Phospho-Regulation of Cancer Causing Human Papillomavirus (HPV) E6 Oncoproteins

Cervical cancer develops through the combined activity of the Human Papillomavirus (HPV) E6 and E7 oncoproteins. A defining characteristic of the E6 oncoproteins derived from cancer-causing HPV types is the presence of a PDZ binding motif (PBM) at the extreme carboxy terminus of the protein, which is absent from E6 proteins derived from the so-called low risk (LR) HPV types. This PDZ binding potential of the high risk (HR) HPV E6 oncoproteins is important for their ability to support the viral life cycle and to cooperate in the induction of malignancy. However, PDZ interactions can be negatively regulated by phosphorylation within the E6 PBM. In this study, I have investigated the differential regulation of diverse HR HPV E6 PBMs. Depending on the HPV type, PDZ binding activity can be regulated by phosphorylation with PKA or AKT. This in turn inhibits PDZ recognition whilst conferring direct association with 14-3-3 family members. Such regulation is highly conserved between E6 proteins derived from HPV-16, HPV-18 and HPV-58, whilst being somewhat weaker or absent from other types such as HPV-31, -33 and -51. Phosphorylation is important for maintaining the steady state levels of HPV-18 E6 and this is also can be affected by its association with 14-3-3. I also show that HPV-18 E6 phosphorylation occurs primarily during the G2/M phase of the cell cycle whereas HPV-16 E6 phosphorylation occurs during S phase. This cell cycle-dependent phosphorylation in turn regulates the levels of E6 expression and confers enhanced interaction with multiple 14-3-3 isoforms. E6 does not degrade 14-3-3, but it alters the subcellular distribution of 14-3-3 and as a consequence, inhibits p53/14-3-3 transcriptional transctivation in an E6 PBM dependent manner. These studies reveal unexpected differences in the regulation of HPV-16 and HPV-18 E6 function and have important implications for how phosphorylation of E6 might be expected to play a role during the respective viral life cycles and tumour development

Cancer-causing human papillomavirus E6 proteins display major differences in the phospho-regulation of their PDZ interactions

Previous studies have shown that the cancer-causing high-risk human papillomavirus (HPV) E6 oncoproteins have PDZ binding potential, an activity which is important for their ability to support the viral life cycle and to cooperate in the induction of malignancy. However, PDZ interactions are not constitutive, and they can be negatively regulated by phosphorylation within the E6 PDZ binding motif (PBM). In this study, we have investigated the differential regulation of the HPV E6 PBMs from diverse high-risk HPV types. We show that, depending on the HPV type, PDZ binding activity can be regulated by phosphorylation with protein kinase A (PKA) or AKT, which, in turn, inhibits PDZ recognition. Such regulation is highly conserved between E6 proteins derived from HPV-16, HPV-18, and HPV-58 while being somewhat weaker or absent from other types such as HPV-31, HPV-33, and HPV-51. In the case of HPV31, PKA phosphorylation occurs within the core of the E6 protein and has no effect on PDZ interactions, and this demonstrates a surprising degree of heterogeneity among the different high-risk HPV E6 oncoproteins in how they are regulated by different cellular signaling pathways. IMPORTANCE This study demonstrated that the cancer-causing HPV E6 oncoproteins are all subject to posttranslational modification of their extreme C-terminal PDZ binding motifs through phosphorylation. However, the identities of the kinase are not the same for all HPV types. This demonstrates a very important divergence between these HPVs, and it suggests that changes in cell signaling pathways have different consequences for different high-risk virus infections and their associated malignancies

The Invasive Capacity of HPV Transformed Cells Requires the hDlg-Dependent Enhancement of SGEF/RhoG Activity

A major target of the HPV E6 oncoprotein is the human Discs Large (hDlg) tumour suppressor, although how this interaction contributes to HPV-induced malignancy is still unclear. Using a proteomic approach we show that a strong interacting partner of hDlg is the RhoG-specific guanine nucleotide exchange factor SGEF. The interaction between hDlg1 and SGEF involves both PDZ and SH3 domain recognition, and directly contributes to the regulation of SGEF's cellular localization and activity. Consistent with this, hDlg is a strong enhancer of RhoG activity, which occurs in an SGEF-dependent manner. We also show that HPV-18 E6 can interact indirectly with SGEF in a manner that is dependent upon the presence of hDlg and PDZ binding capacity. In HPV transformed cells, E6 maintains a high level of RhoG activity, and this is dependent upon the presence of hDlg and SGEF, which are found in complex with E6. Furthermore, we show that E6, hDlg and SGEF each directly contributes to the invasive capacity of HPV-16 and HPV-18 transformed tumour cells. These studies demonstrate that hDlg has a distinct oncogenic function in the context of HPV induced malignancy, one of the outcomes of which is increased RhoG activity and increased invasive capacity

