Exploring hypoxic biology to improve radiotherapy outcomes.

Ionising radiotherapy is a well-established, effective cancer treatment modality, whose efficacy has improved with the application of newer technological modalities. However, patient outcomes are governed and potentially limited by aspects of tumour biology that are associated with radioresistance. Patients also still endure treatment-associated toxicities owed to the action of ionising radiation in normoxic tissue adjacent to the tumour mass. Tumour hypoxia is recognised as a key component of the tumour microenvironment and is well established as leading to therapy resistance and poor prognosis. In this review, we outline the current understanding of hypoxia-mediated radiotherapy resistance, before exploring targeting tumour hypoxia for radiotherapy sensitisation to improve treatment outcomes and increase the therapeutic window. This includes increasing oxygen availability in solid tumours, the use of hypoxia-activated prodrugs, targeting of hypoxia-regulated or associated signalling pathways, as well as the use of high-LET radiotherapy modalities. Ultimately, targeting hypoxic radiobiology combined with precise radiotherapy delivery modalities and modelling should be associated with improvement to patient outcomes.

Replication stress and chromatin context link ATM activation to a role in DNA replication.

ATM-mediated signaling in response to DNA damage is a barrier to tumorigenesis. Here we asked whether replication stress could also contribute to ATM signaling. We demonstrate that, in the absence of DNA damage, ATM responds to replication stress in a hypoxia-induced heterochromatin-like context. In certain hypoxic conditions, replication stress occurs in the absence of detectable DNA damage. Hypoxia also induces H3K9me3, a histone modification associated with gene repression and heterochromatin. Hypoxia-induced replication stress together with increased H3K9me3 leads to ATM activation. Importantly, ATM prevents the accumulation of DNA damage in hypoxia. Most significantly, we describe a stress-specific role for ATM in maintaining DNA replication rates in a background of increased H3K9me3. Furthermore, the ATM-mediated response to oncogene-induced replication stress is enhanced in hypoxic conditions. Together, these data indicate that hypoxia plays a critical role in the activation of the DNA damage response, therefore contributing to this barrier to tumorigenesis.

Polypoidal Choroidal Vasculopathy in Caucasians: Morphological Findings from Multimodal Retinal Imaging.

PURPOSE: The aim of the study was to characterize the morphological features of polypoidal choroidal vasculopathy (PCV) in a large Caucasian population. METHODS: We conducteda multicenter, cross-sectional study of treatment-naïve patients with PCV. Baseline fundus photography, spectral-domain optical coherence tomography (SD-OCT), fluorescein angiography (FA), and indocyanine green angiography (ICGA) were assessed by trained medical graders. Typical PCV features were explored, and retinal thickness (RT) and choroidal thickness (CT) measurements were performed. RESULTS: Seventy-nine eyes of 73 patients (mean age, 72.6 ± 11.9 years) were included. ICGA identified macular polyps in 89.9% of cases. SD-OCT revealed mostly subretinal fluid (93.6%) and a retinal pigment epithelium (RPE) detachment in 91.4%, with sharp protrusion in 67.0% of cases. Polyp-like structures were seen in 74.3% of cases, mostly adherent to an elevated RPE (69.6%). Type 1 neovascularization (NV) was identified in 74.7% of patients, while 16.5% had a mixed NV. The mean macular CT was 220.9 ± 83.2 µm (range, 67.9-403.6). Diffuse and focal pachychoroid were observed in 26.6 and 30.4% of patients, respectively. Soft drusen were reported in 62.0% of cases, but retinal hemorrhage occurred in only 19.0% of cases. CONCLUSION: The morphological features of PCV in Caucasians are similar to those reported in Asians. Pachychoroid signs were found in nearly half of our cohort. However, the mean age at presentation, high prevalence of soft drusen, and low prevalence of large subretinal hemorrhages make PCV closer to age-related macular degeneration in this ethnic group.

Practical Considerations of Dissolved Oxygen Levels for Platelet Function under Hypoxia.

