Regulation of P16ink4a the Cyclin Dependent Kinase Inhibitor & the Tumor Suppressor

Regulation of p16INK4A The Cyclin Dependent Kinase Inhibitor & the Tumor Suppressor

Gözde Işık Master Thesis Cancer Genomics and Developmental Biology Masters May-2009

Supervised by Dr. Inge The Department of Developmental Biology, Utrecht University

Contents

Abstract….…………………...…………………………………………………………3 Abbreviations………….………………………………………………………………4 Introduction……………………………………………………………………………5 Cell Cycle………………………………………………………………………….…..5 Cell Cycle Phases………………………………………………………………….…..5 Cell Cycle Types………………………………………………………………….…...5 Cyclins & CDKs……………………………………………………………………....6 CDK-4 and Cyclin D………………………………………………………………….8 CDK Inhibitors: INK4 and Cip/Kip Family…………………………………………..8 INK/ARF locus and p16INK4A …………………………………………………….…..9 p16 INK4A and Cancer…………………………………………………………….…11 Regulation of p16INK4A protein…………………………………………………...14 Regulation of p16INK4A (INK4/ARF locus) at Transcriptional Level……………15 Ras and Raf; Members of MAPKK team………………………………………..15 Ets Family: induction or repression? ...... 16 p38: a secondary signal to activate INK4A ……………………………. ……….16 Bmi-1, one stone two birds? …………………………………………………….17 Id1: inhibitor of DNA binding-1………………………………………………....18 Regulatory Domain RDINK4/ARF and CDC6………………………………………18 Regulation of p16INK4A at Post-Transcriptional Level……………………………20 RNA binding proteins: hnRNP A1 and A2….…………………………………20 miR-24 suppresses the translation of p16INK4A ………………………………....23 Post-translational Regulation and Modifications of p16INK4A……………………27 Phosphorylation of p16INK4A……………………………………………………..27 Cytoplasmic Localization of p16INK4A ……………………………………..……27 p16INK4A is targeted via N-terminal Ubiquitination………………………….….29 Concluding Remarks………………………………………………………………30 References……………………………………………………………………………32

Abstract

p16INK4A is a tumor suppressor protein which inhibits the activities of CDK4 and CDK6 INK4A thus leading cell cycle arrest in G1 phase. Therefore, regulation of p16 levels and activity play a crucial role in maintenance of homeostasis. Since p16INK4A has key roles in important cellular physiological processes such as tumor suppression, senescence, apoptosis, it must be tightly regulated. Here, I focus on how p16INK4A is regulated. Most of the literature is focused on the transcriptional regulation of p16INK4A therefore I include transcription factors or DNA binding proteins which lead to either repression or induction of p16INK4A gene. In the following part, post-transcriptional regulation of p16INK4A with RNA binding proteins and miRNAs are discussed. Last but not least the protein modifications of p16INK4A such as phosphorylation and ubiquitination are discussed. This review presents an extensive discussion on regulation of p16INK4A tumor suppressor protein.

Abbreviations

Cyclin-dependent kinases CDKs Retinoblastoma pRb Cyclin dependent kinase inhibitors CKIs CDK inhibitory Protein/ Kinase Inhibitor proteins Cip/Kip INhibitors of CDK4 INK4 Alternative Reading Frame ARF Regulatory Domain of INK/ARF locus RDINK4/ARF Origin Recognition Complex ORC Mini-chromosome Maintenance Complex MCM RNA-binding proteins RBPs microRNAs miRNAs Nuclear localization signals NLS Nuclear export signal NES

Introduction Cell Cycle

The highly regulated processes in which the cells make their exact replicas are essential for the life and development of complex organisms. This is accomplished by the cell division cycle. The cell cycle includes a series of events that take place in a cell leading to its division and duplication which will be described below.

Cell Cycle Phases

The mitotic cell cycle is divided into 4 distinct phases: G1, S, G2, and M phase. The cell cycle consists of the replication of chromosomal DNA during synthesis phase (S phase), the partitioning of the replicated chromosomes during mitosis (M phase) and two gaps

one before and one after S phase, which are referred to as G1 phase and G2 phase respectively (Fig.1). Interphase is a collective term that describes the G1, S, and G2 phases. During interphase, the cell grows and prepares for mitosis (M phase) by accumulating nutrients and replicating DNA (S phase). The M phase is composed of 2 coupled processes called mitosis and cytokinesis. In mitosis, the cell’s chromosomes are partitioned between the 2 daughter cells and this is followed by cytokinesis where the cytoplasm physically divides, forming the 2 daughter cells. The mitotic cell cycle ends after cell division and the cells enter the G1 phase. The cell cycle includes 4 distinct periods in which different metabolic events take place related to the cell division and replication.

Cell Cycle Types

Additional to the mitotic division cycle other types of cell cycles exist as well. Cells in early embryos can have continuous cycles of DNA replication and nuclear division at very high speed. The embryonic cell cycles lack G1 and G2 phases. Following the mitosis, DNA replication starts immediately and a full cycle of cell division can be completed in half an hour. As embryogenesis comes to an end, and the cell’s environment gets

complex, the gaps are inserted. G1 phase occurs between the nuclear division (M phase)

and the DNA synthesis (S phase). In addition, another gap called G2 phase appears

between S and M (Fig.1). G1 phase and these gaps allow for the repair of DNA damage and replication errors.

Another type is the meiotic cell cycle which is seen in the formation of haploid gametes. Meiotic cell cycle ends with reduction in the amount of genetic material. Meiotic cell cycle differs from mitotic cell cycle as there are two cell divisions in meiosis, resulting in cells with a haploid number of chromosomes. In addition, during the development of C.elegans (also in other organism, e.g. Drosophila) endoreduplication (or: "endoreplication") cycles happen in which S phases are not followed by mitosis19 leading to increase of DNA content without cell divisions.

There are also a few terminology related to cell cycle such as quiescent and senescent

cells. Cells may stop diving temporarily and enter to a quiescent state called G0 phase. If the cells get permanently non-dividing, they are called senescent cells.

Molecular events in the cell cycle must progress in a sequential and ordered manner; therefore a strict molecular regulation is evolved. It is very important to make sure that each step and the molecular events in the cell cycle are controlled.

Cyclins & CDKs

The cell cycle which is conserved from yeast to human is controlled by cyclin-dependent kinases (CDKs). The cyclin-dependent kinases (CDKs) are a family of serine/threonine protein kinases. The family members are small proteins (~34–40 kDa) and share a catalytic core shared with all other protein kinases. The activities of CDKs are regulated by several activators (cyclins) and CDK inhibitors (INK4, and Cip and Kip inhibitors)23. Cyclins act as regulatory subunits of CDKs. The binding of CDKs and cyclins is necessary for CDKs activity and cell cycle progression. Human cells have multiple loci encoding CDKs and cyclins (13 and 25 loci, respectively)24. Despite the high number of CDKs and cyclin loci, only several CDK-cyclin complexes are directly involved in driving the cell cycle. This group consists of four CDKs: CDK2, CDK4, CDK6 and a mitotic CDK (CDK1) and ten cyclins which are in four different classes: the A-, B-, D-

7 and E-type cyclins. Cyclin and CDK association is essential for cell progression and proper functioning of CDKs.

CDKs act at distinct times in the cell cycle and use specific cyclin partners. In mid G1 phase cyclin D/CDK4 and CDK6 activity rise thus promotes progression through G1 phase. The activity of cyclin E/CDK2 increases entering to the S phase while cyclin A1-

A2/CDK2 is associated with the end of S phase and the entry to the G2 . CDK1 promotes M phase entry/progression (Fig. 1)19. The synthesis and degradation of each specific type of cyclin take place in different and specific phase during the cell cycle which regulates the CDK activity in a timely manner and take place in different and specific phases during the cell cycle.

Figure 1. Cell phases are of a typical (somatic) cell cycle are indicated around the circle with different colors. The cell cycle can be divided in four phases: G1 phase, S phase, G2 phase, and M phase. The nuclear division and cytoplasmic division take place in M phase (mitosis). Different combinations of cyclins and CDKs are depicted with the approximate time of activity in the cell cycle. Shapes outside the cycle indicate increase and reduction of corresponding CDK/cyclin activity 19.

Progression of the cell cycle properly is supervised by check-points25. Check-points are able to sense the defects during DNA synthesis and chromosome segregation. When these check-point mechanisms are activated the cell cycle arrest is induced which occurs

by the altered CDK activity. The cell cycle arrest allows cells to repair defects which prevent the transmission of the abnormalities to the daughter cells.

CDK-4 and Cyclin D

A vast body of biochemical evidence shows that in the early G1 increase in the synthesis of D type cyclin synthesis occurs, which is the end point of several signal transduction pathways26. D type cyclins are necessary for the action of CDK4 and the association of cyclin D with CDK4 mediates the phosphorylation of retinoblastoma (pRb) family 27 protein . pRB is active in its hypophosphorylated form in the G1 phase and able to repress the function of the E2F transcription factor. E2F responsive genes are necessary 28 for progression through the G1-S phase transition and for S phase as well . In addition, cyclin D/CDK4 complex binds to a family of cell cycle inhibitors such as the Cip/Kip complexes which are able to bind and inactivate cyclin E/CDK2 complex29. It has been proposed that cyclin D–CDK4/6 activity requires binding of Cip/Kip inhibitors as the mouse embryonic fibroblast (MEFs) which lack p21Cip1 and p27Kip1 don’t have CDK4 activity30. D type cyclin-CDK4 activity is essential for the progression of cell cycle through S phase either by inhibition of pRB or inhibition of cell cycle inhibitors.

Cyclin D class of cyclins are the cyclins which are involved in tumorigenesis, known to date. It is thought either CDK4 or cyclin D plays a significant role in tumorigenesis31. For example, up to 50% of breast cancers exhibits elevated levels of cyclin D132. The alterations in expression and activities of D cyclin and CDK4, by signal transduction or other regulatory proteins may cause tumor formation.

CDK Inhibitors: INK4 and Cip/Kip Family

CDK inhibitor proteins serve as an important mechanism which prevents the premature entry into the S phase. Cyclin dependent kinase inhibitors (CKIs) are able to bind to CDKs and thus inactivate CDKs’ kinase activity33, leading to a negative regulation of the cell cycle. CDK activity is regulated by two families of inhibitors. The first family of CKIs is the INhibitors of CDK4 (INK4) proteins which are INK4A, INK4B, INK4C and INK4D. The second family includes the Cip and Kip family which is CDK inhibitory

protein/ Kinase inhibitor proteins (Cip/Kip) family in mammals. This family is composed of p21Cip1, p27Kip1 and p57Kip2 34,35,36,37 which are CDK inhibitors. Thus, the CDK inhibitors functions as a break for the premature entry to the S phase of the cell cycle by inhibiting the CDKs.

INK4A INK4/ARF Locus and p16

The INK4 gene family encodes p16INK4A, p15INK4B, p18INK4C, and p19INK4D, all of which bind to CDK4 and CDK6 and inhibit their kinase activities by interfering with their association with D-type cyclins29. Specific polypeptide inhibitors of CDK4 and CDK6, so-called the INK4 proteins (inhibitors of CDK4), can directly block cyclin D-dependent kinase activity and cause G1 phase arrest 38.

INK4B INK4A The INK4/ARF locus encodes three tumor suppressors, p15 , p16 and p19ARF. p16INK4A and p19ARF are transcribed using alternative exons 1α and 1β and spliced onto the same exons (Fig.2) but in different open reading frames38. Despite overlapping coding regions, p16INK4A and p19ARF have unrelated amino acid sequences and distinct functions. Both p16INK4A and p19ARF are able to inhibit the cell cycle progression and therefore referred to as tumor suppressors 39-42. p16INK4A and p19ARF are transcribed from the same gene with different open reading frames which makes them distinct in sequence and function.

p16INK4A(also called CDKN2A) regulates the cell cycle by inhibiting the CDK4 or CDK6 43 cyclin dependent kinases . Inhibition of CDK4 or CDK6 prevents the phosphorylation of pRb proteins and progression into the S phase. pRb is a key gatekeeper in cell cycle

transition. In normal cycling cells, entry to the S-phase from G1 phase is related to the functional inactivation of pRB by phosphorylation and the phosphorylation is performed by cyclin dependent kinases44.

The second protein which is also a growth inhibitory protein as p16INK4A is p19ARF. ARF abbreviation originated from alternative reading frame whereas p19 is the molecular weight of the 19kDa, 169 amino acid mice protein. However, in humans ARF is

translated into the 14kDa, 132 amino acid: p14ARF protein 45. The resulting ARF protein is also acts as potent tumor suppressor46. p19ARF neutralizes MDM2 protein which facilitates the degradation of p53. This condition enhances p53 levels and p53-related functions47. Another consequence of p19ARF expression is induction of p21CIP1-mediated cell-cycle arrest. p19ARF expression leads to induction of both p53 and p21CIP1 protein therefore functioning as a tumor suppressor protein.

Figure 2: INK4A/ARF locus encodes two distinct products. Exon1α encodes part of p16INK4A protein which is a cyclin dependent kinase inhibitor (CDK). Exon1β is spliced onto an acceptor site common to 1α but encodes a non- homologous protein called p19ARF, in an alternative reading frame. p16INK4A inhibits the CDK 4/6 thus leading to inhibition of E2F transcription factors. On the other hand, p19ARF stabilizes p53 by neutralizing the MDM2 protein and induction of p21CIP1-mediated cell-cycle arrest. INK4A/ARF locus encodes two distinct proteins functioning upstream of both RB and p53 pathways in the cell 48.

The INK4A/ARF locus represents a unique phenomenon by encoding two distinct tumor suppressor proteins inhibiting the cell cycle progression in different manner. p16INK4A acts by inhibiting CDK4/6 and keeping pRB active while p19ARF leads to stabilization of p53. Functions of these proteins upstream of both pRb and p53 make this locus a ‘keystone gene’48.

p16INK4A and CANCER

Cancer cells show as a hallmark their ability to divide in situations where they should remain quiescent or senescent. The alterations in their signaling pathways and genes involved in DNA repair, cell cycle or apoptotic response machinery give them the advantage to escape from the normal control of many cellular processes. Mammalian cells require extracellular signals to proliferate, differentiate or die. However, when a cell becomes malignant its dependence to these signals disappears and these transformed cells can proliferate without any mitogenic stimulation. In addition, cancer cells do not enter a quiescent state in response to growth inhibitory signals. These properties of cancer cells are directly or indirectly connected to cell cycle control.

It is well defined that deregulation of progression into interphase of cell cycle contributes to human cancers. For example, deregulation of CDK4 and CDK6 found to be related to many tumor varieties49,24,31. In addition, alterations in CDK activity such as hyperactivity, misregulation of cyclins or CDK regulators, and CKI activity may lead to tumor formation50. Indeed, the analysis of human tumors showed that certain molecules are highly related to cancer progression. For example, the alterations of proteins involved in

G1/S transition in cell cycle are an important class of these tumor related proteins. Cyclin D/ p16INK4A /retinoblastoma/E2F cascade of which one crucial component is coded by the INK4/ARF locus is of interest to many researchers. Either the mutations on the genes coding one of the components of Cyclin D/p16INK4A/retinoblastoma/E2F cascade or the alterations of their regulator proteins may lead to human cancers. In particular, many tumors are found to have deletion in 9p21 chromosome where INK4/ARF locus is located51. This finding draws high attention to the products of INK4/ARF locus and their relationships with tumor suppression.

The function of proteins encoded by the INK4/ARF locus and the regulation of the locus are studied intensively in relationship to the tumorigenesis. INK4 proteins have similar activities as all of them are related to tumor suppression. The cells express the INK4 52 proteins ectopically and rest in G1 phase, but only when the pRb protein is functional . The mechanism underlying this phenotype is related to the inhibition of CDK4/6 kinases

and the structural basis is well understood 33. INK4/ARF locus is involved in a large number of cancer types which makes this locus center of interest of many studies.

As many human cancers carry a deletion in the 9p21, it became very important to define the relevant tumor suppressor for the specific tumor types. The unusual structure of INK4/ARF locus raises questions such as which protein(s) at the locus are related to the phenotype? Or can we find more specific tumor types or lesions related to only one of these proteins? Genetic studies revealed that thousands of human cancers are related to specific somatic loss of p16INK4A and point mutation or small deletions53. In addition, at least 56 germline mutations only in p16INK4A but not in p15INK4B or in ARF are found in unrelated kindred54, supporting the idea that p16INK4A is the most important tumor suppressor coded by the INK4/ARF locus in humans.

In addition to the knowledge obtained from the human tumors, mice models are made in order to study the role of INK4/ARF. One of these models includes the "super INK4A/ARF" mouse which has overexpression of the p16INK4A and p19ARF proteins. The mice are made using a bacterial artificial chromosome and a strain carrying an additional entire INK4A/ARF locus. The primary cell cultures have shown that the super INK4A/ARF mice have increased resistance to oncogenic transformation and in vitro immortalization. In addition, these mice have 3-fold reduction in the incidence of spontaneous cancers while they have normal aging and lifespan55. The authors claim an increase in tumor suppression can be achieved by increasing the activity of INK4A/ARF.

Consistent with the super INK4A/ARF mice observations, the knockout mice models of ARF gene or p15INK4B gene or p16INK4A gene are all more prone to spontaneous cancers than wild-type littermates. The most tumor prone phenotype for spontaneous cancers are observed in mice which lack both p16INK4A and ARF genes56,57. There are more mice models created to study the role of p16INK4A and ARF in specific conditions and in compound backgrounds58 whereas, the mice which lack the entire INK4A/ARF locus has not been reported yet51. In summary, the role of the INK4A/ARF locus also supports the role of the locus and the encoded proteins in tumor suppression.

In murine cancer, the data show that the loss of ARF is potently oncogenic46, 57, but selective inactivation of ARF, in the absence of a concomitant loss of p15INK4B and p16INK4A, has only been reported in a small number of cases of human cancer51. It is thought that the relatively infrequent finding of lesions selectively targeting ARF indicates species differences in the relative importance of and ARF in tumor suppression; which is, ARF is more important in mice, and p16INK4A more important in humans. However, it is also suggested that these data can also be explained by a consideration of the biochemical nature of the ARF-MDM2 interaction51, which only requires the activity of a small portion of the highly basic N terminus of ARF59. Therefore, the chance that ARF’s anticancer activity is disabled by missense mutations may be very improbable. The human and murine genetic data considered as a whole establish that the INK4A/ARF locus encodes at least two (p16INK4A and ARF), and probably three (p15INK4B), tumor suppressor proteins, although their relative and combinatorial importance in any given tumor type has not been fully resolved.

