Tracking the origins and drivers of subclonal metastatic expansion in prostate cancer.

Tumour heterogeneity in primary prostate cancer is a well-established phenomenon. However, how the subclonal diversity of tumours changes during metastasis and progression to lethality is poorly understood. Here we reveal the precise direction of metastatic spread across four lethal prostate cancer patients using whole-genome and ultra-deep targeted sequencing of longitudinally collected primary and metastatic tumours. We find one case of metastatic spread to the surgical bed causing local recurrence, and another case of cross-metastatic site seeding combining with dynamic remoulding of subclonal mixtures in response to therapy. By ultra-deep sequencing end-stage blood, we detect both metastatic and primary tumour clones, even years after removal of the prostate. Analysis of mutations associated with metastasis reveals an enrichment of TP53 mutations, and additional sequencing of metastases from 19 patients demonstrates that acquisition of TP53 mutations is linked with the expansion of subclones with metastatic potential which we can detect in the blood.

Percutaneous image-guided biopsy of prostate cancer metastases yields samples suitable for genomics and personalised oncology.

Personalised oncology through mutational profiling of cancers requires the procurement of fresh frozen tumour samples for genomics applications. While primary cancers are often surgically excised and therefore yield such tissue, metastases in the setting of a known cancer diagnosis are not routinely sampled prior to systemic therapy. Our study aimed to determine the suitability of extracted nucleic acids for genomics applications using distant metastatic prostate cancer samples obtained via percutaneous or surgical biopsy. Patients with metastatic prostate cancer were recruited for image-guided biopsy of metastases. Patients undergoing surgical procedures for the complications of metastases were also recruited. Tissue samples were flash frozen and cryosectioned for histological examination. DNA and RNA were simultaneously extracted and genomic DNA hybridised onto SNP arrays for genome-wide copy number analysis. 37 samples of metastatic tissue from seven patients with prostate cancer were obtained. Five of these underwent image-guided biopsies whilst two had therapeutic surgical procedures performed. 22 biopsy samples were obtained across the image-guided biopsy patients with 80 % of samples being successfully processed for downstream analysis. Nucleic acid yield from these samples were satisfactory for genomics applications. Copy number analysis revealed a median estimated tumour purity of 53 % and all samples showed chromosomal abnormalities suggestive of malignancy. The procurement of osseous metastatic prostate cancer from live patients, including the use of image-guided biopsy, is safe and feasible. Sufficient tissue can be obtained in a manner such that extracted nucleic acids are suitable for genomics research.

Loss of APKC expression independently predicts tumor recurrence in superficial bladder cancers.

OBJECTIVES: Epithelial-mesenchymal transition (EMT) is known to play an important role in the development of tumor invasion and progression in tumors of epithelial origin. Our aim was to investigate the role of tight junction proteins, Par3/Par6/atypical protein kinase C (APKC), Discs large (Dlg), and Scribble in human bladder pathogenesis. METHODS: We evaluated levels of APKC, Dlg, and Scribble in 92 superficial bladder tumors using tissue microarrays and immunohistochemistry, and correlated expression with pathologic variables and clinical outcomes. RESULTS: There was a slight apparent enrichment in strong vs. weak staining for APKC (54.9% vs. 45.1%), Dlg (65.7% vs. 34.3%), and a marked enrichment for Scribble (75% vs. 25%) in the superficial bladder tumors. Univariate analysis determined that both tumor focality and APKC expression were significantly associated with tumor recurrence (P < 0.05). Multivariate analysis using the Cox's proportional hazards model revealed that only APKC (P = 0.025) as well as tumor focality (P = 0.018) were independent and significant prognostic factors for tumor recurrence in all patients. We found that no immunohistochemical staining of any of the cell polarity proteins significantly predicted for tumor progression on either univariate or multivariate analysis. CONCLUSIONS: Loss of APKC expression in superficial bladder tumors is a strong predictor of tumor recurrence.

Dynamin II function is required for EGF-mediated Stat3 activation but not Erk1/2 phosphorylation.

Signalling from receptor tyrosine kinases is elicited by ligand binding which initiates the activation of many downstream signalling cascades. Endocytosis has been widely accepted as one mechanism in which cells inactivate signalling by internalising and subsequently degrading activated receptors. However, it is now evident that endocytosis of signalling receptors is important in initiation and sustaining downstream signalling. We and others have previously shown that epidermal growth factor receptor (EGFR) overexpression and activation of signal transducer and activator of transcription 3 (Stat3) are associated with tumourigenicity. Here, we examine the role of endocytosis in EGFR signal attenuation and differential signalling. Inhibition of dynamin II (Dyn II), a GTPase required for endocytosis, with a small molecular weight inhibitor, led to reduced EGF-mediated Stat3 phosphorylation and transcriptional activity in the A431 and HN5 human tumour cell lines. However, Dyn II inhibition had minimal effect on EGF-mediated EGFR and Erk1/2 phosphorylation, which is often regarded responsible for the tumourigenic function of the EGFR. Interestingly, this effect on Stat3 activation was not due to reduced EGFR/Stat3 association. Likewise, cells transfected with Dyn II siRNA or stably transfected with Dyn II shRNA had reduced EGF-mediated phospho-Stat3 levels but similar EGF-mediated phospho-EGFR and phospho-Erk1/2 levels compared with controls. Dyn II siRNA also reduced Stat3 transcriptional reporter activity and inhibits Stat3 accumulating into the nucleus. Taken together, our data suggest that the activation status of Stat3 and Erk1/2 and the sustainability of these signals are potentially due to the spatial and temporal control of the EGFR within the cell. This notion may have implications on therapeutic targeting and efficacy when using inhibitors to proteins either regulating endocytosis and/or signalling.