Investigating human platelet function in low-oxygen environments is important in multiple settings, including hypobaric hypoxia (e.g., high altitude), sea level hypoxia-related disease, and thrombus stability. These studies often involve drawing blood from which platelets are isolated and analysed at atmospheric conditions or re-exposed to low oxygen levels in hypoxia chambers before testing. However, it remains unknown how the in vitro handling of the samples itself changes their dissolved oxygen concentration, which might affect platelet function and experimental results. Here, we prepared healthy donor platelet-rich plasma and washed platelet (WP) suspensions and exposed them to 2% oxygen. We found that the use of hypoxia pre-equilibrated tubes, higher platelet concentrations (>2 × 108/mL versus 2 × 107/mL), smaller volumes (600 µL versus 3 mL), and presence of plasma reduced the time for samples to reach 2% oxygen. Notably, oxygen levels decreased below 2% in most suspensions, but also in WP maintained at atmospheric 21% oxygen. Additionally, platelet spreading on fibrinogen was decreased when using hypoxic fibrinogen-coated culture plates regardless of the oxygen percentage (2% or 21%) in which platelet incubation took place. Thus, sample handling and experimental conditions should be carefully monitored in platelet-hypoxia studies as they might compromise results interpretation and comparison across studies.

WSB-1 regulates the metastatic potential of hormone receptor negative breast cancer.

BACKGROUND: Metastatic spread is responsible for the majority of cancer-associated deaths. The tumour microenvironment, including hypoxia, is a major driver of metastasis. The aim of this study was to investigate the role of the E3 ligase WSB-1 in breast cancer biology in the context of the hypoxic tumour microenvironment, particularly regarding metastatic spread. METHODS: In this study, WSB-1 expression was evaluated in breast cancer cell lines and patient samples. In silico analyses were used to determine the impact of WSB-1 expression on distant metastasis-free survival (DMFS) in patients, and correlation between WSB1 expression and hypoxia gene expression signatures. The role of WSB-1 on metastasis promotion was evaluated in vitro and in vivo. RESULTS: High WSB1 expression was associated with decreased DMFS in ER-breast cancer and PR-breast cancer patients. Surprisingly, WSB1 expression was not positively correlated with known hypoxic gene expression signatures in patient samples. Our study is the first to show that WSB-1 knockdown led to decreased metastatic potential in breast cancer hormone receptor-negative models in vitro and in vivo. WSB-1 knockdown was associated with decreased metalloproteinase (MMP) activity, vascular endothelial growth factor (VEGF) secretion, and angiogenic potential. CONCLUSIONS: Our data suggests that WSB-1 may be an important regulator of aggressive metastatic disease in hormone receptor-negative breast cancer. WSB-1 could therefore represent a novel regulator and therapeutic target for secondary breast cancer in these patients.

Investigating the impact of the interstitial fluid flow and hypoxia interface on cancer transcriptomes using a spheroid-on-chip perfusion system.

Solid tumours are complex and heterogeneous systems, which exist in a dynamic biophysical microenvironment. Conventional cancer research methods have long relied on two-dimensional (2D) static cultures which neglect the dynamic, three-dimensional (3D) nature of the biophysical tumour microenvironment (TME), especially the role and impact of interstitial fluid flow (IFF). To address this, we undertook a transcriptome-wide analysis of the impact of IFF-like perfusion flow using a spheroid-on-chip microfluidic platform, which allows 3D cancer spheroids to be integrated into extracellular matrices (ECM)-like hydrogels and exposed to continuous perfusion, to mimic IFF in the TME. Importantly, we have performed these studies both in experimental (normoxia) and pathophysiological (hypoxia) oxygen conditions. Our data indicated that gene expression was altered by flow when compared to static conditions, and for the first time showed that these gene expression patterns differed in different oxygen tensions, reflecting a differential role of spheroid perfusion in IFF-like flow in tumour-relevant hypoxic conditions in the biophysical TME. We were also able to identify factors primarily linked with IFF-like conditions which are linked with prognostic value in cancer patients and therefore could correspond to a potential novel biomarker of IFF in cancer. This study therefore highlights the need to consider relevant oxygen conditions when studying the impact of flow in cancer biology, as well as demonstrating the potential of microfluidic models of flow to identify IFF-relevant tumour biomarkers.

The Role of Glucosinolates from Cruciferous Vegetables (Brassicaceae) in Gastrointestinal Cancers: From Prevention to Therapeutics.

The gastrointestinal (GI) tract is composed of rapidly renewing cells, which increase the likelihood of cancer. Colorectal cancer is one of the most frequently diagnosed GI cancers and currently stands in second place regarding cancer-related mortality. Unfortunately, the treatment of GI is limited, and few developments have occurred in the field over the years. With this in mind, new therapeutic strategies involving biologically active phytocompounds are being evaluated as anti-cancer agents. Vegetables such as broccoli, brussels sprouts, cabbage, cauliflower, and radish, all belonging to the Brassicaceae family, are high in dietary fibre, minerals, vitamins, carotenoids, polyphenols, and glucosinolates. The latter compound is a secondary metabolite characteristic of this family and, when biologically active, has demonstrated anti-cancer properties. This article reviews the literature regarding the potential of Cruciferous vegetables in the prevention and/or treatment of GI cancers and the relevance of appropriate compound formulations for improving the stability and bioaccessibility of the major Cruciferous compounds, with a particular focus on glucosinolates.

Predicting personalised and progressive adaptive dose escalation to gross tumour volume using knowledge-based planning models for inoperable advanced-stage non-small cell lung cancer patients treated with volumetric modulated arc therapy.

Objectives. Increased radiation doses could improve local control and overall survival of lung cancer patients, however, this could be challenging without exceeding organs at risk (OAR) dose constraints, especially for patients with advanced-stage disease. Increasing OAR doses could reduce the therapeutic ratio and quality of life. It is therefore important to investigate methods to increase the dose to target volume without exceeding OAR dose constraints.Methods. Gross tumour volume (GTV) was contoured on synthetic computerised tomography (sCT) datasets produced using the Velocity adaptive radiotherapy software for eleven patients. The fractions where GTV volume decreased compared to that prior to radiotherapy (reference plan) were considered for personalised progressive dose escalation. The dose to the adapted GTV (GTVAdaptive) was increased until OAR doses were affected (as compared to the original clinical plan). Planning target volume (PTV) coverage was maintained for all plans. Doses were also escalated to the reference plan (GTVClinical) using the same method. Adapted, dose-escalated, plans were combined to estimate accumulated dose, D99(dose to 99%) of GTVAdapted, PTV D99and OAR doses and compared with those in the original clinical plans. Knowledge-based planning (KBP) model was developed to predict D99of the adapted GTV with OAR doses and PTV coverage kept similar to the original clinical plans; prediction accuracy and model verification were performed using further data sets.Results. Compared to the original clinical plan, the dose to GTV was significantly increased without exceeding OAR doses. Adaptive dose-escalation increased the average D99to GTVAdaptiveby 15.1Gy and 8.7Gy compared to the clinical plans. The KBP models were verified and demonstrated prediction accuracy of 0.4% and 0.7% respectively.Conclusion. Progressive adaptive dose escalation can significantly increase the dose to GTV without increasing OAR doses or compromising the dose to microscopic disease. This may increase overall survival without increasing toxicities.

Validation of in-house knowledge-based planning model for predicting change in target coverage during VMAT radiotherapy to in-operable advanced-stage NSCLC patients.

Objectives. anatomical changes are inevitable during the course of radiotherapy treatments and, if significant, can severely alter expected dose distributions and affect treatment outcome. Adaptive radiotherapy (ART) is employed to maintain the planned distribution and minimise detriment to predicted treatment outcome. Typically, patients who may benefit from adaptive planning are identified via a re-planning process, i.e., re-simulation, re-contouring, re-planning and treatment plan quality assurance (QA). This time-intensive process significantly increases workload, can introduce delays and increases unnecessary stress to those patients who will not actually gain benefit. We consider it crucial to develop efficient models to predict changes to target coverage and trigger ART, without the need for re-planning.Methods.knowledge-based planning (KBP) models were developed using data for 20 patients' (400 fractions) to predict changes in PTV V95coverageΔV95PTV.Initially, this change in coverage was calculated on the synthetic computerised tomography (sCT) images produced using the Velocity adaptive radiotherapy software. Models were developed using patient (cell death bio-marker) and treatment fraction (PTV characteristic) specific parameters to predictΔV95PTVand verified using five patients (100 fractions) data.Results. three models were developed using combinations of patient and fraction specific terms. The prediction accuracy of the model developed using biomarker (PD-L1 expression) and the difference in 'planning' and 'fraction' PTV centre of the mass (characterised by mean square difference, MSD) had the higher prediction accuracy, predicting theΔV95PTVwithin ± 1.0% for 77% of the total fractions; with 59% for the model developed using, PTV size, PD-L1 and MSD and 48% PTV size and MSD respectively.Conclusion. the KBP models can predictΔV95PTVvery effectively and efficiently for advanced-stage NSCLC patients treated using volumetric modulated arc therapy and to identify patients who may benefit from adaption for a specific fraction.

Spheroid-on-chip microfluidic technology for the evaluation of the impact of continuous flow on metastatic potential in cancer models in vitro.

The majority of cancer deaths are linked to tumor spread, or metastasis, but 3D in vitro metastasis models relevant to the tumor microenvironment (including interstitial fluid flow) remain an area of unmet need. Microfluidics allows us to introduce controlled flow to an in vitro cancer model to better understand the relationship between flow and metastasis. Here, we report new hybrid spheroid-on-chip in vitro models for the impact of interstitial fluid flow on cancer spread. We designed a series of reusable glass microfluidic devices to contain one spheroid in a microwell under continuous perfusion culture. Spheroids derived from established cancer cell lines were perfused with complete media at a flow rate relevant to tumor interstitial fluid flow. Spheroid viability and migratory/invasive capabilities were maintained on-chip when compared to off-chip static conditions. Importantly, using flow conditions modeled in vitro, we are the first to report flow-induced secretion of pro-metastatic factors, in this case cytokines vascular endothelial growth factor and interleukin 6. In summary, we have developed a new, streamlined spheroid-on-chip in vitro model that represents a feasible in vitro alternative to conventional murine in vivo metastasis assays, including complex tumor environmental factors, such as interstitial fluid flow, extracellular matrices, and using 3D models to model nutrient and oxygen gradients. Our device, therefore, constitutes a robust alternative to in vivo early-metastasis models for determination of novel metastasis biomarkers as well as evaluation of therapeutically relevant molecular targets not possible in in vivo murine models.

Predicting personalised optimal arc parameter using knowledge-based planning model for inoperable locally advanced lung cancer patients to reduce organ at risk doses.

Objectives. Volumetric modulated arc therapy (VMAT) allows for reduction of organs at risk (OAR) volumes receiving higher doses, but increases OAR volumes receiving lower radiation doses and can subsequently increasing associated toxicity. Therefore, reduction of this low-dose-bath is crucial. This study investigates personalizing the optimization of VMAT arc parameters (gantry start and stop angles) to decrease OAR doses.Materials and Methods. Twenty previously treated locally advanced non-small cell lung cancer (NSCLC) patients treated with half-arcs were randomly selected from our database. These plans were re-optimized with seven different arcs parameters; optimization objectives were kept constant for all plans. All resulting plans were reviewed by two clinicians and the optimal plan (lowest OAR doses and adequate target coverage) was selected. Furthermore, knowledge-based planning (KBP) model was developed using these plans as 'training data' to predict optimal arc parameters for individual patients based on their anatomy. Treatment plan complexity scores and deliverability measurements were performed for both optimal and original clinical plans.Results.The results show that different arc geometries resulted in different dose distributions to the OAR but target coverage was mostly similar. Different arc geometries were required for different patients to minimize OAR doses. Comparison of the personalized against the standard (2 half-arcs) plans showed a significant reduction in lung V5(lung volume receiving 5 Gy), mean lung dose and mean heart doses. Reduction in lung V20and heart V30were statistically insignificant. Plan complexity and deliverability measurements show the test plans can be delivered as planned.Conclusions.Our study demonstrated that personalizing arc parameters based on an individual patient's anatomy significantly reduces both lung and heart doses. Dose reduction is expected to reduce toxicity and improve the quality of life for these patients.

Cytotoxic effects of Ridolfia segetum (L.) Moris phytoproducts in cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The past few years have witnessed an increasing interest in essential oils (EOs) as potential therapeutic agents against a wide variety of pathologies, including cancer. EOs extracted from Ridolfia segetum (L.) Moris (R. segetum) are a clear example of a phytoproduct with therapeutic applications, as it is widely used in traditional medicine due to its antioxidant and anti-inflammatory properties, and these properties were already validated by previous studies. Although, it is well established that inflammation is a key hallmark of cancer, with a key role promoting tumorigenesis, and being chronic inflammation often associated with tumorigenic processes, there are no previous studies regarding the assessment of the antitumoural potential of R. segetum EOs. AIM OF THE STUDY: The present study intends to be the first to evaluate the antitumoural proprieties of R. segetum EO phytoproducts in cancer cell models. MATERIALS AND METHODS: For this, R. segetum EOs were extracted from plants collected at either flowering (RS_Fl) or fruiting (RS_Fr) stage. The impact on proliferation and viability of treatment with R. segetum EO extracts was assessed using in vitro 2D and 3D models. RESULTS: Both R. segetum EOs presented effective antiproliferative/viability effects, evidence noted by low IC50 values in 2D models, and significant reduction of spheroid size in 3D in vitro models. Mechanistically, treatment with R. segetum EOs was associated with an altered G1 (associated with p21 stabilisation), and subsequent induction of apoptosis. CONCLUSIONS: Overall, these results indicate that R. segetum EOs have potential as suitable antitumoural therapeutic agents.

PI3K inhibition as a novel therapeutic strategy for neoadjuvant chemoradiotherapy resistant oesophageal adenocarcinoma.

OBJECTIVE: Neoadjuvant chemoradiotherapy (neo-CRT) prior to surgery is the standard of care for oesophageal adenocarcinoma (OAC) patients. Unfortunately, most patients fail to respond to treatment. MiR-187 was previously shown to be downregulated in neo-CRT non-responders, whist in vitro miR-187 overexpression enhanced radiosensitivity and upregulated PTEN. This study evaluates the role of miR-187 and downstream PI3K signalling in radiation response in OAC. METHODS: The effect of miR-187 overexpression on downstream PI3K signalling was evaluated in OAC cell lines by qPCR and Western blotting. PTEN expression was analysed in OAC pre-treatment biopsies of neo-CRT responders and non-responders. Pharmacological inhibition of PI3K using GDC-0941 was evaluated in combination with radiotherapy in two-dimensional and three-dimensional OAC models in vitro and as a single agent in vivo. Radiation response in vitro was assessed via clonogenic assay. RESULTS: PTEN expression was significantly decreased in neo-CRT non-responders. MiR-187 overexpression significantly upregulated PTEN expression and inhibited downstream PI3K signalling in vitro. GDC-0941 significantly reduced viability and enhanced radiation response in vitro and led to tumour growth inhibition as a single agent in vivo. CONCLUSION: Targeting of PI3K signalling is a promising therapeutic strategy for OAC patients who have repressed miR-187 expression and do not respond to conventional neo-CRT. ADVANCES IN KNOWLEDGE: This is the first study evaluating the effect of PI3K inhibition on radiosensitivity in OAC, with a particular focus on patients that do not respond to neo-CRT. We have shown for the first time that targeting of PI3K signalling is a promising alternative therapeutic strategy for OAC patients who do not respond to conventional neo-CRT.

