p19ARF In Mouse Research Articles

Expanding Opportunities in Viral Oncolysis.

Expanding Opportunities in Viral Oncolysis.

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.

Helicobacter pylori pathogen inhibits cellular responses to oncogenic stress and apoptosis.

Helicobacter pylori (H. pylori) is a common gastric pathogen that infects approximately half of the world’s population. Infection with H. pylori can lead to diverse pathological conditions, including chronic gastritis, peptic ulcer disease, and cancer. The latter is the most severe consequence of H. pylori infection. According to epidemiological studies, gastric infection with H. pylori is the strongest known risk factor for non-cardia gastric cancer (GC), which remains one of the leading causes of cancer-related deaths worldwide. However, it still remains to be poorly understood how host-microbe interactions result in cancer development in the human stomach. Here we focus on the H. pylori bacterial factors that affect the host ubiquitin proteasome system. We investigated E3 ubiquitin ligases SIVA1 and ULF that regulate p14ARF (p19ARF in mice) tumor suppressor. ARF plays a key role in regulation of the oncogenic stress response and is frequently inhibited during GC progression. Expression of ARF, SIVA1 and ULF proteins were investigated in gastroids, H. pylori-infected mice and human gastric tissues. The role of the H. pylori type IV secretion system was assessed using various H. pylori isogenic mutants. Our studies demonstrated that H. pylori infection results in induction of ULF, decrease in SIVA1 protein levels, and subsequent ubiquitination and degradation of p14ARF tumor suppressor. Bacterial CagA protein was found to sequentially bind to SIVA1 and ULF proteins. This process is regulated by CagA protein phosphorylation at the EPIYA motifs. Downregulation of ARF protein leads to inhibition of cellular apoptosis and oncogenic stress response that may promote gastric carcinogenesis.

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

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. Our objective is to study the anticancer and antimicrobial properties of ARF-derived peptides and their cell-penetrating and NrLS conjugates. 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. 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. 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.

Investigating the molecular basis of ARF tumor suppressor function in the nucleolus: how ARF disrupts ribosome biogenesis and function

the ARF tumor suppressor (Alternative Reading Frame, p19Arf in mouse, p14Arf in human), is an arginine (Arg)-rich intrinsically disordered protein. During interphase, ARF is expressed at low levels and is localized to the granular component of the nucleolus through interactions with NPM1. NPM1-ARF complexes are required for the maintenance of ribosome biogenesis homeostasis. p14Arf and NPM1 associate with the nucleolar 60S preribosomal particle. ARF deletion results in a NPM1-dependent surge in ribosome biogenesis, protein synthesis and increased nucleolar size, three phenotypic hallmarks of cancer cells.

C-Myc inactivation of p53 through the pan-cancer lncRNA MILIP drives cancer pathogenesis

The functions of the proto-oncoprotein c-Myc and the tumor suppressor p53 in controlling cell survival and proliferation are inextricably linked as “Yin and Yang” partners in normal cells to maintain tissue homeostasis: c-Myc induces the expression of ARF tumor suppressor (p14ARF in human and p19ARF in mouse) that binds to and inhibits mouse double minute 2 homolog (MDM2) leading to p53 activation, whereas p53 suppresses c-Myc through a combination of mechanisms involving transcriptional inactivation and microRNA-mediated repression. Nonetheless, the regulatory interactions between c-Myc and p53 are not retained by cancer cells as is evident from the often-imbalanced expression of c-Myc over wildtype p53. Although p53 repression in cancer cells is frequently associated with the loss of ARF, we disclose here an alternate mechanism whereby c-Myc inactivates p53 through the actions of the c-Myc-Inducible Long noncoding RNA Inactivating P53 (MILIP). MILIP functions to promote p53 polyubiquitination and turnover by reducing p53 SUMOylation through suppressing tripartite-motif family-like 2 (TRIML2). MILIP upregulation is observed amongst diverse cancer types and is shown to support cell survival, division and tumourigenicity. Thus our results uncover an inhibitory axis targeting p53 through a pan-cancer expressed RNA accomplice that links c-Myc to suppression of p53.

