Resistance to mesenchymal reprogramming sustains clonal propagation in metastatic breast cancer.

The acquisition of mesenchymal traits is considered a hallmark of breast cancer progression. However, the functional relevance of epithelial-to-mesenchymal transition (EMT) remains controversial and context dependent. Here, we isolate epithelial and mesenchymal populations from human breast cancer metastatic biopsies and assess their functional potential in vivo. Strikingly, progressively decreasing epithelial cell adhesion molecule (EPCAM) levels correlate with declining disease propagation. Mechanistically, we find that persistent EPCAM expression marks epithelial clones that resist EMT induction and propagate competitively. In contrast, loss of EPCAM defines clones arrested in a mesenchymal state, with concomitant suppression of tumorigenicity and metastatic potential. This dichotomy results from distinct clonal trajectories impacting global epigenetic programs that are determined by the interplay between human ZEB1 and its target GRHL2. Collectively, our results indicate that susceptibility to irreversible EMT restrains clonal propagation, whereas resistance to mesenchymal reprogramming sustains disease spread in multiple models of human metastatic breast cancer, including patient-derived cells in vivo.

Prenatal exposure to endocrine disrupting chemicals is associated with altered DNA methylation in cord blood.

Prenatal exposure to endocrine disrupting chemicals can interfere with development, and has been associated with social-cognitive functioning and adverse health outcomes later in life. Exposure-associated changes of DNA methylation (DNAm) patterns have been suggested as a possible mediator of this relationship. This study investigated whether prenatal low-dose exposure to polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) is associated with altered DNAm patterns across the genome in a Western urban-industrial population. In 142 mother-infant pairs from the Duisburg Birth Cohort Study, PCBs and PCDD/Fs levels were quantified from maternal blood during late pregnancy and associated with DNAm levels in cord blood using the Illumina EPIC beadchip. The epigenome-wide association studies (EWAS) identified 32 significantly differentially methylated positions (DMPs) and eight differentially methylated regions (DMRs) associated with six congeners of PCB and PCDD in females or males (FDRs < 0.05). DMPs and DMRs mapped to genes involved in neurodevelopment, gene regulation, and immune functioning. Weighted gene correlation network analysis (WGCNA) showed 31 co-methylated modules (FDRs < 0.05) associated with one congener of PCDF levels in females. Results of both analytical strategies indicate that prenatal exposure to PCBs and PCDD/Fs is associated with altered DNAm of genes involved in neurodevelopment, gene expression and immune functioning. DNAm and gene expression levels of several of these genes were previously associated with EDC exposure in rodent models. Follow-up studies will clarify whether these epigenetic changes might contribute to the origin for adverse mental and health outcomes.

Aggressive PDACs Show Hypomethylation of Repetitive Elements and the Execution of an Intrinsic IFN Program Linked to a Ductal Cell of Origin.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive desmoplasia, which challenges the molecular analyses of bulk tumor samples. Here we FACS-purified epithelial cells from human PDAC and normal pancreas and derived their genome-wide transcriptome and DNA methylome landscapes. Clustering based on DNA methylation revealed two distinct PDAC groups displaying different methylation patterns at regions encoding repeat elements. Methylationlow tumors are characterized by higher expression of endogenous retroviral transcripts and double-stranded RNA sensors, which lead to a cell-intrinsic activation of an interferon signature (IFNsign). This results in a protumorigenic microenvironment and poor patient outcome. Methylationlow/IFNsignhigh and Methylationhigh/IFNsignlow PDAC cells preserve lineage traits, respective of normal ductal or acinar pancreatic cells. Moreover, ductal-derived Kras G12D/Trp53 -/- mouse PDACs show higher expression of IFNsign compared with acinar-derived counterparts. Collectively, our data point to two different origins and etiologies of human PDACs, with the aggressive Methylationlow/IFNsignhigh subtype potentially targetable by agents blocking intrinsic IFN signaling. SIGNIFICANCE: The mutational landscapes of PDAC alone cannot explain the observed interpatient heterogeneity. We identified two PDAC subtypes characterized by differential DNA methylation, preserving traits from normal ductal/acinar cells associated with IFN signaling. Our work suggests that epigenetic traits and the cell of origin contribute to PDAC heterogeneity.This article is highlighted in the In This Issue feature, p. 521.

Novel Non-integrating DNA Nano-S/MAR Vectors Restore Gene Function in Isogenic Patient-Derived Pancreatic Tumor Models.

