p14ARF Expression Research Articles

Acceleration of Gastric Tumorigenesis Through MKRN1-Mediated Posttranslational Regulation of p14ARF

BackgroundWe investigated whether Makorin ring finger protein 1 (MKRN1), an E3 ligase, affects p14ARF-associated cellular senescence and tumorigenesis by posttranslational modification in gastric tumorigenesis.MethodsA link between MKRN1 and ARF was examined in MKRN1 null mouse embryonic fibroblasts (MEFs) and in human fibroblasts and gastric cancer cells by silencing MKRN1 using small interfering RNA (siRNA) and short hairpin RNA (shRNA). Ubiquitination and proteasomal degradation assays were used to assess p14ARF degradation associated with MKRN1. MKRN1 and p14ARF expression levels were analyzed with immunohistochemistry in malignant and normal tissues from gastric cancer patients and with χ2 tests. The tumor growth of gastric cancer cells stably expressing MKRN1 shRNA, p14ARF shRNA, or both was examined in mouse xenograft models (n = 4–6) and analyzed with unpaired t tests. All statistical tests were two-sided.ResultsMKRN1 knockout MEFs exhibited premature senescence and growth retardation with increased p19ARF protein expression. Similar results were obtained for human fibroblasts or gastric cancer cell lines by MKRN1 knockdown. Biochemical analyses confirmed that MKRN1 targets p14ARF for ubiquitination and subsequent proteasome-dependent degradation. A statistically significant association was shown between MKRN1 overexpression and p14ARF underexpression (P = .016). Xenograft analyses using p53-functional AGS or -dysfunctional SNU601 cells displayed statistically significant tumor growth retardation by silencing MKRN1, which was reversed under depletion of p14ARF (AGS cells, MKRN1 knockdown tumors vs MKRN1 and p14ARF knockdown tumors: 164.6 vs 464.8mm3, difference = 300.2mm3, 95% CI = 189.1 to 411.3mm3, P < .001).ConclusionsWe demonstrated that MKRN1 functions as a novel E3 ligase of p14ARF and that it potentially regulates cellular senescence and tumorigenesis in gastric cancer.

TBX3 Promotes Human Embryonic Stem Cell Proliferation and Neuroepithelial Differentiation in a Differentiation Stage-Dependent Manner

Abstract T-box 3 (Tbx3) is a member of the T-box family of genes. Mutations that result in the haploinsufficiency of TBX3 cause ulnar mammary syndrome in humans characterized by mammary gland hypoplasia as well as other congenital defects. In mice, homozygous mutations are embryonic lethal, suggesting that Tbx3 is essential for embryo development. Studies in mice have shown that Tbx3 is essential in the maintenance of mouse embryonic stem cell (ESC) self-renewal and in their differentiation into extraembryonic endoderm (ExEn). The role TBX3 plays in regulating human ESCs (hESCs) has not been explored. Since mouse and hESCs are known to represent distinct pluripotent states, it is important to address the role of TBX3 in hESC self-renewal and differentiation. Using overexpression and knockdown strategies, we found that TBX3 overexpression promotes hESC proliferation possibly by repressing the expression of both NFκBIB and p14ARF, known cell cycle regulators. During differentiation, TBX3 knockdown resulted in decreased neural rosette formation and in decreased expression of neuroepithelial and neuroectoderm markers (PAX6, LHX2, FOXG1, and RAX). Taken together, our data suggest a role for TBX3 in hESC proliferation and reveal an unrecognized novel role of TBX3 in promoting neuroepithelial differentiation. Our results suggest that TBX3 plays distinct roles in regulating self-renewal and differentiation in both hESCs and mouse ESCs.

