Wild-type p16INK4A Research Articles

Abstract 2883: Impact of novel CDKN2A/p16INK4a 5’UTR variants predisposing to melanoma on p16 translational regulation

Abstract CDKN2A/p16INK4a is an important tumor-suppressor gene whose dysregulation is associated with melanoma. We have recently demonstrated that the p16INK4a expression can be modulated by IRES-dependent mRNA translation and this regulation might have an important role during tumorigenesis. We also identified YBX1 as RNA binding protein targeting and stimulating p16 mRNA translation efficiency. The identified IRES function was particularly active in hypoxia and inhibition of mTOR. This post-transcriptional regulatory mechanism would potentially be a target for mutational inactivation during melanomagenesis. Indeed, we previously showed that p16INK4a 5’UTR variants found in melanoma patients with a family predisposition can have a negative effect on p16 translation. Single Nucleotide Variants (SNVs) have been investigated during routine testing of CDKN2A/p16INK4a in Italian, English and French melanoma patients followed by GenoMEL, the International Melanoma Genetics Consortium and a total of 17 germline variants were identified in a cohort of nearly 6000 patients. These p16INK4a 5’UTR 17 variants were studied with multiple approaches, that included mono- and bi-cistronic reporter assays, western blot of endogenous protein, and quantification of allelic representation after polysomal profiling to investigate their impact on p16INK4a mRNA translation regulation. We devised a classification score based on the concordance between functional assays and the extent of dysfunction displayed by each variant compared to the wild type. Variants are classified as neutral (score 0) when no difference was observed in at least 3 assays. This applied to: c.-14C>T, c.-20A>G, c.-25C>T+c.-180G>A, c.-30G>A, c.-40C>T, c.-45G>A, c.-59C>G, c.-87T>A, c.-252A>T. Variants were considered as potential mutations when a defect was measured in either one or two assays (score 1-2; c.-42T>A and c.-67G>C variants). Finally, we classified variants as causal mutations when three or more assays showed impairment (score >3). This applied to: c.-27del23, c.-56G>T, c.-93-91delAGG, as well as to c.-21C>T and c.-34G>T, which were already considered as a causal mutations. We have also determined the structure of the wild type p16INK4a 5’UTR by Selective Hydroxyl Acylation or SHAPE assay. The variant c.-42T>A encompassing the predicted YBX1 binding site was also studied and shown to induce a local change in conformation. The structure of all p16INK4a 5’UTR variants examined so far is being investigated as well as their impact on the interaction of RNA binding proteins such as YBX1, SRSF1 and RBM4. Our data indicate that the sequencing of the entire p16INK4a 5’UTR should be included in routine screening of melanoma families as nearly half of SNVs tested so far displayed a negative impact on the p16 mRNA translation efficiency. Citation Format: Alessandra Bisio, Elisa Latorre, Virginia Andreotti, Brigitte Bressac-de Paillerets, Mark Harland, Odile Cabaret, Julia Newton-Bishop, Lorenza Pastorino, William Bruno, Roberto Bertorelli, Veronica De Sanctis, Chiara Menin, Gilberto Fronza, Paola Queirolo, Giovanna Bianchi Scarrà, Robert C. Spitale, Alessandro Provenzani, Alberto Inga, Paola Ghiorzo. Impact of novel CDKN2A/p16INK4a 5’UTR variants predisposing to melanoma on p16 translational regulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2883.

Prevalence ofMITFp.E318K in Patients With Melanoma Independent of the Presence ofCDKN2ACausative Mutations

