Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: Effects of FGFR1 and FGFR4 on lung squamous carcinoma cell lines. See also Supplementary Figure S1. Growth curves in 10% FBS (a) and soft agar assays (b) of FGFR4-overexpressing lung squamous carcinoma cell lines. (c) Western blot analysis of the activation of FGFR-related signalling pathways in FGFR4-overexpressing lung squamous carcinoma cell lines compared to empty-vector-expressing cell lines after stimulation with FBS. Growth curves in 0.5% FBS (d) and soft agar assays (e) of FGFR1- and FGFR4-silenced H520 cells (lung squamous cell carcinoma). (f) Western blot analysis of the activation of FGFR-related signalling pathways in FGFR1- and FGFR4-silenced H520 cells. All experiments were reproduced a minimum of three times in the laboratory, and three technical replicates were obtained for each experiment. For growth curves and western blots, a representative figure/image is shown. On the growth curves, the means and standard deviations of the technical replicates are shown. In the soft agar assays, all values were normalised to the empty vector control, and the mean and standard deviation of all the normalised replicates are presented. Silencing of either gene was performed using two different shRNAs, referred to as “a” and “b”. p-values were obtained with the two-sided Mann-Whitney U test and are indicated by asterisks (* p <0.05; ** p <0.01; *** p <0.001). ADC = Adenocarcinoma, SCC = Squamous cell carcinoma, I = Immortalised, KRAS = KRAS-mutated, EGFR = EGFR-mutated, ALK = ALK translocation bearer, TN = “Triple negative” (referring to the absence of alterations in KRAS, EGFR and ALK), EV = empty vector control, FGFR1 = FGFR1-overexpressing, FGFR4 = FGFR4-overexpressing, scramble = scrambled shRNA control, shFGFR1 = FGFR1 shRNA, shFGFR4 = FGFR4 shRNA, FBS = foetal bovine serum. Western blot molecular weight references are indicated to the right of the images.

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Western Blot, Activation Assay, Plasmid Preparation, Expressing, Control, Standard Deviation, MANN-WHITNEY, Translocation Assay, shRNA, Molecular Weight

Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: Effects of FGFR1 and FGFR4 on non-squamous lung cell lines. See also Supplementary Figure S2. Growth curves in 10% FBS (a) and soft agar assays (b) of the FGFR1- or FGFR4-overexpressing lung adenocarcinoma cell lines H2009 and H3122. (c) Western blot analysis of the activation of FGFR-related signalling pathways in FGFR1-overexpressing H2009 and H3122 cells compared to empty-vector-expressing cells. Growth curves in 10% FBS (d) and soft agar assays (e) of FGFR1- and FGFR4-silenced A549 cells (adenocarcinoma cell line). (f) Western blot analysis of the activation of FGFR-related signalling pathways in FGFR1- and FGFR4-silenced A549 cells. All experiments were reproduced a minimum of three times in the laboratory, and three technical replicates were obtained for each experiment. For growth curves and western blots, a representative figure/image is shown. On the growth curves, the means and standard deviations of the technical replicates are shown. In the soft agar assays, all values were normalised to the empty vector control, and the mean and standard deviation of all the normalised replicates are presented. For western blots, cells were serum-starved for five hours prior to protein extraction. For the serum-stimulated conditions, serum-starved cells were incubated in serum-containing complete medium for 15 min before protein extraction. Silencing of either gene was performed using two different shRNAs, referred to as “a” and “b”. p-values were obtained with the two-sided Mann-Whitney U test and are indicated by asterisks (* p <0.05; ** p <0.01; *** p <0.001). EV = empty vector control, FGFR1 = FGFR1-overexpressing, FGFR4 = FGFR4-overexpressing, scramble = scrambled shRNA control, shFGFR1 = FGFR1 shRNA, shFGFR4 = FGFR4 shRNA, FBS = foetal bovine serum. Western blot molecular weight references are indicated to the right of the images.

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Western Blot, Activation Assay, Plasmid Preparation, Expressing, Control, Standard Deviation, Protein Extraction, Incubation, MANN-WHITNEY, shRNA, Molecular Weight

Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: Effects of N-cadherin on the pro-oncogenic role of FGFR1 and FGFR4. See also Supplementary Figures S3 and S4. (a) Western blots of N-cadherin and E-cadherin protein expression in our lung cell line panel. To assess the expression of these proteins in the 18 cell lines, different blots were performed in parallel with an internal reference sample and the assembled images are shown. 10% FBS growth curves (b) and soft agar assays (c) of H2009 and H3122 cells overexpressing N-cadherin and either FGFR1 or FGFR4. (d) Western blot analysis of the activation of FGFR-related signalling pathways in these cell lines. All experiments were reproduced a minimum of three times in the laboratory and three technical replicates were obtained for each experiment. For growth curves and western blots, a representative figure/image is shown. On the growth curves, the means and standard deviations of the technical replicates are shown. In the soft agar assays, all values were normalised to the empty vector control, and the mean and standard deviation of all the normalised replicates are presented. p-values were obtained with the two-sided Mann-Whitney U test and are indicated by asterisks (* p <0.05; ** p <0.01; *** p <0.001) non-SCC = non-Squamous, SCC = Squamous cell carcinoma, I = Immortalised, KRAS = KRAS-mutated, EGFR = EGFR-mutated, ALK = ALK translocation bearer, TN = “Triple negative” (referring to the absence of alterations in KRAS, EGFR and ALK), EV1 = empty vector 1, EV2 = empty vector 2, FGFR1 = FGFR1-overexpressing, FGFR4 = FGFR4-overexpressing, CDH2 = N -cadherin-overexpressing. Western blot molecular weight references are indicated to the right of the images.

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Western Blot, Expressing, Activation Assay, Plasmid Preparation, Control, Standard Deviation, MANN-WHITNEY, Translocation Assay, Molecular Weight

Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: Effects of N-cadherin on the pro-oncogenic role of FGFR1 and FGFR4 and the interaction of N-cadherin with FGFR1 and FGFR4. See also Supplementary Figure S4. (a) 0.5% FBS growth curves for FGFR1-overexpressing and N-cadherin-silenced (left) or FGFR4-overexpressing and N-cadherin-silenced (right) NL20 cells. (b) Soft agar assays of FGFR-overexpressing and N-cadherin-silenced NL20 cells. (c) Western blot analysis of the activation of FGFR-related signalling pathways in these cell lines. (d) Xenograft tumour volumes of the FGFR1, FGFR4 and N-cadherin interaction models in the immortalised NL20 cell line. (e) Proximity ligation assays (PLA) to assess the physical interaction of N-cadherin with FGFR1 (upper panel) or FGFR4 (lower panel). The interactions detected were quantified and normalised by cell number for each condition. As controls to distinguish signal from noise, interactions were quantified in the single antibody conditions (labelled as “N-cadherin”, “FGFR1” and “FGFR4”). To assess the interaction between the two proteins, both antibodies were used, either N-cadherin + FGFR1 (upper panel) or N-cadherin + FGFR4 (lower panel), in the condition labelled as “combo”. Representative images and quantifications are shown. (f) Co-immunoprecipitation of N-cadherin with FGFR1 and with FGFR4 in the H520 cell line. (g) Kaplan-Meier curves of overall survival (OS) for the entire NSCLC patient cohort ( N = 109). Patients were grouped based on FGFR1 and N-cadherin expression levels or on FGFR4 and N-cadherin expression levels. (h) OS curve of patients in the cohort with high expression of FGFR1 and/or FGFR4 stratified by N-cadherin expression levels. In each analysis, for the FGFR1 and N-cadherin genes, the cut-off point was the median mRNA expression value for that variable. For FGFR4, the cut-off point was the first-quartile mRNA expression value in the TCGA adenocarcinoma cohort. The Kaplan-Meier method was used for survival analyses of the clinical data and cell line xenograft experiments, with a Cox proportional hazards model used to adjust for explanatory variables. A log Rank analysis was used to analyse differences in survival between groups. To obtain the hazard ratio values, the Cox proportional hazards model was used. All in vitro experiments were reproduced a minimum of three times in the laboratory, and three technical replicates were obtained for each experiment. For growth curves and western blots, a representative figure/image is shown. On the growth curves, the means and standard deviations of the technical replicates are shown. In the soft agar assays, all values were normalised to the empty vector control, and the mean and standard deviation of all the normalised replicates are presented. N-cadherin silencing was performed using two different shRNAs. Results generated with the alternative shRNA are shown in Supplementary Figure S3 c-e. p-values were obtained with the two-sided Mann-Whitney U test and are indicated by asterisks (* p <0.05; ** p <0.01; *** p <0.001). EV1 = empty vector 1, EV2 = empty vector 2, FGFR1 = FGFR1-overexpressing, FGFR4 = FGFR4-overexpressing, CDH2 = N -cadherin-overexpressing, scramble = scrambled shRNA control, shCDH2 = silenced with N-cadherin shRNA. Western blot molecular weight references are indicated to the right of the images.

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Western Blot, Activation Assay, Ligation, Immunoprecipitation, Expressing, In Vitro, Plasmid Preparation, Control, Standard Deviation, Generated, shRNA, MANN-WHITNEY, Molecular Weight

Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: RNAseq analysis of TCGA lung adenocarcinoma and squamous cell carcinoma datasets. (a) Differential gene expression analysis in FGFR1/4high-CDH2high ( n = 145) versus FGFR1/4high-CDH2low ( n = 94) patients. The gene dataset was filtered by discarding genes whose expression was dependant on CDH2 high/low status irrespective of FGFR1 and/or FGFR4 expression (FGFR1/4low-CDH2high patients, n = 21). Parameters were set up as logFC>1, B >0. (b) Query of defined gene expression signature against Gene Ontology. The results shown here are based in whole or in part upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/ .

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Gene Expression, Expressing, Generated

Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: Predictive potential of N-cadherin expression for anti-FGFR therapy in vitro . See also Supplementary Figure S5. Treatment for 72 h with AZD4547 or BGJ398 at a concentration of 0.5 or 1 µM was applied to cells with high endogenous expression of FGFR1 and/or FGFR4, with high or low endogenous expression of N-cadherin (a) and to cells either exogenously expressing FGFR1 or FGFR4, alone or in combination with N-cadherin, or to cells with high endogenous expression of the three genes with N-cadherin downregulation (b). All experiments were reproduced a minimum of three times in the laboratory. For growth curves, a representative figure is shown and the mean and standard deviation for the technical replicates are indicated. N-cadherin silencing was performed using two different shRNAs to avoid off-target effects. p-values were obtained with the two-sided Mann-Whitney U test and are indicated by asterisks (* p <0.05; ** p <0.01; *** p <0.001). EV1 = empty vector 1, EV2 = empty vector 2, FGFR1 = FGFR1-overexpressing, FGFR4 = FGFR4-overexpressing, CDH2 = N -cadherin-overexpressing, scramble = scrambled shRNA control, shCDH2 = silenced with N-cadherin shRNA.

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Expressing, In Vitro, Concentration Assay, Standard Deviation, MANN-WHITNEY, Plasmid Preparation, shRNA, Control

Journal: EBioMedicine

Article Title: FGFR1 and FGFR4 oncogenicity depends on n-cadherin and their co-expression may predict FGFR-targeted therapy efficacy

doi: 10.1016/j.ebiom.2020.102683

Figure Lengend Snippet: FGFR efficacy in N-cadherin, FGFR1 and/or FGFR4 co-expressing patient-derived xenografts (PDXs). See also Supplementary Figure S6. (a) Western blot showing FGFR1, FGFR4 and N-cadherin protein expression in five different lung PDXs. AZD4547 treatment of low (b) and high (c) N-cadherin-expressing PDXs. (d) Results of the PDX treatments in terms of tumour versus control volume (T/C), and complete regressions. T/C values are expressed as percentages. (e) Graph showing the median variation in tumour volume from the initial volume, for every model, calculated as the increase or decrease in volume and expressed as a percentage. (f) Western blot showing the effects of AZD4547 treatment on FGFR-related signalling pathways in one low-N-cadherin-expressing (TP13) and one high-N-cadherin-expressing (TP114) adenocarcinoma PDX. p-values were obtained with the two-sided Mann-Whitney U test and are indicated by asterisks (* p <0.05; ** p <0.01; *** p <0.001). Western blot molecular weight references are indicated to the right of the images.

Article Snippet: FGFR1 (RC202080) and FGFR4 (RG204230) cDNA clones were obtained from Origene in the pCMV6 plasmid (PS100001).

