The EMT-activator Zeb1 is a key factor for cell plasticity and promotes metastasis in pancreatic cancer

TL;DR: It is shown that different EMT-TFs have complementary subfunctions in driving pancreatic tumour metastasis, and that Depletion of Zeb1 suppresses stemness, colonization capacity and in particular phenotypic/metabolic plasticity of tumour cells, probably causing the observed in vivo effects.

Abstract: Metastasis is the major cause of cancer-associated death. Partial activation of the epithelial-to-mesenchymal transition program (partial EMT) was considered a major driver of tumour progression from initiation to metastasis. However, the role of EMT in promoting metastasis has recently been challenged, in particular concerning effects of the Snail and Twist EMT transcription factors (EMT-TFs) in pancreatic cancer. In contrast, we show here that in the same pancreatic cancer model, driven by Pdx1-cre-mediated activation of mutant Kras and p53 (KPC model), the EMT-TF Zeb1 is a key factor for the formation of precursor lesions, invasion and notably metastasis. Depletion of Zeb1 suppresses stemness, colonization capacity and in particular phenotypic/metabolic plasticity of tumour cells, probably causing the observed in vivo effects. Accordingly, we conclude that different EMT-TFs have complementary subfunctions in driving pancreatic tumour metastasis. Therapeutic strategies should consider these potential specificities of EMT-TFs to target these factors simultaneously.

Summary

Zeb1 depletion reduces grading, invasion and distant metastasis in PDAC

• KPC-mice develop metastatic pancreatic cancers with an almost 100% penetrance 9 .
• Progeny were born in expected ratios and showed no obvious functional defects of the pancreas.
• Next the authors analysed whether depletion of Zeb1 affects malignant tumour progression.
• A major finding was that the capacity for distant metastasis was strongly reduced in KPCZ tumours (Fig. 1f , Supplementary Table 1 ).

Zeb1 depletion reduces stemness, tumourigenic and colonisation capacities

• To further investigate the consequences of Zeb1 depletion, the authors isolated primary tumour cells from KPC and KPCZ mice.
• Tumourigenicity of the cell lines was significantly reduced in KPCZ cell lines, particularly when compared to the KPC cell lines with a similar epithelial phenotype (Supplementary Fig. 5b ).
• In addition, depletion of Zeb1 almost completely reduced the sphere forming capacity, a surrogate test for stemness competence (Fig. 3e and Supplementary Fig. 5c ).
• Strongly reduced Sox2 expression upon Zeb1 depletion was also reflected in the primary KPC tumours (Supplementary Fig. 2 ).
• Together their data indicate that Zeb1 increases the tumourigenic capacity and is crucial for colonisation of distant organs.

Zeb1 is crucial for cancer cell plasticity

• Zeb1 does not affect expression of single genes or small gene clusters but thousands of genes, leading to a complete reprogramming of cells 31 and the authors have shown that Zeb1 exerts pleiotropic effects on many different programs and pathways [31] [32] [33] .
• Thus, the authors hypothesized that the presence of Zeb1 allows adaptations of gene expression patterns and that loss of cellular plasticity is an important consequence of Zeb1 depletion in cancer cells.
• The genes associated with metastatic progression including Pdgfrb, which were not present in epithelial KPC cells, were also upregulated by TGFβ in a Zeb1-dependent manner (Fig. 6d ).
• The authors exemplified this by modulating the two basic energy consumption pathways: glycolysis and oxidative phosphorylation .
• Finally, high phenotypic plasticity of epithelial KPC cells was also detected in vivo after grafting into syngeneic mice.

DISCUSSION

• Here, the authors describe a key role for the EMT-TF Zeb1 in the in vivo progression of pancreatic cancer from early precursor lesions towards metastasis.
• Based on their results, Zheng et al. claimed that EMT is dispensable for metastasis.
• The authors postulate two major effects of Zeb1 inactivation as the underlying molecular mechanism: the block in cellular plasticity, considered as a major driving force of tumour progression towards metastasis and the reduction of stemness, a crucial property underlying tumourigenicity and colonisation.
• Differentiated KPC as well as KPCZ cancer cells only expressed low levels of metastasis-associated genes.
• Again, mutated p53 might enhance the generation of such a genetically driven metastasis 30 .

Ethic statement

• Animals were kept on a 12:12 h light-dark cycle and provided with food and water ad libitum.
• Animal husbandry and all experiments were performed according to the European Animal Welfare laws and guidelines.
• The protocols were approved by the committee on ethics of animal experiments of the states Baden-Württemberg and Bavaria (Regierungspräsidium Freiburg and Regierung Unterfranken, Würzburg).

Mice

• The Pdx1-Cre transgene (Tg(Pdx1-cre)6Tuv), the conditional Kras LSL.
• In brief, exon6 was flanked by loxP sites to remove sequences coding for large parts of the protein and to induce a premature translational stop.
• Once the tumour reached a maximum tolerated size (tumour diameter of 1 cm), mice were sacrificed, perfused and organs, tumour and macroscopic metastases were isolated.

