Objective The aim of the study was to evaluate the antiproliferative potential of simple phenylboronic acid and benzoxaborole derivatives as well as to provide preliminary insight into their mode of action in cancer cells in vitro. Methods The antiproliferative activity was assessed in five diverse cancer cell lines via the SRB method (sulforhodamine B) or MTT -2,5-diphenyltetrazolium bromide) method after 72 h of treatment. Further studies of the mechanism of action consisted of the influence of the compounds on cell cycle progression and apoptosis induction, which was assessed by flow cytometry, caspase-3 enzymatic activity, fluorescence microscopy and western blot analysis. Results A clear structure-activity relationship was observed for both groups of compounds with several representatives evaluated as highly active antiproliferative agents with low micromolar 18) and 3-morpholino-5-fluorobenzoxaborole (27) exhibited strong cell cycle arrest induction in G2/M associated with caspase-3 activation in an A2780 ovarian cancer cell line. These events were accompanied by a mitotic catastrophe cell morphology and an increased percentage of aneuploid and tetraploid cells. Further experiments indicated that the compounds were phase cycle-specific agents since cells co-treated with hydroxyurea were less sensitive. The observed cell cycle arrest resulted from significant p21 accumulation and was associated neither with cyclin B1 nor β-tubulin degradation. Conclusion Phenylboronic acid and benzoxaborole derivatives were found to be highly promising antiproliferative and proapoptotic compounds with a cell cycle-specific mode of action. The presented data support their candidacy for further studies as a novel class of potential anticancer agents. Camptothecin  was stored as a 1 mg/mL stock solution in DMSO at −20 °C. Cisplatin  was maintained as a ready-to-use stock solution (1 mg/mL) at room temperature.v/v) FBS and 2 mM L-glutamine . The MCF-7 line was cultured in Eagle’s Minimal Essential Medium  supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 1% (v/v) non-essential amino acids, and 8 μg/mL insulin . All culture media contained antibiotics – 100 U/mL penicillin and 100 μg/mL streptomycin . All cell lines were cultured in a humidified atmosphere at 37 °C with 5% CO2 and passaged twice a week using EDTA-Trypsin solution  as a detachment agent (adherent cell lines only).The 5637  cell line was purchased from RIKEN BRC . The A2780 (ovarian carcinoma), A-549  and MCF7 (mammary gland carcinoma) cell lines were purchased from the European Collection of Authenticated Cell Cultures . The MV-4-11 (biphenotypic B myelomonocytic leukemia) cell line was purchased from the American Type Culture Collection . All cell lines were tested for mycoplasma contamination using Venor GeM Classic  with negative results in all cases. All cell lines are maintained at Hirszfeld Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences , Wrocław, Poland. The 5637, A2780 and MV-4-11 cell lines were cultured in RPMI-1640 medium w/GlutaMAX®  supplemented with 10% fetal bovine serum . The MV-4-11 culture medium was additionally supplemented with 1 mM sodium pyruvate . The A-549 cell line was cultured in a 1:1 (v/v) mixture of RPMI-1640 and Opti-MEM  supplemented with 5%  were further prepared using a culture medium containing 0.5 mM HU; in samples described as a pretreatment (± ), after 16 h of HU treatment, the culture medium was replaced with a fresh one without HU.For all 96-well plate-based assays, cells were seeded at a pre-optimized density as follows: 1050  ± standard deviation (SD), which was calculated using the Prolab-3 system based on the Cheburator 0.4 software [At 24 h after seeding the cells in 96-well plates , the tested compounds at concentrations ranging from 200 to 5 μM or cisplatin (10–0.01 μg/mL) were added. At the desired points in time, the plates were subjected to the SRB assay , the tested compounds were applied at various concentrations. At the desired time points, the cells were trypsinized, fixed with ice-cold 70% (v/v) ethanol and analyzed for DNA content according to a previously described procedure , using aAt 24 h after seeding the cells in 24-well plates , the tested compounds were applied at various concentrations. At the desired time points, the apoptosis rate was analyzed using a previously described procedure  with a BAt 24 h after seeding the cells on 50-mm petri dishes , the tested compounds were applied at various concentrations. After 48 h of treatment, the cells were washed with PBS  and lysed with RIPA Buffer supplemented with protease and phosphatase cocktails  for 30 min at 4 °C. After centrifuging the samples , the collected supernatant was assessed for protein content using Pierce™ Coomassie Plus  and stored at −80 °C.Cell lysates  were resolved by SDS-PAGE  under reducing conditions and transferred onto a nitrocellulose membrane  using a semi-dry blotting system . The membrane was washed with Tris-buffered saline  and blocked with 5% skim milk in TBS with 0.1% Tween-20 (TBST) for 1 h at room temperature. Subsequently, the membrane was washed with TBST  and incubated with antigen-specific rabbit IgG antibodies  diluted 1:1000 in 5% BSA in TBST . Next, the membrane was washed with TBST  and incubated with goat anti-rabbit IgG antibodies conjugated to horseradish peroxidase  diluted 1:1000 in 5% BSA in TBST. Following a one-hour incubation at room temperature, the membrane was washed in TBST , chemiluminescent peroxidase substrate was added  and bands were visualized using the blot imaging system .www.imagej.nih.gov) and GraphPad Prism 7.03.Subsequently, the membrane was washed with 10 mM phosphate-buffered saline with 0.05% Tween  and incubated with mouse anti-β-actin IgG antibodies  diluted 1:2500 in 0.5% skim milk in PBST . After washing with PBST , the membrane was incubated with detection HRP-labeled rabbit anti-mouse IgG antibodies  diluted 1:2500 in 0.5% skim milk in PBST. After a one-hour incubation at 37 °C, the membrane was washed  with PBST, and the signal was developed as previously described. The obtained results were analyzed using ImageJ 1.8.0 , the tested compounds were applied at various concentrations. After 48 h of treatment, the cells were gently washed with PBS, fixed with 4% paraformaldehyde  in PBS , permeabilized with 0.1% Triton X-100  in PBS , labeled with DyLight™ 554 Phalloidin  for 10 min and DAPI  for 5 min, visualized using an Olympus IX81 fluorescence microscope equipped with a XC10 camera and analyzed with CellSens Dimension software .p-values less than 0.05 were considered statistically significant.Statistical analyses were performed using GraphPad Prism 7.03. All results are reported as the mean ± SD. One-way ANOVA analyses with the appropriate post hoc tests described in the figure captions were performed. A series of eighteen phenylboronic acid and nine benzoxaborole structurally diverse derivatives was preliminarily evaluated for antiproliferative activity in five cancer cell lines Table . The act1) was almost completely inactive in all cell lines used, even at concentration as high as 200 μM. Among all PBA modifications introduced at the 2-position, the formyl moiety (4) was the only group that significantly increased the biological activity. Further studies indicated that the substituent position affected the antiproliferative potential of the compound. For example, the introduction of the formyl group at position “4” in 11 resulted in an 4 with the greatest increase observed in the A2780 cell line. Concomitantly, modifications utilizing trifluoromethyl group at various positions (6–8) revealed the 4-position as the most promising. Further studies applying various moieties indicated that the mercapto group (9) was the most potent enhancer of 1 activity among the para-modified derivatives in all cell lines. It should be noted that 9 was the only PBA-derived compound that showed at least moderate antiproliferative activity in the MCF7 cell line and was the most active derivative in the A-549 cell line.Phenylboronic acid  did not influence the compound antiproliferative activity or even lowered it significantly in the A-549 cell line . Simultaneously, fluorine incorporation provided 2-fluoro-6-formylphenylboronic acid (18), which showed high antiproliferative activity in MV-4-11, 5637 and A2780. Excluding the markedly less sensitive MCF7 and A549 cell lines, 18 was the most active PBA derivative tested. It is worth noting the striking influence of the fluorine position in 15–18. Compound 16 with fluorine at the 4-position had almost equal activity to 18, whereas 15 with fluorine at the 3-position showed markedly lower activity, especially in 5637 cells (17 (fluorine at 5-position) was more than 10 times less active in the A2780 cell line and approximately 4 times less active in MV-4-11 and 5637 in comparison to 18.Di-substituted derivatives of 20–23. Moderate activity of unsubstituted benzoxaborole (19) was abrogated when fluorine was present at the 4-, 6-, and 7-positions . However, the presence of that substituent at the 5-position (21) resulted in a pronounced rise in activity towards all cell lines, with the highest 21) was the most active benzoxaborole-based compound tested on the MCF7, 5637 and A-549 cell lines. The benzoxaborole substitution at the 3-position (compounds 24–26) had little (on A2780 and A-549 cell lines) or no effect (on MCF7 and 5637 cell lines). MV-4-11 was the only cell line that responded positively to such modifications. Finally, di-substituted 3-morpholino-5-fluorobenzoxaborole (27) proved to be the most active benzoxaborole-based compound in A2780 and MV-4-11 cell lines. None of the tested compounds showed activity comparable to a widely used cytostatic – cisplatin (CDDP) or an additional reference compound – benzyl isothiocyanate (BITC). However, it should be noted that these reference agents plausibly do not share a common mechanism of action with the tested compounds.An even more notable impact of fluorine position on compound antiproliferative activity was observed among benzoxaborole-derived compounds 18 and 27 as the most active representatives of phenylboronic acids and benzoxaboroles, respectively. The A2780 ovarian cancer cell line was used in all subsequent studies because of its high sensitivity to the tested compounds. First, the influence of the compound on the cell cycle was assessed using a standard, RNAse/propidium iodide-based protocol  significantly increased the percentage of cells in G2/M phase even at the lowest 5 μM concentration used (40.4 ± 8.8% in comparison to 21.0 ± 4.6% in the Ctrl), with highest increase observed for 10 μM (59.12 ± 6.56%). In both cases, the G2/M phase arrest was accompanied by a pronounced decrease in G0/G1 and S phase cells percentage and significant increase in the percentage of tetraploid cells (>4 N) – from 0.9 ± 0.1% in the control to 15.6 ± 2.8% in the sample treated with 18 at a 10 μM concentration. Surprisingly, at a high 25 μM concentration, the cell cycle progression arrest caused by 18 shifted towards significant S phase arrest (53.9 ± 6.3% cells in comparison to 18.9 ± 2.8% in the Ctrl) accompanied by almost complete decay of G0/G1 phase (6.8 ± 1.2% in comparison to 59.2 ± 6.9% in the Ctrl). The tetraploid cell population decreased in comparison to the sample treated with 18 at the 10 μM concentration, but they remained significantly higher than in control samples (5.1 ± 2.8% in comparison to 0.9 ± 0.1% in Ctrl). The 3-morpholin-5-fluorobenzoxaborole (27) influenced the cell cycle by significantly increasing the percentage of cells in the G2/M phase and the number of tetraploid cells when A2780 cells were treated with 10 μM (31.6 ± 5.2% cells in G2/M phase and 5.4 ± 2.1% tetraploid cells) and 25 μM (61.3 ± 3.7% cells in G2/M phase and 8.3 ± 0.6% tetraploid cells), but not with 5 μM. In both cases, the G2/M phase arrest was associated with a significant decrease in G0/G1 cell percentage, with the highest of more than 3.8-times decay in the sample treated with 27 at a concentration of 25 μM (13.2 ± 2.6% in comparison to 50.5 ± 6.9% in the Ctrl).Further studies focused on the basic mechanism of action of 18 at 25 μM concentration (9.7 ± 1.9 – fold caspase-3 activity