The main high-penetrance melanoma susceptibility gene is CDKN2A, encoding p16INK4A and p14ARF. The gene MITF variant p.E318K also predisposes to melanoma and renal cell carcinoma. To date, the prevalence of MITF p.E318K and its clinical and phenotypical implications has not been previously assessed in a single cohort of Spanish patients with melanoma or in p16INK4A mutation carriers. To evaluate the prevalence of MITF p.E318K in Spanish patients with melanoma and assess the association with clinical and phenotypic features. A hospital-based, case-control study was conducted at the Melanoma Unit of Hospital Clinic of Barcelona, with MITF p.E318K genotyped in all patients using TaqMan probes. We included 531 patients: 271 patients with multiple primary melanoma (MPM) without mutations affecting p16INK4A (wild-type p16INK4A); 191 probands from melanoma-prone families with a single melanoma diagnosis and without mutations affecting p16INK4A, and 69 probands from different families carrying CDKN2A mutations affecting p16INK4A. A population-based series of 499 age- and sex-matched cancer-free individuals from the Spanish National Bank of DNA were included as controls. Patients were recruited between January 1, 1992, and June 30, 2014; data analysis was conducted from September 1 to November 30, 2014. The genetic results of the MITF p.E318K variant were correlated with clinical and phenotypic features. Among the 531 patients, the prevalence of the MITF p.E318K variant was calculated among the different subsets of patients included and was 1.9% (9 of 462) in all melanoma patients with wild-type p16INK4A, 2.6% (7 of 271) in those with MPM, and 2.9% (2 of 69) in the probands of families with p16INK4A mutations. With results reported as odds ratio (95% CI), the MITF p.E318K was associated with an increased melanoma risk (3.3 [1.43-7.43]; P < .01), especially in MPM (4.5 [1.83-11.01]; P < .01) and high nevi count (>200 nevi) (8.4 [2.14-33.19]; P < .01). Two fast-growing melanomas were detected among 2 MITF p.E318K carriers during dermatologic digital follow-up. In addition to melanoma risk, MITF p.E318K is associated with a high nevi count and could play a role in fast-growing melanomas. Testing for MITF p.E318K should not exclude patients with known mutations in p16INK4A. Strict dermatologic surveillance, periodic self-examination, and renal cell carcinoma surveillance should be encouraged in this context.

The 5'-untranslated region of p16INK4a melanoma tumor suppressor acts as a cellular IRES, controlling mRNA translation under hypoxia through YBX1 binding.

CDKN2A/p16INK4a is an essential tumor suppressor gene that controls cell cycle progression and replicative senescence. It is also the main melanoma susceptibility gene. Here we report that p16INK4a 5'UTR mRNA acts as a cellular Internal Ribosome Entry Site (IRES). The potential for p16INK4a 5'UTR to drive cap-independent translation was evaluated by dual-luciferase assays using bicistronic and monocistronic vectors. Results of reporters' relative activities coupled to control analyses for actual bicistronic mRNA transcription, indicated that the wild type p16INK4a 5'UTR could stimulate cap-independent translation. Notably, hypoxic stress and the treatment with mTOR inhibitors enhanced the translation-stimulating property of p16INK4a 5'UTR. RNA immunoprecipitation performed in melanoma-derived SK-Mel-28 and in a patient-derived lymphoblastoid cell line indicated that YBX1 can bind the wild type p16INK4a mRNA increasing its translation efficiency, particularly during hypoxic stress. Modulation of YBX1 expression further supported its involvement in cap-independent translation of the wild type p16INK4a but not a c.-42T>A variant. RNA SHAPE assays revealed local flexibility changes for the c.-42T>A variant at the predicted YBX1 binding site region. Our results indicate that p16INK4a 5'UTR contains a cellular IRES that can enhance mRNA translation efficiency, in part through YBX1.

Abstract B05: Targeting BRAF and CDK4 in BRAF mutant melanoma induces sustained tumor regression

