Intratumoural evolutionary landscape of high-risk prostate cancer: the PROGENY study of genomic and immune parameters.

BACKGROUND: Intratumoural heterogeneity (ITH) is well recognised in prostate cancer (PC), but its role in high-risk disease is uncertain. A prospective, single-arm, translational study using targeted multiregion prostate biopsies was carried out to study genomic and T-cell ITH in clinically high-risk PC aiming to identify drivers and potential therapeutic strategies. PATIENTS AND METHODS: Forty-nine men with elevated prostate-specific antigen and multiparametric-magnetic resonance imaging detected PC underwent image-guided multiregion transperineal biopsy. Seventy-nine tumour regions from 25 patients with PC underwent sequencing, analysis of mutations, copy number and neoepitopes combined with tumour infiltrating T-cell subset quantification. RESULTS: We demonstrated extensive somatic nucleotide variation and somatic copy number alteration heterogeneity in high-risk PC. Overall, the mutational burden was low (0.93/Megabase), but two patients had hypermutation, with loss of mismatch repair (MMR) proteins, MSH2 and MSH6. Somatic copy number alteration burden was higher in patients with metastatic hormone-naive PC (mHNPC) than in those with high-risk localised PC (hrlPC), independent of Gleason grade. Mutations were rarely ubiquitous and mutational frequencies were similar for mHNPC and hrlPC patients. Enrichment of focal 3q26.2 and 3q21.3, regions containing putative metastasis drivers, was seen in mHNPC patients. We found evidence of parallel evolution with three separate clones containing activating mutations of ß-catenin in a single patient. We demonstrated extensive intratumoural and intertumoural T-cell heterogeneity and high inflammatory infiltrate in the MMR-deficient (MMRD) patients and the patient with parallel evolution of ß-catenin. Analysis of all patients with activating Wnt/ß-catenin mutations demonstrated a low CD8+/FOXP3+ ratio, a potential surrogate marker of immune evasion. CONCLUSIONS: The PROGENY (PROstate cancer GENomic heterogeneitY) study provides a diagnostic platform suitable for studying tumour ITH. Genetic aberrations in clinically high-risk PC are associated with altered patterns of immune infiltrate in tumours. Activating mutations of Wnt/ß-catenin signalling pathway or MMRD could be considered as potential biomarkers for immunomodulation therapies. CLINICAL TRIALS.GOV IDENTIFIER: NCT02022371.

6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 is essential for p53-null cancer cells.

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-4 (PFKFB4) controls metabolic flux through allosteric regulation of glycolysis. Here we show that p53 regulates the expression of PFKFB4 and that p53-deficient cancer cells are highly dependent on the function of this enzyme. We found that p53 downregulates PFKFB4 expression by binding to its promoter and mediating transcriptional repression via histone deacetylases. Depletion of PFKFB4 from p53-deficient cancer cells increased levels of the allosteric regulator fructose-2,6-bisphosphate, leading to increased glycolytic activity but decreased routing of metabolites through the oxidative arm of the pentose-phosphate pathway. PFKFB4 was also required to support the synthesis and regeneration of nicotinamide adenine dinucleotide phosphate (NADPH) in p53-deficient cancer cells. Moreover, depletion of PFKFB4-attenuated cellular biosynthetic activity and resulted in the accumulation of reactive oxygen species and cell death in the absence of p53. Finally, silencing of PFKFB4-induced apoptosis in p53-deficient cancer cells in vivo and interfered with tumour growth. These results demonstrate that PFKFB4 is essential to support anabolic metabolism in p53-deficient cancer cells and suggest that inhibition of PFKFB4 could be an effective strategy for cancer treatment.

Detection of ubiquitous and heterogeneous mutations in cell-free DNA from patients with early-stage non-small-cell lung cancer.

BACKGROUND: The aim of this pilot study was to assess whether both ubiquitous and heterogeneous somatic mutations could be detected in cell-free DNA (cfDNA) from patients with early-stage non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Three stage I and one stage II primary NSCLC tumors were subjected to multiregion whole-exome sequencing (WES) and validated with AmpliSeq. A subset of ubiquitous and heterogeneous single-nucleotide variants (SNVs) were chosen. Multiplexed PCR using custom-designed primers, coupled with next-generation sequencing (mPCR-NGS), was used to detect these SNVs in both tumor DNA and cfDNA isolated from plasma obtained before surgical resection of the tumors. The limit of detection for each assay was determined using cfDNA from 48 presumed-normal healthy volunteers. RESULTS: Tumor DNA and plasma-derived cfDNA was successfully amplified and sequenced for 37/50 (74%) SNVs using the mPCR-NGS method. Twenty-five (68%) were ubiquitous and 12 (32%) were heterogeneous SNVs. Variant detection by mPCR-NGS and WES-AmpliSeq in tumor tissue was well correlated (R(2) = 0.8722, P < 0.0001). Sixteen (43%) out of 37 SNVs were detected in cfDNA. Twelve of these were ubiquitous SNVs with a variant allele frequency (VAF) range of 0.15-23.25%, and four of these were heterogeneous SNVs with a VAF range of 0.28-1.71%. There was a statistically significant linear relationship between the VAFs for tumor and cfDNA (R(2) = 0.5144; P = 0.0018). For all four patients, at least two variants were detected in plasma. The estimated number of copies of variant DNA present in each sample ranged from 5 to 524. The average number of variant copies required for detection (VCRD) was 3.16 (range: 0.2-7.6 copies). CONCLUSIONS: The mPCR-NGS method revealed intratumor heterogeneity in early-stage NSCLC tumors, and was able to detect both ubiquitous and heterogeneous SNVs in cfDNA. Further validation of mPCR-NGS in cfDNA is required to define its potential use in clinical practice.

miR-19-Mediated Inhibition of Transglutaminase-2 Leads to Enhanced Invasion and Metastasis in Colorectal Cancer.

UNLABELLED: Transglutaminase-2 (TG2) is a critical cross-linking enzyme in the extracellular matrix (ECM) and tumor microenvironment (TME). Although its expression has been linked to colorectal cancer, its functional role in the processes that drive disease appears to be context dependent. There is now considerable evidence of a role for microRNAs (miRNA) in the development and progression of cancer, including metastasis. A cell model of metastatic colon adenocarcinoma was used to investigate the contribution of miRNAs to the differential expression of TG2, and functional effects on inflammatory and invasive behavior. The impact of TG2 in colorectal cancer was analyzed in human colorectal tumor specimens and by manipulations in SW480 and SW620 cells. Effects on invasive behavior were measured using Transwell invasion assays, and cytokine production was assessed by ELISA. TG2 was identified as a target for miR-19 by in silico analysis, which was confirmed experimentally. Functional effects were evaluated by overexpression of pre-miR-19a in SW480 cells. Expression of TG2 correlated inversely with invasive behavior, with knockdown in SW480 cells leading to enhanced invasion, and overexpression in SW620 cells the opposite. TG2 expression was observed in colorectal cancer primary tumors but lost in liver metastases. Finally, miR-19 overexpression and subsequent decreased TG2 expression was linked to chromosome-13 amplification events, leading to altered invasive behavior in colorectal cancer cells. IMPLICATIONS: Chromosome-13 amplification in advanced colorectal cancer contributes to invasion and metastasis by upregulating miR-19, which targets TG2.

SREBP maintains lipid biosynthesis and viability of cancer cells under lipid- and oxygen-deprived conditions and defines a gene signature associated with poor survival in glioblastoma multiforme.

Oxygen and nutrient limitation are common features of the tumor microenvironment and are associated with cancer progression and induction of metastasis. The inefficient vascularization of tumor tissue also limits the penetration of other serum-derived factors, such as lipids and lipoproteins, which can be rate limiting for cell proliferation and survival. Here we have investigated the effect of hypoxia and serum deprivation on sterol regulatory element-binding protein (SREBP) activity and the expression of lipid metabolism genes in human glioblastoma multiforme (GBM) cancer cells. We found that SREBP transcriptional activity was induced by serum depletion both in normoxic and hypoxic cells and that activation of SREBP was required to maintain the expression of fatty acid and cholesterol metabolism genes under hypoxic conditions. Moreover, expression of stearoyl-CoA desaturase, the enzyme required for the generation of mono-unsaturated fatty acids, and fatty acid-binding protein 7, a regulator of glioma stem cell function, was strongly dependent on SREBP function. Inhibition of SREBP function blocked lipid biosynthesis in hypoxic cancer cells and impaired cell survival under hypoxia and in a three-dimensional spheroid model. Finally, gene expression analysis revealed that SREBP defines a gene signature that is associated with poor survival in glioblastoma.

