Journal: Oncogene

Article Title: Targeting epiregulin in the treatment-damaged tumor microenvironment restrains therapeutic resistance

doi: 10.1038/s41388-022-02476-7

Figure Lengend Snippet: a Transcriptome-wide profiling of gene expression changes in primary normal human prostate stromal cell line (PSC27) by microarray. Cell lysates were collected for analysis 7 days after treatment. CTRL control. H 2 O 2 hydrogen peroxide. BLEO bleomycin. RAD radiation. Red highlighted, EREG. Agilent microarray data adapted from Sun et al. with permission from Nature Medicine , copyright 2012, Springer Nature . b Representative immunofluorescence staining images (γH2AX and p-53BP1 co-staining, left) and comparative statistics (right) of DNA damage response (DDR) in PSC27 cells treated by DOX (doxorubicin), MIT (mitoxantrone), BLEO (bleomycin), DTX (docetaxel), PTX (paclitaxel) and VBL (vinblastine). DDA DNA-damaging agents (DDAs). NDDA non-DNA-damaging agents. DDR were classified into four sub-categories including 0 foci, 1–3 foci, 4–10 foci and >10 foci per cell. Scale bars, 15 μm. c SA-β-Gal staining of PSC27 cells treated by various agents used in b . Cells were stained 7 days after in vitro treatments. Scale bars, 30 μm. Right, comparative statistics. d BrdU staining of stromal cells treated by different agents as indicated in b and c . Scale bars, 15 μm. Right, comparative statistics. e Quantitative RT-PCR of EREG expression after treatment of PSC27 cells by various agents. Cell lysates were collected for measurement 7 days after treatment. Signals normalized to CTRL. f Immunoblot analysis of EREG expression in stromal cells 7 days after treatments performed as indicated. IC intracellular samples. CM conditioned media. GAPDH, loading control. g Time course expression assessment of a subset of EREG and other typical SASP factors (CXCL8, CSF2, WNT16B, IL6 and MMP3) after drug treatment of stromal cells in vitro. Numeric numbers indicate the individual days after treatment. h Immunoblot measurement of EREG expression at the protein level in the time course described in g . i Comparative appraisal of EREG transcript expression in stromal cells (PSC27) versus cancer epithelial cells (PC3, DU145, LNCaP and M12). Signals normalized to untreated sample per cell line. j Immunoblot assessment of EREG expression in protein lysates of stromal and epithelial cells after bleomycin treatment as performed in i . Data are representative of three independent experiments. ^ p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. p values were calculated by Student’s t test ( c – e , g ) and two-way ANOVA ( b , i ). ^ p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Primary normal human prostate stromal cell line PSC27, breast stromal cell line HBF1203 and lung stromal cell line HFL1 (ATCC) were maintained in stromal complete medium as described [ ].

Techniques: Gene Expression, Microarray, Control, Immunofluorescence, Staining, In Vitro, BrdU Staining, Quantitative RT-PCR, Expressing, Western Blot

Journal: Oncogene

Article Title: Targeting epiregulin in the treatment-damaged tumor microenvironment restrains therapeutic resistance