Prognostication of Vulvar Cancer Based on p14ARF Status: Molecular Assessment of Transcript and Protein

This study was designed to determine the prognostic role of p14ARF in vulvar squamous cell carcinoma (VSCC). Immunohistochemistry for p14ARF and p53 and fluorescent in situ hybridization (FISH) for TP53 were performed in 139 cases of VSCC. Human papillomavirus (HPV) genotyping by hybridization was employed in 100 cases. qRT-PCR for p14ARF and p53 transcript assessment was performed in 16 cases. All results were correlated with clinicopathological variables. Immunohistochemistry analysis showed p14ARF and p53 positivity in 16.4% and 53% cases respectively. Positive p14ARF expression was significantly associated with the following variables: shorter cancer-specific survival (P=0.04) and shorter disease-free survival (P=0.02), presence of perineural invasion (P=0.037), vascular invasion (P=0.047), and node metastasis (P=0.031). Also, p14ARF-positive HPV-negative cases had the shortest cancer-specific survival (P=0.03) and disease-free survival (P=0.04). HPV infection was detected in 32.8% of the cases; HPV16 was the most prevalent type. Viral infection was more common in poorly differentiated tumors (P=0.032). qRT-PCR demonstrated that CDKN2A (p14ARF) had higher expression in tumor samples compared with paired noncancerous samples (P<0.001). The opposite relationship was seen in TP53 expression evaluation (P<0.001). FISH demonstrated 4 cases with deleted TP53 (6.3%). p14ARF represents an important marker of poor prognosis in VSCC. p53 and HPV infection did not show any prognostic importance. Further clinical trials concerning p14ARF positivity may result in important contributions due to its relationship with poor outcome. Mainly due to the relationship of p14ARF with lymph node metastasis, the immunohistochemistry evaluation of this marker may help to identify a subset of patients more suitable to less radical procedures.

P14ARF Post-Transcriptional Regulation of Nuclear Cyclin D1 in MCF-7 Breast Cancer Cells: Discrimination between a Good and Bad Prognosis?

As part of a cell’s inherent protection against carcinogenesis, p14ARF is upregulated in response to hyperproliferative signalling to induce cell cycle arrest. This property makes p14ARF a leading candidate for cancer therapy. This study explores the consequences of reactivating p14ARF in breast cancer and the potential of targeting p14ARF in breast cancer treatment. Our results show that activation of the p14ARF-p53-p21-Rb pathway in the estrogen sensitive MCF-7 breast cancer cells induces many hallmarks of senescence including a large flat cell morphology, multinucleation, senescence-associated-β-gal staining, and rapid G1 and G2/M phase cell cycle arrest. P14ARF also induces the expression of the proto-oncogene cyclin D1, which is most often associated with a transition from G1-S phase and is highly expressed in breast cancers with poor clinical prognosis. In this study, siRNA knockdown of cyclin D1, p21 and p53 show p21 plays a pivotal role in the maintenance of high cyclin D1 expression, cell cycle and growth arrest post-p14ARF induction. High p53 and p14ARF expression and low p21/cyclin D1 did not cause cell-cycle arrest. Knockdown of cyclin D1 stops proliferation but does not reverse senescence-associated cell growth. Furthermore, cyclin D1 accumulation in the nucleus post-p14ARF activation correlated with a rapid loss of nucleolar Ki-67 protein and inhibition of DNA synthesis. Latent effects of the p14ARF-induced cellular processes resulting from high nuclear cyclin D1 accumulation included a redistribution of Ki-67 into the nucleoli, aberrant nuclear growth (multinucleation), and cell proliferation. Lastly, downregulation of cyclin D1 through inhibition of ER abrogated latent recurrence. The mediation of these latent effects by continuous expression of p14ARF further suggests a novel mechanism whereby dysregulation of cyclin D1 could have a double-edged effect. Our results suggest that p14ARF induced-senescence is related to late-onset breast cancer in estrogen responsive breast cancers and/or the recurrence of more aggressive breast cancer post-therapy.

The histone acetyltransferase PCAF regulates p21 transcription through stress-induced acetylation of histone H3

