Signalling of Apoptin Bullenkamp, Jessica; Tavassoli, Mahvash
Advances in experimental medicine and biology,
2014, Letnik:
818
Journal Article
Recenzirano
The virus-derived protein Apoptin has the ability to induce p53-independent apoptosis in a variety of human cancer cells while leaving normal cells unharmed. It thus represents a potential ...anti-cancer therapeutic agent of the future but a proper understanding of Apoptin-induced signalling events is necessary prior to clinical application. The tumor-specific nuclear translocation and phosphorylation of Apoptin by a cellular kinase such as protein kinase C seem to be required for its function but otherwise the mode of tumor selectivity remains unknown. Apoptin has been shown to interact with several cellular proteins including Akt and the anaphase-promoting complex that regulate its activity and promote caspase-dependent apoptosis. This chapter summarizes the available data on tumor-specific pathways sensed by Apoptin and the mechanism of Apoptin-induced cell death.
Hypoxia imaging is a promising tool for targeted therapy but the links between imaging features and underlying molecular characteristics of the tumour have not been investigated. The aim of this ...study was to compare hypoxia biomarkers and gene expression in oropharyngeal squamous cell carcinoma (OPSCC) diagnostic biopsies with hypoxia imaged with
Cu-ATSM PET/CT.
Cu-ATSM imaging, molecular and clinical data were obtained for 15 patients. Primary tumour SUV
, tumour to muscle ratio (TMR) and hypoxic volume were tested for association with reported hypoxia gene signatures in diagnostic biopsies. A putative gene signature for hypoxia in OPSCCs (hypoxic volume-associated gene signature (HVS)) was derived.
Hypoxic volume was significantly associated with a reported hypoxia gene signature (rho=0.57, P=0.045), but SUV
and TMR were not. Immunohistochemical staining with the hypoxia marker carbonic anhydrase 9 (CA9) was associated with a gene expression hypoxia response (rho=0.63, P=0.01). Sixteen genes were positively and five genes negatively associated with hypoxic volume (adjusted P<0.1; eight genes had adjusted P<0.05; HVS). This signature was associated with inferior 3-year progression-free survival (HR=1.5 (1.0-2.2), P=0.047) in an independent patient cohort.
Cu-ATSM-defined hypoxic volume was associated with underlying hypoxia gene expression response. A 21-gene signature derived from hypoxic volume from patients with OPSCCs in our study may be linked to progression-free survival.
Apoptin, the VP3 protein from chicken anaemia virus (CAV), induces tumour cell-specific cell death and represents a potential future anti-cancer therapeutic. In tumour but not in normal cells, ...Apoptin is phosphorylated and translocates to the nucleus, enabling its cytotoxic activity. Recently, the β isozyme of protein kinase C (PKCβ) was shown to phosphorylate Apoptin in multiple myeloma cell lines. However, the exact mechanism and nature of interaction between PKCβ and Apoptin remain unclear. Here we investigated the physical and functional link between PKCβ and CAV-Apoptin as well as with the recently identified Apoptin homologue derived from human Gyrovirus (HGyV). In contrast to HCT116 colorectal cancer cells the normal colon mucosa cell lines expressed low levels of PKCβI and showed reduced Apoptin activation, as evident by cytoplasmic localisation, decreased phosphorylation and lack of cytotoxic activity. Co-immunoprecipitation and proximity ligation assay studies identified binding of both CAV- and HGyV-Apoptin to PKCβI in HCT116 cells. Using Apoptin deletion constructs the N-terminal domain of Apoptin was found to be required for interacting with PKCβI. FRET-based PKC activity reporter assays by fluorescence lifetime imaging microscopy showed that expression of Apoptin in cancer cells but not in normal cells triggers a significant increase in PKC activity. Collectively, the results demonstrate a novel cancer specific interplay between Apoptin and PKCβI. Direct interaction between the two proteins leads to Apoptin-induced activation of PKC and consequently activated PKCβI mediates phosphorylation of Apoptin to promote its tumour-specific nuclear translocation and cytotoxic function.
