Apoptosis: Targets in Pancreatic Cancer

Pancreatic adenocarcinoma is characterized by poor prognosis, because of late diagnosis and lack of response to chemo- and/or radiation therapies. Resistance to apoptosis mainly causes this insensitivity to conventional therapies. Apoptosis or programmed cell death is a central regulator of tissue homeostasis. Certain genetic disturbances of apoptotic signaling pathways have been found in carcinomas leading to tumor development and progression. In the past few years, the knowledge about the complex pathways of apoptosis has strongly increased and new therapeutic approaches based on this knowledge are being developed. This review will focus on the role of apoptotic proteins contributing to pancreatic cancer development and progression and will demonstrate possible targets to influence this deadly disease.


Review
Pancreatic cancer is one of the most malignant tumors with a very poor prognosis. Although pancreatic cancer has an incidence of about 10 cases/100,000 persons it is still the fourth male and fifth female leading cause of cancer-related death in the Western world [1]. Most of the newly diagnosed patients present with an already unresectable tumor stage. The 5-year survival rate of patients with pancreatic cancer receiving surgery and chemotherapy ranges from 1%-2% [2]. One of the reasons for this low survival rate is the insensitivity of pancreatic cancer to most oncologic therapies like chemotherapy, radiotherapy and immunotherapy [3][4][5][6][7][8][9][10]. Tumor development and progression as well as resistance to most oncologic therapies result mainly from lacking response to apoptotic stimuli.
Apoptosis or programmed cell death is a central regulator of tissue homeostasis [reviewed in [11]]. Multicellular organisms eliminate redundant, damaged or infected cells by apoptosis. Because chemotherapy and radiotherapy act primarily by inducing apoptosis, defects in the apoptotic pathway can cause cancer cell resistance [12,13]. Tumor cells utilize multiple pathways to down-modulate apoptosis [14].
Apoptosis mediated by death receptors belonging to the tumor-necrosis factor (TNF) receptor superfamily is the best-studied pathway in cells ( Figure 1) [15,16]. Members of the TNF receptor family, TNF, Fas (Apo-1, CD95) and TRAIL (TNF-related apoptosis-inducing ligand)-R [16] share a common internal domain, the so-called death domain [17]. These receptors are activated by their natural ligands TNFα, FasL, and TRAIL, respectively. The interaction between receptor and ligand causes trimerization of receptor followed by recruitment of FADD (Fas-associated death domain protein) and procaspase-8 to the death domain forming the DISC (death-inducing signaling complex) [18]. At the DISC, cleavage of procaspase-8 yields the active form of this protease [18]. In type I cells, the amount of activated caspase-8 is sufficient to initiate apoptosis via direct activation of the central effector caspase,

Figure 1
Apoptosis mediated by death receptors of the TNF family in type I and type II cells. Apoptosis can be initiated by two alternative pathways: in type I cells the amount of initiator caspases is sufficent to induce executioner caspases directly or in type II cells the enhancing effect of mitochondria is necessary. Active executioner caspases cleave the death substrates, which results in apoptosis.

Executioner caspases
Caspase-3,-6,-7 caspase-3. In type II cells, the signal enhancing-effect of mitochondria is needed to induce apoptosis [19]. The Bcl-2 family member BID mediates activation of mitochondria in response to death receptor activation. BID is cleaved by active caspase-8 producing tBID, which is translocated to the mitochondria [19]. tBID becomes integrated into the mitochondria membrane and induces release of cytochrome c and other apoptogenic factors from the intermembranous space of mitochondria [20,21]. In the cytoplasm, cytochrome c forms a complex with Apaf-1 (apoptotic protease activating factor-1), ATP and procaspase-9 termed the apoptosome. Like caspase-8, caspase-9 can be considered an initiator caspase, which is activated by cleavage at the apoptosome and activates in turn executioner caspases, mainly caspase-3, -6 and -7 [22]. Cleavage of death substrates, DNA fragmentation, and cleavage of cytoskeletal proteins finally lead to cell death [22].
This complex pathway is controlled and influenced by a variety of different pro-and anti-apoptotic factors. The balance of these effectors is important to ensure tissue homeostasis. Activation or downregulation of pro-and anti-apoptotic genes influence cancer cell viability, cancer cell sensitivity to chemotherapy and radiotherapy, and tumor development and progression ( Figure 2). This review will focus on the knowledge about deregulation of apoptotic proteins and pathways in pancreatic cancer and possible therapeutic approaches based on these findings.

