Anke Van den broeck

Human pancreatic cancer contains a side population expressing cancer stem cell-associated and prognostic genes

In many types of cancers, a side population (SP) has been identified based on high efflux capacity, thereby enriching for chemoresistant cells as well as for candidate cancer stem cells (CSC). Here, we explored whether human pancreatic ductal adenocarcinoma (PDAC) contains a SP, and whether its gene expression profile is associated with chemoresistance, CSC and prognosis. After dispersion into single cells and incubation with Hoechst dye, we analyzed human PDAC resections specimens using flow cytometry (FACS). We identified a SP and main population (MP) in all human PDAC resection specimens (n = 52) analyzed, but detected immune (CD45+) and endothelial (CD31+) cells in this fraction together with tumor cells. The SP and MP cells, or more purified fractions depleted from CD31+/CD45+ cells (pSP and pMP), were sorted by FACS and subjected to whole-genome expression analysis. This revealed upregulation of genes associated with therapy resistance and of markers identified before in putative pancreatic CSC. pSP gene signatures of 32 or 10 up- or downregulated genes were developed and tested for discriminatory competence between pSP and pMP in different sets of PDAC samples. The prognostic value of the pSP genes was validated in a large independent series of PDAC patients (n = 78) using nCounter analysis of expression (in tumor versus surrounding pancreatic tissue) and Cox regression for disease-free and overall survival. Of these genes, expression levels of ABCB1 and CXCR4 were correlated with worse patient survival. Thus, our study for the first time demonstrates that human PDAC contains a SP. This tumor subpopulation may represent a valuable therapeutic target given its chemoresistance- and CSC-associated gene expression characteristics with potential prognostic value.

Human pancreatic adenocarcinoma contains a side population resistant to gemcitabine.

BackgroundTherapy resistance remains one of the major challenges to improve the prognosis of patients with pancreatic cancer. Chemoresistant cells, which potentially also display cancer stem cell (CSC) characteristics, can be isolated using the side population (SP) technique. Our aim was to search for a SP in human pancreatic ductal adenocarcinoma (PDAC) and to examine its chemoresistance and CSC(−like) phenotype.MethodsHuman PDAC samples were expanded in immunodeficient mice and first-generation xenografts analyzed for the presence of a Hoechst dye-effluxing SP using flow cytometry (FACS). To investigate chemoresistance of the SP, mice bearing PDAC xenografts were treated with gemcitabine and SP proportion determined. In addition, the SP and the main tumour cell population (MP) were sorted by FACS for RNA extraction to profile gene expression, and for culturing under sphere-forming conditions.ResultsA SP was identified in all PDAC samples, analyzed. This SP was more resistant to gemcitabine than the other tumour cells as examined in vivo. Whole-genome expression profiling of the SP revealed upregulation of genes related to therapy resistance, apoptotic regulation and epithelial-mesenchymal transition. In addition, the SP displayed higher tumourigenic (CSC) activity than the MP as analyzed in vitro by sphere-forming capacity.ConclusionWe identified a SP in human PDAC and uncovered a chemoresistant and CSC-associated phenotype. This SP may represent a new therapeutic target in pancreatic cancer.Trial registrationClinicaltrials.gov NCT00936104

The Human Melanoma Side Population Displays Molecular and Functional Characteristics of Enriched Chemoresistance and Tumorigenesis

