Primers on Molecular Pathways —Cycling toward Pancreatic Cancer

Molecular Pathways Pancreatology 2010;10:6–13 DOI: 10.1159/000283646

Published online: March 19, 2010

Primers on Molecular Pathways – Cycling toward Pancreatic Cancer Yang Liu Sherine F. Elsawa Luciana L. Almada  Schulze Center for Novel Therapeutics, Mayo Clinic, Rochester, Minn., USA

Key Words Cell division ⴢ Cell cycle dysregulation ⴢ DNA damage ⴢ Pancreatic adenocarcinoma ⴢ Cyclin-dependent kinases

Abstract Human cells divide and proliferate during the early stages of life to support development, and throughout adult life to support normal cellular turnover. Each dividing cell follows an orderly and tightly regulated series of events known as the cell cycle. This process ensures proper cellular division that maintains DNA and chromosomal integrity and responds appropriately to external signals which communicate the level of demand for new cells. In cancer, genetic mutations leading to the overexpression of proteins which support cell cycle progression, or the downregulation of proteins involved in cell cycle inhibition contributes to the dysregulated cellular division and proliferation of malignant cells. The resulting uninhibited cellular proliferation provides ample opportunity for additional genetic mutations that lead to tumor progression. In the following review, we provide a brief introduction to the cell cycle and a discussion of the mechanism underlying the dysregulation of the cell cycle in human cancer. We pay particular attention to pancreatic adenocarcinoma, an aggressive tumor that has a 5-year survival rate of 3–5%, and is the fourth leading cause of cancer mortality in the US. Copyright © 2010 S. Karger AG, Basel and IAP

© 2010 S. Karger AG, Basel and IAP 1424–3903/10/0101–0006$26.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/pan

What Is the Cell Cycle?

During embryonic development and throughout the stages of adult life, human cells must proliferate to either produce functional systems or support normal cellular turnover. In order to do this, each individual cell must successfully synthesize a new copy of its DNA and segregate the two copies into two separate daughter cells while maintaining DNA and chromosomal integrity. Furthermore, the cell must be able to respond to internal and external signals in order to assess whether or not proliferation is needed and adequate resources are available to support the energy-consuming process of DNA replication and cellular division. To accomplish this task, the replicating cell undergoes an ordered set of coordinated and regulated events which have come to be known as the cell cycle. The cell cycle consists of four phases, the mitotic (M) phase, the growth 1 (G1) phase, the synthesis (S) phase, and the growth 2 (G2) phase (fig. 1). Cell division occurs during the M phase, DNA synthesis occurs during the S phase, while the G1 and G2 phases provide additional checkpoints in which damaged DNA, replication and cellular division errors may be repaired. Together, the G1, S and G2 phases prepare the cell for cellular and nuclear division during mitosis, and are collectively called interphase. In addition to providing time for cell growth and repairs, the G1 and G2 phases incorporate different signals from the cell’s microenvironment, as well as its metabolic and stress states to determine wheth-

Luciana L. Almada, PhD Mayo Clinic, Schulze Center for Novel Therapeutics Gonda 19-306A, 200 First Street SW Rochester, MN 55905 (USA) Tel. +1 507 293 0876, Fax +1 507 293 0107, E-Mail almada.luciana @ mayo.edu

p15 p16 p19

Mitogenic signal

DNA replication stress

G0

DNA damage by irradiation Cyclin D Rb

CDC25

PCNA CDK4/6

E2F

Cyclin E

Active Sic1 Active Hct1-APC

p21

p53 ATM

CDK2

ATR P P Rb P

E2F

CDC25

G1

CHK1 CDC25

M

Cyclin B

Cyclin A

CDK1

G2

Cyclin A

CHK2

S

CDK2

CDK1 Wee1/Myt1

Cyclin A CDC25

CDK2

Checkpoints

Fig. 1. Schematic representation of the cell cycle.

