EMBO/soda-nel huggingface dataset:
IFNα gene/cell therapy curbs colorectal cancer colonization of the liver by acting on the hepatic microenvironment (2015)
Figure 5

Figure 5. Effective intrahepatic CRC cells growth inhibition and improved survival following Tie2-IFNα HSPCs transplantation in mice with established metastatic liver tumors.

A. Schematic representation of the experimental procedure.

B. Tumor volume quantification measured by MRI analysis of Tie2-GFP (n=6) or Tie2-IFNα mice (n=9) at the indicated time points post-transplant, one additional Tie2-IFNα mouse showed a tumor volume at day 21 of more than 820 mm3 and was statistically rejected by the Grubbs’ test and not further analyzed; mean values are shown; error bars=S.E.M.; p=0.051, by Mann-Whitney test.

C. Images (top panels) and corresponding contrast-enhanced MRI (middle and bottom panels) of the liver from representative Tie2-GFP mice or Tie2-IFNα mice that were intrahepatically injected with 5x103 CT26. White dashed lines identify macroscopic lesions. Red and green dashed lines identify hepatic or extrahepatic CRC metastases respectively from representative MRI z-stacks. Tumors detected by MRI analysis appeared as hypointense and slightly-hyperintense regions in T1 and T2 weighted sequences, respectively. Scale bars=5mm.

D. Percentage of mice with peritoneal carcinomatosis (defined by the presence of multiple intra-peritoneal lesions) determined by MRI analysis from the same mice described in B. Note that while intrahepatic number of lesions remained similar between time points, extrahepatic tumor spreading was significantly reduced in Tie2-IFNα mice. P-values were calculated by Fisher’s exact test.

E. Confocal immunofluorescence images of representative liver sections from Tie2-GFP mice at distal (upper panels) or proximal (bottom panels) areas to CRC liver metastasis, 30 days post HSPC transplant. Note that TEMs were identified as GFP+ cells and by the concomitant expression of different levels of the myeloid cell marker CD11b+ as highlighted in the inset (merge panels, white arrows); the dashed line identifies the metastasis margin; ✱=CRC metastatic area; scale bars=100μm; insets depict the corresponding areas identified by dashed squares in the merge panels, magnified 1.5-folds.

F, G. Quantitative real-time PCR analyses of the relative expression levels of the interferon inducible genes Irf7 and Oas1 within liver of Tie2-GFP (n=4) or Tie2-IFNα (n=4) mice euthanized 30 days post-transplant. The basal expression of Irf7 estimated in liver of control mice (i.e. Tie2-GFP injected with saline), was set to 1 and utilized to calculate the fold increase values observed. Mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test.

H. Kaplan-Meier survival curves of the indicated groups of mice. Tie2-GFP (n=3); Tie2-IFNα (n=5); p=0.007 by log-rank/Mantel-Cox test.
IFNα gene/cell therapy curbs colorectal cancer colonization of the liver by acting on the hepatic microenvironment (2015)
Figure 6

Figure 6. TEMs preferentially accumulate in the peritumoral area of human colorectal cancer liver metastases.

A. IHL and TEM characterization in the liver of patients described in Appendix Fig S6A. Liver samples from surgical resections (n=9) were weighted and IHLs were isolated, counted and normalized over tissue weight (left panel). Flow cytometry analysis revealed that TEMs, identified as 7AAD-/CD45+/CD11b+/CD14+/Tie2+ cells, are detectable within the total IHL populations and accumulate preferentially in the proximity of the metastatic lesions (right panel). Control=hepatic hemangioma; Distal=distal to CRC liver metastasis (>1 cm from the lesion); Proximal=proximal to CRC liver metastasis (<1 cm from the lesion); p-values were calculated by Wilcoxon matched pairs Test.