Specific Deletion of p16INK4a with Retention of p19ARF Enhances the Development of Invasive Oral Squamous Cell Carcinoma

The cyclin-dependent kinase inhibitor 2A (CDKN2A)/alternate reading frame (ARF) locus consists of two overlapping tumor suppressor genes, p16INK4a and p14ARF (p19ARF in mice), encoding two unrelated proteins in alternative reading frames. Previous reports suggest that p16INK4a and p14ARF alterations independently exhibit differential roles, and p16INK4a is more closely associated with a poor prognosis in oral cancer. However, the role of p16INK4a-specific loss in oral squamous cell carcinogenesis remains unclear. The authors assessed chemical carcinogen 4-nitroquinoline 1-oxide (4NQO)-induced multistep oral squamous cell carcinogenesis in mice carrying p16INK4a-specific loss with retention of the p19ARF gene (p16INK4a-/-). 4NQO-treated p16-/- mice exhibited a higher incidence and multiplicity of oral squamous cell carcinoma (OSCC) development relative to 4NQO-treated wild-type mice. 4NQO-treated p16INK4a-/- OSCC cells exhibited higher proliferation and up-regulation of Arf, transcription factor E2f1, tumor protein p63 (tp63), and oncogenic ΔNp63, an isoform p63, compared with observations in 4NQO-treated wild-type OSCC cells. Furthermore, the overexpression of oncogenic ΔNp63 was associated with human OSCC. In conclusion, these results in mice indicate the biological significance of p16INK4a-specific loss with retention of p19ARF in oral squamous cell carcinogenesis, and ΔNp63 may be a potential target for OSCC.

A cis-element within the ARF locus mediates repression of p16INK4A expression via long-range chromatin interactions

Loss of function of CDKN2A/B, also known as INK4/ARF [encoding p16INK4A, p15INK4B, and p14ARF (mouse p19Arf)], confers susceptibility to cancers, whereas its up-regulation during organismal aging provokes cellular senescence and tissue degenerative disorders. To better understand the transcriptional regulation of p16INK4A, a CRISPR screen targeting open, noncoding chromatin regions adjacent to p16INK4A was performed in a human p16INK4A-P2A-mCherry reporter cell line. We identified a repressive element located in the 3' region adjacent to the ARF promoter that controls p16INK4A expression via long-distance chromatin interactions. Coinfection of lentiviral dCas9-KRAB with selected single-guide RNAs against the repressive element abrogated the ARF/p16INK4A chromatin contacts, thus reactivating p16INK4A expression. Genetic CRISPR screening identified candidate transcription factors inhibiting p16INK4A regulation, including ZNF217, which was confirmed to bind the ARF/p16INK4A interaction loop. In summary, direct physical interactions between p16INK4A and ARF genes provide mechanistic insights into their cross-regulation.

Dual Role of the Alternative Reading Frame ARF Protein in Cancer.

The CDKN2a/ARF locus expresses two partially overlapping transcripts that encode two distinct proteins, namely p14ARF (p19Arf in mouse) and p16INK4a, which present no sequence identity. Initial data obtained in mice showed that both proteins are potent tumor suppressors. In line with a tumor-suppressive role, ARF-deficient mice develop lymphomas, sarcomas, and adenocarcinomas, with a median survival rate of one year of age. In humans, the importance of ARF inactivation in cancer is less clear whereas a more obvious role has been documented for p16INK4a. Indeed, many alterations in human tumors result in the elimination of the entire locus, while the majority of point mutations affect p16INK4a. Nevertheless, specific mutations of p14ARF have been described in different types of human cancers such as colorectal and gastric carcinomas, melanoma and glioblastoma. The activity of the tumor suppressor ARF has been shown to rely on both p53-dependent and independent functions. However, novel data collected in the last years has challenged the traditional and established role of this protein as a tumor suppressor. In particular, tumors retaining ARF expression evolve to metastatic and invasive phenotypes and in humans are associated with a poor prognosis. In this review, the recent evidence and the molecular mechanisms of a novel role played by ARF will be presented and discussed, both in pathological and physiological contexts.