er or not the cell should commit its resources toward cell cycle progression. The length of the G1 phase is particularly sensitive to these conditions. If the cell receives signals that are not conducive to cell division, it will enter a resting phase called G0 and remain there for any period of time until the cell again receives signals to proliferate [1]. There are at least three cell-cycle checkpoints that regulate progression through the phases of the cell cycle, and together they compose the cell-cycle control system. These include the DNA damage checkpoint at the G1/S transition (G1 restriction point), which induces cell cycle arrest upon sensing DNA damage upon exposure to chemicals, free radicals, ionizing radiation or other mutagenic stimuli, the intra-S checkpoint, the G2/M checkpoint, and the spindle assembly checkpoint (SAC) which

senses proper chromosomal segregation after successful DNA replication in preparation for cellular division [2]. Upon damage, these checkpoints rely on the activation of signaling pathways that are able to inhibit a family of protein kinases called cyclin-dependent kinases (CDKs), which phosphorylate and activate proteins that support cell cycle progression in actively dividing cells. When mutations in the cell produce ineffective cell-cycle checkpoints, cells are able to proliferate despite damaged DNA or ineffective chromosomal separation, leading to genomic instability or chromosomal aberrations that facilitate tumor development in cancer. CDKs have active kinase activity only after successful binding to another family of proteins called cyclins. Cyclins have two functions: they partially activate corre-

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sponding CDKs, and direct them toward the phosphorylation of stage-appropriate proteins. CDKs are then fully activated by CDK-activating kinases, which phosphorylate a threonine residue near the active site of the CDK resulting in full exposure of the enzyme’s active site for binding to effector proteins [3]. While the level of CDK remains relatively constant throughout the cell cycle, cyclin proteins undergo synthesis and degradation, and these rising and falling levels of cyclin serve as important regulators of CDK activity. In the classical model of cell cycle control, a different class of cyclins is present in high levels at each cell cycle stage, and these cyclins are targeted for degradation by the ubiquitin-proteosome pathway after stage completion [4]. Consequent cyclical changes in CDK activation result in oscillations in the phosphorylation of the intracellular proteins that are essential for the cell cycle functions of each particular stage, such as DNA replication in the S phase, and mitosis in the M phase. Three different CDKs participate in interphase (CDK2, CDK4 and CDK6), while CDK1 is active during mitosis. These complex with ten known cyclins of four classes (A-, B-, D-, and E-type cyclins), although only certain complexes are involved in cell cycle progression [5]. Cyclin-CDK complexes are inhibited by two families of proteins, the CIP/KIP family and the INK4 gene family, which are tumor suppressors with frequently altered expression in human cancers [6]. Cell cycle inhibitors of the CIP/KIP family are best known as cell cycle regulators that bind to all the cyclin-CDK complexes, albeit with differing affinity, and inhibit their activity [7]. Members of the CIP/KIP family, including p21CIP/WAF1/SD1 (p21), p27KIP1 (p27), and p57KIP2 (p57), share an N-terminal domain that facilitates binding to both the cyclin and CDK subunits, while differences in the remaining sequences explain differences in their regulation and additional functions [6]. p21 is transcribed in the presence of p53, a protein that directs cell cycle arrest and apoptosis upon DNA damage. Active p21 primarily disrupts substrate binding to CDK2, preventing cell cycle progression from the G1 into the S phase [8]. As explained in more detail later, p27 is active under normal cell cycle conditions to prevent premature DNA replication, as well as in quiescent cells [9]. Both p21 and p27 may play a key regulatory role in the proliferation of self-renewing neural, intestinal and hematopoietic stem cells. p57 is an important cell cycle regulator during embryonic development in certain tissues [10]. Phosphorylation of these proteins at certain residues can change their binding specificity, and binding to other proteins can alter the potency of their inhibitory function. 8

