Bladder cancer (BC) in dogs is often lethal at the time of diagnosis. Therefore, there is a constant need for novel research on improvements of its characterization and treatment. Due to high cost and limited number of available dog patients, in vitro models of canine BC have been increasingly used for the last 25 years. In the present article, we present existing in vitro models of canine BC, including available simple  and more complex  models.Bladder cancer (BC) constitutes approximately 2% of all spontaneously occurring cancers in dogs. It is characterized by a devastating clinical course in most cases, which emphasizes a constant need for the development of novel methods of disease characterization and treatment. Over the past years, advances in cell engineering have resulted in the development of various canine in vitro models of BC, emerging as complements for in vivo research. In this article, we aimed to review the available data on existing in vitro models of canine BC, focusing primarily on their characteristics, applications in veterinary medicine, as well as advantages and disadvantages. The most commonly used in vitro models of canine BC comprise immortalized cell lines grown as adherent monolayers. They provide an unlimited supply of research material, however, they do not faithfully reflect the conditions prevailing in vivo, since the spatial cellular interactions are lost. The importance of the three-dimensional (3D) features of solid tumors in relation to carcinogenesis or drug response process has resulted in the development of the first canine 3D models of BC available for in vitro research. So far, results obtained with in vitro and in vivo research should be interpreted together. With the constantly growing complexity of in vitro models of BC cancer, animal-based research might be reduced in the future. Bladder cancer (BC) constitutes approximately 2% of all spontaneously occurring cancers in dogs . With esThough obvious benefits that could be gained from the research of canine BC in vivo, there are some inevitable limitations , making extensive in vivo research difficult ,2. CurreIn the present article, we aimed to review the available data on existing in vitro models of canine BC as a tool in veterinary research, focusing primarily on their characteristics, potential applications in veterinary medicine, and advantages and disadvantages. To the best of our knowledge, this is the first review summarizing the current evidence regarding this topic. We conducted a literature search using two electronic databases, namely, Pubmed and Scopus. The most recent search was performed on 28 January 2022. Screening of the literature was conducted using the following search string:  AND (“cancer” OR “carcinoma” OR “neoplasm”) AND (“in vitro” OR “model” OR “cell*” OR “culture”) AND (“dog” OR “canine”). Auto-alerts in Medline were run, as well as reference lists of original articles and review articles for further eligible data. We exclusively included data regarding in vitro models of canine bladder TCC. Only papers in English were considered eligible without restrictions on publication year. The flow diagram of the study selection process is presented in The most commonly used two dimensional (2D) models of canine BC comprise immortalized cell lines and primary cell cultures grown as adherent monolayers in appropriate media. The first immortalized canine BC cell line, called K9TCC, was established by Knapp et al. in 1995 . Since tThe majority of canine BC cell lines express specific cancer-related markers, resembling those presented by the primary tumors in vivo. Due to differences in the development process (mainly related to primary tumor characteristics), the available canine TCC cell lines differ in terms of expressed biomarkers . This alTo date, only a few studies have provided data regarding high-throughput molecular characterization of existing canine BC cell lines. Initial molecular characterization (including genotypic data) of several canine BC cell lines was performed by the Flint Animal Cancer Center (FACC) . SubsequIn vitro studies using BC cell lines play a significant role in the novel drug discovery and development process, providing crucial data on drug effects in the early preclinical stages. Such information is of paramount importance in the decision-making process for drugs moving forward into more expensive and time-consuming in vivo clinical trials. Initial studies using canine TCC cell lines were extensively focused on non-selective cyclooxygenase inhibitors  and various chemotherapeutic agents . FollowiCanine BC cell lines can be also used for investigation of other forms of anti-cancer therapy. As an example, Parfitt et al. investigated and characterized the radiosensitivity and capacity for cellular damage repair of canine BC cell lines . AuthorsConventional 2D cultures have several advantages supporting their important role in preclinical research. Research using cell lines is significantly less expensive than in vivo animal studies and provide an unlimited supply of material, which is widely available and easy to propagate under completely controlled and reproducible environmental conditions . NeverthThe importance of the three-dimensional (3D) features of solid tumors in relation to carcinogenesis or drug response process has prompted efforts to develop in vitro models mimicking in vivo tumor growth more precisely. Examples of 3D culture systems include multicellular aggregates grown as spheroids, scaffold-based models grown within polymer networks, and organoids defined as stem cell-containing self-organizing structures possessing multiple features of the original tumor ,32. DespThe first 3D models of canine BC were established by Elbadawy et al. . They ge3D cell culture approaches hold great potential and offer complex systems for various purposes, such as disease modeling and investigation of anticancer drug efficacy. The similarities in the drug responsiveness among the 3D in vitro models and the in vivo models might largely be due to their similarities in enhanced cellular interactions via adhesion and secretion of soluble factors of tumors ,36. ThesAlthough significant advances have been made over the past years, modeling the complexity of canine BC in in vitro models has not been completely successful. There are still various challenges, including the need for extensive molecular characterization of existing cell lines or creation of new reliable 3D models, incorporating multicellular cultures and diverse cellular microenvironments. So far, results obtained by in vitro and in vivo research should be interpreted together. With the constantly growing complexity of in vitro models of BC cancer, animal-based research might be reduced in the future


Eupatorium japonicum Thunb. of the plant family Asteraceae is a popular traditional herb in Vietnam. However, its chemical constituents as well as bioactive principles have not been investigated yet. We investigated the phytochemistry of E. japonicum in Vietnam and isolated seventeen compounds (1–17) including phytosterols, terpenoids, phenolic acids, flavonoids, fatty alcohols, and fatty acids. They were structurally determined by MS and NMR analysis. Except for compounds 6 and 12, all the other compounds were identified for the first time from E. japonicum. Since many sesquiterpene lactones with α-methylene γ-lactone ring are reported as anti-inflammatory and anticancer agents, eupatoriopicrin (10), 1-hydroxy-8-eudesma-4(15),11(13)-dien-6,12-olide (11) were selected among the isolates for biological assays. Compound 10 was identified as the main bioactive sesquiterpene lactone of E. japonicum showing its potent anti-inflammatory and cytotoxic activity through inhibiting NO production and the growth of HepG2 and MCF-7 human cancer cell lines. For the first time, eupatoriopicrin (10) was demonstrated to strongly inhibit NTERA-2 human cancer stem cell (CSC) line in vitro. It is noticeable that the cytotoxicity of eupatoriopicrin against NTERA-2 cells is mediated by its apoptosis-inducing capability of 10 as demonstrated by the results of Hoechst 33342 staining, flow cytometry apoptosis analysis, and caspase-3 activity assays. The biological activities of the main bioactive constituents 1–7, 10, 12, and 15 supported the reported anti-inflammatory and anticancer properties of extracts from E. japonicum. Eupatorium species used in Vietnamese traditional medicine such as E. fortunei -prop-2-en-oic acid (13) [p-menth-1-ene-3,6-diol (14) [O-rutinoside (rutin) (15) [O-methyl ether (16) [17) [1H-NMR, and 13C-NMR) with reported literature values. Compounds 1–5, 7–11, and 13–17 were isolated for the first time from E. japonicum (The MeOH extract from the leaves of tate (1) , ß-amyritate 2) , α-amyriyrin 3) , ß-amyriyrin (4) , ß-sitoserol (5) , stigmas (5) [6) , β-sitos , α-amyr , ß-amyr (7) [8) , stigmasside (9) , eupator(9) [10) , 28, 1-h10) [11) , 30, caf11) [12) , (2E)-3-iol (14) , quercetin) (15) , quercether (16) , and kae16) [17)  which weaponicum .10 and 11 is shown in 10 strongly inhibited NO production in LPS-stimulated RAW 264.7 cells in a dose-dependent manner with an IC50 value of 7.53 ± 0.28 µg/mL. The positive control L-NMMA exhibited its IC50 value of 8.21 ± 0.84 µg/mL. Cytotoxicity of 10 was observed at the active concentrations; the cell viability was 43.27% at 4 µg/mL (NO inhibition: 93.83%), but up to 85.64% at 0.8 µg/mL (NO inhibition: 85.02%) (10 was reported to show strong anti-inflammatory activity through the inhibition of IL-8 and TNF-α release in lipopolysaccharide- (LPS-) stimulated human neutrophils [10 as a lead compound towards inflammation. Compound 11 did not display potent suppression on NO production at all concentrations tested . The highest inhibition of NO production was 48.02% at 100 µg/mL with the cell viability reaching 95.25%. Among the isolates 1–17, the inhibition of NO generation, inflammatory cytokines, tumor necrosis factor-α, or COX-2 enzyme was reported for triterpenes 1–4 [5–7 [12 and 15 [Nitric oxide (NO) participates in various responses of host resistance to various pathogens and joins to adjust various aspects of life such as vascular integrity, maintains hemostasis, or modulates neural activity , 37. The 85.02%) . Therefotrophils . Altogetenes 1–4 –42, phyt2 and 15 , 45. Thein vitro and in discovering novel anticancer compounds against CSCs, a research area of great interest nowadays. The cytotoxicity of compounds 10 and 11 on human breast cancer cell line (MCF7), human hepatocellular carcinoma (HepG2), and pluripotent human embryonal carcinoma cell line (NTERA-2) was accessed using SRB assay. As shown in 10) showed strong cytotoxic activity on all tested cell lines with IC50 values of 1.22 ± 0.10 µg/mL, 0.94 ± 0.12 µg/mL, and 