B-C. Representative TIE2 immunohistochemistry staining from the liver of a patient with liver CRC metastases from distal (>1 cm; left panels) or proximal (<1 cm; right panels) sites from the CRC liver lesion. C. Higher magnification of the panels identified by a rectangle in panel B. Negative control (bottom panels) is obtained by omitting primary anti-TIE2 antibodies. TIE2 immunostaining, indicates that sites that are distal from the lesion contain only TIE2+ cells with an apparent endothelial morphology (characterized by an elongated appearance, arrows) while sites that are proximal to the lesion also contain additional TIE2+ cells with an apparent monocyte-like morphology (characterized by a round appearance, arrowheads). The dashed lines identifies the metastasis margin; ✱=CRC metastatic area. Scale bars of upper panels=50μm; middle and bottom panels=20 μm.
HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer (2020)
Figure 1

Figure 1.

HNF1A-deficiency leads to increased proliferation and promotes Kras-induced oncogenesis

A. Representative immunofluorescence images and quantifications showing that 3 months old Hnf1apKO mice have increased number of KI67+ (red) acinar cell nuclei co-staining with DAPI (blue) and Amylase (green). Arrows point to KI67+ acinar cells in Hnf1apKO mouse. Scale bar is 200 μm. Acinar proliferation is represented as the average of the KI67+/Amylase+ cell ratio. Quantifications were performed on 3 random fields from 3 Pdx1Cre and 3 Hnf1apKO mice. P-values are from two-tailed Student's t test. Representative H&E stainings of pancreata from KrasG12D and Hnf1apKO;KrasG12D mice.

B-D. KrasG12D and Hnf1apKO;KrasG12D mice have normal morphology at 7 days.

E-J. At 21 days Hnf1apKO;KrasG12D mice show acinar-to-ductal metaplasia (dashed encircled areas) and regions with desmoplastic reaction (asterisk), which are not observed in KrasG12D mice (E, H).

K-P. At 8 weeks KrasG12D pancreas show occasional abnormal ductal structures (dashed encircled areas in N which is a magnification of squared dotted box in K) and Hnf1apKO;KrasG12D mice (L,M,O,P) present mucinous tubular complexes (black arrows), and more advanced PanINs with luminal budding (open arrows) including foci of spindle cell proliferation (asterisks) and incipient infiltrative growth (black dashed box area in O).

Data information: Black dashed boxes in (E,F, K, L and O) indicate magnified areas in (H, G, N, M and P) respectively. Scale bars indicate 100 µm (C,E,F,K,L), 50 µm (O) and 20 µm (B,D,G,H-J,M,N,P).
HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer (2020)
Figure 2

Figure 2.

HNF1A regulates gene programs essential to acinar cell identity, metabolism and proliferation

A. Fold change (FC) in transcripts in Hnf1aaKO vs. control pancreas, plotted against significance (-Log10 q; genes significant at q<0.05 are shown as colored dots above the horizontal line).

B. GSEA showing that genes specific to differentiated acinar cells were downregulated in Hnf1aaKO pancreas, but not genes specific to islets or duct cells. Upregulated genes were enriched in genes specific to mesenchymal cells. Lineage-enriched genes were obtained from Muraro et al. (Muraro et al., 2016).

C. Top functional annotations for differentially expressed genes in Hnf1aaKO pancreas.

D. GSEA revealed that Hnf1aaKO pancreas showed increased transcripts involved in oncogenic pathways such as EMT, MAPK, KRAS, PI3K-AKT.

E. HNF1A promotes transcriptional activation of direct target genes. Left: HNF1A bound-genes were enriched among genes that showed downregulation in Hnf1a mutants, but not among upregulated genes. P-values and odds ratios (O.R.) calculated by Fisher's exact test. Right: Venn diagrams showing overlap of HNF1A-bound genes with genes that were downregulated and upregulated in Hnf1a mutant pancreas.
HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer (2020)
Figure 4

Figure 4.

Kdm6a-dependent Kras-induced oncogenesis

H&E staining of pancreata from Kdm6apKO;KrasG12D and KrasG12D mice.

A-C. 7 day-old KrasG12D and Kdm6apKO;KrasG12D mice showed normal morphology.