Death receptors
As described above, apoptosis is mediated mainly by members of the TNF death receptor superfamily proteins including Fas (Apo-1, CD95) and TRAIL-R and their natural ligands. Deregulation of these pathways may contribute to abnormal tumor growth [23,24].
The Fas-FasL system is believed to represent one of the main apoptotic cell death-signaling pathway [25]. Fas or FasL over-and under-expression has been shown in a variety of human carcinomas including lung [26], renal [27] and colon cancer [28]. Findings concerning Fas receptor expression in pancreatic cancer are contradictorily. It has been demonstrated recently that Fas mRNA was increased in pancreatic carcinomas [29]. Contrarily, an in vivo study revealed that both membranous Fas and cytoplasmic Fas receptors could not be detected invasive ductal-type pancreatic adenocarcinomas [30].

The role of mitochondria
In type II cells, the apoptosis-enhancing effect of mitochondria is necessary to induce the full apoptotic phenotype. It has been demonstrated that pancreatic cancer cell lines are type II cells [19,32,38]. The Bcl-2 family members play the major role in this pathway.

Bcl-2 family
At the center of the cell's decision to live or to die in response to an apoptotic signal is the Bcl-2 family of apoptotic regulators [39]. The Bcl-2 family is the bestcharacterized group of apoptosis-mediating factors and can be divided into two main groups according to their functional properties; anti-apoptotic proteins like Bcl-x L and Bcl-2 and pro-apoptotic proteins, such as Bax, Bak and Bad. Bcl-2 proteins interact with other molecules through an α-helical domain termed BH-3 domain. This interaction is believed to be important for regulation of apoptosis [40].   Bcl-x exists in two distinct isoforms in humans. Bcl-x L , the longer form, functions in an anti-apoptotic manner. Bclx S , the shorter form, in contrast, functions as an apoptosis promoter. Like Bcl-2, Bcl-x L also prevents cytochrome c release from mitochondria [reviewed in [47]]. Different studies showed that most likely every cell type is protected by at least one member of anti-apoptotic Bcl-2-like proteins. In pancreatic carcinoma cells Bcl-x L plays the most important role in protecting from Fas and TRAIL-mediated apoptosis [32]. Furthermore, Bcl-x L is believed to bind to Apaf-1 and may therefore inhibit the association of Apaf-1 with procaspase-9 and thereby prevent caspase-9 activation [48]. Unlike Bcl-2, Bcl-x L is constitutively overexpressed in pancreatic cancer cell lines highly resistant to Fas and TRAIL-mediated apoptosis [32]. Therefore Bcl-x L may be an ideal target for pancreatic cancer therapy. Overexpression of Bcl-x L in cell lines with low Bcl-x L expression like Colo357, showed complete suppression of apoptosis. On the other hand, inhibition of Bcl-x L function by overexpression of Bax or administration of antisense oligonucleotides against Bcl-x L mRNA resulted in sensitization of cells expressing high levels of Bcl-x L like Panc-1 or Panc-TuI [32]. Additionally, another study revealed that Bcl-x L antisense oligonucleotide inhibited pancreatic cancer cell growth and caused apoptosis by reducing Bcl-x L protein levels in different pancreatic cancer cell lines [49]. Bcl-x L antisense nucleotides also increased the sensitivity to chemotherapeutics like gemcitabine. This was confirmed by another study [50]. Activation of Bcl-2 family member Bcl-x L after repeated exposure to the chemotherapeutic drugs 5-FU and gemcitabine contribute to chemo-resistance of pancreatic cancer cells [50]. Correlation of the molecular data with clinical patient parameters revealed that patients whose tumors exhibited no, faint, or weak Bcl-x L expression lived significantly longer after tumor resection than patients whose tumors exhibited moderate Bcl-x L mRNA expression [51].
Bax is a pro-apoptotic member of the Bcl-2 family that resides in the cytosol and translocates to mitochondria upon induction of apoptosis Another member of pro-apoptotic proteins of Bcl-2 family is Bak. In pancreatic cancer, Bak expression and apoptosis occur in regions of chronic inflammation surrounding the cancer cells but not in the tumor cells themselves [57]. This may facilitate tumor growth and spread. Recent studies have demonstrated that Bad, a typical pro-apoptotic protein of Bcl-2 family, binds with its BH3 domain to both Bcl-2 and Bcl-x L but mediates its pro-apoptotic functions through inhibition of Bcl-x L , but not Bcl-2 [58].
To restore the function of downregulated pro-apoptotic Bcl-2 family members, there are many efforts to develop peptides and non-peptide agents that mimic the function of these proteins [59].
The key enzymes for arachidonic acid metabolism, lipoxygenases (LOXs) and cyclooxygenases (COXs) influence the development and progression of several human cancers including pancreatic cancer [60]. It has been shown that both COXs and LOXs are upregulated in human pancreatic cancer tissues. Inhibitor studies revealed that blocking these enzymes inhibit pancreatic cancer growth and induce apoptosis. Further investigations pointed out that blockade of LOXs induce apoptosis through cytochrome c release, caspase-9 activation and changes in the levels of Bcl-2 family proteins [61].
In summary, the deregulation of Bcl-2 family both the pro-and the anti-apoptotic proteins play a crucial role in development, growth and expansion of pancreatic cancer. Several approaches based on these findings have been demonstrated to be a tool potentially influencing pancreatic cancer also in a clinical setting.