0.88 ± 0.05 µg/mL, respectively. Compound 11 did not show cytotoxic effects on HepG2, MCF-7, and NTERA-2 cell lines at the concentrations tested. Hitherto compound 10 expressed its cytotoxic activity against KB, HeLa, HL-60, EN19, EAT, P388, FIO 26, and L5178Y cell lines [10 may be associated with its α-methylene-γ-butyrolactone moiety [in vivo study showing the tumor growth delay after administration of eupatoriopicrin [10 against human CSCs (NTERA-2) as well as human cancer cell lines HepG2 and MCF-7. Glutathione (GSH) depletion is a central signaling event that regulates the activation of cell death pathways [in vitro study by Woerdenbag et al. showed that significant GSH reduction began to occur with concentrations of eupatoriopicrin ≥1 μg/mL in FIO 26 cells [10, we assayed further the apoptosis-inducing activity of 10 in NTERA-2 cells.NTERA-2 is an embryonal carcinoma cell line, which expresses a gene profile related to pluripotency of stem cells such as OCT3/4 and SSEA-4. The cell line presents a lot of early embryo antigens that could be used to identify undifferentiated human embryonic stem cell (hESC) , 47. In ll lines , 48, 49.iopicrin . The prepathways . An in v26 cells . Eupator10 on NTERA-2 CSC line, 10 was further evaluated for its apoptosis-inducing activity to better understand the possible apoptosis pathway. We examined the possible induction of apoptotic cell death by 10 using flow cytometry analysis and activation of caspase-3 in CSCs. Thus, NTERA-2 cancer cells were treated with 10 and stained with Hoechst 33342. Using this nuclear staining dye, apoptotic cells with condensed and fragmented nuclei were detected, while most negative control cells showed nuclei with homogeneously staining morphology  activation (Franco and Cidlowski). Caspase-3 is one of the key enzymes in apoptosis and is activated in both the intracellular and extracellular apoptotic pathways [10 significantly induced caspase-3 production at all tested concentrations (p < 0.05) . Higher or lower concentrations also mediated caspase-3 production but the induction was not as strong as that at 1.0 µg/mL. These results indicated that this concentration might be the optimized concentration of 10 to induce apoptosis in the NTERA-2 CSC line through the activation of caspace-3.In order to confirm the apoptosis-inducing activity of compound pathways , 55. Thipathways . Compoun < 0.05) . Thus, c10 expressed the potential activity against CSCs by apoptosis and/or necrosis induction. A challenge of cancer therapy is multidrug resistant, which is closely related to CSCs [10 that reduce survival rates of CSCs and induce apoptosis attract considerable attention.Throughout,  to CSCs . Multidr to CSCs –60. TherE. japonicum. Belonging to the privileged group of anti-inflammatory and anticancer structures, two sesquiterpene lactones 10 and 11 were selected for biological assays. Eupatoriopicrin (10) was found to strongly inhibit NO production, MCF-7, HepG2, and NTERA-2 cancer cell lines; meanwhile, compound 11 was not active. The cytotoxicity on the NTERA-2 cancer cell line is related to significant apoptosis induction by the action of 10 in the concentration- and caspase-dependent manners. The importance of the findings is that the study provides a chemical and biological basis for developing new approaches to explore therapeutic values of extracts or herbal medicines containing the herb E. japonicum. In addition, anti-inflammatory and anticancer botanical drugs enriched with bioactive constituents can be formulated and prepared from E. japonicum. Eupatoriopicrin (10) is also exposed in the study as a lead compound targeting inflammation or cancer diseases. Further projects on the synthesis of chemical derivatives or analogs of 10 addressing optimization of drug safety and effectiveness in combating inflammatory or cancer diseases are underway.In this study, 15 compounds out of 17 isolates were isolated for the first time from


TET2 mRNA and miR-92a-3p and miR-92a-5p mature miRNAs in NHL cell lines, tumour samples, and the whole blood gDNA of an NHL case control cohort. Increased expression of both miR-92a-3p and miR-92a-5p and aberrant expression of TET2 was observed in NHL cell lines and tumour tissues, as well as disparate levels of dysfunctional promoter CGI methylation. Both miR-92a and TET2 may play a concerted role in NHL malignancy and disease pathogenesis.MicroRNAs (miRNAs) are well known for their ability to regulate the expression of specific target genes through degradation or inhibition of translation of the target mRNA. In various cancers, miRNAs regulate gene expression by altering the epigenetic status of candidate genes that are implicated in various difficult to treat haematological malignancies such as non-Hodgkin lymphoma by acting as either oncogenes or tumour suppressor genes. Cellular and circulating miRNA biomarkers could also be directly utilised as disease markers for diagnosis and monitoring of non-Hodgkin lymphoma (NHL); however, the role of DNA methylation in miRNA expression regulation in NHL requires further scientific inquiry. In this study, we investigated the methylation levels of CpGs in CpG islands spanning the promoter regions of the miR-17–92 cluster host gene and the TET2 gene and correlated them with the expression levels of  Non-Hodgkin Lymphoma (NHL) is a class of cancer that originates in the lymphatic system, caused by an over-proliferation of malignant B-cells. NHL is one of the most common cancers in the United States, and lymphoma is the fifth most common cancer in Australasia , therefoUnderstanding the cell of origin also plays a significant role in understanding NHL classification, progression, and prognosis . B-cell Dysregulation of miRNAs is a hallmark of both cancer initiation and metastasis, through the regulation of gene expression via post-transcriptional repression and mRNA degradation . It is wTET2 gene function in bone marrow cells leads to increased immature B-cells resulting in lymphomagenesis  differentiation, balanced B-cell terminal differentiation, and oxidative demethylation , with tumour tissue from 11 NHL cases  with a confirmed diagnosis of NHL (5 DLBCL and 6 FL) . A cohor2 incubator. Cell count and viability were assessed using Trypan Blue exclusion method in a 1:1 ratio of cell suspension to dye and measured on a TC10™ automated cell counter. Cells were split into a 1:2 ratio, with fresh media was added every second day until >80% confluence and 90% viability were achieved.Studies were conducted using four commercial immortalised cell lines: Raji (BL), Toledo (DLBCL), SUDLH4 (DLBCL), and Mino (Mantle cell lymphoma (MCL)). Vials were stored in liquid nitrogen and rapidly thawed and seeded in a T-75 culture flask in 25 ml RPMI-1640, supplemented with 10% FBS and 1% Penicillin-Streptomycin antibiotics. The cells were incubated at 37°C in a humidified 5% COMIR17HG) promoter region and the miR-92a to TET2 promoter was performed using MethHC 2.0 software.Computational analysis of miRNA and target gene prediction was performed using miRbase , miRTarB® SV Genomic DNA Purification System (Promega). DNA quality and quantity were quantified using a Nanodrop spectrophotometer, determined by A260/A280 ratio .gDNA was previously extracted from whole blood, collected into EDTA tubes, using an in-house salting-out method as evaluated by Chacon et al. . Both NHBisulfite conversion of 500 ng of NHL B-cell line and control PBMC DNA and 750 ng of DNA from each patient/control DNA sample and NHL tumour sample was performed using the EZ DNA Methylation™ Kit (Zymo Research) according to the manufacturer’s protocol, with a modification to the elution volume, from 10 to 40 µL.® Reagent (ThermoFisher) and the Direct-zol RNA MiniPrep Kit (Zymo Research) from cell lines, control PBMCs, and NHL tumour samples. Total RNA, including miRNA, was reverse transcribed using the MiScript II RT Kit (Qiagen) for all samples.Total RNA was extracted using TRIzol® Green PCR, Qiagen) and mRNA transcripts . Customised forward and reverse primers  were designed for the miRNAs and TET2 mRNA transcripts of interest (TET2 expression was measured under the following conditions: 50°C for 2 min (x1 cycle), 95°C for 3 min (x1 cycle), 95°C for 3 s, 60°C for 30 s (x50 cycles), melt curve analysis 60–95°C. Endogenous controls were miR103 for the miRNA assays and 18 S ribosomal RNA for the TET2 assays. Specific amplification of targets was confirmed via melt curve analysis and gene expression was calculated using the relative quantification method (ΔΔCt).Analyses were performed by real-time quantitative PCR (Q-PCR) to measure miR-92a mature miRNAs  as per the manufacturer’s instructions using PyroMark Q96 Gold reagents (Qiagen). Pyrosequencing output analysis was performed using the Qseq software .The UCSC genome browser was used to identify CGI regions in the  regions . Pyromar regions . PrimersStatistical analysis of both expression and methylation data was performed in GraphPad Prism, with a Kruskal–Wallis (KW) test of significance and a post-hoc Dunn test for comparisons of individual groups.TET2 mRNA were assayed by RT-qPCR, and upstream promoter CGI methylation for each gene was assayed by bisulfite pyrosequencing, in 4 biological replicates of NHL cell lines SU-DHL-4, Mino, Raji, and Toledo, and in PBMCs of 6 healthy controls (p = 0.0145) and miR-92a-5p expression increased in Mino (p = 0.0013), Raji (p = 0.0016), and Toledo (p = 0.0200) cells compared to controls . Differential methylation was also observed between SU-DHL-4 and Raji cells (p = 0.0237). and between Mino cells and Raji (p = 0.0029) and Toledo (p = 0.0278). Toledo (p = 0.0329) cells showed increased methylation in region 2 when compared to controls  and Toledo (p = 0.0045) cells compared to controls (p = 0.0016), Raji (p = 0.0385), and Toledo (p = 0.0004) cells. Upstream promoter hypermethylation was observed in SU-DHL-4 and Raji cell lines when compared to controls. (p < 0.0001). SU-DHL-4 cells were also significantly different to Mino and Toledo cells (p < 0.0001), and Raji cells were significantly different to Mino and Toledo cells (p < 0.0001).controls . DiffereTET2 mRNA were assayed in NHL patient tumour biopsies and compared to healthy control PBMCs by RT-qPCR (p = 0.0017), and differential methylation was observed between controls and FL tumours in region 1 of the MIR17HG upstream promoter CGI (p = 0.0203).The expression of miR-92a-3p and miR-92a-5p mature miRNAs and  RT-qPCR . PromoteTET2 mRNA expression was observed in both DLBCL (p = 0.0164) and FL (p = 0.0240) patient tumour biopsies compared to controls (p = 0.0300).Increased controls , and decMIR17HG promoter CGI was observed in the NHL cohort compared to the controls (p < 0.0001). Furthermore, differential DNA methylation was observed between healthy controls and NHL patient subtypes, with increased levels of DNA methylation observed in the FL (p = 0.0004), MCL (p = 0.0046), BL (p = 0.0062), and LBL (p = 0.0004) patient groups (MIR17HG upstream promoter CGI (p = 0.0041) and MCL (p = 0.0398) subtypes when the specific diagnosis was considered (TET2 promoter CGI methylation was observed in NHL cases compared to controls (p = 0.0159), and between healthy controls and FL subtype (p = 0.0210).DNA methylation of CpGs in each region was assayed by bisulfite pyrosequencing of whole blood gDNA from an NHL case-control cohort, comprised of 80 cases with age and sex-matched controls . Increast groups . No diffoter CGI ; howevernsidered . DecreasTET2 gene were quantified by bisulfite pyrosequencing and correlated with the expression levels of TET2 mRNA and mature miR-92a-3p and miR-92a-5p miRNAs. Gene expression was assayed in NHL B-cell lines, NHL tumour samples, and healthy PBMCs. Methylation of promoter CGIs was measured in the DNA of several NHL B-cell lines, in NHL tumour samples, in healthy PBMCs, and in whole blood gDNA of a retrospective NHL case-control cohort.In the last decade, both the role of miRNAs in malignancy and their viability as biomarkers for malignancy has been a rapidly expanding area of research. The mechanisms behind miRNA expression are not fully understood; however, aberrant methylation of promoter regions of miRNA genes has been previously implicated in aberrant miRNA expression in several cancers , includiIncreased expression of both the −3p and −5p mature miRNAs was observed in Mino cells when compared to controls, and additionally, upregulation of −5p was observed in Raji and Toledo cells lines. Previous studies have identified miR-92a overexpression in BL, MCL, and DLBCL cell lines, and our study replicates these findings . Higher MIR17HG between cell lines, and between cell lines and healthy controls; however, this difference may not be biologically significant as the difference between mean methylation is within the detection limit margin of error (MIR17HG promoter region contribute to the differential miR-92a expression between NHL cell lines. A similar trend was identified in the NHL tumour samples; however, again the difference between mean methylation is within the detection limit margin of error. Aberrant methylation of the MIR17HG promoter has been previously correlated with dysfunctional expression of miRNAs in the miR-17–92 cluster in lung biopsies of individuals diagnosed with pulmonary fibrosis (MIR17HG CGI when compared to healthy controls. Although the mean difference between the two cohorts was not substantial, several individuals in the NHL patient cohort presented high levels of genomic DNA methylation across these targeted regions. Aberrant methylation of levels were observed in individuals diagnosed with specific subtypes of NHL when compared to controls, BL being the most prominent. An aggressive subtype of NHL, the BL cell line Raji was identified to exhibit increased methylation in these same regions in the cell line populations, and previous studies have reported a similar increase in methylation levels to those identified in this study in other miRNA promoter regions, in both BL cell lines and BL tumours (MIR17HG, and a previous study identified that aberrant methylation of miRNA promoter regions in circulating B-cells of CLL patients was associated with abnormal miRNA expression when compared to healthy B-cells. Increased expression of miR-92a has been implicated in CLL malignancy, specifically in lymph node proliferation centres (Differential methylation was observed in the upstream promoter CGI of of error . Therefofibrosis , and in fibrosis . The epi tumours . Increas centres . As the TET2 was seen across NHL cell lines and malignant tissues compared to controls, with specifically increased expression of TET2 in Mino, Raji, and Toledo cell lines alongside DLBCL and FL tumour tissue compared to controls. However, when taking into account the increased miR-92a-5p expression in the cell lines, NHL tumour tissue, and increased miR-92a-3p expression in the Mino cell line compared to controls, it is suggestive that miR-92a-5p and −3p may not be strong negative regulators of TET2 within these subtypes. Further assessment of the functional role of miR-92a on TET2 expression is required, feasibly with the utilisation of miRNA mimics and inhibitors, to determine the effect of miR-92a-5p and −3p expression and inhibition in NHL cell models.The findings of this study support previous conclusions regarding the significance of miR-92a in B-cell malignancy and reinforce the importance of investigating its specific role in NHL. Several previous studies have identified miR-92a as being implicated in dysregulation and suppression of tumour suppressors, including PTEN  and PHLPTET2 upstream promotor region when compared to controls and to Mino and Raji cell lines. This novel finding correlated with expected downregulation in TET2 expression in the SU-DHL-4 cells, along with a similar trend observed in the Raji cells compared to Mino and Toledo cell lines in previous studies (TET2 mRNA generally reported increased expression in cell lines and tumours, with the exception of SU-DHL-4, when compared to controls in our findings, TET2 methylation in our target region negatively correlated with expression in the NHL cell lines, indicating that the CpGs assayed in this region may be involved in the regulation of TET2 transcription and expression. TET2 overexpression in NHL is not well explored when compared to well-documented trends of downregulation in TET2 within various haematological malignancies (TET2 over-expression in CLL as observed in our study (TET2 overexpression in cells of some aggressive and indolent NHL subtypes is an innate immune response, involving tumour suppressor and DNA repair pathways, to the specific subtype of malignancy. Examination of TET2 protein and of downstream pathways of TET2 related expression regulation may assist in determining the translational consequence of TET2 overexpression in these NHL samples and cell lines.SU-DHL-4 and Raji cell lines showed significant hypermethylation in the studied CpGs within the  studies . Althouggnancies ; howeverur study . A possiTET2 variants in DLBCL as being associated with malignancy via the hypermethylation of pro-tumorigenic genes (TET2 gene in the specific NHL cell models and tumours samples may therefore provide greater understanding into the potential presence of disease-causing TET2 mutations, which may contribute to disease phenotypes; as highlighted by differential expression DLBCL cell lines SU-DHL-4 and Toledo, both of which are DLBCL cell models. DNA methylation studies of CpGs in the TET2 upstream promotor region across both NHL tumour-derived and control PBMC samples showed consistently low levels of methylation, and a similar trend was observed in the methylation of genomic DNA in the NHL case-control cohort. Differential methylation was observed in the TET2 regulatory region between the case and control cohort; however, this difference may again not be biologically significant (Previous studies have identified ic genes . Genotypnificant . It is tTET2 and miR-92a expression were considered (TET2 expression compared to other DLBCL and FL tumour samples, as well as compared controls considered (TET2 expression in this outlier is the presence of a functional mutation in TET2. Reed-Sternberg-like cells have been previously associated with TET2 mutations (TET2 is the cause of downregulated TET2 levels, thereby further supporting previous findings regarding TET2 as a potential marker and driver of malignancy in NHL.Clinical data from specific DLBCL tumour samples presented interesting characteristics and relationships when both nsidered . One tumnsidered . Hypermeutations  along wiutations . GenotypTET2 in NHL has been previously documented; however, the mechanisms driving TET2 expression regulation, such as DNA methylation, are not well explored. We identified in this study that aberrant methylation of several CpGs in the regulatory region of TET2 correlated with TET2 mRNA expression in NHL cell models. These findings are novel and may indicate that methylation of these regions may play a functional role in TET2 expression. This same relationship was not observed in NHL tumour samples or in NHL patient genomic DNA. Although a target of miR-92a, and aberrant miR-92a-3p and miR-92a-5p expression was observed in NHL cell models and NHL tumours, an association between miR-92a and TET2 was not observed in this study. Further assessment of the functional role of miR-92a-TET2 in human cell models is required; as is, ultimately, assessment in clinical cohorts. Although TET2 may not be comprehensively regulated by miR-92a, these novel findings regarding dysregulated methylation of TET2 regulatory regions in NHL is an intriguing relationship warranting further investigation and reinforces the prominence of TET2 as an element in NHL pathogenesis and malignancy.The role of


TSC1 or TSC2 cause Tuberous Sclerosis Complex, an autosomal dominant disorder, characterized by multi-system tumor and hamartoma development. Mutation and loss of function of TSC1 and/or TSC2 also occur in a variety of sporadic cancers, and rapamycin and related drugs show highly variable treatment benefit in patients with such cancers. The TSC1 and TSC2 proteins function in a complex that inhibits mTORC1, a key regulator of cell growth, which acts to enhance anabolic biosynthetic pathways. In this study, we identified and validated five cancer cell lines with TSC1 or TSC2 mutations and performed a kinase inhibitor drug screen with 197 compounds. The five cell lines were sensitive to several mTOR inhibitors, and cell cycle kinase and HSP90 kinase inhibitors. The IC50 for Torin1 and INK128, both mTOR kinase inhibitors, was significantly increased in three TSC2 null cell lines in which TSC2 expression was restored. Rapamycin was significantly more effective than either INK128 or ganetespib (an HSP90 inhibitor) in reducing the growth of TSC2 null SNU-398 cells in a xenograft model. Combination ganetespib-rapamycin showed no significant enhancement of growth suppression over rapamycin. Hence, although HSP90 inhibitors show strong inhibition of TSC1/TSC2 null cell line growth in vitro, ganetespib showed little benefit at standard dosage in vivo. In contrast, rapamycin which showed very modest growth inhibition in vitro was the best agent for in vivo treatment, but did not cause tumor regression, only growth delay.Inactivating mutations in either  TSC1 or TSC2. Mutations in either gene cause the same phenotype, although mutations in TSC1 are associated with milder clinical severity in multiple respects [Tuberous sclerosis