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

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. Methylation(low) tumors are characterized by higher expression of endogenous retroviral (ERV) transcripts and dsRNA sensors which leads to a cell intrinsic activation of an interferon signature (IFNsign). This results in a pro-tumorigenic microenvironment and poor patient outcome. Methylation(low)/IFNsign(high) and Methylation(high)/IFNsign(low) 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 to acinar-derived counterparts. Collectively, our data point to two different origins and etiologies of human PDACs, with the aggressive Methylation(low)/IFNsign(high) subtype potentially targetable by agents blocking intrinsic IFN-signaling

Regulation of ABCB1 expression is a potential therapeutic target in drug resistant pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal disease with a miserable prognosis. Chemotherapeutic regimens, like FOLFIRINOX or gemcitabine plus nab-paclitaxel remain the standard treatment of care in patients diagnosed with PDAC while only achieving a modest increase in overall survival. Transcriptional subtyping of PDAC discerns tumors into two broad lineages which provides the opportunity to improve patient stratification and treatment, but has not been translated into clinical practice yet. Meanwhile, resistance to chemotherapy continues to be the limiting factor that prevents a patient’s cure from cancer and finding improved therapeutic options could overcome resistance of PDAC to current treatment regimens. With our previously established culturing model for patient-derived PDAC cells, we could investigate the development of resistance to paclitaxel and/or gemcitabine in the different subtypes. We developed a long-term treatment regime that enabled the generation of drug-resistant cells which were analyzed in a multi-omics approach. ATP-binding cassette (ABC) transporter B1 (ABCB1), a membrane-bound glycoprotein that is predominantly expressed in excretory tissue and a common resistance mechanism against paclitaxel in various tumor entities, was found to be overexpressed in all paclitaxel-resistant cells. CRISPR/Cas9-guided knockout and pharmacological inhibition re-sensitized these drug resistant cancer cells to paclitaxel. We found ABCB1 to be heterogeneously expressed in the paclitaxel resistant cell population and expression was lost after prolonged absence of paclitaxel treatment. Short-term drug treatment dynamically increased the proportion of ABCB1-expressing cells in former paclitaxel resistant cell population, re-acquiring paclitaxel-resistance. In of the paclitaxel resistant cell lines, expression of ABCB1 was further enhanced by de novo generation of extrachromosomal DNA (ecDNA), carrying the ABCB1 gene. Similar to ABCB1 gene expression, number ecDNA inside paclitaxel resistant cells was dynamically increased upon paclitaxel treatment. These findings describe a dual mechanism for acquired ABCB1 expression that is dependent on paclitaxel treatment and leads to the induction of ABCB1 expression and amplification of ABCB1-carrying ecDNA

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.ISSN:2666-3864ISSN:2211-124

The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features

INTRODUCTION: Chemotherapy resistance resulting in incomplete pathologic response is associated with high risk of metastasis and early relapse in breast cancer. The aim of this study was to identify and evaluate biomarkers of treatment-resistant tumor cells. METHODS: We performed a cell surface marker screen in triple-negative breast cancer patient-derived xenograft models treated with standard care genotoxic chemotherapy. Global expression profiling was used to further characterize the identified treatment-resistant subpopulations. RESULTS: High expression of sialyl-glycolipid stage-specific embryonic antigen 4 (SSEA4) was found in residual tumor cells surviving chemotherapy and in samples from metastatic patients who relapsed after neoadjuvant chemotherapy. Gene and microRNA (miRNA) expression profiling linked SSEA4 positivity with a mesenchymal phenotype and a deregulation of drug resistance pathways. Functional assays demonstrated a direct link between epithelial-mesenchymal transition (EMT) and SSEA4 expression. Interestingly, SSEA4 expression, EMT, and drug resistance seemed to be regulated posttranscriptionally. Finally, high expression of CMP-N-acetylneuraminate-β-galactosamide-α-2,3-sialyltransferase 2 (ST3GAL2), the rate-limiting enzyme of SSEA4 synthesis, was found to be associated with poor clinical outcome in breast and ovarian cancer patients treated with chemotherapy. CONCLUSIONS: In this study, we identified SSEA4 as highly expressed in a subpopulation of tumor cells resistant to multiple commonly used chemotherapy drugs, as well as ST3GAL2, the rate-limiting enzyme of SSEA4 synthesis, as a predictive marker of poor outcome for breast and ovarian cancer patients undergoing chemotherapy. Both biomarkers and additionally identified regulatory miRNAs may be used to further understand chemoresistance, to stratify patient groups in order to avoid ineffective and painful therapies, and to develop alternative treatment regimens for breast cancer patients

Boletín de Segovia: Número 30 - 1918 marzo 11

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

Additional file 1: of The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features

Supplemental experimental procedures. Detailed description of materials and methods. (DOCX 62 kb

Additional file 12: Figure S8. of The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features

SSEA4-positive breast cancer cells show decreased expression of miRNAs inhibiting EMT inducers. Expression ratios of the 12 miRNAs targeting the key mesenchymal regulator and indicator genes ZEB1, ZEB2, fibronectin 1, Snail1, Snail2, and Twist. Each bar represents the log2 expression ratio of the SSEA4-positive fraction relative to the SSEA4-negative fraction for the respective tumor model. (TIFF 201 kb

Additional file 3: Figure S1. of The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features

Representative gating strategy for flow cytometry–based marker analysis of dissociated xenograft tumor tissue. Tumor tissue was dissociated to obtain a single-cell suspension while preserving cell surface epitopes. The sample was stained for mouse-specific markers to exclude cells of murine origin from the analysis as well as for the screening candidates and analyzed by multiparametric flow cytometry. Doublets were excluded by forward scatter (FSC) area/FSC height gating (a); debris was excluded by FSC/side scatter gating (b); dead cells were excluded by gating off propidium iodide–positive events (c); and mouse cells were excluded by gating on α-mouse-fluorescein isothiocyanate–negative events (d). When we screened two samples in parallel, we found that one of the samples was labeled using an ultraviolet dye, allowing for subsequent separation of the events of each sample by gating on the VioBlue channel fluorescence intensity (e–h). (PNG 736 kb