether, targeting overexpressed Prame in cancer cells inactivated RBX1-Cullin2-EloB-Prame E3 ligase (CRL2Prame) and halted p14/ARF degradation to restrain tumor growth by inducing G2/M phase cell cycle arrest.

The Yin and Yang-Like Clinical Implications of the CDKN2A/ARF/CDKN2B Gene Cluster in Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is a malignant clonal expansion of lymphoid hematopoietic precursors that exhibit developmental arrest at varying stages of differentiation. Similar to what occurs in solid cancers, transformation of normal hematopoietic precursors is governed by a multistep oncogenic process that drives initiation, clonal expansion and metastasis. In this process, alterations in genes encoding proteins that govern processes such as cell proliferation, differentiation, and growth provide us with some of the clearest mechanistic insights into how and why cancer arises. In such a scenario, deletions in the 9p21.3 cluster involving CDKN2A/ARF/CDKN2B genes arise as one of the oncogenic hallmarks of ALL. Deletions in this region are the most frequent structural alteration in T-cell acute lymphoblastic leukemia (T-ALL) and account for roughly 30% of copy number alterations found in B-cell-precursor acute lymphoblastic leukemia (BCP-ALL). Here, we review the literature concerning the involvement of the CDKN2A/B genes as a prognosis marker of good or bad response in the two ALL subtypes (BCP-ALL and T-ALL). We compare frequencies observed in studies performed on several ALL cohorts (adult and child), which mainly consider genetic data produced by genomic techniques. We also summarize what we have learned from mouse models designed to evaluate the functional involvement of the gene cluster in ALL development and in relapse/resistance to treatment. Finally, we examine the range of possibilities for targeting the abnormal function of the protein-coding genes of this cluster and their potential to act as anti-leukemic agents in patients.