Perspectives for cancer immunotherapy mediated by p19Arf plus interferon-beta gene transfer

While cancer immunotherapy has gained much deserved attention in recent years, many areas regarding the optimization of such modalities remain unexplored, including the development of novel approaches and the strategic combination of therapies that target multiple aspects of the cancer-immunity cycle. Our own work involves the use of gene transfer technology to promote cell death and immune stimulation. Such immunogenic cell death, mediated by the combined transfer of the alternate reading frame (p14ARF in humans and p19Arf in mice) and the interferon-β cDNA in our case, was shown to promote an antitumor immune response in mouse models of melanoma and lung carcinoma. With these encouraging results, we are now setting out on the road toward translational and preclinical development of our novel immunotherapeutic approach. Here, we outline the perspectives and challenges that we face, including the use of human tumor and immune cells to verify the response seen in mouse models and the incorporation of clinically relevant models, such as patient-derived xenografts and spontaneous tumors in animals. In addition, we seek to combine our immunotherapeutic approach with other treatments, such as chemotherapy or checkpoint blockade, with the goal of reducing dosage and increasing efficacy. The success of any translational research requires the cooperation of a multidisciplinary team of professionals involved in laboratory and clinical research, a relationship that is fostered at the Cancer Institute of Sao Paulo.

Small mitochondrial Arf (smArf) protein corrects p53-independent developmental defects of Arf tumor suppressor-deficient mice

The mouse p19Arf (human p14ARF) tumor suppressor protein, encoded in part from an alternative reading frame of the Ink4a (Cdkn2a) gene, inhibits the Mdm2 E3 ubiquitin ligase to activate p53. Arf is not expressed in most normal tissues of young mice but is induced by high thresholds of aberrant hyperproliferative signals, thereby activating p53 in incipient tumor cells that have experienced oncogene activation. The single Arf mRNA encodes two distinct polypeptides, including full-length p19Arf and N-terminally truncated and unstable p15smArf ("small mitochondrial Arf") initiated from an internal in-frame AUG codon specifying methionine-45. Interactions of p19Arf with Mdm2, or separately with nucleophosmin (NPM, B23) that localizes and stabilizes p19Arf within the nucleolus, require p19Arf N-terminal amino acids that are not present within p15smArf We have generated mice that produce either smARF alone or M45A-mutated (smArf-deficient) full-length p19Arf proteins. BCR-ABL-expressing pro/pre-B cells producing smArf alone are as oncogenic as their Arf-null counterparts in generating acute lymphoblastic leukemia when infused into unconditioned syngeneic mice. In contrast, smArf-deficient cells from mice of the ArfM45A strain are as resistant as wild-type Arf+/+ cells to comparable oncogenic challenge and do not produce tumors. Apart from being prone to tumor development, Arf-null mice are blind, and their male germ cells exhibit defects in meiotic maturation and sperm production. Although ArfM45A mice manifest the latter defects, smArf alone remarkably rescues both of these p53-independent developmental phenotypes.

MMP7 interacts with ARF in nucleus to potentiate tumor microenvironments for prostate cancer progression in vivo.

ARF couples with TP53 in a canonical signaling pathway to activate cellular senescence for tumor suppressive function under oncogenic insults. However, the mechanisms on aberrant elevation of ARF in cancers are still poorly understood. We previously showed that ARF (p14ARF in human and p19Arf in mouse) elevation correlates with PTEN loss and stabilizes SLUG to reduce cell adhesion in prostate cancer (PCa). Here we report that ARF is essential for MMP7 expression, E-Cadherin decrease and the anchorage loss to the extracellular matrix (ECM) in PCa in vitro and in vivo. We found that Mmp7 is aberrantly elevated in cytosol and nucleus of malignant prostate tumors of Pten/Trp53 mutant mice. Interestingly, p19Arf deficiency strikingly decreases Mmp7 levels but increases E-Cadherin in Pten/Trp53/p19Arf mice. ARF knockdown markedly reduces MMP7 in human PCa cells. Conversely, tetracycline-inducible expression of ARF increases MMP7 with a decrease of E-Cadherin in PCa cells. Importantly, MMP7 physically binds ARF to show the co-localization in nucleus. Co-expression of MMP7 and ARF promotes cell migration, and MMP7 knockdown decreases wound healing in PCa cells. Furthermore, MMP7 elevation correlates with ARF expression in advanced human PCa. Our findings reveal for the first time that the crosstalk between ARF and MMP7 in nucleus contributes to ECM network in tumor microenvironments in vivo, implicating a novel therapeutic target for advanced PCa treatment.

