Sex has not received enough attention as an important biological variable in basic research, even though the sex of cells often affects cell proliferation, differentiation, apoptosis, and response to stimulation. Knowing and considering the sex of cells used in basic research is essential as preclinical and clinical studies are planned based on basic research results. Cell lines derived from tumor have been widely used for proof-of-concept experiments. However, cell lines may have limitations in testing the effect of sex on cell level, as chromosomal abnormality is the single most characteristic feature of tumor. To examine the status of sex chromosomes in a cell line, 12 commercially available gastric carcinoma (GC) cell lines were analyzed using several different methods. Loss of Y chromosome (LOY) accompanied with X chromosome duplication was found in three  out of the six male-derived cell lines, while one cell line (SNU-638) showed at least partial deletion in the Y chromosome. Two (SNU-5 and MKN-28) out of six female-derived cell lines showed a loss of one X chromosome, while SNU-620 gained one extra copy of the X chromosome, resulting in an XXX karyotype. We found that simple polymerase chain reaction (PCR)-based sex determination gives a clue for LOY for male-derived cells, but it does not provide detailed information for the gain or loss of the X chromosome. Our results suggest that carefully examining the sex chromosome status of cell lines is necessary before using them to test the effect of sex on cell level. According to US Food and Drug Administration (FDA) reports, eight out of ten drugs are withdrawn from the market due to more severe side effects in women than in men. This is partially attributed to the fact that mainly male animals and male patients are used in the preclinical and clinical studies ,2. EvideAmong researchers using experimental animals, the notion that skewed results can arise from sex-biased study has been slow to spread ,2. FurthAMELX; p22.3) and Y  chromosomes as homologous sequences [AMELX and AMELY, respectively) using only one pair of primers [AMELY [SRY) located at Yp11.32 [STS) on Y chromosome was also reported [Amelogenin, existing on X  ,19, chroHere, we assessed the intactness of sex chromosomes of six male and six female-derived carcinoma cell lines which are commercially available and widely used for experiments. Five different methods were used to analyze the status of sex chromosomes of each cell line. In general, the results obtained using different analysis methods matched each other. Sex chromosomes in the cell lines showed various abnormalities, including Y chromosome loss.AMELX on the X chromosome; AMELY on the Y chromosome), sex-determining region Y (SRY), and the first intron site of steroid sulfate (STS-1) -123 bp/Y-130 bp, and STS-1_X-158 bp/Y-166 bp [To check whether commonly used cell lines contain intact sex chromosomes, six male and six female gastric cancer (GC) cell lines were obtained for analysis. PCR amplification was performed for sex chromosome-linked genes previously employed for sex determination : ameloge (STS-1) A. For SRSTS-1 gene for SNU-638 cell line was analyzed, an additional band amplified from the Y chromosome was also detected  showing LOY were not due to cell line mix up, STR profiling was carried out for autosomal STR loci on chromosomes 2 , 3 (D3S1358), 4 (FGA), 5 (D5S818), 7 (D7S820), 8 (D8S1179), 11 (TH01), 12 (vWA), 13 (D13S317), 15 (Penta E), 16 (D16S539), 18 (D18S51), 19 (D19S433), 21 , and sex chromosomes (amelogenin). In general, our STR results matched with the STR information the cell vendor provided for the 10 loci, including amelogenin . In SNU-To directly detect sex chromosomes of the three male-derived cell lines , we performed FISH analysis using centromere evaluation probe X (CEPX) and Y (CEPY). Both the X and Y chromosome centromeres were detected by FISH in SNU-638. Interestingly, two instead of one X chromosome centromeres were observed in SNU-484 and MKN-1, while the Y chromosome centromere was undetectable .Among the six male-derived cell lines, NCI-N87 and SNU-1 contained both the X and the Y chromosomes, matching with the sex of the cell donors. In SNU-638, the short arm seems to be lost from the Y chromosome judging by the log R ratio (LRR) value, which is lower than zero in this region. There was no detectable signal for the Y chromosome in SNU-484, KATO III, and MKN-1, implying LOY. One copy of the X chromosome was detected in NCI-N87, SNU-1, and SNU-638 (LRR = 0), while X chromosome gain may have happened in SNU-484, KATO III, and MKN1 (LRR > 0). Among the six female GC cells, partial X chromosome loss was indicated in SNU-5, MKN-28, and SNU-216 (LRR < 0). A female derived gastric carcinoma cell line, AGS, showed an LRR value of around zero and a value of 0.5 on the B allele frequency (BAF) plot, indicating a heterozygous genotype for the X chromosome in this cell line. For SNU-16 and SNU-620, hetero split on the BAF plot was observed together with near-zero LRR .Karyotyping was performed using chromosome banding techniques for SNU-484, SNU-638, SNU-5, MKN-28, SNU-216, and SNU-620. Giemsa banding revealed chromosomal abnormalities for both autosomes and the sex chromosomes in all the tested GC cell lines . The Y cWe tested the status of sex chromosomes in GC cell lines using several methods and compared the obtained results. In general, comparable results were obtained using five different analysis methods as described below. In the GC cell lines, gross chromosomal abnormality of sex chromosomes such as loss of Y chromosome (LOY) as well as loss or duplication of the X chromosome was observed in addition to small deletion and/or amplification in the sex chromosomes.Our PCR, STR, and CNV analyses suggest that three  male cell lines suffer from LOY. FISH results showed that SNU-484 and MKN-1 cells do not contain the centromere of the Y chromosome, but they had two centromeres of the X chromosome, implying LOY and duplication of the X chromosome. G-banding assay confirmed that SNU-484 did not have the Y chromosome indeed, while two instead of one copy of the X chromosomes were present. LOY has been reported to be frequently observed in GC  as well AMELY is not present in SNU-638. However, the STS-1 but neither the AMELY nor the SRY sequence from the Y chromosome was detected in PCR analysis for this cell line. FISH analysis also showed the presence of the centromere of the Y chromosome in SNU-638. The CNV results indicate that the short arm of the Y chromosome (Yp) where AMELY and SRY are located was lost, while the long arm of the Y chromosome (Yq) where STS-1 is located was present in SNU-638. Collectively, these results imply that SNU-638 had lost Yp but maintained the centromere and Yq where the STS-1 gene is located. Perhaps the remaining fragment of the Y chromosome was not detected by G-banding due to its small size.At a glance, seemingly contradictory results were obtained from different assays we performed for a male cell line SNU-638. The G-banding result showed LOY for SNU-638, which is inconsistent with the report from the researchers who established this cell line . In addiOur PCR results for all the female cell lines show amplicons with appropriate sizes from the X chromosome. However, the PCR-based assay does not provide information about duplication or loss of the X chromosome. The G-banding results show that SNU-5 and MKN-28 had only one copy of the X chromosome even though they are derived from female GC patients. CNV analysis for these cell lines also supported loss of heterozygosity (LOH) with large deletion on the X chromosomes. For SNU-620, three copies of the X chromosomes were detected by G-banding, and the CNV assay indicated gain of the X chromosome.The male cell lines  with LOY seem to have duplicated the X chromosome judged from the CNV analysis and G-banding. Our results for KATO III are consistent with a previous report showing LOY and an XXX karyotype in this cell line . NormallWe found that simple PCR-based sex determination gives a clue for LOY for male-derived cells, but it cannot give detailed information for duplication or loss of the X chromosome. It seems that three  out of the six male-derived cell lines lost the Y chromosome completely, while one cell line (SNU-638) showed at least partial deletion in the Y chromosome. Meanwhile, two (SNU-5 and MKN-28) out of the six female-derived cell lines showed a complete loss of one X chromosome. One female-derived GC cell line SNU-620 had gained a copy of the X chromosome.Our results suggest that cell lines may not be suitable to test the effect of sex on cell level as various abnormalities were found not only on the autosomes but also on the sex chromosomes. Primary cells are better choices than cell lines for sex-balanced cell experiments. However, it takes sizable blocks of time and efforts to isolate primary cells from tissues. The source of human primary cells is usually restricted, and obtaining pure primary cell population is not easy. In addition, primary cells have a finite expansion capacity and a short lifespan ,38. ThusTwelve gastric cancer cell lines  were obtained from Korean Cell Line Bank . All of the cells were cultured in RPMI 1640  supplemented with 10% fetal bovine serum (FBS) and antibiotics .Cells were harvested, and genomic DNA was extracted using phenol:chloroform  for the analysis of sex chromosomes.SRY, STS-1. Two primer sets were used to amplify different regions of the SRY gene—SRYa and SRYb [SRYa primer set amplified homologous sequences on the X or the Y chromosome, while the SRYb primer set amplified homologous sequences on the Y chromosome and the 7th chromosome. Amplified products of the 7th chromosome functioned as a positive control for successful amplification. A single PCR product band for each gene can be detected for DNA from a female-originated cell line as both of the X chromosomes have the same size genes. In contrast, PCR products having two different sizes can be observed for each gene if DNA from a male-originated cell line is used, as genes on the X and the Y chromosomes have different sizes.PCR was conducted to amplify genes showing size differences depend on whether they are located on the X or the Y chromosome:amelogenin, and SRYb . The SRYSRYa; 5′-AGCCCCTGTGGAAAAAATTAGTTTT-3′ and 5′-AAAACACCATAGTTTTATATGGAGTAAAGGAAC-3′, SRYb; 5′-TTGTGCAGCCATCACCTCT-3′ and 5′-AAATCAGATTAATGGTTGCT-3′, STS-1; 5′-GTTTCTTCAGGAACATTTTGGAGCTAT-3′ and 5′-GGTTAGTTCAGGTGTAGACC-3′. Acrylamide gel was prepared using 40% acryl/bisacrylamide solution . PCR products were separated on a 15% acrylamide gel at 100 voltage. The bands in the acrylamide gel were detected by Gel DocTM  after staining with ethidium bromide (EtBr).The sequences of the primers were: amelogenin; 5′-GTTTCTTCCCTGGGCTCTGTAAAGAATAGTG-3′ and 5′-ATCAGAGCTTAAACTGGGAAGCTG-3′, ® 18D System . The PowerPlex® 18D System allowed co-amplification and four-color fluorescent detection of eighteen loci: TPOX, D3S1358, FGA, D5S818, CSF1PO, D7S820, TH01, vWA, D13S317, amelogenin, D16S539, D21S11, D18S51, D8S1179, D2S1338, D19S433, Penta D, and Penta E.We had Cosmogenetech Co.  perform a STR profiling for the cell lines using the PowerPlexhttps://metasystems-international.com/).FISH analysis was performed with Centromere evaluation probe X/Y  in accordance with the manufacturer’s recommendations as previously reported . FluoresDNA samples were hybridized to Infinium Human Omni1M microarrays  in accordance with the manufacturer’s protocols. After whole-genome amplification, the product was fragmented, precipitated with 2-propanol, and resuspended in formamide-containing hybridization buffer. The DNA samples were denatured at 95 °C for 20 min, then placed in a humidified container for a minimum of 16 h at 48 °C allowing SNP loci to hybridize to the 50mer capture probes. For the single-base extension (Infinium II) assay, primers were extended with a polymerase and labeled nucleotide mix. The slides were then stained, washed with low salt wash buffer, immediately coated, and imaged on the Illumina iScan Reader. A single sample mode in which reference values are derived from canonical genotyping clusters  created from clustering on ~120 normal reference samples was used. Individual copy number variation was detected using the software provided by the manufacturer (cnvPartition 3.2.0 plug-in of Illumina Genome Studio 2.0). Log R ratio and B-allele frequency were obtained from GenomeStudio. ASCAT  was then used in R to estimate aberrant cell fraction.Cultured cells were treated with 10 μg/μL colcemide  for 4 h. The cells were harvested and treated with hypotonic 0.075M KCl solution and fixed with Carnoy’s solution . Banding


Germ cell tumours (GCTs) are the most common solid malignancies in young men. Although high cure rates can be achieved, metastases, resistance to cisplatin-based therapy and late toxicities still represent a lethal threat, arguing for the need of new therapeutic options. In this study, we analysed the potential of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors palbociclib and ribociclib (PaRi) as molecular drugs to treat cisplatin-resistant and -sensitive paediatric and adult GCTs.Ten GCT cell lines, including cisplatin-resistant subclones and non-malignant controls, were treated with PaRi and screened for changes in viability (triphenyl tetrazolium chloride (XTT) assay), apoptosis rates , the cell cycle (flow cytometry), the transcriptome  and on protein level (western blot). Expression profiling was performed on paediatric and adult GCT tissues .CDK4, while paediatric GCTs strongly express CDK6 instead. Thus, both GCT types are potentially treatable by PaRi. GCTs presented as highly sensitive towards PaRi, which caused a decrease in viability, cell cycle arrest and apoptosis. Although GCTs mainly arrested in the G1/G0 phase, some embryonal carcinoma cell lines were able to bypass the G1/S checkpoint and progressed to the G2/M phase. We found that upregulation of CDK3 and downregulation of many mitosis regulation factors, like the HAUS genes, might be responsible for bypassing the G1/S checkpoint and termination of mitosis, respectively. We postulate that GCT cells do not tolerate these alterations in the cell cycle and eventually induce apoptosis.We demonstrate that adult GCTs highly express Our study highlights PaRi as therapeutic options for cisplatin-resistant and -sensitive paediatric and adult GCTs. Despite high cure rates exceeding 90% over all stages, ~10–15% of patients with advanced disease relapse despite adequate multimodal first-line and first-salvage treatments and are deemed treatment refractory.9 The role of the most prominent CDK4/6 inhibitors palbociclib (Ibrance), ribociclib  and abemaciclib (Verzenio) in monotherapy and combined therapy is under evaluation in several phase 1, 2 or 3 studies. Palbociclib and ribociclib (PaRi) are reversible orally active pyridopyrimidine, which bind to the adenosine triphosphate binding site of the two CyclinD (CCND)-dependent kinases.10 Trials observed nearly indistinguishable clinical results between palbociclib and ribociclib.11 In 2017, the Food and Drug Administration approved PaRi in combination with aromatase inhibitors and abemaciclib plus fulvestrant as initial endocrine-based therapy for women with hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced or metastatic breast cancer. These inhibitors eventually block the CCND–CDK4/6 complex, resulting in hypophosphorylation of the retinoblastoma (RB) protein.12 As a consequence, the transcription factor E2F cannot be cleaved from the RB complex and transcription of genes involved in cell cycle progression diminishes, resulting in a cell cycle arrest in the G1/G0 phase.13 In GCTs, an ‘unorthodox’ spectrum of defects within the CDK4/6-RB1 pathway has been described, that is, expression of RB1 on the messenger RNA (mRNA) level in all seminomatous and non-seminomatous GCTs, but detectable expression of RB1 on protein level only in teratomas, frequent deletions of RB1 and low expression of P16INK4A in GCNIS, seminomas and embryonal carcinomas (ECs) and overexpression of CyclinD2 (CCND2), as well as expression of CDK4 in GCNIS and invasive GCTs.19Cyclin-dependent kinases (CDK) have been defined as key drivers of cell cycle progression in several cancer entities. An arrest in the cell cycle leads to suppressed tumour growth as well as metastasis. Thus, targeting the cell cycle via selective pharmacological inhibition is a promising therapeutic option. Previous studies proved the CDK4/6 pathway as being active in solid tumours, such as liposarcoma, rhabdomyosarcoma, non-small-cell lung cancer, glioblastoma, oesophageal cancer, melanoma and breast cancer.In this study, we investigated the therapeutic potential of CDK4/6 inhibition in cisplatin-resistant and -sensitive paediatric and adult GCTs.21 All GCT cell lines, MPAF fibroblasts and FS1 Sertoli cells were cultivated as described previously.23 The cell lines HaCat, HEK293 and VHF fibroblasts were cultivated in Dulbecco’s modified Eagle’s medium , 1% penicillin/streptomycin) and THP-1 cells in RPMI medium  at 37 °C and 5% CO2. Short tandem repeat profiles of all cell lines are checked on a regular basis and are available upon request. For CDK4/6 inhibition, PaRi were dissolved in distilled water (pH 3) or dimethyl sulfoxide, respectively.In this study, the following GCT cell lines were used: TCam-2 (seminoma), 2102EP, NCCIT, NT2/D1 , JAR, JEG3 and BeWo . In the cisplatin-resistant GCT sublines 2102EP-R, NCCIT-R and NT2/D1-R, cisplatin resistance was provoked by cultivating these EC cell lines for a prolonged time with increasing sub-lethal cisplatin concentrations as published.23 In summary, 4000 cells were plated onto 96-well plates 24 h before PaRi or solvent application. In a total of 96 h, every day viability was screened by adding 50 μl XTT (1 mg/ml) plus 0.5 μl phenazine methosulfate (1.25 mM)  and measuring absorbance 4 h later in a ultraviolet–visible spectrometer . Each time point/concentration was measured in quadruplicates. LD50  doses were calculated using the GraphPad Prism software version 6. Relative Caspase-3/7 activity was adjusted to the relative cell viability as measured by Caspase-Glo-3/7 Assay and CellTiter-Glo Luminescent Cell Viability Assay , respectively, and performed according to the manufacturer’s protocols.The triphenyl tetrazolium chloride (XTT) assays were performed as described previously.2O) before incubation with 5 μl Annexin V  and 50 μg/ml PI (Sigma-Aldrich). For phospho-H3 (pH3) antibody staining, cells were permeabilised with 0.5% Triton X-100 before an 1 h incubation with a pH3 antibody at RT. Secondary antibodies and 50 μg/ml PI were added subsequently before flow cytometric measurement. For antibody details see Table Flow cytometry analyses of apoptosis rates and cell cycle distribution was performed by Annexin V/propidium iodide (PI) or PI staining, respectively. Briefly, for PI flow cytometry, cells were washed once with phosphate-buffered saline (PBS), fixed with Nicoletti buffer , and stained with 50 μg/ml PI (Sigma-Aldrich) for 10 min at room temperature (RT). For apoptosis measurement, cells were washed once with Annexin V-binding buffer  using the RNAeasy Mini Kit  according to the manual.Proteins were extracted by RIPA buffer containing 10% protease and phosphatase inhibitors (Sigma-Aldrich). Protein concentrations were assessed by the BCA Protein Assay Reagent Kit .Twenty micrograms of whole protein lysates were used for western blotting. Staining with Ponceau S solution (Sigma-Aldrich); 0.5% in 5% acetic acid) confirmed a correct membrane transfer. Membranes were blocked in 5% non-fat milk or 5% bovine serum albumin (BSA) in PBS + 1% Tween-20 (PBST) for 1 h and then incubated with primary antibodies overnight at 4 °C. Secondary horseradish peroxidase (HRP)-conjugated antibodies were incubated for 1 h at RT. The ChemiDoc Imaging System  was used for imaging. For antibody details see Table Cells were seeded in a 96-well plate and fixed with 4% formaldehyde 10 min) before permeabilisation with 0.2% Triton X-100 in PBS (5 min) with subsequent blocking in 1% BSA, 5% goat serum, 0.3 M glycine and 0.1% Tween-20 in PBS (1 h). Ki-67 antibody was incubated overnight at 4 °C before the addition of secondary antibody (1 h) and counterstaining with 0.5 μg 4′,6-diamidino-2-phenylindole (5 min). For antibody details see Table 0 min bef24 Antigen retrieval was carried out at 98 °C in ethylenediaminetetraacetic acid buffer . Primary antibodies were incubated for 30 min at RT. Afterwards, sections were incubated with an HRP-labelled secondary antibody at RT for 25 min. The substrate ‘DAB + Chromogen’ system was applied to visualise the target antigen . For antibody details see Table Immunohistochemical reactions were performed on 4-μm formalin-fixed and paraffin-embedded tissue sections as published previously.1 µg of RNA was in vitro transcribed into complementary DNA (cDNA) using 1 µl dNTP-Mix (10 mM), 1 µl Oligo(dT)18 Primer (0.5 µg/µl), 4 µl RT Buffer (5×), 1 µl Maxima H Minus Reverse Transcriptase 200 U/µl) and 0.5 µl RiboLock RNase Inhibitor (40 U/µl)  on a S1000 cycler (Bio-Rad) at 50 °C for 30 min. Further, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) runs were performed on a 384-well C1000 cycler (both Bio-Rad). In general, all samples were analysed in technical triplicates using 7.34 ng of cDNA for each replicate and the SYBR-green-based Luna Universal qPCR Master Mix . At the end of each run, melting-curve analyses were performed. Oligonucleotide sequences are given in Table 00 U/µl an = 3; seminomas, n = 4; ECs, n = 3; teratomas, n = 3; normal testis tissue (NTT), n = 4) was performed previously and re-analysed in the context of this study.25 Illumina microarray expression data of parental GCT cell lines  was extracted from previous studies available via Gene Expression Omnibus  .29 The Illumina 450k DNA methylation array was generated in a previous study and re-analysed in the context of this study (GSE76709).30The Affymetrix expression array analysis of GCT tissues  (25 May 2017) genome sequence. Statistical differential expression tests were determined using the ‘Differential Expression in Two Groups’ tool (version 1.02). The resulting p values were corrected for multiple testing by false discovery rate and Bonferroni correction. A p value of ≤0.05 was considered significant.RNA samples used for transcriptome analyses were quantified  and quality was measured by capillary electrophoresis using the Fragment Analyser and the ‘Total RNA Standard Sensitivity Assay’ . The library preparation was performed according to the manufacturer’s protocol (VAHTS Stranded mRNA-Seq Library Prep Kit). Three hundred nanograms of total RNA was used for mRNA capturing, fragmentation, synthesis of cDNA, adapter ligation and library amplification. Bead purified libraries were normalised and finally sequenced on the HiSeq 3000/4000 System  with a read setup of 1 × 150 base pairs (bp). The bcl2fastq tool was used to convert the bcl files to fastq files as well for adapter trimming and demultiplexing. Data analyses on fastq files were conducted with CLC Genomics Workbench . The reads of all probes were adapter (Illumina TruSeq) and quality trimmed . Mapping was done against the https://bioinfogp.cnb.csic.es/tools/venny/).31 The STRING algorithm was used to predict interactive networks from RNA-seq data (https://string-db.org).32 Functional annotation analyses were performed by DAVID (https://david.ncifcrf.gov/home.jsp).34 In functional annotation analyses of commonly deregulated genes in GCT cells, only categories (UP_Keywords) with at least five members and p values ≤0.05 were considered significant. For functional annotation analysis of genes deregulated in each GCT cell line, only categories (UP_Keywords) with at least ten members and a p value ≤0.05 was considered significant. Only genes related to an official human gene symbol were included. ‘The Cancer Genome Atlas’ (TCGA) datasets were analysed for isoform/gene expression, DNA methylation and copy number alterations (CNAs) using the UCSC Xena browser (https://xena.ucsc.edu) and the cBioPortal .37Venn diagrams were generated using Venny 2.1  and cell lines   and fibroblasts (MPAF) , ECs (n = 7), yolk-sac tumours (n = 4) and teratomas (n = 3)  and CDK6-201 (ENST00000265734.8) seemed to be the predominantly expressed isoforms in GCT (purple) and normal testis tissue (green)  and an EC expression signature  , demonstrating that AFP positivity is associated with the EC signature (indicative of yolk-sac tumour components), while β-hCG positivity can be found in both expression signatures (indicative of choriocarcinoma component in EC signature and choriocarcinoma/trophoblast component in seminoma signature)  and ribociclib  as therapeutic options for cisplatin-resistant and -sensitive GCTs. First, by re-evaluating microarray data of GCT tissues and cell lines, as well as by western blot analyses of GCT cell lines, we screened for expression of ely Fig. . CDK4/CDas) Fig. . AdditioAF) Fig. . In contsed Fig. . Interes 3) Fig. . Using tCDK4/6 expression in GCTs  and the gene body of CDK4   Fig. . On prot36) , CDKN1B  and MDM2  commonly amplified in seminomas and non-seminomas  and CHEK1 (6.8%) were amplified or deleted in non-seminomas, respectively (Supplementary data We screened three studies of GCT tissues for common CNAs (amplifications and deletions) in seminomas and non-seminomas  than ribociclib (LD50 range: 14.58–43.65 μM), and among the different GCT entities, choriocarcinoma cell lines presented as least sensitive to PaRi   presented as less sensitive towards PaRi than the GCT cells   and NT2/D1(-R) with cisplatin (5 μM/2.5 μM) alone or in combination with 5 μM palbociclib or 15 μM ribociclib Fig. . CompareWe also measured the effects of PaRi on the cell cycle and apoptosis rates by flow cytometry Fig. . As CDK4PaRi considerably induced apoptosis in GCT cell lines Fig. , except Accumulation of GCT cells in either in the G1/G0 or G2/M phase of the cell cycle and the induction of apoptosis pointed at an alternative mode of action of PaRi apart from the canonical CDK4–RB1 axis, prompting us to decipher the molecular mode of action of PaRi in GCT cells in more detail. We performed RNA-sequencing (RNA-seq) analyses of GCT cells ) treated for 16 h with 10 μM palbociclib to measure transcriptome-wide changes in gene expression. High quality  of all sequenced RNAs was verified by Agilent Fragment Analyser  in GCT cell lines after palbociclib treatment compared to solvent-treated cells  consisting of VLDLR, FOS, VEGFA and DDIT4, (2) consisting of SLC7A11 and SLC1A4 and (3) consisting of POLE2, KIF20A, AURKB, UHRF1 and CDCA3. Interestingly, genes also deregulated in MPAF fibroblasts (underlined in red) were mainly found in network (3). Based on a DAVID gene ontology (GO) analysis, the commonly deregulated genes in GCT cells could be associated with (ranked by frequency) cellular growth processes (genes in network (3)), such as mitosis, cell cycle and proliferation (light blue), protein regulatory processes (light green), regulation of transcription (red), drug response (black), developmental processes (yellow), cell surface (pink) and disease (grey)  genes  is a common palbociclib response in GCT cell lines and fibroblasts affecting the cell cycle. We confirmed RNA-seq data by qRT-PCR analysis of FOS, DDIT4, VEGFA, UHFR1 and AURKB in palbociclib-treated GCT cell lines and fibroblasts  Fig. .33,34 Thsts Fig. . Additiosts Fig. . Importasts Fig. .Fig. 5DeNext, we analysed the individual effects of palbociclib on each GCT entity  and MPAF fibroblasts by a DAVID GO analysis   were linked to all mentioned GO categories, six genes  were linked to four out of five detected categories, two genes  were linked to three out of five categories and five genes  were related to two out of five categories  in expression  and NT2/D1(-R) accumulated in the G2/M phase instead of the G1/G0 phase of the cell cycle Fig. . To idenTaken together, some EC cells are able to bypass the PaRi-induced G1/S arrest and reach the G2/M phase of the cell cycle, but downregulation of a plethora of the G2/M phase-regulating genes impedes completion of cell cycle and leads to induction of apoptosis.In this study, we demonstrated the utility of CDK4/6 inhibitors palbociclib and ribociclib in treating cisplatin-resistant and -sensitive GCTs. GCTs presented as more sensitive towards PaRi than analysed control cells (including MCF7 breast cancer cells), opening a putative therapeutic window for patients suffering from GCT. Although, it has to be mentioned that results gathered in this study by comparing GCT cell lines to one breast cancer cell lines is not enough to draw reliable conclusions on the differences between GCTs and breast cancers regarding PaRi treatment. In the future, comparing large tumour cohorts of each entity would be necessary.18 demonstrated that among seminomas and non-seminomas, ECs show highest levels of CDK4 expression, thus in ECs sensitivity to CDK4/6 inhibition correlates to high levels of CDK4 expression.18 Additionally, in our study cisplatin-resistant EC subclones showed a further increased sensitivity compared to parental cells. This suggests that mechanisms causing cisplatin resistance might in turn cause sensitisation towards inhibition of cell cycle regulators, that is, CDK4 or application of PaRi is able to revert the cisplatin-resistant phenotype. In line, combination of cisplatin with PaRi might be a novel therapeutic option for treatment of GCT patients, since combining both drugs considerably increased therapeutic efficacy. It remains elusive if combining PaRi with cisplatin leads to additive or synergistic effects.Among the different GCT entities, choriocarcinoma cells presented as least sensitive and EC cells as most sensitive. Interestingly, Schmidt et al.39 In this study, especially mature teratomas  had a favourable 24-week progression-free survival rate.39 In a follow-up study, Narayan et al.40 performed a retrospective analysis with long-term follow-up data of the patient cohort with unresectable mature teratoma treated with palbociclib and demonstrated that in 12 patients palbociclib might lead to a clinically meaningful delay in disease-related clinical events. Further, a randomised, blinded and placebo-controlled clinical study (NCT02300987) analysed the suitability of ribociclib to treat relapsed, refractory, incurable teratoma with recent progression. In this study, only 10 participants were recruited and thus numbers are quite limited (results pending). Nevertheless, CDK4/6 inhibition seems to be promising in treating especially mature teratomas, including teratomas with malignant transformation. The beneficial effect of palbociclib on the progression-free survival of non-teratoma GCT patients seems to be considerably weaker compared to patients with teratoma.39 Nevertheless, in the study of Vaughn et al.39, only a limited number of patients as well as only RB1-expressing GCT were included and only palbociclib was tested. Based on the result of our study, in GCTs PaRi do not only act via the canonical CDK4–RB1 axis and provoke effects beyond a cell cycle block, that is, apoptosis. Thus, it would be interesting and worth to (a) screen a larger cohort of type I and type II GCT patients with (b) no restrictions to RB1 status and to (c) include ribociclib and abemaciclib (Lilly) as alternatives to palbociclib. Additionally, we found that PaRi treatment led to an upregulation of CDK4 in GCT cell lines, which we postulated might counteract the effects of PaRi and might cause resistance. It would be interesting to screen the patients treated by Vaughn et al.39 for upregulation of CDK4 under therapy. If upregulation of CDK4 would be restricted to non-teratomatous GCT patients, the reduced activity of palbociclib in these patients could be explained.So far, only one early clinical study  tested the potential of the CDK4/6 inhibitor palbociclib in treating incurable, refractory, RB1-expressing GCTs.CDK4 was highly expressed, while CDK6 expression was very low, confirming results of Schmidt et al.,18 who also detected high CDK4 in 41% of GCTs (most strongly in ECs) and reduced CDK6 expression in 64% of seminomas and 23% of non-seminomas. Thus, in adult type II GCTs CDK4 seems to be the primary target of PaRi. In contrast, in all type I paediatric GCTs analysed, CDK6 was expressed considerably higher than CDK4. In conclusion, although differing in expression of PaRi target molecules, PaRi might be a therapeutic option for both type I and type II GCTs.In type II adult GCT cells In type II GCT cell lines, PaRi induced a pseudo-senescent state, mostly leading to accumulation of cells in the G1/G0 phase of the cell cycle Fig. . Neverth43 Thus, induction of apoptosis in response to CDK4/6 inhibition seems to be tumour type dependent, but is in general of benefit in terms of a therapeutic application.Furthermore, PaRi induced apoptosis in cisplatin-resistant and parental GCT cell lines, but not in the tested control cells. Induction of apoptosis after PaRi has also been described in bladder cancer cells and T cell acute lymphoblastic leukaemia, while in pancreatic ductal adenocarcinoma cell lines, CDK4/6 inhibition alone did not induce apoptosis.E2F/1, CDCA3, KIF20A, SPC25, UHRF1, CDKN2D and CDKN3  CDK4. Thus, GCT cells might become resistant to PaRi treatment over time, but we found no upregulation of the CDK4 downstream target CCND2 is commonly amplified in seminomas and non-seminomas (Supplementary data CCND1-amplified patients. Advances in PROteolysis TArgeting Chimeras (PROTACs) suppressing CDK4/6 were recently introduced in breast cancer and glioblastoma cell lines, where CDK4 and CDK6 protein expressions were reduced considerably.46 Thus, a PROTAC targeting CDK4/6 could be used as an alternative to PaRi, if resistance to these drugs might be an issue.In our analysis of CNAs, we found that In conclusion, palbociclib and ribociclib present a new putative therapeutic option for cisplatin-resistant and -sensitive paediatric and adult GCTs. Nevertheless, it has to be shown in the future whether mechanisms and drug kinetics/dynamics found and measured in this study on artificial cell lines can be mirrored to the in vivo situation. Therefore, large type I and II GCT cohorts have to be analysed in detail. Additionally, further experiments will have to reveal whether GCTs are able to acquire resistance towards PaRi and if a combinatorial treatment of cisplatin and PaRi can be of benefit for the patients. Up to now, it remains elusive if CDK4/6 inhibition might revert a cisplatin-resistant phenotype or if mechanisms causing cisplatin resistance in parallel sensitise GCT cells towards drugs that interfere with the cell cycle.Supplemental Figures, combined word-fileData S1


Despite having one of the lowest survival rates of all cancers, there have been no new approved treatments for malignant pleural mesothelioma (MPM) in over a decade. Standard-of-care treatment relies on Cisplatin plus Pemetrexed chemotherapy. Here, we tested a suite of BH3-mimetic drugs targeting BCL-2 pro-survival proteins of the intrinsic apoptotic pathway. We found BCL-XL is the dominant pro-survival protein in a panel of cell lines in vitro, though potent, synergistic cell killing occurred with MCL-1 co-targeting. This correlates with high-level expression of BCL-XL and MCL-1 in cell lines and a large cohort of patient tumour samples. BCL-XL inhibition combined with Cisplatin also enhanced cell killing. In vivo BCL-XL inhibition was as effective as Cisplatin, and the combination enhanced tumour growth control and survival. Genetic ablation of MCL-1 also enhanced the effects of BCL-XL inhibitors, in vivo. Combined, these data provide a compelling rationale for the clinical investigation of BH3-mimetics targeting BCL-XL in MPM. Despite rapid advances across multiple other tumour types, the only progress made in MPM treatment since 2003 is a modest improvement in median overall survival (OS) (by 2 months) with the addition of Bevacizumab to Cisplatin and Pemetrexed chemotherapy following the phase III MAPS study2. Multiple attempts to target some commonly mutated pathways in MPM with novel agents have been unuccessful3, whilst immune checkpoint inhibitors have had variable results, and are associated with increased toxicities4.Malignant pleural mesothelioma (MPM) is an aggressive and incurable, asbestos-related cancer associated with a high global mortality rate5. Accordingly, one avenue for cancer treatment gaining momentum is to directly target the intrinsic apoptosis pathway governed by the BCL-2 protein family6. This is now possible due to the development of small molecule inhibitors, known as BH3-mimetics, which directly target the BCL-2 pro-survival proteins. ABT-737, and its orally bioavailable analogue ABT-263/“Navitoclax”, were the first bona fide BH3-mimetics described. These bind to BCL-2, BCL-XL, and BCL-W with high affinity8. Pro-survival protein-specific inhibitors have since been developed including ABT-199 (“Venetoclax”) which targets BCL-29, A-1331852, a BCL-XL-specific inhibitor10, and S63845 and AZD5991, both MCL-1 inhibitors12, amongst others13. Venetoclax was recently approved by various regulatory bodies for use in the treatment of refractory chronic lymphocytic leukaemia15, and is being trialled in a range of other haematological cancers18.Defective apoptotic signalling is one of the hallmarks of cancer, and is associated with chemo-resistance19. In other solid cancers BCL-XL is frequently identified as a key pro-survival protein, although a common theme that is emerging is their additional co-dependence on MCL-1. This presents issues for translation to the clinic as in vivo co-administration of BCL-XL and MCL-1 inhibitors in mice results in fatal hepatotoxicity20.Notably, single agent efficacy of BH3-mimetics is predominantly observed across haematological malignancies as compared to solid cancers. Navitoclax showed promising activity in small-cell lung cancer mouse xenograft models, but when tested in a phase II human clinical trial, there was limited benefit seen and this was associated with dose-limiting toxicity (thrombocytopenia) due to potent, on-target killing of platelets22, though a recent study demonstrated single-agent efficacy with the BCL-XL inhibitor, A-1331852, which was enhanced by co-treatment with ionising radiation23.Studies on the importance of the intrinsic apoptosis pathway in MPM are relatively limited. Expression analyses on small patient cohorts and cell lines have been contradictory, although expression of BCL-XL and MCL-1 is consistently reported. The investigation of BH3-mimetics in MPM has also been limitedIn this current study, we show MPM cells are predominantly dependent on BCL-XL but MCL-1 is also a barrier to drug responses and, for the first time, in vivo responses can be achieved in MPM cell xenografts with BCL-XL inhibitors. Hence, BH3-mimetics have potential for future clincial application in the treatment of MPM.BCL-2 family member expression was compared in cell lines representative of the major MPM subtypes to their expression in other solid tumour cell lines by Western blot. Of the pro-survival proteins, BCL-XL was strongly expressed in all the MPM cell lines at levels relatively higher than most other tumour cell lines, whilst MCL-1 levels were consistent with the others Fig. . BCL-2 w50 values > 10 μM) . However, potent cell killing of most cell lines was observed with A-1331852 , and to a lesser extent ABT-263 (EC50 < 1 μM). All cell lines were also were relatively resistant to the standard-of-care drug Cisplatin (EC50 > 5 μM) except MSTO-211H (EC50 ~ 1.5 μM). Hence, the sensitivity of the cell lines to A-1331852 and ABT-263, suggests BCL-XL is the more critical pro-survival protein in MPM.Human MPM cell lines were treated with ABT-263 , ABT-199 (targeting BCL-2), A-1331852 (targeting BCL-XL) and S63845 (targeting MCL-1), and cell viability determined using CellTiter-Glo assays that reflect total live cell numbers. All cell lines were relatively resistant to both ABT-199 and S63845  was observed when ABT-263 or A-1331852 were titrated with S63845, with EC50 values decreasing by up to 100-fold  .We next assessed whether the potent single-agent activity of A-1331852 could be recapitulated in vivo, and how it compared with standard-of-care treatment, Cisplatin. Cisplatin and A-1331852 resulted in similar tumour growth control and survival  Fig. , and almDrug-treated tumours had increased apoptotic cell numbers (cleaved Caspase-3-positive), accompanied by reduced proliferating (Ki67-positive) cell numbers Fig. . TreatmeImmunohistochemistry and qRT-PCR showed BIM levels (protein and mRNA) increased in tumours from mice receiving Cisplatin, and unexpectedly, MCL-1 levels were reduced in tumours following A-1331852 treatment Fig. S. As the As A-1331852 has yet to be used in humans, we next tested ABT-263 (Navitoclax) in vivo as it potentially provides an avenue for future clinical studies. Using a similar dosing schedule , ABT-263 mirrored A-1331852 in terms of efficacy alone and in combination with Cisplatin Fig. S, weight 20. To circumvent this issue, we instead evaluated a genetic approach used previously20. MSTO-211H cells were engineered to express Cas9 and doxycycline-inducible sgRNAs targeting MCL-1, which were both effective , but sensitised the cells to A-1331852 by 14-22-fold, via apoptosis induction , and correlated with patient characteristics (Table p = 0.016) but not sarcomatoid histology, which was also observed in another study with a much smaller sample size (n = 54)27. High MCL-1 expression was associated with non-sarcomatoid histology (p < 0.001), while high BCL-2 expression was associated with non-epithelioid histology (p < 0.001). For the pro-apoptotic proteins, BAK expression was associated with age <65 years (p = 0.018) and patients who had received surgery (p = 0.001). BAK and BAX expressions were both associated with non-sarcomatoid pathology (p = 0.004).As our studies to assess BH3-mimetic responses used MPM cell lines, it was important to confirm that their expression pattern of BCL-2 proteins was reflected in patient tumour samples. Accordingly, tissue microarrays representative of 326 individual patients with MPM (see ref. cs Table . As withcs Table . On univp = 0.057) but not high MCL-1 expression (13.5 versus 11.6 months) . Notably, patients with tumours with high BCL-2 expression had a significantly worse OS (9.1 versus 12.8 months) , though high BCL-2 was seen in only a small percentage of samples.BCL-2 family protein expression was also correlated with patient OS Fig. . Patientp = 0.023)   Fig. S. When a 3) Fig. S.These findings confirm that in a large cohort of MPM patients, BCL-2 expression  are associated with poorer survival, regardless of histological subtype, the historically dominant prognostic factor in MPM.Platinum-based chemotherapy, in combination with Pemetrexed, has been the mainstay of MPM treatment for close to two decades. Here, we provide the first compelling in vivo evidence that the intrinsic apoptotic pathway provides an actionable target worthy of consideration for future clinical investigation in MPM patients. Specifically, we demonstrated that BCL-XL is the critical BCL-2 pro-survival family member, though MCL-1 expression also provides a significant barrier to apoptotic induction using BH3-mimetic drugs.23. However, whilst there was no single-agent activity with S63845 23, our studies are the first to demonstrate that MCL-1 and BCL-XL co-targeting with BH3-mimetics synergistically increases MPM cell killing in all MPM subtypes. These data agree with earlier work showing antisense oligonucleotides against BCL-XL induce apoptosis in MPM cells30, and studies showing RNAi against MCL-1 and BCL-XL co-operate to induce MPM cell killing, as does a NOXA peptide (targeting MCL-1) with ABT-73731. Our data are also consistent with the expression pattern of BCL-2 family proteins in our cell lines, as well as a large cohort of patient samples where BCL-XL and MCL-1 are the predominant pro-survival proteins expressed.The impressive MPM cell killing activity by BCL-XL inhibition using A-1331852 or ABT-263 we observed was consistent with recent in vitro studies using the same agents in MPM33. Intriguingly, we found inferior OS in the high BCL-2 patient tumour samples, which unexpectedly was associated with the epithelioid subtype that generally has a better prognosis. Whilst, this represents a small proportion of patients (4.5%), there may be some benefit in future studies examining how higher BCL-2 expression influences responses to Venetoclax in MPM.Notably, BCL-2 inhibition in vitro using Venetoclax was ineffective either alone or in combination with S63845. This is in keeping with the low expression of BCL-2 seen across our MPM cell lines and patient tissues as BCL-2 is a predictive biomarker for Venetoclax response28. Ex vivo analyses of tumours, and in vitro studies on the MSTO-211H cells used in xenografts, showed induction of BH3-only proteins BIM and NOXA, which would account for the improved outcomes seen in the combination studies, as both of these proteins can potently inhibit MCL-1. Unexpectedly, MCL-1 levels were also observed to be lower in tumours treated with BH3-mimetics, though this could be a consequence of apoptosis induction in the cells due to Caspase-3 activation, though it was not apparent in tumours treated with Cisplatin alone.The single-agent activity of both A-1331852 and ABT-263 translated into the in vivo setting where both drugs were as effective as Cisplatin. A caveat here is that both BH3-mimetics were administered almost daily over the treatment period, whilst only a single dose of Cisplatin was used. Nevertherless, the BH3-mimetics were both well-tolerated (other than the expected on-target thrombocytopaenia), and indeed, pilot studies (not shown) using additional doses of Cisplatin had to cease due to excessive weight loss observed in the mice. Most promising was our data showing further improvement in tumour growth control and survival  when both drugs were combined. This supports an earlier study that showed antisense oligonucleotides against BCL-XL promoted responses to Cisplatin in MPM xenograftsplus Cisplatin combinations was reflected in our studies using tumour-specific MCL-1 deletion to by-pass the fatal hepatotoxicity associated with co-administration of BCL-XL and MCL-1 inhibitors20. Although, this outcome is promising, the similarity in the outcomes of both of these “combination” in vivo experiments where indirect approaches were used to target MCL-1 (i.e. through BH3-only protein upregulation via chemotherapy or genetic deletion), suggests a subpopulation of cells within the tumours remain highly resistant to treatment as the tumours failed to regress. Whether this is due to inherent or acquired resistance is unclear at this stage, though will be an important avenue for future investigation. It will also be interesting to see whether these outcomes can be further improved once more targeted BH3-mimetics (e.g. through conjugation to tumour-specific antibodies) are developed and which could potentially allow safe co-treatment with BCL-XL plus MCL-1 direct inhibitors. One potential limitation of our mouse studies is that a single cell line was used, hence, future studies using additional cell lines, or alternatively, patient-derived xenografts, will be informative to provide further insight into the in vivo effect of BH3-mimics in MPM.The improvement we observed with BCL-XL inhibitor 19. Platinum-relapsed small-cell lung cancer remains a notoriously difficult disease to treat and, therefore, this outcome should not be a deterrent towards testing Navitoclax in other cancers, such as MPM. Indeed, Navitoclax is currently being tested in several phase II clinical studies either as monotherapy or in novel combinations . Trials have also just commenced in both haematological and solid malignancies of a novel BH3-mimetic formulation targeting BCL-XL that may lead to reduced toxicity and increased efficacy . Hence, BCL-XL remains an actionable target for MPM treatment.Currently, Navitoclax represents the most feasible BCL-XL inhibitor of those investigated for translation due to its previous (and current) testing in humans. One potential argument against this is the suboptimal responses and high rates of grade III–IV thrombocytopenia observed in the phase II small-cell lung cancer single arm studyIn conclusion, our data suggest that BCL-2 family proteins play a pivotal role in MPM cell survival. Although BCL-XL and MCL-1 co-inhibition provides the most effective strategy to eliminate MPM cells, BCL-XL inhibition alone has encouraging activity, and sensitises cells to Cisplatin chemotherapy, resulting in enhanced cell killing. In a disease with limited therapeutic options and poor survival outcomes, our findings will help direct relevant BH3-mimetic-based clinical trials for MPM patients.All BH3-mimetic drugs and Cisplatin were purchased from Selleckchem.25. Immunohistochemical analyses were performed as described previously34 using antibodies listed in Table S25.Tumour samples were collected from consensual patients with a diagnosis of MPM from the Austin Hospital  between 1 January 2000 and 31 December 2010, under an Austin Hospital ethics committee-approved protocol (H2012/04446) and tissue microarray (TMA) prepared from 326 patient samples, as described previously2.Cell lines were obtained from the ATTC (USA) except 14R-100, which was generated from a consensual Austin Hospital MPM patient  under Austin Health ethics number H2006/02394. All lines were confirmed to be mycoplasma negative based on in-house MycoAlert assays (Lonza). Cells were cultured in RPMI 1640 medium (Gibco) containing 10% (v/v) foetal calf serum , 1% (v/v) GlutaMAX (Gibco) and 100 U/ml penicillin/100 mg/ml streptomycin (Gibco), and maintained at 37 °C with 5% CO34. For FACS-based assays, cells  were seeded into 24-well plates, then 24 h later treated with vehicles, drugs, either alone, or in combination for 72 h. For pan-caspase inhibitor assays, Q-VD-OPh  was added  to respective wells. Live and dead cells were harvested and pelleted by centrifugation then stained with Annexin V-APC (BD Biosciences) and propidium iodide (Sigma Aldrich) in Annexin V-binding buffer (BD Biosciences).CellTiter-Glo viability assays were performed after 72 h incubation with drugs as described previously35.FACS analysis was performed on a BD FACSCanto II flow cytometer . Data was analysed using FlowJo Software Version 10  with the number of viable cells (Annexin V negative/propidium iodide negative) normalised relative to the number of viable cells cultured in the vehicle control. GraphPad Software was used for statistical analysis. Synergy analysis was performed with Combenefit software using the BLISS models36, as described previously34. sgRNAs were designed using MIT CRISPR design software (http://crispr.mit.edu) providing the sequences: (1) 5′-CCGCCCGGGAGGGCGACTTT-3′ and (2) 5′-CTCAAAAGAAACGCGGTAAT-3′.Deletion of MCL-1 was performed using the lentiviral vector system34. Membranes were probed with antibodies listed in Table SCell lysates were prepared and analysed by Western blotting as described previously37.Total RNA was extracted from cell pellets or fresh-frozen tumour samples and analysed by quantitative RT-PCR using previously published methods and primer sets6) in Matrigel  were injected subcutaneously into the right flank of NOD-scid (6–8 weeks) female mice. When tumours reached 150–200 mm3, the mice were randomised to ensure consistent average tumour size across all study arms (n = 8–10 per group based on previous outcomes of drug treatment studies using MPM xenografts) and treated by oral gavage with either A-1331852 (25 mg/kg) or ABT-263 (100 mg/kg) prepared in 60% (v/v) Phosal 50PG, 27.5% (v/v) PEG400, 10% (v/v) ethanol, and 2.5% (v/v) DMSO for 5 consecutive days per week for 3 weeks. Cisplatin  was delivered by single intraperitoneal injection on Day 1. To induce Mcl-1 sgRNA expression, mice were fed with doxycyline chow . Tumours were measured using calipers by a blinded investigator (SA) and three mice per treatment group were culled by CO2 asphyxiation, 1 day after the final dose. The remaining mice were culled at ethical endpoint when tumours reached 1000 mm3. Blood was collected 24 h after treatment on day 10 and analysed on an Advia 2120 (Siemens).All animal experiments were approved by the Austin Health Animal Ethics Committee (A2017_05465/A2018_05584) and carried out in accordance with the guidelines of Austin Health/University of Melbourne and conformed to the National Health and Medical Research Council’s code of practice for the care and use of animals for scientific purposes. Cells . Student’s Supplementary Table LegendsSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6Supplementary Table 7Supplementary Figure LegendsSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Figure 6Supplementary Figure 7Supplementary Figure 8Supplementary Figure 9Supplementary Figure 10Supplementary Figure 11Supplementary Figure 12Supplementary Figure 13Supplementary Figure 14


Hepatocellular carcinoma (HCC) is the most aggressive and frequently diagnosed malignancy of the liver. Despite aggressive therapy, life expectancy of many patients in these cases is extended by only a few months. Hepatocellular carcinoma (HCC) has a particularly poor prognosis and would greatly benefit from more effective therapies.The CCK-8 assay and colony formation assays were used to test the cell proliferation and viability. The effects of combination Biochanin A and SB590885 on apoptosis and cell cycle arrest of HCC cells were analysed by flow cytometry. The expression of ERK MAPK and PI3K/AKT/mTOR signalling as well as apoptosis and cell cycle-related proteins in HCC cells were tested by western blotting. The HCC cell xenograft model was established to test the tumor proliferation. Serum and plasma were tested for liver and kidney safety markers  by using SpectraMax i3X.The combination of natural product Biochanin A with the BRAF inhibitor SB590885 synergistically suppressed proliferation, and promoted cell cycle arrest and apoptosis in vitro. Furthermore, we demonstrated that the combination of Biochanin A and SB590885 led to increased impairment of proliferation and HCC tumour inhibition through disrupting of the ERK MAPK and the PI3K/AKT pathways in vitro. The volumes tumors and the weights of tumours were significantly reduced by the combination treatment compared to the control or single treatments in vivo. In addition, we found that there was no significant hepatorenal toxicity with the drug combination, as indicated by the hepatorenal toxicity test.The results identify an effective combination therapy for the most aggressive form of HCC and provide the possibility of therapeutic improvement for patients with advanced HCC. Hepatocellular carcinoma (HCC) is identified as the fifth most common cause of cancer related mortality in worldwide and the prognosis for HCC remains very poor –6. SorafThe development of drug resistance restricts the efficacy of single therapeutic agents . Thus, nTherefore, to develop a therapeutic strategy that could provide an improvement in the treatment of advanced HCC without increased toxicity, we investigated the effects of combina-tions of biochanin A with the BRAF inhibitor SB590885 on anti-proliferative and survival pathways in HCC cells in vitro and in vivo.2.The HCC cell lines Bel-7402 and SK-Hep-1 were obtained from Shanghai Genechem Co. . The STR genotyping reports of the HCC cell lines Bel‑7402 and SK-Hep-1 are listed in Additional file 3) were seeded into a 96‑well plate and cultured in regular growth medium containing 10% foetal bovine serum. After 24 h, the cells were exposed to serial dilutions of biochanin A , SB590885 . After the cells were incubated at 37 °C for 48 h, the medium was removed and 100 μl of RPMI‑1640 and 10 μl of CCK‑8 reagent were added. The cells were incubated for 2 h at 37 °C. Finally, the absorbance of each well was measured at 450 nm using SpectraMaxi3X . All cell viability assays were performed at least three times. The combination index (CI), which was calculated by the Chou–Talalay equation, indicates synergistic effects at CI < 1, additive effects at CI = 1, and antagonism at CI > 1 [Biochanin A and SB590885 were purchased from Targetmol . Cell viability was tested using the CCK‑8 assay according to the manufacturer’s instructions. Cells  for 30 min at 37 °C in the dark, stained with 400 μl propidium iodide (PI) (50 μg/ml) and placed at 4 °C in the dark for 30 min , 25. TheSK-Hep1 and Bel-7402 cells were plated into 6-well plates for 24 h, and then treated with 75 µM biochaninA,12 µM SB590885 or the combination for 48 h. Total protein from the cells was extracted using RIPA buffer with a proteinase inhibitor . Protein samples were subjected to SDS–PAGE and then transferred to PVDF membranes . The membranes were blocked with 5% BSA for 1 h, and incubated with primary antibodies such as Bcl-2 (1:1000), Bax (1:1000), cleaved PARP (1:1000), cleaved caspase-9 (1:1000), P21 (1:1000), P27 (1:1000), CyclinD1 (1:1000), p-MEK (1:1000), MEK (1:1000), p-ERK (1:1000), ERK (1:1000), AKT (1:1000), p-AKT (1:1000), P70S6K (1:1000), p-P70S6K (1:1000), S6 (1:1000), p-S6 (1:1000), β-actin (1:1000) ) and GAPDH (1:1000) at 4 °C overnight. The membranes were washed three times and incubated with the secondary anti-rabbit or anti-mouse antibody for 1.5 h at room temperature. Protein bands were visualized by Bio-Rad ChemiDoc Imaging System.6 Bel-7402 cells were injected subcutaneously into the right flank near the hind leg of each nude mouse until the tumor volume was ~ 100 mm3. Biochanin A was dissolved in dimethyl sulfoxide (DMSO) at stock concentration of 100 mM, and stored in the dark at − 20 °C. SB590885 was dissolved in a 10 mM stock solution in DMSO and stored in the dark at − 20 °C. Then, we treated male athymic nude mice bearing palpable Bel-7402 xenografts tumours (~ 100 mm3) with control (vehicle-treated mice) 50 μl 0.5% dimethyl sulfoxide (DMSO), biochanin A (25 mg/kg/day), SB590885 (7.5 mg/kg/day) and the combination by intraperitoneal injection for 5 weeks, 7 times/week. The tumour volume was measured every week and was calculated by the following formula: volume = 1/2 (length × width2). After 5 weeks, the mice were euthanized and the tumours were isolated.The HCC cell xenograft model was established as previously described . BrieflyTumor xenografts were formalin-fixed, paraffin-embedded, and sectioned in preadherent slides. The slides were subjected to the indicated primary antibody against PCNA (1:200) ) overnight. The sections were incubated with secondary antibody and then developed with 3,3′-diaminobenzidine chromogen according to the protocol. All samples were visualized, and images were captured using a DM2500 fluorescence microscope . Image‑Pro Plus 4.5 software was used to analyse the staining data.Serum and plasma were tested for liver and kidney safety markers  . Graphs were generated with GraphPad Prism 7.0 software . All experiments were performed three independent times. Statistical significance was determined by Student’s t-test, and a two-sided To determine the effect of the combination treatment on hepatocellular carcinoma, we investigated the effects on cellular morphology using microscopic inspection and the viability of Bel-7402 and SK-Hep-1 cells using the CCK-8 assay. We found that single treatment with biochanin A and SB590885 for 48 h suppressed the viability of Bel-7402 and SK-Hep-1 cells. The combination treatment significantly decreased the cell viability compared with that of the single treatment group  with control (vehicle-treated mice), biochanin A (25 mg/kg/day), SB590885 (7.5 mg/kg/day) and a combination of biochanin A and SB590885 for 5 weeks. The size, the volume and the weight of tumours were significantly reduced by the combination treatment compared to those of the control or single treatments (Fig. To test the effect of the combination treatment in vivo, we treated male athymic nude mice bearing palpable Bel-7402 xenografts tumours (~ 100 mmnts Fig. a–c. Furtnts Fig. e. Westernts Fig. f. In addThe heterogeneity of HCC is characterized by both heritability  and phenChemotherapy is an important treatment option for various clinical factors. Among the chemotherapeutic agents, sorafenib is administered and is significant in the treatment of HCC. However, sorafenib only improves median overall survival by ~ 3 months –34. SingSeveral studies have suggested that HCC cell activation by different factors is known to increase both Ras/Raf/ERK MAPK and PI3K/AKT/mTOR signalling , 40. SorIn conclusion, our results show that combination of the natural product Biochanin A with the BRAF inhibitor SB590885 synergistically suppressed proliferation, and promoted cell cycle arrest and apoptosis in HCC cells. The combination of biochanin A and SB590885 inhibited the HCC cells proliferation through ERK MAPK and PI3K/AKT pathways. We found that there was no significant hepatorenal toxicity with the drug combination, as indicated by the hepatorenal toxicity test. These results indicate that combination Biochanin A and SB590885 is a potential treatment for HCC.Additional file 1: Table S1. Synergistic indexes of combination treatment with Biochanin A and SB590885 in Bel-7402 and Sk-Hep1 hepatocellular carcinoma cell lines. Table S2. Clinical and biological analyses. Table S3. The STR test results of SK-Hep-1 cells. Table S4. The STR test results of Bel‑7402 cells.Additional file 2. Representative photomicrographs of Bel-7402 and SK-Hep1 cells after 48 h of combination treatment biochanin A and SB590885.


