Christopher E. Barbieri

Loss of p63 leads to increased cell migration and up-regulation of genes involved in invasion and metastasis.

p63, a homologue of the tumor suppressor p53, is critical for the development and maintenance of squamous epithelia. p63 is specifically expressed in the basal layers of stratified epithelial tissues and is considered a specific marker for cells of this type. The role of p63 in tumorigenesis remains poorly defined. Numerous studies have highlighted the oncogenic potential of the predominant p63 isoform DeltaNp63alpha; however, data suggest that other p63 proteins can act as tumor suppressors or alter the metastatic potential of tumors. DeltaNp63alpha can act as a transcriptional repressor, but the link between the transcriptional functions of p63 and its biological role is still unclear. In this study, we used a loss-of-function approach to investigate the transcriptional programs controlled by p63. Disruption of p63 in squamous cell lines resulted in down-regulation of transcripts specifically expressed in squamous tissues and a significant alteration of keratinocyte differentiation. Interestingly, we found that disruption of p63 led to up-regulation of markers of nonepithelial tissues (mesenchyme and neural tissue) in both primary and immortalized squamous cells. Many of these up-regulated genes are associated with increased capacity for invasion and metastasis in tumors. Furthermore, loss of p63 expression was accompanied by a shift toward mesenchymal morphology and an increase in motility in primary keratinocytes and squamous cell lines. We conclude that loss of endogenous p63 expression results in up-regulation of genes associated with invasion and metastasis, and predisposes to a loss of epithelial and acquisition of mesenchymal characteristics. These findings have implications for the role of p63 in both development and tumorigenesis.

The Mutational Landscape of Prostate Cancer

CONTEXT Prostate cancer (PCa) is a clinically heterogeneous disease with marked variability in patient outcomes. Molecular characterization has revealed striking mutational heterogeneity that may underlie the variable clinical course of the disease. OBJECTIVE In this review, we discuss the common genomic alterations that form the molecular basis of PCa, their functional significance, and the potential to translate this knowledge into patient care. EVIDENCE ACQUISITION We reviewed the relevant literature, with a particular focus on recent studies on somatic alterations in PCa. EVIDENCE SYNTHESIS Advances in sequencing technology have resulted in an explosion of data regarding the mutational events underlying the development and progression of PCa. Heterogeneity is the norm; few abnormalities in specific genes are highly recurrent, but alterations in certain signaling pathways do predominate. These alterations include those in pathways known to affect tumorigenesis in a wide spectrum of tissues, such as the phosphoinositide 3-kinase/phosphatase and tensin homolog/Akt pathway, cell cycle regulation, and chromatin regulation. Alterations more specific to PCa are also observed, particularly gene fusions of ETS transcription factors and alterations in androgen signaling. Mounting data suggest that PCa can be subdivided based on a molecular profile of genetic alterations. CONCLUSIONS Major advances have been made in cataloging the genomic alterations in PCa and understanding the molecular mechanisms underlying the disease. These findings raise the possibility that PCa could soon transition from being a poorly understood, heterogeneous disease with a variable clinical course to being a collection of homogenous subtypes identifiable by molecular criteria, associated with distinct risk profiles, and perhaps amenable to specific management strategies or targeted therapies.

Prostate cancer-associated mutations in speckle-type POZ protein (SPOP) regulate steroid receptor coactivator 3 protein turnover

The p160 steroid receptor coactivators (SRCs) SRC-1, SRC-2 [nuclear receptor coactivator (NCOA)2], and SRC-3 [amplified in breast cancer 1 (AIB1)/NCOA3] are key pleiotropic “master regulators” of transcription factor activity necessary for cancer cell proliferation, survival, metabolism, and metastasis. SRC overexpression and overactivation occur in numerous human cancers and are associated with poor clinical outcomes and resistance to therapy. In prostate cancer (PC), the p160 SRCs play critical roles in androgen receptor transcriptional activity, cell proliferation, and resistance to androgen deprivation therapy. We recently demonstrated that the E3 ubiquitin ligase adaptor speckle-type poxvirus and zinc finger (POZ) domain protein (SPOP) interacts directly with SRC-3 and promotes its cullin 3-dependent ubiquitination and proteolysis in breast cancer, thus functioning as a potential tumor suppressor. Interestingly, somatic heterozygous missense mutations in the SPOP substrate-binding cleft recently were identified in up to 15% of human PCs (making SPOP the gene most commonly affected by nonsynonymous point mutations in PC), but their contribution to PC pathophysiology remains unknown. We now report that PC-associated SPOP mutants cannot interact with SRC-3 protein or promote its ubiquitination and degradation. Our data suggest that wild-type SPOP plays a critical tumor suppressor role in PC cells, promoting the turnover of SRC-3 protein and suppressing androgen receptor transcriptional activity. This tumor suppressor effect is abrogated by the PC-associated SPOP mutations. These studies provide a possible explanation for the role of SPOP mutations in PC, and highlight the potential of SRC-3 as a therapeutic target in PC.