doi: 10.1038/s41388-022-02476-7

Figure Lengend Snippet: a Schematic of putative NF-κB binding sites in the proximal region of EREG promoter. A set of reporter constructs was generated by sequential cloning of the promoter fragments into a pGL4.22 vector (pGL-EREG-P01 to P05) that expresses firefly luciferase. Numeric numbers on the top denote the core site of each putative NF-κB binding motif, while numbers at the left mark the length of each segmental promoter clone. TSS transcription start site. Lower-left inlet, consensus binding motif of the NF-κB subunit p65. b Assessment of luciferase activities upon exposure of 293F cells pre-transfected with the individual EREG promoter constructs to TNF-α at 40 ng/ml in culture. The empty vector was used as a negative control, while a construct NAT11-Luc2CP encoding multiple copies of typical NF-κB binding sequences and an optimized IL-2 minimal promoter served as a positive control. Signals were presented as relative ratios of firefly/renilla luciferase activities. c Luciferase activity assay with lysates of PSC27 cells pre-transfected with each of the constructs used in b prior to treatment by 50 μg/ml bleomycin (BLEO) in culture. d Chromatin immunoprecipitation (ChIP) was performed to identify potential NF-κB binding sites in the proximal promoter of EREG. Left, EREG-p2/p3/p4/p5 denotes four representative genomic sites in EREG promoter region, while selective NF-κB binding sites from IL6 and CXCL8 served as positive controls. e EREG and MMP3 transcript expression in PSC27 cells exposed to BLEO, MIT (mitoxantrone) or DOX (doxorubicin), with or without the NF-κB inhibitor BAY (Bay 11-7982, 5 μM). Signals were normalized to untreated cells, with MMP3 expression analyzed as positive control. f The reporter construct pGL-EREG-P05 was transiently transfected into PSC27 cells before treatment by BLEO. BAY (5 μM), BA (betulinic acid, 10 μM), T-5224 (10 μM) were applied with BLEO as small molecule inhibitors against NF-κB, C/EBP family and AP-1, respectively. SR (SR 11302, 3 μM), a positive control inhibitor against AP-1. Cells were lysed 7 days after treatment, with lysates subject to luciferase activity assay. g PSC27 cells were treated in the same conditions as described in f , with lysates collected for total RNA preparation and subject to quantitative RT-PCR analysis. Expression of EREG (left), IL6 (mid) or CXCL8 (right) was compared between CTLR (untreated), Mock (PBS-treated), BAY, BA, T-5224 and SR treatment groups. Cells were damaged by BLEO (50 μg/ml) or VBL (vinblastine, 20 nM) treatment. h Immunoblot analysis of DDR signaling (ATM), p38MAPK activation, cellular senescence (p16, p21) and NF-κB activation (p65) in PSC27 cells treated by various chemotherapeutic agents as indicated. GAPDH, loading control. i Immunoblot analysis Expression assay of p65 nuclear translocation in PSC27 cells treated by VBL, PTX, BLEO or MIT, individually. C cytoplasmic, N Nuclear. Histone H3, loading control for nuclear proteins. Note, the relative signal intensities (RSI, presented as percentage) of p65 were quantified as the virtual intensity of an individual sample after scanning, and calculated in relative to that of the strongest signal (BLEO, C for the p65 blot). j Presentation of p65-specific ChIP-seq tracks of the gene locus of several SASP hallmarks and senescence-associated factors. Illustrations were prepared from datasets deposited in the GEO (accession number GSE141992), with raw data available at publicly released sources . Data are representative of three independent experiments. All p values were calculated by Student’s t tests. ^ p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Primary normal human prostate stromal cell line PSC27, breast stromal cell line HBF1203 and lung stromal cell line HFL1 (ATCC) were maintained in stromal complete medium as described [ ].

Techniques: Binding Assay, Construct, Generated, Cloning, Plasmid Preparation, Luciferase, Transfection, Negative Control, Positive Control, Activity Assay, Chromatin Immunoprecipitation, Expressing, Quantitative RT-PCR, Western Blot, Activation Assay, Control, Translocation Assay, ChIP-sequencing

Journal: Oncogene

Article Title: Targeting epiregulin in the treatment-damaged tumor microenvironment restrains therapeutic resistance