increase in comparison to the Ctrl). Similarly to the cell cycle analysis, 27 caused apoptosis of a slightly lower scope, with a 5.6 ± 1.8 – fold caspase-3 activity increase with 25 μM.Subsequently, the pro-apoptotic activity of the compounds was assessed after 48 h of A2780 cell treatment by caspase-3 enzymatic activity analysis  [18, even at the highest concentration used (25 μM), which caused S, not G2/M phase cell cycle arrest. Compound 27 induced a slightly less pronounced increase in the p21 protein level, but similarly to 18, a clear dose-dependence was demonstrated. Cyclin B1 level was significantly increased when cells were treated with 18 at 10 μM (1.8 ± 0.3 – fold level increase) and 27 at 25 μM (1.8 ± 0.3 – fold level increase), but its level remained equal to the control sample when 18 was applied at 25 μM, which correlates with its ability to suppress cell cycle progression in S phase. β-tubulin level did not change significantly as a result of the compound treatment regardless of the concentration used.To provide further insight into the compound mode of action, β-tubulin, p21 and cyclin B1 protein levels were assessed by western blot analysis after 48 h of A2780 cell treatment with various compound concentrations   or as a clin B1) . A remar2/M phase, the ability to induce formation of the tetraploid cell population, and the ability of compounds to induce apoptosis was observed. This finding indicated that the compound mode of action was associated with mitotic catastrophe processes, characterized by aberrant mitosis, mitotic cell death, and as a result of aberrant mitosis slippage, the presence of aneuploid and tetraploid cells [A clear correlation between cell cycle arrest in Gid cells . To addr50 assessment at various time points indicated that prolonged treatment was necessary to reveal the high antiproliferative potential , similarly to the cell cycle specific cytostatic – cisplatin  and in contrast to benzyl isothiocyanate (BITC), which had similar IC50 values after 24, 48 and 72 h of treatment , irrespectively of the concentration used (representative results shown). Such a negative effect was not observed when HU co-treated cells were additionally treated with camptothecin (40 ± 5.8 – fold change compared with the untreated control). In the samples that were pre-treated with HU, the ability of 18 and 27 to induce apoptosis was also significantly reduced. Particularly in the case of 27 at 25 μM, which caused a 3.8 ± 0.8 – fold increase in caspase-3 activity in HU pre-treated cells (2.1 ± 0.4 – fold change for HU pre-treated control cells) in contrast to 6.2 ± 2.0 – fold change in untreated cells.A strong correlation between the compound-induced cell cycle inhibition and apoptosis rate raised the question of whether both events were not only correlated but also dependent. Additionally, the compound ICial Fig. a. The IC S phase . Pre-tre18 and 27 caused a significant increase in G2/M and > 4 N cell percentage, whereas these effects almost completely disappeared when samples were co-treated with HU and were significantly reduced when cells were pre-treated with HU. The described correlation was observed regardless of the used concentrations of compounds (representative results are presented). This phenomenon was most striking for 27, which applied alone at 25 μM caused a massive accumulation of cells in G2/M phase (64.4 ± 1.3% cell percentage) in comparison to the untreated control (22.6 ± 3.5% cells); however, after concurrent usage with HU, its impact was abolished (16.5 ± 2.0% cell percentage in G2/M). Additionally, HU treatment significantly reduced the capability of the tested compounds to induce tetraploidy, e.g., compared with the 16.8 ± 2.5% >4 N cell percentage originally observed for 18-treated cells (at 10 μM), only 3.2 ± 0.3% cells were observed following co-treatment with HU (1.0 ± 0.1% cell percentage in untreated control). Taken together, the above-described observations clearly indicate that phenylboronic acid and benzoxaborole derivatives act as G2/M phase cycle-specific pro-apoptotic agents.The cell cycle analysis confirmed the above-mentioned observation Fig. d, e, f. 1) anticancer properties of phenylboronic acid were studied only by the Plopper group [1 as a cell growth and migration inhibitor, but the required compound concentrations to observe a biological effect exceeded 500 μM in most cases. Recently, moderate anticancer activity of PBA was described in 4T1 murine mammary gland adenocarcinoma and SCCVII squamous carcinoma but also at high concentrations [The first boron-containing anticancer drug was bortezomib – a dipeptide boronic acid that was featured as a strong proteasome inhibitor and approved by FDA for multiple myeloma treatment in 2003. Since then, much effort was undertaken to search for novel boron-containing compounds that are useful in medicinal chemistry, resulting in a large set of bioactive molecules at various stages of development  . In the er group , 37. Thetrations .1 led to highly active phenylboronic acid-derived compounds such as 9, 16 and 18 with low micromolar 9 was observed in all cancer cell lines among all para-substituted derivatives, which indicated that resonance or inductive effects might play an important role in this matter since the mercapto group was the only electron donating group tested. Moreover, compound pKa modulation appeared to be crucial, at least in the case of the di-substituted compounds 15–18, of which the least active was 17 with a pKa = 6.72; the other compounds exhibiting pKa = 5.74, 6.42 and 6.05  were si2/M phases identified as those targeted by 18 and 27.Notable differences were observed in the sensitivity of the cell lines to the tested compounds, with MCF7 breast cancer cells recognized as generally resistant, and ovarian cancer (A2780) as well as leukemia cells (MV-4-11) identified as the most sensitive. The molecular origin of the observed discrepancies remains unknown but identifies biological activity of the compounds as a result of interactions with specific molecular targets rather than their overall toxicity. Moreover, our studies of HU-treated A2780 cells clearly indicated that the compound activity was focused on proliferating cells, with cells in S and/or G21) already approved for the treatment of onychomycosis as the most spectacular example [21), a derivative comprising a single fluorine atom as a substituent.Since the discovery of the sugar-binding properties of benzoxaboroles in 2006 , this cl example . This in example , but fur example , whereas example , similar example . In bothKa lowering from 7.39 and 7.42 for 19 and 23, respectively, to 6.97 observed for 21 was accompanied by a significant increase in biological activity. However, 20 and 22 showed poor antiproliferative potential despite their even lower pKa  [In our studies, we not only observed relatively poor activity of the tested benzoxaboroles in MCF7 but also in some other cancer cell lines (data not shown). Concomitantly, several tested compounds exhibited high antiproliferative activity in A2780 and MV-4-11 cell lines, and, to a lesser extent, the 5637 urinary bladder cell line. Similar to PBA derivatives, the acidity of the compounds appeared to play an important role since pctively) , indicatctively) , which m2/M cell cycle arrest was observed for various cell lines, including DU-145 and PC-3 prostate cancer cells after treatment with 1 [D,D-carboxypeptidase and leucyl-tRNA synthetase (LeuRS) inhibition [21 and some of its derivatives in human osteosarcoma (U2OS) and ovarian cancer (SKOV-3) cells. The apoptotic morphology associated with the ambiguous transcriptional activity of the p21 promotor and no significant impact on the cell cycle was reported [The mode of antiproliferative activity of phenylboronic acids is largely unknown. Inhibition of GTPases by the Rho family in the DU-145 prostate cancer cell line was suggested by McAuley et al. as a plausible mechanism . Additiot with 1 . The antreported .18) and 3-morpholine-5-fluorobenzoxaborole (27) – and the A2780 ovarian cancer cell line were used. A very evident cell cycle arrest at G2/M phase associated with a significantly increased level of p21 protein probably resulted from the binding of p21 to cyclin B1-CDK1 and its inhibition. p21 acts as a cyclin-dependent kinase inhibitor that binds to various cyclin-CDK (cyclin dependent kinase) complexes and regulates cell cycle progression [18 to induce S phase arrest at a concentration of 25 μM. Such activity could also result from p21 binding to the proliferating cell nuclear antigen (PCNA) that is also responsible for S phase arrest [In the present work, we performed a preliminary evaluation of the mechanism of action underlying phenylboronic acid and benzoxaborole derivative antiproliferative activity. The two most potent representatives – 2-fluoro-6-formylphenylboronic acid , a significant increase in cyclin B1 was accompanied by a high level of aberrant tetraploid cells – plausibly a result of failed mitotic death that led to mitotic catastrophe slippage and an increased number of multinucleated cells. Such cells are increasingly susceptible to cell death with every cell cycle progression and eventually undergo apoptosis [Another possible mechanism underlying such strong Glchicine ) or α- alchicine ); howevepoptosis .2 phases. The upstream events caused by 18 and 27 that led to the above discussed image of A2780 cells remain unknown, but based on these preliminary studies, establishment of the exact mechanisms of action of phenylboronic acid and benzoxaborole derivatives should be possible in the future. Cell line features responsible for the specificity of the observed compounds will also be an important part of further studies.In conclusion, we identified phenylboronic acid and benzoxaborole derivatives as potent antiproliferative agents acting as cell cycle arrest and apoptosis inducers. Great possibilities for further modifications makes them a promising new class of anticancer agents. Additionally, the tested compounds were evaluated as phase cycle-specific agents with activity focused on proliferating cells in late S/early G


Dear Editor,Globally, gastric cancer is the most common malignant tumor and the second highest contributor to cancer deaths after lung cancer . Further, expression levels of let-7i were lower in gastric cancer cell lines  as compared to that in normal gastric epithelial cells GES-1, while no significant difference in this respect was observed in the MKN-45 cell lines , and lymph node metastasis . The effects of let-7i restoration on regulation of gastric cancer cell vitality and cell proliferation were assessed by transfecting let-7i mimic or miRNA negative control into two human gastric cancer cell lines, SGC-7901 and MGC-803, which have relatively lower levels of let-7i expression. As expected, ectopic let-7i expression markedly suppressed viability of SGC-7901  as assessed by use of cell counting kits. Furthermore, over expression of let-7i also reduced proliferation of both SGC-7901 , 48 h after transfection, as revealed on colony formation assay. Overexpression of let-7i also reduced invasive and migratory ability of both SGC-7901 . These findings suggest that let-7i reduced cell viability and proliferative ability and inhibited invasive and migratory properties of gastric cancer cells in vitro. SGC-7901 cell lines which stably expressing let-7i and miRNA-control were subcutaneously injected into the dorsal flank of nude mice to evaluate the in vivo effects of let-7i on gastric cancer tumor growth. The result showed reduced tumor volume and tumor weight in nude mice with let-7i mimic injection , foundation of Jilin Province Science and Technology Department (172408GH010234983), and Jilin University-Xinjiang Medical University joint research project.Yue Shi, Zipeng Duan, Xun Zhang, Xiaotian Zhang, Guoqing Wang and Fan Li declare that they have no conflict of interest.All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was obtained from all patients for being included in the study.All institutional and national guidelines for the care and use of laboratory animals were followed.Supplementary material 1 (PDF 614 kb)Below is the link to the electronic supplementary material.


Background: Cutaneous squamous cell carcinoma (cSCC) incidence continues to rise with increasing morbidity and mortality, with limited treatment options for advanced disease. Future improvements in targeted therapy will rely on advances in genomic/transcriptomic understanding and the use of model systems for basic research. We describe here the panel of 16 primary and metastatic cSCC cell lines developed and characterised over the past three decades in our laboratory in order to provide such a resource for future preclinical research and drug screening. Methods: Primary keratinocytes were isolated from cSCC tumours and metastases, and cell lines were established. These were characterised using short tandem repeat (STR) profiling and genotyped by whole exome sequencing. Multiple in vitro assays were performed to document their morphology, growth characteristics, migration and invasion characteristics, and in vivo xenograft growth. Results: STR profiles of the cSCC lines allow the confirmation of their unique identity. Phylogenetic trees derived from exome sequence analysis of the matched primary and metastatic lines provide insight into the genetic basis of disease progression. The results of in vivo and in vitro analyses allow researchers to select suitable cell lines for specific experimentation. Conclusions: There are few well-characterised cSCC lines available for widespread preclinical experimentation and drug screening. The described cSCC cell line panel provides a critical tool for in vitro and in vivo experimentation. Cutaneous squamous cell carcinoma (cSCC) is a common and growing problem, with over 45,000 cases per year in the UK in 2015 ,4. AlthoA total of 16 cell lines were derived from 11 patients with cSCC attending dermatology outpatient clinics. Five of these 11 patients were immunocompetent individuals  and six of these 11 patients were immunosuppressed male organ transplant recipients (OTR) aged 51–87 years and 45–67 years, respectively, at the time of biopsy . One celCell morphology representative of nine of the 16 lines is shown in Fourteen of the 16 cell lines were investigated in xenograft models. The occurrence and growth rate of tumours developing after subcutaneous injection varied between cell lines A. PalpabFifteen STR loci plus amelogenin were genotyped to give tumour-specific profiles . These pWhole exome sequencing (WES) was performed on the 15 lines, for which matched normal samples were available (GSE98780) . Our res−4), chromatin modification , RNA splicing , and the mRNA metabolic process , were significantly overrepresented for these 58 genes, highlighting the potential involvement of these pathways and processes in driving or significantly contributing to cSCC metastasis.The non-silent mutations identified were used to construct evolutionary trees for the two isogenic cell line sets, primary cSCC IC1 and its metastasis IC1MET, as well as for the three related (MET1/MET2/MET4) and two unrelated lines (PM1/T9) derived from the single immunosuppressed transplant recipient male patient A. A majop < 0.0001). In contrast, the proportion of other mutations became less abundant. In particular, there was a >10-fold increase in A > G/T > C transitions during the tumour progression, representing more than 20% of all late mutations for both series (https://cancer.sanger.ac.uk/cosmic/signatures), which often consist of A > G/T > C substitutions, became more dominant after the tumours are fully established and during the tumour progression. Although signature 7 (UV light exposure) remained the most dominant signature throughout, its influence became important after the full establishment and during the progression and metastatic stages.We next investigated the mutational signatures (with respect to transitions and transversions) for early and late mutations, and aimed to identify any significant differences between them. As shown in h series B. This sp-value ≤ 0.01), including known cancer drivers .We then explored the methylation characteristics of six cSCC cell lines  using genome-wide DNA methylation microarray. The cSCC lines were hybridised to the same chip with three normal human keratinocytes (NHK) to account for possible batch effects. Genome-wide methylation profiles reflected the original histologies (cSCC vs. NHK) and also differentiation status subtypes of cSCC based on Pearson’s correlation . Cell liWe then explored the potential enrichment of subsets of differentially methylated genes. Gene-set enrichment analysis revealed significant enrichment in several canonical pathways including MAPK, JAK-STAT, insulin signaling and the p53 pathway. P53 is known to play a critical role in cSCC oncogenesis, and genes involved in p53 signaling, IGF1, IGFBP3, CASP8, SERPINB5, were identified as being differentially methylated. This suggests that the genome-wide methylation profile retained by stable cell lines maintains the original tissue features and reflects the methylation changes in processes inherent to cSCC onset and progression.We describe for the first time a unique panel of 16 cSCC lines which have been extensively characterised in terms of clinical and histological phenotyping; genotyping by STR DNA profiling, exomic sequencing, copy number alteration analysis and phylogenetic analysis; in vitro cell morphology, growth, transwell migration, organotypic invasion; and in vivo tumourigenicity. This panel includes multiple isogenic lines and lines which represent the spectrum of tissue sites and histologies, and is a unique resource for preclinical cSCC research. Indeed, to date, a few of these lines have been used individually in multiple collaborative research efforts .There are few well-established cSCC cell lines. An online search using the cell line catalogue databases ATCC, the European Collection of Authenticated Cell Cultures (ECACC) and the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) revealed that the only SCC lines listed are of head and neck origin. As far as we are aware, the only cutaneous SCC line widely available commercially is A431. This line is of vulval origin ,9,10 andCancer-derived cell lines lack the critical influence in vivo from the tumour microenvironment, but this can be mimicked in organotypical cultures and subcutaneous xenografts. We present the optimal lines from our series, which produce early xenografts and highlight the range of individual tumours from multiple mice in each of the key lines. This illustrates that the optimal line for consistent rapid tumour growth for drug and therapeutic screening appears to be IC1MET. The remarkable conservation of the pathological changes found in the primary tumours is shown in the photomicrographs. Particularly striking are the hyperkeratotic lesions from a field of actinic dysplasia . As the xenografts are in SCID mice, this clearly provides a permissive environment for tumour development, which allows the intrinsic pattern of tumour differentiation to develop. However, in our published studies of organotypical cultures using keratinocytes and fibroblasts from patients who develop cSCC due to the rare genetic disease recessive dystrophic epidermolysis bullosa (RDEB-type VII collagen deficiency), RDEB cancer-associated fibroblasts (CAFs) increased the adhesion and invasion of both tumour (including IC1) and non-tumour keratinocytes . It woulThere have been a number of potential problems in the use of cSCC cell lines derived from tissue collections or by multiple partnering labs using those cell lines for experimentation. For example, analysis of 122 HNSCC lines  authenticated only 85 unique cell lines by STR DNA profiling within this collection and highlighted misidentification and cross-contamination as common problems 3, where L is length and W is width. The time points which ended tumour sampling were determined by the point at which the tumours reached the size requiring sacrifice of the animal (800 mm3) or developed ulceration.Female SCID Balb/c mice or male CD1 nu/nu mice were subcutaneously injected in the right flank with 4 × 10DNA was extracted from cultured cells and blood using the Qiagen DNA Mini kit  according to the manufacturer’s instructions. Quantitation was performed using the double-stranded DNA-specific Qubit dsDNA BR assay kit in conjunction with the Qubit 2.0 fluorometer . Short tandem repeat (STR) genotyping was performed to authenticate the unique identity of each cell line. Sixteen loci distributed across the human genome were amplified using the AmpFLSTR Identifiler PCR Amplification Kit followed by capillary sequencing on the 3730xl DNA Analyzer . For SNP 6.0 analysis, DNA samples from cultured cells and paired non-tumour blood samples were hybridised to the microarrays according to the manufacturer’s protocols . Exome sequencing was performed by Oxford Gene Technology (OGT), using Agilent SureSelect All Exon v5  for exome capture. Briefly, 1 µg DNA from each sample was used to prepare the sequencing library through shearing of the DNA followed by ligation of sequencing adaptors. Sequencing was performed on the Illumina HiSeq platform . Paired-end sequencing (2 × 100 bp) was carried out using HiSeq sequencing instruments.The WES data generation and full analytical procedure has been described previously in detail (accession number GSE98780) .Two independent approaches were applied to the copy number analysis of WES data. First, to generate single nucleotide polymorphism (SNP) and indel variant genotyping information, the tumour-normal pair was processed against the reference genome using the VarScan2 germline variants calling method. The minimum coverage for identified sites was 10 reads for both tumour and normal. Next, the logR and BAF  files were created based on the tumour-normal pair genotyping information, with the depth information normalised by dividing the depth of each variant by the median depth across all variants. The ASCAT R package  was thenhttp://evolution.genetics.washington.edu/phylip.html). The non-silent mutations were used for this analysis. The mutations in the tree trunk were defined as “early” events, and the mutations in the branches after the tree split were “late” events.For two patients where multiple tumour cell lines were generated and sequenced, evolutionary trees were constructed on the basis of distance matrix among normal control and multiple cell line samples, using the neighbour-joining algorithm  implemenA total of 6 cSCC lines characterised within this manuscript were used for the detection of differentially methylated genes. Three early-passage primary normal human keratinocytes derived from 3 different participants served as a normal control for comparison. DNA was extracted as described above and bisulfite modification was carried out as the following: 500–1000 ng of DNA yield from each sample was modified with bisulfite using the EZ DNA Methylation Kit  according to the manufacturer’s instructions. Methylation detection was performed using the Illumina Infinium HumanMethylation27 BeadChip. This platform detects the methylation status of 27,578 CpG sites spread across 14,495 genes by sequencing-based genotyping of bisulfite-converted DNA. Eventual methylation scores  were generated for each site with BeadStudio software  and raw background-corrected values were used for further analysis. The methylation assay was performed according to the manufacturer’s instructions. Briefly, bisulfite-converted DNA was amplified, fragmented and hybridised to the chip arrays, followed by imaging with the Illumina BeadArray reader. Image processing and intensity data extraction were performed according to Illumina’s instructions. All analyses were carried out using Bioconductor and R.We present a definitive analysis of a unique set of cSCC lines that can be used as a model of skin squamous carcinogenesis for in vivo and in vitro studies. We provide details of their extensive characterisation, including clinical, phenotypic, genotypic, in vitro and in vivo properties. In addition to the research reported to date using these cell lines, we anticipate that this unique resource will continue to provide researchers with a powerful preclinical model for future investigations of the molecular drivers of cSCC progression, the identification of novel therapeutic targets and, ultimately, the development of more effective treatment strategies for this common skin cancer.


