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

© 2014 Landes Bioscience. 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

Inhibition of PARP-1 by olaparib (AZD2281) increases the radiosensitivity of a lung tumor xenograft

Poly(ADP-ribose) polymerase-1 is a critical enzyme in the repair of DNA strand breaks. Inhibition of PARP-1 increases the effectiveness of radiation in killing tumor cells. However, while the mechanism(s) are well understood for these radiosensitizing effects in vitro, the underlying mechanism(s) in vivo are less clear. Nicotinamide, a drug structurally related to the first generation PARP-1 inhibitor, 3-aminobenzamide, reduces tumor hypoxia by preventing transient cessations in tumor blood flow, thus improving tumor oxygenation and sensitivity to radiotherapy. Here we investigate whether olaparib, a potent PARP-1 inhibitor, enhances radiotherapy, not only by inhibiting DNA repair but also by changing tumor vascular haemodynamics in non-small cell lung carcinoma. In irradiated Calu-6 and A549 cells, olaparib enhanced the cytotoxic effects of radiation (SER(10)=1.5 and 1.3) and DNA double strand breaks persisted for at least 24 h after treatment. Combination treatment of Calu-6 xenografts with olaparib and fractionated radiotherapy caused significant tumor regression (p=0.007) relative to radiotherapy alone. To determine whether this radiosensitisation was due solely to effects on DNA repair we used a dorsal window chamber model to establish the drug/radiation effects on vessel dynamics. Olaparib alone, when given as single or multiple daily doses, or in combination with fractionated radiotherapy, increased the perfusion of tumor blood vessels. Furthermore, an ex vivo assay in phenylephrine pre-constricted arteries confirmed olaparib to have higher vasodilatory properties than nicotinamide. This study suggests that olaparib warrants consideration for further development in combination with radiotherapy in clinical oncology settings such as NSCLC

Inhibition of HIF-1α expression by DNA damaging agents.

<p>(<b>A</b>) Western blot showing the protein levels of HIF-1α, phosphorylated ERK 1/2 (P-MAPK) and ERK 1/2 (MAPK) in HCT116 cells treated with 1.25 µM U0126 and 0.4 µg/ml doxorubicin, and cultured at 20% or 5% O₂ for 2 days. Cells treated with 500 µM CoCl₂ for 16 hours were used as a positive control for the induction of HIF-1α (H). (<b>B</b>) Western blot showing the protein levels of HIF-1α in HCT116 and HCT116 p53^−/− in lysates collected 24 hours after treatment with 1 µg/ml Actinomicyn D (ActD) or 200 µM tert-Butyl Hydroperoxyde (tBH) for 2hours (<b>C</b>) Western blot showing the protein levels of HIF-1α, p53 and p21 in HCT116 and MCF-7 cells treated with 0.4 µg/ml doxorubicin or 10 Gy γ-radiation for 24 hours at 20% or <1% O₂ (hypoxia).</p

HIF-1α contributes to increased proliferation of cells at physiological oxygen tensions.

<p>(<b>A</b>) Western blot showing the protein levels of HIF-1α in HCT116 HIF^+/+ and HIF^−/−cultured either at 20% O₂ or under hypoxic stress (<0.1% O₂) for 16 hours. (<b>B</b>) Proliferation curves of HIF^+/+ and HIF^−/− HCT116 cells cultured at 20% or 5% O₂ from 2 to 8 days. Values represent ratio of cell numbers normalized to the initial seeded cells (10⁶). (<b>C</b>) Representative colony formation assay for HCT116 HIF^+/+ and HIF^−/−cultured at 20% or 5% O₂. 200 cells were seeded in each plate and 14 days later they were stained with Giemsa. Media was not changed during the process. (<b>D</b>) Percentage of EdU positive HCT116 HIF^+/+ and HIF^−/− cells as assessed by immunofluorescence (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097938#pone.0097938.s002" target="_blank">Figure S2</a>). Cells were incubated with EdU for 30 minutes in the corresponding oxygen tensions. Results represent means of two independent experiments. Two microscope fields were scored in each experiment. Error bars represent standard error. P value (unpaired t-test): 0.0127 (*), (<b>E</b>) Proposed model of the roles of HIF-1 at different oxygen concentrations.</p

Chemical inhibition of MAPK reduces the activation of HIF-1α.