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The INK4 family is encoded by the 9p21 locus and includes p16INK4A (p16), p15INK4B (p15), p18INK4C (p18) and p19INK4D (p19), which disrupt CDK4 and CDK6 binding to D-type cyclins in the G1 phase [11]. Therefore, INK4a binding prevents progression past the G1 phase. p16 causes cell cycle arrest in late G1 by competing with cyclin D1 for binding with CDK4, thus preventing the phosphorylation of retinoblastoma (Rb) and release of E2F transcription factors, which aid in the transcription of proteins required for DNA synthesis in the S phase. It is rarely present in cells under normal conditions but is released under stress conditions, such as in vitro when primary cells are released into new culture. p14ARF (p14) is another tumor suppressor encoded by the 9p21 locus that inhibits the p53 inhibitor M2M, leading to the stabilization of p53. In addition to the cell cycle inhibitory effects of the INK4 and CIP/KIP family of proteins, the three cell cycle checkpoints employ important cell signaling pathways which, when activated upon recognition of DNA damage, result in downstream inhibition of cyclin-CDK complexes. This inhibition provides proliferating cells with the necessary time to properly repair the damage before proceeding with the cell cycle. Two key phosphatidylinositol 3-kinase-related kinases, ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and Rad3 related (ATR), are activated upon recognition of DNA damage from ionizing radiation and DNA replication stress, respectively [12]. While the molecular components of these signaling cascades are under active investigation, it is clear that activation of ATM or ATR leads to the respective phosphorylation and activation of checkpoint kinases CHK2 and CHK1. The downstream phosphorylation by these kinases results in the silencing of CDC25 phosphatases, which under normal conditions removes inhibitory phosphate groups from cyclin-CDK complexes [13]. The phosphorylation of CDC25 phosphatases by CHK1 and CHK2 inhibits its phosphatase activity, thereby inhibiting cyclin-CDK function when DNA damage is detected at any of the cell cycle checkpoints. The ATM-CHK2 cascade is also known to act downstream to increase the transcription of p53 and its consequent transcription of p21. Challenging the classical model of cell cycle control, there is new genetic evidence suggesting that only mitotic CDK1 is required for cell cycle progression and that the interphase CDKs are required for proliferation in only a subset of very specialized cells [14]. CDK1, for example, can sufficiently guide cellular proliferation in the developing mouse embryo until at least mid-gestation [15]. Furthermore, genetic ablation of the interphase cyLiu/Elsawa/Almada

clins, such as D-type cyclins, E-type cyclins and A-type cyclins, results in early embryonic death, suggesting that they play a physiological role outside of their function in cyclin-CDK complexes. Interestingly, while these interphase CDKs are dispensable in normal cellular proliferation, they appear to be necessary for the development of certain tumors, such as mammary gland tumors, in response to activation of upstream oncogenes including ERBB2, HRAS and MYC [16]. Targeting these interphase CDKs may provide a new avenue in cancer therapeutics.

Cell Cycle Dysregulation in Cancer

In human cancer, germline or somatic mutations either activate cell cycle genes that support cell cycle progression, or inactivate cell cycle repressors that are responsible for stalling mitosis in the event of DNA damage or the presence of growth inhibitory signals. These two situations individually produce unregulated cellular proliferation which evades growth inhibitory signals and leads to expansion of a clonal population of cells each housing the original mutation. In the following section, important cell cycle proteins are introduced by cell cycle phase, followed by a brief discussion of the common mutations that have been found to be important contributors to either the origin of tumors or tumor progression. Pancreatic ductal adenocarcinoma (PDAC) is an example of a malignancy involving cell cycle dysregulation. PDAC is an aggressive tumor that has a 5-year survival rate of 3–5%, and is the fourth leading cause of cancer mortality in the US [17, 18]. The first presenting symptom is usually pain, which occurs only when the cancer has metastasized and is beyond cure. In pancreatic cancer, there is a well-established serial progression of genetic lesions resulting in dysregulation of the cell cycle and unregulated cellular proliferation. Histopathologic studies indicate that PDAC begins through precursor lesions called pancreatic intraepithelial neoplasias (PanIN), and develops increasingly atypical DNA and cellular division, progressing eventually to invasive PDAC [19]. Activating mutations of the KRAS signaling cascade are thought to be the initiating mutation and one of the most prevalent mutations in PDAC, occurring in over 90% of cases. However, there are three main cell cycle lesions in PDAC. 95% of PDAC cases involve the hypermethylation and consequent silencing of the p16 tumor suppressor, which usually occurs in the PanIN2 stage of lesions [20]. 50–75% of PDAC cases involve an inactivation of p53, which tends to occur in PanIN3 lesions that have already acquired Primers on Molecular Pathways – Cycling toward Pancreatic Cancer

