hcc cells panel  (ATCC)

Journal: Cancer research

Article Title: Transcriptionally Active Androgen Receptor Splice Variants Promote Hepatocellular Carcinoma Progression

doi: 10.1158/0008-5472.CAN-19-1117

Figure Lengend Snippet: (a, left) Top 100 (of 372) combined per-patient (x-axis) AR-FL (blue) and ligand-independent AR-SVs (red, as described in Supplementary Table 2). Numbers of patients with abundant AR-SV expression noted (inset) (a, right) RNA-Seq data from TCGA LIHC cohort were interrogated for AR-SVs transcript expression in female (n=121) and male (n=251). Statistical significance for AR-Svs expression in males vs females were evaluated using Mann-Whitney test **** p<0.0001 versus female. (b) Analyses of tumor RNA from 12 HCC majority cirrhotic and chronic hepatitis infected patients who underwent liver resection (male=10, female=2). Levels are compared to negative control THLE-2, normal liver cells, and positive control VCaP, PCa cells, to show abundant patient AR-FL and AR-v7 expression. Bars represent average technical duplicates and are matched for each patient. (c) Transcript abundance in transcript per million (TPM) of protein coding androgen receptor transcripts in 2 prostate cancer and 18 HCC cell lines from Cancer Cell Line Encyclopedia (CCLE) database. AR-FL (blue) and AR-SVs (red), as in Figure 1A, are presented. HCCLM3 cell data are not present in the CCLE. HCC cell AR transcript and protein expression were further validated by RT-PCR (d, h) and Western Blot (f, g), respectively. (d). RT-PCR analyses of AR-FL and AR-SVs transcripts in AR-positive prostate cancer (VCaP), AR-negative prostate cancer (DU145), AR-positive HCC (HCCLM3, SNU-423), AR-negative HCC (HepG2, PLC/PRF/5) and immortalized normal liver (THLE2) cell lines. (n=3, geometric mean ± SD). ARv4 and ARv12 were undetectable (supplementary Figure 5A). (e) Comparison of mean AR-FL and AR-v7 mRNA in primary samples as compared to the most abundant AR-SV expressing AR-positive HCC cells, HCCLM3, demonstrating robust AR isoform expression in primary HCC. (f) Western blot analysis with an N-terminal directed monoclonal AR antibody shows abundant AR-FL protein in HCCLM3 and SNU-423 cells and low molecular weight (MW) AR species in HCCLM3 cells migrating similarly to known AR-SVs in VCaP PCa cells. No AR-FL or lower MW species of AR were detected in HepG2, PLC/PRF/5, DU145, or THLE-2 cells. AR-negative HCC cell line, PLC/PRF/5, was transfected with either AR-FL expressing plasmid (PLC5_pAR-FL) or AR-v7 expressing plasmid (PLC5_pAR-v7) as positive controls for AR-FL and AR-v7, respectively. (g) Western blot analysis with a C-terminal directed monoclonal AR antibody shows abundant AR-FL protein in HCCLM3, SNU-423 and PLC5_pAR-FL cells. However, N-terminal directed monoclonal AR antibody detectable AR-SVs in VCaP and HCCLM3 cells are not detectable with c-terminal directed monoclonal AR antibody. WB performed with 35μg total protein lysate for all liver cell lines and 10μg for VCaP and DU145 and with primary N-terminal (CS#5153, Cell Signaling) or C-terminal AR mAb (ab52615, Abcam). (h). RT-PCR analyses of AR-FL and AR-SVs transcripts namely AR-v1, v3, and v7 in AR-positive HCC (HCCLM3, SNU-423, SNU475), AR-negative HCC (PLC/PRF/5) and immortalized normal liver (THLE2) cell lines (performed on low passage cells from ATCC Liver Cancer Panel TCP-1011, n=3, geometric mean ± SD). (i) Western blot analysis with an AR-v7 specific monoclonal AR antibody shows AR-v7 protein in 22Rv1, PLC5_pAR-v7, VCaP and SNU-475 cells. No AR-v7 reactive lower MW species of AR were detected in HCCLM3 cells. No AR-FL protein was detected in any of these cells. (j) To further confirm that the low molecular weight species that were detected by an AR-v7 specific AR mAb are C-terminal truncated splice variants, the blot presented in Figure 1I performed with a C-terminal targeting AR mAb was stripped, blocked and incubated with an N-terminal targeting AR mAb revealing abundant AR-FL in 22Rv1, VCaP and HCCLM3. The GAPDH blot from (i) is presented again here for convenience. No AR-FL isoform was detected in SNU-475 or PLC5_pAR-v7. However, low molecular weight AR species were detected in HCCLM3. (k) WGS of SNU-475 cells revealed a large ~48-kb hemizygous deletion in the AR-locus which included exons 4–8 of the AR-FL gene. This deletion is consistent with AR-v7 but not AR-FL expression and is strongly supported by sequencing data which included 56 read pairs with split reads and/or discordant pair alignments.

