Association between hypoxic volume and underlying hypoxia-induced gene expression in oropharyngeal squamous cell carcinoma.

BACKGROUND: 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 64Cu-ATSM PET/CT. METHODS: 64Cu-ATSM imaging, molecular and clinical data were obtained for 15 patients. Primary tumour SUVmax, 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. RESULTS: Hypoxic volume was significantly associated with a reported hypoxia gene signature (rho=0.57, P=0.045), but SUVmax 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. CONCLUSIONS: 64Cu-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.

Blood transfusion during radical chemo-radiotherapy does not reduce tumour hypoxia in squamous cell cancer of the head and neck.

BACKGROUND: 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. METHODS: This was a prospective study of 20 patients with HNSCC receiving radical CRT undergoing PRCT for Hb<11.5 g dl-1. 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 T2* and Ktrans maps were calculated from the MRI data for primary tumours and cervical lymph node metastases. RESULTS: PRCT produced no change (11 patients) or reduced (1 patient) T2* (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 Ktrans (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. CONCLUSIONS: 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.

Activation of the Chicken Anemia Virus Apoptin Protein by Chk1/2 Phosphorylation Is Required for Apoptotic Activity and Efficient Viral Replication.

UNLABELLED: 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. IMPORTANCE: 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.

ASPP2 suppresses squamous cell carcinoma via RelA/p65-mediated repression of p63.

Squamous cell carcinoma (SCC) is highly malignant and refractory to therapy. The majority of existing mouse SCC models involve multiple gene mutations. Very few mouse models of spontaneous SCC have been generated by a single gene deletion. Here we report a haploinsufficient SCC mouse model in which exon 3 of the Tp53BP2 gene (a p53 binding protein) was deleted in one allele in a BALB/c genetic background. Tp53BP2 encodes ASPP2 (ankyrin repeats, SH3 domain and protein rich region containing protein 2). Keratinocyte differentiation induces ASPP2 and its expression is inversely correlated with p63 protein in vitro and in vivo. Up-regulation of p63 expression is required for ASPP2(Δexon3/+) BALB/c mice to develop SCC, as heterozygosity of p63 but not p53 prevents them from developing it. Mechanistically, ASPP2 inhibits ΔNp63 expression through its ability to bind IκB and enhance nuclear Rel/A p65, a component of the NF-κB transcription complex, which mediates the repression of p63. Reduced ASPP2 expression associates with tumor metastasis and increased p63 expression in human head and neck SCCs. This study identifies ASPP2 as a tumor suppressor that suppresses SCC via inflammatory signaling through NF-κB-mediated repression of p63.

MicroRNA-196a promotes an oncogenic effect in head and neck cancer cells by suppressing annexin A1 and enhancing radioresistance.

Radiotherapy is a major treatment modality for head and neck squamous cell carcinoma (HNSCC). Up to 50% of patients with locally advanced disease relapse after radical treatment and there is therefore a need to develop predictive bomarkers for clinical use that allow the selection of patients who are likely to respond. MicroRNA (miRNA) expression profiling of a panel of HNSCC tumours with and without recurrent disease after surgery and radiotherapy detected miR-196a as one of the highest upregulated miRNAs in the poor prognostic group. To further study the role of miR-196a, its expression was determined in eight head and neck cancer cell lines. Overexpression of miR-196a in HNSCC cells, with low endogenous miR-196a expression, significantly increased cell proliferation, migration and invasion, and induced epithelial to mesenchymal transition. Conversely, miR-196a knockdown in cells with high endogenous expression levels significantly reduced oncogenic behaviour. Importantly, overexpression of miR-196a increased radioresistance of cells as measured by gamma H2AX staining and MTT survival assay. Annexin A1 (ANXA1), a known target of miR-196a, was found to be directly modulated by miR-196a as measured by luciferase assay and confirmed by Western blot analysis. ANXA1 knockdown in HNSCC exhibited similar phenotypic effects to miR-196a overexpression, suggesting the oncogenic effect of miR-196a may at least be partly regulated through suppression of ANXA1. In conclusion, this study identifies miR-196a as a potential important biomarker of prognosis and response of HNSCC to radiotherapy. Furthermore, our data suggest that miR-196a and/or its target gene ANXA1 could represent important therapeutic targets in HNSCC.

Apoptin interacts with and regulates the activity of protein kinase C beta in cancer cells.