KRAS activation and p16 silencing, and results in unregulated cellular proliferation of cells with damaged DNA. Fewer than 5% of cases contain inactivating mutations of Rb1. These genetic lesions will be introduced and discussed below.

G1 Phase: G1 Restriction Point, Cyclin D/CDK4 or -6

In resting cells, or those emerging from recent mitotic activity in the M phase, a protein called retinoblastoma susceptibility protein (Rb), also known as p105, and potentially other ‘pocket protein’ family members (RbL1P107, RbL2-P130), bind to the E2F transcription factors, normally located in the nucleus, and sequester them to the cytoplasm, rendering them inactive [21]. Mitogenic stimuli received in the G1 phase, such as growth factors, send mitogenic signals that synthesize and stabilize D type cyclins and release CDK4 and CDK6 from inhibitory INK4 proteins. Cyclin D then binds to and activates these CDKs to form complexes that phosphorylate and inactivate Rb [22]. This inactivation results in the dissociation of Rb from the E2F transcription factors and the subsequent transcription of E type cyclins, A type cyclins, DNA polymerases and other proteins that are required for the initiation of DNA transcription and passage through the late G1 cell cycle checkpoint (G1 restriction point). The formation and activation of cyclin E-CDK2 complexes in late G1 further phosphorylates Rb resulting in its complete inactivation. Cyclin E-CDK2 complexes are thought to be essential for the G1/S phase transition [23]. The inhibitory member of the CIP and KIP family of CDK inhibitors p27 binds to and silences the cyclin ECDK2 complex early in the G1 phase, thereby preventing premature DNA replication or replication under growth inhibitory conditions. Signals for mitosis lead to the liberation of CDK2 from its binding to p27, either through the inhibition of transcription, translation, or disruption of the nuclear localization of p27. Mitotic signals may also induce cyclin D-CDK4 or CDK 6 complexes to sequester p27. As active levels CDK2 levels increase, CDK2 is able to target p27 for ubiquitination and destruction by proteosomes. Furthermore, activation of downstream effectors of the anaphase-promoting complex/cyclosome (APC) CDC20 during mitosis and into the G1 phase leads to p27 polyubiquitination. APC is an important mediator of chromatin separation during mitosis as well as cell cycle exit from the M phase, and during G1 it targets SKP2, a protein that is essential for the polyubiquitination of Pancreatology 2010;10:6–13