Patients with head and neck squamous cell carcinoma (HNSCC) undergoing radical chemo-radiation (CRT) frequently receive transfusion with packed red cells (PRCT) during radiotherapy on the basis that ...PRCT increases tumour oxygenation and overcomes hypoxia-induced radio-resistance. This is likely to be a significant oversimplification given the fact that tumour hypoxia is the result of several intrinsic and extrinsic factors, including many that are not directly related to serum haemoglobin (Hb). Therefore, we have studied the effect of PRCT on tumour oxygenation in a prospective cohort of patients who developed low Hb during radical CRT for HNSCC.
This was a prospective study of 20 patients with HNSCC receiving radical CRT undergoing PRCT for Hb<11.5 g dl
. Patients underwent pretransfusion and posttransfusion intrinsic susceptibility-weighted (SWI) MRI and dynamic contrast-enhanced (DCE) MRI. Blood samples were obtained at the time of MRI scanning and two further time points for measuring Hb and a panel of serum cytokine markers of tumour hypoxia. 3D T
* and K
maps were calculated from the MRI data for primary tumours and cervical lymph node metastases.
PRCT produced no change (11 patients) or reduced (1 patient) T
* (tumour oxygenation) in 12 of the 16 (75%) evaluable primary tumours. Three of the four patients with improved tumour oxygenation progressed or had partial response following treatment completion. There were variable changes in K
(tumour perfusion or vessel permeability) following PRCT that were of small magnitude for most tumours. Pre- and Post-PRCT levels of measured cytokines were not significantly different.
This study suggests that PRCT during radical CRT for HNSCC does not improve tumour oxygenation. Therefore, oncologists should consider changing practice according to NICE and American Association of Blood Banks guidelines on PRCT for anaemia.
Head and neck squamous cell carcinomas (
HNSCC
) are associated with poor morbidity and mortality. Current treatment strategies are highly toxic and do not benefit over 50% of patients. There is ...therefore a crucial need for predictive and/or prognostic biomarkers to allow treatment stratification for individual patients. One class of biomarkers that has recently gained importance are micro
RNA
(mi
RNA
). Mi
RNA
are small, noncoding molecules which regulate gene expression post‐transcriptionally. We performed mi
RNA
expression profiling of a cohort of head and neck tumours with known clinical outcomes. The results showed miR‐9 to be significantly downregulated in patients with poor treatment outcome, indicating its role as a potential biomarker in
HNSCC
. Overexpression of miR‐9 in
HNSCC
cell lines significantly decreased cellular proliferation and inhibited colony formation in soft agar. Conversely, miR‐9 knockdown significantly increased both these features. Importantly, endogenous
CXCR
4 expression levels, a known target of miR‐9, inversely correlated with miR‐9 expression in a panel of
HNSCC
cell lines tested. Induced overexpression of
CXCR
4 in low expressing cells increased proliferation, colony formation and cell cycle progression. Moreover,
CXCR
4‐specific ligand,
CXCL
12, enhanced cellular proliferation, migration, colony formation and invasion in
CXCR
4‐overexpressing and similarly in miR‐9 knockdown cells.
CXCR
4‐specific inhibitor plerixafor abrogated the oncogenic phenotype of
CXCR
4 overexpression as well as miR‐9 knockdown. Our data demonstrate a clear role for miR‐9 as a tumour suppressor micro
RNA
in
HNSCC
, and its role seems to be mediated through
CXCR
4 suppression. MiR‐9 knockdown, similar to
CXCR
4 overexpression, significantly promoted aggressive
HNSCC
tumour cell characteristics. Our results suggest
CXCR
4‐specific inhibitor plerixafor as a potential therapeutic agent, and miR‐9 as a possible predictive biomarker of treatment response in
HNSCC
.
