Transposable Elements Are Co-opted as Oncogenic Regulatory Elements by Lineage-Specific Transcription Factors in Prostate Cancer.

Transposable elements hold regulatory functions that impact cell fate determination by controlling gene expression. However, little is known about the transcriptional machinery engaged at transposable elements in pluripotent and mature versus oncogenic cell states. Through positional analysis over repetitive DNA sequences of H3K27ac chromatin immunoprecipitation sequencing data from 32 normal cell states, we report pluripotent/stem and mature cell state-specific "regulatory transposable elements." Pluripotent/stem elements are binding sites for pluripotency factors (e.g., NANOG, SOX2, OCT4). Mature cell elements are docking sites for lineage-specific transcription factors, including AR and FOXA1 in prostate epithelium. Expanding the analysis to prostate tumors, we identify a subset of regulatory transposable elements shared with pluripotent/stem cells, including Tigger3a. Using chromatin editing technology, we show how such elements promote prostate cancer growth by regulating AR transcriptional activity. Collectively, our results suggest that oncogenesis arises from lineage-specific transcription factors hijacking pluripotent/stem cell regulatory transposable elements. SIGNIFICANCE: We show that oncogenesis relies on co-opting transposable elements from pluripotent stem cells as regulatory elements altering the recruitment of lineage-specific transcription factors. We further discover how co-option is dependent on active chromatin states with important implications for developing treatment options against drivers of oncogenesis across the repetitive DNA. This article is featured in Selected Articles from This Issue, p. 2293.

Measurement of the Drug Sensitivity of Single Prostate Cancer Cells.

The treatment of cancer faces a serious challenge as cancer cells within patients are heterogeneous and frequently resistant to therapeutic drugs. Here, we introduce a technology enabling the assessment of single cancer cells exposed to different drugs. PCa cells were individually sorted in self-seeding microwells, cultured for 24 h, and then exposed to several drugs to induce (R1881) or inhibit (Enzalutamide/Abiraterone) the secretion of a protein (PSA). Cell viability and PSA secretion of each individual prostate cell were monitored over a 3-day period. The PSA protein secreted by each cell was captured on a PVDF membrane through a pore in the bottom of each well. The basal PSA secretion was found to be 6.1 ± 4.5 and 3.7 ± 1.9 pg/cell/day for LNCaP and VCaP, respectively. After exposure to R1881, the PSA secretion increased by ~90% on average and was not altered for ~10% of the cells. PSA production decreased in the majority of cells after exposure to enzalutamide and abiraterone.

Continued androgen signalling inhibition improves cabazitaxel efficacy in prostate cancer.

BACKGROUND: The androgen receptor (AR) pathway is a key driver of neoplastic behaviour in the different stages of metastatic prostate cancer (mPCa). Targeting the AR therefore remains the cornerstone for mPCa treatment. We have previously reported that activation of AR signalling affects taxane chemo-sensitivity in preclinical models of castration resistant PCa (CRPC). Here, we explored the anti-tumour efficacy of the AR targeted inhibitor enzalutamide combined with cabazitaxel. METHODS: We used the AR positive CRPC model PC346C-DCC-K to assess the in vitro and in vivo activity of combining enzalutamide with cabazitaxel. Subsequent validation studies were performed using an enzalutamide resistant VCaP model. To investigate the impact of AR signalling on cabazitaxel activity we used quantitative live-cell imaging of tubulin stabilization and apoptosis related nuclear fragmentation. FINDINGS: Enzalutamide strongly amplified cabazitaxel anti-tumour activity in the patient-derived xenograft models PC346C-DCC-K (median time to humane endpoint 77 versus 48 days, P<0.0001) and VCaP-Enza-B (median time to humane endpoint 80 versus 53 days, P<0.001). Although enzalutamide treatment by itself was ineffective in reducing tumour growth, it significantly suppressed AR signalling in PC346C-DCC-K tumours as shown by AR target gene expression. The addition of enzalutamide enhanced cabazitaxel induced apoptosis as shown by live-cell imaging (P<0.001). INTERPRETATION: Our study demonstrates that cabazitaxel efficacy can be improved by simultaneous blocking of AR signalling by enzalutamide, even if AR targeted treatment no longer affects tumour growth. These findings support clinical studies that combine AR targeted inhibitors with cabazitaxel in CRPC.

Modeling Prostate Cancer Treatment Responses in the Organoid Era: 3D Environment Impacts Drug Testing.