Article Snippet: For reproducibility experiments, HCC cells panel (HepG2/C3A, PLC/PRF/5, SNU-423, SNU-475) was obtained from ATCC (ATCC® TCP-1011).

Techniques: Expressing, RNA Sequencing, MANN-WHITNEY, Infection, Negative Control, Positive Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Comparison, Molecular Weight, Transfection, Plasmid Preparation, Incubation, Sequencing

Journal: Cancer research

Article Title: Transcriptionally Active Androgen Receptor Splice Variants Promote Hepatocellular Carcinoma Progression

doi: 10.1158/0008-5472.CAN-19-1117

Figure Lengend Snippet: (a) Western blot of AR expression in SNU-423 (left) and HCCLM3 (right) cytoplasmic or nuclear fractions using an N-terminal targeting AR anti-body. Whole cell lysate (WCL), cytoplasmic extract (CE) and nuclear extract (NE) fractions were assayed after vehicle or 1 nM R1881 treatment for 24hours. In vehicle treated SNU-423 cells, the AR is mainly cytoplasmic but becomes predominantly nuclear following treatment with R1881. In contrast with SNU-423 cells, nuclear localized AR-SVs can be detected in untreated HCCLM3 cells. Following treatment with R1881, the expression of all nuclear localized AR species increases. GAPDH and Histone 3 or HDAC1 serve as cytoplasmic and nuclear controls, respectively. (b) Immunofluorescence analysis of AR in HCCLM3 performed using an N-terminal AR antibody (AR-NT, green) with DAPI nuclear counter stain visualized by confocal microscopy (60×). SNU-423 (left) were treated with either vehicle, 1 nM R1881, or androgen antagonist 10 μM enzalutamide (ENZ) with 1 nM R1881 for 24 hours. Matching nuclear fractionation in Figure 2A, AR stain was primarily diffuse and cytoplasmic in untreated SNU-423 but became nuclear following treatment with R1881. HCCLM3 (right) were similarly treated but revealed intense nuclear staining in the absence of androgen. R1881 treatment reduced the minimal cytoplasmic staining that was apparent in untreated cells but co-treatment with ENZ resulted in considerable residual nuclear localized AR. (c) Immunofluorescence analysis of AR in HCCLM3 with a C-terminal AR (AR-CT, green) antibody. In contrast with N-terminal staining in Figure 2B, C-terminal reactive AR is primarily cytoplasmic in the absence of the ligand but becomes nuclear localized when cells were treated with R1881 for 24 hours. (d) Immunofluorescence analysis of SNU-423 cells was performed after transfection of expression vectors encoding AR-v7 (pAR-v7), or plasmid control (pControl). An N-terminal AR monoclonal antibody was used to detect AR localization (green) as in Figure 2. B. Consistent with Figure 2B, AR localization as determined by AR-NT or AR-CT is predominantly cytoplasmic in untreated control plasmid transfected cells. Whereas transfection with pAR-v7, resulted in strong, predominantly nuclear staining with both AR-NT and AR-CT.(e) Immunofluorescence analysis of SNU-475 cells, consistent with Figure 1H–I, ​,ARAR localization as determined by AR-NT is predominantly nuclear. Whereas AR was undetectable by AR-CT antibody. (f) CE and NE fractions of four representative, primary HCC samples analyzed for AR expression using an N-terminal reactive AR antibody. Tumor (T) AR expression is greater than patient matched, adjacent non-tumor (N) samples and multiple patients demonstrate expression of nuclear localized low molecular weight AR species. For Immunofluorescence experiments, AR localization was analyzed using the Olympus FluoView 4.2 program on Olympus FV 1000 spectral confocal microscope (panels B-D). DAPI staining (blue) indicates nuclei. All experiments were carried out in triplicate with representative fields presented.

Article Snippet: For reproducibility experiments, HCC cells panel (HepG2/C3A, PLC/PRF/5, SNU-423, SNU-475) was obtained from ATCC (ATCC® TCP-1011).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Confocal Microscopy, Fractionation, Transfection, Plasmid Preparation, Control, Molecular Weight, Microscopy