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p27 by the SCF (SKP1/CUL1/F-BOX) protein for destruction. Consequently, high levels of CDC20 during the M phase and into the G1 phase inhibit the polyubiquitination of p27, allowing it to inhibit cyclin E/CDK2 activity. This maintains the gap between mitosis and the DNA transcription mediated by CDK2. If DNA damage occurs in the early stages of G1, primarily from ionizing radiation, the ATM-CHK2-p53/ MDM2-p21 pathway is activated. Activation of ATM leads to direct phosphorylation of p53 as well as the degradation of MDM2, an inhibitor of p53 activity. This results in the accumulation and stabilization of p53. The activation of p53 leads to temporary cell cycle arrest while the DNA damage is repaired [24]. At the G1 restriction point, genotoxic stress leads to increased CHK1 and CHK2 activity, which downregulates CDC25a and inhibits the cyclin E(A)-CDK2 complex [25]. Furthermore, CHK2 is believed to be necessary for p53-mediated apoptosis of cells that are damaged beyond repair [26]. Given this review of the G1 cell cycle phase, the following are examples of genetic lesions in cell cycle genes that make a significant contribution to the characteristic cell cycle dysregulation seen in cancer. Overexpression of Cyclin D1 Cyclin D1 overexpression is particularly important in the pathogenesis of mantle cell lymphoma, a form of nonHodgkin’s lymphoma in which the hallmark lesion is the t(11; 14)(q13; 32) translocation that places the CCDN1 gene, the proto-oncogene encoding cyclin D1 of chromosome 11q13, to the Ig heavy chain gene on chromosome 14q32 [27]. This dysregulation of the G1/S checkpoint allows for constitutive activity of cyclin D1 in B lymphocytes and consequently constitutive inactivation of the tumor suppressor Rb, leading to unchecked cellular proliferation. Amplification of the cyclin D1 gene resulting in increased levels of circulating cyclin D1 occurs in over 50% of breast carcinomas, and is also seen in esophageal carcinomas and various squamous cell carcinomas. It is also overexpressed in PDAC [28, 29]. Overexpression of CDK4 and CDK6 Mutations in the catalytic subunits of CDK-cyclin complexes are rare in cancer, but cyclin D-CDK4/CDK6 complex mutations have been seen in certain subgroups of melanoma, such as familial melanoma. This mutation in the cyclin-CDK complex leads to its decreased affinity for the cell cycle inhibitor p16INK4a, leading to dysregulated kinase activity and cellular proliferation. In addition to this mutation, both CDK4 and CDK6 can expe10

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rience gene amplification that leads to their overexpression. This overexpression hyperphosphorylates Rb and increases its frequency of dissociation from E2F transcription factors, thereby promoting progression from the G1 to the S phase. Amplification of CDK4 is seen in sporadic melanoma, glioblastoma, osteosarcoma and breast, cervical and uterine cancers, frequently with coamplification of MDM2 [5]. Amplification of CDK6 is seen in lymphomas, various squamous cell carcinomas and glioma. The role of these amplifying mutations in inducing cancer is under investigation. Dysregulation of Rb In the resting G0 or early G1 state, Rb is hypophosphorylated and binds to E2F transcription factors, forming a complex that can bind to the promoter regions of E2F-associated genes and recruit histone deacetylases that induce chromatin condensation, thereby inhibiting transcription. In human cancers, mutations must inactivate both copies of the Rb gene in order develop a cell with tumor potential. Mutations occur in the ‘Rb pocket’ of the Rb gene, which decrease its binding affinity to the E2F transcription factors. In familial retinoblastoma, individuals acquire a germline mutation in the Rb gene, and then experience inactivation of the second healthy copy through a somatic mutation [30]. Individuals with familial retinoblastoma are also at higher risk for the development of osteoblastoma, and inactivating mutations in Rb are seen in a myriad of cancers, including small cell lung cancer, adenocarcinoma of the breast and bladder carcinoma. About 5% of cases of PDAC contain inactivating mutations of Rb1. Hypermethylation of p14 and p16 In human cancer, epigenetic changes such as the hypermethylation of CpG islands in the promoter region of tumor suppressor genes leads to gene silencing without affecting the fundamental sequence of DNA. In many cancers, the abnormal silencing of p16 transcription removes its inhibition of D-cyclin binding to CDK4 and 6, thus removing one layer of cell cycle regulation from the G1 to S phase transition. The p16 gene can also be inactivated by gene deletions or point mutations. 95% of PDAC cases involve the hypermethylation and consequent silencing of the p16 tumor suppressor, which occurs usually in the PanIN2 stage of lesions [20]. On the other hand, silencing of the p14 gene leads to uninhibited M2M activity which permanently inactivates p53. p14 hypermethylation occurs most frequently in stomach and colon cancers. Liu/Elsawa/Almada