Abstract Background: The p16-cyclinD-CDK4-RB1 pathway (CDK4 pathway) is deregulated in 90% of melanomas. The main outcome of CDK4 activation is the phosphorylation and thus, inhibition of RB1 leading to G1–S cell-cycle transition. In addition, CDK4 directly phosphorylates other proteins that promote cell-cycle progression and inhibit both cell senescence and apoptosis. The MAPK/ERK pathway is deregulated in 90% of melanoma and enhances CDK4 pathway signaling through increasing cyclin D1 expression. We have previously demonstrated in a panel of melanoma cell lines that the majority of cell lines were sensitive to a CDK4/6 inhibitor (Palbociclib) and consistent with the concept of CDK4 pathway-dependent oncogene addiction, cell lines with loss of functional p16INK4A were significantly more sensitive to Palbociclib than cells with wild-type p16INK4A. We hypothesized that dual targeting the MAPK/ERK and CDK4 pathways would lead to robust inhibition of the CDK4/Cyclin D complex and consequently induce greater tumor regression than single agent treatment. Results: In vitro, PLX4720, Palbociclib or their combination inhibited BRAF mutant melanoma cell line proliferation and clonogenic survival, however, only with the combination treatment group was a rapid sustained inhibition achieved. Enlarged flattened cells and a significant increase in SA-β-galactosidase staining indicated that the dual treatment resulted in robust cellular senescence. In a BRAFV600E mutant A375 xenograft model, Palbociclib and PLX4720 initially induced tumor regression and tumor stasis respectively, but resistance eventually developed. In contrast, in the combination treatment, tumors regressed and sustained inhibition of tumor growth was maintained. Preliminary biomarker studies indicate resistance to PLX4720 was due to reactivation MAPK/ERK pathway and resistance to Palbociclib to partial restoration of phosphorylated RB1. Conclusions: Combination therapy of MAPK/ERK pathway inhibitors with CDK4 inhibitors is likely to be an effective treatment for BRAF mutant melanoma and in addition delay resistance to single agent targeted therapy. Citation Format: Karen E. Sheppard, Cullinane Carleen, Laura Kirby, Kelly Waldeck, Richard Young, Claire Martin, Todd VanArsdale, Sophia Randolph, Grant A. McArthur. Targeting BRAF and CDK4 in BRAF mutant melanoma induces sustained tumor regression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B05.

Abstract 3384: An internal ribosomal entry site in the 5’-untranslated region of p16INK4a mRNA provides a novel mechanism for the regulation of its translation

Abstract In mammalian systems, controlled progression through the cell cycle is essential for normal proliferation and its loss is a hallmark of malignancy. p16INK4a inhibits the cyclin-dependent kinases CDK4 and CDK6, thereby keeping the retinoblastoma protein (pRB) in a hypo-phosphorylated state, which can lead to G1/S checkpoint activation. In addition p16INK4a plays a crucial role in the process of replicative senescence. p16INK4a loss or inactivation is associated with predisposition to melanoma, pancreatic cancer and other malignancies, highlighting its recognized function as a tumor suppressor. Given its critical role in cell homeostasis, there is much interest in understanding the molecular regulators of p16INK4a expression. Here we report that p16 belongs to the expanding group of proteins whose translation is influenced by sequence/structural features of the 5’UTR mRNA that are endowed of cellular Internal Ribosome Entry Site (IRES) activity. To study the potential for p16INK4a 5’UTR to drive cap-independent translation we developed a dual-luciferase assay using a bicistronic vector (named pRuF), where wild type or deletion mutants of the p16INK4a 5’UTRs were cloned as intervening sequence between Renilla and Firefly luciferase cDNAs. The p16INK4a 5’UTR sequence in inverted orientation was included as additional control. Results of reporters’ relative activity coupled to control analyses of actual bicistronic mRNA transcription, indicated that the wild type p16INK4a 5’UTR could stimulate Firefly luciferase translation. The IRES-like activity could not be mapped to a specific region of the 5’UTR based on results with deletion constructs, and was two-fold stronger compared to the cMYC 5’UTR, a known cellular IRES used as a control. Notably, hypoxic stress in particular, but also the treatment with mTOR inhibitors, enhanced the translation-stimulating property of the p16INK4a 5’UTR. RNA immuno-precipitation (RIP) assays performed in the p16-positive melanoma-derived cell line SK-Mel-28 suggest that the RNA-binding protein YBX-1, known to act in translation control, can participate in p16 INK4a translation. Experiments were YBX-1 was over-expressed or knocked-down by si-RNA confirmed its involvement in p16INK4a cap-independent translational regulation. Taken collectively, our results suggest that the 5’UTR region can modulate p16INK4a mRNA translation efficiency. Citation Format: Alessandra Bisio, Elisa Latorre, Virginia Andreotti, Alessandro Provenzani, Giovanna Bianchi- Scarrà, Paola Ghiorzo, Alberto Inga. An internal ribosomal entry site in the 5’-untranslated region of p16INK4a mRNA provides a novel mechanism for the regulation of its translation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3384. doi:10.1158/1538-7445.AM2014-3384