We describe herein non-integrating minimally sized nano-S/MAR DNA vectors, which can be used to genetically modify dividing cells in place of integrating vectors. They represent a unique genetic tool, which avoids vector-mediated damage. Previous work has shown that DNA vectors comprising a mammalian S/MAR element can provide persistent mitotic stability over hundreds of cell divisions, resisting epigenetic silencing and thereby allowing sustained transgene expression. The composition of the original S/MAR vectors does present some inherent limitations that can provoke cellular toxicity. Herein, we present a new system, the nano-S/MAR, which drives higher transgene expression and has improved efficiency of establishment, due to the minimal impact on cellular processes and perturbation of the endogenous transcriptome. We show that these features enable the hitherto challenging genetic modification of patient-derived cells to stably restore the tumor suppressor gene SMAD4 to a patient-derived SMAD4 knockout pancreatic cancer line. Nano-S/MAR modification does not alter the molecular or phenotypic integrity of the patient-derived cells in cell culture and xenograft mouse models. In conclusion, we show that these DNA vectors can be used to persistently modify a range of cells, providing sustained transgene expression while avoiding the risks of insertional mutagenesis and other vector-mediated toxicity.

Identification and Validation of Novel Subtype-Specific Protein Biomarkers in Pancreatic Ductal Adenocarcinoma.

OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) has been subclassified into 3 molecular subtypes: classical, quasi-mesenchymal, and exocrine-like. These subtypes exhibit differences in patient survival and drug resistance to conventional therapies. The aim of the current study is to identify novel subtype-specific protein biomarkers facilitating subtype stratification of patients with PDAC and novel therapy development. METHODS: A set of 12 human patient-derived primary cell lines was used as a starting material for an advanced label-free proteomics approach leading to the identification of novel cell surface and secreted biomarkers. Cell surface protein identification was achieved by in vitro biotinylation, followed by mass spectrometric analysis of purified biotin-tagged proteins. Proteins secreted into a chemically defined serum-free cell culture medium were analyzed by shotgun proteomics. RESULTS: Of 3288 identified proteins, 2 pan-PDAC (protocadherin-1 and lipocalin-2) and 2 exocrine-like-specific (cadherin-17 and galectin-4) biomarker candidates have been validated. Proximity ligation assay analysis of the 2 exocrine-like biomarkers revealed their co-localization on the surface of exocrine-like cells. CONCLUSIONS: The study reports the identification and validation of novel PDAC biomarkers relevant for the development of patient stratification tools. In addition, cadherin-17 and galectin-4 may serve as targets for bispecific antibodies as novel therapeutics in PDAC.

CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma.

Although subtypes of pancreatic ductal adenocarcinoma (PDAC) have been described, this malignancy is clinically still treated as a single disease. Here we present patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identify two markers--HNF1A and KRT81--that enable stratification of tumors into different subtypes by using immunohistochemistry. Individuals with tumors of these subtypes showed substantial differences in overall survival, and their tumors differed in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. Cytochrome P450 3A5 (CYP3A5) metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or short hairpin RNA (shRNA)-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Whereas hepatocyte nuclear factor 4, alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and it is highly expressed in several additional malignancies. These findings designate CYP3A5 as a predictor of therapy response and as a tumor cell-autonomous detoxification mechanism that must be overcome to prevent drug resistance.

Defined conditions for the isolation and expansion of basal prostate progenitor cells of mouse and human origin.

Methods to isolate and culture primary prostate epithelial stem/progenitor cells (PESCs) have proven difficult and ineffective. Here, we present a method to grow and expand both murine and human basal PESCs long term in serum- and feeder-free conditions. The method enriches for adherent mouse basal PESCs with a Lin(-)SCA-1(+)CD49f(+)TROP2(high) phenotype. Progesterone and sodium selenite are additionally required for the growth of human Lin(-)CD49f(+)TROP2(high) PESCs. The gene-expression profiles of expanded basal PESCs show similarities to ESCs, and NF-kB function is critical for epithelial differentiation of sphere-cultured PESCs. When transplanted in combination with urogenital sinus mesenchyme, expanded mouse and human PESCs generate ectopic prostatic tubules, demonstrating their stem cell activity in vivo. This novel method will facilitate the molecular, genomic, and functional characterization of normal and pathologic prostate glands of mouse and human origin.