Validation of in-house knowledge-based planning model for advance-stage lung cancer patients treated using VMAT radiotherapy.

OBJECTIVES: Radiotherapy plan quality may vary considerably depending on planner's experience and time constraints. The variability in treatment plans can be assessed by calculating the difference between achieved and the optimal dose distribution. The achieved treatment plans may still be suboptimal if there is further scope to reduce organs-at-risk doses without compromising target coverage and deliverability. This study aims to develop a knowledge-based planning (KBP) model to reduce variability of volumetric modulated arc therapy (VMAT) lung plans by predicting minimum achievable lung volume-dose metrics. METHODS: Dosimetric and geometric data collected from 40 retrospective plans were used to develop KBP models aiming to predict the minimum achievable lung dose metrics via calculating the ratio of the residual lung volume to the total lung volume. Model accuracy was verified by replanning 40 plans. Plan complexity metrics were calculated using locally developed script and their effect on treatment delivery was assessed via measurement. RESULTS: The use of KBP resulted in significant reduction in plan variability in all three studied dosimetric parameters V5, V20 and mean lung dose by 4.9% (p = 0.007, 10.8 to 5.9%), 1.3% (p = 0.038, 4.0 to 2.7%) and 0.9 Gy (p = 0.012, 2.5 to 1.6Gy), respectively. It also increased lung sparing without compromising the overall plan quality. The accuracy of the model was proven as clinically acceptable. Plan complexity increased compared to original plans; however, the implication on delivery errors was clinically insignificant as demonstrated by plan verification measurements. CONCLUSION: Our in-house model for VMAT lung plans led to a significant reduction in plan variability with concurrent decrease in lung dose. Our study also demonstrated that treatment delivery verifications are important prior to clinical implementation of KBP models. ADVANCES IN KNOWLEDGE: In-house KBP models can predict minimum achievable lung dose-volume constraints for advance-stage lung cancer patients treated with VMAT. The study demonstrates that plan complexity could increase and should be assessed prior to clinical implementation.

Secondary metabolites (essential oils) from sand-dune plants induce cytotoxic effects in cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Despite advances in modern therapeutic strategies, cancer remains the second leading cause of death worldwide. Therefore, there is a constant need to develop more efficient anticancer targeting strategies. The anticancer therapeutic proprieties of medicinal plants and their bioactive compounds have been reported for several years, making natural extracts and/or compounds derived from these a promising source of novel anticancer agents. Sand dune plants are subjected to severe environmental stresses, leading to the development of adaptations, including the production of secondary metabolites with a wide range of bioactivities, such as: anti-inflammatory, analgesic, antiseptic, hypoglycaemic, hypotensive, antinociceptive, antioxidant and anticancer. AIM OF THE STUDY: The anticancer potential of sand dune plants remains under-investigated, so this research describes the characterisation of the composition of bioactive EOs from sand-dune plants of Peniche (Portugal), and assessment of their activity in vitro and potential mechanism of action. MATERIALS AND METHODS: EOs were extracted from six sand-dune species of plants from Peniche sand dunes: Crithmum maritimum L., Seseli tortuosum L., Artemisia campestris subsp. maritima (DC.) Arcang., Juniperus phoenicea var. turbinata (Guss.) Parl., Otanthus maritimus (L.) Hoffmanns. & Link, and Eryngium maritimum L.. EOs composition was fully characterised chemically using Gas Chromatography-Mass Spectrometry (GC-MS). The assessment of anticancer activity and mechanism of action was performed in vitro using breast and colorectal cancer 2D and 3D spheroid cell line models, through cell proliferation assay, western blotting analysis, and cell cycle analysis. RESULTS: EOs from the majority of the species tested (S. tortuosum, A. campestris subsp. maritima, O. maritimus, and E. maritimum) were mainly composed by hydrocarbon compounds (sequisterpenes and monoterpenes), showing antiproliferative activity in both 2D and 3D models. EO extracted from S. tortuosum and O. maritimus were identified as having the lowest IC50 values for both cell lines when compared with the other species tested. Furthermore, this antiproliferative activity was associated with increased p21 expression and induction of apoptosis. CONCLUSIONS: The present study suggests that EOs extracted from S. tortuosum and O. maritimus present promising cytotoxic properties. Further evaluation of the extracts and their key components as potential anticancer agents should therefore be explored.

