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Received: October 22^nd 2014

Accepted: May 15^th 2015

Apoptosis modulation by human papillomavirus

Luis Felipe Jave-Suárez,^a Sarah Ratkovich-González,^a Vicente Olimón-Andalón,^a Adriana Aguilar-Lemarroy^a

División de Inmunología, Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social, Guadalajara, Jalisco, México

Communication with: Luis Felipe Jave-Suárez

Teléfono y fax: (33) 3617 0060, extensión 31926

Email: lfjave@yahoo.com

One of the most important processes to keep the homeostasis in organisms is the apoptosis, also called programmed cell death. This mechanism works through two pathways: The intrinsic or mitochondrial, which responds to DNA damage and extern agents like UV radiation; and the extrinsic or receptor-mediated, which binds to their ligands to initiate the apoptotic trail. The evasion of apoptosis is one of the main causes of cellular transformation to malignity. Many viruses had shown capacity to modify the apoptotic process; among them is the human papillomavirus, which, by means of its oncoproteins, interferes in pathways, reacting with the receptors and molecules and participating in the death mechanism. This creates ideal conditions for cancer development.

Keywords: Apoptosis, Human papillomavirus, Uterine cervical neoplasms.

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The apoptosis process

Avoidance of apoptosis (programmed cell death) is one of the crucial events that a cell develops during the tumorigenic process. Every day new evidence arises corroborating the close relationship between changes in apoptosis and cancer development. The word apoptosis derives from the Greek apo, which means separation or derivation, and ptosis, which means fall. This term was used in ancient Greece to describe the leaves falling from the trees in the fall. This expression highlights the physiological character of apoptosis, since it implies that for an organism to function properly, it must not only have the ability to produce new cells, but also the ability to remove them. Unlike necrosis, which can cause inflammation, apoptosis removes cells silently and gradually without inducing inflammation. The apoptotic process can be divided into three stages: 1) initiation, in which the cell receives the stimulus that leads to death; 2) execution, when most of the morphological and biochemical changes characteristic of apoptosis happen, and 3) elimination, in which the cell debris are engulfed by adjacent macrophages and cells.¹

Pathways inducing apoptosis

The induction of apoptosis is mediated primarily by two mechanisms: 1) activation of "death receptors" in target cells, called the extrinsic pathway, and 2) changes in mitochondria that culminate in the formation and activation of apoptosome, the pathway known as intrinsic or mitochondrial.²

In the extrinsic pathway, the proapoptotic signal is triggered by a death receptor binding with its ligand. The former includes a group of cell surface molecules of the tumor necrosis factor (TNF) receptor superfamily, such as TNFR-1, CD95/Apo-1/Fas, and TRAIL-R (TNF-related apoptosis inducing ligand receptor), which bond to their respective ligands, TNF-alpha, CD95L, and TRAIL, expressed primarily on the surface of cytotoxic T lymphocytes.¹ This subfamily of TNF receptors is characterized by having an intracellular domain called the death domain (DED). When receptors are activated by binding with their ligands, the DED attracts intracellular adapter proteins, especially the FADD (FAS-associated death domain), which is in charge of recruiting procaspase 8. The resulting protein complex is called DISC (death inducing signaling complex);³ DISC formation permits cleavage of procaspase 8, which is activated when cut (caspase 8) and in turn activates procaspases 3 and 7. The latter caspases are termed effectors, as they bind to different target substrates including poly (ADP-ribose) polymerase protein (PARP), which is a nuclear enzyme that detects the occurrence of breaks in DNA. Caspase 3 cleaves PARP in two fragments, one of 24 kDa and one of 89 kDa, thereby eliminating the ability of PARP to function in DNA repair and consequently causing damage accumulation there, further promoting apoptosis.⁴