P16INK4a‐induced senescence is disabled by melanoma‐associated mutations

The p16INK4a-Rb tumour suppressor pathway is required for the initiation and maintenance of cellular senescence, a state of permanent growth arrest that acts as a natural barrier against cancer progression. Senescence can be overcome if the pathway is not fully engaged, and this may occur when p16INK4a is inactivated. p16INK4a is frequently altered in human cancer and germline mutations affecting p16INK4a have been linked to melanoma susceptibility. To characterize the functions of melanoma-associated p16INK4a mutations, in terms of promoting proliferative arrest and initiating senescence, we utilized an inducible expression system in a melanoma cell model. We show that wild-type p16INK4a promotes rapid cell cycle arrest that leads to a senescence programme characterized by the appearance of chromatin foci, activation of acidic β-galactosidase activity, p53 independence and Rb dependence. Accumulation of wild-type p16INK4a also promoted cell enlargement and extensive vacuolization independent of Rb status. In contrast, the highly penetrant p16INK4a variants, R24P and A36P failed to arrest cell proliferation and did not initiate senescence. We also show that overexpression of CDK4, or its homologue CDK6, but not the downstream kinase, CDK2, inhibited the ability of wild-type p16INK4a to promote cell cycle arrest and senescence. Our data provide the first evidence that p16INK4a can initiate a CDK4/6-dependent autonomous senescence programme that is disabled by inherited melanoma-associated mutations.

The PAX3-FKHR Fusion Gene of Rhabdomyosarcoma Cooperates with Loss of p16INK4A to Promote Bypass of Cellular Senescence

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood and adolescence. Despite advances in therapy, patients with a histologic variant of rhabdomyosarcoma known as alveolar rhabdomyosarcoma (ARMS) have a 5-year survival of <30%. ARMS is characterized by a chromosomal translocation generating the PAX3-FKHR fusion gene. However, ectopic expression of PAX3-FKHR often induces inhibition of cell proliferation, or cell death, when expressed in nonmuscle cells. This prompted us to explore the effect of expressing PAX3-FKHR in more relevant cells, specifically primary human skeletal muscle cells because these cells can be converted to a tumorigenic state that mimics rhabdomyosarcoma. PAX3-FKHR expression promoted both fetal and postnatal primary human skeletal muscle cell precursors to bypass the senescence growth arrest checkpoint. This bypass was accompanied by epigenetic DNA methylation of the p16(INK4A) promoter and correspondingly a loss of expression of this tumor suppressor. Knockdown of p16(INK4A) cooperated with PAX3-FKHR to drive proliferation past senescence, whereas reintroduction of wild-type p16(INK4A) in post-senescent cells caused growth arrest. Thus, PAX3-FKHR acts in concert with loss of p16(INK4A) to promote inappropriate proliferation of skeletal muscle cells. This association between PAX3-FKHR expression and p16(INK4A) loss was seen in human ARMS tumor tissue, as both human rhabdomyosarcoma cell lines and tissue microarrays showed a trend toward down-regulation of p16(INK4A) protein in alveolar subsets. We surmise that the generation of the PAX3-FKHR fusion protein may require loss of p16(INK4A) to promote malignant proliferation of skeletal muscle cells as an early step in ARMS tumorigenesis.

Inhibition of the Extracellular Signal-regulated Kinase/Mitogen-activated Protein Kinase Pathway Decreases DNA Methylation in Colon Cancer Cells

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) pathway is a critical intermediary for cell proliferation, differentiation, and survival. In the human colon cancer cell line SW1116, treatment with the DNA methyltransferase 1 (DNMT1) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) or the ERK-MAPK inhibitors PD98059 or rottlerin, or transient transfection with the MAP/ERK kinase (MEK)1/2 small interfering RNA down-regulates DNMT1 and proliferating cell nuclear antigen levels. In this report, we found that drug treatment or small interfering RNA transfection of SW1116 cells induced promoter demethylation of the p16(INK4A) and p21(WAF1) genes, which up-regulated their mRNA and protein expression levels. Flow cytometry revealed that rottlerin treatment induced cell cycle arrest at phase G(1) (p < 0.05). Thus, the ERK-MAPK inhibitor treatment or siRNA-mediated knockdown of ERK-MAPK decreases DNA methylation via down-regulating DNMT1 expression and other unknown mediator(s) in SW1116 colon cancer cells.