Organoid-based studies have revolutionized in vitro preclinical research and hold great promise for the cancer research field, including prostate cancer (PCa). However, experimental variability in organoid drug testing complicates reproducibility. For example, we observed PCa organoids to be less affected by cabazitaxel, abiraterone and enzalutamide as compared to corresponding single cells prior to organoid assembly. We hypothesized that three-dimensional (3D) organoid organization and the use of various 3D scaffolds impact treatment efficacy. Live-cell imaging of androgen-induced androgen receptor (AR) nuclear translocation and taxane-induced tubulin stabilization was used to investigate the impact of 3D scaffolds, spatial organoid distribution and organoid size on treatment effect. Scaffolds delayed AR translocation and tubulin stabilization, with Matrigel causing a more pronounced delay than synthetic hydrogel as well as incomplete tubulin stabilization. Drug effect was further attenuated the more centrally organoids were located in the scaffold dome. Moreover, cells in the organoid core revealed a delayed treatment effect compared to cells in the organoid periphery, underscoring the impact of organoid size. These findings indicate that analysis of organoid drug responses needs careful interpretation and requires dedicated read-outs with consideration of underlying technical aspects.

Generating human prostate cancer organoids from leukapheresis enriched circulating tumour cells.

BACKGROUND: Circulating tumour cell (CTC)-derived organoids have the potential to provide a powerful tool for personalised cancer therapy but are restrained by low CTC numbers provided by blood samples. Here, we used diagnostic leukapheresis (DLA) to enrich CTCs from patients with metastatic prostate cancer (mPCa) and explored whether organoids provide a platform for ex vivo treatment modelling. METHODS: We prospectively screened 102 patients with mPCa and performed DLA in 40 patients with ≥5 CTCs/7.5 mL blood. We enriched CTCs from DLA using white blood cell (WBC) depletion alone or combined with EpCAM selection. The enriched CTC samples were cultured in 3D to obtain organoids and used for downstream analyses. RESULTS: The DLA procedure resulted in a median yield of 5312 CTCs as compared with 22 CTCs in 7.5 mL of blood. Using WBC depletion, we recovered 46% of the CTCs, which reduced to 12% with subsequent EpCAM selection. From the isolated and enriched CTC samples, organoid expansion succeeded in 35%. Successful organoid cultures contained significantly higher CTC numbers at initiation. Moreover, we performed treatment modelling in one organoid cell line and identified substantial tumour heterogeneity in CTCs using single cell DNA sequencing. CONCLUSIONS: DLA is an efficient method to enrich CTCs, although the modest success rate of culturing CTCs precludes large scale clinical application. Our data do suggest that DLA and subsequent processing provides a rich source of viable tumour cells. Therefore, DLA offers a promising alternative to biopsy procedures to obtain sufficient number of tumour cells to study sequential samples in patients with mPCa. TRIAL REGISTRATION NUMBER: NL6019.

Androgen receptor signalling impairs docetaxel efficacy in castration-resistant prostate cancer.

Androgen receptor (AR) signalling drives neoplastic growth and therapy resistance in prostate cancer. Recent clinical data show that docetaxel combined with androgen deprivation therapy improves outcome in hormone-sensitive disease. We studied whether testosterone and AR signalling interferes with docetaxel treatment efficacy in castration-resistant prostate cancer (CRPC). We found that testosterone supplementation significantly impaired docetaxel tumour accumulation in a CRPC model, resulting in decreased tubulin stabilisation and antitumour activity. Furthermore, testosterone competed with docetaxel for uptake by the drug transporter OATP1B3. Irrespective of docetaxel-induced tubulin stabilisation, AR signalling by testosterone counteracted docetaxel efficacy. AR-pathway activation could also reverse long-term tumour regression by docetaxel treatment in vivo. These results indicate that to optimise docetaxel efficacy, androgen levels and AR signalling need to be suppressed. This study lends evidence for continued maximum suppression of AR signalling by combining targeted therapeutics with docetaxel in CRPC.

CETSA-based target engagement of taxanes as biomarkers for efficacy and resistance.

The use of taxanes has for decades been crucial for treatment of several cancers. A major limitation of these therapies is inherent or acquired drug resistance. A key to improved outcome of taxane-based therapies is to develop tools to predict and monitor drug efficacy and resistance in the clinical setting allowing for treatment and dose stratification for individual patients. To assess treatment efficacy up to the level of drug target engagement, we have established several formats of tubulin-specific Cellular Thermal Shift Assays (CETSAs). This technique was evaluated in breast and prostate cancer models and in a cohort of breast cancer patients. Here we show that taxanes induce significant CETSA shifts in cell lines as well as in animal models including patient-derived xenograft (PDX) models. Furthermore, isothermal dose response CETSA measurements allowed for drugs to be rapidly ranked according to their reported potency. Using multidrug resistant cancer cell lines and taxane-resistant PDX models we demonstrate that CETSA can identify taxane resistance up to the level of target engagement. An imaging-based CETSA format was also established, which in principle allows for taxane target engagement to be accessed in specific cell types in complex cell mixtures. Using a highly sensitive implementation of CETSA, we measured target engagement in fine needle aspirates from breast cancer patients, revealing a range of different sensitivities. Together, our data support that CETSA is a robust tool for assessing taxane target engagement in preclinical models and clinical material and therefore should be evaluated as a prognostic tool during taxane-based therapies.