ΔNp63α Expression Is Regulated by the Phosphoinositide 3-Kinase Pathway

p63 is a homologue of p53 that functions to maintain progenitor cell populations in stratified epithelia. ΔNp63α is overexpressed in epithelial cancers and has been shown to have oncogenic properties. We have previously reported that inhibition of epidermal growth factor receptor signaling results in a decrease in ΔNp63α expression. Here, we demonstrate ΔNp63α is a target of the phosphoinositide-3-kinase (PI3K) pathway downstream of the epidermal growth factor receptor. Treatment of keratinocytes with epidermal growth factor results in an increase in ΔNp63α expression at the mRNA level, which is abrogated by inhibition of PI3K but not mitogen-activated protein kinase signaling. Small interfering RNA-mediated knockdown of the p110β catalytic subunit of PI3K results in a decrease in ΔNp63α protein levels in keratinocytes. The results presented herein suggest that regulation of ΔNp63α expression by the PI3K pathway plays a critical role in the survival and proliferative capacity of squamous epithelia.

Ultraviolet radiation induces phosphorylation and ubiquitin-mediated degradation of ΔNp63α

ΔNP63α, a homologue of the tumor suppressor p53, acts as a transcriptional repressor with dominant negative effects towards p53. Additionally, ΔNP63α is overexpressed in a number of squamous cell carcinomas, suggesting a potential role in oncogenesis. However, the mechanisms regulating p63 have yet to be elucidated. The goal of the current study was to determine the effect of various genotoxic stresses on ΔNP63α posttranslational modification and stability in normal and transformed squamous epithelial cells. We found that ΔNP63α protein levels decreased after ultraviolet radiation and paclitaxel treatment of both normal and transformed cells. After UV and paclitaxel treatment, ΔNP63αphosphorylation was significantly modulated. Additionally, ΔNP63α protein levels were regulated in a proteasome-dependent manner in control and UV treated cells with increased ?Np63? ubiquitination after UV treatment or proteasome inhibition. Our studies provide insight to a mechanism for ΔNP63α regulation during normal cell proliferation and, in particular, after stress. Further, the inverse regulation of p53 and ΔNP63α protein levels after cell stress through opposing regulation of proteasome-mediated degradation may allow for rapid transcriptional changes of specific target genes that are consistent with the roles of these family members in tumor suppression and cell growth.

TMPRSS2:ERG Gene Fusion Predicts Subsequent Detection of Prostate Cancer in Patients With High-Grade Prostatic Intraepithelial Neoplasia

PURPOSE High-grade prostatic intraepithelial neoplasia (HGPIN) is considered a precursor lesion of prostate cancer (PCa). The predictive value of ERG gene fusion in HGPIN for PCa was interrogated as a post hoc analysis in the context of a randomized clinical trial. PATIENTS AND METHODS The GTx Protocol G300104 randomly assigned 1,590 men with biopsy-diagnosed HGPIN to receive toremifene or placebo for 3 years or until a diagnosis of PCa was made on prostate biopsy. As part of this phase III clinical trial, a central pathologist evaluated biopsies of patients with isolated HGPIN at baseline and 12, 24, and 36 months of follow-up. ERG immunohistochemistry was performed on biopsies from 461 patients and evaluated for protein overexpression. RESULTS ERG expression was detected in 11.1% of patients (51 of 461 patients) with isolated HGPIN. In the first year and during the 3-year clinical trial, 14.7% and 36.9% of 461 patients were diagnosed with PCa, respectively. Patients with ERG expression were more likely to develop PCa, with 27 (53%) of 51 ERG-positive and 143 (35%) of 410 ERG-negative patients experiencing progression to PCa (P = .014, Fishers exact test). ERG expression was not associated with age, baseline PSA, Gleason score, or tumor volume. CONCLUSION This study underscores the necessity of more stringent follow-up for men with HGPIN that is also positive for ERG overexpression. Clinicians should consider molecular characterization of HGPIN as a means to improve risk stratification.

The prostate cancer genome: Perspectives and potential

OBJECTIVES Prostate cancer has a variable clinical course, and molecular characterization has revealed striking mutational heterogeneity that may underlie the unpredictable clinical behavior of the disease. Advances in technology have resulted in a rapid expansion of our understanding of the genomic events responsible for the development and progression of prostate cancer. In this review, we discuss the genomic alterations underlying prostate cancer, and potential to utilize this knowledge for diagnostic and prognostic benefit. METHODS AND MATERIALS We reviewed the relevant literature, with a focus on recent studies on somatic alterations in prostate cancer. RESULTS Pathways known to affect tumorigenesis across a wide spectrum of tissues are dysregulated, such as the PI3K pathway, cell cycle control, and chromatin regulation. Lesions more specific to prostate cancer include alterations in androgen signaling, gene fusions of ETS transcription factors, and mutations in SPOP. Accumulating data suggests that prostate cancer can be subdivided based on a molecular profile of these genetic alterations. CONCLUSIONS These findings raise the possibility that prostate cancer could transition from a poorly understood, heterogeneous disease with a variable clinical course to a collection of homogenous subtypes, identifiable by molecular criteria, associated with distinct risk profiles, and perhaps amenable to specific management strategies or targeted therapies.

Evidence for Molecular Differences in Prostate Cancer between African American and Caucasian Men

Purpose: The aim of this study was to compare the frequency of ERG rearrangement, PTEN deletion, SPINK1 overexpression, and SPOP mutation in prostate cancer in African American and Caucasian men. Experimental design: Dominant tumor nodules from radical prostatectomy specimens of 105 African American men (AAM) were compared with 113 dominant nodules from Caucasian men (CaM). Clinical and pathologic characteristics of the two groups were similar. SPINK1 overexpression was evaluated by immunohistochemistry, ERG rearrangement and PTEN deletion by FISH, and SPOP mutation by Sanger sequencing. Results: ERG rearrangement was identified in 48 of 113 tumors (42.5%) in CaM and 29 of 105 tumors (27.6%) in AAM (P = 0.024). PTEN deletion was seen in 19 of 96 tumors (19.8%) in CaM and 7 of 101 tumors (6.9%) in AAM (P = 0.011). SPINK1 overexpression was present in 9 of 110 tumors (8.2%) in CaM and 25 of 105 tumors (23.4%) in AAM (P = 0.002). SPOP mutation was identified in 8 of 78 (10.3%) tumors in CaM and 4 of 88 (4.5%) tumors in AAM (P = 0.230). When adjusted for age, body mass index, Gleason score, and pathologic stage, ERG rearrangement and SPINK1 overexpression remain significantly different (P = 0.018 and P = 0.008, respectively), and differences in PTEN deletion and SPOP mutation approach significance (P = 0.061 and P = 0.087, respectively). Conclusions: Significant molecular differences exist between prostate cancers in AAM and CaM. SPINK1 overexpression, an alteration associated with more aggressive prostate cancers, was more frequent in AAM, whereas ERG rearrangement and PTEN deletion were less frequent in this cohort. Further investigation is warranted to determine whether these molecular differences explain some of the disparity in incidence and mortality between these two ethnic groups. Clin Cancer Res; 20(18); 4925–34. ©2014 AACR.

Unraveling the clonal hierarchy of somatic genomic aberrations

Defining the chronology of molecular alterations may identify milestones in carcinogenesis. To unravel the temporal evolution of aberrations from clinical tumors, we developed CLONET, which upon estimation of tumor admixture and ploidy infers the clonal hierarchy of genomic aberrations. Comparative analysis across 100 sequenced genomes from prostate, melanoma, and lung cancers established diverse evolutionary hierarchies, demonstrating the early disruption of tumor-specific pathways. The analyses highlight the diversity of clonal evolution within and across tumor types that might be informative for risk stratification and patient selection for targeted therapies. CLONET addresses heterogeneous clinical samples seen in the setting of precision medicine.

DNA Repair in Prostate Cancer: Biology and Clinical Implications

CONTEXT For more precise, personalized care in prostate cancer (PC), a new classification based on molecular features relevant for prognostication and treatment stratification is needed. Genomic aberrations in the DNA damage repair pathway are common in PC, particularly in late-stage disease, and may be relevant for treatment stratification. OBJECTIVE To review current knowledge on the prevalence and clinical significance of aberrations in DNA repair genes in PC, particularly in metastatic disease. EVIDENCE ACQUISITION A literature search up to July 2016 was conducted, including clinical trials and preclinical basic research studies. Keywords included DNA repair, BRCA, ATM, CRPC, prostate cancer, PARP, platinum, predictive biomarkers, and hereditary cancer. EVIDENCE SYNTHESIS We review how the DNA repair pathway is relevant to prostate carcinogenesis and progression. Data on how this may be relevant to hereditary cancer and genetic counseling are included, as well as data from clinical trials of PARP inhibitors and platinum therapeutics in PC. CONCLUSIONS Relevant studies have identified genomic defects in DNA repair in PCs in 20-30% of advanced castration-resistant PC cases, a proportion of which are germline aberrations and heritable. Phase 1/2 clinical trial data, and other supporting clinical data, support the development of PARP inhibitors and DNA-damaging agents in this molecularly defined subgroup of PC following success in other cancer types. These studies may be an opportunity to improve patient care with personalized therapeutic strategies. PATIENT SUMMARY Key literature on how genomic defects in the DNA damage repair pathway are relevant for prostate cancer biology and clinical management is reviewed. Potential implications for future changes in patient care are discussed.