Abstract 1264: Conditional mouse and zebrafish models of INK4-mediated tumor suppression reveal ARF-independent regulation of cellular senescence

Abstract The INK4b-ARF-INK4a locus on human chromosome 9p21 is a hot spot genomic region that undergoes frequent inactivation or deletion in a wide spectrum of human cancers. The locus encodes three cell cycle inhibitory proteins: p15INK4b encoded by CDKN2b, p16INK4a encoded by CDKN2a and p14ARF (p19Arf in mice) encoded by an alternative reading frame (ARF) of CDKN2a. We have recently identified a homologous INK4 genomic locus in zebrafish that is surprisingly devoid of ARF sequences, and encodes a single zebrafish ink4ab gene that functions to activate stress-induced senescence. Therefore, evolution of the mammalian INK4 locus to include multiple tumor suppressors sharing regulatory features could attribute to stronger mechanisms of tumor suppression in these longer-lived vertebrates. Oxidative and/or oncogenic stress provokes cellular senescence involving the retinoblastoma (Rb) and ARF/p53 pathways, leading to silencing of growth-promoting genes by methylation of histone H3 lysine 9 (H3K9me) with the histone methyltransferase Suv39h1. We found that ink4ab deficient zebrafish had deregulated Rb signaling and senescence responses, and reduced suv39h1 expression levels that led to poor overall survival. Ink4ab deficient zebrafish had increased lymphocyte proliferation, splenomegaly, and developed multiple spontaneous tumors including metastatic melanoma, osteosarcoma, hepatocellular adenoma, leukemias and myelodysplastic disorders. Moreover, zebrafish heterozygous for both p53 and ink4ab mutations displayed significantly higher tumor incidence compared to p53 heterozygotes with wild-type ink4ab, indicating that ink4ab haploinsufficiency promotes tumorigenesis. To this end, the combined deficiency of ink4ab and p53 accelerates tumor latency, likely by reversing cellular senescence responses. To further examine ARF-independent senescence regulatory roles of INK4 products in a mammalian model, we have generated conditional mice deficient for all three INK4 open reading frames in the adult hematopoietic system. Within seven months, homozygous Ink4a/Ink4b/ARF-/- mice exhibited marked splenomegaly and developed spontaneous tumors, including leukemias and soft tissue sarcomas. Peripheral blood, splenic and bone marrow analyses of homozygous mutant mice showed deregulated lymphocyte development, uncontrolled proliferation, apoptosis and Suv39h1-dependent senescence when compared to heterozygous and wild type mice. These results identify ARF-independent and Suv39h1-dependent senescence regulatory and tumor suppressor mechanisms whose inactivation permits tumor formation in response to oncogenic and/or oxidative stress. Our models offer the opportunity to reveal novel paradigms for promoting cellular senescence as an alternative strategy for cancer prevention and therapy. Citation Format: Kathleen Flaherty, Daniel Jones, Shamila Yussuf, Stephani Davis, Eric Huselid, Wei Wang, Monica Bartucci, Hatem E. Sabaawy. Conditional mouse and zebrafish models of INK4-mediated tumor suppression reveal ARF-independent regulation of cellular senescence. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1264. doi:10.1158/1538-7445.AM2015-1264

Role of Ink4a/Arf Locus in Beta Cell Mass Expansion under Physiological and Pathological Conditions