The activity of p53 as a tumor suppressor primarily depends on its ability to transactivate specific target genes in response to genotoxic and other potentially mutagenic stresses. Several histone acetyl transferases (HATs), including p300, CBP, PCAF and GCN5 have been implicated in the activation of p53-dependent transcription of the cyclin-dependent kinase (cdk) inhibitor p21 as well as other target genes. Here we show that PCAF, but not CBP or p300, is a critical regulator of p53-dependent p21 expression in response to multiple p53-activating stresses. PCAF was required for the transcriptional activation of p21 in response to exogenous p53 in p53-null cells, nutlin-3, DNA damaging agents and p14ARF expression, suggesting a broad requirement for PCAF in p53 signaling to p21 after stress. Importantly, cells lacking PCAF failed to undergo cell cycle arrest in response to nutlin-3 treatment or p14ARF expression, consistent with a physiologically important role for PCAF in this p53 function. Surprisingly, the role for PCAF in induction of p21 was independent of p53 lysine 320 acetylation, a previously suggested target of PCAF-mediated acetylation. Though p21 promoter occupancy by p53 was not altered by PCAF knockdown, activation of p21 transcription required an intact PCAF HAT domain, and induction of chromatin marks acetyl-H3K9 and acetyl-H3K14 at the p21 promoter by p53 was dependent upon physiologic levels of PCAF. Together, our experiments indicate that PCAF is required for stress-responsive histone 3 acetylation at the p21 promoter, p53-directed transcription of p21 and the resultant growth arrest.

Mutated N-ras does not induce p19arf in CO25 cell line

The mouse cell line (CO25) used in this study was transfected with a glucocorticoid inducible mutated human N-ras oncogene under transcriptional control of the steroid-sensitive promoter of the mouse mammary tumors virus long terminal repeat MMTV-LTR. This study was aimed to investigate the expression of p19 arf and MDM2 genes under the effect of N-ras oncogene induction and to invent the role of p19 arf , MDM2 in N-ras pathway during various periods (12, 24, 48, 72, 96 h) using western blotting method. The levels of â-actin proteins in the same periods were our control group. The observations showed no increase of p19 arf protein expression in normal, cancer and differentiated CO25 cells. MDM2 was accumulated until 72 h and after 96 h, it showed a dramatical decrease while β-actin levels were increased correlated to the volume of protein loaded to the gel. Because of the role of p19 arf as tumor suppressor and p53-MDM2 linker, it is highly recommended to investigate the relationship between N-ras and p53 and MDM2 in the same system to recognize the molecule that may play a linker molecule between p53 and MDM2 in p19 arf lack system. Key words : Oncogene, N-ras, p19 arf , myoblast, CO25 cells, differentiation, MDM2.

Hypermethylation-mediated silencing of p14ARF in fibroblasts from idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease of unknown etiology. A conspicuous feature is the formation and persistence of fibroblastic/myofibroblastic foci throughout the lung parenchyma. Mechanisms remain unknown, but data indicate that fibroblasts acquire an antiapoptotic phenotype. We hypothesized that transcriptional silencing of proapoptotic genes may be implicated, and accordingly we evaluated the epigenetic regulation of p14(ARF). The expression of p14(ARF) was analyzed by RT-PCR in IPF (n = 8) and normal derived fibroblasts (n = 4) before and after treatment with 5-aza-2'-deoxycytidine (5-aza) and trichostatin A (TSA). p14(ARF) gene promoter methylation was determined by methylation-specific PCR (MS-PCR) and by DNA digestion with endonuclease McrBc, which cleaves 50% of methylated CpG. Apoptosis was evaluated by Annexin-V and nuclear staining. p14(ARF) expression was significantly decreased in four of the eight IPF fibroblasts lines, which was restored after 5-aza treatment. No changes were found with TSA. MS-PCR of bisulfite-treated genomic DNA showed a correlation between the reduced expression of p14(ARF) and the presence of hypermethylated promoter. No amplification was observed in the DNA treated with the McrBc enzyme, corroborating promoter hypermethylation. p14(ARF)-hypermethylated IPF fibroblasts were significantly more resistant to staurosporine-and S-nitrosoglutathione-induced apoptosis compared with normal and nonmethylated IPF fibroblasts (P < 0.01) and showed reduced levels of p53. Resistance to apoptosis was provoked in fibroblasts when p14(ARF) expression was inhibited by siRNA (P < 0.05). These findings demonstrate that many IPF fibroblasts have reduced expression of the proapoptotic p14(ARF) attributable to promoter hypermethylation and indicate that epigenetic mechanisms may underlie their resistance to apoptosis.