Meanwhile, the intrinsic pathway can be induced by a wide range of signals, including radiation, cytotoxic drugs, cellular stress, and lack of cytokines or growth factors. This pathway is tightly regulated by the concerted action of many molecules, including members of the BCL-2 family, which act as proapoptotic agents (proteins with BH3 domains) and antiapoptotic agents (Bcl-2, Bcl-XL, Bcl-w, Mcl -1 Bfl1/A-1, and Bcl-B).⁵ The interactions between these proteins control the process of cytochrome C release from the mitochondrial interior, which is preceded by loss of mitochondrial membrane potential and destabilization of the mitochondrial outer membrane. The permeabilization of this is considered the "point of no return" in death by apoptosis. In the cytosol, cytochrome c binds to Apaf-1 and in the presence of dATP or ATP, the complex known as apoptosome is formed, which recruits and activates caspase 9, which in turn is able to activate the effector caspases (caspases 3, 6, and 7) (Figure 1).¹

The intrinsic pathway plays a significant role in the response to cancer therapy, since the overexpression of anti-apoptotic members of the BLC-2 family has been observed in various chemotherapy-resistant cancers in humans.⁶

Among the factors that can inhibit the apoptotic process are those that directly interfere with the activation of death receptors, and those that act indirectly, causing an intracellular response that alters the apoptotic signaling cascade. Many viruses, including human papilloma virus (HPV), have developed strategies to block apoptosis. HPV’s capacity for persistence in the host for long periods of time without being eliminated attests to the sophistication of its avoidance mechanisms.

Human papillomavirus and cancer

The high percentage of viral DNA in cervical tumors and the presence of messenger RNA and protein of the viral oncogenes E6 and E7 in tumors and cell lines derived from cervical carcinomas has shown the importance of HPV in the pathogenesis of cancer. It has also been shown that viral oncogenes of HPV 16 or 18 are able to immortalize primary human keratinocyte cultures, and that culturing these cells for long periods yields malignant clones. Therefore, the association between cervical cancer and, more recently, oral cancer with high-risk HPV infection is widely accepted today.⁷ Among HPV-associated cancers, cervical cancer (CC) is one of the leading causes of death among women in developing countries. In Mexico, cervical cancer is the second leading cause of cancer death in women. HPV infection is considered a necessary and predisposing factor for the development of cervical cancer. The life cycle of the virus begins with infection of the cells of the basal layer of the cervical epithelium. It is hypothesized that the virus reaches the basal layer of epithelial tissue through microabrasions. Once the viral genome has entered the cell, it is transported to the nucleus and establishes itself with a set number of viral copies per cell. The HPV replication cycle strictly follows the host cell’s differentiation program. Thus the development of cervical cancer follows a series of steps, starting with HPV transmission, viral persistence, progression of precancerous infected cells, and finally invasion.⁸

Modulation of apoptosis by E5

The role of the HPV viral oncoprotein E5 has not received much attention because the open reading frame of E5 is often destroyed when the HPV genome integrates into the DNA of the host cell. The E5 gene plays a central role in tumorigenesis, since it has been shown that its introduction into non-tumorigenic fibroblasts has a transforming effect, causing the formation of colonies in soft agar. Similarly, E5 increases the efficiency of immortalization of E6 and E7 in human keratinocytes.⁹ Indeed, before integration of viral DNA, when the HPV genome is episomal, the most abundant viral mRNA is the one encoding E5 protein.¹⁰ This suggests that in early stages the presence of this protein is critical for modulating cellular events that allow the virus to fulfill its lifecycle.

E5 employs various mechanisms that have the potential to contribute to the malignant transformation of a cell. It has been described that it can modulate the signaling pathway of the epidermal growth factor receptor (EGFR), which regulates gene transcription and modulates important processes such as cellular proliferation, angiogenesis, tumor invasion, and metastasis. E5 protein forms a stable complex with the EGFR receptor, thereby inducing receptor dimerization and activation; once the EGFR pathway is activated, it induces Bax ubiquitination and degradation by the proteasome. This results in inhibiting the intrinsic pathway of apoptosis.¹¹ Through this mechanism, it has been described that E5 impedes apoptosis induced by hydrogen peroxide, which is part of the strategies used by immune cells in our defense. 