Characterisations of human prostate stem cells reveal deficiency in class I UGT enzymes as a novel mechanism for castration-resistant prostate cancer.

BACKGROUND: Evidence increasingly supports that prostate cancer is initiated by the malignant transformation of stem cells (SCs). Furthermore, many SC-signalling pathways are shown to be shared in prostate cancer. Therefore, we planned transcriptome characterisation of adult prostate SCs as a strategy to consider new targets for cancer treatment. METHODS: Intuitive pathway analysis was used for putative target discovery in 12 matched selections of human prostate SCs, transiently amplifying cells and terminally differentiated cells. These were pooled into three groups according to the stage of differentiation for mRNA microarray analysis. Targets identified were validated using uncultured primary tissue (n=12), functional models of prostate cancer and a tissue microarray consisting of benign (n=42) and malignant prostate (n=223). RESULTS: A deficiency in class 1 UDP glucuronosyltransferase (UGT) enzymes (UGT1A) was identified in prostate SCs, which are involved in androgen catabolism. Class 1 UGT enzyme expression was also downregulated in cancer SCs and during progression to metastatic castration-resistant prostate cancer (CRPC). Reduction of UGT1A expression in vitro was seen to improve cell survival and increase androgen receptor (AR) activity, as shown by upregulation of prostate-specific antigen expression. INTERPRETATION: Inactivation of intracellular androgen catabolism represents a novel mechanism to maintain AR activity during CRPC.

Pleiotropic actions of miR-21 highlight the critical role of deregulated stromal microRNAs during colorectal cancer progression.

The oncogene microRNA-21 (miRNA; miR-21) is overexpressed in most solid organ tumours; however, a recent examination of stage II colorectal cancer (CRC) specimens suggests this may be a stromal phenomenon and not only a feature of cancer cells. In vitro and in vivo studies show that miR-21 has potent pro-metastatic effects in various malignant carcinoma cell lines. The tumour microenvironment has also been identified as a key actor during the metastatic cascade; however to date the significance of deregulated miR-21 expression within the cancer-associated stroma has not been examined. In the present study, a quantitative RT-PCR-based analysis of laser microdissected tissue confirmed that miR-21 expression is associated with a four-fold mean increase in CRC stroma compared with normal tissue. In situ hybridisation using locked nucleic acid probes localised miR-21 expression predominantly to fibroblasts within tumour-associated stroma. To study the molecular and biological impact of deregulated stromal miR-21 in CRC, stable ectopic expression was induced in immortalised fibroblasts. This resulted in upregulated α-smooth muscle actin expression implying miR-21 overexpression is driving the fibroblast-to-myofibroblast transdifferentiation. Conditioned medium from miR-21-overexpressing fibroblasts protected CRC cells from oxaliplatin-induced apoptosis and increased their proliferative capacity. 3D organotypic co-cultures containing fibroblasts and CRC cells revealed that ectopic stromal miR-21 expression was associated with increased epithelial invasiveness. Reversion-inducing cysteine-rich protein with kazal motifs, an inhibitor of matrix-remodelling enzyme MMP2, was significantly downregulated by ectopic miR-21 in established and primary colorectal fibroblasts with a reciprocal rise in MMP2 activity. Inhibition of MMP2 abrogated the invasion-promoting effects of ectopic miR-21. This data, which characterises a novel pro-metastatic mechanism mediated by miR-21 in the CRC stroma, highlights the importance of miRNA deregulation within the tumour microenvironment and identifies a potential application for stromal miRNAs as biomarkers in cancer.

mRNA expression profiling of phyllodes tumours of the breast: identification of genes important in the development of borderline and malignant phyllodes tumours.