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 ß isozyme of protein kinase C (PKCß) was shown to phosphorylate Apoptin in multiple myeloma cell lines. However, the exact mechanism and nature of interaction between PKCß and Apoptin remain unclear. Here we investigated the physical and functional link between PKCß 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 PKCßI 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 PKCßI in HCT116 cells. Using Apoptin deletion constructs the N-terminal domain of Apoptin was found to be required for interacting with PKCßI. 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 PKCßI. Direct interaction between the two proteins leads to Apoptin-induced activation of PKC and consequently activated PKCßI mediates phosphorylation of Apoptin to promote its tumour-specific nuclear translocation and cytotoxic function.

A functional assay for microRNA target identification and validation.

MicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes. Due to the incomplete homology of the miRNA to its target site identification of miRNA target genes is difficult and currently based on computational algorithms predicting large numbers of potential targets for a given miRNA. To enable the identification of biologically relevant miRNA targets, we describe a novel functional assay based on a 3'-UTR-enriched library and a positive/negative selection strategy. As proof of principle we have used mir-130a and its validated target MAFB to test this strategy. Identification of MAFB and five additional targets and their subsequent confirmation as mir-130a targets by western blot analysis and knockdown experiments validates this strategy for the functional identification of miRNA targets.

Signalling of Apoptin.

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.

Crosstalk between CAFs and tumour cells in head and neck cancer.

Head and neck squamous cell carcinomas (HNSCCs) are amongst the most aggressive, complex, and heterogeneous malignancies. The standard of care treatments for HNC patients include surgery, radiotherapy, chemotherapy, or their combination. However, around 50% do not benefit while suffering severe toxic side effects, costing the individuals and society. Decades have been spent to improve HNSCC treatment outcomes with only limited success. Much of the research in HNSCC treatment has focused on understanding the genetics of the HNSCC malignant cells, but it has become clear that tumour microenvironment (TME) plays an important role in the progression as well as treatment response in HNSCC. Understanding the crosstalk between cancer cells and TME is crucial for inhibiting progression and treatment resistance. Cancer-associated fibroblasts (CAFs), the predominant component of stroma in HNSCC, serve as the primary source of extra-cellular matrix (ECM) and various pro-tumoral composites in TME. The activation of CAFs in HNSCC is primarily driven by cancer cell-secreted molecules, which in turn induce phenotypic changes, elevated secretive status, and altered ECM production profile. Concurrently, CAFs play a pivotal role in modulating the cell cycle, stemness, epithelial-mesenchymal transition (EMT), and resistance to targeted and chemoradiotherapy in HNSCC cells. This modulation occurs through interactions with secreted molecules or direct contact with the ECM or CAF. Co-culture and 3D models of tumour cells and other TME cell types allows to mimic the HNSCC tumour milieu and enable modulating tumour hypoxia and reprograming cancer stem cells (CSC). This review aims to provide an update on the development of HNSCC tumour models comprising CAFs to obtain better understanding of the interaction between CAFs and tumour cells, and for providing preclinical testing platforms of current and combination with emerging therapeutics.

Targeting BRD4: Potential therapeutic strategy for head and neck squamous cell carcinoma (Review).

As a member of BET (bromodomain and extra-terminal) protein family, BRD4 (bromodomain­containing protein 4) is a chromatin­associated protein that interacts with acetylated histones and actively recruits regulatory proteins, leading to the modulation of gene expression and chromatin remodeling. The cellular and epigenetic functions of BRD4 implicate normal development, fibrosis and inflammation. BRD4 has been suggested as a potential therapeutic target as it is often overexpressed and plays a critical role in regulating gene expression programs that drive tumor cell proliferation, survival, migration and drug resistance. To address the roles of BRD4 in cancer, several drugs that specifically target BRD4 have been developed. Inhibition of BRD4 has shown promising results in preclinical models, with several BRD4 inhibitors undergoing clinical trials for the treatment of various cancers. Head and neck squamous cell carcinoma (HNSCC), a heterogeneous group of cancers, remains a health challenge with a high incidence rate and poor prognosis. Conventional therapies for HNSCC often cause adverse effects to the patients. Targeting BRD4, therefore, represents a promising strategy to sensitize HNSCC to chemo­ and radiotherapy allowing de­intensification of the current therapeutic regime and subsequent reduced side effects. However, further studies are required to fully understand the underlying mechanisms of action of BRD4 in HNSCC in order to determine the optimal dosing and administration of BRD4­targeted drugs for the treatment of patients with HNSCC.

Semisynthesis reveals apoptin as a tumour-selective protein prodrug that causes cytoskeletal collapse.

Apoptin is a small viral protein capable of inducing cell death selectively in cancer cells. Despite its potential as an anticancer agent, relatively little is known about its mechanism of toxicity and cancer-selectivity. Previous experiments suggest that cancer-selective phosphorylation modulates apoptin toxicity, although a lack of chemical tools has hampered the dissection of underlying mechanisms. Here, we describe structure-function studies with site-specifically phosphorylated apoptin (apoptin-T108ph) in living cells which revealed that Thr108 phosphorylation is the selectivity switch for apoptin toxicity. Mechanistic investigations link T108ph to actin binding, cytoskeletal disruption and downstream inhibition of anoikis-resistance as well as cancer cell invasion. These results establish apoptin as a protein pro-drug, selectively activated in cancer cells by phosphorylation, which disrupts the cytoskeleton and promotes cell death. We anticipate that this mechanism provides a framework for the design of next generation anticancer proteins with enhanced selectivity and potency.

A cytosolic mutp53(E285K) variant confers chemoresistance of malignant melanoma.

Malignant melanoma (MM) is known to be intrinsically chemoresistant, even though only ~20% of MM carry mutations of the tumor suppressor p53. Despite improvement of systemic therapy the mortality rate of patients suffering from metastatic MM is still ~70%, highlighting the need for alternative treatment options or for the re-establishment of conventional therapeutic approaches, including chemotherapy. Screening the p53 mutation status in a cohort of 19 patient-derived melanoma samples, we identified one rarely described missense mutation of p53 leading to E285K amino acid exchange (mutp53(E285K)). Employing structural and computational analysis we revealed a major role of E285 residue in maintaining stable conformation of wild-type p53 (wtp53). E285K mutation was predicted to cause interruption of a salt-bridge network affecting the conformation of the C-terminal helix of the DNA-binding domain (DBD) thereby preventing DNA interaction. In this context, a cluster of frequently mutated amino acid residues in cancer was identified to putatively lead to similar structural effects as E285K substitution (E285 cluster). Functional analysis, including knockdown of endogenous p53 and reconstitution with diverse p53 missense mutants confirmed mutp53(E285K) to have lost transcriptional activity, to be localized in the cytosol of cancer cells, by both means conferring chemoresistance. Re-sensitization to cisplatin-induced cell death was achieved using clinically approved compounds aiming to restore p53 wild-type function (PRIMA1-Met), or inhibition of AKT-driven MAPK survival pathways (afuresertib), in both cases being partially due to ferroptosis induction. Consequently, active ferroptosis induction using the GPX4 inhibitor RSL3 proved superior in tumorselectively fighting MM cells. Due to high prevalence of the E285-cluster mutations in MM as well as in a variety of other tumor types, we conclude this cluster to serve an important function in tumor development and therapy and suggest new implications for ferroptosis induction in therapeutic applications fighting MM in particular and cancer in general.

p53 polymorphism influences response in cancer chemotherapy via modulation of p73-dependent apoptosis.

Intact p73 function is shown to be an important determinant of cellular sensitivity to anticancer agents. Inhibition of p73 function by dominant-negative proteins or by mutant p53 abrogates apoptosis and cytotoxicity induced by these agents. A polymorphism encoding either arginine (72R) or proline (72P) at codon 72 of p53 influences inhibition of p73 by a range of p53 mutants identified in squamous cancers. Clinical response following cisplatin-based chemo-radiotherapy for advanced head and neck cancer is influenced by this polymorphism, cancers expressing 72R mutants having lower response rates than those expressing 72P mutants. Polymorphism in p53 may influence individual responsiveness to cancer therapy.

EGFR-induced suppression of HPV E6/E7 is mediated by microRNA-9-5p silencing of BRD4 protein in HPV-positive head and neck squamous cell carcinoma.