Silencing of p27 Evidence from both clinical and animal model studies suggests that p27 is a tumor suppressor via its inhibition of cyclin-CDK complexes in the nucleus. In fact, p27 is used as a clinical diagnostic marker in a variety of cancers, in which low nuclear levels of p27 are associated with increased tumor aggressiveness [6]. While the CDKN1b gene encoding this protein is rarely inactivated in these cancers, downregulation occurs through increased degradation, decreased transcription or mislocalization in the cytoplasm. In carcinomas of the breast, cervix, esophagus, ovary, uterus and some leukemias, lymphomas and melanomas, increased levels of p27 localized in the cytoplasm is a negative indicator of prognosis [31]. New directions in research include investigations into the other functions of this protein and how the dysregulation of these functions may contribute to an oncogenic potential of p27. Inactivation of p53 p53 is the most frequently mutated tumor suppressor in human cancers, including lung, colon and breast carcinomas, and most mutations occur in somatic cells. Under normal conditions, most cells contain little to no p53, but increase their concentrations in response to stress, especially after exposure to ionizing radiation. When present, p53 induces the transcription of cell cycle inhibitor p21, which binds to and inhibits the cyclin-CDK2 complexes in G1. Inhibition of this cyclin-CDK complex leads to cell cycle arrest in G1 before commitment to the S phase, which provides time for DNA repair. If the damage cannot be successfully impaired, p53 induces the transcription of proapoptotic proteins such as BAX, leading to apoptosis. Mutations in the p53 protein most frequently alter the shape of its DNA binding domain, preventing it from inducing the transcription of key apoptotic or cell cycle arrest genes. Another type of mutation leads to the overexpression of MDM2, a protein that targets p53 for destruction by the ubiquitin-proteosome complex under normal conditions. 33% of human sarcomas and 50% of leukemias express p53 inactivation. 50– 75% of PDAC express p53 inactivation, which tends to occur in PanIN3 lesions that have already acquired KRAS activation and p16 silencing, and results in unregulated cellular proliferation of cells with damaged DNA. The loss of function of p53 sets the stage for the proliferation of cells that have acquired DNA damage, which would otherwise have experienced cell cycle arrest or apoptosis.

Primers on Molecular Pathways – Cycling toward Pancreatic Cancer

S Phase: Cyclin E/CDK2, Intra-S-Phase Checkpoint

At the end of the G1 phase, increasing levels of E-type cyclins bind to and activate CDK2. Active CDK2-cyclin E complexes continue to phosphorylate Rb proteins, engaging in a positive feedback loop that commits the cell to DNA synthesis and facilitates the G1-S phase transition. Furthermore, CDK2 phosphorylates the origins of replication in the chromosome as well as newly docked prereplicative complexes, facilitating the recruitment of DNA helicases, primases, and polymerases. Phosphorylation of Rb in this phase also promotes the dissociation of histone acetylases, which causes the DNA to unwind and become accessible to these transcription proteins [32]. In response to DNA damage incurred during DNA synthesis, the intra-S-phase cell cycle checkpoint causes a temporary cell cycle arrest by inhibiting the firing from origins of DNA replication that have not yet been used. This inhibition employs the activation of the CHK2-CDC25a cascade, which inhibits CDK2 activity and blocks the chromatin loading of CDC45, a protein required for the recruitment of DNA polymerase a. Another signaling branch of this checkpoint is ATM-mediated, but is less well understood [12]. Overexpression of Cyclin E, Inactivation of CIP/KIP Cell Cycle Regulators While there is no indication that CDK2 is involved in tumorigenesis, overexpression of cyclin E or inactivation of p27 and p21 inhibitors leads to increasing levels of activated CDK2, and is present in melanoma, osteosarcoma, ovarian carcinoma, pancreatic cancer and sarcoma [14]. Loss of ATM and ATR Function Loss of function of ATM and ATR, important components in the recognition of DNA damage in various cell cycle checkpoints, is seen in a variety of cancers. Inactivation of these components compromises the integrity of cell cycle checkpoints, leading to decreased cell cycle inhibition in the event of DNA damage. Loss of function of ATM that occurs by truncation or by missense mutations can be seen in lymphoid tumors, such as thymic lymphomas [33], while loss of function that occurs by nonsense mutations are seen in breast cancers and other tumors [34]. Furthermore, loss of ATR through truncation or missense mutations leads to decreased phosphorylation of p53 and is seen in stomach, breast and endometrial cancers [35]. Pancreatology 2010;10:6–13