Fanconi anemia and homologous recombination gene variants are associated with functional DNA repair defects in vitro and poor outcome in patients with advanced head and neck squamous cell carcinoma.

Mutations in Fanconi Anemia or Homologous Recombination (FA/HR) genes can cause DNA repair defects and could therefore impact cancer treatment response and patient outcome. Their functional impact and clinical relevance in head and neck squamous cell carcinoma (HNSCC) is unknown. We therefore questioned whether functional FA/HR defects occurred in HNSCC and whether they are associated with FA/HR variants. We assayed a panel of 29 patient-derived HNSCC cell lines and found that a considerable fraction is hypersensitive to the crosslinker Mitomycin C and PARP inhibitors, a functional measure of FA/HR defects. DNA sequencing showed that these hypersensitivities are associated with the presence of bi-allelic rare germline and somatic FA/HR gene variants. We next questioned whether such variants are associated with prognosis and treatment response in HNSCC patients. DNA sequencing of 77 advanced stage HNSCC tumors revealed a 19% incidence of such variants. Importantly, these variants were associated with a poor prognosis (p = 0.027; HR = 2.6, 1.1-6.0) but favorable response to high cumulative cisplatin dose. We show how an integrated in vitro functional repair and genomic analysis can improve the prognostic value of genetic biomarkers. We conclude that repair defects are marked and frequent in HNSCC and are associated with clinical outcome.

Testosterone Diminishes Cabazitaxel Efficacy and Intratumoral Accumulation in a Prostate Cancer Xenograft Model.

Inactivation of the androgen receptor (AR) pathway by androgen deprivation therapy (ADT) is the mainstay of (metastatic) prostate cancer therapy. Ultimately, the AR pathway will be re-activated despite castrate levels of circulating androgens. Thereby, maintaining its role even in castration resistant prostate cancer (CRPC). The recent STAMPEDE and CHAARTED trials showed that docetaxel in combination with ADT increased survival in hormone sensitive prostate cancer patients, suggesting cross-talk between AR signaling and chemotherapy efficacy. We hypothesized that a similar interaction may also apply for CRPC that is treated with cabazitaxel. We studied the impact of androgen status on the efficacy, pharmacodynamics and -kinetics of cabazitaxel in a unique and clinically relevant patient derived xenograft model of castration resistant disease. We found that cabazitaxel is highly effective in a castrate setting with strongly reduced AR activation, while tumor growth inhibition by cabazitaxel was completely abolished in the presence of high AR pathway activity. Moreover, additional experiments showed that intratumoral cabazitaxel levels were 3.5 times higher in tumors from castrated mice as compared to tumors from androgen-supplemented animals. We confirmed that cabazitaxel pharmacokinetics were not affected by testosterone, suggesting that androgen status might influence cabazitaxel tumor uptake directly. This study reveals the impact of androgen status on cabazitaxel efficacy and supports the potential of combination of taxane chemotherapeutics with AR axis targeting agents.

0.5 Mb array as a first-line prenatal cytogenetic test in cases without ultrasound abnormalities and its implementation in clinical practice.

Using whole-genome array testing instead of karyotyping in prenatal diagnosis for all indications may be desirable because of the higher diagnostic yield and shorter reporting time. The goal of this research was finding the optimal array resolution that could replace routine prenatal karyotyping in cases without ultrasound abnormalities, for example, referred for advanced maternal age or abnormal first trimester screening. As variants of unknown clinical significance (VOUS), if reported, might complicate decision-making about continuation of pregnancy, such an optimal array resolution should have a high abnormality detection rate and reveal a minimal amount of VOUS. The array data of 465 fetuses were retrospectively evaluated with several resolution levels, and the Decipher microdeletion/microduplication syndrome list was reviewed to assess what could be theoretically missed with a lower resolution. A 0.5-Mb resolution showed a high diagnostic yield potential and significantly minimized the number of VOUS. Based on our experience, we recommend genomic SNP array as a first-tier test in prenatal diagnosis. The resolution should be chosen based on the indication. In cases of fetal ultrasound abnormalities or intrauterine fetal death (IUFD), high-resolution analysis should be done. In other cases, we advise replacing karyotyping by SNP array analysis with 0.5 Mb resolution.