Transcriptional Regulation of the Platelet-Derived Growth Factor B-receptor by p53 Family Members

Aims: The platelet-derived growth factor β-receptor (PDGFRB) is critically involved in embryonic development and has a role in many diseases. In cells, signaling through PDGFRB affects growth, migration and death. The role of PDGFRB in these crucial processes necessitates strict regulation and therefore it is regulated in many ways, including transcription, dephosphorylation, internalization, and degradation. Of these, transcriptional regulation is the least studied. Previously, PDGFRB transcription has been shown to be under the control of the transcription factors nuclear factor y (NF-Y), specificity protein 1 (Sp1) and the p53 family member p73. In the present thesis we investigated the role of p53 family members and their mechanisms for transcriptional regulation of PDGFRB. Results: In search for the mechanism behind p73α-mediated repression of the PDGFRB, we found that p73α competed with histone acetyltransferases for binding to NF-Y. The recruitment of p73α and ∆Np73 to the PDGFRB promoter corresponded with PDGFRB expres¬sion. In repression of the PDGFRB promoter, p73 was recruited with the co-repressor HDAC1. Binding of ∆Np73 and the co-activator p300, on the other hand, corresponded to PDGFRB promoter induction. Overexpression of the p53 interacting viral large T antigen (LT) in NIH3T3 fibroblasts resulted in repressed PDGFRB promoter activity and decreased expression of PDGFRB protein and mRNA. The same type of overexpression in c-Myc–/– HO15.19 fibroblasts, Rb–/–NIH3T3, and pRb- and p53-lacking osteosarcoma, Saos2 did not repress PDGFRB promoter activity, showing the importance of these molecules for LT-mediated repression of PDGFRB. In order to identify the role of p53, we overexpressed p53 in mouse embryonic fibroblasts (MEF), p53–/– MEF, and Saos2, which induced repression of PDGFRB promoter activity and decreased mRNA and pro¬tein expression. Endogenous p53 activated by mitomycin treatment also downregulated PDGFRB expres¬sion. Experiments showed that p53 could bind the PDGFRB promoter region surrounding the CCAAT-motif. Upon p53 induction, when PDGFRB expression was repressed, p53 and HDAC1 bound the PDGFRB promoter and the co-activator p300 was dismissed. The role of ∆Np73 in PDGFRB expression was investigated using the neuroblastoma cell line IMR-32 which had dysregulated PDGFRB expression and SH-SY5Y which had regulated expression. Silencing of ∆Np73 repressed PDGFRB promoter activity and protein expression in IMR-32 but not in SH-SY5Y and ∆Np73 was constitutively bound to the PDGFRB promoter only in IMR-32. Treatment with the anticancer drug cisplatin decreased PDGFRB protein, mRNA and promoter activity in both cell lines. In IMR-32, cisplatin was found to dismiss ∆Np73 and p300 from the PDGFRB promoter and recruit HDAC4. Conclusions: Results presented in this thesis suggest a role for p53 family members in downregulation of PDGFRB expression upon growth stimulation or in response to DNA damage. In addition, we demonstrated that ∆Np73 have a role in dysregulated PDGFRB expression. Also, we propose a potential for tyrosine kinase inhibitors in the treatment of neuroblastoma with high ∆Np73 and PDGFR expression

Blood-based liquid biopsies for prostate cancer: clinical opportunities and challenges

Liquid biopsy has been established as a powerful, minimally invasive, tool to detect clinically actionable aberrations across numerous cancer types in real-time. With the development of new therapeutic agents in prostate cancer (PC) including DNA repair targeted therapies, this is especially attractive. However, there is unclarity on how best to screen for PC, improve risk stratification and ultimately how to treat advanced disease. Therefore, there is an urgent need to develop better biomarkers to help guide oncologists’ decisions in these settings. Circulating tumour cells (CTCs), exosomes and cell-free DNA/RNA (cfDNA/cfRNA) analysis, including epigenetic features such as methylation, have all shown potential in prognostication, treatment response assessment and detection of emerging mechanisms of resistance. However, there are still challenges to overcome prior to implementing liquid biopsies in routine clinical practice such as preanalytical considerations including blood collection and storage, the cost of CTC isolation and enrichment, low-circulating tumour content as a limitation for genomic analysis and how to better interpret the sequencing data generated. In this review, we describe an overview of the up-to-date clinical opportunities in the management of PC through blood-based liquid biopsies and the next steps for its implementation in personalised treatment guidance