doi: 10.1038/s41388-022-02476-7

Figure Lengend Snippet: a Immunoblot analysis of EGFR-associated pathways in PC3 and DU145 cells treated by the CM from PSC27 cells transduced with the empty vector or EREG construct, or alongside the EGFR inhibitor AG-1478 (2 μM). Antibodies of p-EGFR (Y845), p-Akt (S473), p-mTOR (S2448), p-MEK (S217/S221) and p-ERK (T202/Y204) were applied to probe the individual molecules. Total protein per molecule and GAPDH were used as loading control. b Schematic diagram of the construct encoding the mature chain of EREG (upper) and immunoprecipitation (IP, lower) followed by immunoblot assay of EGFR and His-EREG (fusion protein) in the whole lysates of PC3 cells. PC3 was treated by the CM of PSC27 Vector and PSC27 His-EREG for 3 days. Antibodies including IgG and anti-EGFR were used for IP, with both EGFR and His-EREG in inputs analyzed. c Measurement of cellular senescence by quantification of SA-β-Gal staining positivity. Stromal cells were pre-transduced with shRNAs and treated by BLEO. Upper, statistics. Lower, representative images. Scale bar, 20 μm. d PCa cells were treated with the CM from PSC27 sublines for 3 days, and subject to cell proliferation assay. Native and shRNA-transduced PSC27 cells as indicated were treated by bleomycin (BLEO), with the conditioned media (CM) collected 7 days after drug treatment and used for PCa cell culture. The CM were collected from equal number of cells per condition, with a starting DMEM that contains 0.5% FBS to make the CM. e Migration assay of PCa cells seeded within transwells in 6-well plates, with cells cultured for 3 days in the CM from PSC27 sublines depicted in d . f Invasiveness appraisal of PCa cells across the transwell membrane upon culture with the CM from PSC27 sublines described in d . g Chemoresistance assay of PCa cells cultured with the CM from PSC27 sublines described in d . MIT (mitoxantrone) was applied at the concentration of IC50 value pre-determined per cell line. AG-1478 (2 μM), cetuximab (50 μg/ml) or EREG mAb (1 μg/ml) were applied alongside with PSC27 CM. h Dose-response curves (non-linear regression/curve fit) plotted from drug-based survival assays of PC3 cells cultured with the CM of PSC27 native or damaged by bleomycin (PSC27-BLEO), and concurrently treated by a wide range of concentrations MIT. AG-1478, cetuximab or EREG mAb (1 μg/ml) were applied with PSC27 CM. Data are representative of three independent experiments. All p values were calculated by Student’s t tests. ^ p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary normal human prostate stromal cell line PSC27, breast stromal cell line HBF1203 and lung stromal cell line HFL1 (ATCC) were maintained in stromal complete medium as described [ ].

Techniques: Western Blot, Transduction, Plasmid Preparation, Construct, Control, Immunoprecipitation, Staining, Proliferation Assay, shRNA, Cell Culture, Migration, Membrane, Concentration Assay

Journal: Oncogene

Article Title: Targeting epiregulin in the treatment-damaged tumor microenvironment restrains therapeutic resistance

doi: 10.1038/s41388-022-02476-7

Figure Lengend Snippet: a Heatmap depicting differentially expressed human transcripts in PC3 cells after a 3-days culture with EREG-containing CM collected from PSC27 cells. In contrast to cancer cells cultured with control CM (CTRL), 970 and 1362 genes were upregulated and downregulated, respectively, in those treated with the CM from EREG-expressing PSC27 cells (EREG). b Graphical visualization of pathways by GO profiling. Those significantly enriched genes in the upregulated list were sorted according to their fold change in PC3 cells exposed to the CM of EREG-expressing PSC27 cells. c Venn diagram displaying the overlap of 39 transcripts upregulated in PC3 and DU145 cells upon treatment with EREG-containing CM from stromal cells (970 and 309 genes with unique annotations for PC3 and DU145, respectively). d Statistics of transcripts differentially expressed (fold change either ≥2 or ≤0.5, with p < 0.05) in PC3 and DU145 upon EREG stimulation, and classified into typical categories according to functional annotations mapped by Genecode (V27). e Heatmap showing the top 39 transcripts upregulated by both PC3 and DU145 cells, sorted according to their expression fold change in PC3. f Pie chart depicting the biological processes associated with transcripts upregulated by EREG after GO analysis of the 39 transcripts in PC3. g Quantitative RT-PCR measurement of the expression of KIF20A, MARCHF4 and SPNS2 in both PCa lines upon exposure to CM of stromal cells expressing EREG. Signals normalized to those of cells exposed to PSC27 cells transduced with vector. h Dose-response curves (non-linear regression/curve fit) plotted from drug-based survival assays of PC3 cells transduced with vector or MARCHF4 construct and treated by a range of concentrations of MIT. i Dose-response curves (non-linear regression/curve fit) plotted from drug-based survival assays of DU145 cells treated in a manner similar to that of PC3 cells. j Immunoblot assessment of protein expression of EMT-associated molecules. CD81, a downstream target of MARCHF4. β-actin, loading control. k Immunoblot profiling of apoptosis-related factors of self-cleavage activity in both PCa cell lines pre-transduced with vector or MARCHF4 construct and exposed to MIT for 72 h. β-actin, loading control. Data in g – k are representative of three independent experiments. All p values were calculated by Student’s t tests. ^ p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Primary normal human prostate stromal cell line PSC27, breast stromal cell line HBF1203 and lung stromal cell line HFL1 (ATCC) were maintained in stromal complete medium as described [ ].