It has been described that cervical tumors with HPV infection have decreased expression levels of CD95, leading to deficient apoptosis.¹² The cause of this action may also be due to the E5 protein; in this respect, in vitro studies have shown that exogenous introduction of E5 from HPV-16 into keratinocytes reduces expression of CD95, which increases cell resistance to apoptosis mediated by the extrinsic pathway. Similarly, the presence of E5 confers resistance to apoptosis mediated by the TRAIL pathway; however, the expression of the TRAIL receptor is not affected by the presence of E5 in the cells, but rather this prevents the formation of DISC.¹³ Moreover, the E5 of HPV-16 is able to protect cells from apoptosis induced by UV radiation; this effect is mediated by activation of MAP kinases and the PI3K- Akt pathway.¹⁴     

Therefore, E5 may interfere with the ability of the immune system to eliminate infected cells by altering the signaling mediated by death receptors and activating survival pathways. Together, the results of the studies mentioned here provide strong evidence that E5 contributes to avoidance of immune surveillance during the early stages of HPV infection.

Modulation of apoptosis by E6

The E6 protein of HPV has been widely studied due to its strong oncogenic properties. E6 is produced in two versions, a long form of approximately 16 kDa, and a shorter version of approximately 8 kDa.¹⁵ Both proteins have as a common feature the presence of four Cys-XX-Cys motifs which are able to bind to zinc and which are involved in various functions. The oncogenic activities described for E6 include the immortalization of human epithelial cells, the transformation of established fibroblast lines from mice, transcriptional activation, resistance to terminal differentiation of human keratinocytes, and finally modulation of apoptosis and tumorigenesis in animals.¹⁶

One of the main targets of the E6 protein is the tumor suppressor gene p53. In the early stages of high-risk HPV infection, E7 induces a significant increase in cellular proliferation as a result of its interaction with retinoblastoma protein (pRB); this causes an overexpression of p53, which in turn leads to cell cycle arrest and induction of apoptosis. To reverse this situation, E6 binds to p53 with the help of the E6AP protein, and prevents p53 from inducing cell cycle arrest and apoptosis. By binding to E6, p53 is flagged for degradation via the proteasome.¹⁷

Another important target for the action of E6 is the BAK protein (BCL-2 family), which is responsible for inducing apoptosis through activation of the intrinsic pathway. E6 binds directly to BAK and induces its degradation. It has been determined that the ability to bind to this protein is conserved among E6 proteins in low and high risk viruses, indicating that inactivation of the BAK protein is crucial for the virus replication cycle.¹⁸ Additionally, it has been observed that E6 inhibits TNF-mediated apoptosis by reducing the expression of BAK and increasing BCL-2 without affecting expression levels of caspases 3 and 8.¹⁹

Like for p53, BAK and MYC proteins are degraded by E6 through joint action with E6AP, which marks them by ubiquitination for degradation via the proteasome.²⁰ Decreased BAK mediated by E6 and E6AP allows infected cells to be resistant to UV radiation. Furthermore, it has been found that E6 decreases epithelial stratification and inhibits apoptosis during differentiation induced by serum and calcium in human foreskin keratinocytes. These facts are correlated with elevated BCL-2 expression and a reduction in BAX expression.²¹ 

Using human fibroblasts it has been shown that the presence of E6 induces a significant increase in survivin promoter activity. Survivin expression is negatively regulated by p53, as E6 negatively regulates p53 and probably influences the regulation of survivin transcription, so is postulated that the survivin gene might be relevant in the antiapoptotic function of E6.²²

There is a link between p53-dependent apoptosis and the CD95-mediated extrinsic pathway; CD95 receptor expression increases in the cell membrane in response to treatment with chemotherapy agents, and this increase is dependent on the action of p53.²³ In human keratinocytes immortalized with E6, apoptosis levels decrease when treated with a CD95 agonist, which indicates that E6 protein regulates CD95 expression through p53 degradation. In this sense, by inhibiting proteasome activity in cells expressing E6, new sensitivity is generated to apoptosis mediated by CD95 or TRAIL.²⁴ This highlights the fundamental role of E6AP protein and the proteasome pathway in the activity of the E6 oncoprotein. Additionally, in malignant cervical carcinomas positive for HPV-16 that are insensitive to the ligands for CD95 and TNF-alpha, the formation of nonfunctional DISC has been documented. This suggests that some viral proteins may be interacting with death receptors and thus protecting cells from apoptosis. In vitro studies have shown that E6 protects cells from TNF-mediated death through a mechanism independent of p53. Inhibition of this pathway could be because E6 binds to a C-terminal fraction of the TNF-R1 receptor, blocking apoptotic signal transduction.²⁵ Additionally, it has been observed that E6 induces an increase in NF-kB activity and target genes of this transcription factor, such as the inhibitor of apoptosis cIAP-2.²⁶  

Another feature of E6 is that it has the ability to bind to the DED domains of the FADD adapter protein and procaspase-8, whereby it stimulates their degradation. The decrease of FADD and caspase-8 prevents proper response to apoptotic signals.²⁷

Modulation of apoptosis by E7

Like E6, E7 is an important oncoprotein by which HPV modulates the cell cycle and survival of the infected cell. One of the mechanisms by which E7 promotes cell growth and proliferation is through partnership with the protein pRB.²⁸ Normally, pRB forms a complex with histone deacetylase (HDAC) and binds to E2F transcription factor in the G1 phase of the cell cycle. This prevents E2F from inducing activation of genes that are required for proliferation until the cell enters phase S. However, when E7 is expressed in the cells, it binds to pRB and HDAC, liberating E2F from the repression exerted by pRB. This results in constitutive activation of E2F-regulated genes. Inactivation of pRB’s ability to arrest the cell cycle is critical for cell transformation, uncontrolled cell growth, and proliferation induced by viral infection. Additionally, E7 is a potent inhibitor of the activity of p21CIP1 and p27KIP1, which avoids the checkpoint in the G1 phase of the cell cycle and allows DNA synthesis.²⁹ The stimulation of progression from phases G1 to S allows the virus to efficiently use cellular DNA replication machinery to achieve viral genome replication. E7 also interferes with histone deacetylation mediated by HDAC1 and HDAC2, leading to activation of transcription.³⁰ As antiapoptotic functions for pRb have been described, the degradation of this protein by the E7-mediated ubiquitin pathway suggests a pro-apoptotic role for E7. In this regard, it has been observed that E7 induces apoptosis in the retinas of transgenic animals that express it. Similarly, mouse fibroblasts in which the expression of E7 from high and low risk HPV was induced present apoptosis.³¹ According to these observations, the co-expression of E7 and p21 in U2OS cells induced apoptosis. Furthermore, overexpression of E7 in primary human keratinocytes caused spontaneous cell death and sensitized the cells to TNF-mediated apoptosis.³² Furthermore, there are also reports of anti-apoptotic activity of E7. One example is a recent study of Yuan et al., which suggests that E7 can inhibit TNF-mediated apoptosis in keratinocytes by regulating the expression of c-IAP2.³³ Another study shows that E7 expression in fibroblasts delays apoptosis via CD95 and prevents TNF-mediated apoptosis, using a mechanism involving inhibition of caspase-8 activation.³⁴ In this context, it has recently been observed that E7 from HPV-16 interacts with proapoptotic factor Siva-1 to inhibit apoptosis induced by UV radiation in keratinocyte cell lines.³⁵ This evidence shows that E7 has the dual property of inducing or inhibiting apoptosis, depending on the cell type and the type of HPV.      

Conclusions

HPV, like many other viruses, has developed sophisticated mechanisms to evade host defenses. A very important one among these is blocking apoptosis to prevent the destruction of infected cells. To do this, HPV has within its arsenal viral oncoproteins E5, E6, and E7. These proteins attack both apoptotic pathways, while stimulating survival pathways. These actions together allow the virus to establish a successful infection and in the case of integration of viral genome into host DNA (an undesired consequence of the HPV life cycle), cellular transformation and the onset of cancer.

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