Caspases and caspase inhibitors Caspases
The central component of apoptosis is a proteolytic system involving a family of cysteine proteases termed caspases. All caspases are expressed as proenzymes. Recent studies suggest that FLIP is not simply an inhibitor of death-receptor-induced apoptosis but it is involved in activation of NF-κB by recruiting adaptor proteins like TRAF [74]. NFκ-B is known to regulate several genes that mediate tumorigenesis and metastasis [reviewed in [75]]. This may also contribute to tumor growth. Downregulation of FLIP in prostate cancer cell line resulted in sensitization to Fas-mediated apoptosis [76]. Recently, it has been demonstrated that natural and synthetic ligands of the transcription factor PPARγ (peroxysome proliferator-activated receptor γ) sensitize tumor cells to apoptosis by decreasing the level of FLIP [77]. Equivalent data concerning pancreatic cancer are eagerly awaited. It has been shown that caspase expression is more or less normal in pancreatic carcinoma. In contrast, effectors blocking caspase activation or function like FLIPs are deregulated in pancreatic cancer leading to resistance to death receptor mediated apoptosis.
A new therapeutic approach uses somatostatin receptor subtype 2 (sst2) to activate caspase-3 and therefore to induce apoptosis in pancreatic cancer [78]. Likewise, sst2 treatment of human cancer cell lines with Diospyrin, a bisnaphthoquinonoid natural product, resulted in induction of apoptosis mediated via activation of caspase-3 and caspase-8 [79].

IAP family
The inhibitor of apoptosis (IAP) family including cIAPs, XIAP and survivin can block apoptosis through interaction with members of the caspase family. IAPs are characterized by a domain termed the baculoviral IAP repeat (BIR) necessary for caspase interaction [81,82]. At least one BIR domain is necessary for suppression of apoptosis [83]. Another structural feature of IAPs is the presence of a carboxy terminal RING zinc finger domain [83]. But it seems that this domain is not always strictly required for inhibition of apoptosis [84][85][86][87]. Overexpression of XIAP, cIAP1, cIAP2 and survivin has been demonstrated to suppress apoptosis [reviewed in [83]]. The cellular function of the IAP family members cIAP1, cIAP2 and XIAP is largely unclear. These proteins seem to be involved in pathogenesis of small-cell lung cancer [88], non-small cell lung cancer [89], myeloid leukemia cells [90] and prostrate cancer [91]. Concerning pancreatic cancer, there are only few data on the involvement of cIAP1, cIAP2 and XIAP in the pathogenesis of this type of cancer. Our own results indicate increased levels of cIAP1, cIAP2 and XIAP in pancreatic cancer cell lines highly resistant to Fas-and TRAILinduced apoptosis (A. Trauzold, unpublished data). Therefore, changes of expression of cIAP1, cIAP2 and XIAP may also have a role in the pathogenesis also in pancreatic cancer.

Survivin
Survivin has been identified as a new member of the inhibitor of apoptosis (IAP) family. Survivin is characterized by a unique structure that discriminates it from all other members of the IAP family. It contains only a single BIR repeat and lacks a carboxy terminal RING finger domain. Survivin is expressed in the G 2 /M phase of the cell cycle in a cycle-regulated manner [92]. It directly binds to and inhibits both caspase-3 and caspase-7 activity leading to arrest of apoptosis [93].
Survivin expression is not detectable in differentiated normal adult cells of any organ [94] but it is highly expressed in a wide range of cancer tissues [95] including neuroblastoma [96], colorectal [97] and stomach [98] carcinoma. It has been demonstrated recently that survivin is also frequently expressed in malignant pancreatic ductal tumors [99]. Because survivin is a potent caspase-inhibitor, its overexpression in cancer cells is implicated in the resistance to different apoptotic stimuli including chemotherapy. Molecular manipulation of survivin expression may enhance chemotherapy and radiation therapy not only in pancreatic cancer.