Techniques: Cell Culture, Control, Expressing, Functional Assay, Quantitative RT-PCR, Transduction, Plasmid Preparation, Construct, Western Blot, Activity Assay

Journal: Oncogene

Article Title: Targeting epiregulin in the treatment-damaged tumor microenvironment restrains therapeutic resistance

doi: 10.1038/s41388-022-02476-7

Figure Lengend Snippet: a Schematic workflow of experimental procedure for severe combined immunodeficient (SCID) mice. Two weeks after subcutaneous implantation and in vivo uptake of tissue recombinants, animals received either single or combinational agents administered as metronomic treatments composed of several cycles. b Statistical profiling of tumor end volumes. PC3 cells were xenograted alone or together with PSC27 cells to the hind flank of SCID mice. Prior to implantation, PSC27 cells were transduced with the control vector or EREG construct to make stable sublines. MIT was administered to induce tumor regression. Right, representative tumor images. c Transcript assessment of several canonical SASP factors expressed in stromal cells isolated from the tumors of SCID mice. Tissues from animals implanted with both stromal and cancer cells were subject to LCM isolation, total RNA preparation and expression assays. d Representative IHC images of EREG expression in tissues isolated from placebo or MIT-treated animals. Square regions in the upper images were zoomed into lower images. Scale bars, 100 μm. e Statistical comparison of tumor growth in animals that underwent several different treatment modalities. Mice were implanted with PC3 alone or in combination with PSC27, before treated by the chemotherapeutic drug (MIT) or combinational agents (MIT/cetuximab or MIT/EREG mAb). Tumor volumes were measured at the end of an 8-week preclinical regimen. f Representative bioluminescence images (BLI) of PC3/PSC27 tumor-bearing animals in the preclinical trial. Digital signals were proportional to in vivo luciferase activities measured by an IVIS device. g Statistical assessment of DNA-damaged and apoptotic cells in the tumor specimens analyzed in e . Values are presented as percentage of cells positively stained by IHC with antibodies against γH2AX/p-53BP1 (co-staining) or caspase 3 (cleaved). h Representative IHC images of caspase 3 (cleaved) in tumors at the end of therapeutic regimens. Biopsies of placebo-treated animals served as negative controls for MIT-treated mice. Scale bars, 50 μm. i EREG concentration assessment in circulating blood of experimental mice treated by chemotherapy and/or EREG mAb. Data were derived from human EREG-specific ELISA assays. Data are representative of three independent experiments. Animal studies were performed with ten mice per group ( n = 10). All p values were calculated by Student’s t tests. ^ p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary normal human prostate stromal cell line PSC27, breast stromal cell line HBF1203 and lung stromal cell line HFL1 (ATCC) were maintained in stromal complete medium as described [ ].

Techniques: In Vivo, Transduction, Control, Plasmid Preparation, Construct, Isolation, Expressing, Comparison, Luciferase, Staining, Concentration Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay