A Comparative Endocrine Trans-Differentiation Approach to Pancreatic Ductal Adenocarcinoma Cells with Different EMT Phenotypes Identifies Quasi-Mesenchymal Tumor Cells as Those with Highest Plasticity

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and therapy-resistant cancer types which is largely due to tumor heterogeneity, cancer cell de-differentiation, and early metastatic spread. The major molecular subtypes of PDAC are designated classical/epithelial (E) and quasi-mesenchymal (QM) subtypes, with the latter having the worst prognosis. Epithelial–mesenchymal transition (EMT) and the reverse process, mesenchymal-epithelial transition (MET), are involved in regulating invasion/metastasis and stem cell generation in cancer cells but also early pancreatic endocrine differentiation or de-differentiation of adult pancreatic islet cells in vitro, suggesting that pancreatic ductal exocrine and endocrine cells share common EMT programs. Using a panel of PDAC-derived cell lines classified by epithelial/mesenchymal expression as either E or QM, we compared their trans-differentiation (TD) potential to endocrine progenitor or β cell-like cells since studies with human pancreatic cancer cells for possible future TD therapy in PDAC patients are not available so far. We observed that QM cell lines responded strongly to TD culture using as inducers 5′-aza-2′-deoxycytidine or growth factors/cytokines, while their E counterparts were refractory or showed only a weak response. Moreover, the gain of plasticity was associated with a decrease in proliferative and migratory activities and was directly related to epigenetic changes acquired during selection of a metastatic phenotype as revealed by TD experiments using the paired isogenic COLO 357-L3.6pl model. Our data indicate that a QM phenotype in PDAC coincides with increased plasticity and heightened trans-differentiation potential to activate a pancreatic β cell-specific transcriptional program. We strongly assume that this specific biological feature has potential to be exploited clinically in TD-based therapy to convert metastatic PDAC cells into less malignant or even benign cells.

Activation of a Ductal-to-Endocrine Transdifferentiation Transcriptional Program in the Pancreatic Cancer Cell Line PANC-1 Is Controlled by RAC1 and RAC1b through Antagonistic Regulation of Stemness Factors

“…We have previously shown that the RAC1 splice isoform, RAC1b, acts as an endogenous inhibitor of RAC1 in the regulation of EMT and cell migration in PDAC-derived cells, consistent with low or absent expression of RAC1b in the QM cell lines PANC-1, MIA PaCa-2, and PaTu 8988s [ 23 , 30 , 32 ]. To study if RAC1b also impacts the deTDtP, we ectopically expressed in a stable or transient fashion an HA-tagged version of RAC1b in PANC-1 or MIA PaCa-2 cells, respectively.…”

“…The expression and functional activity of the N17 mutant were verified here by immunoprecipitation ( Figure S1, Figure S12, left-hand panel ) and its ability to reduce basal migration of PANC-1 cells ( Figure S2 ) mimicking the loss in migratory activity after siRNA-mediated RAC1 knockdown [ 36 ]. Following application of an FGFb + TF-based protocol [ 14 ] for induction of a β cell transcriptional program—assessed by expression of INS, SLC2A2, and MAFA [ 23 ]—we noted that the induction of these markers was clearly impaired upon dn inhibition of RAC1 activation ( Figure 1 A). Similar results for INS expression were obtained with MIA PaCa-2 cells transiently transfected with an RAC1 N17 -encoding expression vector ( Figure 1 B and Figure S1, middle panel ).…”

“…To confirm the inhibitory function of RAC1 N17 we employed an alternative protocol for deTD that used a 3-day treatment with a combination of inflammatory cytokines IFNγ, IL1β, and TNFα (IIT) [ 6 , 23 ]. This cocktail was originally shown to induce expression of the endocrine progenitor marker NGN3 (encoded by NEUROG3 ) in (parental) PANC-1 cells [ 6 ].…”

“…Based on the idea that exocrine ductal and endocrine β cells share a common stem cell, the PDAC-derived cell line, PANC-1, was successfully converted to insulin-producing/expressing cells using a variety of protocols [ 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ]. To reveal whether this ability was confined to only this cell line, ductal cells with a QM phenotype, or was a general feature of pancreatic ductal cells regardless of their subtype, we compared in a separate study the response of various PDAC cell lines of either E or QM phenotype to pancreatic endocrine- or pancreatic β cell-specific transdifferentiation using 5′-Aza and FGFb + TF-based protocols.…”

“…We have recently compared a panel of E and QM PDAC cell lines for their capacity to activate a ductal-to-endocrine transdifferentiation transcriptional program (deTDtP) using a combination of fibroblast growth factor-basic (FGFb) and transferrin (TF) as inducers [ 14 ]. While E type cell lines were not or only weakly responsive with regard to induction of the genes for Insulin (encoded by INS ), Glucose transporter-2 (GLUT-2, encoded by SLC2A2 ), and MafA (encoded by MAFA ), the QM lines PANC-1 and MIA PaCa-2 reacted strongly [ 23 ]. However, the molecular basis of this high deTD potential of PANC-1 (and MIA PaCa-2) cells, i.e., with regard to the involvement of stem cell markers or their upstream regulators, remains obscure.…”