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. 2022 Sep 2;8(1):378.
doi: 10.1038/s41420-022-01165-4.

Pharmacological inhibition and reversal of pancreatic acinar ductal metaplasia

Lais da Silva #  1 , Jinmai Jiang #  1 , Corey Perkins #  1 , Kalina Rosenova Atanasova  2   3 , Julie K Bray  4 , Gamze Bulut  1 , Ana Azevedo-Pouly  5 , Martha Campbell-Thompson  4 , Xiaozhi Yang  2 , Hesamedin Hakimjavadi  4 , Srikar Chamala  4 , Ranjala Ratnayake  2   3 , Raad Z Gharaibeh  6   7 , Chenglong Li  2   3 , Hendrik Luesch  2   3 , Thomas D Schmittgen  8
Affiliations

Pharmacological inhibition and reversal of pancreatic acinar ductal metaplasia

Lais da Silva et al. Cell Death Discov. .

Abstract

Pancreatic acinar cells display a remarkable degree of plasticity and can dedifferentiate into ductal-like progenitor cells by a process known as acinar ductal metaplasia (ADM). ADM is believed to be one of the earliest precursor lesions toward the development of pancreatic ductal adenocarcinoma and maintaining the pancreatic acinar cell phenotype suppresses tumor formation. The effects of a novel pStat3 inhibitor (LLL12B) and the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) were investigated using 3-D cultures from p48Cre/+ and p48Cre/+LSL-KrasG12D/+ (KC) mice. LLL12B and TSA inhibited ADM in both KC and p48Cre/+ mouse pancreatic organoids. Furthermore, treatment with LLL12B or TSA on dedifferentiated acini from p48Cre/+ and KC mice that had undergone ADM produced morphologic and gene expression changes that suggest a reversal of ADM. Validation experiments using qRT-PCR (p48Cre/+ and KC) and RNA sequencing (KC) of the LLL12B and TSA treated cultures showed that the ADM reversal was more robust for the TSA treatments. Pathway analysis showed that TSA inhibited Spink1 and PI3K/AKT signaling during ADM reversal. The ability of TSA to reverse ADM was also observed in primary human acinar cultures. We report that pStat3 and HDAC inhibition can attenuate ADM in vitro and reverse ADM in the context of wild-type Kras. Our findings suggest that pharmacological inhibition or reversal of pancreatic ADM represents a potential therapeutic strategy for blocking aberrant ductal reprogramming of acinar cells.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. The pStat3 inhibitor LLL12B attenuates ADM.
A p48Cre/+ mouse acinar cells were treated with LLL12B or untreated control for 4 days and the amount of ADM was microscopically quantified. B KC mouse pancreatic acini were treated with LLL12B or untreated control for 2.5 days and the amount of ADM was calculated microscopically. The viability of p48Cre/+ and KC mice treated with 500 nM LLL12B or untreated control for 4 and 2.5 days, respectively, was determined by C calcein AM staining or D MTT assay. The effects of LLL12B on the mRNA expression of Cpa2 and Krt19 in E p48Cre/+ (4-day exposure of 500 nM) or F KC (2.5-day exposure of 500 nM) mouse organoids as determined by qRT-PCR. The control group represents untreated control while basal represents the untreated day one cultures. G Western blot of KC mouse organoids from untreated (UnTx) or 1000 nM LLL12B treatment (Tx) for 6 h. The phosphorylation site for pStat3 is Tyr705. Mean ± SD from triplicate treatments. **P < 0.01, ***P < 0.005. ****P < 0.001.
Fig. 2
Fig. 2. The HDAC inhibitor TSA attenuates ADM.
p48Cre/+ (A) and KC (B) primary mouse pancreatic acini were treated with TSA for 3 and 2 days, respectively, and the amount of ADM was calculated microscopically. p48Cre/+ (C) and KC (D) acinar cultures were exposed to 500 nM TSA or untreated control for 3 and 2 days, respectively, and the expression of Cpa2 and Krt19 was determined using qRT-PCR. Mean ± SD from triplicate treatments. *P < 0.05; ***P < 0.001, n.s. not significant.
Fig. 3
Fig. 3. Reversal of ADM by LLL12B in p48Cre/+ mouse acini.