Tumor Suppressor p16INK4A Regulates Polycomb-mediated DNA Hypermethylation in Human Mammary Epithelial Cells

Alterations in DNA methylation are important in cancer, but the acquisition of these alterations is poorly understood. Using an unbiased global screen for CpG island methylation events, we have identified a non-random pattern of DNA hypermethylation acquired in p16-repressed cells. Interestingly, this pattern included loci located upstream of a number of homeobox genes. Upon removal of p16(INK4A) activity in primary human mammary epithelial cells, polycomb repressors, EZH2 and SUZ12, are up-regulated and recruited to HOXA9, a locus expressed during normal breast development and epigenetically silenced in breast cancer. We demonstrate that at this targeted locus, the up-regulation of polycomb repressors is accompanied by the recruitment of DNA methyltransferases and the hypermethylation of DNA, an endpoint, which we show to be dependent on SUZ12 expression. These results demonstrate a causal role of p16(INK4A) disruption in modulating DNA hypermethylation, and identify a dynamic and active process whereby epigenetic modulation of gene expression is activated as an early event in breast tumor progression.

Impaired inhibition of NF-κB activity by melanoma-associated p16INK4a mutations

The cyclin-dependent kinase inhibitor p16INK4a has been identified as tumor suppressor and melanoma predisposition gene. While its cell cycle inhibitory ability is important in protecting cells from uncontrolled growth and possible tumor formation, other functions of p16INK4a are likely to contribute to its nature as a tumor suppressor. p16INK4a binding and inhibition of the transcription factor NF-κB has been shown and is consistent with the reports of abnormally increased NF-κB activity in various cancers including melanoma. Here, we present evidence that wild type p16INK4a binds to the NF-κB subunit RelA more efficiently than melanoma-associated p16INK4a mutations. Furthermore, whereas wild type p16INK4a strongly inhibits NF-κB transcriptional activity, a subset of melanoma-associated p16INK4a mutants show reduced NF-κB inhibitory function. p16INK4a does not affect NF-κB nuclear translocation or DNA binding, suggesting a mechanism that reduces NF-κB transactivation activity.

The melanoma-associated 24 base pair duplication in p16INK4a is functionally impaired

Melanoma-associated germline mutations affecting the tumor suppressor and cyclin-dependent kinase (CDK) inhibitor, CDKN2A/p16INK4a, have been identified in over 100 melanoma-prone families worldwide. To predict the melanoma risk for carriers of specific mutations, mutant p16INK4a can be tested in biochemical and cellular assays. In most cases, p16INK4a mutations with predicted disease relation, due to segregation with melanoma, are functionally impaired in such assays. The N-terminal 24 base pair duplication of CDKN2A, however, encodes a p16INK4a variant previously shown to have wild-type function, despite segregating with melanoma in at least 5 melanoma families. To clarify whether the duplication mutation has a cell cycle regulatory defect or behaves like wild-type p16INK4a, we reanalyzed the cell cycle-inhibitory activity of this mutation. Stable cell clones of the p16-null WMM1175 melanoma cell line inducible for ectopic p16INK4a were used in this study. In these cells, p16INK4a expression can be controlled at physiologic levels. Our results show that in comparison to wild-type p16INK4a, the duplication mutant induced weaker S-phase inhibition and cells expressing this mutant form of p16INK4a retained colony formation ability. We also show that the cell cycle-regulatory defect of the p16INK4a duplication mutant was associated with decreased inhibition of pRb phosphorylation even though it retained significant binding to CDK4.

Cellular characteristics of primary and immortal canine embryonic fibroblast cells.