D-I. At 21 days, KrasG12D mice showed normal pancreas morphology (dotted area at in D is shown at large magnification in G) whereas Kdm6apKO;KrasG12D mice already show acinar-to-ductal metaplasia (dashed encircled area in F), spindle cell proliferation (asterisks in E and H), sarcomatoid architecture (I) and desmoplastic reaction (black arrowhead in H).

J-O. KrasG12D mice present occasional acinar-to-ductal metaplasia and low grade PanINs at 8 weeks (M is a magnification of squared dotted box in J and see Fig 1N) whereas at the same age pancreas from Kdm6apKO;KrasG12D mice show massive remodeling (K), extensive acinar-to-ductal metaplasia (L and dashed encircled area in N), cancer with prominent spindle/mesenchymal proliferation, infiltrative growth (black arrows and asterisk respectively in O) and abundant stroma (black arrowheads in N).

Data information: Scale bars: 100 µm (B,D,E,J,K) or 20 µm (A,C,F-I, L-N).
HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer (2020)
Figure 5

Figure 5.

KDM6A promotes acinar cell differentiation gene programs and suppresses oncogenic pathways

A. Fold change (FC) and significance for transcripts in Kdm6apKO vs. control pancreas. Genes significant at q<0.05 are shown as colored dots above the horizontal line.

B. GSEA showing that genes specific to differentiated acinar cells were downregulated in KDM6A-deficient pancreas, but not genes specific to islets or duct cells. Upregulated genes were enriched in genes specific to mesenchymal cells. Lineage-enriched genes were obtained from (Muraro et al., 2016).

C. Most enriched functional annotations in genes differentially expressed in Kdm6apKO.

D. GSEA showed increased expression of indicated oncogenic pathway genes in Kdm6apKO pancreas.

E. KDM6A functions as a transcriptional activator of direct target genes in pancreatic cells. Left: Downregulated, but not upregulated, genes in Kdm6apKO pancreas were enriched for KDM6A binding. P-values and odds ratios (O.R.) were calculated by Fisher's exact test. Right: Venn diagrams showing overlap of down and upregulated genes with KDM6A binding.

F. KDM6A-bound enhancers and promoters showed increased H3K27me3 and decreased H3K27ac in Kdm6apKO pancreas.

G. Genes that were downregulated in Kdm6apKO pancreas (KDM6A-dependent genes) showed greatest changes in histone marks. Box plots show fold-changes of H3K27me3 (left) and H3K27ac (right) signals in Kdm6apKO compared to control. Signals were analyzed in KDM6A bound regions in promoters and enhancers of genes that were KDM6A dependent (grey; n=420 regions) and independent (white; n=8035 regions). The signals are average values from ChIP-seq experiments in two biological replicates. The horizontal central line marks the median. Box limits indicate the first and third quartiles and whiskers extend to highest and lowest data points within 1.5 x IQR outside box limits. P-values were determined by two-tailed Mann-Whitney U test.

H. Changes in KDM6A, H3K27me3 and H3K27ac ChIP-seq profiles in the Ppp4r4-Serpina10 locus in Kdm6apKO pancreas.

I. qPCR of H3K27me3, H3K27ac and H3K4me1 changes in promoter and enhancer regions highlighted in (H) (R1, R2, R3), and Serpin10 mRNA. Error bars show +/-SD and P-values are from two-tailed Student's t test, n=3.

J. KDM6A-bound genes that showed decreased expression in Kdm6apKO pancreas (functional KDM6A binding sites) showed simultaneous gain of H3K27me3 and loss and of H3K27ac.
HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer (2020)
Figure 6

Figure 6.

KDM6A and HNF1A regulate shared target genes

A. Motif analysis in functional KDM6A-bound regions, showing top ten de novo motifs ranked by P-value determined by HOMER software.

B. Co-binding analysis in functional KDM6A-bound enhancer and promoter regions revealed that HNF1A was the most enriched co-bound TF amongst three other acinar cell TFs. Binding regions of TAL1 in a non-pancreatic cell type and random binding sites were used as negative controls. P-values was determined by Fisher's exact test for peak comparisons using all enhancer and promoter regions as background.