Co-expression of MET and CD47 is a novel prognosticator for survival of luminal breast cancer patients.

Although luminal-type primary breast cancer can be efficiently treated, development of metastatic disease remains a significant clinical problem. We have previously shown that luminal-type circulating tumor cells (CTCs) co-expressing the tyrosine-kinase MET and CD47, a ligand involved in cancer cell evasion from macrophage scavenging, are able to initiate metastasis in xenografts. Here, we investigated the clinical relevance of MET-CD47 co-expression in 255 hormone receptor positive breast tumors by immunohistochemistry and found a 10.3- year mean overall-survival difference between MET-CD47 double-positive and double-negative patients (p<0.001) MET-CD47 co-expression defined a novel independent prognosticator for overall-survival by multivariate analysis (Cox proportional hazards model: HR: 4.1, p<0.002) and CD47 expression alone or in combination with MET was strongly associated with lymph node metastasis. Furthermore, flow cytometric analysis of metastatic patient blood revealed consistent presence of MET+CD47+ CTCs (range 0.8 - 33.3% of CTCs) and their frequency was associated with increased metastatic spread. Finally, primary uncultured CTCs with high MET+CD47+ content showed an enhanced capacity to initiate metastasis in mice. Detection and targeting of MET and CD47 may thus provide a rational basis for risk stratification and treatment of patients with luminal-type breast cancer.

Expression and prognostic significance of cancer stem cell markers CD24 and CD44 in urothelial bladder cancer xenografts and patients undergoing radical cystectomy.

OBJECTIVES: To evaluate CD24/CD44/CD47 cancer stem cell marker expressions in bladder cancer (BCa) and provide data on their prognostic significance for clinical outcome in patients undergoing radical cystectomy (RC). MATERIAL AND METHODS: Primary BCa tissue was used for xenograft studies. A tissue microarray was prepared using specimens from a cohort of 132 patients. All patients underwent RC for urothelial BCa between 2001 and 2010. Expression of CD24, CD44, and CD47 was examined in primary samples and xenografts by fluorescence-activated cell sorting. Populations of CD24(low)- and CD24(high)-expressing cells were sorted and evaluated for tumorigenicity in vivo. Tissue microarray was analyzed for CD24/CD44 staining intensity and tumor-specific vs. stromal cell staining. Associations with BCa survival, BCa stage, and lymph node status were evaluated by univariate and multivariate analyses. RESULTS: CD24 and CD44/CD47 expressions mark distinct cell populations within the normal urothelium as well as in BCa. CD24(high/low) expression was not sufficient to characterize CD24 as a BCa-initiating marker in in vivo primary xenotransplants. CD24 and CD44 expressions correlated with lower cancer-specific survival in patients. However, multivariate analyses of CD24 or CD44 did not demonstrate significantly increased hazards for cancer-specific death if analyzed together with stage, grade, and nodal status of patients. CONCLUSIONS: Cancer stem cell markers CD24/CD44/CD47 are differentially expressed in cells of urothelial BCa in patients undergoing RC and influence cancer-specific survival of patients. Further evaluation of CD24/CD44/CD47 protein expression could be of high therapeutic value in BCa. However, both CD24 and CD44 expressions cannot be regarded as independent prognostic parameters for patients undergoing RC.

Identification of a population of blood circulating tumor cells from breast cancer patients that initiates metastasis in a xenograft assay.

It has been hypothesized that carcinoma metastasis is initiated by a subpopulation of circulating tumor cells (CTCs) found in the blood of patients. However, although the presence of CTCs is an indicator of poor prognosis in several carcinoma entities, the existence and phenotype of metastasis-initiating cells (MICs) among CTCs has not been experimentally demonstrated. Here we developed a xenograft assay and used it to show that primary human luminal breast cancer CTCs contain MICs that give rise to bone, lung and liver metastases in mice. These MIC-containing CTC populations expressed EPCAM, CD44, CD47 and MET. In a small cohort of patients with metastases, the number of EPCAM(+)CD44(+)CD47(+)MET(+) CTCs, but not of bulk EPCAM(+) CTCs, correlated with lower overall survival and increased number of metastasic sites. These data describe functional circulating MICs and associated markers, which may aid the design of better tools to diagnose and treat metastatic breast cancer.

Gaboxadol -- a different hypnotic profile with no tolerance to sleep EEG and sedative effects after repeated daily dosing.