Using normal canine embryonic fibroblasts (CaEF) that were shown to be senescent at passages 7th-9th, we established two spontaneously immortalized CaEF cell lines (designated CGFR-Ca-1 and -2) from normal senescent CaEF cells, and an immortal CaEF cell line by exogenous introduction of a catalytic telomerase subunit (designated CGFR-Ca-3). Immortal CGFR- Ca-1, -2 and -3 cell lines grew faster than primary CaEF counterpart in the presence of either 0.1% or 10% FBS. Cell cycle analysis demonstrated that all three immortal CaEF cell lines contained a significantly high proportion of S-phase cells compared to primary CaEF cells. CGFR-Ca-1 and -3 cell lines showed a loss of p53 mRNA and protein expression leading to inactivation of p53 regulatory function, while the CGFR-Ca-2 cell line was found to have the inactive mutant p53. Unlike the CGFR-Ca-3 cell line that down-regulated p16INK4a mRNA due to its promoter methylation but had an intact p16INK4a regulatory function, CGFR-Ca-1 and -2 cell lines expressed p16INK4a mRNA but had a functionally inactive p16INK4a regulatory pathway as judged by the lack of obvious differences in cell growth and phenotype when reconstituted with wild-type p16INK4a. All CGFR-Ca-1, -2 and -3 cell lines were shown to be untransformed but immortal as determined by anchorage-dependent assay, while these cell lines were fully transformed when overexpressed oncogenic H-rasG12V. Taken together, similar to the nature of murine embryo fibroblasts, the present study suggests that normal primary CaEF cells have relatively short in vitro lifespans and should be spontaneously immortalized at high frequency.

Wild-type p16INK4a suppresses cell growth, telomerase activity and DNA repair in human breast cancer MCF-7 cells.

p16INK4a, a cell cycle inhibitor that inhibits cyclin-dependent kinase 4 (cdk4) and cdk6, has been found as the tumor suppressor gene and is frequently deleted, methylated or mutated in many malignancies. Since p16INK4a is also a key element controlling cellular senescence and other functions, we hypothesized that p16INK4a induced tumor suppression may not be limited to the inhibition of cdks. To investigate the role of p16INK4a in tumor suppression and the potential interaction between p16INK4a and other cellular controlling elements, such as telomerase activity and DNA repair ability, the full-length of p16INK4a cDNA was cloned into a retroviral vector and introduced into human breast cancer MCF-7 cells that were previously demonstrated to harbor homozygous deletions of the p16INK4a gene. Stable expression of p16INK4a suppressed the malignant phenotype in MCF-7 cells, including cell proliferation, anchorage-independent growth, G1/G0 cell cycle arrest, and the blockage of pRB phosphorylation. In addition, expression of p16INK4a suppressed telomerase activity and restored the telomere shortening process, and decreased cell DNA repair ability and sensitized cells to the DNA damage reagent. Our data suggest that the wild-type p16INK4a plays an important role in suppression of tumor malignancy, not only by inhibiting cell proliferation through cell cycle arrest, but also by inhibiting other cellular controlling mechanisms, such as telomerase activity and DNA repair capacity.

Inactivation mechanisms and growth suppressive effects of p16INK4a in Asian esophageal squamous carcinoma cell lines

The inactivation mechanisms and functional role of p16INK4a in three Asian esophageal squamous cell carcinoma (ESCC) cell lines were investigated by polymerase chain reaction (PCR) amplification, DNA sequencing, methylation-specific PCR analysis, reverse transcription-PCR, Western blotting, and colony formation assays. The p16INK4a was inactivated by promoter hypermethylation in all three cell lines, a homozygous deletion of exons 2 and 3, and a frameshift deletion on exon 1, leading to transcriptional silencing or the production of mutant p16INK4a protein. Two ESCC cell lines transfected with wild type p16INK4a show significantly reduced cell growth properties. The results of the present studies support the suppressive role of p16INK4a in ESCC development.