C. The most downregulated genes in Kdm6apKO pancreas are shown ranked by q-value, and are almost invariably bound by HNF1A and down regulated in Hnf1aaKO pancreas, or known to be direct HNF1A-dependent target genes from other studies (red and purple, respectively).

D,E. GSEA analysis on the Hnf1aaKO and Kdm6apKO ranked-ordered gene lists vs. their reciprocal up- or downregulated gene sets, demonstrated that KDM6A and HNF1A regulate similar genes.

F. Expression changes in Hnf1aaKO and Kdm6apKO pancreas, showing that genes bound by KDM6A and downregulated in Kdm6apKO pancreas (red dots) were generally downregulated in Hnf1aaKO pancreas.

G. HNF1A and KDM6A co-occupy the same regions in Pah, which is downregulated in Hnf1a and Kdm6a knock-out pancreas.

H. Genes that were co-bound by KDM6A and HNF1A showed greatest downregulation in Kdm6apKO pancreas, compared with KDM6A-bound genes that were not bound by HNF1A. Box plots show median and IQR of Log2 TPM fold-changes and whiskers extend to highest and lowest data points within 1.5 x IQR outside box limits. P-values were determined by two-tailed Student's t tests and n=4 replicates per condition.
HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer (2020)
Figure 7

Figure 7.

HNF1A recruits KDM6A to activate transcription of its target genes

A. Co-immunoprecipitation of endogenous HNF1A and KDM6A followed by western blot demonstrated that HNF1A is in the same complex as KDM6A.

B. Western blot showing loss of HNF1A and unchanged KDM6A in Hnf1a-/- pancreas.

C. Differential binding analysis of KDM6A in Hnf1a-/- vs. wild type pancreas. Pink dots below zero (1873 sites) show regions with reduced KDM6A binding and pink dots above zero (118 sites) are regions with increased binding at FDR<0.05.

D,E. Regions that show reduced KDM6A binding in Hnf1a-/- chromatin are strongly bound by HNF1A and are highly enriched in HNF1 motifs. P-values in (D) were calculated with two-tailed Mann-Whitney U test and in (E) with Fisher's exact test.

F. KDM6A binding is markedly reduced in HNF1A and KDM6A co-bound regions in Hnf1a-/- pancreas, but not in other KDM6A bound regions.

G. Genes that loose KDM6A binding in Hnf1a-mutant pancreas are predominantly downregulated in Hnf1aaKO pancreas and are direct HNF1A target genes (red dots).

H. Summary model depicting that HNF1A recruits KDM6A to genomic binding sites, activating an acinar differentiation program that indirectly suppresses core oncogenic pathways. Defective HNF1A or KDM6A function results in failure of this shared program, with increased activity of pathways that, in the presence of KRAS mutations, promote high-grade non-classical PDAC with sarcomatoid features.

Expanded View Figure Legends:
Beclin–phosphatidylinositol 3‐kinase complex functions at the trans‐Golgi network (2001)
Figure 1

Beclin and PtdIns 3‐kinase form a complex. (A) Cell lysates prepared from HeLa cells were solubilized with Triton X‐100 and subjected to immunoprecipitation using preimmune sera (lane 1) or anti‐Beclin antibodies (lanes 2 and 3). The immunoprecipitates were incubated with phosphatidylinositol, [γ‐32P]ATP and 60 μM cold ATP in the presence of Mn2+ (lanes 1 and 2) or Mg2+ (lane 3) for 5 min at 30°C. (B) Immunoprecipitates of anti‐Beclin antibodies prepared as described in (A) were assayed for PI 3‐kinase activity in the presence of wortmannin at concentrations of 0 nM (lane 1), 1 nM (lane 2), 10 nM (lane 3), 100 nM (lane 4). The labeled lipids were extracted and separated by thin layer chromatography, followed by detection by autoradiography using a bioimaging analyzer BAS2000 (Fuji Film). (C) Cell lysates prepared from HeLa cells were solubilized with Triton X‐100 and incubated with protein A‐immobilized anti‐Beclin (lane 2) or anti‐PtdIns 3‐kinase antibodies (lane 4). As controls, preimmune sera of the respective antibodies (lanes 1 and 3) were used. Retained proteins were separated by SDS–PAGE and detected by immunoblotting with anti‐PtdIns 3‐kinase (upper panels) and anti‐Beclin (lower panels) antibodies.
Beclin–phosphatidylinositol 3‐kinase complex functions at the trans‐Golgi network (2001)
Figure 2