REGULATION OF p16INK4A PROTEIN

Given the importance of products of the INK4A/ARF locus in tumor suppression and senescence, regulation of the locus has been an area of intense study. Regulation of p16INK4A protein may be in different stages: regulation in transcriptional level, post transcriptional level, post translational level. The regulation of p16INK4A transcriptionally is determined by the changes in chromatin and activities of transcription factors. Posttranscriptionally, the regulation is through the changes in mRNA stability and translation while posttranslational regulation may be by phosphorylation and ubiquitination.

Several lines of evidence suggest that INK4/ARF expression increases in the early stages of cancer progression (reviewed in 60), however which exact stimuli induces the expression of the locus relevant to the cancer is not known. In rodents, most stimuli results in coregulation of the two genes (p16INK4A and p19ARF) in the locus although some stimuli are inducing only p16INK4A or only p19ARF 61, 62. On the other hand, in human cells it is not very common to have coregulation of both p16INK4A and p19ARF proteins 51. INK4/ARF locus can be activated in cancer progression by stimuli which may result in coregulation or regulation of each gene individually in the locus.

Many noxious stimuli have been shown to induce p16INK4A and/or ARF expression in vitro and in vivo. Oxygen radicals, ionizing radiation, chemotherapeutic agents, telomere dysfunction and UV light are included in this stimuli list 51. The increase of p16INK4A in these stress conditions is often associated with MAPK activation63,64,65. MAPKs (mitogen activated protein kinases) play a key role in transduction extracellular signals to cellular responses and also are related to various biological events in the cell including their development, proliferation and apoptosis 66. Ras which is a key element in this cascade may lead to induction in INK4/ARF expression by ERK-mediated activation of Ets1/2 to induce p16INK4A 67 (discussed below) and Jun-mediated activation of Dmp1 to induce ARF68. Expression of p16INK4A can be induced by different stimuli which are connected to cellular signaling pathways which I will describe below in more detail.

Regulation of p16INK4A (INK4/ARF locus) at Transcriptional Level

Most our knowledge about the regulation of p16INK4A is focused in the transcriptional level. In addition to noxious stimuli, several tumor-relevant or/and stress signaling pathways are associated with INK4A/ARF expression. Several molecules which function to induce the growth and proliferation have been shown to regulate one or more products of INK4A/ARF 51.

Ras and Raf; members of MAPKK team

The relationship between the oncogenic signaling and the transcriptional activation of INK4A was first suggested with the observations in primary fibroblasts that activated oncogenic RAS is able to induce premature senescence69. The signaling pathway underlying this effect is thought to be the Raf-Mek-Erk kinase cascade, with the supporting observations done in a tumor with a BRAF mutation which is relevant to human naevi (Box 1)13, 67, 70, 71. According to the model, activation of the Raf-Mek-Erk cascade will activate a key transcriptional factor of p16INK4A gene.

In order to investigate the signaling pathways Box 1. Human naevi (moles) are benign INK4A which regulate p16 expression, the tumors of melanocytes that frequently response of p16INK4A promoter to activated Ras harbour oncogenic mutations in BRAF or Mek are tested using a luciferase assay. In (predominantly V600E, where valine is substituted for glutamic acid)1, a protein these assays, different length of promoter kinase and downstream effector of Ras. sequences (869, 530, 343, 247 nucleotides) Nonetheless, naevi typically remain in a INK4A upstream of the p16 translation site are growth-arrested state for decades and only used. It is found that H-RasV12 (a constitutively rarely progress into malignancy (melanoma) 5,6,9 V600E active mutant in which Glycine 12 is mutated to . It is shown that sustained BRAF Valine) or constitutively active Mek are able to expression in human melanocytes induces cell cycle arrest, which is accompanied by stimulate the promoter activity up to 5-fold in the induction of p16INK4A and senescence 13. SV-40 immortalized human fibroblasts even if

16 the shortest length promoter sequence is used. On the other hand, it is shown that this effect could be blocked by an inhibitor of Mek pathway67. These results indicate that the p16INK4A promoter activity shows induction when Ras-Raf-Mek cascade is activated.

Ets Family: induction or repression?

Ets family including Ets-1, Ets-2, Pea3, Sap1 and Elk1 transcription factors are known to be downstream targets of Ras-Raf-Mek signaling and therefore can get activated by MAPK-mediated phosphorylation72. The promoter activity of p16INK4A gene in response to different Ets family members is measured in human diploid fibroblasts 67. Both Ets-1 and Ets-2 are able to induce the promoter activity through an Ets binding site in p16INK4A promoter up to 5-10 fold whereas Pea3, Sap1 and Elk1 caused an opposite affect and caused 2- to 3- fold reduction in p16INK4A promoter activity. Although, it is shown that activation of the Raf-Mek-Erk cascade induces the promoter activity of p16INK4A, Ets family members may act in an opposing manner; Ets-1 and Ets-2 may induce the promoter activity while other family members may repress the p16INK4A promoter activity.

p38: a secondary signal to activate INK4A

p38 appeared as an candidate for activation of INK4A when the senescence inducing experiments are conducted in fibroblasts using RAS. p38 is one of the three modules of the MAPK pathway and contains three kinases (MAPKKK, MAPK kinase kinase; MAPKK, MAPK kinase; and MAPK). p38 appears to be activated indirectly by oncogenic Ras and it is a secondary signal leading to activation of p16INK4A. Currently, we lack the understanding how the p38 pathway can be activated by several stress conditions which are related with promotion of senescence65,73. In addition, the inhibitors of p38 signaling can modulate in a negative way or delay the senescence 64. As the p38 family is able to influence the cell-cycle progression in several ways 74, we need more research in order to understand how the secondary signal p38 signals for the activation of p16INK4A 65, 73, 75, 76.

Bmi-1, one stone two birds?

