Dysregulation of the PRUNE2/PCA3 genetic axis in human prostate cancer: from experimental discovery to validation in two independent patient cohorts.

Background: We have previously shown that the long non-coding (lnc)RNA (; formerly ) functions as a trans-dominant negative oncogene by targeting the previously unrecognized prostate cancer suppressor gene (a homolog of the gene), thereby forming a functional unit within a unique allelic locus in human cells. Here, we investigated the / regulatory axis from early (tumorigenic) to late (biochemical recurrence) genetic events during human prostate cancer progression.

Methods: The reciprocal and gene expression relationship in paired prostate cancer and adjacent normal prostate was analyzed in two independent retrospective cohorts of clinically annotated cases post-radical prostatectomy: a single-institutional discovery cohort (n=107) and a multi-institutional validation cohort (n=497).

MAPKAPK2 (MK2) inhibition mediates radiation-induced inflammatory cytokine production and tumor growth in head and neck squamous cell carcinoma.

Radiation therapy (RT) is a cornerstone of treatment in the management of head and neck squamous cell carcinomas (HNSCC), yet treatment failure and disease recurrence are common. The p38/MK2 pathway is activated in response to cellular stressors, including radiation, and promotes tumor inflammation in a variety of cancers. We investigated MK2 pathway activation in HNSCC and the interaction of MK2 and RT in vitro and in vivo.

Recommendations for determining HPV status in patients with oropharyngeal cancers under TNM8 guidelines: a two-tier approach.

Background: TNM8 staging for oropharyngeal squamous cell carcinomas (OPSCC) surrogates p16 immunohistochemistry for HPV testing. Patients with p16+ OPSCC may lack HPV aetiology. Here, we evaluate the suitability of TNM8 staging for guiding prognosis in such patients.

ΔNp63γ/SRC/Slug Signaling Axis Promotes Epithelial-to-Mesenchymal Transition in Squamous Cancers.

To investigate the regulation of epithelial-to-mesenchymal transition (EMT) in head and neck squamous cell carcinoma (HNSCC) and its importance in tumor invasion. We use a three-dimensional invasive organotypic raft culture model of human foreskin keratinocytes expressing the E6/E7 genes of the human papilloma virus-16, coupled with bioinformatic and IHC analysis of patient samples to investigate the role played by EMT in invasion and identify effectors and upstream regulatory pathways. We identify SNAI2 (Slug) as a critical effector of EMT-activated downstream of TP63 overexpression in HNSCC.

The IGF axis in HPV associated cancers.

Human papillomaviruses (HPV) infect and replicate in stratified epithelium at cutaneous and mucosal surfaces. The proliferation and maintenance of keratinocytes, the cells which make up this epithelium, are controlled by a number of growth factor receptors such as the keratinocyte growth factor receptor (KGFR, also called fibroblast growth factor receptor 2b (FGFR2b)), the epithelial growth factor receptor (EGFR) and the insulin-like growth factor receptors 1 and 2 (IGF1R and IGF2R). In this review, we will delineate the mutation, gene transcription, translation and processing of the IGF axis within HPV associated cancers.

HPV-Related Oropharynx Cancer in the United Kingdom: An Evolution in the Understanding of Disease Etiology.

A rising incidence of oropharyngeal squamous cell carcinoma (OPSCC) incidence has occurred throughout the developed world, where it has been attributed to an increasing impact of human papillomavirus (HPV) on disease etiology. This report presents the findings of a multicenter cross-sectional retrospective study aimed at determining the proportion of HPV-positive and HPV-negative OPSCC within the United Kingdom. Archival tumor tissue blocks from 1,602 patients previously diagnosed with OPSCC (2002-2011) were collated from 11 centers.

HPV16 Down-Regulates the Insulin-Like Growth Factor Binding Protein 2 to Promote Epithelial Invasion in Organotypic Cultures.

Cervical cancer is a multi-stage disease caused by human papillomaviruses (HPV) infection of cervical epithelial cells, but the mechanisms regulating disease progression are not clearly defined. Using 3-dimensional organotypic cultures, we demonstrate that HPV16 E6 and E7 proteins alter the secretome of primary human keratinocytes resulting in local epithelial invasion. Mechanistically, absence of the IGF-binding protein 2 (IGFBP2) caused increases in IGFI/II signalling and through crosstalk with KGF/FGFR2b/AKT, cell invasion.

p63 drives invasion in keratinocytes expressing HPV16 E6/E7 genes through regulation of Src-FAK signalling.

