The Quasimesenchymal Pancreatic Ductal Epithelial Cell Line PANC-1—A Useful Model to Study Clonal Heterogeneity and EMT Subtype Shifting

Abstract

Intratumoral heterogeneity (ITH) is an intrinsic feature of malignant tumors that eventually allows a subfraction of resistant cancer cells to clonally evolve and cause therapy failure or relapse. ITH, cellular plasticity and tumor progression are driven by epithelial–mesenchymal transition (EMT) and the reverse process, MET. During these developmental programs, epithelial (E) cells are successively converted to invasive mesenchymal (M) cells, or back to E cells, by passing through a series of intermediate E/M states, a phenomenon termed E–M plasticity (EMP). The induction of MET has clinical potential as it can block the initial EMT stages that favor tumor cell dissemination, while its inhibition can curb metastatic outgrowth at distant sites. In pancreatic ductal adenocarcinoma (PDAC), cellular models with which to study EMP or MET induction are scarce. Here, we have generated single cell-derived clonal cultures of the quasimesenchymal PDAC-derived cell line, PANC-1, and found that these differ strongly with respect to cell morphology and EMT marker expression, allowing for their tentative classification as E, E/M or M. Interestingly, the different EMT phenotypes were found to segregate with differences in tumorigenic potential in vitro, as measured by colony forming and invasive activities, and in circadian clock function. Moreover, the individual clones the phenotypes of which remained stable upon prolonged culture also responded differently to treatment with transforming growth factor (TGF)β1 in regard to regulation of growth and individual TGFβ target genes, and to culture conditions that favour ductal-to-endocrine transdifferentiation as a more direct measure for cellular plasticity. Of note, stimulation with TGFβ1 induced a shift in parental PANC-1 cultures towards a more extreme M and invasive phenotype, while exposing the cells to a combination of the proinflammatory cytokines IFNγ, IL1β and TNFα (IIT) elicited a shift towards a more E and less invasive phenotype resembling a MET-like process. Finally, we show that the actions of TGFβ1 and IIT both converge on regulating the ratio of the small GTPase RAC1 and its splice isoform, RAC1b. Our data provide strong evidence for dynamic EMT–MET transitions and qualify this cell line as a useful model with which to study EMP.

Suppressive Role of ACVR1/ALK2 in Basal and TGFβ1-Induced Cell Migration in Pancreatic Ductal Adenocarcinoma Cells and Identification of a Self-Perpetuating Autoregulatory Loop Involving the Small GTPase RAC1b

“…We employed the xCELLigence ® DP system (ACEA Biosciences, San Diego, CA, USA) to measure random/spontaneous cell migration in a chemokinesis setup according to previous descriptions [ 13 , 14 , 18 ]. Each well of the CIM plates-16 received 60,000–80,000 cells in a standard growth medium supplemented with 1% FBS.…”

“…We have recently identified RAC1b, a splice isoform of human RAC1 , as an important negative regulator of TGFβ signaling in PDAC-derived cells by its ability to suppress the expression of ALK5 and, as a consequence, the activation of SMAD3 [ 13 ]. Since, conversely, RAC1b protein levels are downregulated by the TGFβ1 via the ALK5 kinase in the PDAC line PANC-1 [ 14 ], the mutual inhibition of ALK5 and RAC1b constitutes a double-negative feedback loop. Although the downregulation of the TGFBR1 by RAC1b—and, as a consequence, the reduced ALK5-dependent activation of ALK2—is likely to be effective in blocking TGFβ signaling to SMAD1/5, the ALK5-independent negative regulation of ALK2 by RAC1b, if operating, would make this inhibition even more efficient.…”

“…We have recently established the PDAC-derived cell line PANC-1 as a suitable model to study EMTs/METs with a particular focus on the control of migration/invasion. Originally classified as quasimesenchymal [ 15 ], we were able to show that this cell line is heterogeneous in composition, consisting of cells with both mixed and mesenchymal phenotypes [ 14 ]. Induction of the EMT in these cells by the TGFβ1 was associated with a strong increase in migratory activity, while the MET induction was evident by a concomitant decrease [ 14 ].…”

“…Originally classified as quasimesenchymal [ 15 ], we were able to show that this cell line is heterogeneous in composition, consisting of cells with both mixed and mesenchymal phenotypes [ 14 ]. Induction of the EMT in these cells by the TGFβ1 was associated with a strong increase in migratory activity, while the MET induction was evident by a concomitant decrease [ 14 ]. Concomitantly, we observed a downregulation of the mesenchymal markers with promigratory functions, such as RAC1, vimentin, and SNAIL, and an upregulation of the epithelial markers, such as Claudin-4, GRHL2, and OVOL2, most of which also represent TGFβ target genes [ 14 ].…”

Grape-seed proanthocyanidin extract (GSPE) modulates diurnal oscillations of key hepatic metabolic genes and metabolites alleviating hepatic lipid deposition in cafeteria-fed obese rats in a time-of-day-dependent manner

“…For bioluminescence data, rhythmicity was assessed using the Circasingle algorithm as previously described [44] using the normalized bioluminescent data per hour. CircaSingle algorithm was set to consider amplitude a decay factor and period was not pre-established.…”