Inhibitor of caspase-activated DNase expression enhances caspase-activated DNase expression and inhibits oxidative stress-induced chromosome breaks at the mixed lineage leukaemia gene in nasopharyngeal carcinoma cells

BACKGROUND: Nasopharyngeal carcinoma (NPC) is commonly found in Asia, especially among the Chinese ethnic group. Chromosome rearrangements are common among NPC patients. Although the mechanism underlying the chromosome rearrangements in NPC is unclear, various mechanisms including activation of caspase-activated DNase (CAD) were proposed to contribute to chromosome rearrangements in leukaemia. Activation of CAD can be initiated by multiple agents, including oxidative stress, which is well implicated in carcinogenesis. CAD is the main enzyme that causes DNA fragmentation during apoptosis, and CAD is also implicated in promoting cell differentiation. In view of the role of oxidative stress in carcinogenesis and CAD activation, and since CAD was suggested to contribute to chromosome rearrangement in leukaemia, we hypothesise that oxidative stress-induced CAD activation could be one of the mechanisms that leads to chromosome rearrangements in NPC. METHODS: SUNEI cells were treated with various concentrations of H(2)O(2) for different period of time to ensure that cells undergo H(2)O(2)-induced MLL gene cleavage. Transfections with hCAD, mCAD, mutant hCAD, or cotransfection with hCAD and mICAD, and cotransfection with mutant hCAD and mICAD were performed. Gene expression was confirmed by Western blotting and MLL gene cleavage was assessed by inverse polymerase chain reaction (IPCR). RESULTS: Treatment with H(2)O(2) clearly induces cleavages within the MLL gene which locates at 11q23, a common deletion site in NPC. In order to investigate the role of CAD, CAD was overexpressed in SUNE1 cells, but that did not result in significant changes in H(2)O(2)-induced MLL gene cleavage. This could be because CAD requires ICAD for proper folding. Indeed, by overexpressing ICAD alone or co-expressing ICAD with CAD, Western blotting showed that CAD was expressed. In addition, ICAD overexpression also suppressed H(2)O(2)-induced MLL gene cleavage, suggesting a possible role of CAD in initiating chromosome cleavage during oxidative stress. CONCLUSIONS: Oxidative stress mediated by H(2)O(2) induces cleavage of the MLL gene, most likely via the caspase-activated DNase, CAD, and CAD expression requires ICAD. Since the MLL gene is located at 11q23, a common deletion site in NPC, thus stress-induced CAD activation may represent one of the mechanisms leading to chromosome rearrangement in NPC

The Role Of Caspase-Activated Dnase (Cad) In Chromosome Breaks During Oxidative Stressinduced Apoptosis

Chromosomal rearrangement, such as additions, deletions, translocations and inversions are phenomena commonly observed in various types of cancers including leukaemia and nasopharyngeal carcinoma (NPC). In leukaemia, structural rearrangements of the Mixed Lineage Leukaemia (MLL) gene at 11q23 have been reported extensively. In NPC, chromosomal deletions and additions are observed where one of the common deletion sites is at 11q23. However, the precise site of deletion has not been mapped to the gene level. Furthermore, mechanism leading to chromosome rearrangements in NPC is unknown. In the case of leukaemia, there are various mechanisms being proposed where apoptotic nuclease is one of them. Harmful or mutated cells undergo autonomous elimination through programmed cell death or apoptosis. However, dysregulation of apoptosis can result in many diseases such as cancer. Morphologically, apoptotic cells are characterised by cells dislodgement, membrane blebbing, condensation of cytoplasm and nucleus, as well as cellular fragmentation into membrane apoptotic bodies. In addition, apoptotic cells DNA integrity is changed where chromatin loop domains are released and may be followed by internucleosomal cleavage. This leads to DNA ladder formation typically seen on agarose gel. During apoptosis, there are a few DNases responsible for DNA fragmentation, in which one of them is the Caspaseactivated DNase (CAD). In normal growing cells, CAD co-exists with its inhibitor, ICAD, and remains inactive. ICAD also functions as a chaperone for CAD and is essential for the correct folding of CAD protein. When triggered by apoptotic stimuli, ICAD is cleaved by caspase-3, releasing the active CAD. Activated CAD will then cleave the genomic DNA. Mutation of CAD and ICAD may result in reduction of internucleosomal cleavage. This is because mutated CAD does not form stable complex with the DNA. Moreover, mutated ICAD, which is resistant to caspase-3 cleavage, is bound to CAD permanently, thus, in spite of the presence of apoptotic stimuli, CAD does not become activated. In vitro studies showed that a variety of cells, including tumour cell lines and normal cell types undergo apoptosis when they are exposed to oxidative stress. Oxidative stress occurs when cells are injured due to elevated production of reactive oxygen species (ROS), such as hydrogen peroxide, H2O2. Excessive ROS production leads to direct DNA damage, which can result in induction of apoptosis in severely damaged cells. We hypothesized that, during stress-induced apoptosis, CAD may cause the initial chromosome break which eventually leads to result in chromosomal rearrangement of the MLL breakpoint cluster region (bcr). Our dose response experiments showed that, NPC (SUNE1) and cervical cancer (HeLa) cell line treated with 50 μM and 100 μM H2O2 respectively for 20 hours showed apoptotic features. Cleavage within the MLL bcr was further confirmed via Inverse Polymerase Chain Reaction (IPCR). In order to study the role of CAD and ICAD, these genes were expressed transiently and stably in mammalian cell lines. Human and murine CAD genes as well as murine ICAD genes were subcloned into 2 expression vectors, namely pcDNA and pTracer. The role of CAD was studied by using four approaches. Firstly, CAD was overexpressed in mammalian cell line but the result was inconclusive. This may be due to the CAD expressed was not functional. Secondly, mutant ICAD was overexpressed to inhibit CAD directly. Mutant ICAD which was resistant to caspase-3 cleavage bound to endogenous CAD and hence CAD could not execute its DNase activity. However, the IPCR result showed that mutant ICAD did not reduce cleavage within the MLL bcr. Therefore, third approach was designed where CAD was inhibited indirectly by using caspase inhibitor. Caspase inhibitor should block the activation of caspase-3 and thus block CAD activation. However, our result showed no significant difference between cells with and without caspase inhibition. This could be due to inefficient uptake of caspase inhibitor into the cells. There might be other DNase responsible to DNA cleavage within the MLL bcr, such as Endonuclease G (Endo G), Apoptosis-induced Factor (AIF) and so on. Chromosomal breakage within the MLL bcr might not solely dependent on the caspase pathway upon oxidative stress-induced apoptosis. In the last approach, CAD and/or ICAD was overexpressed. As confirmed by Western blotting, coexpression of CAD and ICAD resulted in CAD expression. Overexpression of ICAD alone was sufficient to induce high levels of endogenous CAD expression. From the IPCR result, it showed that CAD expression enhances cleavage within the MLL bcr upon oxidative stress. In conclusion, oxidative stress can induce apoptosis in mammalian cell lines. CAD may be involved directly in the cleavage of the MLL bcr. Chromosome rearrangements via the apoptotic process may be dependent on the chromatin structure. In order to further strengthen the hypothesis, involvement of CAD in the cleavage of other genes should also be studied

Inhibitor of caspase-activated DNase expression enhances caspase-activated DNase expression and inhibits oxidative stress-induced chromosome breaks at the mixed lineage leukaemia gene in nasopharyngeal carcinoma cells

Nasopharyngeal carcinoma (NPC) is commonly found in Asia, especially among the Chinese ethnic group. Chromosome rearrangements are common among NPC patients. Although the mechanism underlying the chromosome rearrangements in NPC is unclear, various mechanisms including activation of caspase-activated DNase (CAD) were proposed to contribute to chromosome rearrangements in leukaemia. Activation of CAD can be initiated by multiple agents, including oxidative stress, which is well implicated in carcinogenesis. CAD is the main enzyme that causes DNA fragmentation during apoptosis, and CAD is also implicated in promoting cell differentiation. In view of the role of oxidative stress in carcinogenesis and CAD activation, and since CAD was suggested to contribute to chromosome rearrangement in leukaemia, we hypothesise that oxidative stress-induced CAD activation could be one of the mechanisms that leads to chromosome rearrangements in NPC

Review of the Standard and Advanced Screening, Staging Systems and Treatment Modalities for Cervical Cancer

Cancer arising from the uterine cervix is the fourth most common cause of cancer death among women worldwide. Almost 90% of cervical cancer mortality has occurred in low- and middle-income countries. One of the major aetiologies contributing to cervical cancer is the persistent infection by the cancer-causing types of the human papillomavirus. The disease is preventable if the premalignant lesion is detected early and managed effectively. In this review, we outlined the standard guidelines that have been introduced and implemented worldwide for decades, including the cytology, the HPV detection and genotyping, and the immunostaining of surrogate markers. In addition, the staging system used to classify the premalignancy and malignancy of the uterine cervix, as well as the safety and efficacy of the various treatment modalities in clinical trials for cervical cancers, are also discussed. In this millennial world, the advancements in computer-aided technology, including robotic modules and artificial intelligence (AI), are also incorporated into the screening, diagnostic, and treatment platforms. These innovations reduce the dependence on specialists and technologists, as well as the work burden and time incurred for sample processing. However, concerns over the practicality of these advancements remain, due to the high cost, lack of flexibility, and the judgment of a trained professional that is currently not replaceable by a machine

Current Updates on Cancer-Causing Types of Human Papillomaviruses (HPVs) in East, Southeast, and South Asia

Human papillomavirus (HPV) infection remains one of the most prominent cancer-causing DNA viruses, contributing to approximately 5% of human cancers. While association between HPV and cervical cancers has been well-established, evidence on the attribution of head and neck cancers (HNC) to HPV have been increasing in recent years. Among the cancer-causing HPV genotypes, HPV16 and 18 remain the major contributors to cancers across the globe. Nonetheless, the distribution of HPV genotypes in ethnically, geographically, and socio-economically diverse East, Southeast, and South Asia may differ from other parts of the world. In this review, we garner and provide updated insight into various aspects of HPV reported in recent years (2015–2021) in these regions. We included: (i) the HPV genotypes detected in normal cancers of the uterine cervix and head and neck, as well as the distribution of the HPV genotypes by geography and age groups; (ii) the laboratory diagnostic methods and treatment regimens used within these regions; and (iii) the oncogenic properties of HPV prototypes and their variants contributing to carcinogenesis. More importantly, we also unveil the similarities and discrepancies between these aspects, the areas lacking study, and the challenges faced in HPV studies

Role of polycyclic aromatic hydrocarbons as a co-factor in human papillomavirus-mediated carcinogenesis

Abstract Background Human papillomavirus (HPV) is an etiological agent of cervical cancer. Yet co-factors are believed to be involved in HPV-mediated carcinogenesis. Polycyclic aromatic hydrocarbons (PAHs) are considered as one of these co-factors. Epidemiologic studies have associated high PAH exposure with increased risk for cancer development. To date, many studies focus on benzo[a]pyrene, however, the role of other PAHs should not be neglected. This study aimed to compare the potential of different PAHs as a co-factor in HPV-mediated carcinogenesis, and to investigate the possible mechanisms involved. Methods The effect of 17 PAHs on high-risk HPV (HPV16) were examined in this study. HPV16 E7 oncogene was expressed in primary cells extracted from baby rat kidney and treated with PAHs. The co-transforming ability of PAHs were measured by colony formation index according to the number and size of transformed colonies. Effects of PAHs on proliferation of HPV-null (C33A) and –infected (CaSki) were examined using CCK-8 assay. Wound healing assay and matrigel invasion chambers were used to investigate effects of PAHs on cell motility and invasivion of HPV-null (MCF7, C33A) and –infected (SiHa) cells. Results Benzo[a]pyrene (BaP), dibenz[a,h]anthracene (DBA) and indeno[1,2,3-cd]pyrene (IDP) showed the greatest co-transforming potential in the baby rat kidney cell system. Short-term exposure to BaP, DBA, IDP and pyrene (PR) did not affect proliferation of C33A or CaSki cells, however, long-term exposure of these four PAHs led to dramatic increase in growth rate of CaSki cells by 120–140%. Besides, exposure of PAHs has an effect on cell motility and invasiveness of C33A and SiHa cells, but not for MCF7 cells. Exposure of BaP and DBA enhanced migration (1.26 to 1.40-fold) and invasion (1.68 to 1.94-fold) capacity of C33A cells. Intriguingly, exposure of all four types of PAHs boosted the migration (1.12 to 1.28-fold) and invasion (1.26 to 1.40-fold) capacity of SiHa cells. Conclusions Our results indicate that exposure to PAHs can be a key co-factor in HPV-related cancer development. They could act on all three stages, namely initiation, promotion and progression. Further study is needed to unveil the mechanisms by which PAHs interact with HPV to cause malignancy