Gaboxadol, a selective extra synaptic GABA(A) receptor agonist, has been in clinical development for the treatment of insomnia. Development of tolerance to therapeutic effects (e.g. hypnotic and anticonvulsant and sedative) and withdrawal symptoms (e.g. REM sleep rebound and reduced seizure threshold) upon treatment discontinuation is reported for GABA(A) receptor allosteric modulators acting via the benzodiazepine binding site, e.g. zolpidem and indiplon. We conducted a head to head comparison in rats of the hypnotic (sleep EEG after 21 daily doses and 24 and 48 h after the last dose) and seizure threshold modifying (bicuculline assay 24 h after 28 daily doses) effects of gaboxadol and benzodiazepine ligands. Furthermore, we investigated in further details a previously reported apparent rapid development of tolerance to gaboxadol's effects in a rat rotarod motor coordination assay and related this effect to CNS exposure levels and in vitro potency at extra synaptic GABA(A) receptors. Sleep EEG studies demonstrated lack of tolerance and withdrawal effects after 28 daily doses with gaboxadol, whereas zolpidem produced both tolerance and withdrawal effects under a similar dosing regimen. Daily dosing with gaboxadol, zolpidem or indiplon for 28 days and acute discontinuation of treatment left the threshold to bicuculline-induced seizures unchanged. The rapidly attenuated effect of repeated gaboxadol dosing was confirmed in the rotarod model. However, re-challenge of gaboxadol insensitive animals with gaboxadol produced a maximum response, ruling out that receptor desensitisation accounts for these effects. By comparing CNS exposure at rotarod responses and concentration response relation at cloned GABA(A) receptors expressed in Xenopus oocytes it appears that the decline in response in the rotarod model coincides with the steep part of the concentration response curve for gaboxadol at extra synaptic GABA(A) receptors. In conclusion, rat sleep EEG repeated dose studies of gaboxadol confirm a hypnotic-like profile and no withdrawal effects, whereas tolerance and withdrawal effects were shown with zolpidem. Withdrawal from gaboxadol, zolpidem and indiplon did not affect the seizure threshold to bicuculline. Gaboxadol's apparent rapid development of tolerance in the rotarod assay appears to be kinetically determined.

Measurement of cortical and hippocampal epileptiform activity in freely moving rats by means of implantable radiotelemetry.

Implanted radiotelemetry has been used for the measurement of cortical electroencephalogram (EEG), locomotor activity, body temperature and cardiovascular parameters. This technique allows high quality data acquisition from freely moving animals with no complications of externalised apparatus. This paper focuses on the methodology for short and long-term monitoring of epileptiform activity by simultaneous cortical EEG, hippocampal (HC) EEG and electromyogram (EMG) in rats. The circadian rhythm of temperature (CRT) was monitored after surgery to estimate the need for post surgical recovery of animals. Different placements of EMG electrodes were assessed in order to minimise artefacts and increase sensitivity. The occurrence of epileptiform ictal and interictal activity following an acute injection of either 40 mg/kg pentylenetetrazole (PTZ) or 13.8 mg/kg kainic acid (KA) was investigated. The occurrence of spontaneous seizures was also monitored 5-8 weeks after administration of KA. The present study demonstrated a sensitive method for monitoring cortical EEG, hippocampal EEG and EMG short and long-term by implantable radiotelemetry in freely moving rats.

EEG measurements by means of radiotelemetry after intracerebroventricular (ICV) cannulation in rodents.

The utility of implanted radiotelemetry transmitters for the measurement of electroencephalogram (EEG), locomotor activity, body temperature and cardiovascular parameters has been well documented. This paper focuses on the methodology, of combining radiotelemetry with intracerebroventricular (ICV) cannulation. The two excitatory neuropeptides, orexin-A and orexin-B, can only be given by ICV injection, therefore we examined their effects on the normal sleep-wake cycle of rodents in the present study. The effects of orexins on sleep architecture have been extensively studied in tethered models demonstrating increased levels of wakefulness. In this study, both orexin neuropeptides, especially orexin-A, increase wakefulness within the first sleep period followed by an increase in slow wave (SW) sleep and paradoxical sleep (PS), towards the end of a 5-h recording period which may be a rebound phenomena. The present study has demonstrated that ICV cannulation can be used effectively in studying the effects of pharmacological agents on the sleep-wake cycle of rodents by measuring EEG and EMG by radiotelemetry.