The Bmi-1 is a polycombcomb group (PcG) protein which are a conserved group of proteins required for stable repression of specific target genes during development, such as homeobox-cluster genes77,78. Bmi-1 is found as an oncogene in mouse lymphomas cooperating with the c-myc oncogene79, 80 and it is shown that Bmi-1 deficient primary mouse embryonic fibroblasts are not able to progress through the S phase of the cell cycle. These fibroblasts have raised amounts of p16INK4A and p19ARF proteins which are related to retention in the G1 phase and a premature senescence phenotype (Box 2). Oppositely, the overexpression of Bmi-1 protein downregulates the expression of p16INK4A and p19ARF and causes fibroblast immortalization. In summary, BMI-1 acts as a suppressor of p16INK4A/cyclin D/retinoblastoma protein pathway and also the p19ARF/MDM2/p53 pathway leading the cell entering to the S phase81.

Box 2: p16INK4A and Senescence: The p16INK4A functions as an inhibitor of CDK4 and CDK6 which initiates the phosphorylation of pRb protein. Therefore, p16INK4A has the capacity to arrest the cells in INK4A the G1 phase of the cell cycle (discussed in the Introduction Chapter). p16 also has a physiological role in irreversible growth arrest termed as cellular senescence. Cellular senescence is eventual growth arrest defined by the termination of cell proliferation of normal cells in culture4. The cells which are

senescent are irreversibly arrested in G1 phase and no longer able to divide although they are metabolically active and still viable. Most of the tumors are lack the mechanism to undergo senescence and are able to go through the cell division without the proper checkpoints. It was proposed that the cellular senescence may act as a mechanism to prevent cancer 8.

The senescent cells remain in the G1 phase and CKI’s function in cell cycle arrest linked this cellular process with these proteins. In accordance with this, cell culture models implicated the importance of CDK inhibitors in cellular senescence. Senescent human and murine fibroblasts, which are used in senescence studies intensively 11, have increased expression of p16INK4A 14,16,18. The cells are able to bypass this arrest by transcriptional silencing of INK4A/ARF. Fibroblasts in which p16INK4A activity has been reduced or ablated; exhibited an extended lifespan21. This is probably due to the requirement INK4A INK4A of p16 for senescence. As the p16 levels are high, more G1-S phase CDKs will be inhibited and thus leading to cell cycle arrest.

Id1: inhibitor of DNA binding 1

Id1 protein is a member of Id protein family which are helix–loop–helix (HLH) transcriptional regulatory proteins. This family has an important role in regulation of cell 82 growth in mammalian cells and the G1–S phase transition . Id1-null primary mouse embryo fibroblasts undergo premature senescence. These cells have increased expression of p16INK4 protein and decreased CDK2 and CDK4 kinase activity 83,71. On the other hand, overexpression of Id-1 has been reported to be related with several human primary cancers including pancreatic84, breast85, prostate86, cervical87 and colorectal adenocarcinoma88. Evidences indicate that Id-1 may be associated with an aggressive phenotype of human cancers.

It is proposed that Id1 is important for DNA-binding motif (E-box)-mediated repression of the p16INK4A promoter. The direct relationship between inhibition of p16INK4A and Id1 protein is shown in NIH 3T3 cells which are transfected with 1.2 kb of the p16INK4A promoter possessing two E-box motifs (-349 and -615 bp). In the performed Northern blot experiments repression of p16INK4A promoter up to 75% by full-length Id1 or a C- terminal Id1 deletion mutant (with an intact HLH domain) is observed 83. Lack of Id-1 protein causes a senescent phenotype whereas overexpression is related with many cancer types. In other words, Id1 is important factor in repression of p16INK4A promoter activity.

Regulatory Domain RDINK4/ARF and CDC6

A conserved DNA element with the capacity to regulate the INK4/ARF locus has been identified with an approach that is based on the localized induction of heterochromatinization using the RNA interference89-92. Using this technique, basically, heterochromatinization of a site in the genome is possible by transfection or transduction of RNA molecules. This leads to the disruption of the normal function of the targeted DNA region. Inactivation of a DNA element which was 350bp long by heterochromatization results in global silencing of the INK4/ARF locus and the conserved DNA element is then referred as RDINK4/ARF for the Regulatory Domain of INK4/ARF 93.

Regulatory domain of INK4/ARF: RDINK4/ARF plays role in regulation and activation of INK4/ARF locus.

RDINK4/ARF is a 350bp genomic segment located near the promoter of p15INK4B. It functions both as a transcription regulatory element and a replication origin94. CDC6 ATPase is a replication protein which plays role in the activation of replication origins by binding to the Origin Recognition Complex (ORC) and recruits the Mini-chromosome Maintenance (MCM) complex95. When CDC6 binds to the RDINK4/ARF the histone deacetylases are recruited. This results in heterochromatinization and repression of the INK4/ARF locus94. The association of endogenous and overexpressed CDC6 protein with the RDINK4/ARF is proven by chromatin immunoprecipitation experiments94. A global repression of INK4/ARF locus is observed when CDC6 was overexpressed93. The overexpression of CDC6 exhibits the same situation similar to the other oncogenes, such as BMI1 leading to the repression of INK4/ARF locus. The overexpression of CDC6 which lead to suppression of INK4/ARF locus through binding to the RDINK4/ARF and therefore transcriptional repression p16INK4A and p14ARF might be relevant to some class of lung cancers which have high levels of CDC694.

The regulation of p16INK4Ais complicated which is achieved with the action of many different transcription factors. Some stimuli and transcription factors and also genomic segments induce the transcription of INK4/ARF locus whereas other factors may cause silencing.

Regulation of p16INK4A at Post-Transcriptional Level

Although many stimuli or genetic alternations regulate the mRNA expression of INK4A/ARF locus, less is known about the post-transcriptional regulation of the p16INK4A. Here, I will discuss two ways of p16INK4A regulation on mRNA level; regulation with the RNA binding proteins hnRNPA1 and A2, and with microRNAs: miR-24.