Exogenous wild-type p16INK4A gene induces delayed cell proliferation and promotes chemosensitivity through decreased pRB and increased E2F-1 expressions

Human gastric cancer SNU 484 cells express mutant p16, which migrates slower than the wild-type p16. We constructed an expression vector containing human p16 cDNA to evaluate the cytotoxic effects of exogenous p16 expression on SNU 484 cell proliferation and to explore the potential use of p16 in cancer gene therapy. The stable transfectant expressing wild-type p16, showed a 2-fold slower growth rate than mock and non-infected cells through down-regulation of CDK4-dependent kinase activity. When cells were transiently transfected with mock or p16 encoded vector, the mock cells showed larger survival colonies than those of wild-type p16. Furthermore, p16-expressing stable transfectant was readily progressed into cell death by combination with treatment of chemotherapeutic drug in a dose-dependent manner. According to western blot analysis, both decreased expression of pRB and increased expression of E2F-1 may contribute to the susceptibility of cell death. Our data indicate that exogenous wild-type p16 induces delayed cell proliferation and promotes chemo-sensitivity in the gastric cancer cell line, implying the promise of p16 in cancer gene therapy.

Obligate roles for p16(Ink4a) and p19(Arf)-p53 in the suppression of murine pancreatic neoplasia.

Epithelial tumors of the pancreas exhibit a wide spectrum of histologies with varying propensities for metastasis and tissue invasion. The histogenic relationship among these tumor types is not well established; moreover, the specific role of genetic lesions in the progression of these malignancies is largely undefined. Transgenic mice with ectopic expression of transforming growth factor alpha (TGF-alpha) in the pancreatic acinar cells develop tubular metaplasia, a potential premalignant lesion of the pancreatic ductal epithelium. To evaluate the cooperative interactions between TGF-alpha and signature mutations in pancreatic tumor genesis and progression, TGFalpha transgenic mice were crossed onto Ink4a/Arf and/or p53 mutant backgrounds. These compound mutant mice developed a novel pancreatic neoplasm, serous cystadenoma (SCA), presenting as large epithelial tumors bearing conspicuous gross and histological resemblances to their human counterpart. TGFalpha animals heterozygous for both the Ink4a/Arf and the p53 mutation showed a dramatically increased incidence of SCA, indicating synergistic interaction of these alleles. Inactivation of p16(Ink4a) by loss of heterozygosity, intragenic mutation, or promoter hypermethylation was a common feature in these SCAs, and correspondingly, none of the tumors expressed wild-type p16(Ink4a). All tumors sustained loss of p53 or Arf, generally in a mutually exclusive fashion. The tumor incidence data and molecular profiles establish a pathogenic role for the dual inactivation of p16(Ink4a) and p19(Arf)-p53 in the development of SCA in mice, demonstrating that p16(Ink4a) is a murine tumor suppressor. This genetically defined model provides insights into the molecular pathogenesis of SCA and serves as a platform for dissection of cell-specific programs of epithelial tumor suppression.

Expression of Cell-Cycle Mediators in Ovarian Cancer Cells after Transfection with p16INK4a, p21WAF1/Cip-1, and p53

Objective.The purpose of this study was to determine whether transfection of ovarian cancer cell lines with recombinant adenoviral vectors containing wild-type p16INK4a, p21WAF1/Cip-1, and p53 caused growth inhibition and induction of apoptosis. We also measured the expression of the cell-cycle mediators Bax, Bcl-2, pRb, and mdm-2.Methods. We introduced the wild-type p16INK4a, p21WAF1/Cip-1, and p53 genes into the ovarian cancer cell lines SK-OV-3 (p16INK4a and p53 null) and OVCA-420 (p16INK4a and p53 wild-type) by adenoviral transfection. Cell growth inhibition was measured over a 10-day period. Induction of apoptosis was tested for both cell lines 48 h after cell transfection. Expression of cell-cycle mediators was evaluated by Western blot analysis and densitometry.Results. Growth inhibition was documented after transfection with p16INK4a, p21WAF1/Cip-1, and p53 in both SK-OV-3 cells and OVCA-420 cells. Apoptosis was greatest in SKOV-3 cells after transfection with p53. A significant expression of Bax was only seen in the SKOV-3 cells transfected with p53. The bcl-2 protein was poorly expressed in both cell lines. Expression of pRb was suppressed in OVCA-420 cells transfected with p16INK4a and p21WAF1/Cip-1. Infection with Adp16INK4a and Adp53 led to an increase in the level of mdm-2 in the SK-OV-3 cell line only.Conclusions. In the ovarian cancer cell lines studied, cell growth was inhibited after transfection with p16INK4a, p21WAF1/Cip-1, and p53. Cell cycle arrest was highest with p53 transfection. The expression of pro-apoptosis proteins was primarily a function of p53 expression.