Techniques: Expressing, Derivative Assay, Western Blot, Control, MANN-WHITNEY, Molecular Weight

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A Workflow showing the selection strategy for KHDRBS1 among DNA-damage response genes transcriptionally activated by Myc and significantly associated to breast cancer prognosis. Venn diagram showing the overlap between Myc-transcriptionally activated genes, DNA-damage response genes and genes associated to breast cancer. Specifically, genes were retrieved from: (i) microarray data of Myc-overexpressing mammospheres (M2) (GSE86407); (ii) published dataset (MD Anderson Human-DNA Repair Genes, https://www.mdanderson.org/documents/Labs/Wood-Laboratory/human-dna-repair-genes.html ), BioRad DNA-damage signaling pathway (SAB Target List H96) and recently published DNA-damage-associated genes (Supplementary Table ); and (iii) breast cancer versus normal breast tissues TCGA BRCA and GTeX gene expression data (Supplementary Table ). Genes were further selected for association to the worse relapse-free survival probability in breast cancer (Supplementary Table ) and novelty in the field, excluding known genes associated with BRCAness . B Box plot representing the distribution of log2 gene expression of KHDRBS1 retrieved from TCGA BRCA ( n = 1212) and GTeX ( n = 179) gene expression data (RNASeq2GeneNorm). p value was calculated with Wilcoxon rank sum test. C Kaplan–Meier plots of relapse-free survival (RFS) probability of BC patients stratified by high or low KHDRBS1 expression levels. D GSEA of DNA-repair gene signatures in IMEC-WT versus M2 ( n =3). E Scheme showing MYC and H3K4me3 PCR amplicons localization (red box) on IMEC-WT and M2 cells and layered H3K27ac signals on KHDRBS1 ( SAM68 ) promoter from ENCODE. Chromatin state was assessed by ChromHMM from ENCODE. MYC-MAX binding on multiple cell lines was assessed by ChIP-seq from ENCODE. F ChIP-qPCR estimating MYC binding at SAM68 promoter in IMEC-WT and M2 cells. Data are mean ± SEM ( n = 3). G qRT-PCR analysis of SAM68 gene expression in IMEC-WT and M2 cells. Data are mean ± SEM ( n = 3). H ChIP-qPCR of H3K4me3 deposition at KHDRBS1 ( SAM68 ) promoter in IMEC-WT and M2 cells. Data are mean ± SEM ( n = 3).

Article Snippet: For immunoprecipitation experiments, an equal amount of protein lysates was incubated overnight at 4 °C with 2 μg of anti-Sam68 antibody (H-4, mouse IgG 1 , Santa Cruz Biotechnology) mixed with protein G-Sepharose (Sigma-Aldrich).

Techniques: Selection, Microarray, Gene Expression, Expressing, Binding Assay, ChIP-sequencing, ChIP-qPCR, Quantitative RT-PCR

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A Kaplan–Meier plots of distant relapse-free survival (DRFS) of BC patients stratified by high or low Sam68 protein expression levels. Patients were categorized according to all molecular subtypes ( n = 211) and Luminal-A ( n = 91), Luminal-B ( n = 61), HER2 + ( n = 27), TNBC ( n = 32), HER2 + + TNBC ( n = 59) BCs. B Box plot representing the distribution of log2 gene expression of KHDRBS1 retrieved from TCGA BRCA gene expression data (RNASeq2GeneNorm). p value was calculated with Wilcoxon rank sum test. The indicated statistics refer to each molecular subtype versus basal subtypes. * p value ≤ 0.05; ** p value ≤ 0.01; **** p value ≤ 0.0001. C ChIP-qPCR estimating MYC and MAX binding at SAM68 promoter in BCSphCs (#4 and #15). Data are mean ± SEM of two independent experiment for each BCSphCs. D Expression of Myc (green color) and Sam68 (red color) on paraffin-embedded sections on parental BC and corresponding PDX tissue. Nuclei were counterstained with Toto-3 (blue color). Scale bar represents 40 µm. E Relative mRNA expression levels of MYC and KHDRBS1 on BCSphCs (#4, #13, and #21) expressing a MycER fusion protein induced by 50 nM of OHT. Data are represented as fold mRNA level changes of OHT-treated cells over vehicle. Data are represented as mean ± SD of three independent experiments. * p value ≤ 0.05; ** p value ≤ 0.01. F Cell proliferation analysis of ER+ (MCF7), TNBC (BT549), TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#1, #4, #13, and #21) transduced with doxycyclin-inducible non-targeting (nt) and short hairpin Sam68 (shSam68). Data are represented as fold variation of shSam68 over scr. ns not significant; ** p value ≤ 0.01. G Size of tumors generated by orthotopic injection of ER+ (MCF7), TNBC (BT549), TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#4, #13) in immunocompromised mice (NOD/SCID) at the indicated time points. Data are expressed as mean ± SD ( n = 5 mice per group). ns not significant, *** p value ≤ 0.001.

Article Snippet: For immunoprecipitation experiments, an equal amount of protein lysates was incubated overnight at 4 °C with 2 μg of anti-Sam68 antibody (H-4, mouse IgG 1 , Santa Cruz Biotechnology) mixed with protein G-Sepharose (Sigma-Aldrich).

Techniques: Expressing, Gene Expression, ChIP-qPCR, Binding Assay, Transduction, Generated, Injection

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A MYC binding on DNA-damage related genes transcription start sites (TSS) on IMEC-WT and M2 breast cells. B Representative immunofluorescence analysis of Rad51 foci formation in ER+ (MCF7), TNBC (BT549), TNBC BRCA mut (HCC1937) BC established cell lines and BCSphCs (#4) untreated (UT) and after 6 h of 8 Gy single dose γ-irradiation (IR). Nuclei were counterstained by Toto-3 (blue). Scale bar represents 10 µm. C Waterfall plot analysis of doxorubicin (DOX, 200 nM, left panel ), paclitaxel (PTX, 10 nM, middle panel ) and carboplatin (CARB, 100 µM, left panel ) response at 72 h in ER+ and TNBC BC established cell lines and BCSphCs. D Response rate distribution to chemotherapy for ER+ and TNBC BC established cell lines and BCSphCs treated as in ( C ). Middle line shows the median value of response per group, while single points represent the average value of BC cell response to DOX, PTX and CARB. Data are mean of three independent experiments. Statistical analysis was performed by using Kruskal–Wallis test. Ns not significant, * p value ≤ 0.05; ** p value ≤ 0.01. E Immunoblot analysis of PARP and Sam68 (input) and after immunoprecipitation (IP) with Sam68 antibody in BCSphCs (#15) treated for 4 h with vehicle, doxorubicin (DOX), paclitaxel (PTX) and carboplatin (CARB). Lamin-B was used as loading control. F Immunoblot analysis of nuclear PAR, PARP, and Sam68 in scramble (scr) and short hairpin Sam68 (shSam68) ER+ (MCF7), TNBC (BT549), and TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#4) treated with vehicle, doxorubicin (DOX), paclitaxel (PTX) and carboplatin (CARB) for 4 h. H3 was used as loading control. G Cell proliferation analysis of ER+ (MCF7), TNBC (BT549), and TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#1, #4, #13, #21) transduced with scramble and short hairpin Sam68 (shSam68) treated with vehicle, doxorubicin (DOX), paclitaxel (PTX) and carboplatin (CARB) for 72 h. Data are represented as fold variation of shSam68 over scramble. Data are mean ± SD of three independent experiments. ns not significant; * p value ≤ 0.05; ** p value ≤ 0.01. H , I Relative mRNA expression levels of RAD51 (H) and MYC (I) on scramble (scr) and short hairpin Sam68 (shSam68) ER+ (MCF7), TNBC (BT549), and TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#12 and #13) treated with vehicle, doxorubicin (DOX), paclitaxel (PTX), and carboplatin (CARB) for 24 h. Data are represented as fold mRNA level changes of treated scr and shSam68 cells over vehicle. Data are represented as mean ± SD of three independent experiments. Ns not significant, * p value ≤ 0.05; ** p value ≤ 0.01; *** p value ≤ 0.001.

Article Snippet: For immunoprecipitation experiments, an equal amount of protein lysates was incubated overnight at 4 °C with 2 μg of anti-Sam68 antibody (H-4, mouse IgG 1 , Santa Cruz Biotechnology) mixed with protein G-Sepharose (Sigma-Aldrich).

Techniques: Binding Assay, Immunofluorescence, Irradiation, Western Blot, Immunoprecipitation, Control, Transduction, Expressing