Background: Microsatellite and chromosomal instability have been investigated in Hodgkin lymphoma (HL). Materials and Methods: We studied seven HL cell lines (five Nodular Sclerosis (NS) and two Mixed Cellularity (MC)) and patient peripheral blood lymphocytes (100 NS-HL and 23 MC-HL). Microsatellite instability (MSI) was assessed by PCR. Chromosomal instability and telomere dysfunction were investigated by FISH. DNA repair mechanisms were studied by transcriptomic and molecular approaches. Results: In the cell lines, we observed high MSI in L428 (4/5), KMH2, and HDLM2 (3/5), low MSI in L540, L591, and SUP-HD1, and none in L1236. NS-HL cell lines showed telomere shortening, associated with alterations of nuclear shape. Small cells were characterized by telomere loss and deletion, leading to chromosomal fusion, large nucleoplasmic bridges, and breakage/fusion/bridge (B/F/B) cycles, leading to chromosomal instability. The MC-HL cell lines showed substantial heterogeneity of telomere length. Intrachromosmal double strand breaks induced dicentric chromosome formation, high levels of micronucleus formation, and small nucleoplasmic bridges. B/F/B cycles induced complex chromosomal rearrangements. We observed a similar pattern in circulating lymphocytes of NS-HL and MC-HL patients. Transcriptome analysis confirmed the differences in the DNA repair pathways between the NS and MC cell lines. In addition, the NS-HL cell lines were radiosensitive and the MC-cell lines resistant to apoptosis after radiation exposure. Conclusions: In mononuclear NS-HL cells, loss of telomere integrity may present the first step in the ongoing process of chromosomal instability. Here, we identified, MSI as an additional mechanism for genomic instability in HL. TP53 is still a matter of debate [Hodgkin lymphoma (HL) is a unique disease in which the neoplastic Hodgkin and Reed–Sternberg (HRS) cells constitute only 1–2% of the tumor tissue, the remaining tumor microenvironment being composed of various inflammatory cells ,4 and thf debate . Howeverf debate ,8,9. TheTwo important models have been implicated in the genomic instability observed in cancer cells: microsatellite instability (MSI) and chromosomal instability, of which the latter inversely correlates with MSI in hereditary non-polyposis colorectal cancer  and deteOnly a single study has previously investigated the implication of MSI in HL, and concluded that MSI is not involved in the oncogenic process . NeverthThe aim of this study was to investigate mechanisms underlying genomic instability in HL through combined cytogenetic and molecular approaches. We demonstrate, for the first time, the involvement of MSI in HL cell lines. The loss of the protective function of telomeres in NS-HL cell lines induced chromosomal fusions (dicentric chromosome formation) and “breakage-fusion-bridges” (B/F/B) cycles, resulting in a series of chromosomal breaks and duplications, which can lead to chromosome imbalances, gene amplification, non-reciprocal translocation, and altered gene expression. In MC-HL cell lines, the high level of spontaneous double strand breaks (DSB) within and outside telomeres, detected using 53BP1 foci, induced the formation of dicentric chromosomes and the lagging of acentric chromosomes (micronuclei with only telomere sequences) and B/F/B cycles. Transcriptome analysis demonstrated the difference in DNA repair mechanisms between the NS-HL and MC-HL cell lines. Finally, a NS-HL cell line exhibited high radiation sensitivity compared to MC-HL cell line. In addition, we validated our findings in a large cohort of NS-HL and MC-HL patients.TP53 and the clonal homogeneity of these cell lines  showed the presence of 100% red colonies generated from three of the cell lines , indicating the nonfunctional status of ll lines . Sequencangement . We observed telomere shortening (less than 6 kb) in three cell lines  A. Only KThe use of Q-FISH permits the analysis of telomere aberrations: telomere loss, telomere deletions, and interstitial telomeres in metaphases. We observed a high rate of telomere loss and deletion in the HL cell lines and a lymphoblastoid cell line (REMB) with short telomeres C. NevertAll HL cell lines demonstrated numerical chromosomal aberrations A. In theThe second common characteristic was the presence of clonal and sporadic dicentric chromosomes B. Only tJAK2-specific FISH revealed the presence of several gene copies  and found the subtelomeric sequences of 15q to be in the breakpoint of the dicentric chromosome A,B, showWe used the same approach to study the MC-HL cell lines, and lack of sub-telomeric sequences in dicentric chromosome breakpoints was observed also in L1236 and KHM2 cells . NeverthClonal dicentric chromosomes in KMH2 cells were characterized by a specific configuration with the pericentrometric breakpoints and the two centromeres in close proximity . In addiThe clonal structural chromosomal aberrations identified in each cell line are summarized in Numerical chromosomal aberrations were present in all HL cell lines, with a higher frequency of gains than losses. We observed the loss of chromosome Y in four cell lines derived from men. Chromosome 13 was lost from five cell lines and chromosomes 15 and 8 from two. There were gains for chromosomes 19 (6/7), 9(6/7), 5 (5/7), 11 (5/7), 18 (5/7), and 17 (4/7) .MYC, in all the NS -HL cell lines (JAK2 in 6/7 cell lines. HDLM2 exhibited a particularly high copy number for JAK2 (eight copies)  , includiTP53 mutation nor MSI instability and telomere shortening in the HL cell lines. Nevertheless, the NS HL cell lines with high MSI showed a high frequency of CCRs and B/F/B aberrations. In addition, KMH2, a MC HL cell line, showed a high frequency of CCRs relative to L1236 cells. There was no correlation between chromosomal instability and p < 10−16; p < 10−16; p < 10−9, respectively) and (2) telomere length, telomere loss, and telomere deletions in NS-HL and MC-HL patients  (p < 10−5). These characteristics reflect our observations in the HL cell lines and the long telomeres observed in KMH2 cells.We examined telomere dysfunction in circulating lymphocytes from 100 NS-HL and 23 MC-HL patients, as previously published ,22, to vctively) . InteresWe restricted the transcriptome analysis in this study to the comparison of NS HL with MC HL cell lines. We found 3,755 transcripts and 2,415 unique genes to be differentially expressed between the NS-HL  and MC-HL (L1236 and KHM2) cell lines . The affp = 0.005)  .MRE11 by immunofluorescence.DNA repair ontology and the affected pathways are part of a Reactome pathway . DNA repThe presence of two different mechanisms underlying genomic and chromosomal instability in HL cells suggests that the existence of a different pathway in DNA repair may be an important biological event that influences treatment resistance in HL. Therefore, we explored the radiation sensitivity of the HL cell lines. The HL cell lines and two lymphoblastoid B cell lines (NAD and REMB) were exposed to ionizing radiation and two approaches were used to assess radiation sensitivity: (a) apoptosis, including cell cycle arrest after 6Gy of irradiation by FACs analysis, and (b) mRNA expression of PUMA, P21, ZMAT3, Sesterin1 (SESN1), and CCNG1 at two and 24 h after 2Gy of irradiation . These gThe mechanisms underlying genomic instability and the primary transforming events of HL are still obscure ,9. In thWe report, for the first time, that MSI is involved in HL. We confirmed the results obtained for L1236 concerning the absence of MSI ,26,27.TP53 status of HL has been the subject of debate and remains controversial [TP53 mutations in lymph nodes contrasts with the levels of its expression which are often high [TP53 in three cell lines and DNA sequencing revealed the same mutations previously described in L1236, HDLM2, and L428 [TP53 status in HL using a sensitive technique reinforces the concept that TP53 mutation may be involved in the pathology of some cases of HL [TP53 status in lymph nodes derived from relapsing or refractory HL patients. The correlation between TP53 status and high-grade progression in B-cell lymphoma has been previously established [TP53 mutation were characterized by high MSI. For MC-HL, we did not detect MSI in the L1236 cell line, which carries mutated TP53, and conversely, we detected a high frequency of MSI in the KMH2 cell line, without a TP53 mutation.The oversial . The lacten high . The disten high . In thisand L428 . This rees of HL  and perhes of HL  or HL asablished ,32, as wChromosomal instability, a second mechanism of genomic instability, has been defined as an accumulation of point mutations, translocations, chromosomal gains and losses, telomere shortening, and defects in the nuclear architecture, which may cause genome instability .Here, we demonstrate the involvement of telomere dysfunction in chromosomal instability in HL. We first confirmed previous published data using seven HL cell lines and a large cohort of HL patients ,15 and vSecond, we demonstrate, for the first time, a significant difference between telomere length in NS-HL and MC-HL patients and cell lines. MC-HL patients showed high heterogeneity and a subset of patients showed long telomeres, as well as the KMH2 cell line. All NS-HL patients and cell lines presented telomere shortening associated with a high frequency of telomere aberrations (loss of one or two telomeres). Mononuclear cells presented longer telomeres than RS cells, but a higher frequency of telomere loss. We speculated that the loss of telomere integrity of mononuclear cells in NS-HL cell lines may represent the first step in the ongoing process of chromosomal instability. Such telomere dysfunction induced the formation of dicentric chromosomes, which have been observed in oncogenic processes, particularly hematological disorders .Jak2 in the HDLM2 cell line. The findings reported here suggest that chromosomal instability in NS-HL cell lines can arise as a consequence of telomere dysfunction in the early stages of HL tumorigenesis. HRS cells, the end of the proliferative stage, were characterized by telomere shortening, the accumulation of complex chromosomal rearrangements, and fewer telomere aberrations, resulting in a stable stage. Of note TP53 mutation, detected in L428 and HDLM2 cell lines, play a role in telomere shortening as well as the accumulation of chromosomal rearrangements and chromosomal instability [In the HDLM2 and L428 cell lines, we identified the subtelomeric sequences at the breakpoint of clonal dicentric chromosomes, in addition to the presence of interstitial telomere sequences. These dicentric chromosomes developed into large chromatin bridges connecting the daughter cells. The chromatin bridges did not appear to give rise to micronuclei , but contability ,38,39.TP53 mutations plays a major role in telomere dysfunction [Nevertheless, the mechanisms of dicentric formation in MC-HL cell lines are not related exclusively to telomere dysfunction. The lack of sub-telomere sequences at the dicentric breakpoints which were usually localized to the pericentromeric region, discards the possibility that telomere dysfunction exclusively induced the formation of dicentric chromosomes in the MC HL cell lines. We propose that spontaneous DNA DSBs detected by IF-FISH and associated with a higher frequency of 53BP1 foci, were produced in pericentromeric regions due to cellular metabolism or a genetic program, and induced the formation of dicentric chromosomes with specific configurations. Nucleoplasmic bridges without signals associated with the presence of micronuclei with only telomere sequences  may lend support to this hypothesis. These dicentric chromosomes contributed to the subsequent accumulation of CCRs and alterations related to B/F/B cycles. This profile was found in KMH2 cells. However, in L1236, the function  and the Dicentric chromosomes that have been observed in oncogenic processes, in particular in hematological disorders ,41,42,43TP53 status and MSI and thus the type of HL cell line. We demonstrate, for the first time, the radiation sensitivity of NS-HL cell lines and confirm the relevance of radiation therapy in HL treatment [The response to ionizing radiation is associated with the reatment . The L54reatment . Neverthreatment .Transcriptome analysis confirmed this difference between the NS and MC HL cell lines with respect to DNA repair processes, telomere maintenance, and cell cycle and apoptotic process.The human HL-derived cell lines L428 , L591 5, SUP-HD1Peripheral blood lymphocytes were obtained from 100 patients with NS HL and 100 healthy donors following informed consent . The col2 in RPMI 1640 medium  supplemented with 10% fetal bovine serum  and 1% antibiotic-antimycotic (Gibco).The HL cell lines and in vitro Epstein-Barr virus (EBV) infected cell lines NAD, REMB, G36, and RV10 were cultured at 37 °C in an atmosphere containing 5% CO2, in a humidified atmosphere to block them in metaphase. After harvesting the cells and centrifugation for 7 min at 1400 RPM at room temperature, the supernatant was removed and the cells were re-suspended in a solution of warm (37 °C) 0.075 M potassium chloride (KCl)  and incubated for 20 min in a 37 °C water bath (hypotonic shock). For pre-fixation of the cells, approximately five drops of fixative (3:1 ethanol/acetic acid) was added to each tube under permanent agitation and the tubes centrifuged for 7 min at 1400 RPM at room temperature. The supernatant was removed and the cells suspended in fixative solution and centrifuged using the same parameters. After two additional rounds of these fixative steps, the cells were stored in fixative solution at 4 °C overnight and metaphases were spread on cold, wet slides the next day. Slides spread with metaphases were dried overnight at room temperature and stored at −20 °C until further use.Cells were exposed to colcemid (0.1 µg/mL)  for 2 h at 37 °C, 5% COImmuno-phenotyping of Hodgkin lymphoma cell lines was performed on a LSRII flow cytometer (BD) using FITC- and APC-conjugated mouse anti-human specific monoclonal antibodies for CD30 (Ber-H83) and CD15 (HI98), respectively. Both antibodies were obtained from BD Biosciences . Staining for CD15 and CD30 was performed according to the manufacturer’s instructions. After the addition of 1 μg/mL Hoechst  to discriminate dead cells, the cells were analyzed by FACS as previously described.Cell-cycle fractions were determined by Hoechst nuclear staining. Briefly, cells were harvested, washed in PBS, and incubated in Hoechst solution for 30 min at room temperature. Data were collected using a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo Version 7.5.5 . The results represent the mean value of three independent experiments.+ cells, leading to the generation of white colonies. Thus, colonies containing wild-type TP53 were white (Ade2+) and those containing the TP53 mutation red. After identification of non-functional TP53 samples, red colonies were cloned and sequenced by the Sanger method to characterize the TP53 mutation. Finally, we classified the mutations using the IARC p53 database according to their impact on the protein.To study the functionality of the TP53 gene, a functional assay was performed in accordance with the method described by Flaman et al. . The assThe Revised Bethesda Guidelines were used to determine the MSI status. Analysis of MSI was performed by five-plex polymerase chain reaction (PCR) using a panel of five quasimonomorphic mononucleotide repeat markers  that map to intron 15, intron 16, the 5′ untranslated region (UTR), 3′ UTR, and 3′ UTR of MSH2, c-kit, SLC7A8, transmembrane protein precursor B5, and ZNF2 genes. A single 5-plex polymerase chain reaction (PCR) allowed co-amplification of all five markers, which were subsequently analyzed using a genetic analyzer  and computerized fragment analysis for each of the five primer pairs used in the PCR.The denatured PCR products were separated by capillary electrophoresis using the genetic analyzer and were further analyzed using commercially available software . HL cell lines with instability of three markers were defined as MSI-H, those with < 3 as MSI-L, or MSS if instability was not identified .l-lysine-coated glass slides at 700 rpm for 4 min, fixed with 10% formalin for 10 min, and treated with 0.25% Triton X-100 solution for 10 min. After blocking with 5% bovine serum albumin , the cells were incubated overnight in 4 °C with primary antibody. After washing in PBS, cells were treated with Cyanine 3 anti-mouse IgG  or FITC anti rabbit  secondary antibody at 37 °C for 45 min. cells were mounted with p-phenylene diamine after counterstaining with 4,6-diamidino-2-phenylindole (Sigma-Aldrich). As a negative control, staining was carried out in the absence of primary antibody.Cells were cytospun onto poly-g after 5 h of colchicine (0.09 mg/mL) treatment at 37 °C in a humidified atmosphere of 5% CO2. The pellet was washed in 1× PBS at 37 °C and re-centrifuged. The cells were subjected to hypertonic shock by resuspension in 34 mM citrate at 37 °C to obtain a suspension of cells at a concentration of 60,000 cells/mL and incubated for 1 h at 37 °C. The suspension (200 μL) was subsequently applied to polylysine slides by cytospin. Following fixation  3%, sucrose 2%), cells were immunostained as described. Prior to telomere hybridization with the PNA probe (CCCTAA)3-FITC, cells were successively fixed , washed in PBS, and dehydrated (50/70/100 ethanol).IF-FISH was performed using a protocol similar to one described previously ,64. CellTelomere quantification was performed using the Q-FISH technique with a Cy-3-labelled PNA probe specific for (TTAGGG) . Two approaches were developed. The first approach consisted of the quantification of telomere length in interphase cells, permitting the investigation of intercellular variation in a large number of scored cells. Quantitative image acquisition and analysis were performed using Metacyte software . The mean fluorescence intensity (FI) of telomeres was automatically quantified in 10,000 nuclei on each slide. Settings for exposure and gain remained constant between captures. The experiment was performed in triplicate. The second approach consisted of quantifying telomere length in metaphases using automated acquisition module Autocapt software  and a ZEISS Plan-Apochromat 63×/1.40 oil and CoolCube 1 Digital High Resolution CCD Camera with constant settings for exposure and gain. The mean telomere length was measured and telomere loss and telomere doublet scoring were performed. This approach allows the study of intra-cellular variation of telomere length. The experiment was performed in triplicate. Telomere length, measured as mean fluorescence intensity (FI), strongly correlated with telomere length measured by Southern blot analysis using the telomeric restriction fragment (TRF). The mean telomere length is expressed in kb.Cytogenetic slides were subjected to telomere and centromere  staining followed by M-FISH  technique. The protocol was described previously ,52.Micronucleus assay was performed in the absence of cytochalasineB. The protocol was previously described . In addiTRF1, TRF2 and Ku 86 expressions were assessed by western blotting on lysates of HL cell lines. Cells were sonicated in 500 µL of a buffer containing 8 M urea, 150 mM β-mercaptoethanol, 50 mM Tris-HCl (pH 7.2) and centrifuged for 30 min at 4 °C to remove cellular debris. Samples were subjected to electrophoresis on 12% (p53 and Ku) or 6% (DNA-PKcs) SDS–polyacrylamide gels, blotted onto nitrocellulose membranes, and developed using the ECL system . To verify that equivalent amounts of each sample were loaded, the filters were additionally probed with anti-actin antibody . Densitometry was performed to evaluate the intensity of Ku70, p53, DNA-PKcs, and actin bands.6 cells were irradiated at 6Gy and harvested 24-h after irradiation. The protocol was previously published [To evaluate the sensitivity of cell lines to apoptosis, 10ublished .Cell-cycle fractions were determined by Hoechst nuclear staining. Briefly, cells were harvested, washed in PBS, and incubated in Hoechst solution for 30 min at room temperature. Data were collected using a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo Version 7.5.5. The results represent the mean value of three independent experiments.Basal and post-irradiation expression of five radiation-responsive genes  was determined by quantitative real-time PCR in HL cell lines at 2 h and 24 h after 2Gy irradiation. The presented data show the rapport between basal gene expression and that after exposure. The protocol was previously published .Total RNA was extracted from frozen HL cells and controls (REMB lymphoblastoide cell line) using the AllPrep DNA/RNA/protein Mini kit , quality-controlled using a 2100 BioAnalyzer , and quantified using a Nanodrop 2000c spectrophotometer . The WT Expression Kit  was used to prepare cDNA from 10 μg purified cRNA, originally synthesised and purified from 0.25 μg of total RNA, following the manufacturer’s instructions.Micronucleus (MN) scoring was performed after telomere and centromere staining. Automated scoring of mononuleated cells was performed using Metafer 4 image analyser software  and a Zeiss Axioplan 2 imager . Ten thousand mononucleated cells per culture  were processed in the image analyzer. Following telomere and centromere staining, MN were classified based on the detection of centromeric regions and telomeric sequences. We detected MN with telomeric and centromeric sequences , MN with only telomeric sequences (acentric chromosome or chromatid fragments), and MN without any sequences . In addition, nucleoplasmic bridges (NPBs) were assessed following telomere and centromere staining (dicentric chromosomes caused by telomere end fusion) or without (dicentric chromosomes resulting from misrepair of DNA strand breaks). Nuclear buds (NBUDs) were also assessed to study the process of elimination of amplified DNA possibly generated via BFB cycles (with telomere sequences or without) or the process of elimination of excess chromosomes may occur in polyploidy cells to facilitate aneuploidy rescue. The scoring of chromosomal aberrations was performed after telomere and centromere staining to score dicentric chromosomes and various types of acentric fragments