Histology, histopathology and immunohistochemistry

• PFA-fixed tissues were embedded into paraffin, sectioned at 4-5µm and stained with Mayer's Haematoxylin and Eosin solution G (HE).
• For histopathological scoring, tumours were classified using the standard pathological grading scheme into either well differentiated (grade 1), moderately differentiated (grade 2), poorly differentiated (grade 3) and anaplastic or sarcomatoid (grade 4).
• Masson's trichrome staining (MTS) was performed according to the manufacturer's instructions (Sigma-Aldrich, HT15) and counterstained by Weigert's Iron Haematoxylin.
• After blocking in 3% BSA/PBS, tissue was incubated with anti-Zeb1 antibody (Sigma, HPA027524, 1:100) followed by Alexa594-conjugated secondary antibody (Life technologies).
• No statistical method was used to predetermine sample size and the experiments were not randomized.

Primary cell lines

• Successful and complete recombination of cell line deprivation was confirmed by PCR.
• For partial knockdown of Zeb1, cells were infected with lentivirus containing a pGIPZ shZeb1 knockdown (V2LMM_18639) or a pGIPZ non-silencing shRNA control construct.
• All experiments using primary cells in vitro were done at least in triplicates (n=3).
• Only primary cells from mouse tumours were used and these were not further authenticated nor tested for mycoplasma contamination.

Immunoblotting, RNA isolation and quantitative RT-PCR

• Protein was extracted with RIPA buffer and Western blotting was carried out as described 31, 32 with the exception that protein detection on the nitrocellulose membrane was done by incubation in Western Lightning Plus-ECL (Perkin Elmer, NEL103001EA) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, 34095) and a ChemiDoc imaging system .
• Western blots were done for at least three individual experiments and one representative blot is shown.
• Total RNA was isolated and reversely transcribed using the RNeasy Plus Mini Kit (Qiagen, 74136) and the RevertAid First Strand cDNA Synthesis Kit (Thermo, K1622) for mRNA and the miRCURY universal cDNA synthesis kit II (Exiqon, 203301) for miRNA.
• MiRNAs were analysed with the miRCURY ExiLENT SYBR Green Kit (Exiqon, 203421) with specific primer sets according to the manufacturer's instructions.
• All samples were run in a LightCycler 480 and values were normalised to Gapdh and Mir16-1 levels where appropriate and expressed relative to controls.

Cell viability (MTT) and BrdU cell proliferation assays

• IC50 values were calculated with GraphPad Prism using logarithmic transformed 5 data and nonlinear regression.
• For proliferation analysis 1,000 cells were plated in 96-well plates and BrdU incorporation was measured after a 2-h pulse with BrdU using the Cell Proliferation ELISA Kit (Roche, 11647229001) according to the manufacturer's instructions.

Immunofluorescence staining

• Cells were seeded on coverslips and fixed with 4% PFA, followed by permeabilization with 0.1% Triton X-100/PBS.
• After blocking in 3% BSA/PBS, cells were incubated with primary antibodies overnight at 4°C (polyclonal rabbit anti-Zeb1 (Sigma, HPA027524, 1:300); monoclonal mouse anti-E-Cadherin (BD Transduction Laboratories, 610182, Clone 36, 1:200), followed by appropriate Alexa594-and Alexa488-conjugated secondary antibodies (Life technologies) for 1 hour at RT.
• All images were acquired with a DM5500B microscope and the LAX software .
• All IF experiments were performed in at least three individual experiments and one representative image is shown.

Lung colonization/tumourigenicity

• Tumour cell colonisation and metastasising capacities to the lung were analysed by tail vein injections into syngeneic mice or NMRI-Foxn1 nu/nu mice.
• Mice were sacrificed after 18 days and analysed for 6 lung metastasis by HE staining.
• For short-term colonisation analysis cells were infected with pCDH-MSCV-LUC_EF1-GFP-T2A-Puro, selected by puromycin and sorted for medium to high levels of GFP expression.
• After tail vein injection mice were sacrificed after 2 h.
• For calculating tumourigenicity and analysis of tumour growth upon subcutaneous engraftment 500, 2,500, 12,500 and 100,000 cells were injected into flanks of C57BL/6 mice.

Microarray analysis, pre-processing, GSEA and data availability

• Gene expression of three epithelial, three mesenchymal KPC, six KPCZ, two TGFβ-treated epithelial KPC and two TGFβ-treated KPCZ cell lines was measured using Illumina Mouse WG6 v2 beadarrays (Illumina, San Diego, CA, USA).
• Total RNA was isolated, labelled and hybridised according to the manufacturer's protocol in two separate experiments.
• Illumina probes were mapped to Entrez IDs using the IlluminaMousev2 annotation (v. 1.26) from Bioconductor.
• Gene Set Enrichment analysis (GSEA) was performed using the Broad Institute platform (http://www.broadinstitute.org/gsea/index.jsp; Version 2.2.2).

Metabolic parameters

• Bioenergetics of epithelial KPC and KPCZ cell lines was determined using the XFe96 Extracellular Flux Analyzer (Seahorse Bioscience/Agilent Technologies, North Billerica, MA).
• For the determination of glycolytic parameters a glycose stress test was performed: basal extracellular acidification rate (ECAR; indicative of glycolysis) was first determined under glucose-free conditions.
• Finally, glycolytic capacity and glycolytic reserve were calculated after inhibition of mitochondrial respiration via oligomycin (Sigma, 75351, 1 µM) and hexokinase activity via 2-deoxy-glucose (2DG, Sigma, D6134, 100 mM). ).
• For the determination of respiratory parameters a mito stress test was performed: basal oxygen consumption rate (OCR, indicator for mitochondrial respiration) was measured.

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