Abstract 802: A key role for ARF in drug resistance in invasive bladder cancer

Abstract Although most superficial bladder cancers can be removed transurethrally with excellent prognosis, clinical outcome is much more problematic for patients with muscle-invasive disease. Indeed, invasive bladder cancer is a major clinical challenge since it is highly associated with postoperative recurrence and metastasis. Current treatments for those lethal forms of bladder cancer include systemic chemotherapy and molecular targeted therapy; however, survival is poor since most patients develop resistance to the drugs within a short timeframe. Clearly, there is a need to identify novel therapeutic options for invasive bladder cancer as well as a greater understanding of the molecular mechanisms of drug resistance. We have been investigating mechanisms of drug resistance using genetically-engineered mouse models of invasive bladder cancer based on the combinatorial deletion of p53 and Pten in bladder epithelium. p53 and PTEN are frequently inactivated in human bladder cancers, particularly those with poor prognosis. Indeed, allograft tumors established from these mice responded initially to the treatment with cisplatin, docetaxel, or rapamycin but eventually developed resistance. We have observed that these p53; PTEN deficient tumors express robust levels of p19Arf, while targeted deletion of Arf retards the aquisition of resistance following drug treatment. The significance of this expression was further suggested by analysis of a published gene expression profile of human bladder cancers which revealed that high Arf expression is an independent predictor of poor survival of invasive bladder cancer patients. Furthermore, following drug treatment in the mouse model, the Arf-positive, compared to the Arf-negative, tumors were significantly enriched in extracellular matrix/integrin signaling gene signature consistent with activation of downstream PI3K-mTOR pathway, suggesting that a model for drug resistance is via PI3K pathway activation. We have observed similar results in human bladder cancer cell lines either following knock-down of p14Arf in J82 cells that express endogenous p14Arf or following forced expression of p14Arf in ARF-negative UMUC3 human bladder cancer cells, and in both cases it was coincident with deregulated activation of PI3K-mTOR pathway. Thus, we propose that Arf confers drug resistance on bladder cancer by activating PI3K-mTOR pathway, highlighting a potential therapeutic target for advanced invasive bladder cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 802. doi:1538-7445.AM2012-802

Aberrant expression of cell cycle regulatory genes predicts overall and disease free survival in malignant pleural mesothelioma patients

BackgroundMalignant pleural mesothelioma (MPM) is a highly aggressive disease with a generally poor prognosis. Since escape from cell cycle checkpoint control is common in several solid tumors, the present study was performed to evaluate the role of some cell cycle regulatory genes in the development and progression of MPM. Patients and methodsAberrant expression of p14ARF, p16INK4A, p21waf, p27KIP, p53, mdm2 and Rb was assessed in 55 MPM cases from Egypt using immunohistochemistry and PCR techniques. Results were correlated with clinico-pathological prognostic factors, overall and disease free survival (OS&DFS). ResultsAltered expression of p14ARF, p16INK4A, p21waf, p27KIP1, Rb, p53 and mdm2 proteins was detected in 50.9%, 54.5%, 53.3%, 61.8%, 53.3%, 58.2%, and 50.8% of cases, respectively. SV40 infection significantly correlated with p14ARF, 16INK4A, p27kip1 and Rb aberrations (p=0.014, p=0.02, p=0.01, p=−0.01). Asbestos exposure significantly correlated with p53, p21waf and mdm2 aberrations (p=0.001, p=0.03, p=0.02). On multivariate analysis PS≥2, p27KIP1 and Rb aberrations were independent prognostic factors for OS (p=0.016, p=0.011, p=0.003) whereas on tumor recurrence, p27KIP1 and Rb aberrations were independent prognostic factors for DFS (p=0.002, p=0.03, p=0.01). ConclusionsMPM is a complex disease characterized by multiple genetic aberrations; some of them involve cell cycle regulatory genes. p14ARF, p16INK4A, Rb and p27KIP1 seem to be involved in SV40-associated MPM whereas mdm2, p53 and p21WAF are related to asbestos exposure. In addition to recurrence and PS, only p27KIP1and Rb could be used as molecular prognostic markers in MPM.

Hypermethylation‐Mediated Silencing of p14ARF in Fibroblasts from Idiopathic Pulmonary Fibrosis (IPF)

IPF is a lethal lung disease characterized by the formation and persistence of fibroblast foci and matrix accumulation. Evidence suggests that fibroblasts acquire an anti‐apoptotic phenotype and we hypothesized that epigenetic silencing of the pro‐apoptotic gene p14ARF may be involved. p14ARF expression was analyzed by RT‐PCR and Western blot in IPF and normal human lung fibroblasts. p14ARF gene promoter methylation was determined by methylation specific PCR (MS‐PCR) and by DNA digestion with endonuclease McrBc. Apoptosis was evaluated by annexin‐V and nuclear staining. p14ARF expression was significantly decreased in 4 of 8 IPF fibroblasts lines, which was restored after 5‐aza‐2′‐deoxycytidine treatment. MS‐PCR of bisulfite‐treated genomic DNA showed a correlation between the reduced expression of p14ARF and the presence of hypermethylated promoter. No amplification was observed in the DNA treated with McrBc confirming promoter hypermethylation. p14ARF hypermethylated IPF fibroblasts were more resistant to staurosporine‐induced apoptosis compared with normal and non‐methylated IPF fibroblasts (p&lt;0.01) and showed reduced levels of p53. Apoptosis resistance was induced in fibroblasts inhibiting p14ARF expression by siRNA (p&lt;0.05). These findings reveal that many IPF fibroblasts have reduced expression of p14ARF indicating that epigenetic mechanisms may cause their resistance to apoptosis.

554 A KEY ROLE FOR ARF IN DRUG RESISTANCE IN INVASIVE BLADDER CANCER

INTRODUCTION AND OBJECTIVES: Although most superficial bladder cancers can be removed transurethrally with excellent prognosis, clinical outcome is much more problematic for patients with muscle-invasive disease. Indeed, invasive bladder cancer is a major clinical challenge since it is highly associated with postoperative recurrence and metastasis. Current treatments for those lethal forms of bladder cancer include systemic chemotherapy and molecular targeted therapy; however, survival is poor since most patients develop resistance to the drugs within a short timeframe. Clearly, there is a need to identify novel therapeutic options for invasive bladder cancer as well as a greater understanding of the molecular mechanisms of drug resistance. METHODS: We have been investigating mechanisms of drug resistance using genetically-engineered mouse models of invasive bladder cancer based on the combinatorial deletion of p53 and Pten in bladder epithelium. p53 and PTEN are frequently inactivated in human bladder cancers, particularly those with poor prognosis. Indeed, allograft tumors established from these mice responded initially to the treatment with cisplatin, docetaxel, or rapamycin but eventually developed resistance. RESULTS: We have observed that these p53; PTEN deficient tumors express robust levels of p19Arf, while targeted deletion of Arf retards the aquisition of resistance following drug treatment. The significance of this expression was further suggested by analysis of a published gene expression profile of human bladder cancers which revealed that high Arf expression is an independent predictor of poor survival of invasive bladder cancer patients. Furthermore, following drug treatment in the mouse model, the Arf-positive, compared to the Arf-negative, tumors were significantly enriched in extracellular matrix/ integrin signaling gene signature consistent with activation of downstream PI3K-mTOR pathway, suggesting that a model for drug resistance is via PI3K pathway activation. We have observed similar results in human bladder cancer cell lines either following knock-down of p14Arf in J82 cells that express endogenous p14Arf or following forced expression of p14Arf in ARF-negative UMUC3 human bladder cancer cells, and in both cases it was coincident with deregulated activation of PI3K-mTOR pathway. CONCLUSIONS: Thus, we propose that Arf confers drug resistance on bladder cancer by activating PI3K-mTOR pathway, highlighting a potential therapeutic target for advanced invasive bladder cancer patients.

PIWIL4 regulates cervical cancer cell line growth and is involved in down-regulating the expression of p14ARF and p53

Recently, PIWIL4 has been identified as a functional protein involved in tumorigenesis. Cervical cancer is the second most prevalent form of cancer worldwide. The relationship between PIWIL4 and cervical cancer is still unknown. Here, we found that PIWIL4 is up-regulated in human cervical cancer tissues in comparison to adjacent normal tissues, and it promotes cell growth and proliferation by inhibiting apoptosis through the p14ARF/p53 pathway. PIWIL4 can also promote the invasion of cervical cancer cells. These results suggest that PIWIL4 might play an oncogenic role in cervical cancer and be useful as a new therapeutic target in the future.