Fission yeast Dss1 associates with the proteasome and is required for efficient ubiquitin-dependent proteolysis.

Human DSS1 associates with BRCA2, a tumour suppressor protein required for efficient recombinational DNA repair, but the biochemical function of DSS1 is not known. Orthologues of DSS1 are found in organisms such as budding yeast and fission yeast that do not have BRCA2-related proteins, indicating that DSS1 has a physiological role independent of BRCA2. The DSS1 orthologue in Saccharomyces cerevisiae has been shown to associate with the 26 S proteasome and, in the present paper, we report that in the distantly related fission yeast Schizosaccharomyces pombe, Dss1 associates with the 19 S RP (regulatory particle) of the 26 S proteasome. A role for S. pombe Dss1 in proteasome function is supported by three lines of evidence. First, overexpression of two components of the 19 S RP, namely Pad1/Rpn11 and Mts3/Rpn12, rescued the temperature-sensitive growth defect of the dss1 mutant. Secondly, the dss1 mutant showed phenotypes indicative of a defect in proteasome function: growth of the dss1 mutant was inhibited by low concentrations of L-canavanine, an amino acid analogue, and cells of the dss1 mutant accumulated high molecular mass poly-ubiquitylated proteins. Thirdly, synthetic growth defects were found when the dss1 mutation was combined with mutations in other proteasome subunit genes. These findings show that DSS1 has an evolutionarily conserved role as a regulator of proteasome function and suggest that DSS1 may provide a link between BRCA2 and ubiquitin-mediated proteolysis in human cells.

HIF-1α-independent hypoxia-induced rapid PTK6 stabilization is associated with increased motility and invasion.

PTK6/Brk is a non-receptor tyrosine kinase overexpressed in cancer. Here we demonstrate that cytosolic PTK6 is rapidly and robustly induced in response to hypoxic conditions in a HIF-1-independent manner. Furthermore, a proportion of hypoxic PTK6 subsequently re-localized to the cell membrane. We observed that the rapid stabilization of PTK6 is associated with a decrease in PTK6 ubiquitylation and we have identified c-Cbl as a putative PTK6 E3 ligase in normoxia. The consequences of hypoxia-induced PTK6 stabilization and subcellular re-localization to the plasma membrane include increased cell motility and invasion, suggesting PTK6 targeting as a therapeutic approach to reduce hypoxia-regulated metastatic potential. This could have particular significance for breast cancer patients with triple negative disease.

Use of the xCELLigence system for real-time analysis of changes in cellular motility and adhesion in physiological conditions.

Investigation of the mechanisms behind the regulation of cellular motility and adhesion is key to understanding metastasis and the biology of tumor spreading. There are many technologies available for these studies, but the majority of them are either dependent on the use of labels or limited to endpoint analysis. The xCELLigence RTCA (real-time cell analysis) provides a platform for label free and operator independent investigation of the migration, invasion and adhesion proprieties of cells in physiologically relevant conditions. The real-time kinetic data acquisition also allows for a more accurate characterization of short-lived cellular events. In this chapter we describe the use of the xCELLigence Real-Time Cell Analyzer to investigate changes in cellular adhesion and motility in real time.