complex (TSC) is an autosomal dominant neurocutaneous disorder, which is caused by inactivating mutation either in respects . Althougrespects .TSC1 and TSC2 mutations also occur rarely in multiple cancer types. Cancers with higher rates of TSC1/TSC2 mutation include: urothelial carcinoma of the bladder and upper tract, with 6–10% incidence of TSC1 mutations [TSC2 and TSC1 mutations [Inactivating utations  and periThe mechanistic target of rapamycin (mTOR) is a large  protein kinase that occurs in cells in either of two complexes, mTOR complex 1 (mTORC1) and mTORC2, that have overlapping as well as distinct components. They have different roles, and mTORC1 regulates cell growth in part by enhancing anabolic biosynthetic pathways , 8.TSC1 encodes TSC1/hamartin, TSC2 encodes TSC2/tuberin, and with TBC1D7 the three proteins form the TSC protein complex [ complex . This TS complex . Loss of complex . mTORC1  complex , 12. Bot complex , 13.Rapamycin, also called Sirolimus, has antiproliferative and immunosuppressive activities. Rapamycin binds to FK-506-binding protein (FKBP12) with high affinity, and rapamycin-FKBP12 binds to mTORC1 to inhibit its kinase activity in an allosteric manner . RapamycClinically, rapamycin is FDA-approved for both prevention of allograft rejection, and for treatment of lymphangioleiomyomatosis, Drugs closely related to rapamycin are termed rapalogs, and include temsirolimus, everolimus, and deforolimus. Rapalogs have very similar if not identical activity in vivo .Heat shock protein 90 (HSP90) is an ATP-dependent molecular chaperone, which is highly expressed and helps to maintain proteostasis. HSP90 regulates the proper conformation, function and activity of multiple proteins (about 200 ‘client’ proteins) by protecting them from proteasome-mediated degradation. HSP90 expression is upregulated in many forms of cancer, and is thought to promote/enable malignant transformation, tumor progression, invasion, metastasis, and/or angiogenesis , 18.HSP90 inhibition results in proteasome-mediated degradation of protein substrates –22. Lumi2. For serum starvation, cells were incubated with standard medium without FBS for 24 hours. For serum stimulation, FBS was added back with a final concentration of 10% for 30 minutes.All cell lines were obtained from the Broad Institute of Harvard and MIT. PEER (T-cell acute lymphoblastic leukemia), SNU-878 (hepatocellular carcinoma), SNU-886 (hepatocellular carcinoma), CW-2 (large intestine adenocarcinoma), 23132/87 (stomach adenocarcinoma), MEF-319 (endometrium adenosquamous carcinoma), KM12 (large intestine adenocarcinoma), HEC-151 (endometrium adenocarcinoma), DV-90 (lung adenocarcinoma), OVK18 (ovarian endometrioid carcinoma) and HCC-95 (lung squamous cell carcinoma) were cultured in RPMI 1650 with 10% fetal bovine serum (FBS); CAL-72 (osteosarcoma) and NCI-H1651 (lung adenocarcinoma) in DMEM/F-12 with 10% FBS; MGH-U1 (urinary bladder carcinoma) and HEK-293 (embryonic kidney cells) in DMEM with 10% FBS; and SNU-398 (hepatocellular carcinoma) in RPMI 1650 GlutaMAX medium with 10% FBS. All media was supplemented with 1% penicillin- streptomycin-amphotericin B (Corning). All cell culture was done in a 37°C humidified incubator in 5% COTSC1 and TSC2 on the cell lines, where mutations were reported, were designed with Primer3 [For DNA purification Gentra Puregene Tissue Kit was used (Qiagen). Oligonucleotide primers for sequencing mutated regions of  Primer3 . FastStaLKB1, PTEN, CDKN2A, TSC1, and TSC2. 57 of 425 cancer cell lines showed some degree of reduction in signal for one more TSC1 or TSC2 probes, and were subject repeat analysis using probe sets specific for either TSC1 or TSC2, the SALSA MLPA probemix P124 TSC1, and the SALSA MLPA probemix P046-D1 TSC2, respectively. Amplification products were run on an ABI 3100Genetic Analyzer  and electrophoregrams were generated. Peak heights were loaded into a standard Excel file to determine copy number for each probe and sample pair.MLPA was performed by standard methods  using a probe set that covered five tumor suppressor genes: Cells were lysed in cell lysis buffer  with added protease and phosphatase inhibitor cocktails (Sigma-Aldrich). The protein concentration was determined using a Bradford assay (Boston BioProducts). For immunoblotting, proteins were loaded in 4–12% gradient NuPAGE bis-tris gels (Life Technologies) or home-made 15% polyacrylamide gels, separated by SDS–PAGE and transferred to PVDF membranes. For detection the following primary antibodies were used: mTOR , TSC2, C-20 , TSC1 , pAKT-Ser473 , AKT1, C-20 , pS6K-Thr389 , S6 kinase, C-18 , pS6-Ser235/236 , pS6-Ser240/244 , GAPDH , 4E-BP1 , p4E-BP1-Ser65, , p4E-BP1-Thr37/T46 , peIF2α-Ser51 , BiP , cleaved caspase-3  and β-Actin .Secondary antibodies were anti-mouse, anti-rabbit, and anti-goat (Santa Cruz Biotechnology) conjugated to horseradish peroxidase, and were used at 1:3000 dilution. Immunoreactive bands were detected by chemiluminescence  using the G:Box:iChemiXT imager (Syngene).50 values were calculated using Graphpad Prism as the drug concentration that reduced cell viability by 50% compared to untreated cells. Rapamycin was purchased from LC laboratories and ganetespib from Synta Pharmaceuticals.The cell lines SNU-878, SNU-886, SNU-398, CAL-72, and PEER were screened for kinase inhibitors using a kinase inhibitor-focused library (LINCS). The LINCS library contained 197 kinase inhibitors, from a diverse ATP competitive kinase inhibitor set. These kinase inhibitors were shown to be relatively potent and selective towards a narrow range of targets. 2000 adherent cells and 3000 suspension cells were plated with 50 μL medium in each well of a 384 microplate. Drugs with a concentration of 660 nM were added the same day. After 48 hours cellular proliferation was determined using CellTiter-Glo (Promega). Drugs identified as having a significant effect on this screen were tested in greater detail. 2000 adherent or 4000 suspension cells were plated on day 0 on a 96 well plate with 100 μL medium, except on the marginal wells. Drugs were added on day 1, which were serially diluted three-fold from 10 μM to 1.5 nM. Cell viability was determined after 72 hours of treatment by adding 20 μL CellTiter-Glo. Cell viability was determined using XLfit4.0 software. ICTo add back TSC2 to cells lacking a functional TSC2 expression , pMSCV and pMSCV-TSC2 (containing the TSC2 cDNA) plasmids were used. HEK293T cells were transfected with pCL Ampho retrovirus packaging vector (Imgenex) and pMSCV-EV (empty vector) or pMSCV-TSC2, with selection to generate retrovirus. Retrovirus was then added to each of the SNU cell lines. Neomycin was used as a selection antibiotic.All procedures were carried out according to the Guide for the Humane Use and Care of Laboratory Animals, and the experiments were approved by the Animal Care and Use Committee of the Children’s Hospital, Boston, MA, USA (protocol reference number: 1589).Igh-1b/IcrTac-Prkdcscid) mice were used to generate xenograft tumors. Mice were purchased at the age of 3–4 weeks from Taconic, Germantown, NY, USA. Mice were 6 to 8 weeks old and their weight was between 17 to 25 g when 3 x 106 tumor cells of the cancer cell line SNU-398 in 100 μL medium mixed with 100 μL matrigel were injected subcutaneously in both back flank regions.Immunodeficient strain CB17SC-M scid . For drug administration, INK128 powder was dissolved daily in vehicle ; and was administered by gavage 5 days/week at a dose of 1mg/kg and a volume of 100 μL. Rapamycin was prepared as a 20 mg/mL stock solution using 100% ethanol, and was mixed daily with sterile vehicle /80% dextrose (D5W). Ganetespib was administered by tail vein injection once/week in a dose of 50 mg/kg. Control mice received vehicle treatment for one of these three drugs.Xenograft tumor nodules were followed 3x weekly, and when they reached a minimum diameter of 4mm (14–30 days after injection), drug treatments were initiated. Mice were treated either with INK128 (Intellikine Inc.), rapamycin (LC laboratories), or ganetespib ; or vehicle; or a combination of ganetespib and INK128, or ganetespib and rapamycin. Bodyweight was measured at least 3 times a week. Tumor size was measured with an electronic digital caliper in 2 dimensions . For IHC tumors were fixed with 10% formalin overnight and stored in 70% ethanol solution until paraffin embedding. Paraffin-embedded sections, both unstained and stained (hematoxylin and eosin stain), were prepared by the Rodent Histopathology Core, Harvard Medical School.For IHC, slides were rinsed with a series of xylene, ethanol, 95% ethanol, 80% ethanol, and PBS. To unmask the epitopes slides were boiled in 10 mM sodium citrate for 60 minutes and cooled to room temperature. Slides were washed with PBS. Slides were put into peroxidase blocking reagent  for 10 minutes, and washed with PBS. Afterward, slides were washed with distilled water, sections were counterstained with hematoxylin and washed under running water.Cell proliferation was assessed using an antibody against PCNA and the HistoMouse kit- plus kit (Invitrogen/Thermo Fisher Scientific), using AEC single solution chromogen, and then counterstained with hematoxylin. Sections were mounted with cover glass and Fluoromount G media.For TSC2 and pS6 IHC primary antibodies against pS6 (Ser235/236)  at a dilution of 1:250 and TSC2  at a dilution of 1:100 were used and incubated at 4°C overnight, rinsed, and then treated with anti-rabbit-HRP secondary antibody at room temperature for 60 minutes, followed by rinsing and slide preparation as above.To identify apoptotic cells, ApopTag plus peroxidase in situ apoptosis detection kit (Merck Millipore) was used according to the manufacturer’s instructions.The quantitative data of the xenograft tumor experiments are reported as the mean ± standard deviation for at least 5 tumors. Tumor sizes for the different treatment groups were compared using the Wilcoxon Rank Sum test using GraphPad Prism software. P-values less than 0.05 were considered statistically significant.TSC1/TSC2 are known recessive oncogenes whose loss is known to activate mTORC1. Here we sought to identify and characterize cancer cell lines with complete loss of either TSC1 or TSC2, and then to examine their sensitivity to a broad panel of kinase inhibitors, with a goal to identify additional inhibitors beyond rapalogs.TSC1 or TSC2. Among 1457 cell lines, 49 were reported to have mutations in TSC1 and 111 to have mutations in TSC2. These mutations were reviewed to identify those with nonsense mutations, frameshift deletions or insertions, or in-frame deletions in either TSC1 or TSC2, yielding 4 cell lines with probable mutations in TSC1 and 10 with probable mutations in TSC2 (Data from the Broad Institute Cancer Cell Line Encyclopedia (CCLE)  was revi in TSC2 .S1 Fig inTSC1; SNU-878 and SNU-886 showed homozygous nonsense mutations in TSC2. All other cell lines had mutations with allele ratios around 0.5.All of these cell lines were obtained, and mutations were confirmed by Sanger sequencing  and pS6 (Ser240/244) expression in serum starvation, in PEER, MFE-319, OVK18, SNU-886, and SNU-887 cells. GAPDH was used as a loading control.TSC1 and TSC2 genes by the MLPA method , in serum absence and serum add back conditions . This haVariable 4E-BP1 expression was seen among these cell lines, but all TSC1 or TSC2 deficient cell lines showed similar levels of expression of p4E-BP1-Thr37/Thr46 in serum-deprived and serum add back conditions. Expression of p-eIF2α (Ser51) was also higher in TSC1 or TSC2 deficient cells in comparison to the control cell line .The cell lines SNU-878, SNU-886, SNU-398, CAL-72, and PEER were screened for sensitivity to kinase inhibition using a kinase inhibitor-focused library (LINCS), which contained 197 selective kinase inhibitors . This liA wide variety of inhibitors showed a positive signal in this initial assay, including multiple mTOR, CDK, PLK, CHK, Aurora, and HSP90 inhibitors .S2 Fig inS3-S7 Figs inS3-S7 Figs inCandidate inhibitors were studied against most or all of the TSC1/TSC2-deficient cell lines in standard 96 well plate growth assays over a dose range of 1 nM to 10μM . We focu50 values ranging from 68 to 179 nM  levels were decreased in the absence of serum conditions in the TSC2 add back cells, indicating return to normal regulation of mTORC1. pAKT (Ser473) expression was quite low in the TSC2 add back cells, but increased significantly in response to serum stimulation in the cell lines SNU-886 and SNU-878, compared to the levels of native or empty vector-transfected cells. SNU-398 did not show a pAKT (Ser473) expression increase in TSC2 add back cells .S5 and S7 Figs inS3, S4 and S6 Figs inS2, S8-S12, S15 and S17-S21 Figs inThese three TSC2 add back cell lines were used in drug testing experiments similar to what was done above. Interestingly, TSC2 add back cells had a 3 to 6-fold higher IC50 for both Torin1 and INK 128 than their empty vector controls or original unmanipulated cells  and pS6 S240/244) to a major degree in the absence of serum, as well as with serum stimulation . These f to a majGanetespib at lower doses, up to 10 nM, had little or no effect on expression of key mTOR signaling proteins, or activation of mTORC1, as assessed by pS6 (Ser240/244) levels . At 100 We also examined the effects of ganetespib on phosphorylation of 4E-BP1 and induction of apoptosis as assessed by cleaved caspase 3 levels  Fig 7)..Fig 7). The observation that there was little effect on mTORC1 signaling or protein expression in any of the cell lines near the IC50 dose of ganetespib (3 to 35nM) suggests that the growth inhibition effects were being mediated by other client proteins of HSP90 whose expression was likely reduced to some extent at doses near 10nM, and likely a collective effect on multiple HSP90 client proteins.6 SNU-398 cells were injected subcutaneously into the flank region of immunodeficient CB17SC-M (C.B-Igh-1b/IcrTac-Prkdcscid) mice. After approximately 10 days palpable and measurable tumors with a diameter of 3–5 mm were noted. Mice were treated with rapamycin, INK 128, or ganetespib, using doses described previously as being the maximal tolerated dose, in studies by ourselves and others [Given the evidence of some synergy in the growth inhibition of the TSC1/TSC2 null cell lines in response to combined mTOR and HSP90 inhibition, and evidence that they were impacting growth through different mechanisms, we explored the potential synergistic effect of treatment with these compounds in vivo using a subcutaneous xenograft tumor model with SNU-398 cells. 3.0x10d others , 46.3, after a mean (SD) of 18 (4.8) days after tumor cell injection. Treatment with the individual drugs was assigned randomly.Mice were treated when tumors had a mean (SD) diameter of 7.8 (1.7) mm and a mean (SD) volume of 186 (110) mmS22a-S22d Fig inGrowth of xenograft tumor nodules was reduced by each of the three drugs, with ganetespib, 50 mg/kg by tail vein injection 1 time/week, having the least effect; and rapamycin, 3mg/kg given intra-peritoneally 3 days per week, having the greatest effect; and INK 128, 1 mg/kg by gavage 5 days/week, being intermediate , and immunoblotting of tumor lysates. Histologically, the tumors were not homogeneous, with necrotic areas centrally and high proliferation in the periphery of nodules, regardless of treatment, as is commonly seen in xenograft models.IHC against proliferating cell nuclear antigen (PCNA) was used to assess proliferation , and theIHC showed no TSC2 expression in the tumors. Vehicle and ganetespib-treated tumors were pS6 (Ser235/236)+. In contrast rapamycin-treated tumors had much reduced pS6 (Ser235/236) expression, and this was also lower in INK 128-treated tumors, in comparison to the tumors treated with ganetespib or vehicle .Immunoblots of harvested tumors, carefully dissected for viable and not necrotic regions, and livers, of mice treated with vehicle, rapamycin, INK 128, ganetespib, and the combinations were performed to examine the effect of the drugs on protein expression and the mTORC1 pathway in vivo. None of the treated livers showed major changes in expression of AKT or S6K . SimilarThe current and accelerating trend in cancer therapy is the use of personalized  cancer treatment. Such targeted therapies may be directed at genetic mutations which are driver events in cancer; or at expressed proteins, such as the estrogen receptor in breast cancer .TSC1 or TSC2 from this resource. From a large set of candidate cell lines, subjected to sequencing and immunoblotting, we identified 5 cell lines with a total loss of either TSC1 or TSC2, and which also showed constitutively upregulated mTORC1 signaling, as indicated by high expression of pS6K (Thr389) and pS6 (Ser240/244) in the absence of serum. Two other cell lines, MFE-319 and OVK18, also showed constitutively upregulated mTORC1 signaling as well as upregulated pAKT (Ser473) expression, likely due to loss of PTEN by mutation [One resource that has been developed for a better understanding of cancer development, and to investigate potential targeted therapies are cancer cell lines, including the Cancer Cell Line Encyclopedia of 947 human cancer cell lines . In thismutation .The mTORC1 signaling pathway is one of the main regulators of cell growth, which acts by enhancing anabolic biosynthetic pathways in both normal and cancer cells. The mTOR signaling pathway is involved in many important processes including proliferation, autophagy, protein and lipid synthesis , 51, lysmTOR is an atypical serine/threonine kinase, and forms the key component of two protein complexes, mTORC1 and mTORC2. The TSC1 and TSC2 proteins function in a complex with a third component, TBC1D7 , as one TSC1 or TSC2 in the germ line cause Tuberous Sclerosis Complex, an autosomal dominant neurocutaneous disorder, characterized by skin lesions, neuropsychiatric disorders and mainly benign tumors of the brain, kidney, lung, heart, and skin [TSC1 and/or TSC2 have also been shown to occur consistently in a variety of sporadic cancers including bladder, kidney, pancreatic neuroendocrine tumors, and PEComa. More rarely TSC1/TSC2 mutations have been identified in many other cancer types, though whether they are important driver events or passenger mutations is not clear in many instances.Inactivating mutations in either and skin . MutatioTSC1 or TSC2, we performed drug screen using 197 kinase inhibitors. The cell lines were sensitive to several mTOR inhibitors, but also HSP90 and cell cycle inhibitors. In addition, PEER cells were sensitive to Aurora kinase inhibitors.Following our identification of five cell lines, PEER, CAL-72, SNU-886, SNU-878, and SNU-398, with bi-allelic mutation and loss of either We showed that the sensitivity of these cell lines to mTOR inhibitors like Torin1 and INK 128 correlated strongly with the deficiency of TSC2 and could be reversed by add back of TSC2 expression. However, we did not see this correlation with WYE-125132, AZD8055, and Torin2, which may reflect the activities of these compounds on kinases other than mTOR.Various rapalogs have been approved for treatment of various cancers, but complete responses are rare, and the PR rate is typically 5–10% –57. In aOne potential reason for the limited therapeutic benefit of rapalogs, is their lack of effect on many mTORC1 phosphorylation targets, including 4E-BP1 , 61. In Potentially, newer ATP kinase pocket directed mTOR inhibitors could or should be more effective in controlling the growth of cells and tumors with TSC1/TSC2 inactivation. Torin1, for example, showed complete inhibition of phosphorylated 4E-BP1 and AKT. However, an apoptotic effect was still lacking. In addition, in our xenograft assay, rapamycin was more effective than the dual mTORC1/2 inhibitor INK 128 in inhibition of growth of the TSC2 null S-398 cell line. However, this may reflect the consequence of dual mTORC1 and mTORC2 inhibition by INK 128, leading to a lower tolerable dose, and less effective suppression of each of mTORC1 and mTORC2. Other off-target effects of this drug may also contribute to in vivo toxicity, limiting effective targeting of mTOR.The HSP90 inhibitors ganetespib and NVP-AUY922 both showed strong inhibition of growth of all five TSC1/TSC2 null cell lines in vitro, with IC50’s in the range of 2 to 35 nM. Ganetespib was studied in greater detail. Ganetespib reduced mTOR, AKT, and S6K, as well as pS6, levels in all cell lines, but at 100 nM, a dose higher than the IC50 dose. These results suggest that ganetespib’s effect was not due directly to effects on mTOR signaling, but rather likely to broad effects in lowering expression of HSP90’s client proteins.Based on these findings of growth inhibition by ganetespib in vitro, we performed tumor xenograft experiments using the TSC2 null SNU-398 cell line. This cell line showed robust growth in immunodeficient CB17SC-M scid mice as subcutaneous xenografts. All three of rapamycin, INK 128, and ganetespib, when given at previously determined doses near the maximally tolerated dose, showed a significant effect on the growth of the SNU-398 xenografts . RapamycNext, we examined the potential of combination therapy using ganetespib with each of the other two drugs. Ganetespib combined with INK 128 showed greater reduction in tumor growth than either drug alone, although this did not quite meet statistical significance . In contTSC1 or TSC2. Blenis and colleagues found that the combination of glutaminase (GLS) and HSP90 inhibition selectively triggered the death of TSC1/TSC2 deficient cells [Multiple past studies have examined the interaction between mTOR signaling and HSP90, and the potential benefit of HSP90 inhibition on growth of cell lines lacking nt cells , likely nt cells . They thnt cells .In addition, the combination of rapamycin and HSP90 inhibitors has been previously studied for treatment of hepatocellular cancer (HCC) . Lang ethttps://www.clinicaltrials.gov/).Numerous inhibitors of HSP90 have been developed as potential anticancer drugs. Many such drugs, including ganetespib, have been evaluated in clinical trials, but none have been approved by the FDA, due to lack of benefit. The main reason for the limited efficacy appears to be that at effective doses of HSP90 inhibition, there is release of the HSF1 transcription factor from HSP90, which enters the nucleus leading to a prosurvival heat shock response . NonetheTSC1 or TSC2, and consequent constitutive mTORC1 activation. Through our kinase inhibitor screen, we identified a number of inhibitors that have some activity on these cell lines, including cell cycle kinase and HSP90 kinase inhibitors. In vitro, the HSP90 inhibitors NVP-AUY922 and ganetespib had major inhibitory effects on the growth of all five lines in the nanomolar range. However, this appeared to be due to global effects on HSP90 client protein expression, and not a specific effect on components of the mTOR signaling pathway. In vivo analysis of an HCC subcutaneous xenograft model using the TSC2 null SNU-398 cell line showed that ganetespib at usual doses had minimal effects on tumor growth both alone and in combination with rapamycin and INK 128. Rapamycin showed activity superior to that of INK 128 in vivo.In conclusion, we have identified and validated five cancer cell lines that have complete loss of either S1 Raw images(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


Recent research has suggested that the endocannabinoid system offers several pharmacotherapeutic targets for drug administration as new options for the treatment and prophylaxis of skin cancer. This review focused on the anticarcinogenic mechanisms of cannabinoids at the different levels of skin cancer progression, such as inhibition of tumour growth, proliferation, invasion and angiogenesis, as well as inducing apoptosis and autophagy.Drugs targeting the endocannabinoid system are of interest as potential systemic chemotherapeutic treatments and for palliative care in cancer. In this context, cannabinoid compounds have been successfully tested as a systemic therapeutic option in preclinical models over the past decades. Recent findings have suggested an essential function of the endocannabinoid system in the homeostasis of various skin functions and indicated that cannabinoids could also be considered for the treatment and prophylaxis of tumour diseases of the skin. Cannabinoids have been shown to exert their anticarcinogenic effects at different levels of skin cancer progression, such as inhibition of tumour growth, proliferation, invasion and angiogenesis, as well as inducing apoptosis and autophagy. This review provides an insight into the current literature on cannabinoid compounds as potential pharmaceuticals for the treatment of melanoma and squamous cell carcinoma. Skin cancer essentially comprises three tumour categories, namely, cutaneous melanoma, basal cell carcinoma and squamous cell carcinoma. The latter two are grouped under the term non-melanoma skin cancer, which also includes other primary skin neoplasms such as cutaneous lymphoma, adnexal carcinoma, dermatofibrosarcoma protuberans and Merkel cell carcinoma. Therefore, “keratinocyte carcinoma” is the preferred term, combining basal cell carcinomas and cutaneous squamous cell carcinomas because of their common descent from epidermal keratinocytes . Basal cRegarding some previous studies, the treatment of cutaneous melanoma with classical chemotherapeutic agents such as the Dartmouth regimen   and withBRAF) is found in half of all cases of skin tumours, whereas mutations in the neuroblastoma RAS (rat sarcoma) viral oncogene homologue (NRAS) occur in 21% GTPγS binding assays [As early as 1984, Allyn C. Howlett demonstrated that Δactivity . The rec and CB2 ,32. Witht of THC ,34. Furtactivity . A studyreceptor . On the receptor ,36. Anotreceptor . The aut agonist . Accordima cells . Finallyg assays .i values, while the non-psychoactive phytocannabioid cannabidiol (CBD) has only weak cannabinoid receptor affinity with Ki values in the micromolar range . Competectively . In anotagonists . In a ree of THC . This coe of THC ).1 and CB2 receptors [1 and weak CB2 receptor agonist properties [The extended endocannabinoid system likewise includes other receptors modulated by cannabinoids, such as transient receptor potential vanilloid 1 (TRPV1), which is activated by AEA  and CBD eceptors . Moreoveeceptors . It was operties , acts asoperties . From thoperties .50 values for AEA, 2-AGE, 2-AG, PEA, OEA, abnormal CBD (synthetic regioisomer of CBD) and O-1602  were registered that were lower than the corresponding EC50 values in CB1 and CB2 activity assays, while for CBD an antagonistic effect at GPR55 was demonstrated [q and G12 proteins. The human G protein-coupled receptor 55 (GPR55), which was first isolated and cloned in 1999, is also activated by various cannabinoids in a cell-type- and tissue-dependent manner. Only two studies on this topic are outlined here. In GPR55-transfected cells, ECnstrated . In anotFinally, the proliferator-activated receptor (PPAR) α was reported to be activated by different endocannabinoid-like substances. In this context, OEA  and PEA Among the cannabinoid compounds, ajulemic acid, a synthetic analogue of the THC metabolite THC-11-oic acid, was first found to bind directly to PPARγ and activate its transcriptional activity . Moreove2 ethanolamide [2 glycerol ester [2 glycerol ester is subsequently converted to PGE2 glycerol ester by PGE synthase and to PGD2 glycerol ester by PGD synthase. Later, the same group discovered that AEA and 2-AG can be further metabolised by thromboxane and prostacyclin (PGI) synthase to thromboxane and PGI ethanolamides and glycerol esters, respectively [The synthesis of AEA is mainly carried out by N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD). 2-AG, on the other hand, is synthesised by phospholipase C or by diacylglycerol lipase α and β . The denolamide . Regardiol ester . Using cectively . AccordiThe presence of the endocannabinoid system in skin tissue was demonstrated in the mid-1990s by the discovery of the endocannabinoid AEA in rat skin, where it was found in concentrations similar to those in the rat brain . Later, 1/CB2 receptor double knockout mice showed increased allergic inflammation, while conversely, FAAH-deficient mice with correspondingly higher AEA levels exhibited fewer allergic skin reactions. In line with this, numerous studies on the role of endocannabinoids in various skin functions have led to these compounds now being considered important neuroendocrine regulators in the maintenance of skin homeostasis [The great importance of cannabinoid receptors for the homeostasis of the skin was clarified in a comprehensive study that demonstrated an involvement of these receptors in the pathogenesis of allergic contact dermatitis . The auteostasis . Accordieostasis , regenereostasis ,74 and meostasis . Current2 receptor is upregulated in melanoma tissue [1 receptor-positive squamous cell carcinomas of the oesophagus were associated with poorer patient survival compared with patients with CB1 receptor-negative tissue [2 receptor immunoreactivity in biopsies from patients with squamous cell carcinoma of the head and neck was significantly associated with a reduced disease-specific survival rate [2 receptor expression and concurrent CB1/CB2 receptor expression occurring more frequently in female than male patients. An increase in cannabinoid receptors was also observed more frequently in older than in younger patients. Therefore, it is difficult to make a statement about whether the regulation of cannabinoid receptors in tumour tissue in melanoma or squamous cell carcinoma is predictive of disease progression or even of the functional role of these receptors in the pathogenesis of these cancers.Recently, several studies have investigated possible regulations of cannabinoid receptors and other elements of the endocannabinoid system in skin cancer tissues, as well as a possible link between these factors and skin tissue malignancy. In this context, one study showed that the CBa tissue . Other ee tissue . Anotherval rate . In contval rate . This stRegarding the regulation of other elements of the endocannabinoid system in skin cancer, one study found upregulation of MAGL in melanoma tissue, with increased expression associated with higher tumour aggressiveness .1 and CB2 receptors. In 2006, Blázquez et al. showed that activation of CB1 and CB2 leads to a reduction in growth, proliferation, and metastasis of melanoma cells, as well as neovascularisation of melanoma xenografts [1 and CB2 antagonists. A similar decrease in viability by cannabinoids could not be detected in non-transformed melanocytic cell lines. The authors also reported a pronounced inhibitory effect of WIN 55,212-2 or JWH-133 at 50 µg per day on the growth of xenografts generated by injection of B16 mouse melanoma cells into C57BL/6 mice. In another work, WIN 55,212-2 was shown to have an antiproliferative effect on the metastatic skin melanoma cell line SK-MEL28 and the non-metastatic ocular uveal melanoma cell line OCM-1 via a receptor-independent pathway involving lipid rafts [1 and CB2 antagonists did not rescue melanoma cells from cannabinoid-induced cell death, whereas disruption of lipid rafts by methyl-β-cyclodextrin protected cancer cells. A recent study tested the effect of CBD on malignant melanoma cell growth and metastasis in a mouse model in which B16F10 mouse melanoma cells were injected into C57BL/6 mice [There have been numerous investigations into the antiproliferative and proapoptotic effects of cannabinoids on human melanoma cancer cells expressing CBnografts . Part ofnografts . In addiid rafts . Here, CL/6 mice . CompareL/6 mice . A furthL/6 mice . HoweverCannabis sativa extract alone or in combination with a single dose of radiation significantly inhibited melanoma cell viability and proliferation in vitro in a concentration-dependent manner. Remarkably, this inhibition of melanoma cell viability was accompanied by an increase in necrosis but not apoptosis [As for combinations with radiation treatments, a standardised poptosis .1 receptors partially reduced the cytotoxicity of AEA. In contrast, methyl-β-cyclodextrin, tested at a concentration sufficient to destroy membrane lipid rafts without affecting viability, completely reversed cytotoxicity by AEA and by the GPR55 agonist O-1602, suggesting a possible role of lipid rafts and GRP55 in the viability-impairing effect of AEA [A number of publications further found endocannabinoids and inhibitors of endocannabinoid-degrading enzymes to influence melanoma growth. Accordingly, antitumour activity of AEA on A375 melanoma cells was reported in a study showing concentration-dependent cytotoxicity of AEA in association with a caspase-dependent apoptotic signalling pathway . Here, ct of AEA . Anothert of AEA . The stut of AEA . In addition, MAGL has been shown to be a factor that decisively influences the malignancy of melanoma cells. One study showed that the melanoma cell line C8161, in which MAGL was permanently downregulated by transfection with a small hairpin (sh) RNA, exhibited reduced viability. Interestingly, the authors could not confirm inhibition of cell survival when using the MAGL inhibitor JZL184. However, both MAGL shRNA and JZL184 inhibited xenograft growth in vivo, with the effect of MAGL shRNA being abolished by a high-fat diet . Consist2 ethanolamide [12,14-PGJ2 ethanolamide [Finally, PGDnolamide  and 15-dnolamide , both prnolamide .1 agonist AM-251 has been associated in vitro with arrest of the G2-M cell cycle, inhibition of the expression of anti-apoptotic proteins such as B-cell lymphoma 2 (BCL2) and survivin, and increased expression of the proapoptotic Bcl-2-associated X protein (BAX) [i/o protein-coupled CB1 receptor, a 40% increase in basal cAMP levels was observed after treatment of A375 melanoma cells with AM-251, although its causal involvement in cell cycle arrest was not further investigated [1 using shRNAs, led to a slowing in the cell cycle in the G1-S phase, which was accompanied by reduced activation of protein kinase B (Akt) and ERK, suggesting a possible function of the CB1 receptor as a tumour-promoting signal in human skin melanomas [1 receptor antagonist/inverse agonist AM-251.It is also noteworthy that cannabinoid receptor activation is not a process that has been uniformly described as growth inhibitory. For example, in A375 melanoma cells, the inverse CBin (BAX) . Consiststigated . In anotelanomas . In a fuelanomas . Paradox1 agonist ACEA and 2-AG, induced melanogenesis via CB1 activation up to a concentration of 3 µM, whereas apoptosis of primary normal human epidermal melanocytes occurred via activation of TRPV1 at high concentrations of Met-AEA (≥4 µM) [It can be assumed that the mode of action of cannabinoids under certain conditions can induce concentration-dependent opposite effects with low, proliferatively effective concentrations and high antiproliferative and proapoptotic concentrations. Thus, Met-AEA and other cannabinoids, such as AEA, the CB (≥4 µM) .2 agonist JWH-133 induce apoptosis in a mouse spindle cell squamous cell carcinoma cell line (PDVC 57B cells) in vitro and reduce tumour growth in a murine xenograft model represents the first ever report of anticarcinogenic cannabinoid effects in skin cancer.As for melanomas, there are several studies on possible antiproliferative and proapoptotic effects of (endo)cannabinoids on squamous cell carcinomas. In this context, the finding published by Casanova et al. in 2003  that the2 and PGD2-ethanolamide as well as PGJ2 and 15-deoxy-Δ12,14-PGJ2 [2 ethanolamide, Δ12-PGJ2 ethanolamide and 15-deoxy-Δ12,14-PGJ2 ethanolamide, metabolites of AEA formed via COX-2 that cause stress in the endoplasmic reticulum, finally leading to apoptotic cell death [2 ethanolamide leads to apoptotic cell death of skin cancer cells via both oxidative and endoplasmic reticulum stress [It was later shown that cannabinoids in squamous cell carcinomas lead to the death of the cancer cells not only through their effect on the cannabinoid receptors, but also through other mechanisms of action. In this context, the fact that most non-melanoma skin cancers as well as other epithelial tumours overexpress COX-2 plays an important role. According to van Dross et al., treatment with AEA in the murine COX-2-overexpressing keratinocyte squamous cell carcinoma cell line JWF2 led to cell death due to the metabolism of AEA to cytotoxic PGD,14-PGJ2 . The aut,14-PGJ2 . Further,14-PGJ2 . This wall death . Finallym stress . At the 1 receptor and TRPV1 activation and instead partially abrogated by antioxidants and 5-LOX inhibition. Further experiments showed that in addition to the growth inhibitory effects, the ROS-increasing and Akt phosphorylation-inhibiting effects of the PUFAs tested were reduced by inhibition of 5-LOX, suggesting that ROS generated by the 5-LOX pathway mediate the anticancer effects of DHEA and NALA [Furthermore, AEA, but not 2-AG, was found to exert an anticancer effect on head and neck squamous cell carcinoma cell lines  via a receptor-independent mechanism mediated by an increase in ROS . It is nand NALA . Regarding the anticancer effects of phytocannabinoids, CBD was demonstrated to significantly reduce cell viability of the human tongue carcinoma cell line SCC-25 and induce apoptosis . In thisTo determine the contribution of GPR55 as another cannabinoid-driven target to squamous cell carcinoma carcinogenesis, a study was conducted using GPR55-deficient mice. Here, GPR55 was shown to increase the number of papillomas and carcinomas in a mouse skin carcinogenesis model. Using the murine transformed mouse keratinocyte cell line PDV, the authors also found that cells with stable suppression of GPR55 had lower viability, colony formation and invasion in vitro, and lower xenograft tumour growth in vivo . However1 agonist ACEA, the CB2 agonist HU308 and THC were found to increase proliferation in three out of five different HPV-positive head and neck squamous cell carcinoma cell lines at concentrations up to 1 µM after an incubation period of three days. ACEA and HU308 left proliferation virtually unchanged in the other two cell lines, while THC inhibited proliferation in one cell line and had no effect in another cell line. ACEA and HU308, at concentrations of 10 µM each, resulted in additional inhibition of cell growth in another of the five cell lines (UM-SCC-47). The selective CB1 receptor antagonist rimonabant and the CB2 receptor antagonist SR144528 each inhibited proliferation in three of these five cell lines and had no effect on cell growth in the other two cell lines. The authors also confirmed the trend of these results in vivo with rimonabant, SR144528 and suppression of CB1 and CB2 by shRNA transfection, each of which reduced the growth of xenografts of UD-SCC-2 cells in nude mice. The administration of THC accelerated tumour growth accordingly [1 receptor expression and the degree of metastasis to lymph nodes. Consistent with this notion, the authors found that the inverse CB1 agonist AM-251 significantly suppressed the proliferation of several oesophageal cancer cell lines [As with the effects of cannabinoid receptor-associated signalling pathways in melanoma cells, antagonisation rather than activation of cannabinoid receptors has been shown to mediate anticarcinogenic effects in squamous cell carcinoma cells. Accordingly, using the human papillomavirus (HPV)-positive squamous cell carcinoma cell lines UD-SCC-2, UPCI-SCC-090, UM-SCC-47 and 93VU147T, it has been reported that cannabinoid receptor silencing inhibits the proliferation of these cancer cells . In the ordingly . Hijiya ll lines .2 receptor antagonist [Finally, it should be mentioned that with regard to cannabinoid effects, other skin cancer types have been investigated in addition to melanoma and squamous cell carcinoma cells. For example, cannabinoids have been successfully tested preclinically for their anticarcinogenic effects on cells of Kaposi’s sarcoma, a tumour of endothelial origin with multiple lesions of the skin, the etiological agent of which is a Kaposi’s sarcoma-associated herpes virus (KSHV), also known as human herpesvirus 8 (HHV-8). In this context, CBD was shown to inhibit proliferation and induce apoptosis in KSHV-infected human dermal microvascular endothelial cells . The opptagonist . In a retagonist . TherebyBRAF wild-type melanoma xenografts generated with CHL-1 melanoma cells or treatment with a combination of THC and CBD resulted in a stronger reduction in cancer growth than treatment with temozolomide [As a further mode of action, treatment of melanoma cells with cannabinoids resulted in loss of cell viability due to activation of autophagy in several publications. The induction of autophagy in the melanoma cell lines HL-1, A375 and SK-MEL-28 after treatment with THC and the resulting apoptosis was prevented by simultaneous treatment with chloroquine, a classical inhibitor of autophagy that acts by changing the acidic content of the lysosomes . THC-indzolomide .Triggering of autophagy processes by cannabinoids in squamous cell carcinoma cells was demonstrated only in those of the head and neck treated with CBD . In the Tumours require an adequate supply of oxygen and nutrients to grow. To meet this need, tumours are able to promote the formation of new blood vessels, a condition that represents a potential target for therapeutic agents. In xenografts of the epidermal tumour cell line PDV C57B, derived from spindle cell squamous cell carcinoma of mouse skin, Casanova et al. found an impairment of tumour vascularisation and decreases in several growth factors associated with angiogenesis, such as vascular epithelial growth factor (VEGF), placenta-derived growth factor (PlGF) and angiopoietin-2, after treatment of nude mice with WIN 55,212-2 and JWH-133. In addition, both cannabinoids reduced epidermal growth factor (EGF) receptor activation in tissues derived from PDV C57B xenografts. . An anti2 receptor with JWH-133 was shown to reduce melanoma cell attachment to brain endothelial cells and transendothelial migration of melanoma cells [2 receptor can reduce the transmigration of melanoma cells across the blood–brain barrier in the context of the formation of parenchymal brain metastases. Further in vitro work revealed that migration of B16F10 melanoma cells was reduced by treatment with a standardised cannabis extract [Several publications have indicated that cannabinoids inhibit melanoma cell motility, invasion, and metastasis. In this context, activation of the CBma cells . These i extract .In vivo studies in mice found that treatment with WIN 55,212-2 reduced the number of metastatic nodules in the lungs and livers of immunocompetent C57BL/6 and immunodeficient nude mice injected with B16 melanoma cells . In anot1 or CB2 receptor antagonists, restored migration suppressed by MAGL shRNA, suggesting that the mechanism of MAGL-mediated cancer aggressiveness in these cells is mediated by free fatty acids rather than by reduction in the