Plasma Androgen Receptor and Docetaxel for Metastatic Castration-resistant Prostate Cancer

Càncer de pròstata; Receptor d'andrògens; DocetaxelCáncer de próstata; Receptor de andrógenos; DocetaxelProstate cancer; Androgen receptor; DocetaxelPlasma androgen receptor (AR) gain identifies metastatic castration-resistant prostate cancer (mCRPC) patients with worse outcome on abiraterone/enzalutamide, but its relevance in the context of taxane chemotherapy is unknown. We aimed to evaluate whether docetaxel is active regardless of plasma AR and to perform an exploratory analysis to compare docetaxel with abiraterone/enzalutamide. This multi-institutional study was a pooled analysis of AR status, determined by droplet digital polymerase chain reaction, on pretreatment plasma samples. We evaluated associations between plasma AR and overall/progression-free survival (OS/PFS) and prostate-specific antigen (PSA) response rate in 163 docetaxel-treated patients. OS was significantly shorter in case of AR gain (hazard ratio [HR]=1.61, 95% confidence interval [CI]=1.08-2.39, p=0.018), but not PFS (HR=1.04, 95% CI 0.74-1.46, p=0.8) or PSA response (odds ratio=1.14, 95% CI=0.65-1.99, p=0.7). We investigated the interaction between plasma AR and treatment type after incorporating updated data from our prior study of 73 chemotherapy-naïve, abiraterone/enzalutamide-treated patients, with data from 115 first-line docetaxel patients. In an exploratory analysis of mCRPC patients receiving first-line therapies, a significant interaction was observed between plasma AR and docetaxel versus abiraterone/enzalutamide for OS (HR=0.16, 95% CI=0.06-0.46, p<0.001) and PFS (HR=0.31, 95% CI=0.12-0.80, p=0.02). Specifically, we reported a significant difference for OS favoring abiraterone/enzalutamide for AR-normal patients (HR=1.93, 95% CI=1.19-3.12, p=0.008) and a suggestion favoring docetaxel for AR-gained patients (HR=0.53, 95% CI=0.24-1.16, p=0.11). These data suggest that AR-normal patients should receive abiraterone/enzalutamide and AR-gained could benefit from docetaxel. This treatment selection merits prospective evaluation in a randomized trial. PATIENT SUMMARY: We investigated whether plasma androgen receptor (AR) predicted outcome in metastatic castration-resistant prostate cancer (mCRPC) patients treated with docetaxel, and we performed an exploratory analysis in patients treated with docetaxel or AR-directed drugs as first-line mCRPC therapy. We showed that plasma AR normal favored hormonal treatment, whilst plasma AR-gained patients may have had a longer response to docetaxel, suggesting that plasma AR status could be a useful treatment selection biomarker.Funding/Support and role of the sponsor: V. Conteduca was funded by a European Society of Medical Oncology Translational Clinical Research Fellowship. A. Jayaram is supported by a grant from the Medical Research Council (MR/P002072/1). G. Attard is supported by a Cancer Research UK Advanced Clinician Scientist Grant (A22744). This work was funded in part by Prostate Cancer UK (PG12-49), the “Instituto de Salud Carlos III” (ISCII) PI16/01565 grant. E. Gonzalez-Billalabeitia was funded by a grant from the “Instituto de Salud Carlos III” (ISCIII) PI15/01499. N. RomeroLaorden was funded by a grant from the “Instituto de Salud Carlos III” (CM14-00200). E. Castro is supported by a Prostate Cancer Foundation Young Investigator Award (2017). E. Castro and D. Olmos are supported by grants from the Ministerio de Economía, Industria y Competitividad (JCI-2014-19129 to E.C., RYC-2015-18625 to D.O.). B. Mellado and M. Marin-Aguilera work were supported by the Instituto de Salud Carlos IIISubdirección General de Evaluación y Fomento de la Investigación (PI12/ 01226 and PI15/676) and co-funded by the European Regional Development Fund. Funding from CERCA Programme/Generalitat de Catalunya is gratefully acknowledged. During the conduct of the study, E. Castro was supported by a grant from the Ministerio de Educación, Cultura y Deportes (CAS17/00182). The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding authors had full access to all data and had the final responsibility for the decision to submit for publication

Allele-informed copy number evaluation of plasma DNA samples from metastatic prostate cancer patients: the PCF_SELECT consortium assay.

