Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. impartial (drug-tolerant) states and thus escape extinction. We further demonstrate that inhibition of KRAS signaling alone via co-targeting the MAPK and PI3K pathways fails to induce extensive tumor cell death and, therefore, has limited efficacy against PDAC. However, the addition of histone deacetylase (HDAC) inhibitors greatly improves outcomes, reduces the self-renewal of cancer cells, and blocks cancer metastasis Rabbit polyclonal to ZNF286A in vivo. Our results suggest that targeting HDACs in combination with KRAS or its effector pathways provides an effective strategy for the treatment of PDAC. model system to investigate the origins and evolution of pancreatic cancer cells. As a proof of concept, we isolated the main epithelial cell types from which PDAC originates and characterized their propensity to form metastases [10, 11]. In this study, we explore the relative importance of oncogenic KRAS signaling pathways for tumor maintenance and in conferring therapy resistance. Our analysis reveals that oncogenic KRAS dependency can be relinquished in KRAS-initiated tumors, and that some cancer cells can shuttle between the KRAS-dependent (drug-sensitive) and impartial (drug-tolerant) says. We further demonstrate that therapeutic targeting of KRAS signaling alone has limited efficacy against PDAC. However, clinically available drugs, used at clinically achievable doses, can be effective against PDAC when co-administered with epigenetic modifiers, such as inhibitors of histone deacetylases. Our data suggest that targeting HDACs in combination with KRAS effector pathways provides an effective strategy for the treatment of PDAC. RESULTS Pancreatic cancer metastases display morphological and phenotypic heterogeneity Using genetically designed mice carrying KRAS and p53 mutations, we recently identified two main epithelial cell types from which PDAC originates and characterized their propensity to form metastases [10, 11]. The population of less mature cells bears the phenotype of EpCAM+CD24+CD44+SCA1? (referred to as SCA1-) that distinguishes them from a more mature populace of EpCAM+CD24+CD44+CD133+SCA1+ cells (referred to as SCA1+) (Fig. S1). The majority of tumors derived from SCA1? cells showed features of undifferentiated (sarcomatoid) carcinoma, whereas the histology of tumors derived from SCA1+ cells exhibited a pattern of well-differentiated adenocarcinoma GSK963 (Fig. S1). To explore factors contributing to GSK963 PDAC heterogeneity and therapeutic outcomes, we established clonal cell lines from the respective metastatic foci. The cell lines were assessed for the expression of pancreatic duct specific genes (PDX1, KRT19) and epithelial cell markers (EpCAM, CDH1, CD133). We categorized the cell lines into three groups. Class A cell lines (referred to as CLA) are the real spindle cell carcinomas exhibiting the EpCAM-CD24+CD44+CD133? surface phenotype (Fig. ?(Fig.1A,1A, ?,1B).1B). Class B cell lines (CLB) are adenocarcinomas exhibiting a real epithelial morphology and the EpCAM+CD24+CD44+CD133+ phenotype (Fig. ?(Fig.1A,1A, ?,1B).1B). Class C carcinomas (CLC) are morphologically heterogeneous and comprise interconvertible EpCAM+CD133+ epithelial and EpCAM?CD133? mesenchymal cells (Fig. ?(Fig.1A,1A, ?,1B).1B). Based on these features, class C tumors represent reversible epithelial-mesenchymal transition (EMT). Western blot analysis confirmed that CLA carcinomas were Vimentin (VIM) positive, KRT19/CDH1 unfavorable, while CLB carcinomas were VIM unfavorable, KRT19/CDH1 positive (Fig. ?(Fig.1C).1C). Injection of CLA, CLB or CLC cell lines into nude mice led to the development GSK963 of tumors maintaining the histological appearance of their parental neoplasms (Fig. ?(Fig.1A).1A). CLB clones are representative of the predominant form of human metastatic PDAC  and GSK963 hence we focused our analysis mainly on this cell type. Open in a separate windows Physique 1 Pancreatic cancer metastases display morphological and phenotypic heterogeneityA. Morphological appearance of CLA, CLB and CLC carcinomas derived from KrasG12D p53KO pancreatic cells. Representative H&E-stained sections made up of metastatic foci are shown. B. FACS analysis of CLA, CLB and CLC carcinomas. C. Immunoblot analysis of control pre-tumor cells and representative carcinomas. KRT19 (keratin 19), CDH1 (E-Cadherin), and VIM GSK963 (vimentin) are shown. ERK1/2 is the loading control. D. Western blot analysis of human PDAC cell lines maintained in defined serum-free medium for epithelial cells. A mouse B6-PDAC cell line is shown for comparison. Oncogenic KRAS signaling in primary and metastatic PDAC Signaling through the RAS/MAPK and PI3K pathways plays a causative role in pancreatic carcinogenesis [1, 2]. To assess the contribution of these pathways to PDAC maintenance, we evaluated the growth of the different subtypes in defined serum-free medium for epithelial cells, in the presence of exogenous growth factors, or under non-adherent culture conditions that mimic malignancy cell dissemination . Analysis confirmed that this KRAS oncogene activates the MAPK signaling (as assessed by phosphorylated ERK1/2) and PI3K/PDK1 signaling (as assessed by phosphorylated PDK1), but not PI3K/AKT signaling (Fig. ?(Fig.1C).1C). Addition of various growth factors further potentiated MAPK/ERK signaling, and.