Cross‐linking of Beclin and PtdIns 3‐kinase. (A) Total cell lysates were treated with DSP (lanes 3, 4, 7 and 8) or its solvent, dimethylsulfoxide (lanes 1, 2, 5 and 6), at 4°C for 2 h. Proteins were solubilized with SDS and subjected to immunoprecipitation using protein A‐immobilized anti‐PtdIns 3‐kinase (PtdIns 3‐K) (lanes 1–4) or Beclin (lanes 5–8) antibodies. Immunoprecipitates were eluted with 1× SDS sample buffer (62.5 mM Tris–HCl pH 6.8, 2% SDS, 10% glycerol, a trace amount of Bromophenol blue). After treatment with 5% 2‐mercaptoethanol, proteins were separated by SDS–PAGE, followed by detection by immunoblotting with anti‐Beclin (upper panels) and anti‐PtdIns 3‐kinase (lower panels) antibodies. (B) Cell lysates prepared from HeLa cells were subjected to subcellular fractionation by centrifugation at 100 000 g for 1 h. The membrane (lanes 1 and 3) and supernatant (lanes 2 and 4) fractions were subjected to SDS–PAGE, followed by immunoblotting with anti‐PtdIns 3‐kinase (lanes 1 and 2) and anti‐Beclin (lanes 3 and 4) antibodies. (C) Membrane (lanes 1–4; 100 000 g, 30 min, pellet) and soluble fractions (lanes 5–8; 100 000 g, 30 min, supernatant) prepared from HeLa cells were treated with DSP (lanes 3, 4, 7 and 8) or dimethylsulfoxide (lanes 1, 2, 5 and 6). Proteins were solubilized with SDS and subjected to immunoprecipitation using protein A‐immobilized anti‐PtdIns 3‐kinase antibodies. Immunoprecipitates were separated by SDS–PAGE under a reducing condition, followed by detection by immunoblotting with Beclin antibodies. F and E indicate flow‐through and elute fracions, respectively.
Beclin–phosphatidylinositol 3‐kinase complex functions at the trans‐Golgi network (2001)
Figure 3

Beclin localizes to the TGN. HeLa (A, D, E and F), BNL CL.2 (B) and H‐4‐II‐E cells (C) were grown on cover slips. Cells were permeabilized with digitonin before (C, D and E) or after (A, B and F) fixation with 3% paraformaldehyde. Cells were double‐labeled with anti‐Beclin (A–F, left panels) and either anti‐syntaxin 6 (A and B, middle panels), anti‐TGN38 (C, middle panel), anti‐EEA1 (D, middle panel), anti‐LBPA (E, middle panel) or anti‐TfR (F, middle panel) antibodies. In the merged images (right panels), yellow indicates co‐localization. Bar, 10 μm.
Beclin–phosphatidylinositol 3‐kinase complex functions at the trans‐Golgi network (2001)
Figure 4

Immunofluorescence localization of PtdIns 3‐kinase. HeLa (A and C) and H‐4‐II‐E cells (B) were permeabilized, fixed, and then subjected to immunostaining. Cells were double‐labeled with anti‐PtdIns 3‐kinase (A–C, left panels) and either anti‐syntaxin 6 (A, middle panel), anti‐TGN38 (B, middle panel) or anti‐LBPA (C, middle panel) antibodies. Bar, 10 μm.
Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis (2006)
figf1