The ARF/INK4A (Cdkn2a) locus includes the linked tumour suppressor genes p16INK4a and p14ARF (p19ARF in mice) that trigger the antiproliferative activities of both RB and p53. With beta cell self-replication being the primary source for new beta cell generation in adult animals, the network by which beta cell replication could be increased to enhance beta cell mass and function is one of the approaches in diabetes research. In this review, we show a general view of the regulation points at transcriptional and posttranslational levels of Cdkn2a locus. We describe the molecular pathways and functions of Cdkn2a in beta cell cycle regulation. Given that aging reveals increased p16Ink4a levels in the pancreas that inhibit the proliferation of beta cells and decrease their ability to respond to injury, we show the state of the art about the role of this locus in beta cell senescence and diabetes development. Additionally, we focus on two approaches in beta cell regeneration strategies that rely on Cdkn2a locus negative regulation: long noncoding RNAs and betatrophin.

In the race for protection, ARF comes second

In the race for protection, ARF comes second

ARF represses androgen receptor transactivation in prostate cancer.

Androgen receptor (AR) signaling is essential for prostate cancer (PCa) development in humans. The initiation of prostate malignancy and progression to a castration-resistant stage are largely contributed by the modulation of AR activity through its coregulatory proteins. We and others previously reported that p14 alternative reading frame (ARF) expression is positively correlated with the disease progression and severity of PCa. Here, we provide evidence that p14ARF physically interacts with AR and functions as an AR corespressor in both an androgen-dependent and androgen-independent manner. Endogenous ARF (p14ARF in human and p19ARF in mouse) and AR colocalize in both human PCa cells in vitro and PCa tissues of mouse and human in vivo. Overexpression of p14ARF in PCa cells significantly attenuates the activities of androgen response region (ARR2)-probasin and prostate-specific antigen (PSA) promoters. The forced expression of p14ARF in cells resulted in a suppression of PSA and NK transcription factor locus 1 (NKX3.1) expression. Conversely, knockdown of endogenous p14ARF in human PCa cells with short hairpin RNA enhanced AR transactivation activities in a dose-dependent and p53-independent manner. Furthermore, we demonstrated that p14ARF binds to both the N-terminal domain and the ligand-binding domain of AR, and the human double minute 2 (HDM2)-binding motif of p14ARF is required for the interaction of p14ARF and AR proteins. p14ARF perturbs the androgen-induced interaction between the N terminus and C terminus of AR. Most importantly, we observed that the expression of PSA is reversely correlated with p14ARF in human prostate tissues. Taken together, our results reveal a novel function of ARF in modulation of AR transactivation in PCa.

Abstract C23: Blocking endogenous TBX2 expression abrogates prostate cancer bone metastasis in a xenograft mouse model