<p>Western blot showing the protein levels of HIF-1α and phosphorylated (active) ERK 1/2 MAPK in HCT116 cultured at 20% or 5% O₂for 12 to 48 hours, in the presence of 1.25 µM U0126. U0126 was added at the same time cells were transferred to 5%O₂. Total MAPK levels are provided as loading control.</p

Investigation of radiosensitivity gene signatures in cancer cell lines

Intrinsic radiosensitivity is an important factor underlying radiotherapy response, but there is no method for its routine assessment in human tumours. Gene signatures are currently being derived and some were previously generated by expression profiling the NCI-60 cell line panel. It was hypothesised that focusing on more homogeneous tumour types would be a better approach. Two cell line cohorts were used derived from cervix [n = 16] and head and neck [n = 11] cancers. Radiosensitivity was measured as surviving fraction following irradiation with 2 Gy (SF2) by clonogenic assay. Differential gene expression between radiosensitive and radioresistant cell lines (SF2</> median) was investigated using Affymetrix GeneChip Exon 1.0ST (cervix) or U133A Plus2 (head and neck) arrays. There were differences within cell line cohorts relating to tissue of origin reflected by expression of the stratified epithelial marker p63. Of 138 genes identified as being associated with SF2, only 2 (1.4%) were congruent between the cervix and head and neck carcinoma cell lines (MGST1 and TFPI), and these did not partition the published NCI-60 cell lines based on SF2. There was variable success in applying three published radiosensitivity signatures to our cohorts. One gene signature, originally trained on the NCI-60 cell lines, did partially separate sensitive and resistant cell lines in all three cell line datasets. The findings do not confirm our hypothesis but suggest that a common transcriptional signature can reflect the radiosensitivity of tumours of heterogeneous origins

Assessment of established radiosensitivity gene signatures.

<p><b>A)</b> PCA of the Tewari radiosensitivity gene signature. The original signature consists of 49 genes, with mapping to the NCI-60 (60 Plus2 probesets) HNSCC (60 Plus2 probesets) and cervix cell line (48/49 genes) datasets. The x-axis shows PC1, accounting for the largest amount of variation in the experiment and the y-axis shows the second principal component (PC2). Colouring based on median SF2, blue data-points are radiosensitive cell lines (below the median SF2) with red data-points being the radioresistant lines (above the median SF2). <b>B)</b> Implementation of the Eschrich radiosensitivity model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086329#pone.0086329-Eschrich2" target="_blank">[12]</a>. Applied to a training set of 16 samples from the NCI-60 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086329#pone.0086329-Eschrich3" target="_blank">[13]</a>. xy-scatterplot with the x-axis showing reported SF2 values, generated with these cell lines on a earlier array type (U95) against values generated by implementing the model in the current U133 plus 2.0 dataset (y-axis). Line indicates perfect correlation. <b>C)</b> Applied to the HNSCC and cervix cancer cell line cohorts. The y-axis indicates the predicted SF2 determined from the radiosensitivity model. The x-axis shows the empirically derived SF2 values. <b>D)</b> Principal component analysis of the Amundson radiosensitivity gene signature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086329#pone.0086329-Amundson1" target="_blank">[10]</a>. The original signature consists of 22 genes (33 Plus2 probesets), with mapping to the NCI-60 (33 Plus2 probesets), HNSCC (33 Plus2 probesets) and cervix cell line (21/22 genes) datasets. The x-axis shows PC1, accounting for the largest amount of variation in the experiment and the y-axis shows the second principal component (PC2). In the NCI-60 data colouring is based a threshold of 0.2 (previously defined <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086329#pone.0086329-Hall1" target="_blank">[21]</a> where the HNSCC and cervix cell line datasets are coloured by median SF2. In all cases blue data-points are radiosensitive cell lines (below the median SF2) with red data-points being the radioresistant lines (above the median SF2).</p

Characterisation of a head and neck squamous cell carcinoma (HNSCC) cell line cohort.

