Karl Mulligan

Development and Independent Validation of a Prognostic Assay for Stage II Colon Cancer Using Formalin-Fixed Paraffin-Embedded Tissue

PURPOSE Current prognostic factors are poor at identifying patients at risk of disease recurrence after surgery for stage II colon cancer. Here we describe a DNA microarray-based prognostic assay using clinically relevant formalin-fixed paraffin-embedded (FFPE) samples. PATIENTS AND METHODS A gene signature was developed from a balanced set of 73 patients with recurrent disease (high risk) and 142 patients with no recurrence (low risk) within 5 years of surgery. RESULTS The 634-probe set signature identified high-risk patients with a hazard ratio (HR) of 2.62 (P < .001) during cross validation of the training set. In an independent validation set of 144 samples, the signature identified high-risk patients with an HR of 2.53 (P < .001) for recurrence and an HR of 2.21 (P = .0084) for cancer-related death. Additionally, the signature was shown to perform independently from known prognostic factors (P < .001). CONCLUSION This gene signature represents a novel prognostic biomarker for patients with stage II colon cancer that can be applied to FFPE tumor samples.

Cortactin underpins CD44-promoted invasion and adhesion of breast cancer cells to bone marrow endothelial cells

Using a validated tetracycline (tet)-regulated MCF7-founder (MCF7F) expression system to modulate expression of CD44 standard form (CD44s), we report the functional importance of CD44s and that of a novel transcriptional target of hyaluronan (HA)/CD44s signaling, EMS1/cortactin, in underpinning breast cancer metastasis. In functional experiments, tet-regulated induction of CD44s potentiated the migration and invasion of MCF7F cells through HA-supplemented Matrigel. EMS1/cortactin was identified by expression profiling as a novel transcriptional target of HA/CD44 signaling, an association validated by quantitative PCR and immunoblotting experiments in a range of breast cancer cell lines. The mechanistic basis underpinning CD44-promoted transcription of EMS1/cortactin was shown to be dependent upon a NFκB mechanism, since pharmacological inhibition of IκKinase-2 or suppression of p65 Rel A expression attenuated CD44-induced increases in cortactin mRNA transcript levels. Overexpression of a c-myc tagged murine cortactin construct in the weakly invasive, CD44-deficient MCF7F and T47D cells potentiated their invasion. Furthermore, the functional importance of cortactin to CD44s-promoted metastasis was demonstrated by selective suppression of cortactin in CD44-expressing MCF7F-B5 and MDA-MB-231 breast cancer cells using RNAi, which was shown to result in attenuated CD44-promoted invasion and CD44-promoted adhesion to bone marrow endothelial cells (BMECs).

BRCA1 and c-Myc Associate to Transcriptionally Repress Psoriasin, a DNA Damage–Inducible Gene

Evidence is accumulating to suggest that some of the diverse functions associated with BRCA1 may relate to its ability to transcriptionally regulate key downstream target genes. Here, we identify S100A7 (psoriasin), S100A8, and S100A9, members of the S100A family of calcium-binding proteins, as novel BRCA1-repressed targets. We show that functional BRCA1 is required for repression of these family members and that a BRCA1 disease-associated mutation abrogates BRCA1-mediated repression of psoriasin. Furthermore, we show that BRCA1 and c-Myc form a complex on the psoriasin promoter and that BRCA1-mediated repression of psoriasin is dependent on functional c-Myc. Finally, we show that psoriasin expression is induced by the topoisomerase IIalpha poison, etoposide, in the absence of functional BRCA1 and increased psoriasin expression enhances cellular sensitivity to this chemotherapeutic agent. Therefore, we identified a novel transcriptional mechanism that is likely to contribute to BRCA1-mediated resistance to etoposide.

Epidermal growth factor up-regulates CD44-dependent astrocytoma invasion in vitro

CD44/hyaluronan interactions and epidermal growth factor (EGF) stimulation are both known to enhance tumour invasion in vitro. The frequent amplification of the EGF receptor (EGFR) in high‐grade astrocytomas led to the examination of the hypothesis that CD44‐dependent astrocytoma invasion is regulated by EGF. It has been shown that human astrocytoma cells express only the standard (haemopoietic) form of CD44 (CD44s) and that EGF up‐regulates CD44 mRNA and protein in a time‐ and dose‐dependent (10–100 ng/ml) manner. EGF stimulation did not result in induction of additional splice variants. No EGF‐induced increase in CD44s was observed after treatment of cells with the wild‐type EGFR tyrosine kinase inhibitor Tyrphostin AG1478 (30 nM). Up‐regulation of CD44 by EGF is also prevented by the transcriptional inhibitor actinomycin D (5 µg/ml) and by blocking the MAP kinase (MAPK) pathway using the MEK inibitor U0126 (100 µM). CD44 up‐regulation was associated with a 50% increase in invasion through hyaluronan‐supplemented Matrigel™, which was abrogated by ligating CD44 with the specific antibody KM201. These results suggest that increased CD44 expression in response to EGF stimulation plays a significant role in astrocytoma invasion. Copyright

Generation of a non-small cell lung cancer transcriptome microarray

BackgroundNon-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. At present no reliable biomarkers are available to guide the management of this condition. Microarray technology may allow appropriate biomarkers to be identified but present platforms are lacking disease focus and are thus likely to miss potentially vital information contained in patient tissue samples.MethodsA combination of large-scale in-house sequencing, gene expression profiling and public sequence and gene expression data mining were used to characterise the transcriptome of NSCLC and the data used to generate a disease-focused microarray – the Lung Cancer DSA research tool.ResultsBuilt on the Affymetrix GeneChip platform, the Lung Cancer DSA research tool allows for interrogation of ~60,000 transcripts relevant to Lung Cancer, tens of thousands of which are unavailable on leading commercial microarrays.ConclusionWe have developed the first high-density disease specific transcriptome microarray. We present the array design process and the results of experiments carried out to demonstrate the arrays utility. This approach serves as a template for the development of other disease transcriptome microarrays, including non-neoplastic diseases.