Abstract Identifying factors that mediate the metastatic spread and subsequent remodeling of the bone is highly relevant to successful therapeutic intervention in advanced human Prostate Cancer (PCa). TBX2, a transcription factor that belongs to the T-box family is known to play critical roles in the embryonic development of the organisms. TBX2 acts as a transcriptional repressor by negatively regulating p21 in humans and p19 ARF in mice. Further, TBX2 has been reported to interact with Retinoblastoma (Rb) and mediates its functional specificity. Previously, TBX2 was found to be over-expressed in BRCA1 and BRCA2 associated breast tumors and melanoma and pancreatic cancer cell lines. Interestingly, a recent report found that TBX2 expression is increased 10-fold at the mRNA level in an androgen-independent bone lesion from a human PCa patient when compared to a bone lesion from an androgen-responsive PCa patient. TBX2 was found to be over-expressed in more aggressive human PCa cell lines and that TBX2 expression correlates with BMP2 expression in these cell lines. Addition of BMP2 to androgen-responsive LAPC4 human prostate cancer cells robustly induces TBX2 expression. Androgen-independent PC3 cells, expressing wild-type TBX2, when infected with the dominant negative TBX2 retroviral construct (PC3-TBX2 DN), display decreased PTHrP, RANKL and SMAD 1,5,8. These results suggest that TBX2, a downstream BMP2 target, csignificantly controls cell signaling network in prostate cancer cells. In vivo, PC3-TBX2 DN cells tagged with firefly luciferase when injected intra-cardiacally in athymic mice lost their ability to metastasize to bone and soft tissues in mice when compared with the control vector infected cells. These results are supported by reduced invasive ability of the PC3-TBX2 DN tumors at the interface between tumor and kidney when grafted under the sub-renal capsule of the male athymic nude mice. Further, utilizing the intra-tibial mouse model, we have found that blocking endogenous TBX2 in PC3 cells significantly reduces their ability to grow in the bone microenvironment. Interestingly, blocking TBX2 does not affect tumor size suggesting that TBX2 is a metastasis suppressor gene. Further, the non-metastatic TrampC2 mouse prostate cancer cells stably over-expressing RANKL show increased Tbx2 expression and develop soft tissue metastases when injected intra-cardiacally in C56/Bl6 immune competent mice. These results suggest that Tbx2 and RANKL pathways cross-talk with each other in prostate cancer metastasis. In summary, we have found that blocking endogenous TBX2 levels in human PCa cells leads to decreased invasion, metastasis and growth in the bone microenvironment, and that TBX2 potentially mediates its effect through BMP2 and RANKL pathways. TBX2, therefore, is a tumor metastasis suppressor and therapeutic target for PCa metastasis. Citation Format: Srinivas Nandana, Manisha Tripathi, Murali Gururajan, Gina Chu, Robert J. Matusik, Leland W.K. Chung. Blocking endogenous TBX2 expression abrogates prostate cancer bone metastasis in a xenograft mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C23.

REGγ Mediated Regulation of p21<sup>Waf/Cip1</sup>, p16<sup>INK4a</sup> and p14<sup>ARF</sup>/p19<sup>ARF</sup> <i>in Vivo</i>

p21Waf/Cip1, p16INK4a and p14ARF (p19ARF in mice) have been demonstrated to be degraded by REGγ-proteasome pathway in an ATP- and ubiquitin-independent manner in vitro. However, the in vivo roles of REGγ mediated-degradation of p21Waf/Cip1, p16INK4a and p14ARF remain unclear. In this study, we showed enhanced expression of p21Waf/Cip1, p16INK4a and p19ARF in multiple tissues from REGg–/– mice compared to REGg+/+ mice. Furthermore, we examined the expression of p21Waf/Cip1, p16INK4a and p14ARF in different cancer tissues and observed that the REGγ protein levels were highly expressed in different human cancers while the level of p21Waf/Cip1, p16INK4a and p14ARF appears to be inversely correlated. These results demonstrate that REGγ may exert its function in physiological and pathological conditions through degradation of p21Waf/Cip1, p16INK4a and p14ARF in vivo.