A p48Cre/+ mouse pancreatic acini underwent ADM over 3 days of culture and were then exposed to increasing concentrations of LLL12B for 3 additional days. B KC mouse pancreatic acini underwent ADM over 2 days of culture and were then exposed to increasing concentrations of LLL12B for 2 additional days. Expression of acinar and ductal genes from LLL12B treated p48Cre/+ (C) or KC (D) mouse pancreatic organoids following the identical treatment as in (A) and (B), respectively. Data are presented as fold-change normalized to 18S rRNA and relative to the untreated control. Mean ± SD from duplicate experiments. *P < 0.05; **P < 0.01.
Fig. 4
Fig. 4. Reversal of ADM by TSA in p48Cre/+ mouse acini.
p48Cre/+ mouse pancreatic acini underwent ADM over 3 days of culture (A) and were then exposed to DMSO (B) or 1 µM TSA (C) for 3 additional days followed by Calcein AM viability staining. Shown are the high content images, with the enlarged sections (dashed lines) in the lower panels. Scale bars in the top images represent 500 µm and in the lower images 100 µm. D Acini and ducts were microscopically counted from four sections of the high content images. E Expression of acinar and ductal genes from TSA treated p48Cre/+ mouse pancreatic organoids following the identical treatment as in (A–C). Data are presented as fold-change normalized to 18S rRNA and relative to the untreated control. Mean ± SD from duplicate experiments. *P < 0.05; ***P < 0.001.
Fig. 5
Fig. 5. Reversal of ADM by TSA in KC mouse acini.
KC mouse pancreatic acini underwent ADM over 2 days of culture (A) and were then exposed to DMSO (B) or 1 µM TSA (C) for 2 additional days followed by Calcein AM viability staining. Shown are the high content images, with the enlarged sections (dashed lines) in the lower panels. Scale bars in the top images represent 500 µm and in the lower images 100 µm. D Acini and ducts were microscopically counted from four sections of the high content images. E Expression of acinar and ductal genes from TSA treated KC mouse pancreatic organoids following the identical treatment as in (A–C). Data are presented as fold-change normalized to 18S rRNA and relative to the untreated control. Mean ± SD from duplicate experiments. *P < 0.05; **P < 0.01.
Fig. 6
Fig. 6. Acinar and ductal changes during dedifferentiation of KC mouse acini by TSA.
KC mouse pancreatic acini were plated onto Matrigel and were fixed following one day of culture (A). Yellow arrows, ducts formed following ADM. After 2 complete days of culture the cells were either untreated (B) or exposed to 500 nM TSA (C) and cultured for 2 additional days before fixation and immunostaining. Scale bars: 50 µm.
Fig. 7
Fig. 7. Reversal of ADM by TSA in primary human acinar cultures.
Primary, human acini were cultured in Matrigel on glass chamber slides for 5 days. On day 5 of culture, 500 nM of TSA was added and the cells were cultured for 5 additional days. The cells were processed for immunohistochemistry and imaged for amylase (red) and KRT19 (green) using confocal microscopy.
Fig. 8
Fig. 8. Volcano plot of differentially expressed genes from TSA-induced ADM reversal in KC mouse acini.
KC mouse pancreatic acini underwent ADM over 2 days of culture and were then exposed to 500 nM of TSA for 2 additional days. RNA isolated from the treated and day 6 untreated control were subjected to Illumina NovaSeq6000 whole transcriptome sequencing. Shown are the expression of a selected set of 82 genes which are the mouse equivalents to those previously identified as associated with the pancreatic acinar/ductal phenotype or genes associated with the onset or progression of PDAC as described in the “Methods” section.
Fig. 9
Fig. 9. Pathway and upstream analysis during the reversal of ADM by TSA in KC acinar cells.
RNA sequencing data from the ADM reversal in KC mouse acini following 500 nM TSA treatment was analyzed using Ingenuity Pathway Analysis. A, C Top 10 transcription factors involved in the upstream regulation ranked by Z-score. B, D Most highly ranked signaling pathways ranked by Z-score. E Top 10 chemical drugs predicted to regulate the signaling pathways. Blue, inhibited and orange, activated pathways.
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