EGFR upregulation is an established biomarker of treatment resistance and aggressiveness in head and neck cancers (HNSCC). EGFR-targeted therapies have shown benefits for HPV-negative HNSCC; surprisingly, inhibiting EGFR in HPV-associated HNSCC led to inferior therapeutic outcomes suggesting opposing roles for EGFR in the two HNSCC subtypes. The current study aimed to understand the link between EGFR and HPV-infected HNSCC particularly the regulation of HPV oncoproteins E6 and E7. We demonstrate that EGFR overexpression suppresses cellular proliferation and increases radiosensitivity of HPV-positive HNSCC cell lines. EGFR overexpression inhibited protein expression of BRD4, a known cellular transcriptional regulator of HPV E6/E7 expression and DNA damage repair facilitator. Inhibition of EGFR by cetuximab restored the expression of BRD4 leading to increased HPV E6 and E7 transcription. Concordantly, pharmacological inhibition of BRD4 led to suppression of HPV E6 and E7 transcription, delayed cellular proliferation and sensitised HPV-positive HNSCC cells to ionising radiation. This effect was shown to be mediated through EGFR-induced upregulation of microRNA-9-5p and consequent silencing of its target BRD4 at protein translational level, repressing HPV E6 and E7 transcription and restoring p53 tumour suppressor functions. These results suggest a novel mechanism for EGFR inhibition of HPV E6/E7 oncoprotein expression through an epigenetic pathway, independent of MAPK, but mediated through microRNA-9-5p/BRD4 regulation. Therefore, targeting EGFR may not be the best course of therapy for certain cancer types including HPV-positive HNSCC, while targeting specific signalling pathways such as BRD4 could provide a better and potentially new treatment to improve HNSCC therapeutic outcome.

Hypoxia-induced therapy resistance: Available hypoxia-targeting strategies and current advances in head and neck cancer.

Most solid tumors, such as head and neck cancers, feature a hypoxic microenvironment due to angiogenic dysregulation and the consequent disruption of their vascular network. Such nutrient-deprived environment can induce genomic changes in several tumor cell populations, conferring survival and proliferative advantages to cancer cells through immunosuppression, metabolic switches and enhanced invasiveness. These transcriptional changes, together with the selective pressure hypoxia exerts on cancer cells, leads to the propagation of more aggressive and stress-resistant subpopulations increasing therapy resistance and worsening patient outcomes. Although extensive preclinical and clinical studies involving hypoxia-targeted drugs have been performed, most of these drugs have failed late-stage clinical trials and only a few have managed to be implemented in clinical practice. Here, we provide an overview of three main strategies to target tumor hypoxia: HIF-inhibitors, hypoxia-activated prodrugs and anti-angiogenic agents; summarizing the clinical advances that have been made over the last decade. Given that most hypoxia-targeted drugs seem to fail clinical trials because of insufficient drug delivery, combination with anti-angiogenic agents is proposed for the improvement of therapy response via vascular normalization and enhanced drug delivery. Furthermore, we suggest that using novel nanoparticle delivery strategies might further improve the selectivity and efficiency of hypoxia-targeted therapies and should therefore be taken into consideration for future therapeutic design. Lastly, recent findings point out the relevance that hypoxia-targeted therapy is likely to have in head and neck cancer as a chemo/radiotherapy sensitizer for treatment efficiency improvement.

EGFR overexpression increases radiotherapy response in HPV-positive head and neck cancer through inhibition of DNA damage repair and HPV E6 downregulation.

High-risk Human Papillomavirus (HPV) infections have recently emerged as an independent risk factor in head and neck squamous cell carcinoma (HNSCC). There has been a marked increase in the incidence of HPV-induced HNSCC subtype, which demonstrates different genetics with better treatment outcome. Despite the favourable prognosis of HPV-HNSCC, the treatment modality, consisting of high dose radiotherapy (RT) in combination with chemotherapy (CT), remains similar to HPV-negative tumours, associated with toxic side effects. Epidermal growth factor receptor (EGFR) is overexpressed in over 80% of HNSCC and correlates with RT resistance. EGFR inhibitor Cetuximab is the only FDA approved targeted therapy for both HNSCC subtypes, however the response varies between HNSCC subtypes. In HPV-negative HNSCC, Cetuximab sensitises HNSCC to RT improving survival rates. To reduce adverse cytotoxicity of CT, Cetuximab has been approved for treatment de-escalation of HPV-positive HNSCC. The results of several recent clinical trials have concluded differing outcome to HPV-negative HNSCC. Here we investigated the role of EGFR in HPV-positive HNSCC response to RT. Remarkably, in HPV-positive HNSCC cell lines and in vivo tumour models, EGFR activation was strongly indicative of increased RT response. In response to RT, EGFR activation induced impairment of DNA damage repair and increased RT response. Furthermore, EGFR was found to downregulate HPV oncoproteinE6 expression and induced p53 activity in response to RT. Collectively, our data uncovers a novel role for EGFR in virally induced HNSCC and highlights the importance of using EGFR-targeted therapies in the context of the genetic makeup of cancer.