The aim of this study was to identify genes involved in the development of borderline and malignant phyllodes tumours of the breast (PTs). Expression profiling of 23 PTs (12 benign, 11 borderline/malignant) was performed using Affymetrix U133A GeneChips. mRNA expression in the borderline/malignant PTs was compared to the benign PTs. A group of 162 genes was over-expressed in the borderline/malignant group with a fold change > 2 and FDR < 0.1. Four of these genes were chosen for further investigation: PAX3, SIX1, TGFB2 and HMGA2. Over-expression was validated in a separate set of formalin-fixed, paraffin-embedded (FFPE) tumours, using either in situ hybridization or immunohistochemistry. This confirmed that expression of PAX3, SIX1, TGFB2 and HMGA2 in the stromal component of PTs was associated with the borderline/malignant phenotypes (p = 8.7 x 10(-5), p = 0.05, p = 0.009, p = 0.003, respectively; Fisher's exact test). The functional consequences of down-regulating these genes were studied using siRNA in short-term cultures and cell lines established from PTs. mRNA 'knock-down' of PAX3 resulted in significantly decreased cell proliferation in both a malignant and a borderline PT cell culture. mRNA 'knock-down' of SIX1 and HMGA2 resulted in decreased cell proliferation only in the malignant PT cell line, and 'knock-down' of TGFB2 resulted in decreased cell proliferation only in the borderline PT cell culture. This study shows that these four genes are involved in the development of borderline/malignant PTs. SIX1 over-expression was most marked in the highly malignant PTs, with particularly high expression in one case of metastatic PT. PAX3, TGFB2 and HMGA2 were expressed predominantly in borderline/malignant PTs, but showed some expression in benign tumours; they may be important in the transition from the benign to borderline/malignant phenotype.

The atypical Rho GTPase RhoBTB2 is required for expression of the chemokine CXCL14 in normal and cancerous epithelial cells.

The Rho family of small GTPases control cell migration, cell invasion and cell cycle. Many of these processes are perturbed in cancer and several family members show altered expression in a number of tumor types. RhoBTB2/DBC2 is an atypical member of this family of signaling proteins, containing two BTB domains in addition to its conserved Rho GTPase domain. RhoBTB2 is mutated, deleted or silenced in a large percentage of breast and lung cancers; however, the functional consequences of this loss are unclear. Here we use RNA interference in primary human epithelial cells to mimic the loss of RhoBTB2 seen in cancer cells. Through microarray analysis of global gene expression, we show that loss of RhoBTB2 results in downregulation of CXCL14-a chemokine that controls leukocyte migration and angiogenesis, and whose expression is lost through unknown mechanisms in a wide range of epithelial cancers. Loss of RhoBTB2 expression correlates with loss of CXCL14 secretion by head and neck squamous cell carcinoma cell lines, whereas reintroduction of RhoBTB2 restores CXCL14 secretion. Our studies identify CXCL14 as a gene target of RhoBTB2 and support downregulation of CXCL14 as a functional outcome of RhoBTB2 loss in cancer.

A comprehensive genetic profile of phyllodes tumours of the breast detects important mutations, intra-tumoral genetic heterogeneity and new genetic changes on recurrence.

The aims of this study were to identify genetic changes associated with malignant progression of the fibroepithelial neoplasms, phyllodes tumours of the breast (PTs), and to ascertain whether genetic progression occurs when PTs recur locally. A further aim was to assess whether the genetic data support the classification of these tumours into three subtypes, benign, borderline and malignant. 126 PTs (37 benign, 41 borderline, 48 malignant) were analysed by either array-CGH or the Illumina Goldengate assay. The large-scale genetic changes associated with malignant/borderline phenotypes were +1q, +5p, +7, +8, -6, -9p, -10p and -13. Cluster analysis of the array-CGH data supported the division of malignant and borderline PTs into two separate groups, one comprising almost all malignant lesions and the other, benign and borderline tumours. Interstitial deletions of 9p21 that involved the p16INK4a locus were present in many malignant/borderline PTs, and some of these appeared to cause homozygous loss. Loss of expression of p16INK4a was found frequently and this was associated with 9p deletion; we also identified one p16INK4a mutation and evidence of methylation of p16INK4a in malignant PTs. Our evidence shows that inactivation of this gene is important in the development of malignant PTs. In selected PTs, multiple areas of stroma were isolated and analysed separately by array-CGH. We found considerable intra-tumoral genetic heterogeneity. Analysis of paired primary and recurrent tumours showed that recurrent tumours often acquired new genetic changes; in particular, benign tumours tended to acquire changes characteristic of the malignant/borderline phenotype. We believe it likely that unfavourable sub-clones not easily identified by histology account for the unpredictable clinical behaviour of these tumours.