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A Schematic model of DNA-repair signaling pathways mediating the resistance of BC stem-like cells to chemotherapy. B Workflow of purification of sphere cells from serially transplanted BC PDX and their use for in vitro and in vivo drug toxicity testing. C Size of tumors generated by orthotopic injection of scramble (scr) and short hairpin Sam68 (shSam68) BCSphCs treated with vehicle (veh) and BO2. Arrows indicate the start and the end of treatment. Data are expressed as mean of tumors generated by the injection of BCSphCs (#4, #13, and #21) ± SEM ( n = 5 mice per group). D Size of tumors generated by orthotopic injection of scramble (scr) and short hairpin Sam68 (shSam68) BCSphCs (#4, #13, #21) treated with vehicle, olaparib, BO2, cisplatin and olaparib plus BO2 and olaparib plus cisplatin and BO2. Arrows indicate the beginning and the end of treatment. Data are expressed as mean of tumors generated by the injection of BCSphCs (#4, #13, and #21) ± SEM ( n = 5 mice per group). **** p value ≤ 0.0001. E Immunoblot analysis of Rad51 in BCSphCs (#15) treated with dinaciclib for 24 h at the indicated concentration. Β-actin was used as loading control. F Cell viability percentage of scramble (scr) and short hairpin Sam68 (shSam68) BCSphCs (#4, #13, #15, and #21) treated with vehicle and dinaciclib (10 nM) for 6 days. Data are represented as mean ± SEM ( n = 2). * p value ≤ 0.05; *** p value ≤ 0.001. G Representative images ( left panel ) and quantification of area ( right panel ) of BC sphere cells (#21), transduced with scramble (scr) and short hairpin Sam68 (shSam68) lentiviral vectors, treated with vehicle and dinaciclib for 6 days. Data are represented as mean ± SEM ( n = 3). Ns not significant, ** p value ≤ 0.01; *** p value ≤ 0.001. Scale bar represents 100 µm. H Size of tumors generated by orthotopic injection of scramble (scr) and short hairpin Sam68 (shSam68) BCSphCs treated with vehicle (veh) and dinaciclib (din). Arrows indicate the start and the end of treatment. Data are expressed as mean of tumors generated by the injection of BCSphCs (#4, #7, #13) ± SEM ( n = 5 mice per group). **** p value ≤ 0.0001. I Cell viability percentage of BCSphCs (#4, #13, #14, #15, #21) treated with vehicle, olaparib and dinaciclib, alone or in combination, at the indicated concentrations for 6 days. Data are represented as mean ± SD ( n = 3). J Synergy plot representing the combination index (CI), computed in CompuSyn by using Chou-Talalay method, for each olaparib and dinaciclib dose pair, calculated from cell viability data of BCSphCs (#13). K Size of tumors generated by orthotopic injection of BCSphCs treated with vehicle, olaparib, dinaciclib and olaparib plus dinaciclib. Arrows indicate the start and the end of treatment. Data are expressed as mean of tumors generated with BCSphCs (#4, #7, #13) ± SEM ( n = 5 mice per group). *** p value ≤ 0.001.

Article Snippet: For immunoprecipitation experiments, an equal amount of protein lysates was incubated overnight at 4 °C with 2 μg of anti-Sam68 antibody (H-4, mouse IgG 1 , Santa Cruz Biotechnology) mixed with protein G-Sepharose (Sigma-Aldrich).

Techniques: Protein-Protein interactions, Purification, In Vitro, In Vivo, Generated, Injection, Western Blot, Concentration Assay, Control, Transduction

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A Cell viability percentage of scramble (scr) and short hairpin Sam68 (shSam68) ER+ R (MCF7) BC cell line treated with vehicle and dinaciclib (10 nM) for 6 days. Data are represented as mean ± SEM ( n = 4). * p value ≤ 0.05; ** p value ≤ 0.01; *** p value ≤ 0.001. B Relative mRNA expression levels of RAD51 and MYC on scramble (scr) and short hairpin Sam68 (shSam68) ER+ R (MCF7) BC cells treated with vehicle and dinaciclib for 6 days. Data are represented as fold mRNA level changes of treated scr and shSam68 over vehicle ( n = 3). C Cell viability percentage in ER+ R (MCF7) BC cells treated with vehicle, olaparib and dinaciclib, alone or in combination, at the indicated concentrations for 6 days. Data are represented as mean ± SD ( n = 3). D Kaplan–Meier plots of relapse-free survival (RFS) probability of BC patients of all molecular subtypes stratified by high or low MYC , KHDRBS1 , and RAD51 expression levels. E Schematic model showing the persistence of a BC stem-like population, characterized by high expression levels of MYC, SAM68 , and RAD51 , following standard anticancer therapies.

Article Snippet: For immunoprecipitation experiments, an equal amount of protein lysates was incubated overnight at 4 °C with 2 μg of anti-Sam68 antibody (H-4, mouse IgG 1 , Santa Cruz Biotechnology) mixed with protein G-Sepharose (Sigma-Aldrich).

Techniques: Expressing