Journal: Cancer research

Article Title: Transcriptionally Active Androgen Receptor Splice Variants Promote Hepatocellular Carcinoma Progression

doi: 10.1158/0008-5472.CAN-19-1117

Figure Lengend Snippet: (a) HCC (HepG2, SNU-423 and HCCLM3) and PCa (VCaP and DU145) cells were transiently transfected with an androgen responsive inducible reporter construct (MMTV-LUC) along with constitutively active renilla luciferase (RN-LUC) transfection control. Cells were maintained for 24 hours in charcoal-stripped FBS containing media (csFBS) then treated with vehicle, 1 nM R1881 or 10 μM enzalutamide (ENZ) with 1 nM R1881 for 24 hours. In SNU-423 and VCaP cells there was significant promoter and androgen-dependent induction of transcriptional activation in R1881-treated cells that was also reversible by co-treatment with ENZ. By contrast, there was no significant activation in HCCLM3, HepG2 and DU145 cells. (b) Comparing basal MMTV-LUC activity to pGL4.24 controls (in the absence of ligand) revealed a constitutive, ligand-independent transcriptional response for VCaP and HCCLM3 cells (left). This activity was significantly reduced by siRNA targeting AR-FL and AR-SV isoforms (AR exons 3 and 7). Successful AR knockdown was confirmed by WB in VCaP and HCCLM3 using N-terminal AR mAb (right). (c) AR-SV expressing HCC cells SNU-475 shows constitutive transcriptional activity similar to HCCLM3 (as determined in Figure 3B). This activity was significantly reduced by 3 different siRNA targeting AR-FL and AR-SV isoforms (left). Successful knock down of AR-v7 in SNU-475 was confirmed by WB with an N-terminal AR mAb (right). (d) Constitutive transcriptional activity in VCaP and HCCLM3 cells (as determined in Figure 3B) was insensitive or only weakly sensitive, respectively, to 24 hour 10 μM ENZ treatment. However, the AR-dependence of the transcriptional signal was demonstrated by knockdown of AR using siRNA targeting AR-FL and AR-SV isoforms (as in Figure 3B, 24 hours). (e) AR expressing SNU-423 HCC cells were transiently transfected with pGL4.24 LUC control or MMTV-LUC and an increasing amount of AR-v7 expressing plasmid (left). Successful overexpression of AR-v7 in SNU-423 was confirmed by WB with an N-terminal AR mAb (right). Exogenous AR-v7 expression in SNU-423 cells demonstrated a concentration dependent ability to increase constitutive MMTV-LUC activation. (f) SNU-423 cells were transiently cotransfected with MMTV-LUC and 10 μg pAR-v7 or empty expression vector control (pcw107) and treated as indicated for 24 hours. Relative to the control construct (pcw107), cells demonstrated increased AR-v7-dependent transcriptional activity (red bars) that was only weakly responsive to treatment with R1881 and insensitive to antagonism with ENZ. (g) C3A cells were transiently cotransfected with pGL4.24 LUC control (black bar) or MMTV-LUC (red bars) and 10 μg pAR-v7 or empty expression vector control (pcw107) for 24 hours. Relative to the control construct (pcw107), cells demonstrated a significant promotor and AR-v7-dependent transcriptional activity. All panels: Dual Luciferase Assay (Promega) with triplicate FF/RN values reported as fold versus vehicle treated control (a, d, f), basal promoter control (b, c, e), or expression vector control (g) as mean+STD. One-way ANOVA with Dunnett’s multiple comparisons test. * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001 versus vehicle treated cells (a, d, f), basal promoter transfected cells (b,e) siRNA controls (c) and empty expression vector controls (g), respectively.

Article Snippet: For reproducibility experiments, HCC cells panel (HepG2/C3A, PLC/PRF/5, SNU-423, SNU-475) was obtained from ATCC (ATCC® TCP-1011).

Techniques: Transfection, Construct, Luciferase, Control, Activation Assay, Activity Assay, Knockdown, Expressing, Plasmid Preparation, Over Expression, Concentration Assay

Journal: Cancer research

Article Title: Transcriptionally Active Androgen Receptor Splice Variants Promote Hepatocellular Carcinoma Progression