We report significant ROS-specific metabolic alterations in sphingolipid metabolism, fatty acid oxidation, purine metabolism, amino acid homeostasis and glycolysis. These data provide further evidence of ROS directed metabolic changes in AML and the potential for metabolic targeting as novel therapeutic arm to combat this disease.Acute myeloid leukemia (AML) is a heterogeneous disease with poor clinical outcomes. We have previously shown that constitutive activation of NADPH oxidase 2 (NOX2), resulting in over-production of reactive oxygen species (ROS), occurs in over 60% of AML patients. We have also shown that increased ROS production promotes increased glucose uptake and proliferation in AML cells, mediated by changes in carbohydrate metabolism. Given that carbohydrate, lipid, and protein metabolisms are all intricately interconnected, we aimed to examine the effect of cellular ROS levels on these pathways and establish further evidence that ROS rewires metabolism in AML. We carried out metabolomic profiling of AML cell lines in which NOX2-derived ROS production was inhibited and conversely in cells treated with exogenous H We furt2 (NOX2) . Indeed,pression . To undepression . We demopression .2O2), we report significant metabolic alterations in sphingolipid metabolism, fatty acid oxidation (FAO), purine metabolism, amino acid homeostasis and glycolysis.In addition to carbohydrate metabolism, there are two other main classes of molecules involved in metabolism, proteins, and lipids. These can be either catabolized to produce energy or synthesized to molecules such as nucleotides and structural proteins for the generation of cell membranes. Given that carbohydrate, lipid, and protein metabolisms are all intricately interconnected, we aimed to examine the effect of ROS on these pathways and establish further evidence that ROS rewires metabolism in AML. Using metabolomic profiling of AML cell lines (in which ROS production was inhibited by knocking down NOX2 expression) or using a cell line incubated with glucose oxidase  at purchase. THP-1 and NOMO-1 cells were lentivirally transduced with shRNA complementary to NOX2 mRNA and encoding puromycin resistance (THP-1 NOX2-KD or NOMO-1 NOX KD) and control cells (shTHP-1 or shNOMO-1) which had been transfected with shRNA coding for a non-mammalian target sequence as previously described . Viability was tested using 7-AAD (1 μg/ml) and analyzed using flow cytometry; viable cells were used in subsequent superoxide and metabolic assays.Cell lines were cultured according to recommended conditions at 37°C, 5% COescribed . Additioescribed . To mimiTo determine expression levels of NOX2, cells were incubated with NOX2 PE conjugated antibody (5 ng/μl) or an isotype matched control (MBL), incubated for 45 min at 4°C and analyzed by flow cytometry. All flow cytometric data were acquired using an Accuri C6 flow cytometer . A minimum of 3,000 events was collected in the region of interest. Data analysis was performed using FCS express v6.Cell cultures were adjusted for viable cell number and superoxide measurement carried out using the chemiluminescent probe Diogenes as previously described .http://www.metabolon.com). Cell line samples were analyzed using ultra-high-performance liquid chromatography mass spectrometry (UPLC-MS), utilizing Waters ACQUITY UPLC and Thermoscientific Q-Exactive high resolution mass spectrometer interfaced with a heated electrospray ionization source and Orbitrap mass analyzer. Raw data was extracted, peaks identified, and quality control processed using proprietary Metabolon™ hardware, software, and biochemical library database. Following normalization to Bradford protein concentration, log transformation and imputation of missing values with the minimum observed value for each compound, Welch unequal variance two-sample t test was performed to identify significant differences between the experimental groups. To account for potentially high false discovery rate (because of multiple comparisons), a q-value was also calculated, where a lower q-value is an indication of higher confidence in the result.Metabolomic analysis was performed on quadruplicate samples of the AML cell lines THP-1, NOMO-1 and Mv4;11 by Metabolon™ (2O2) in an AML cell line  that does not generate NOX2 derived ROS. To establish further evidence that ROS affects metabolism in AML, we have now analyzed the whole metabolome of AML cell lines producing different levels of NOX-2 derived ROS. Since Mv4;11 cells have very low levels of ROS, we treated these cells with GOX. Conversely, we have also analyzed the metabolome of lines generating NOX2 derived ROS (THP-1 and NOMO-1) and have examined the impact of knockdown or inhibition of NOX2 in this context using shRNA vectors and DPI, respectively. To compare NOX2-KD to an appropriate control, we created control lines infected with non-mammalian target (labeled ‘sh’). The levels of DMSO in treated samples were less than 0.01%. Given the very low levels of DMSO, we did not treat control samples with this proportion of DMSO as the effects would be negligible. The knockdown of NOX2 expression/superoxide production (>90% reduction) in THP-1 NOX2 KD as well as the impact of DPI on THP-1 cells has been previously described (2O2 (mediated by incubation with GOX) resulted in several significant changes of biochemical metabolites when cultured with 10 and 20 mU/ml, respectively. Analysis of the overall biochemical variations between each sample was performed using principal component analysis (PCA) . Due to the limited use of three cell lines, it was not possible to correlate changes with cell line abnormalities . However, NOX2 KD and those cells treated with DPI showed significant differences of biochemical metabolite levels compared to shTHP-1/NOMO-1 controls. The largest effects were observed with DPI inhibition. The global differences between Mv4;11 cells treated/untreated with GOX were much more modest  analyses of cellular metabolic profiles  and in free carnitine and its metabolic precursor (deoxycarnitine). Many of the latter metabolites were lower in the NOMO-1 NOX2 KD cells but fell below the cut-off for statistical significance   GOX dosage  than AML blasts exhibiting low levels of ROS  that constitutively produce extracellular superoxide (ROS) and a cell line with negligible ROS production . These models permitted the reciprocal approach to reduce ROS levels (by knocking down NOX2) or to add exogenous ROS (H2O2) by incubating cells with GOX.Previous work by our group linked mutational RAS activation with increased NOX2 derived ROS production and cellular proliferation in normal human hematopoietic cells . This wand NF-κB . More rend NF-κB . We now When NOX2 was knocked down in THP-1 and NOMO-1 cell lines, both cell types exhibited alterations in metabolites linked to FAO and complex lipid homeostasis. Treatment of both these cell lines with DPI also resulted in changes consistent with the effect of NOX2 derived ROS on metabolites linked to glucose utilization and amino acid homeostasis. These ROS induced changes in concentrations of glycolytic metabolites are commensurate with the idea that ROS induces increase in glucose uptake in these cell lines which leads to metabolic changes and redox adaptation that supports the enhanced proliferation of leukemia cells . However2O2 is produced as a co-product by the enzyme xanthine oxidase . In addition to these changes, THP-1 and NOMO-1 cells also exhibited decreases in the purine synthetic intermediate AICA ribonucleotide and increases in the pyrimidine synthetic intermediate orotate  and serine synthesis pathway, and discrepancies here may simply be reflective of differing relative cellular utilization of alternative antioxidant generating pathways. Overall, the more modest changes in this cell line may be reflective of the smaller variation between the untreated and treated samples, as revealed by the PCA analysis  dose demonstrated changes to both sphingolipid metabolism and amino acid homeostasis consistent with the data generated in the THP-1 and NOMO-1 cell lines. Culture with GOX did not dose dependently increase the various sphingolipids and other sphingomyelins. It suggests higher GOX levels maybe toxic, but we did not observe changes in cell viability. Alternatively, it is interesting to speculate that higher levels of GOX are activating a negative feedback loop. Little effect was observed on purine synthesis and the impact on FAO. FAO is known to be negatively correlated with ROS production  and is aThe biochemical changes arising from the DPI treatment noted several common alterations; however, there were some degree of differences within the THP-1 and NOMO-1 cells. Specifically, both cell lines exhibited alterations in metabolites linked to glucose utilization, TCA cycle activity, lipid availability, nucleotide turnover, nicotinamide metabolism, and amino acid homeostasis. It is recognized that at micro-molar concentrations, DPI inhibits not only NOX but also mitochondrial respiration through the inhibition of NAPDH cytochrome P450 oxidoreductase, as well as, nitric oxide synthase, and xanthine oxidase [reviewed in ]. AdditiOtto Warburg initially ascribed his observation that cancer cells exhibited increased aerobic glycolysis to defective mitochondrial function in these cells , althougIn summary, exposure of cells to NOX2 derived ROS is consistent with cellular alterations in protein degradation rates, amino acid utilization, lipid metabolism and energy production. Changes of this nature may correlate with alterations in growth and proliferation rates.The original contributions presented in the study are included in the article/Conceptualization, AT and RD. Investigation, AR, SD, RD, and AT. Writing—Original Draft, AR and AT. Writing—Review & Editing, AT, RD, and AR. Funding acquisition, AT and RD. Resources, SD. Supervision, AT, RD, and SD. All authors contributed to the article and approved the submitted version.This work was supported by grants from Tenovus Cancer Care (AR: PhD2013/L19) and Blood Cancer UK (13029). Welcome ISSF supported AT and RD.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HDAC5 expression has been associated with poor prognosis in breast cancer (BC) tissue. We aimed to analyze HDAC5 expression in BCVY and older patients and their correlation with clinical features, also studying the potential of HDAC5 inhibition in BC cell lines. Methods: HDAC5 expression in 60 BCVY and 47 older cases were analyzed by qRT-PCR and correlated with clinical data. The effect of the HDAC5 inhibitor, LMK-235, was analyzed in BC cell lines from older and young patients. We performed time and dose dependence viability, migration, proliferation, and apoptosis assays. Results: Our results correlate higher HDAC5 expression with worse prognosis in BCVY. However, we observed no differences between HDAC5 expression and pathological features. Our results showed greatly reduced progression in BCVY cell lines and also in all triple negative subtypes when cell lines were treated with LMK-235. Conclusions: In BCVY, we found higher expression of HDAC5. Overexpression of HDAC5 in BCVY correlates with lower survival rates. LMK-235 could be a potential treatment in BCVY.Background: Breast cancer in very young women (BCVY) defined as <35 years old, presents with different molecular biology than in older patients. High  Breast cancer is the most common cancer among women worldwide . As a peEpigenetic modifications are reported to play an important role in the onset and progression of many diseases. Carcinogenesis can be explained by genetic alterations, but epigenetic processes such as DNA methylation, histone modifications, and non-coding RNA deregulation are also involved . HDAC5 has been correlated with worse prognosis in patients with breast cancer (BC) [Histone deacetylases are essential for global acetylation patterns in the nucleus and the epigenetic regulation of gene expression . Histonecer (BC) .Histone deacetylase inhibitors (HDACi) are currently acquiring importance in cancer treatment, being the only approved epigenetic therapy in clinics altering histone proteins to date. HDACi have been classified into four groups depending on their structure: Hydroxamic acids, cyclic peptides, benzamides, and short chain fatty acids. The hydroxamic acids  have been approved by the FDA as anticancer treatments. However, they are limited by their nonspecificity, affecting all HDACs . These aA previous study based on evaluation of new HDAC inhibitors identified LMK235 (N-((6-(hydroxyamino)-6-oxohexyl)oxy)-3,5-dimethylbenzamide) as the most cytotoxic compound, displaying equipotent HDAC inhibition in pan-HDAC assay when compared to vorinostat. In contrast to vorinostat, LMK235 showed a novel HDAC isoform selectivity profile with a preference for HDAC4 and HDAC5 .HDAC5 expression in BCVY, and this methylation was related to significant HDAC5 overexpression in BCVY patients [HDAC5 expression in a large cohort of BC patients, to clarify the correlation between HDAC5 overexpression and relapse and survival in BCVY and BCO and the inhibitory effect of LMK-235 in breast cancer cell lines from very young and older women with BC. Previous group studies showed significant differences in miRNA expression  and methpatients . In the A total of 107 patients were included, 60 were young women under 35 years (BCVY) and 47 samples from women over 45 years (BCO). The median age at breast cancer diagnosis in the young patient’s group was 32 years , and in the old patient’s group was 69 years . Hereditary cases with BRCA1 and BRCA2 mutations were excluded from the study. In BCVY cohort, the immunohistochemical analyses showed 39.9% (n = 24) of luminal patients, 11.6% (n = 7) HER2-positive, 21.6% (n = 13) luminal/HER2 and 23.3% (n = 14) triple negative. In BCO group, 59.5% (n = 28) of patients were luminal, 10.6% (n = 5) HER2-positive, 10.6% (n = 5) luminal/HER2 and 17% (n = 8) presented triple negative subtype. Median follow-up was 93.4 months .HDAC5 was significantly overexpressed in BCVY patients   where the lower grades presented higher HDAC5 expression  A. We foupression . Regardi to BCVY . DespiteHDAC5 was significantly overexpressed in BCVY patients that died in comparison with BCO  and a significant HDAC5 overexpression was observed for BCO patients that survive when compare with BCO patients that died   and 13.3% (n = 6), respectively. However,  = 0.01) B. These xpressed A whereasepressed B. In terBreast cancer cell lines were exposed to increasing concentrations of HDAC5 inhibitor LMK-235  for 24, 48, and 72 h. We observed that relative proliferation decreased in a dose- and time-dependent manner for some breast cancer cell lines . After 4p-value = 0.01), HCC1806  and MDA-MB-231  breast cancer cell lines after 48 h of treatment. Specifically, we observed ~23% less cell migration in the HCC1937 cell line, ~14.7% for MDA-MB-231, and also HCC1806 cell line showed a substantial reduction. HCC1500 cells treated with LMK-235 presented cell migration reduced by ~5.3%. However, the last cell line exhibited considerable cell death at 48 h of cell treatment making wound-healing analysis difficult. In contrast, breast cancer cell lines MCF-7 and BT474, both from luminal old BC patients, showed insignificant cell migration reduction when cells were treated with LMK-235 , independently of age. However, we observed markedly diminished viability in the young and luminal HCC1500 cell line treated with HDAC5 inhibitor, which could not be detected in the luminal subtypes from older cell lines (MCF7 and BT474). To confirm the results obtained by MTT, we performed an apoptosis assay by flow cytometry. After 48 h of treatment, similar results to the MTT assay were observed. Results show increasingly early apoptosis in most BC cells lines after 48 h of LMK-235 (10 and/or 20 µM) treatment. Interestingly, and in line with previous proliferation and migration results, the most important increase in apoptosis was observed in triple negative cell lines from both BCVY and BCO. Intriguingly, LMK-235 induced a more profound increase in HCC1500 cell death for higher concentrations than in MTT assay A. These HDAC5 mRNA expression in all breast cancer cell lines that were treated with LMK-235  for 48 h. The levels of HDAC5 protein were barely detectable for all cell lines, as previously observed in other studies [To investigate whether the changes in  studies ,24 that HDAC5 was notably overexpressed in BCVY. We validate this issue analyzing HDAC5 overexpression in a larger cohort of young patients under 35 years and over 45, as well as studying the correlation of HDAC5 expression levels with different clinic-pathological features. Additionally, we analyzed the effects of an HDAC5 inhibitor, LMK-235, on apoptosis, proliferation, and migration in breast cancer cell lines.BCVY can be understood as a unique and different entity from breast cancer in older people, not only because of patients’ characteristics (where aspects such as fertility preservation must take into account) but also in its biological and molecular tumor characteristics. Our group has focused for years on the study of these differential molecular characteristics in breast cancer tumors of young and old women. Previous results  showed uHistones are acetylated on lysine side chains, which neutralizes lysine’s positive charge, leading to open chromatin structure by reducing interaction between histone and negatively charged DNA ,26. ThusHDAC5, high expression has been correlated with poorer prognosis in patients with BC or pancreatic neuroendocrine tumors and has been attributed to oncogenic effects [HDAC5 overexpression has been correlated with triple negative BC tumors [HDAC5 expression in BCVY than BCO samples. Next, we wonder whether HDAC5 overexpression was correlated with relapse and survival, and other clinical and pathological data. BCVY patients with HDAC5 overexpression showed a higher risk of exitus, whereas results were contrary for BCO that showed a significant risk of death when HDAC5 was repressed. In agreement with these results, higher HDAC5 expression was observed in BCVY samples that died whereas lower expression was found in BCO patients that die. Our results reinforce the hypothesis that HDAC5 overexpression in BCVY patients is related with a poor outcome observed in this group of age. BCVY is not a usual diagnosis, so there is a limitation in the number of BCVY samples and the present study is actually the first report that includes one of the highest cohort of BCVY patients in comparison with other larger datasets . HDAC5 expression and its correlation with different clinical and pathological features . We found no correlation between HDAC5 expression and clinical features in BCVY. The only meaningful association observed was between HDAC5 downregulation and higher tumor grades (II and III) in BCO. Higher tumor grades are associated with poorer prognosis and in our study with HDAC5 repression for BCO, which agrees with survival tendencies observed at this age group where HDAC5 repression correlates with poorer survival in BCO. Next, we evaluated HDAC inhibitors are the most widely investigated epigenetic drugs in clinical studies . In vitrAdditionally, LMK-235 response was lower in ER-positive cell lines, except for HCC1500 which presented an intermedium response in comparison with the rest of ER-positive cells, as we previously mentioned. Previous studies found LMK-235 response in triple negative BC cell lines ,34,35,36HDAC5 is involved in tumorigenesis and cancer progression reducing survival, specifically in BCVY. We propose studying HDAC5 inhibitors like LMK-235 in a larger cohort of breast cancer cell lines from young and old women and from different molecular subtypes in order to evaluate their inhibitory effect. Additionally, cell lines studies show a significant response to HDAC5i of triple negative BCO in addition to all BCVY cell lines independently of their molecular subtype.These results reinforce the hypothesis that All samples included in the study were archived formalin-fixed paraffin-embedded (FFPE) BC tissues stored at the Hospital Clínico Universitario of Valencia, Spain. We selected 60 breast cancer samples from women under 35 years old (BCVY) and 47 samples from women over 45 (BCO). The clinical characteristics of patients are included in All patients’ clinical data and tumor pathological characteristics were collected at their first visit and information about relapse and survival on subsequent visits. Tumor size and lymph node involvement were assessed using the eighth edition of the American Joint Committee on Cancer staging manual . Patient2 . Cell lines were cultured in RPMI 1640 or DMEM medium supplemented with 1% L-glutamine and 10% fetal bovine serum . The culture conditions were identical in all cell lines: 37 °C and 5% CO2 . Cells wThe protocol used is based on a colorimetric MTT -2,5-diphenyltetrazolium bromide) assay. We seeded and cultured 2000 cells in 96-well plates. Next, cells were treated with a specific drug dose, and MTT assay was performed 24, 48, and 72 h after treatment. The absorbance of this colored solution was quantified by measuring at 590 by spectrophotometer. Each experiment was performed in triplicate and repeated at least twice. Average values for triplicates were calculated. Absorbance observed at different drug concentrations was compared with the respective non-treated controls and cell viability was calculated.5 cells/well and incubated overnight until they reached 70% confluence. A pipette tip was used to generate a wound in the cell layer. Cells were then treated with LMK-235 (20 μM). Each experiment was performed in triplicate and repeated at least twice. Images were obtained at 0, 24 and 48 h at the same position and percentage of cell migration was evaluated using ImageJ .Cells were seeded in six-well plates at a density of 5 × 10We plated 100,000 cells in 12-well plates. After 48 h of treatment, trypsinized and floating cells collected from the supernatant were centrifuged and incubated with Annexin V-FITC  and DAPI for 15 min. Apoptosis was detected using Flow Cytometry BD LSRFortessa™ .We then prepared the nuclear protein fraction of breast cancer cell lines using a nuclear extraction kit . A total of 40 µg of protein were resolved by 12% SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were blocked in 5% BSA and hybridized with antibodies against aceyl-H3 (1:1000) and GAPDH (1:1000) as a loading control. Immunoreactive bands were visualized by chemiluminescence  and captured by Image Quant™ LAS4000 . The band densities, normalized to the GAPDH, were analyzed with ImageJ software.Total RNA from BC cell lines was isolated using the High Pure RNA Isolation Kit , following the manufacturer’s protocol. Total RNA from FFPE samples was isolated using RecoverAll Total Nucleic Acid Isolation Kit , following the manufacturer’s protocol. RNA concentration was measured using a NanoDrop ND 2000-UV-vis Spectrophotometer .HDAC5 and GAPDH as endogenous control was carried out by quantitative real time-PCR (qRT-PCR) in RNA extracted from FFPE samples. We used the TaqMan® Gene Expression Assays . Normalization was done with GAPDH. The data were managed using the QuantStudio Desing & Analysis Software v1.4 . Relative expression was calculated using the comparative Ct method and obtaining the fold-change value (ΔΔCt). Gene expression of https://www.bioconductor.org/). To determine differences between HDAC5 expression and breast cancer patients/cells from different ages we performed a Wilcoxon Rank Sum test. Results were considered significant when p-value < 0.05. OS and RFS were performed using follow-up data from BC patients to analyze the prognostic value of HDAC5 expression in terms of relapse and survival. Patients were divided into three groups according to the distribution of the log10 HDAC5 Fold change: high expression (> 0.2 log10 Fold change), medium (> −0.2 and <0.2) and low (< −0.2). RFS and OS studies were performed using survival R package . All statistical analysis was performed using R Bioconductor (HDAC5. Our findings are consistent with those previously published by Li et al. [HDAC5 promotes proliferation, invasion, and migration in human breast cancer. We also demonstrate that HDAC5 overexpression in BCVY correlates with lower survival rates.Taken together, the results provide insights into the biology of breast cancer in younger women and support our hypothesis that breast tumors of younger women activate molecular pathways related to increased aggressiveness, such as i et al. , confirmIn our study, we show that targeted treatment with HDAC5 inhibitor LMK-235 reduces migration and proliferation of tumor cells and increases apoptosis in triple negative breast cancer cell lines, as previous results demonstrated, and as a novelty in luminal cell lines from younger BC patients. In summary, our findings show, for the first time, a potential treatment specific for breast cancer affecting very young patients. However, more efforts should be addressed to analyze its therapeutic role.