RNA binding proteins: hnRNP A1 and A2

RNA-binding proteins (RBPs) are an important class of trans-acting factors. These proteins associate with RNA and regulate gene expression after gene transcription. The regulation may be on different levels such as pre-mRNA splicing and maturation, mRNA transport, storage, stability, and translation 96.

hnRNP A1 and A2 RNA binding proteins have many functions and may influence the expression of mRNA isoforms during development. Both RBPs may play important roles in the biogenesis of mRNA in vitro and in vivo 97 and may affect gene expression of eukaryotes in a tissue-specific manner98,99,100. hnRNP A1, hnRNPA2 and human pre- mRNA splicing factor SF2/ASF are able to modulate 5’splice site selection in a concentration dependent manner101,102. In addition to splice site modifications, the alternations in the ratios of hnRNP A1 and A2 to human pre-mRNA splicing factor SF2/ASF can influence exon skipping103. They also have been shown to shuttle between the nucleus and cytoplasm in order to regulate the mRNA transport104. In summary, hnRNP A1 and A2 is shown to participate in a variety of cellular functions, including exon skipping, mRNA splicing, trafficking, and turnover. However, their importance in aging and/or replicative senescence has not been determined.

hnRNP A1 and A2 RNA binding proteins are reported to be lower and less active in senescent human fibroblasts. As both p16INK4A and ARF proteins are also found to be related to cellular senescence (Box 2), the relationship between hnRNP A1 and A2 RNA binding proteins and p16INK4A mRNA is investigated 105. Increasing the protein levels of hnRNP A1 or A2 favors the selection of transcription of the distal 5’site the DNA both in vivo and in vitro101,102. Therefore, changes in the mRNA ratios of ARF and p16INK4A are

thought to be influenced by the changes in the expression of either hnRNP A1 or A2 proteins since ARF and p16INK4A mRNAs are generated by alternative splicing (reviewed in106). As the alternations in the ratios of hnRNP A1 and A2 to SF2/ASF can influence exon skipping103, when a constitutive decrease in the ratio of hnRNP A1 and A2 to SF2/ASF takes place in the nucleus, this would lead a shift to the more proximal splice site resulting in the predominance of the p16INK4A than p14ARF, in senescent human fibroblasts.

On the other hand, increasing the levels of A1 or A2 relative to SF2/ASF may result in preferential generation of the ARF isoform105. Since expression of hnRNP A1 and A2 is lower in senescent fibroblasts than in young cells107, the ratio of ARF to that of p16INK4A should decrease. The decrease in the ratio of ARF mRNA to p16INK4A mRNA suggests that p16INK4A protein levels may be higher than that of p14ARF in senescent cells. The ratio of hnRNP A1 or A2 proteins to SF2/ASF splicing factors plays an important role in alternative splicing of INK/ARF locus. Increase in hnRNP A1 or A2 to SF2/ASF ratio will favor in production of ARF mRNA while decrease will favor for p16INK4A mRNA.

It is shown that the overexpression of both A1 and A2 RNA binding proteins increase the steady state of p16INK4A mRNA (and also ARF mRNA), suggesting that in addition to alternative splicing, hnRNPA1 and A2 may affect the mRNA stability105. Accumulation of hnRNP A1 or A2 protein in the cytoplasm (albeit at low level) may interact directly with the mRNAs of ARF and p16INK4A conferring mRNA stability. Indirectly, they may bind mRNAs of transcription factors that modulate the transcription rates of ARF and p16INK4A mRNA. Either hnRNP A1/A2 increase the mRNA stability of p16INK4A or the mRNA stability of transcription factors responsible for p16INK4A expression, hnRNP A1/ A2 increase the steady state levels of ARF and p16INK4A 105. A summary of the possible mechanisms how the localization of hnRNP A1 and A2 may influence their activities and the levels of p16INK4A and ARF are presented in Figure 3. These are the first studies showing that the post-transcriptional processes may play role in regulation of p16INK4A mRNA, during the senescence. It is suggested that hnRNP A1 and A2 may play an important role during replicative senescence by altering expression of cell cycle regulatory proteins such as p16INK4A through mRNA metabolism.

Figure 3. Cellular localization may influence the function and activities of hnRNP A1 and A2 RNA binding proteins 105

Low levels of A1 and A2 relative to that of SF2/ASF protein in the nucleus would cause a shift to the more proximal splice site resulting in the predominance of the p16INK4A isoform. Accumulation of hnRNP A1 or A2 protein in the cytoplasm (albeit at low level) may interact directly with the mRNAs of p14ARF and p16INK4A conferring mRNA stability. Indirectly, they may bind mRNAs of transcription factors that modulate the transcription rates of p14ARF and p16INK4A mRNA. In either case, one would observe an increase in the steady state levels of p14ARF and p16INK4A.

23 miR-24 suppresses the translation of p16INK4A

In addition to RBPs, microRNAs Box 3. miRNAs are a collection of short (21-26-nt (miRNAs) (Box 3) represent another long), single strand, non coding RNAs which are major class of posttranscriptional mediated the suppression of gene expression. miRNAs are described in many organisms and the regulatory factors. miR-24-2 (hereafter molecular details how they function are rapidly miR-24) is identified as a microRNA emerging. miRNAs are assembled into RNA- INK4A which suppresses p16 translation in induced silencing complexes (RISC) which recruit cultured human cells. In order to obtain a target mRNA to processing bodies (P-bodies). P- more understanding on the regulation of bodies function as cytoplasmic foci of mRNA degradation and translational repression (reviewed p16INK4A expression levels of miR-24, in reference 2). HeLa cells are used for polysome If the mRNA-miRNA complex is extensively fractionation followed by RT-qPCR complementarily, miRNAs are able to promote the analysis. In HeLa cells, miR-24 is cleavage of the target mRNA. However, in the case localized predominantly in fractions 1 and of less complete base-pairing, miRNAs can suppress mRNA translation but not mRNA 2 (Fig 4) , and hence dissociated from the degradation4,7,10,12. The precise mechanism of translational apparatus 108. inhibition of mRNA by the mi-RNA is

controversial. According to some studies, miRNAs block the initiation of translation by triggering the formation of large ribonucleoprotein (RNP) ‘pseudo-polysomes’. On the other hand, other studies indicate that miRNAs suppress post initiation steps by repressing translational elongation by promoting ribosome drop-off or by

15,17,20,22 Figure 4. Cytoplasmic lysates from untreated cells inhibiting the termination . It is also were prepared and fractionated through sucrose possible that multiple mechanisms exist for the gradients. Representative miR- 24 levels in each fraction, visualized after RT-PCR (30 cycles) and miRNA functioning: such as the untranslated electrophoresis (1.5% agarose). miR-24 is mostly regions and the cellular context. abundant in 1st and 2nd fractions of cytoplasmic lysates of untreated cells.

Overexpression of miR-24 in HeLa cells by transfecting premiR-24 and monitoring its abundance in cells by RT-qPCR reveal that overexpression of miR-24 in HeLa cells do not significantly alter the relative distribution of miR-24 on polysome gradients (Fig. 5A)

24 or affect the p16INK4A mRNA levels (Fig. 5B) . However, a decrease in protein levels of p16INK4A is observed comparing to the control group (Fig. 5C). As the p16INK4A mRNA distribution profiles in polysome gradient between the control and the premiR-24 are highly similar (Fig. 5D), the decrease in protein levels do not result from lower p16INK4A translational initiation. Instead, it is suggested that miR-24 overexpression reduces the elongation p16INK4A during translation 108.

Figure 5. Increasing miR-24 reduces p16INK4A levels in HeLa cells. (A) HeLa cells were transfected with either Ctrl. siRNA or pre-miR-24 (100 nM) and 48 hr later cytoplasmic lysates were prepared and fractionated through sucrose gradients. The levels of miR-24 were then measured by RT-qPCR in each of the fractions and shown as relative (fold) miR-24 levels in the pre-miR-24 transfection group relative to the levels in the Ctrl. siRNA transfection group (left) and also as a percentage of the total miR-24 in each of the two transfection groups (right). (B) The comparisons were made between populations transfected with pre- miR-24 and Ctrl. siRNA. The levels of mRNAs encoding p16INK4A and housekeeping controls GAPDH (for normalization) and SDHA were calculated by RT-qPCR and represented (means+SD from 3 independent experiments) as fold differences relative to control transfected cells. (C) Representative Western blot analysis of p16INK4A (and loading control α-Tubulin) levels in each transfection group. p16INK4A protein signals were measured by densitometry and represented as a percent of the p16INK4A levels in the control transfection group. (D) Representative distribution of p16INK4A and GAPDH mRNAs on polysome gradients 108.

In order to obtain more insights on the influence of miR-24 upon the translation of p16INK4A, a transcript antisense (AS) to miR-24 is used. HeLa cells overexpressing AS- miR24 show a small increase in p16INK4A mRNA levels comparing to the controls (Fig. INK4A 6A). On the other hand, p16 protein levels show increase up to three fold (Fig. 6B). When the transcriptional status of p16INK4A mRNA are tested by comparing its relative distribution in sucrose gradients prepared from both transfection groups, the cells exhibit a moderate but a distinct shift towards the actively translating fractions (Fig. 6C). Despite its reduced abundance, the remaining miR-24 shows a comparable polysome gradient distribution between the two transfection groups (Fig. 6D)108. This indicates that the p16INK4A mRNA is associated with larger polysomes when the miR-24 is inhibited. These results suggest that translational initiation was enhanced after miR-24 levels are reduced.

Figure 6. Lowering miR-24 increases p16INK4A levels in HeLa cells. (A) Cells were transfected with either a control (Ctrl.) siRNA or antisense (AS) miR-24 (100 nM) and the levels of mRNAs encoding p16INK4A, and controls GAPDH (for normalization) and SDHA were calculated by RT-qPCR and represented as the means+SD from 3 independent experiments; the levels of miR-24 and U6 RNA were also measured. (B) Representative Western blot analysis of p16INK4A (and loading control α-Tubulin) levels in each transfection group. p16INK4A signals were measured by densitometry and represented as % p16INK4A levels relative to the control transfection group. (C) The levels of p16INK4A and control GAPDH mRNAs on polysome gradients were measured. (D) The levels of miR-24 were measured by RT-qPCR in each of the fractions and shown as relative miR-24 levels in the AS-miR-24 transfection group relative to the levels in the Ctrl. siRNA transfection group (left) and also as a percentage of the total miR-24 in each of the two transfection groups (right). Note that the abundance of miR-24 was preferentially reduced in fractions 1 and 2 (left), but the remaining miR-24 was found distributed among all of the fractions of the polysome gradient (right) 108.

Alterations of miR-24 directly affect the expression levels of p16INK4A. miR-24 is predicted to bind both to the coding region and the 39–untranslated region (UTR) of p16INK4A mRNA. It does not affect the p16INK4A mRNA levels but it suppresses p16INK4A translation. This observation is explained by the property of mammalian miRNAs that these miRNAs are able to suppress protein biosynthesis rather than target mRNA degradation. The results presented above provide mechanistic support for the notion that miR-24 suppresses p16INK4A production by inhibiting both the initiation and elongation of p16INK4A translation 108.

Post-translational Regulation and Modifications of p16INK4A

Despite our increasing knowledge about the role of p16INK4A in cell proliferation and in induction of tumorigenesis, not much is known about the modifications and the regulation of p16INK4A.

Phosphorylation of p16INK4A

p16INK4A can be phosphorylated109 and it is shown in immunoprecipitation (IP) experiments that the phosphorylated form of p16INK4A is preferentially associated with CDK4 in human fibroblasts. The phosphorylation sites in the purified protein are mapped to the serines 7, 8, 140, 152 109. The same researchers find that the endogenous p16INK4A associated with CDK4 is phosphorylated at serine 152. The phosphorylated sites are not located in the protein kinase-binding domain. Interestingly, these sites are found to be mutated in familial and sporadic cancers. Phosphorylation of p16INK4A might be an important modification for its regulation and function. The phosphorylation may influence its affinity and binding to CDK4 and CDK6 or the localization in the cell and further research is needed to clarify the importance of phosphorylation.

Cytoplasmic Localization of p16INK4A

It is well established that p16INK4A protein acts as a cell cycle inhibitor in the nucleus. Therefore, cytoplasmic localization of p16INK4A usually is regarded as nonspecific. As all nuclear factors are produced in the cytoplasm and transferred to the nucleus (cytoplasmic to nucleus shuttling), they all may exhibit cytoplasmic staining theoretically. Under normal conditions, shuttling from the cytoplasm to nucleus ratio would favor the distribution to the nucleus and therefore in many cases weak cytoplasmic staining is observed110. As p16INK4A functions as a cell cycle inhibitor, it is expected to be localized in the nucleus whereas a weak cytoplasmic staining was is referred to as nonspecific.

In cancer cells, p16INK4A is not only expressed in the nucleus but also in the cytoplasm 111,112. It is demonstrated that cytoplasmic staining of p16INK4A is specific and it may represent a mechanism of p16INK4A inactivation similar to which is observed in other tumor suppressor proteins such as BRCA1110. It is reported that cytoplasmic

accumulation of p16INK4A can be used for identification of a subset breast tumors with accelerated proliferation and other unfavorable properties. p16INK4A immunohistochemistry can be used to classify the breast tumors (Fig.7) and it is shown that cytoplasmic p16INK4A overexpression in breast cancer was associated with a highly malignant tumor phenotype3. In contrast, cytoplasmic overexpression of p16INK4A in colon cancers indicates a good prognosis113. Cytoplasmic localization of p16INK4A is observed in different types of cancers and may be a good prognosis for tumorigenesis and malignancy.

Figure 7 p16INK4A immunohistochemistry. Representative examples of mammary carcinomas with weak (A), moderate (B) or strong p16INK4A expression (C) Magnification: 250x. A: weak cytoplasmic staining in the tumor cells. Around the tumor, several p16INK4A-positive fibroblasts are shown. B: weak cytoplasmic and stronger nuclear p16INK4A immunoreactivity in around 50% tumor cells. C: strong cytoplasmic and partly nuclear staining throughout the tumor 3.

Unfortunately, we do not know the mechanism(s) lying under the cytoplasmic accumulation of p16INK4A. It is possible that the mutations on p16INK4A may affect the cytoplasmic shuttling as seen in BRCA1 mutations. In case of BRCA1, the mutations in the region coding for the nuclear localization signals (NLS) or the HN2- terminal (nuclear export signal, NES) may lead to the cytoplasmic accumulation of this nuclear protein114. Although p16INK4A lack the NLS and NES, a mutation may cause defects in its association with nuclear chaperone groups115. Another mechanism might be that p16INK4A functions as a protection for overexpression of CDK4. In a condition that CDK4 is

overexpressed, a portion of p16INK4A might be binding to CDK4 in the cytoplasm. Because of the large size of the complex, it cannot pass to the nucleus and remain in the cytoplasm while the unbound p16INK4A can enter to the nucleus and act as a tumor suppressor113. Therefore, the sequestration of p16INK4A in the cytoplasm may represent an alternative mechanism of p16INK4A inactivation. The mutations affecting its cytoplasm to nucleus shuttling can be related to this phenotype. On the other hand, overexpression of CDK4 leading to huge amounts of CDK4-p16INK4A complex may be an alternative mechanism to explain the cytoplasmic staining of p16INK4A.

p16INK4A is targeted via N-terminal Ubiquitination

The initial step of ubiquitylation is conjugation of C-terminal carboxylate residue of a small (76 amino acid residue) protein called ubiquitin to an internal lysine for the degradation of the majority of the substrates of the ubiquitin system. It is shown for several substrates that the first ubiquitine is conjugated to the N-terminal residue of the protein. However, in all these substrates the internal lysines may also play a role for the stability of the ubiquitines. Despite experimental data which supports the existence of N- terminal ubiquitination (NTU), a direct evidence for this modification have been missing 116. In order to better understand the physiological importance of this novel mode of modification, the identification of proteins whose degradation is completely dependent on N-terminal ubiquitination have been appealing. Since all of the studied proteins contain internal lysines which can still be ubiquitinated, naturally occurring lysine less proteins (NOLLPs) became an important potential substrate for N-terminal ubiquitination. Through the efforts to prove N-terminal ubiquitination, p16INK4A which is a lysine-less protein found as targeted via N-terminal ubiquitination 116. In addition, it is shown that p16INK4A is degraded in a cell density-dependent manner. The protein is degraded only in sparse cells and is stable in confluent cells116. In correlation with this finding, the observation that ubiquitination of the protein also occurs only in sparse cells strongly links ubiquitination to degradation of the protein. However, it is unknown which signals targets p16INK4A for degradation only in sparse cells and stabilize the protein in confluent cells116. In summary, p16INK4A is ubiquitinated and degraded only in sparse cells while it is stable in dense cells by N-terminal uibiquitination.

Concluding Remarks

In mammalian systems, the maintenance of homeostasis requires a strict regulation of cell proliferation. Cell cycle is regulated by cyclin-CDK complexes and CDK associated cell cycle inhibitors such as p16INK4A. Inhibiting the activities of CDK4 and CDK6 kinases by p16INK4A keeps the retinoblastoma protein (pRb) in hypophosphorylated form and cells

arrest in the G1 phase of the cell cycle.

INK4A G1 cell cycle arrest plays an important role in cancer prevention. p16 and p19ARF,

two different tumor suppressor proteins are both related to G1 cell cycle arrest and encoded by a single locus; INK/ARF locus. Deletions, point mutations or small deletions in this locus are related to thousands of cancer cases. p16INK4A plays a role in cellular senescence, apoptosis and stem-cell self-renewal74 and this is the reason for p16INK4A’s role in tumor suppression network. Since p16INK4A protein is an important key for many physiological processes, its regulation is tightly controlled.

Most of our knowledge about the regulation of p16INK4A is on transcriptional level. There are many transcriptional factors identified or found to be related to INK4/ARF locus. These factors are able to induce or repress the transcription downstream of certain signaling pathways in mouse cell lines. However as p16INK4A promoter sequences are not well conserved between human and mouse, therefore it is still unclear whether the mechanisms that regulate expression of p16INK4A are similar in both species31. p16INK4A can be also regulated in mRNA level (post-transcriptional level) by action of RNA binding proteins and also by microRNAs. As a post-translational control, p16INK4A protein is ubiquitinated for destruction and has different phosphorylation sites. Although we know where the phosphorylation sites are in the protein, we do not know the importance of this modification since past studies have determined that p16INK4A is not phosphorylated117. The phosphorylation of p16INK4A might be important for its efficient functioning or for its location. There is lack of information in current literature regarding the regulation of localization of p16INK4A; further studies should be conducted to that direction. Since the regulation of p16INK4A can be at different levels with different factors, it is difficult to make a linear relationship between these factors, the conditions and

p16INK4A however, giving the importance of p16INK4A in tumor suppression there is little doubt that we will soon have a clear picture regarding to the regulation of p16INK4A.

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