(a) Cell death was analysed after the activation of death pathways in the presence and absence of enforced Atg5 expression (18 h cultures). Values are means ± s.e.m. of three independent experiments. (b) The cell death regulated by Atg5 is apoptosis. All data presented are from 5 h cultures. Caspase-3-like activity analysed in HeLa cells (graph). Morphology of Jurkat cells is also shown. The numbers in the lower right corner indicate the percentage of cell death, which was determined in parallel experiments using flow cytometry. (c) Staurosporine induced redistribution of phosphatidylserine was enhanced in Atg5 overexpressing Jurkat cells and blocked by z-VAD-fmk. Numbers indicate the results of the quantitative analysis (percentage) of each dot blot. (d) Reduction of drug-induced cell death by silencing of Atg5. Values are means ± s.e.m. of three independent experiments (18 h cultures). The effects of the Atg5 siRNA on Atg5 and GAPDH levels were analysed by immunoblotting. The asterisks indicate P 0.05. n.s., no significant difference.
Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis (2006)
figf2

(a) Spontaneous neutrophil apoptosis. Apoptosis was associated with the appearance of a 24K anti-Atg5-reactive protein. 33K Atg5 disappeared during apoptosis in a time-dependent manner. HeLa cells overexpressing Atg5 served as positive control. (b) Anti-CD95-induced apoptosis of Jurkat cells. Apoptosis was associated with the appearance of a 24K anti-Atg5-reactive protein and a reduction of 33K Atg5 in a time-dependent manner. The 24K anti-Atg5-reactive protein was also visible in Jurkat cells after treatment with actinomycin D (Act. D) or cycloheximide (CHX; 5 h treatment in both cases). GAPDH expression indicates equal loading in each panel. All immmunoblots are representative of at least three independent experiments.
Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis (2006)
figf3

(a) Caspases-3, -6, -7, -8 and -9 did not cleave 35S-Atg5. To control the reaction conditions, 35S-Bid was cleaved by caspase-8. The enzymatic activity of caspases-3, -6, -7 and -9 were controlled by incubating them together with lysates of Jurkat cells and subsequent caspase-3 immunoblotting experiments (data not shown). (b) Cathepsins B and D did not cleave 35S-Atg5. To control the reaction conditions, 35S-Bid was cleaved by cathepsin B. (c) Calpains-1 and -2 cleaved 35S-Atg5. The calpain inhibitors calpastatin, ALLN and E64-d completely prevented calpain-mediated cleavage of 35S-Atg5. (d) Following calpain-mediated cleavage of recombinant Atg5 in vitro, the sample was fractionated by preparative reversed-phase HPLC and analysed by mass spectrometry. The exact relative molecular masses for full-length and truncated Atg5 are indicated. (e) Recombinant Atg5 was digested with calpain and the products sequenced by Edman degradation. Calpain cleaved Atg5 at Thr 193. Comparison of Atg5 sequences of different species demonstrates the presence of the same calpain cleavage site in mice. The sequence alignment was perfromed with Clustal W using sequence data obtained from GenBank. The position of the six-amino-acid deletion causing calpain resistance is indicated in red. (f) A six-amino-acid deletion surrounding the cleavage site (35S-Atg5Δ191–196) led to a loss of the calpain cleavage fragment. (g) Calpain inhibition by ALLN blocked both cleavage of Bax and Atg5 in spontaneous and Fas receptor-mediated apoptosis of neutrophils. In contrast, Atg6 did not show evidence for cleavage and its levels remained unchanged in this system. Results of 7 h cultures are shown. (h) Atg5 is not cleaved in apoptotic HeLa cells (5 h staurosporine treatment) lacking the small subunit of calpain, which was silenced by using specific siRNA. (i) Calpain resistant Atg5Δ191–196 was unable to sensitize HeLa cells lacking endogeneous Atg5 (due to gene silencing, see Fig. 1c) to staurosporine-induced cell death. Values are means ± s.e.m. of three independent experiments (18 h cultures).