MAGL substrate 2-AG. Consistent with this, the addition of free fatty acids also abolished the migration inhibition induced by JZL184 in MAGL-overexpressing MUM2C melanoma cells [The results of another study suggested that MAGL may play an important role in the malignancy of melanomas . The melma cells .1 receptor agonist AM-251 inhibited the invasion of the oesophageal cancer cell lines TE-1, TE-8 and T-Tn [1 antagonist rimonabant and the CB2 antagonist SR144528 at 1 µM each resulted in inhibition of migration of UD-SCC-2, UM-SCC-47 (rimonabant only), UPCI-SCC-090 (SR144528 only) and 93VU147T cells [Data on a possible impact of cannabinoids on squamous cell carcinoma migration, invasion, and metastasis are limited to a few studies. In one study, CBD was reported to reduce invasion of squamous cell carcinoma of the oral tongue cell line SCC15 in a concentration-dependent manner at low micromolar concentrations (4–8 µM) . Remarkaand T-Tn . Consist7T cells . It is known that cannabinoid compounds influence the functional activities of immune cells. Therefore, the role of cannabinoids in the immune defence of tumours has been critically discussed. About 30 years ago, it was already proven that THC and 11-hydroxy-THC suppress the cytolytic activity of cultured natural killer cells ,115. LatR24C mice, with melanoma growth induced by a single epicutaneous application of DMBA. In view of these data, the role of cannabinoid receptors in chemically induced skin cancer appears to be of secondary importance. However, as a consequence of an antagonistic effect on the proinflammatory microenvironment, THC inhibited tumour growth of HCmel12 melanoma xenografts, which was associated with reduced infiltration of protumorigenic myeloid immune cells into the tumour microenvironment [To further deepen the knowledge about the effect of cannabinoids on the immunological tumour defence of melanoma cells, the effect of systemically administered THC on the growth of the melanoma cell line HCmel12 was investigated in a mouse model . Flow cyironment . Interesironment . In summ9-tetrahydrocannabivarin (THCV) were found to downregulate lipopolysaccharide-induced release of the proinflammatory cytokines IL-1α, IL-1β, IL-6, IL-8 and tumour necrosis factor (TNF)-α in sebocytes [2 receptor agonist that showed an inhibitory effect on 1-fluoro-2,4-dinitrobenzene (DNFB)-induced ear inflammation in Balb/c mice [2 receptor and phospholipase A2. Subsequently, increased concentrations of 15-deoxy-Δ12,14-PGJ2 are formed via COX-2, which leads to apoptosis and the subsiding of chronic inflammation via caspase-3 release. A LOX-mediated pathway is also thought to mediate the production of anti-inflammatory and proresolving lipoxin A4 and other proresolving mediators. However, the corresponding conditions of both possible signalling pathways and their interaction have not yet been clearly defined. PPARγ activation as a possible mechanism of anti-inflammatory action of lenabasum has been referred to earlier in this review [The results of some experimental setups demonstrating a chemopreventive effect of the test substances used give hope that cannabinoids could possibly exert a skin tumour-preventive effect. The successful preclinical testing of cannabinoids in inflammatory skin diseases supports this hypothesis insofar as inflammatory processes are associated with tumour-promoting effects. Accordingly, the non-psychoactive cannabinoids cannabichromene (CBC), cannabidivarin (CBDV), cannabigerol (CBG), cannabigerovarin (CBGV) and Δebocytes . Other cebocytes , is a nob/c mice . Lenabasb/c mice , enzymess review .1 and CB2 phosphorylation and internalisation and subsequent activation of MAPK signalling pathways [1 and CB2 receptor double knockout mice showed less pronounced inflammation with lower activation of the transcription factor nuclear factor-κB (NF-κB), lower production of TNF-α and consequently a lower incidence of subcorneal pustules and epidermal erosions. Consistent with this finding, wild-type mice were four times more likely to develop papillomas than CB1 and CB2 double knockout mice. Inhibition of cannabinoid receptors could therefore be a prophylactic option to prevent skin cancer caused by UV radiation.Regarding the effect of cannabinoids on cancerous or precancerous lesions of the skin, some preclinical data have already suggested the potential of cannabinoids as preventive agents for skin cancer. For example, in a two-stage model of skin carcinogenesis in mice, in which skin carcinogenesis was induced by DMBA and promoted by TPA, the synthetic cannabinoid naphthoylindoles JWH-018, JWH-122, and JWH-210 were shown to reduce the promotion of TPA-induced carcinogenesis in mouse skin when applied topically 30 min before each TPA treatment. Thus, a topical chemopreventive effect for cannabinoids was demonstrated, at least in animal experiments . Howeverpathways . In the 4, while proinflammatory and protumorigenic lysophospholipids, as well as PGE2 and thromboxane B2, were downregulated [In the case of CBD, one study further showed that this cannabinoid increases the activity of antioxidant enzymes such as superoxide dismutase and thioredoxin reductase in keratinocytes exposed to UV radiation, suggesting that CBD may serve as a protective agent for skin cancer prevention . The antegulated . In summclinicaltrials.gov website that addressed the systemic effects of cannabinoids in cancer patients . HoweveIn a systematic review and meta-analysis of note, the quality of clinical studies on the use of cannabinoids for various medical indications was assessed with regard to chronic pain  and improvement of nausea and vomiting due to chemotherapy (28 studies) . For theAnother recent systematic review of randomised controlled trials showed with moderate- to high-certainty evidence that compared with placebo, treatment with non-inhaled medicinal cannabis or cannabinoids results in a very small increase in the proportion of patients with chronic cancer and non-cancer pain who experience significant improvement in pain relief, physical functioning and sleep quality, along with some adverse side effects . FinallyIt is also worth noting that in a study of cannabis use in patients with head and neck cancer undergoing radiotherapy, medical marijuana was reported to contribute to benefits in altered cognition, weight maintenance, depression, pain, loss of appetite, dysphagia, xerostomia, muscle cramps and sticky saliva . HoweverIn recent years, some clinical research has been conducted on the effects of cannabis use on chemotherapy outcomes. For example, a recent retrospective observational study showed a lower response rate of cancer patients with advanced melanoma, non-small cell lung cancer and clear cell renal carcinoma to treatment with nivolumab when cannabis was used concomitantly. Therefore, the use of cannabis in connection with immunotherapies has to be evaluated critically . HoweverAnother study reached a more concerning conclusion regarding the effect of cannabis use during immunotherapies. Here, cannabis reduced some of the adverse effects associated with immunotherapy, such as skin toxicity, colitis and thyroid disorders. On the other hand, cannabis users showed a less favourable prognosis in terms of time to tumour progression and overall survival than patients who did not use cannabis . It is np = 0.065), which became statistically significant  after removing the data on testicular cancer, which showed an increased risk [Although not comparable with each other, there have also been studies that showed that marijuana use generally reduces the risk of cancer. For example, a population-based case-control study from 2009 found that 10 to 20 years of marijuana use was associated with a lower risk of head and neck squamous cell carcinoma after adjustment for potential confounding factors such as smoking and alcohol consumption . Anothersed risk . NotablyIn order to be able to make reasonable predictions about the systemic efficacy of cannabinoids, more prospective studies must be conducted in the future, which would then also allow statements about the correct application and dosage.Currently, clinical data on the effect of cannabinoids on cancer progression from comprehensive clinical trials is lacking. Nevertheless, the data on the systemic anticancer effect of nabiximols in high-grade glioblastoma  also givWith regard to possible use of cannabinoid-based medicinal products in connection with skin cancer, different forms of application and indications of these substances have to be considered and discussed in a differentiated way. This concerns the systemic application of cannabinoids and the prophylactic topical application of cannabinoids for the prevention of sun-related and chemically induced skin cancers. Concerning the latter, the beneficial effects of CBD on reducing inflammation need to be further investigated in order to fathom the potential inhibitory effect of CBD on the development of skin cancer. On the adverse side effects of topical cannabinoid compounds in general, it should be noted that caution is also advised in eczema patients, as many topical cannabinoid-containing formulations contain a number of known irritants . In factIn terms of the systemic effects of cannabinoids in the context of skin cancer, the preclinical data on the effect of CBD on melanoma  and squaAs a matter of fact, cannabinoids could be promising drugs for the treatment and prophylaxis of skin cancer. However, in view of the recent observations mentioned above that cannabis use does not improve the efficacy of tumour immunotherapies but rather impairs it ,138, it A major threat to the potentially valuable therapeutic use of cannabinoids is that cannabis product providers are increasingly making and promoting numerous unsubstantiated claims to consumers , therebyPreclinical results of recent years, especially with regard to antiproliferative and growth inhibitory effects, show that cannabinoids could be a promising option for the systemic treatment of melanoma and squamous cell carcinoma of the skin. Important questions in the clinical context will be which cannabinoid or combination of cannabinoids is actually most effective for which type of skin cancer and how high the dosage can be chosen without compromising patients’ quality of life through adverse drug effects. Another crucial question is in which combination with other cancer therapies cannabinoids show a synergistic anticancer effect and thereby possibly also reduce cytostatic-typical side effects and prevent chemoresistance. A consistent continuation of the previous preclinical studies as well as the implementation of randomised clinical studies on the effect of cannabinoids in skin cancer is encouraged on the basis of the data available so far.