Using microarray information from oro-pharyngeal data sets and results from primary human foreskin keratinocytes (HFK) expressing Human Papilloma Virus (HPV)-16 E6/E7 proteins, we show that p63 expression regulates signalling molecules which initiate cell migration such as Src and focal adhesion kinase (FAK) and induce invasion in 3D-organotypic rafts; a phenotype that can be reversed by depletion of p63. Knockdown of Src or FAK in the invasive cells restored focal adhesion protein paxillin at cell periphery and impaired the cell migration. In addition, specific inhibition of FAK (PF573228) or Src (dasatinib) activities mitigated invasion and attenuated the expression/activity of matrix metalloproteinase 14 (MMP14), a pivotal MMP in the MMP activation cascade.

IGF-Binding Protein 2 - Oncogene or Tumor Suppressor?

The role of insulin-like growth factor binding protein 2 (IGFBP2) in cancer is unclear. In general, IGFBP2 is considered to be oncogenic and its expression is often observed to be elevated in cancer. However, there are a number of conflicting reports in vitro and in vivo where IGFBP2 acts in a tumor suppressor manner.

Genome-wide characterization reveals complex interplay between TP53 and TP63 in response to genotoxic stress.

In response to genotoxic stress the TP53 tumour suppressor activates target gene expression to induce cell cycle arrest or apoptosis depending on the extent of DNA damage. These canonical activities can be repressed by TP63 in normal stratifying epithelia to maintain proliferative capacity or drive proliferation of squamous cell carcinomas, where TP63 is frequently overexpressed/amplified. Here we use ChIP-sequencing, integrated with microarray analysis, to define the genome-wide interplay between TP53 and TP63 in response to genotoxic stress in normal cells.

Identification of a BRCA1-mRNA splicing complex required for efficient DNA repair and maintenance of genomic stability.

Mutations within BRCA1 predispose carriers to a high risk of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through the assembly of multiple protein complexes involved in DNA repair, cell-cycle arrest, and transcriptional regulation. Here, we report the identification of a DNA damage-induced BRCA1 protein complex containing BCLAF1 and other key components of the mRNA-splicing machinery.

miR-24 and miR-205 expression is dependent on HPV onco-protein expression in keratinocytes.

A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miR-24 and miR-205. We investigated how expression of Human Papilloma Virus Type-16 (HPV16) onco-proteins E6 and E7 affected expression of miR-24 and miR-205 during proliferation and differentiation of HFKs. We show that the induction of both miR-24 and miR-205 observed during differentiation of HFKs is lost in HFKs expressing E6 and E7.

AKT in stromal fibroblasts controls invasion of epithelial cells.

The tumour microenvironment has an important role in cancer progression and recent reports have proposed that stromal AKT is activated and regulates tumourigenesis and invasion. We have shown, by immuno-fluorescent analysis of oro-pharyngeal cancer biopsies, an increase in AKT activity in tumour associated stromal fibroblasts compared to normal stromal fibroblasts. Using organotypic raft co-cultures, we show that activation of stromal AKT can induce the invasion of keratinocytes expressing the HPV type 16 E6 and E7 proteins, in a Keratinocyte Growth Factor (KGF) dependent manner.

Regulation of epithelial differentiation and proliferation by the stroma: a role for the retinoblastoma protein.

Signaling between the epithelium and stromal cells is crucial for growth, differentiation, and repair of the epithelium. Although the retinoblastoma protein (Rb) is known to regulate the growth of keratinocytes in a cell-autonomous manner, here we describe a function of Rb in the stromal compartment. We find that Rb depletion in fibroblasts leads to inhibition of differentiation and enhanced proliferation of the epithelium.

Inactivation of Rb in stromal fibroblasts promotes epithelial cell invasion.

Stromal-derived growth factors are required for normal epithelial growth but are also implicated in tumour progression. We have observed inactivation of the retinoblastoma protein (Rb), through phosphorylation, in cancer-associated fibroblasts in oro-pharyngeal cancer specimens. Rb is well known for its cell-autonomous effects on cancer initiation and progression; however, cell non-autonomous functions of Rb are not well described.

Genome-wide analysis of p63 binding sites identifies AP-2 factors as co-regulators of epidermal differentiation.

The p63 transcription factor (TP63) is critical in development, growth and differentiation of stratifying epithelia. This is highlighted by the severity of congenital abnormalities caused by TP63 mutations in humans, the dramatic phenotypes in knockout mice and de-regulation of TP63 expression in neoplasia altering the tumour suppressive roles of the TP53 family. In order to define the normal role played by TP63 and provide the basis for better understanding how this network is perturbed in disease, we used chromatin immunoprecipitation combined with massively parallel sequencing (ChIP-seq) to identify >7500 high-confidence TP63-binding regions across the entire genome, in primary human neonatal foreskin keratinocytes (HFKs).

Evidence for alteration of EZH2, BMI1, and KDM6A and epigenetic reprogramming in human papillomavirus type 16 E6/E7-expressing keratinocytes.