doi: 10.1158/0008-5472.CAN-19-1117

Figure Lengend Snippet: (a) We performed differential gene expression (DGE) analysis of 8 AR-positive relative to 14 AR-negative HCC cell lines (as listed in Figure 1C) and obtained 1058 differentially expressed genes. Gene set enrichment analysis (GSEA) on this set of genes using molecular signature database (MSigDB) revealed significant enrichment of the EMT pathway among the top 10 molecular pathways in AR-positive HCC cells. P-value < 0.01 (Fisher exact test). (b) Transcript abundance from CCLE data show a positive correlation (Spearman correlation coefficient) between SNAI2 and AR expression in AR-positive (red) relative to AR-negative (black) cell lines suggesting a putative role for AR:SNAI2(Slug) mediated migration and invasion in HCC. (c) RT-PCR of SNAI2 mRNA in SNU-423 cells treated with 1 nM R1881 for 3, 8 and 24 hours (left) as well as by dose response at 24 hours (right). SNAI2 mRNA demonstrated both time- and concentration-dependent, androgen-dependent regulation. (d) SNU-423 cells were treated with vehicle, 1 nM R1881 or 10 μM enzalutamide with 1 nM R1881 for 3 and 24hours. AR and slug protein were assessed by western blot (left) revealing androgen-dependent slug regulation (densitometry, right). (e) The cellular localization of AR and slug in SNU-423 cells were determined by immunofluorescence in the presence of 1 nM R1881 alone and in combination with 10 μM enzalutamide for 24 hours. AR and slug are cytoplasmic in the absence of androgen, but both became predominantly nuclear upon stimulation with 1 nM R1881 for 24 hours. This androgen-mediated nuclear translocation of slug was inhibited in part upon co-treatment with enzalutamide. (f) Androgen treatment with 1 nM R1881 for 48 hours promoted invasion that was both AR- and SNAI2-dependent as demonstrated by the Matrigel invasion assay (performed and analyzed as described in Figure 4E, quantification bottom right). Both AR and SNAI2 were successfully knocked down using siRNA targeting AR (as in Figure 3B) or SNAI2 (western blot inset, top right). (g) 48 hours Matrigel invasion assays were performed on SNU-423 cells transfected with either 10 μg AR-v7 expressing plasmid (pAR-v7) or control (pcw107, pControl) demonstrating increased invasive capacity for AR-v7 expressing cells.(h) 48 hours post transfection, immunofluorescence analysis of AR-v7 or control transfected cells showed AR (anti-AR mAb targeting N-terminal region of AR, red) and slug (green) were cytoplasmic in the presence of control plasmid. Upon the addition of exogenous, constitutively active AR-v7, both AR and slug staining became predominantly nuclear. Cells were also harvested and analyzed for AR and slug protein content by western blot (inset bottom left) revealing an AR-v7 mediated increase in slug protein (western blot inset, bottom). (i) Immunofluorescence analysis of HCCLM3 cells shows AR and slug co-nuclearization in the absence of androgen stimulation. (j) Correlation analysis of AR and SNAI2 expression in the HCC cohort in TCGA demonstrates a relationship between AR and SNAI2 mRNA levels in liver cancer tissue (TCGA) but not normal tissue (TCGA and GTEx). Spearman correlation analyses showed statistically significant positive correlations between AR and SNAI2 in primary samples (372 patients, left) but no correlation in matched normal samples (50 patients, middle) or normal liver tissues (150 donors, right) (43). One-way ANOVA with Dunnett’s multiple comparisons test. All panels: * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001 versus vehicle (c, f), and expression plasmid controls (g)

Article Snippet: For reproducibility experiments, HCC cells panel (HepG2/C3A, PLC/PRF/5, SNU-423, SNU-475) was obtained from ATCC (ATCC® TCP-1011).

Techniques: Gene Expression, Expressing, Migration, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Western Blot, Immunofluorescence, Translocation Assay, Invasion Assay, Transfection, Plasmid Preparation, Control, Staining

Journal: Cancer research

Article Title: Transcriptionally Active Androgen Receptor Splice Variants Promote Hepatocellular Carcinoma Progression

doi: 10.1158/0008-5472.CAN-19-1117

Figure Lengend Snippet: (a) SNU-423 were transfected with siRNA control or siRNA against AR (as in 5F). Relative to siControl cells, siRNA AR-transfected cells demonstrated upregulation of both phosphorylated mTOR and AKT with no change in total mTOR and AKT. (b) AR expressing HCC cells SNU-423 were treated with vehicle, 1 nM R1881 or 10uM enzalutamide with 1 nM R1881 for 3 and 24 hours. Relative to vehicle-treated cells, no change in protein expression of total or phosphorylated mTOR or AKT was apparent following treatment. (c) AR-negative, C3A, and AR-expressing SNU-423 HCC cells were transiently transfected with either 10 μg AR-v7 expressing plasmid (pAR-v7) or control (pcw107, pControl). Relative to pControl, AR-v7-overexpressing cells showed an upregulation of phosphorylated mTOR and AKT with no change in the total levels of mTOR and AKT. AR protein levels in C3A also shown in Figure 3G (d) AR-Sv expressing HCC cells SNU-475 were transfected with control siRNA (siControl) or 3 different siRNA against AR and compared to AR-v7 transfected SNU-423 cells. Relative to siControl, siRNA AR-transfected SNU-475 cells showed a downregulation of both phosphorylated mTOR and AKT with no change in the protein levels of total mTOR and AKT. (e) Graphical depiction of potential AR signaling to modulate EMT in HCC, androgen-dependent AR-FL homodimers (left), androgen-independent AR-Svs homodimers (middle) and androgen-independent AR-FL and AR-Svs heterodimers (right).

Article Snippet: For reproducibility experiments, HCC cells panel (HepG2/C3A, PLC/PRF/5, SNU-423, SNU-475) was obtained from ATCC (ATCC® TCP-1011).

Techniques: Transfection, Control, Expressing, Plasmid Preparation