Chicken anemia virus (CAV) is a single-stranded circular DNA virus that carries 3 genes, the most studied of which is the gene encoding VP3, also known as apoptin. This protein has been demonstrated ...to specifically kill transformed cells while leaving normal cells unharmed in a manner that is independent of p53 status. Although the mechanistic basis for this differential activity is unclear, it is evident that the subcellular localization of the protein is important for the difference. In normal cells, apoptin exists in filamentous networks in the cytoplasm, whereas in transformed cells, apoptin is present in the nucleus and appears as distinct foci. We have previously demonstrated that DNA damage signaling through the ataxia telangiectasia mutated (ATM) pathway induces the translocation of apoptin from the cytoplasm to the nucleus, where it induces apoptosis. We found that apoptin contains four checkpoint kinase consensus sites and that mutation of either threonine 56 or 61 to alanine restricts apoptin to the cytoplasm. Furthermore, treatment of tumor cells expressing apoptin with inhibitors of checkpoint kinase 1 (Chk1) and Chk2 causes apoptin to localize to the cytoplasm. Importantly, silencing of Chk2 rescues cancer cells from the cytotoxic effects of apoptin. Finally, treatment of virus-producing cells with Chk inhibitor protects them from virus-mediated toxicity and reduces the titer of progeny virus. Taken together, our results indicate that apoptin is a sensor of DNA damage signaling through the ATM-Chk2 pathway, which induces it to migrate to the nucleus during viral replication.
The chicken anemia virus (CAV) protein apoptin is known to induce tumor cell-specific death when expressed. Therefore, understanding its regulation and mechanism of action could provide new insights into tumor cell biology. We have determined that checkpoint kinase 1 and 2 signaling is important for apoptin regulation and is a likely feature of both tumor cells and host cells producing virus progeny. Inhibition of checkpoint signaling prevents apoptin toxicity in tumor cells and attenuates CAV replication, suggesting it may be a future target for antiviral therapy.
The chicken anemia virus-derived protein apoptin induces apoptosis in a variety of human malignant and transformed cells but not in normal cells. However, the mechanisms through which apoptin ...achieves its selective killing effects are not well understood. We developed a lentiviral vector encoding a green fluorescent protein-apoptin fusion gene (LV-GFP-AP) that can efficiently deliver apoptin into hematopoietic cells. Apoptin selectively killed the human multiple myeloma cell lines MM1.R and MM1.S, and the leukemia cell lines K562, HL60, U937, KG1, and NB4. In contrast, normal CD34(+) cells were not killed and maintained their differentiation potential in multilineage colony formation assays. In addition, dexamethasone-resistant MM1.R cells were found to be more susceptible to apoptin-induced cell death than the parental matched MM1.S cells. Death susceptibility correlated with increased phosphorylation and activation of the apoptin protein in MM1.R cells. Expression array profiling identified differential kinase profiles between MM1.R and MM1.S cells. Among these kinases, protein kinase Cβ (PKCβ) was found by immunoprecipitation and in vitro kinase studies to be a candidate kinase responsible for apoptin phosphorylation. Indeed, shRNA knockdown or drug-mediated inhibition of PKCβ significantly reduced apoptin phosphorylation. Furthermore, apoptin-mediated cell death proceeded through the upregulation of PKCβ, activation of caspase-9/3, cleavage of the PKCδ catalytic domain, and downregulation of the MERTK and AKT kinases. Collectively, these results elucidate a novel pathway for apoptin activation involving PKCβ and PKCδ. Further, they highlight the potential of apoptin and its cellular regulators to purge bone marrow used in autologous transplantation for multiple myeloma.
Apoptin has been described to induce apoptosis in various human cancer cell lines, but not in normal cells, thus making it an interesting candidate for the development of novel therapeutic ...strategies. Apoptin was generated and cloned into several mammalian expression vectors. Transfection or microinjection of apoptin cDNA resulted in its expression, initially in the cytoplasm with a filamentous pattern. Subsequently, apoptin entered the nucleus and efficiently induced apoptosis in several cancer cell lines. Nuclear localization was shown to be required for induction of apoptosis. Apoptin expression level was found to be an important determinant of the efficiency of induction of apoptosis. Surprisingly, expression of apoptin or GFP-apoptin cDNA induced apoptosis in some normal cells. When fused to the HIV-TAT protein transduction domain and delivered as a protein, TAT-apoptin was transduced efficiently (>90%) into normal and tumour cells. However, TAT-apoptin remained in the cytoplasm and did not kill normal 6689 and 1BR3 fibroblasts. In contrast TAT-apoptin migrated from the cytoplasm to the nucleus of Saos-2 and HSC-3 cancer cells resulting in apoptosis after 24 h. This study shows that apoptin is a powerful apoptosis-inducing protein with a potential for cancer therapy.
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