A number of epigenetic alterations occur in both the virus and host cellular genomes during human papillomavirus (HPV)-associated carcinogenesis, and investigations of such alterations, including changes in chromatin proteins and histone modifications, have the potential to lead to therapeutic epigenetic reversion. We report here that transformed HPV16 E6/E7-expressing primary human foreskin keratinocytes (HFKs) (E6/E7 cells) demonstrate increased expression of the PRC2 methyltransferase EZH2 at both the mRNA and protein levels but do not exhibit the expected increase in trimethylated H3K27 (H3K27me3) compared to normal keratinocytes. In contrast, these cells show a reduction in global H3K27me3 levels in vitro, as well as upregulation of the KDM6A demethylase.

p63 maintains keratinocyte proliferative capacity through regulation of Skp2-p130 levels.

p63 is a master regulator of proliferation and differentiation in stratifying epithelia, and its expression is frequently altered in carcinogenesis. However, its role in maintaining proliferative capacity remains unclear. Here, we demonstrate that hypoproliferation and loss of differentiation in organotypic raft cultures of primary neonatal human foreskin keratinocytes (HFKs) depleted of the α and β isoforms of p63 result from p53-p21-mediated accumulation of retinoblastoma (Rb) family member p130.

The DeltaNp63 proteins are key allies of BRCA1 in the prevention of basal-like breast cancer.

Little is known about the origin of basal-like breast cancers, an aggressive disease that is highly similar to BRCA1-mutant breast cancers. p63 family proteins that are structurally related to the p53 suppressor protein are known to function in stem cell regulation and stratified epithelia development in multiple tissues, and p63 expression may be a marker of basal-like breast cancers. Here we report that ΔNp63 isoforms of p63 are transcriptional targets for positive regulation by BRCA1.

Role of DeltaNp63gamma in epithelial to mesenchymal transition.

Although members of the p63 family of transcription factors are known for their role in the development and differentiation of epithelial surfaces, their function in cancer is less clear. Here, we show that depletion of the ΔNp63α and β isoforms, leaving only ΔNp63γ, results in epithelial to mesenchymal transition (EMT) in the normal breast cell line MCF10A. EMT can be rescued by the expression of the ΔNp63α isoform.

Acetylation of Rb by PCAF is required for nuclear localization and keratinocyte differentiation.

Although the retinoblastoma protein (Rb) functions as a checkpoint in the cell cycle, it also regulates differentiation. It has recently been shown that Rb is acetylated during differentiation; however, the role of this modification has not been identified. Depletion of Rb levels with short hairpin RNA resulted in inhibition of human keratinocyte differentiation, delayed cell cycle exit and allowed cell cycle re-entry.

Compromised spindle assembly checkpoint due to altered expression of Ubch10 and Cdc20 in human papillomavirus type 16 E6- and E7-expressing keratinocytes.

Cells expressing human papillomavirus type 16 (HPV-16) E6 and E7 proteins exhibit deregulation of G2/M genes, allowing bypass of DNA damage arrest signals. Normally, cells with DNA damage that override the G2 damage checkpoint would precociously enter mitosis and ultimately face mitotic catastrophe and apoptotic cell death. However, E6/E7-expressing cells (E6/E7 cells) have the ability to enter and exit mitosis in the presence of DNA damage and continue with the next round of the cell cycle.

MicroRNA 203 expression in keratinocytes is dependent on regulation of p53 levels by E6.

A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miRNA 203 (miR-203), which has previously been shown to play an important role in epithelial cell biology by regulating p63 levels. We investigated how expression of human papillomavirus type 16 (HPV16) oncoproteins E6 and E7 affected miR-203 expression during proliferation and differentiation of HFKs. We demonstrated that miR-203 expression is reduced in HFKs where p53 function is compromised, either by the viral oncoprotein E6 or by knockout of p53 using short hairpin RNAs (p53i).

Human papillomavirus type 16 E6/E7 upregulation of nucleophosmin is important for proliferation and inhibition of differentiation.

The E6 and E7 oncoproteins of high-risk human papillomaviruses (HPVs) are together sufficient to cause cellular transformation. Nucleophosmin (NPM) was identified as a protein with increased levels in two-dimensional (2-D) gel analysis of human foreskin keratinocytes (HFKs) expressing E7 following methylcellulose-induced differentiation. Analysis of NPM expression in E7-expressing cells and E6- and E7-expressing cells in culture and in organotypic rafts confirmed the increased levels observed in 2-D gel analysis.

p300 alters keratinocyte cell growth and differentiation through regulation of p21(Waf1/CIP1).

Background: p300 functions as a transcriptional co-activator to regulate many cellular responses such as cell growth, transformation, development and differentiation. It has been shown to affect the transcriptional activity of p53 which regulates p21(Waf1/CIP1) expression, however, the role of p300 in differentiation remains unclear.

Methodology And Principal Findings: Knockdown of p300 protein with short hairpin RNA (shRNA) molecules delays human neonatal foreskin keratinocyte (HFKs) differentiation.