with four telomeres (resulting from a fusion event), two telomeres, representing terminal deletions, and acentric fragments without any telomere sequence, representing interstitial deletions. Karyotype analysis was then performed using the M-FISH technique on the same slide. The introduction of telomere and centromere staining in the establishment of complex karyotypes allows easy detection and robust classification of chromosomal abnormalities. Complex chromosomal rearrangements were defined as those chromosomes involved in three or more chromosomal exchanges.Similarly BFB chromosomes were defined as those chromosomes that were involved in multiple cycles of repetitive fusions and breakage following the loss of a telomere .Metaphase images were acquired using automated acquisition module Autocapt software  and a ZEISS Plan-Apochromat 63×/1.40 oil and CoolCube 1 Digital High Resolution CCD Camera. The analysis was carried out using Isis software . Automatic scoring of MN following TC staining was performed using MN-score software . Spectral karyotype analysis was performed using Isis software .Linear regression, supported by the least squares method and the Fisher test, was performed to describe the relationship between the chosen factors. The normality of signal distribution was verified with the Shapiro-Wilk test. Because of the significant departure from normality, the non-parametric tests: Wilcoxon rank sum test and exact Wilcoxon-Mann-Whitney test were used to compare telomere length, telomere loss and telomere deletion in NS-HL and MC-HL patients versus healthy donors.For transcriptome analysis, the KMH2 and L1236 cell lines (MC) were considered as one group (6 samples) and L428, L591, SUP, HDLM2 cell lines as another . The Shapiro-Wilk test was applied to check on distribution normality. The hypotheses on the equality of median values between groups were tested by nonparametric exact Wilcoxon-Mann-Whitney test due to non-normal signal distributions. Benjamini-Hochberg procedure was used to control False Discovery Rate caused by multiple testing. For each transcript, the effect size (ES) translated to the Spearman’s r correlation coefficient was calculated using a formula dedicated for non-normal distribution and the U-Mann-Whitney test . AccordiHere, we provide the first evidence for the involvement of MSI to the genomic instability observed in HL. The second main finding in this study is the existence of two different mechanisms that effect chromosomal instability in HL, reflecting the clinical differences observed in terms of remission and survival of NS and MC-HL patients . This stIn the MC HL cell lines, we demonstrate that chromosomal instability is not only related to telomere dysfunction, but that dicentric formation was induced through a specific non-homologous end joining (NHEJ) pathway of DSB repair, essentially in the pericentromeric region. These processes involve the formation of specific dicentric chromosomes with both centromeres in close proximity. This may result from difference in radiation sensitivity. In line with HL cell lines findings, we report telomere dysfunction in circulating lymphocytes from NS-HL patients and high telomere heterogeneity in MC-HL patients. Altogether, our results suggest that telomere dysfunction in SN-HL induces chromosomal instability necessary for HL initiation.


Muehrcke's lines are a type of apparent leukonychia that are common in patients receiving chemotherapeutic agents. They are self‐limited, and there is no need to more workup. Muehrcke's lines are a type of apparent leukonychia that are common in patients receiving chemotherapeutic agents. They are self‐limited, and there is no need to more workup. There was no significant point in the past medical history. The laboratory data including renal function test, liver function test, and serum albumin levels were normal. Treatments with acitretin 25 mg daily were initiated. After 5 months of treatments, paired transverse white lines were noted involving all the fingernails and made patient anxious. On each finger, there were two pale lines 1 mm in width, parallel to the lunula with regular border. The lines were disappeared with pressing on the nail plate, and they did not move distally during nail growth (Figure After 2 months of discontinuation of acitretin, patient represented with white lines faded Figure B.3Firstly, Muehrcke described a nail condition as two parallel lines of pallor in the nail bed in the patients with low serum albumin,Muehrcke’s lines are double white band parallel to the lunulaThe most important differential diagnosis is Mee’s line in which there is disturbance in nail plate but not in nail bed.Muehrcke's lines could be present in the absence of hypoalbuminemia.This condition is asymptomatic and resolves after drug withdrawal.The authors declare that they have no competing interests.FM: designed and collected the data and wrote, read, and approved the final manuscript. FD: collected the data and wrote, read, and approved the final manuscript.This study does not require any approval from the ethics committee.The authors have consent for publication.


The aggressive B-cell non-Hodgkin lymphomas diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are characterised by a high proliferation rate. The anaphase-promoting complex/cyclosome (APC/C) and its co-activators Cdc20 and Cdh1 represent an important checkpoint in mitosis. Here, the role of the APC/C and its co-activators is examined in DLBCL and MCL.The expression and prognostic value of Cdc20 and Cdh1 was investigated using GEP data and immunohistochemistry. Moreover, the therapeutic potential of APC/C targeting was evaluated using the small-molecule inhibitor proTAME and the underlying mechanisms of action were investigated by western blot.We demonstrated that Cdc20 is highly expressed in DLBCL and aggressive MCL, correlating with a poor prognosis in DLBCL. ProTAME induced a prolonged metaphase, resulting in accumulation of the APC/C-Cdc20 substrate cyclin B1, inactivation/degradation of Bcl-2 and Bcl-xL and caspase-dependent apoptosis. In addition, proTAME strongly enhanced the anti-lymphoma effect of the clinically relevant agents doxorubicin and venetoclax.We identified for the first time APC/C as a new, promising target in DLBCL and MCL. Moreover, we provide evidence that Cdc20 might be a novel, independent prognostic factor in DLBCL and MCL. Although the standard-of-care regimen R-CHOP  has significantly improved the survival rates, MCL remains incurable and up to one-third of the DLBCL patients are or become refractory.4 However, these agents induce severe toxicity and are associated with multidrug resistance development.5 Clinical studies are currently ongoing to find more selective cell cycle targets by blocking either the interphase (e.g. Cdk inhibitors) or mitotic entry .6 In B-cell NHL, the anti-lymphoma effects of these agents were either disappointing or the trials were suspended early due to toxicity issues.9 Recent preclinical studies show that targeting molecules involved in the mitotic exit, such as the anaphase-promoting complex/cyclosome (APC/C), is a better strategy since it provokes a more permanent mitotic arrest, thereby reducing the chance of mitotic slippage and thus enhancing mitotic cell death.10 The activation of the APC/C, an E3-ubiquitin ligase, depends on the interaction with its co-activators Cdc20 or Cdh1. The spindle assembly checkpoint (SAC) prevents APC/C activation in early mitosis. When bi-orientation of the chromosomes is achieved in the metaphase, the SAC pathway is inactivated and APC/C–Cdc20 binding occurs. This interaction leads to proteasomal degradation of the substrates cyclin B1 and securin, subsequently allowing the onset of the anaphase.11 Cdc20 is thereafter replaced by Cdh1 and the APC/C–Cdh1 targets Cdc20, aurora kinases and Plk-1 during mitotic exit. In early G1 phase, Skp2 and mitotic cyclins are degraded, resulting in increased p21, p27 and cyclin D levels, which is necessary to maintain the G1 phase.12The clinical use of cell cycle targeting agents, such as the microtubule-targeting agent vincristine, has proven that this is an interesting approach in the treatment of high-grade B-cell NHL.13 Wang et al. were the first to demonstrate APC/C mutations in human cancer cells, showing that a disruption of some of the APC/C subunits contributes to tumorigenesis by deregulation of key cell cycle regulators.14 Increased Cdc20 expression is correlated with poor prognosis in several human cancers,21 and depletion of Cdc20 in various cancer cell lines resulted in tumour metaphase arrest and induction of apoptosis.23 Moreover, inhibition of Cdh1 has been associated with tumorigenesis and decreased Cdh1 expression is observed in breast and colon cancer.25 The small molecule TAME (tosyl-L-arginine methyl ester) was discovered as a specific APC/C inhibitor in the past decade. It mimics the IR-tail of the co-activators and blocks the interaction between APC/C and Cdc20 or Cdh1.26 Previously, we and others demonstrated that proTAME (permeable variant of TAME) treatment results in a reduced viability, a growth arrest and apoptosis of malignant plasma cells.27Several studies have already suggested the involvement of the APC/C and its co-activators in tumorigenesis of different cancers and their potential as a new therapeutic target.Currently, however, little is known about the therapeutic potential of targeting the APC/C and its co-activators in DLBCL and MCL. The aim of this study is to investigate the expression of the APC/C co-activators Cdc20 and Cdh1 and the therapeutic potential of APC/C targeting in DLBCL and MCL.29 , GSE5631530 , GSE1645531 (containing gene expression data of 7 indolent and 15 aggressive MCL patients) and GSE3613332 (containing gene expression data of 13 human DLBCL cell lines and 5 MCL cell lines) were used. Raw CEL files were obtained from the Gene Expression Omnibus (GEO) and gcrma-normalisation was performed in R using bioconductor. Survival analysis of GEP microarray data was done using Genomicscape (http://genomicscape.com). The following probe sets were used: 202870-s-at (Cdc20) and 209416-s-at (Cdh1).The publicly available GEP datasets GSE1084633 All samples were stained with Cdc20 antibody  using an automated immunostainer Benchmark XT . As a positive control, tonsils with secondary follicles were used. Two hundred tumour cells were counted and the percentage of tumour cells with a staining of any intensity was determined at a magnification ×400 on a Leica DM2000 microscope. The counting was performed in hot-spot areas by means of an ocular grid. Based on the R-IPI of the DLBCL patients, they were divided into good and poor prognosis groups. Cells were scored MYC and Bcl-2 positive as we described previously.33Patient samples were collected at the Department of Pathology in Brussels  and the Department of Biopathology in Montpellier . Paraffin-embedded samples available from seven DLBCL patients were selected in Brussels and 3-μm-thick sections from tissue microarrays containing three representative 0.6-mm cores of routinely processed tissues from 27 DLBCL patients were included from Montpellier.2atmosphere. All cell lines were obtained from ATCC and regularly tested for mycoplasma contamination. They were authenticated by STR profiling.All the MCL  and ABC–DLBCL cell lines (U2932 and RI-1) were maintained in the RPMI-1640 medium  supplemented with 10% foetal calf serum (FCS)  and 2 mM glutamine . The GCB–DLBCL cell lines  were maintained in the IMDM medium (Life Technologies) supplemented with 10% FCS and 2 mM glutamine. Cells were cultured at 37 °C in a humidified 5% COThe APC/C inhibitor proTAME was obtained from R&D Systems . Venetoclax and rituximab were obtained from Selleckchem , doxorubicin hydrochlorate was purchased from Sigma-Aldrich  and apcin was kindly provided by Dr. R.W. King .p-value of p < 0.05 was considered significant) was determined by a Mann–Whitney U test (to compare two groups) and a one-way ANOVA with Bonferonni correction for multiple testing.Prognostic significance of Cdc20 and Cdh1 gene expression was calculated using the MaxStat R package. Statistical differences in overall survival were calculated by a log-rank test and survival curves were plotted using Kaplan–Meier method. Multivariate analysis was performed using the Cox proportional hazards model. Graphical and statistical analysis was performed using GraphPad Prism 5.01 software. Statistical significance (More details on the ‘Methods' section are described in n = 33), ABC–DLBCL (n = 190), GCB–DLBCL (n = 212) and unclassified DLBCL patients (n = 67). The gene expression levels of Cdc20 were significantly increased in all DLBCL subtypes compared with the B-cell samples. In contrast, the Cdh1 gene expression levels were not significantly different in the GCB–DLBCL and ABC–DLBCL patients compared with the B-cell samples  datasets. The expression of Cdc20 and Cdh1 mRNA was assessed in DLBCL patients, using GEP data from B-cell samples   and molecular DLBCL subtypes. Univariate COX analysis demonstrated that all these factors have a prognostic value in DLBCL Table . In the 42) Fig. . Consist42) Fig.  nor doub42) Fig. . TogetheNext, the APC/C was investigated as a potential target in DLBCL and MCL using human cell lines. Therefore, the Cdc20 and Cdh1 expression was first determined using gene expression profiling data of a large panel of DLBCL and MCL cell lines. Similar to the primary patient samples, we found a significantly higher Cdc20 mRNA expression in both the DLBCL and MCL cell lines compared with B-cell samples and a lower Cdh1 mRNA expression .ine Fig. . In contine Fig. . To furtine Fig. . These f35 The effect of proTAME treatment on the expression and phosphorylation of Mcl-1, Bcl-2 and Bcl-xL was evaluated during mitotic arrest by western blot. No clear difference in the total Bcl-2, Bcl-xL and Mcl-1 protein was observed. Nevertheless, consistent with the prolonged activation of Cdk1, an increase in phosphorylation of Bcl-2 and Bcl-xL was observed during the mitotic arrest  of both agents were used to test the combinatory effect 44 and apcin.45 The small molecule proTAME is the only true/selective APC/C inhibitor known so far.26 ProTAME has already been described to cause a prolonged metaphase in cancer cells.26 Consistent with these observations, synchronised DLBCL and MCL cells released into proTAME treatment were found to accumulate in the G2/M phase. Western blot analysis of these cells demonstrated an increase in cyclin B1 protein levels during this mitotic arrest, while Skp2 levels are not or only slightly affected. The induced G2/M-phase arrest was further investigated, and a significant increase in the percentage of cells in the metaphase was observed. It is known that cells in a prolonged metaphase can either undergo cell death or re-enter the cell cycle to exit mitosis. Cdc20 is believed to be a critical factor in this decision-making. When Cdc20 levels increase during the metaphase, cyclin B1 will be degraded, resulting in Cdk1 inactivation and mitotic exit.46 On the other hand, a prolonged activation of the cyclin B1–Cdk1 complex occurs when cells are arrested in the metaphase, resulting in the phosphorylation and inactivation of Bcl-2 and Bcl-xL, and subsequent activation of the intrinsic apoptotic pathway.48 Consistent with the above, the proTAME-induced prolonged metaphase in DLBCL and MCL cells is accompanied by a dose-dependent decrease in viability and increase in caspase-mediated apoptosis. Western blot analysis confirmed the phosphorylation of both Bcl-2 and Bcl-xL. In contrast, Mcl-1 and Cdc4 levels remained mostly unchanged during the mitotic arrest, indicating that the Cdc4–Mcl-1 axis is most likely not involved in proTAME-induced cell death. These findings provide evidence that APC/C targeting using proTAME induces a metaphase arrest in DLBCL and MCL cells, resulting in the accumulation of cyclin B1 and a prolonged activation of Cdk1, followed by phosphorylation of Bcl-2 and Bcl-xL and eventually cell death.The therapeutic potential of targeting the APC/C, either by blocking the activation of the complex or the co-activators, has been examined in cancer using proTAME,36 ProTAME blocks the interaction between the APC/C and its co-activators, while apcin inhibits the interaction between the co-activators and their substrates.36 This combination resulted in a synergistic anti-lymphoma effect in all cell lines. Other studies in myeloma also showed a combinatory effect with the anti-myeloma agents melphalan, vincristine, etoposide and the topoisomerase II inhibitor doxorubicin.27 Topoisomerase IIα is identified as a Cdh1 substrate and both proTAME and knockdown of Cdh1 significantly enhance the sensitivity of cancers cells to topoisomerase II inhibitors.49 Consistent with these findings, proTAME was also found to sensitise all lymphoma cell lines tested to doxorubicin. Combining proTAME with rituximab did not demonstrate any additional effect. This could be explained by the minor effects that rituximab alone had on the different cell lines in vitro. Consistently with our data, others also observed no or only minor effects when using rituximab as monotherapy in DLBCL cell lines.50 The anti-lymphoma effect of proTAME combined with a novel agent in clinical development in DLBCL and MCL, namely the selective Bcl-2 inhibitor venetoclax, was also tested. ProTAME strongly and significantly potentiated the anti-lymphoma activity of venetoclax in all cell lines tested, even in the highly sensitive ABC–DLBCL cell line U2932.To further ensure that cells will not re-enter the cell cycle after the metaphase arrest induced by proTAME, combination studies were performed. Previously, we and others demonstrated that inactivation of the APC/C is more effective when multiple protein interactions are simultaneously disrupted using proTAME and apcin.26 These enzymes can be present in the bloodstream, thus limiting the in vivo use of proTAME. A possible solution could be to pack (pro)TAME in a liposomal vesicle, such as the vesicle used for liposomal doxorubicin in the treatment of aggressive B-cell lymphoma.51 Another general concern is that inhibition of the APC/C will most likely affect all dividing cell types. To avoid these off-target effects, liposomal (pro)TAME could be altered, so that it can only be recognised by CD20+B cells. This would give the advantage of specifically targeting the lymphoma cells like rituximab.To further validate the therapeutic potential of targeting of APC/C in DLBCL and MCL, we acknowledge that in vivo studies are required. Unfortunately, when it comes to the in vivo use of proTAME, there are some concerns. ProTAME has not yet been optimised for in vivo studies, since this prodrug is activated intracellularly by esterases and the active form (TAME) is not cell permeable.In conclusion, this study demonstrates that high Cdc20 expression correlates with a poor prognosis in the aggressive B-cell malignancy DLBCL. We also provide evidence that APC/C targeting in DLBCL and MCL cells results in a prolonged metaphase followed by apoptosis and sensitises the lymphoma cells to the clinically relevant agents doxorubicin and venetoclax. Together, this study suggests that the APC/C is a potential new target in these cancer types, making it a promising target for further preclinical research.Supplementary information


For decades, retinoids and their synthetic derivatives have been well established anticancer treatments due to their ability to regulate cell growth and induce cell differentiation and apoptosis. Many studies have reported the promising role of retinoids in attaining better outcomes for adult or pediatric patients suffering from several types of cancer, especially acute myeloid leukemia and neuroblastoma. However, even this promising differentiation therapy has some limitations: retinoid toxicity and intrinsic or acquired resistance have been observed in many patients. Therefore, the identification of molecular markers that predict the therapeutic response to retinoid treatment is undoubtedly important for retinoid use in clinical practice. The purpose of this review is to summarize the current knowledge on candidate markers, including both genetic alterations and protein markers, for retinoid resistance and sensitivity in human malignancies. Defective or aberrant cell differentiation is a hallmark of many human malignancies. The initial step in an aberrant tumor cell phenotype involves various mutations that alter signaling pathways, epigenetic modifiers, and transcription factors, leading to the deregulated expression of proteins required for cell differentiation.During the 1970s and 1980s, as an elegant alternative to killing cancer cells by cytotoxic therapies, several scientific achievements popularized the strategy of inducing malignant cells to overcome differentiation inhibition and to enter apoptotic pathways .In general, differentiation therapy aims to reactivate the endogenous differentiation program in transformed cells to resume the mutation process and eliminate the tumor phenotype. Thus, this strategy offers the prospect of a less aggressive treatment that limits damage to the normal cells in the organism.trans retinoic acid (ATRA), 9-cis retinoic acid (9-cis-RA), and 13-cis retinoic acid (13-cis-RA), have been tested for potential use in cancer therapy and chemoprevention  functions as a transcriptional activator targeting PLZF-binding sites, leading to cellular retinoic acid-binding protein 1 (CRABP1) upregulation. The CRABP1 protein is structurally similar to the cellular retinol-binding proteins, sequesters retinoic acid to limit its access to the nucleus [PLZF-RARA fusion transcript only were sensitive to ATRA treatment under in vitro conditions, and these results correlated with clinical remission after ATRA administration in this patient [Molecular analysis of the possible mechanisms of retinoid resistance suggested that the reciprocal  nucleus , and is  nucleus –51. Simi nucleus , 52, 53. patient . Moreove patient . Further patient . NeverthPML-RARA fusion gene and the therapeutic response to ATRA [PML sequence, which contains heterogeneous breakpoint cluster regions (bcrs) at three different sites E6(−) isoform with exons 5 and 6 deleted, is associated with the ATRA-resistant phenotype [PML-RARA fusion gene might be helpful for predicting a patient’s response to ATRA treatment.Sequencing analysis of the henotype . A subsePML and RARA mRNA fusion partners. Subsequent in vitro studies confirmed these results, revealing that spacer deletion restored ATRA sensitivity [Similarly, APL cells with the V-type splicing isoform, characterized by exon 6 truncation, were also reported to be less sensitive to ATRA treatment. In this group of APL patients, a subset with lower ATRA sensitivity presented with a relatively long “spacer” with a cryptic coding sequence inserted into the joining sites between the truncated sitivity .RARβ expression is downregulated in PDAC [The vitamin A metabolism disturbances that result in a decreased intracellular ATRA concentration were originally described in pancreatic ductal adenocarcinoma (PDAC)  and late in PDAC –68, whichighCRABP2null PDAC lines were resistant to ATRA-mediated growth inhibition and apoptosis and also exhibited an increased migration and invasion phenotype. In contrast, FABP5nullCRABP2high cell lines retained ATRA sensitivity. These results were also confirmed in vivo using xenograft models [ATRA typically induces cell differentiation and growth arrest in most epithelial cell types. However, experiments in Capan-1 cell line have shown that in addition to an antiproliferative effect, retinoids increase cell migration, resulting in an invasive phenotype . This eft models . Immunoht models . Since tBreast carcinoma is a heterogenous disease classified into subtypes according to the expression of biological markers, such as estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2) –74. AccoIndeed, several studies have demonstrated that the average RARα receptor level is significantly higher in ATRA-sensitive than ATRA-resistant breast carcinoma cell lines –77. FurtERBB2 transfection in ATRA-sensitive breast carcinoma cells induced ATRA resistance. When Her2/neu was blocked by trastuzumab, the cells exhibiting induced ATRA resistance became ATRA sensitive again. This study also hypothesized that Her2/neu may induce ATRA resistance in breast carcinoma cells by suppressing RARA expression and/or by deregulating the G1 checkpoint of the cell cycle.Another potential marker of ATRA resistance was suggested by a study describing Her2/neu-induced ATRA resistance in breast cancer cell lines . ERBB2 tFABP5 is preferentially upregulated in estrogen receptor-negative (ER-) and triple-negative breast carcinoma cells (TNBC), and an increased FABP5 mRNA level is associated with poor patient prognosis and high tumor grade [FABP5 normalized signal intensity scores were categorized into high versus low using cut-off point of 0.768. In this cohort, 61% of patients showed high FABP5 expression and these patients had a significantly decreased survival rate if compared with those with low FABP5 expression. Moreover, FABP5 silencing in Hs578T breast carcinoma cell line resulted in approximately 40% reduction in proliferation activity. However, although breast cancer cells with an increased FABP5/CRABP2 ratio present with increased ATRA resistance, this ratio does not always accurately predict the breast cancer cell response to ATRA, indicating that other factors are also involved in the mechanism of retinoid resistance development. Another recent study identified CRABP1 as the third key player that potentially influences the breast cancer cell response to ATRA. This protein has been identified as a retinoid inhibitor and probably sequesters retinoic acid in the cytoplasm, thereby preventing RAR activation in the nucleus. Similarly to FABP5, CRABP1 is also preferentially expressed in ER- and TNBC tumor tissues that are prone to ATRA resistance [As described in the PDAC section in this review, the abundance of the intracellular retinoic acid transporters CRABP2 and FABP5 within the cell can indicate breast carcinoma cell response to ATRA, since these molecules have been shown to play opposing roles in mediating the cellular response to retinoids . Accordior grade . In thissistance . AccordiThese findings provide molecular tools to predict and eventually overcome ATRA resistance in breast carcinoma therapy. CRABP1 and FABP5 co-expression may serve as a predictive biomarker of ATRA resistance in this tumor type, and the downregulation may be a key step in (re)sensitizing breast carcinoma cells to retinoid therapy. A novel mechanism for resensitizing ATRA-resistant cells to ATRA-mediated apoptosis was recently introduced: the phytochemical curcumin is able to upregulate CRABPII, RARβ and RARγ expression in TNBC cell lines and thereby sensitizes cells to ATRA-induced apoptosis. This reversed resistance to ATRA-induced apoptosis in TNBC cells was dependent on the curcumin dose and treatment length . OverallAnother recent study compared the phosphoproteome and transcriptome of established ATRA-sensitive and ATRA-resistant cell lines derived from breast carcinoma . One of the most interesting results was that ATRA did not regulate the phosphorylation of the same proteins in both cell lines, i.e., the ATRA-resistant cell line exhibited a deregulated kinome. High-throughput sequencing experiments revealed that 80% of the genes regulated by ATRA in MCF7 cells were not regulated in BT474 cells and vice versa. Additionally, 40% more genes were regulated by ATRA in the MCF7 cells than in the BT474 cells. Moreover, this study indicates that ATRA induced RARα phosphorylation in resistant cell lines only, which may cause kinome deregulation and consequences in other intracellular metabolic pathways .Neuroblastoma (NBL) is a neuroectodermal tumor arising from elements of the neural crest and represents the most common extracranial solid tumor in children. In a subset of high-risk NBL patients with minimal residual disease, retinoid administration was proven effective as a part of postconsolidation therapy after intensive multimodal treatment. Unfortunately, approximately 50% of this patient population is resistant to this treatment or develops resistance during therapy . Moreovecis-RA, PBX1 mRNA and protein expression levels are both induced in 13-cis-RA-sensitive cell lines only. After treatment with 13-cis RA, all 6 RA-sensitive cell lines showed a significant increase in PBX1 expression, whereas RA-resistant cell lines exhibited no such effect. These studies also revealed that reduced PBX1 protein levels result in an aggressive growth phenotype and 13-cis-RA resistance. Finally, the authors demonstrated that in primary NBL tumor tissue, PBX1 expression correlated with the histological NBL subtype, with the highest PBX1 expression in benign ganglioneuromas and the lowest expression in high-risk NBL [PBX1 belongs to the three-amino-acid loop extension (TALE) family of atypical homeodomain proteins and interacts with other homeodomain-containing nuclear proteins, such as HOX and MEIS, to form heterodimeric transcription complexes. PBX1 is involved in a variety of biological processes including cell differentiation and tumorigenesis , 87. Recrisk NBL .HOXC9 promoter is epigenetically primed in an active state in ATRA-sensitive NBL cell lines and in a silenced state in ATRA-resistant NBL cell lines. Moreover, HOXC9 protein levels were significantly higher in differentiated NBL cells than in NBL cells undergoing ATRA-induced differentiation [Homeobox (HOX) proteins function as regulators of morphogenesis and cell fate specification and are key mediators of retinoid action in nervous system development. Among members of the HOX family of transcription factors, HOXC9 seems to play an important role in neuronal differentiation. A recent study revealed that the ntiation .NF1 knockdown continued to proliferate when exposed to RA in contrast to the control cells. Subsequent experiments showed downregulation of RA target genes in NF1 knockdown cells. These results may indicate the role of NF1 in maintaining RA resistant phenotype.The protein neurofibromin 1 (NF1) is known to antagonize the activation of RAS proteins but is also involved in other signaling pathways, such as the cAMP/PKA pathway . NF1 conNF1 gene were found in 6% of primary NBL representing a subset of cases where the loss of NF1 gene could be caused by gene mutation.In further research, genomic aberrations of the cis-RA (isotretinoin) treatment. Moreover, decreased NF1 and ZNF423 gene expression, reflecting hyperactivated RAS/MAPK signaling, is correlated with a very poor clinical outcome in NBL patients and was detected in 78% of patients with relapsed NBL [ZNF423 mRNA levels and the sensitivity of MPNST cells to ATRA [A connection between NF1-RAS-MEK signaling and retinoic acid action was demonstrated by the finding that the NF1-RAS-MEK cascade suppresses ZNF423 protein expression, which functions as a RAR/RXR coactivator. Additionally, tumors with activated RAS signaling and low ZNF423 expression present with a poor response to 13-psed NBL , whereaspsed NBL . Another to ATRA . These rHMGA2 is completely absent in adult organisms; its expression is restricted to rapidly dividing embryonic cells and tumors with epithelial and mesenchymal origins [HMGA2 was also detected in some retinoid-resistant NBL cell lines. In NBL cell lines, a causal link between HMGA2 expression and retinoid-induced growth arrest inhibition was proven using exogenous HMGA2 expression, which was sufficient to convert HMGA2-negative, retinoid-sensitive cells into retinoid-resistant cells [HMGA1 was found to be expressed at different levels in all NBL cell lines [High-mobility group A (HMGA) proteins function as ancillary transcription factors and regulate gene expression through direct DNA binding or protein-protein interactions and play important functions in controlling cell growth and differentiation.  origins . HMGA2 wnt cells . In contll lines , indicatUNC45A gene, a member of UNC45-like genes, which are evolutionarily highly conserved, and the resulting protein products are involved in muscle development and myosin assembly [UNC45A, another potential marker of retinoid resistance, is a protein encoded by the assembly . The UNCassembly . In NBL assembly . When UNThis review was aimed to summarize the current knowledge, both clinical and experimental, on predictive markers in human cancers that are treated with retinoids as a part of the therapeutic regimen. This review demonstrated that each described cancer type seems to have a unique pattern of altered signaling pathways, resulting in a set of predictive biomarkers that indicate retinoid resistance or sensitivity, which is typical for this malignancy. Many of the research studies mentioned in this review are only initial, and the acquired results require further detailed investigation and clinical validation of the proposed predictive biomarkers. However, these studies demonstrate the promising future for differentiation therapies that use retinoids, especially in identifying reliable markers that predict the response of each individual patient to this type of treatment. Hopefully, the personalized approach will be a new milestone in anticancer differentiation therapy.