Sequencing of cell-free DNA (cfDNA) in cancer patients' plasma offers a minimally-invasive solution to detect tumor cell genomic alterations to aid real-time clinical decision-making. The reliability of copy number detection decreases at lower cfDNA tumor fractions, limiting utility at earlier stages of the disease. To test a novel strategy for detection of allelic imbalance, we developed a prostate cancer bespoke assay, PCF_SELECT, that includes an innovative sequencing panel covering ∼25 000 high minor allele frequency SNPs and tailored analytical solutions to enable allele-informed evaluation. First, we assessed it on plasma samples from 50 advanced prostate cancer patients. We then confirmed improved detection of genomic alterations in samples with <10% tumor fractions when compared against an independent assay. Finally, we applied PCF_SELECT to serial plasma samples intensively collected from three patients previously characterized as harboring alterations involving DNA repair genes and consequently offered PARP inhibition. We identified more extensive pan-genome allelic imbalance than previously recognized in prostate cancer. We confirmed high sensitivity detection of BRCA2 allelic imbalance with decreasing tumor fractions resultant from treatment and identified complex ATM genomic states that may be incongruent with protein losses. Overall, we present a framework for sensitive detection of allele-specific copy number changes in cfDNA

Allele-informed copy number evaluation of plasma DNA samples from metastatic prostate cancer patients: the PCF_SELECT consortium assay

Sequencing of cell-free DNA (cfDNA) in cancer patients' plasma offers a minimally-invasive solution to detect tumor cell genomic alterations to aid real-time clinical decision-making. The reliability of copy number detection decreases at lower cfDNA tumor fractions, limiting utility at earlier stages of the disease. To test a novel strategy for detection of allelic imbalance, we developed a prostate cancer bespoke assay, PCF_SELECT, that includes an innovative sequencing panel covering ∼25 000 high minor allele frequency SNPs and tailored analytical solutions to enable allele-informed evaluation. First, we assessed it on plasma samples from 50 advanced prostate cancer patients. We then confirmed improved detection of genomic alterations in samples with <10% tumor fractions when compared against an independent assay. Finally, we applied PCF_SELECT to serial plasma samples intensively collected from three patients previously characterized as harboring alterations involving DNA repair genes and consequently offered PARP inhibition. We identified more extensive pan-genome allelic imbalance than previously recognized in prostate cancer. We confirmed high sensitivity detection of BRCA2 allelic imbalance with decreasing tumor fractions resultant from treatment and identified complex ATM genomic states that may be incongruent with protein losses. Overall, we present a framework for sensitive detection of allele-specific copy number changes in cfDNA

Copy number architectures define treatment-mediated selection of lethal prostate cancer clones

Despite initial responses to hormone treatment, metastatic prostate cancer invariably evolves to a lethal state. To characterize the intra-patient evolutionary relationships of metastases that evade treatment, we perform genome-wide copy number profiling and bespoke approaches targeting the androgen receptor (AR) on 167 metastatic regions from 11 organs harvested post-mortem from 10 men who died from prostate cancer. We identify diverse and patient-unique alterations clustering around the AR in metastases from every patient with evidence of independent acquisition of related genomic changes within an individual and, in some patients, the co-existence of AR-neutral clones. Using the genomic boundaries of pan-autosome copy number changes, we confirm a common clone of origin across metastases and diagnostic biopsies, and identified in individual patients, clusters of metastases occupied by dominant clones with diverged autosomal copy number alterations. These autosome-defined clusters are characterized by cluster-specific AR gene architectures, and in two index cases are topologically more congruent than by chance (p-values 3.07 × 10-8 and 6.4 × 10-4). Integration with anatomical sites suggests patterns of spread and points of genomic divergence. Here, we show that copy number boundaries identify treatment-selected clones with putatively distinct lethal trajectories