P14ARF inhibits the growth of lung adenocarcinoma cells harbouring an EGFR L858R mutation by activating a STAT3-dependent pro-apoptotic signalling pathway

Epidermal growth factor receptor (EGFR) stimulates proliferative and survival signals. Activating mutations of EGFR are involved in the aetiology and maintenance of the malignant phenotype of lung tumours. We previously described the frequent association of these mutations with the decreased expression of the p14(ARF) tumour suppressor, another common feature of lung cancer. Based on these data, we postulated that p14(ARF) could protect cells against untimely or excessive mitotic signals induced by mutant EGFR. In this study, we demonstrate that p14(ARF) promotes apoptosis in lung tumour cells harbouring the EGFR L858R mutation through the accumulation of phosphorylated signal transducer and activator of transcription 3 (STAT3) on Tyr 705 residue, which leads to Bcl-2 downregulation. Using siRNA against PTP-RT, the phosphatase that specifically targets Tyr 705 residue, we show that accumulation of pSTAT3-Tyr705 promotes EGFR L858R mutant cell death, thereby confirming the existence of a STAT3-dependent pro-apoptotic pathway in these cells. Finally, we show that the expression of the EGFR L858R mutant represses p14(ARF) expression and inhibits STAT3/Bcl-2 signalling. These results identify a novel link between the p14(ARF) and EGFR pathways and suggest that EGFR L858R counteracts the pro-apoptotic function of p14(ARF) by downregulating its expression to promote carcinogenesis.

Expression of p14ARF, p15INK4b, p16INK4a and skp2 increases during esophageal squamous cell cancer progression

Esophageal carcinoma is the sixth most common cause of cancer-related mortality in the world. Senescence and apoptosis are assumed to be two main mechanisms that inhibit age-related carcinogenesis. p14(ARF), p15(INK4b) and p16(INK4a), which are known to induce senescence by regulating G(1) cell cycle arrest, have been identified as senescence markers. However, the mechanism by which senescence and apoptosis causes neoplasia in esophageal squamous cell carcinoma (ESCC) has not been identified. In this study, 20 cases of normal esophageal tissues, 11 cases of esophageal intraepithelial dysplasia (EID) and 60 cases of ESCC were obtained and pathologically diagnosed. Immunohistochemical staining was performed to assess the expression of p14(ARF), p15(INK4b), p16(INK4a), skp2, bcl-2 and ki-67. The senescence markers p14(ARF) and p16(INK4a) were found to be expressed in 15 and 10% of the normal tissues, 82 and 73% of the EID cases and 100 and 88% of the ESCC cases, respectively. The expression of p15(INK4b) was low in normal tissues, while 92% of the ESCC specimens were diffusely and markedly stained, involving the basal, middle and upper portion of the epithelium. The nuclear expression markers ki-67 and skp2 were highly expressed in ESCC tissues (100 and 72%, respectively). bcl-2 was expressed weakly in normal tissues (10%) and demonstrated various staining patterns in carcinoma specimens (strong in 60%, negative in 40%). MI was 0.09% in normal tissues and 0.95% in the ESCC specimens. Apart from the increased proliferation in esophageal carcinogenesis, as indicated in the ki-67 and skp2 indices, there was an increased expression of senescence-associated molecular markers in the ESCC specimens, which indicates that the senescence pathway may be activated and become a part of cancer development. Of greatest interest to us was that, when compared with clinical information, the expression of the senescence markers was markedly high in the poorly differentiated specimens with lymph node metastasis, indicating that senescence markers may have diagnostic potential in clinical settings.

HIF1α is required for survival maintenance of chronic myeloid leukemia stem cells

AbstractHypoxia-inducible factor-1α (HIF1α), a master transcriptional regulator of the cellular and systemic hypoxia response, is essential for the maintenance of self-renewal capacity of normal HSCs. It is still unknown whether HIF1α has a role in survival regulation of leukemia stem cells (LSCs) in chronic myeloid leukemia (CML). Using a mouse model of CML, here we report that HIF1α plays a crucial role in survival maintenance of LSCs. Deletion of HIF1α impairs the propagation of CML through impairing cell-cycle progression and inducing apoptosis of LSCs. Deletion of HIF1α results in elevated expression of p16Ink4a and p19Arf in LSCs, and knockdown of p16Ink4a and p19Arf rescues the defective colony-forming ability of HIF1α−/− LSCs. Compared with normal HSCs, LSCs appear to be more dependent on the HIF1α pathway. Together, these results demonstrate that HIF1α represents a critical pathway in LSCs and inhibition of the HIF1α pathway provides a therapeutic strategy for eradicating LSCs in CML.