Abstract 3850: CDKN2a deficiency exposes a requirement for RhoA in maintenance of non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related death world-wide. Tumorigenesis is a multistep process that involves several genetic aberrations. For example, K-RAS (onc-K-RAS), the most commonly mutated oncogene in human cancer, is expressed in about 25% of NSCLC. In addition, NSCLC often carry mutations or epigenetic modifications that affect the expression of the CDKN2a locus, which comprises the p16INK4a and the p14ARF (p19ARF in mice) tumor suppressor genes, which in turn regulate the retinoblastoma and the p53 tumor suppressor pathways. NSCLC tumorigenesis is faithfully recapitulated in transgenic mice where onc-K-Ras expression invariably induces adenomas and, after a long latency, adenocarcinomas. To characterize the consequence of concomitant onc-K-RAS expression and CDKN2a inactivation, we generated p16CDK4/p19ARF null mice expressing onc-K-Ras in the respiratory epithelium. We determined that Cdkn2a deficiency promotes rapid progression of lung adenomas to adenocarcinomas, which display aggressive features. We determined the status of activation of oncogenic signaling networks downstream of onc-K-Ras with phosphospecific antibodies and by expression profiling. We discovered that NSCLC that arises in CDKN2A deficient mice display dysregulation of RhoA, a small GTPase implicated in the regulation of the cytoskeleton, cell adhesion, and motility of cancer cells in culture. These activities are pertinent to tumorigenesis, however it is not known whether RhoA plays a role in the maintenance of established tumors. Surprisingly, we determined that RhoA is required for the survival of NSCLC derived lines expressing onc-K-RAS and deficient for either INK4a or the p53 tumor suppressor both when cultured in vitro and as xenografts in vivo. Cell death was due to a proapoptotic program and was accompanied by disruption of the cytoskeleton. Moreover, by performing loss and gain of function experiments we identified focal adhesion kinase (FAK) protein as the principal downstream target of RhoA responsible for this effect. Experiments in vivo with specific inhibitors are ongoing and will be presented at the meeting. Our findings reveal a previously unappreciated context dependent requirement of the RhoGTPase to maintain viability of established NSCLC. We propose that RhoA and its signaling network are novel therapeutic targets in NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3850. doi:10.1158/1538-7445.AM2011-3850

Abstract 2958: Cyclin D1 regulates the ARF and INK4a promoters

Abstract Cyclin D1 is a member of cyclin protein family, which drives the cell cycle transition from G1 to S phase. The cyclin D1 protein is overexpressed in ∼50% of human breast cancers and is associated with short survival of patients when the gene is amplified. Cyclin D1 forms holoenzymes with cyclin-dependent protein kinase (Cdk) 4 and 6, and the Cyclin D-Cdk4/6 complex phosphorylates and inactivates the pRb retinoblastoma tumor suppressor, releasing E2F transcriptional factors from Rb inhibition. The pRb phosphorylation is blocked by p16INK4a, encoded by the CDKN2A locus, which inhibits the kinase activity of Cyclin D-Cdk4/6 complex. The CDKN2A locus also encodes another tumor suppressor protein named p14ARF (p19Arf in mouse), which stabilizes p53 by inhibiting MDM2-dependent degradation. Previous studies from our laboratory showed that the cyclin D1 collaborated with the Dmp1 (cyclin D binding myb-like protein 1) tumor suppressor to activate the Arf promoter. To further demonstrate the effect of cyclin D1 on p19Arf and p16Ink4a promoter activity, NIH 3T3 cells and MEF cells were transfected a construct expressing the luciferase reporter gene linked with the Arf promoter together with vector or cyclin D1. Using luciferase reporter assay, we found that the Arf promoter was activated upon cyclin D1 overexpression in both cell lines, and the activation was abrogated when cells were co-transfected with E2F-DB, which displaces endogenous E2Fs and acts in a dominant-negative manner. Consistent with reporter assays, the Arf mRNA was increased in MEFs overexpressing cyclin D1, compared to those infected with vector only. By performing chromatin immunoprecipitation in tumors of mammary gland from MMTV-neu transgenic mice that overexpress cyclin D1, we found that cyclin D1 can interact with the Arf promoter although it cannot directly bind to the Arf promoter region in electrophoretic mobility shift assay using recombinant cyclin D1 protein. To identify the effect of cyclin D1 on p16Ink4a promoter, we performed luciferase reporter assay in NIH 3T3 cells by co-transfecting Ink4a promoter together with vector or cyclin D1. We found that the Ink4a promoter activity was increased upon cyclin D1 overexpression, and the activation was not abrogated when cells were transfected with cyclin D1-K114E that does not bind to CDK4. Taken together, our data suggest that overexpression of cyclin D1 activates both p19Arf and p16Ink4a promoters to prevent neoplastic growth of incipient cancer cells, and the activation is mediated by indirect binding of cyclin D1 to the promoter regions. We are currently mapping the cyclin D1-responsive elements on the Arf and Ink4a promoters to identify transcription factors responsible for Arf/Ink4a induction by cyclin D1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2958. doi:10.1158/1538-7445.AM2011-2958