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Loss of CHK1 and CHK2 Function Frameshift mutations in the CHK1 gene lead to truncated or defective CHK1 and are seen in colorectal, gastric, endometrial and small cell lung cancers [16], while truncation or missense mutations in CHK2 lead to unstable CHK2 protein. The loss of this CHK2 kinase activity decreases downstream phosphorylation of p53 and CDC25a, which removes important inhibitors of cell cycle progression.

G2 and M Phase: Cyclin A/CDK2, Cyclin B/CDK1, G2 Checkpoint and SAC

In the S phase, E2F transcription factors also induce the transcription of cyclin A, which complexes with CDK2 to enter the M phase and regulate the events of mitotic prophase, such as chromosomal condensation and microtubule formation. After nuclear envelope breakdown, cyclin A is degraded and cyclin B, which is activated by the protein phosphatase CDC25a during the late stages of prophase, complexes with CDK1 to facilitate the rest of the migration through the M phase. At this point, the G2 checkpoint identifies DNA damage incurred during previous G1 and S phases and activates the ATM/ATR, CHK1/CHK2 and/or p38-kinase-mediated inhibition of CDC25a, thereby inhibiting the cyclin B-CDK1 complex [36]. Unlike at the G1 checkpoint, G2 proteins are rarely mutated in cancers [37]. At the end of the M phase, CDK1 undergoes proteolysis by the APC or cyclosome (APC-C), a component of the SAC. If the chromosomes are not properly attached to the mitotic spindle, the myriad of proteins in the SAC work together to inactivate APC-C and prevent the early progression of sister chromatids, the latter of which would result in chromosomal instability. After successful cell division, the cell can either enter quiescence or undergo another round of replication. A decrease in the level of CDK1 at the end of the M phase allows the chromosomal origins of replication in the DNA to be loaded with prereplicative complex proteins (ORC, CDC6/18, CTD1) as well as MCM (mini-chromosomal maintenance) proteins, which prepare the DNA again for the initiation of transcription once again [38]. Overexpression of Cyclin B1 The amplification of cyclin B1 can lead to increased activation of CDK1 in a number of cancers, including breast, colon, prostate, oral, lung and esophageal cancers. In some cases, cyclin B1 is used as a marker with a positive correlation to poor prognosis [39]. 12

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Dysregulation of SAC Proteins Aneuploidy is a prevalent feature of highly dysplastic neoplasms. While the specific molecular workings of the SAC are out of the scope of this review, several key SAC proteins are dysregulated in cancers, leading to a loss of integrity in chromosomal separation during mitosis and resultant aneuploidic nuclei. Aurora A is an important serine-threonine protein kinase that plays an important role in the coordination of the centrosome cycle, including entry into mitosis and assembly of the spindle. When Aurora A is upregulated in breast, colorectal and bladder cancers through gene amplification, the cells experience premature separation of the sister chromatids and aneuploidy [40]. Other dysregulated proteins in human cancer include PLK1, BUB1 and BUB1B, all serine threonine protein kinases that maintain the integrity of the SAC [16].

Conclusions

Given the important contribution of cell cycle dysregulation in tumor development and progression, and the increased knowledge base regarding cell cycle progression in malignancies, finding ways to reverse this dysregulation will open up significant new avenues in targeted cancer therapeutics and patient care. Certainly, research will continue to identify complex interactions between cell cycle pathways and potentially other signaling pathways that will expand our understanding of the mechanisms that contribute to tumorigenesis. In particular in PDAC, finding targeted molecular therapeutics to reverse Rb and p53 inactivation, p16 silencing or cyclin D1 overexpression may provide new avenues for addressing this aggressive form of cancer and improve patient outcomes.

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