<p><b>A)</b> Graph showing the mean SF2 (log10) (y-axis) for each of the 11 cervix cancer cell lines (x-axis). Error bars show the standard error of mean of 2–3 independent experiments. <b>B)</b> Graph showing that there is no difference in TP63 expression between the SF2 high and low groups. Bar shows the median expression. <b>C)</b> Unsupervised hierarchical clustering of the top 1000 genes ranked by coefficient of variation (from U133 array data). Heatmap colouring is by log₂ expression value. Rows represent genes and columns are cell lines. x-axis dendrogram (clusters) indicates the similarity of the cell lines and y-axis dendrogram the similarity of genes. Cluster 1 represents two samples with the lowest TP63 values (p63 negative). Cluster 2 shows the grouping of the other p63− cell line, along with low TP63 expressing lines. Cluster 3 groups together all HNSCC lines with >6.0 (log2 expression) TP63 expression. <b>D)</b> Diagram to represent the integrated SF2 analysis of the cervix and HNSCC cell lines. Rank product analysis (FDR <0.05) identified 96 genes in the cervix cohort differentially expressed between SF2 low and high cell lines. An identical analysis in the HNSCC cell lines identifies 97 probesets (42 genes) differentially expressed between SF2 low and high cell lines. PCA of the cervix genes shows that they are capable of separating the cell lines by SF2. PCA of the HNSCC genes is equally capable of separating the samples based on SF2. The Venn diagram shows that only 4/138 genes are common between the two cohorts and of these only 2/138 are “congruent” and associated with the same directionality (high SF2/low SF2 in both HNSCC and cervix). PCA shows probeset expression of these two “common” and “congruent” genes (MGST1 and TFPI) in the NCI-60 dataset. The NCI-60 upper PCA shows data-points coloured for median SF2 and lower PCA coloured for 0.2, used previously to partition radiosensitive and radioresistant cell lines in this cohort.</p

ZeptoMARK protein profiling of the cervix cancer cell lines.

<p><b>A)</b> Histogram displaying the ZeptoMARK protein-array derived abundance for the 16 cervix cancer cell lines. The y-axis displays E-cadherin protein level (relative fluorescent intensity (RFI) for each of the cell lines (x-axis). Cell lines are ranked based on TP63 expression. Grouping into p63 negative and p63 positive cell lines confirms the association of E-cadherin with p63. The p value is T-test derived comparing the difference in E-cadherin expression between the p63 positive and negative groups, error bars display standard deviation of two biological replicates. <b>B)</b> x–y scatterplot showing E-cadherin gene expression (Exon array) on the y-axis against E-cadherin protein expression on the x-axis. Dashed line represents perfect correlation. Exon array data-points represent the average of multiple exonic probesets (n = 19) from a single Exon expression array, where protein data are the mean of two biological replicates. <b>C)</b> Heatmap showing clustering of proteins with similar expression (y-axis) in the ZeptoMARK protein profiling data. Cell lines ranked by SF2. Heatmap colouring is based on row Z-score. <b>D)</b> xy-scatter plot showing the expression (y-axis) of the top 5 proteins from LIMMA against SF2 (x-axis). Table summarises the results of Limma differential protein expression analysis between high and low SF2 groups and Pearson correlation of protein expression (RFI) against SF2. p values denote those proteins with differential expression (* p<0.05 or ** p<0.01) between SF2 low and high groups according to LIMMA analysis. However these fail to pass false discovery rate correction.</p