Adenovirus-mediated p16INK4a gene expression radiosensitizes non-small cell lung cancer cells in a p53-dependent manner.

We examined the influence of adenovirus-mediated wild-type p16INK4a (Ad/p16) expression on the radiation sensitivity of NSCLC cell lines, all of which lacked constitutive p16INK4a but each of which varied in p53 status: A549 (-p16INK4a/ +pRb/wt-p53), H322 (-p16INK4a/ +pRb/mt-p53), and H1299 (-p16INK4a/ +pRb/deleted-p53). The in vitro clonogenic survival results indicate that Ad/p16 enhanced the radiosensitivity of A549 but not H322 or H1299. Further analysis indicated that the apoptosis induced by combination therapy using Ad/p16 plus irradiation was dependent on the endogenous p53 status of the cancer cells. We performed Western blotting to analyse the p53 protein expression of A549 cells treated with either Ad/p16 or Ad/Luc. Endogenous p53 protein levels were higher in A549 cells transfected with Ad/p16 than in those transfected with Ad/Luc. Furthermore, when wt-p53 protein expression was restored in H1299 using Ad/ p53, Ad/p16 stabilized p53 protein expression and radiosensitized the cells. These results suggest that Ad/ p16-induced stabilization of p53 protein may play an important role in Ad/p16 mediated radiosensitization by enhancing or restoring apoptosis properties. Thus, Ad/ p16 plus radiation in combination may be a useful gene therapy strategy for tumors that have wt-p53 but nonfunctional p16INK4a.

Induction of apoptosis in p16INK4A mutant cell lines by adenovirus-mediated overexpression of p16INK4A protein.

The tumor suppressor gene p16INK4A is a cyclin-dependent kinase inhibitor (CDKI) and an important cell cycle regulator. We have previously constructed a recombinant adenovirus which expresses p16 (Adp16) and shown that infection in a variety of human tumor cell lines with this recombinant virus results in high levels of p16INK4A protein expression resulting in cell cycle arrest and loss of cyclin-cdk activity. Furthermore, adenoviral-mediated overexpression of wild-type p16INK4A is more toxic in cancer cells which express mutant forms of p16INK4A compared to cancer cell lines containing endogenous wild-type p16. TUNEL assay and DAPI staining following infection of MDA-MB 231 breast cancer cells with Adp16 indicate that p16INK4A-mediated cytotoxicity was associated with apoptosis. This is supported by studies demonstrating a decrease in cpp32 and cyclinB1 protein levels and induction of poly (ADP-ribose) polymerase (PARP) cleavage following infection of MDA-MB-231 cells with Adp16. These results suggest that gene therapy using Adp16 may be a promising treatment option for human cancers containing alterations in p16 expression.

Features of replicative senescence induced by direct addition of antennapedia-p16 INK4A fusion protein to human diploid fibroblasts

The p16 INK4A cyclin-dependent kinase (Cdk) inhibitor is now recognized as a major tumor suppressor that is inactivated by a variety of mechanisms in a wide range of human cancers. It is also implicated in the mechanisms underlying replicative senescence since p16 INK4A RNA and protein accumulate as cells approach their proscribed limit of population doublings in tissue culture. To obtain further evidence of its role in senescence, we have sought ways of overexpressing p16 INK4A in primary human diploid fibroblasts (HDF). To circumvent the low transfection efficiency of primary cells we have exploited a recombinant form of the full-length p16 INK4A protein fused to a 16 amino acid peptide from the Drosophila antennapedia protein. This peptide has the capacity to cross both cytoplasmic and nuclear membranes allowing the direct introduction of the active protein to primary cells. Here, we show that antennapedia-tagged wild-type p16 INK4A protein, but not a functionally compromised tumor-specific variant, causes G1 arrest in early passage HDFs by inhibiting the phosphorylation of the retinoblastoma protein. Significantly, the arrested cells display several phenotypic features that are considered characteristic of senescent cells. These data support a role for p16 INK4A in replicative senescence and raise the possibility of using the antennapedia-tagged protein therapeutically.