ImmunoCluster provides a computational framework for the nonspecialist to profile high-dimensional cytometry data.

High-dimensional cytometry is an innovative tool for immune monitoring in health and disease, and it has provided novel insight into the underlying biology as well as biomarkers for a variety of diseases. However, the analysis of large multiparametric datasets usually requires specialist computational knowledge. Here, we describe ImmunoCluster (https://github.com/kordastilab/ImmunoCluster), an R package for immune profiling cellular heterogeneity in high-dimensional liquid and imaging mass cytometry, and flow cytometry data, designed to facilitate computational analysis by a nonspecialist. The analysis framework implemented within ImmunoCluster is readily scalable to millions of cells and provides a variety of visualization and analytical approaches, as well as a rich array of plotting tools that can be tailored to users' needs. The protocol consists of three core computational stages: (1) data import and quality control; (2) dimensionality reduction and unsupervised clustering; and (3) annotation and differential testing, all contained within an R-based open-source framework.

Delivery of therapeutic proteins as secretable TAT fusion products.

The trans-acting activator of transcription (TAT) protein transduction domain (PTD) mediates the transduction of peptides and proteins into target cells. The TAT-PTD has an important potential as a tool for the delivery of therapeutic agents. The production of TAT fusion proteins in bacteria, however, is problematic because of protein insolubility and the absence of eukaryotic post-translational modification. An attractive alternative, both for in vitro protein production and for in vivo applications, is the use of higher eukaryotic cells for secretion of TAT fusion proteins. However, the ubiquitous expression of furin endoprotease (PACE or SPC1) in the Golgi/endoplasmic reticulum, and the presence of furin recognition sequences within TAT-PTD, results in the cleavage and loss of the TAT-PTD domain during its secretory transition through the endoplasmic reticulum and Golgi. In this study, we show the development of a synthetic TATkappa-PTD in which mutation of the furin recognition sequences, but retention of protein transduction activity, allows secretion of recombinant proteins, followed by successful uptake of the modified protein, by the target cells. This system was used to successfully secrete marker protein, green fluorescent protein (GFP), and apoptin, a protein with tumor-specific cytotoxicity. Detection of GFP, phosphorylation, and induction of cell death by TATkappa-GFP-apoptin indicated that the secreted proteins were functional in target cells. This novel strategy therefore has important potential for the efficient delivery of therapeutic proteins.

Inside the hypoxic tumour: reprogramming of the DDR and radioresistance.

The hypoxic tumour is a chaotic landscape of struggle and adaption. Against the adversity of oxygen starvation, hypoxic cancer cells initiate a reprogramming of transcriptional activities, allowing for survival, metastasis and treatment failure. This makes hypoxia a crucial feature of aggressive tumours. Its importance, to cancer and other diseases, was recognised by the award of the 2019 Nobel Prize in Physiology or Medicine for research contributing to our understanding of the cellular response to oxygen deprivation. For cancers with limited treatment options, for example those that rely heavily on radiotherapy, the results of hypoxic adaption are particularly restrictive to treatment success. A fundamental aspect of this hypoxic reprogramming with direct relevance to radioresistance, is the alteration to the DNA damage response, a complex set of intermingling processes that guide the cell (for good or for bad) towards DNA repair or cell death. These alterations, compounded by the fact that oxygen is required to induce damage to DNA during radiotherapy, means that hypoxia represents a persistent obstacle in the treatment of many solid tumours. Considerable research has been done to reverse, correct or diminish hypoxia's power over successful treatment. Though many clinical trials have been performed or are ongoing, particularly in the context of imaging studies and biomarker discovery, this research has yet to inform clinical practice. Indeed, the only hypoxia intervention incorporated into standard of care is the use of the hypoxia-activated prodrug Nimorazole, for head and neck cancer patients in Denmark. Decades of research have allowed us to build a picture of the shift in the DNA repair capabilities of hypoxic cancer cells. A literature consensus tells us that key signal transducers of this response are upregulated, where repair proteins are downregulated. However, a complete understanding of how these alterations lead to radioresistance is yet to come.