Melanoma remains the most lethal skin cancer, mainly because of high resistance to therapy. Side population (SP) cells are found in many types of cancer and are usually enriched in therapy-resistant as well as tumorigenic cells. Here, we identified a Hoechst dye-effluxing SP in a large series of human melanoma samples representing different progression phases. The SP size did not change with disease stage but was correlated with the prognostic “Breslow’s depth” in the primary (cutaneous) tumors. When injected into immunodeficient mice, the SP generated larger tumors than the bulk “main population” (MP) melanoma cells in two consecutive generations, and showed tumorigenic capacity at lower cell numbers than the MP. In addition, the SP reconstituted the heterogeneous composition of the human A375 melanoma cell line, and its clonogenic activity was 2.5-fold higher than that of the MP. Gene-expression analysis revealed upregulated expression in the melanoma SP (versus the MP) of genes associated with chemoresistance and anti-apoptosis. Consistent with these molecular characteristics, the SP increased in proportion when A375 cells were exposed to the melanoma standard chemotherapeutic agent dacarbazine, and to the aggravating condition of hypoxia. In addition, the SP showed enhanced expression of genes related to cell invasion and migration, as well as to putative (melanoma) cancer stem cells (CSC) including ABCB1 and JARID1B. ABCB1 immunoreactivity was detected in a number of tumor cells in human melanomas, and in particular in clusters at the invasive front of the primary tumors. Together, our findings support that the human melanoma SP is enriched in tumorigenic and chemoresistant capacity, considered key characteristics of CSC. The melanoma SP may therefore represent an interesting therapeutic target.

Molecular markers associated with outcome and metastasis in human pancreatic cancer

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a heterogeneous cancer in which differences in survival rates might be related to a variety in gene expression profiles. Although the molecular biology of PDAC begins to be revealed, genes or pathways that specifically drive tumour progression or metastasis are not well understood.MethodsWe performed microarray analyses on whole-tumour samples of 2 human PDAC subpopulations with similar clinicopathological features, but extremely distinct survival rates after potentially curative surgery, i.e. good outcome (OS and DFS > 50 months, n = 7) versus bad outcome (OS < 19 months and DFS < 7 months, n = 10). Additionally, liver- and peritoneal metastases were analysed and compared to primary cancer tissue (n = 11).ResultsThe integrin and ephrin receptor families were upregulated in all PDAC samples, irrespective of outcome, supporting an important role of the interaction between pancreatic cancer cells and the surrounding desmoplastic reaction in tumorigenesis and cancer progression. Moreover, some components such as ITGB1 and EPHA2 were upregulated in PDAC samples with a poor outcome, Additionally, overexpression of the non-canonical Wnt/β-catenin pathway and EMT genes in PDAC samples with bad versus good outcome suggests their contribution to the invasiveness of pancreatic cancer, with β-catenin being also highly upregulated in metastatic tissue.ConclusionsComponents of the integrin and ephrin pathways and EMT related genes, might serve as molecular markers in pancreatic cancer as their expression seems to be related with prognosis.

Prognostic relevance of molecular subtypes and master regulators in pancreatic ductal adenocarcinoma

BackgroundPancreatic cancer is poorly characterized at genetic and non-genetic levels. The current study evaluates in a large cohort of patients the prognostic relevance of molecular subtypes and key transcription factors in pancreatic ductal adenocarcinoma (PDAC).MethodsWe performed gene expression analysis of whole-tumor tissue obtained from 118 surgically resected PDAC and 13 histologically normal pancreatic tissue samples. Cox regression models were used to study the effect on survival of molecular subtypes and 16 clinicopathological prognostic factors. In order to better understand the biology of PDAC we used iRegulon to identify transcription factors (TFs) as master regulators of PDAC and its subtypes.ResultsWe confirmed the PDAssign gene signature as classifier of PDAC in molecular subtypes with prognostic relevance. We found molecular subtypes, but not clinicopathological factors, as independent predictors of survival. Regulatory network analysis predicted that HNF1A/B are among thousand TFs the top enriched master regulators of the genes expressed in the normal pancreatic tissue compared to the PDAC regulatory network. On immunohistochemistry staining of PDAC samples, we observed low expression of HNF1B in well differentiated towards no expression in poorly differentiated PDAC samples. We predicted IRF/STAT, AP-1, and ETS-family members as key transcription factors in gene signatures downstream of mutated KRAS.ConclusionsPDAC can be classified in molecular subtypes that independently predict survival. HNF1A/B seem to be good candidates as master regulators of pancreatic differentiation, which at the protein level loses its expression in malignant ductal cells of the pancreas, suggesting its putative role as tumor suppressor in pancreatic cancer.Trial registrationThe study was registered at ClinicalTrials.gov under the number NCT01116791 (May 3, 2010).