P16INK4a and p14ARF mRNA expression in Pap smears is age-related

Expression of high-risk HPV oncogenes results in a strong overexpression of cellular protein p16INK4a. Immunohistochemical staining for p16INK4a is widely used as diagnostic marker. However, p16INK4a upregulation was also described as a biomarker of age. Here we analyzed p16INK4a expression in cervical smears to investigate if patient age may influence p16INK4a-based cervical cancer diagnosis. p14ARF was analyzed as a related supportive biomarker. Cervical scrapes were taken and stored in RNAlater. Total RNA was extracted, and cDNA was analyzed for expression of p16INK4a and p14ARF relative to β-actin, by real-time reverse transcriptase PCR SYBR-Green I assays. Patient-derived smears referred as HSIL (n=45) had 6.27-fold higher p16INK4a mRNA expression than smears of cytologically normal and HPV-negative persons (n=48). Expression of p14ARF was 4.87-fold higher. When women with normal diagnoses were stratified for age, a significantly enhanced p16INK4a (2.88-fold) and p14ARF (1.9-fold) expression was observed as a consequence of ageing. A significant age-dependent upregulation was also observed in older HSIL patients (2.54-fold). Our study revealed significantly enhanced expression of p16INK4a/p14ARF mRNA in cervical scrapes referred to as HSIL compared with normal women. An age-dependent bias has to be considered when quantifying these tumor suppressor genes, with respect to cervical cancer development.

Regulation of p14ARF expression by miR-24: a potential mechanism compromising the p53 response during retinoblastoma development

BackgroundMost human cancers show inactivation of both pRB- and p53-pathways. While retinoblastomas are initiated by loss of the RB1 tumor suppressor gene, TP53 mutations have not been found. High expression of the p53-antagonist MDM2 in human retinoblastomas may compromise p53 tumor surveillance so that TP53 mutations are not selected for in retinoblastoma tumorigenesis. We previously showed that p14ARF protein, which activates p53 by inhibiting MDM2, is low in retinoblastomas despite high mRNA expression.MethodsIn human fetal retinas, adult retinas, and retinoblastoma cells, we determined endogenous p14ARF mRNA, ARF protein, and miR-24 expression, while integrity of p53 signalling in WERI-Rb1 cells was tested using an adenovirus vector expressing p14ARF. To study p14ARF biogenesis, retinoblastoma cells were treated with the proteasome inhibitor, MG132, and siRNA against miR-24.ResultsIn human retinoblastoma cell lines, p14ARF mRNA was disproportionally high relative to the level of p14ARF protein expression, suggesting a perturbation of p14ARF regulation. When p14ARF was over-expressed by an adenovirus vector, expression of p53 and downstream targets increased and cell growth was inhibited indicating an intact p14ARF-p53 axis. To investigate the discrepancy between p14ARF mRNA and protein in retinoblastoma, we examined p14ARF biogenesis. The proteasome inhibitor, MG132, did not cause p14ARF accumulation, although p14ARF normally is degraded by proteasomes. miR-24, a microRNA that represses p14ARF expression, is expressed in retinoblastoma cell lines and correlates with lower protein expression when compared to other cell lines with high p14ARF mRNA. Transient over-expression of siRNA against miR-24 led to elevated p14ARF protein in retinoblastoma cells.ConclusionsIn retinoblastoma cells where high levels of p14ARF mRNA are not accompanied by high p14ARF protein, we found a correlation between miR-24 expression and low p14ARF protein. p14ARF protein levels were restored without change in mRNA abundance upon miR-24 inhibition suggesting that miR-24 could functionally repress expression, effectively blocking p53 tumor surveillance. During retinal tumorigenesis, miR-24 may intrinsically compromise the p53 response to RB1 loss.