Accumulation of copy number alterations and clinical progression across advanced prostate cancer

Background: Genomic copy number alterations commonly occur in prostate cancer and are one measure of genomic instability. The clinical implication of copy number change in advanced prostate cancer, which defines a wide spectrum of disease from high-risk localised to metastatic, is unknown. Methods: We performed copy number profiling on 688 tumour regions from 300 patients, who presented with advanced prostate cancer prior to the start of long-term androgen deprivation therapy (ADT), in the control arm of the prospective randomised STAMPEDE trial. Patients were categorised into metastatic states as follows; high-risk non-metastatic with or without local lymph node involvement, or metastatic low/high volume. We followed up patients for a median of 7 years. Univariable and multivariable Cox survival models were fitted to estimate the association between the burden of copy number alteration as a continuous variable and the hazard of death or disease progression. Results: The burden of copy number alterations positively associated with radiologically evident distant metastases at diagnosis (P=0.00006) and showed a non-linear relationship with clinical outcome on univariable and multivariable analysis, characterised by a sharp increase in the relative risk of progression (P=0.003) and death (P=0.045) for each unit increase, stabilising into more modest increases with higher copy number burdens. This association between copy number burden and outcome was similar in each metastatic state. Copy number loss occurred significantly more frequently than gain at the lowest copy number burden quartile (q=4.1 × 10−6). Loss of segments in chromosome 5q21-22 and gains at 8q21-24, respectively including CHD1 and cMYC occurred more frequently in cases with higher copy number alteration (for either region: Kolmogorov–Smirnov distance, 0.5; adjusted P<0.0001). Copy number alterations showed variability across tumour regions in the same prostate. This variance associated with increased risk of distant metastases (Kruskal-Wallis test P=0.037). Conclusions: Copy number alteration in advanced prostate cancer associates with increased risk of metastases at diagnosis. Accumulation of a limited number of copy number alterations associates with most of the increased risk of disease progression and death. The increased likelihood of involvement of specific segments in high copy number alteration burden cancers may suggest an order underlying the accumulation of copy number changes

A correlative biomarker study and integrative prognostic model in chemotherapy-naïve metastatic castration-resistant prostate cancer treated with enzalutamide

There is a considerable need to incorporate biomarkers of resistance to new antiandrogen agents in the management of castration-resistant prostate cancer (CRPC). We conducted a phase II trial of enzalutamide in first-line chemo-naïve asymptomatic or minimally symptomatic mCRPC and analyzed the prognostic value of TMPRSS2-ERG and other biomarkers, including circulating tumor cells (CTCs), androgen receptor splice variant (AR-V7) in CTCs and plasma Androgen Receptor copy number gain (AR-gain). These biomarkers were correlated with treatment response and survival outcomes and developed a clinical-molecular prognostic model using penalized cox-proportional hazard model. This model was validated in an independent cohort. Ninety-eight patients were included. TMPRSS2-ERG fusion gene was detected in 32 patients with no differences observed in efficacy outcomes. CTC detection was associated with worse outcome and AR-V7 in CTCs was associated with increased rate of progression as best response. Plasma AR gain was strongly associated with an adverse outcome, with worse median prostate specific antigen (PSA)-PFS (4.2 vs. 14.7 m; p < 0.0001), rad-PFS (4.5 vs. 27.6 m; p < 0.0001), and OS (12.7 vs. 38.1 m; p < 0.0001). The clinical prognostic model developed in PREVAIL was validated (C-Index 0.70) and the addition of plasma AR (C-Index 0.79; p < 0.001) increased its prognostic ability. We generated a parsimonious model including alkaline phosphatase (ALP); PSA and AR gain (C-index 0.78) that was validated in an independent cohort. TMPRSS2-ERG detection did not correlate with differential activity of enzalutamide in first-line mCRPC. However, we observed that CTCs and plasma AR gain were the most relevant biomarkers

Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS

The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin