Molecular analysis of archival diagnostic prostate cancer biopsies identifies genomic similarities in cases with progression post-radiotherapy, and those with de novo metastatic disease.

BACKGROUND: It is important to identify molecular features that improve prostate cancer (PCa) risk stratification before radical treatment with curative intent. Molecular analysis of historical diagnostic formalin-fixed paraffin-embedded (FFPE) prostate biopsies from cohorts with post-radiotherapy (RT) long-term clinical follow-up has been limited. Utilizing parallel sequencing modalities, we performed a proof-of-principle sequencing analysis of historical diagnostic FFPE prostate biopsies. We compared patients with (i) stable PCa (sPCa) postprimary or salvage RT, (ii) progressing PCa (pPCa) post-RT, and (iii) de novo metastatic PCa (mPCa). METHODS: A cohort of 19 patients with diagnostic prostate biopsies (n = 6 sPCa, n = 5 pPCa, n = 8 mPCa) and mean 4 years 10 months follow-up (diagnosed 2009-2016) underwent nucleic acid extraction from demarcated malignancy. Samples underwent 3'RNA sequencing (3'RNAseq) (n = 19), nanoString analysis (n = 12), and Illumina 850k methylation (n = 8) sequencing. Bioinformatic analysis was performed to coherently identify differentially expressed genes and methylated genomic regions (MGRs). RESULTS: Eighteen of 19 samples provided useable 3'RNAseq data. Principal component analysis (PCA) demonstrated similar expression profiles between pPCa and mPCa cases, versus sPCa. Coherently differentially methylated probes between these groups identified ~600 differentially MGRs. The top 50 genes with increased expression in pPCa patients were associated with reduced progression-free survival post-RT (p < 0.0001) in an external cohort. CONCLUSIONS: 3'RNAseq, nanoString and 850k-methylation analyses are each achievable from historical FFPE diagnostic pretreatment prostate biopsies, unlocking the potential to utilize large cohorts of historic clinical samples. Profiling similarities between individuals with pPCa and mPCa suggests biological similarities and historical radiological staging limitations, which warrant further investigation.

Glucocorticoid receptor and Klf4 co-regulate anti-inflammatory genes in keratinocytes.

The glucocorticoid (GC) receptor (GR) and Kruppel-like factor Klf4 are transcription factors that play major roles in skin homeostasis. However, whether these transcription factors cooperate in binding genomic regulatory regions in epidermal keratinocytes was not known. Here, we show that in dexamethasone-treated keratinocytes GR and Klf4 are recruited to genomic regions containing adjacent GR and KLF binding motifs to control transcription of the anti-inflammatory genes Tsc22d3 and Zfp36. GR- and Klf4 loss of function experiments showed total GR but partial Klf4 requirement for full gene induction in response to dexamethasone. In wild type keratinocytes induced to differentiate, GR and Klf4 protein expression increased concomitant with Tsc22d3 and Zfp36 up-regulation. In contrast, GR-deficient cells failed to differentiate or fully induce Klf4, Tsc22d3 and Zfp36 correlating with increased expression of the epithelium-specific Trp63, a known transcriptional repressor of Klf4. The identified transcriptional cooperation between GR and Klf4 may determine cell-type specific regulation and have implications for developing therapies for skin diseases.