Stress-Regulated Transcription Factor ATF4 Promotes Neoplastic Transformation by Suppressing Expression of the INK4a/ARF Cell Senescence Factors

Many cancers overexpress ATF4, a stress-induced transcription factor that promotes cell survival under hypoxic conditions and other stresses of the tumor microenvironment, but the potential contributions of ATF4 to oncogenesis itself have been little explored. Here, we report that ATF4 promotes oncogene-induced neoplastic transformation by suppressing the expression of cellular senescence-associated genes. Strikingly, primary embryo fibroblasts from ATF4-deficient mice were resistant to transformation by coexpression of H-ras(V12) and SV40 large T antigen. In wild-type cells these oncogenes induced expression of the murine Atf4 gene along with the cyclin-dependent kinase inhibitor Cdkn2a, which encodes the cell senescence-associated proteins p16INK4 and p19ARF. Elevated levels of ATF4 were sufficient to suppress expression of these proteins and drive oncogenic transformation. Conversely, genetic ablation of ATF4 led to constitutive expression of p16INK4a and p19ARF, triggering cellular senescence. Our findings define a central function for ATF4 in promoting oncogenic transformation by suppressing a central pathway of cellular senescence.

ErbB380 kDa, a nuclear variant of the ErbB3 receptor, binds to the Cyclin D1 promoter to activate cell proliferation but is negatively controlled by p14ARF

EGFR family members are tyrosine kinase transmembrane receptors that, in response to specific extracellular ligands, activate cytoplasmic pathways involved in cell proliferation, migration and differentiation. More recently, a pivotal role for EGF receptors has emerged, through the description of their nuclear localization.We report here the characterization of a nuclear variant of the kinase-defective ErbB3 receptor, ErbB380kDa, spanning the intracytoplasmic domain of the receptor. We assessed the putative transcriptional functions of ErbB380KDa in cancer cells, through the regulation of the proliferative Cyclin D1 gene, an already known target of the ErbB3 cytoplasmic signaling. We demonstrate here that the binding of ErbB380KDa on the promoter activates Cyclin D1 transcription and subsequent protein expression, leading to an increased cell proliferation. This mechanism can be balanced in response to the ectopic expression of the tumor suppressor p14ARF that physically interacts with ErbB3100kDa and sequesters it into nucleoli.Our data also show that ErbB380kDa increases the transcription of proliferative genes even though the cytoplasmic pathways are not activated. This nuclear ErbB3 pathway and the target genes concerned need to be further studied. Indeed, such mechanism could explain the tumor relapse observed in response to treatments aimed at blocking the receptor activation in response to ligand binding.

Fibroid explants reveal a higher sensitivity against MDM2-inhibitor nutlin-3 than matching myometrium

BackgroundSpontaneous cessation of growth is a frequent finding in uterine fibroids. Increasing evidence suggests an important role of cellular senescence in this growth control. Deciphering the underlying mechanisms of growth control that can be expected not only to shed light on the biology of the tumors but also to identify novel therapeutic targets.MethodsWe have analyzed uterine leiomyomas and matching normal tissue for the expression of p14Arf and used explants to see if reducing the MDM2 activity using the small-molecule inhibitor nutlin-3 can induce p53 and activate genes involved in senescence and/or apoptosis. For these studies quantitative real-time RT-PCR, Western blots, and immunohistochemistry were used. Statistical analyses were performed using the student's t test.ResultsAn in depth analysis of 52 fibroids along with matching myometrium from 31 patients revealed in almost all cases a higher expression of p14Arf in the tumors than in the matching normal tissue. In tissue explants, treatment with the MDM2 inhibitor nutlin-3 induced apoptosis as well as senescence as revealed by a dose-dependent increase of the expression of BAX as well as of p21, respectively. Simultaneously, the expression of the proliferation marker Ki-67 drastically decreased. Western-blot analysis identified an increase of the p53 level as the most likely reason for the increased activity of its downstream markers BAX and p21. Because as a rule fibroids express much higher levels of p14Arf, a major negative regulator of MDM2, than matching myometrium it was then analyzed if fibroids are more sensitive against nutlin-3 treatment than matching myometrium. We were able to show that in most fibroids analyzed a higher sensibility than that of matching myometrium was noted with a corresponding increase of the p53 immunopositivity of the fibroid samples compared to those from myometrium.ConclusionsThe results show that uterine fibroids represent a cell population of advanced cellular age compared to matching myometrium. Moreover, the data point to members of the p53-network as to potential novel therapeutic targets for the treatment of uterine fibroids.