SMAC/DIABLO
IAPs are inhibited by a protein named SMAC/DIABLO (Second mitochondria-derived activator of caspase/direct IAP binding protein with low pI) [100,101]. SMAC/DIA-BLO is synthesized as a precursor protein and is imported into mitochondria [100]. Upon cellular stress, SMAC/DI-ABLO is released into the cytosol. In the cytosol it promotes cell death by preventing IAP inhibition of caspases [102]. Like cytochrome c, release of SMAC/DIABLO from the mitochondria is inhibitable by Bcl-2 [100]. SMAC/DI-ABLO could have a therapeutic application in enhancing the effect of chemotherapeutics by binding IAPs that are overexpressed in a variety of carcinoma cells including pancreatic cancer. Recently it has been shown that SMAC/ DIABLO fusion peptide was able to enhance apoptosis induced by diverse anticancer agents including paclitaxel, etoposide and others in MCF-7 breast cancer cells [103].

Growth Factors
Growth factor independence is a hallmark of tumors and is known to give cells a selective growth advantage [104].

Figure 3
Effects causing pancreatic cancer and possible therapeutic approaches. Multiple changes of apoptotic proteins contribute to pancreatic cancer development and progression. But many therapeutic approaches are developed to restore normal sensitivity to apoptotic stimuli and therefore to repress pancreatic cancer.

Other factors and signaling pathways
Nuclear factor of κB (NF-κB) Nuclear factor κB (NFκB) is a sequence-specific transcription factor that is involved in many cellular activities including the inflammatory and immune response. NF-κB also inhibits apoptosis by activation of several anti- Survival signaling through PI3K/AKT Survival signals like growth factors, cytokines and hormones activate phosphatidylinositol 3-kinase (PI3K) [155]. Subsequently, PI3K activates AKT/PKB [156] that interferes with the apoptotic machinery. Activated AKT/ PKB mediate cell survival via the regulation of numerous apoptotic relevant proteins such as the Bcl-2 family members BAD and Bcl-x L and the transcription factor NF-κB [157,158]. Survival signaling by AKT is counteracted by PTEN that antagonizes the action of PI3K. PI3K and AKT are overexpressed in a variety of cancers [159,160]. In addition, PTEN is frequently deleted in advanced tumors [161,162]. These alterations lead to a 'constitutively active' survival-signaling pathway that enhances the insensitivity of tumor cells to apoptosis induction. Additionally, EGFR directly influences PI3K [163]. Because PI3K mediates survival signals, EGFR overexpression leads to a decrease in the apoptotic response and therefore a stronger survival of cancer cells. Recent work has shown that the IGF-1R suppresses apoptosis by signaling through PI3K and AKT [120]. Activated AKT in turn phosphorylates the intracellular transducer, Bad, which modulates the activity of the apoptosis suppressors Bcl-2 and Bcl-x L [164]. Additionally, AKT can directly phosphorylate caspase-9, which leads to inactivation of caspase-9 [165]. Strategies are pursued that aim to block the enzymatic activity of PI3K and PKB/AKT, in order to prevent inactivation of pro-apoptotic Bad. Wortmannin was shown to be a potent inhibitor of PI3K [160]. In several cancers including non-small-cell lung cancer [160] and pancreatic cancer [166], treatment of cells with wortmannin leads to inhibition of proliferation and increased apoptosis. Additionally, wortmannin enhanced gemcitabine-induced apoptosis in human pancreatic cells in vitro [167] and in vivo [168]. In contrast, studies investigating different pancreatic cancer cell lines pointed out that the PI3K/AKT pathway is not involved in gemcitabine-resistance [A. Arlt, unpublished data]. Nei-ther did the basal AKT-activity correlate with the sensitivity towards gemcitabine treatment, nor did the inhibition of PI3K/AKT alter gemcitabine-induced apoptosis. p53 The p53 pathway represents the molecular connection between the cell cycle and apoptosis. The p53 gene encodes a 53-kDa nuclear phosphoprotein. p53 inhibits cell growth through activation of cell cycle arrest and apoptosis [169]. The p53 gene is mutated in over 50% of human cancers. Pancreatic cell lines showed mutations in p53 at frequencies of 95% [170]. This enhances tumor development and progression. Currently, many therapeutic approaches were attempted to normalize p53 function in a variety of cancers including pancreatic cancer. Transfer of wild-type p53 induces apoptosis and produces significant growth suppression of pancreatic cancer in vitro and in vivo [170][171][172].

Conclusion
In the past, many efforts were made to cure patients from pancreatic cancer. Changes of expression and mutations of apoptotic proteins are common in pancreatic cancer cells and contribute to tumor development and progression. Consequently, pancreatic cancer has developed multiple resistance mechanisms to apoptosis. Many efforts to restore apoptosis and thereby reducing tumor growth were made with considerable success at least under preclinical conditions (Figure 3). Future therapies have to translate this knowledge into the clinic. They need to combine various therapeutic strategies and have to modulate selectively the sensitivity of pancreatic cancer cells to apoptosis without affecting normal cells.