Esophageal cancer (EC) is a common malignant tumor, which brings heavy economic burden to patients and society. Therefore, it is important to understand the molecular mechanism of recurrence, metastasis, and drug resistance of esophageal cancer.  Human esophageal cancer cell line TE13 (poorly differentiated squamous cell carcinoma) and normal human esophageal epithelial cell line het-1a were selected for aseptic culture. At the same time, 6 bottles of TE13 cell line were inoculated in logarithmic phase. Cell apoptosis was analyzed by flow cytometry (FCM). Cell clone formation assay was used to analyze the proliferation. Fibronectin-coated dishes were used to detect the characteristics of cell adhesion to extracellular matrix. The Transwell method was used to detect the cell invasion ability. Western blot was used to analyze the expression of Yap1, PTPN14, FAT1, and Myc.  Results showed that FAT1 and PTPN14 were downregulated, while Yap1 was upregulated in esophageal cancer tissues. FAT1 inhibited the proliferation, adhesion, and invasion of human esophageal cancer cell lines, which might be associated with the upregulation of PTPN14 and the inhibition of Yap1 and Myc.  The results suggested that PTPN14 and FAT1 could regulate malignant progression and chemotherapy resistance of esophageal cancer based on the Hippo signaling pathway. Esophageal cancer (EC) is a common malignant tumor of the digestive tract, which brings heavy economic burden to patients and society . Due to The Hippo pathway is first found in the process of functional genetic screening in Drosophila, which is highly conservative and plays a role in regulating organ size and maintaining the dynamic balance of cell proliferation or apoptosis –7. Yes-aProtein tyrosine phosphatase nonreceptor type 14 (PTPN14) is a member of the protein tyrosine phosphatase (PTPs) family encoded by 14 genes. PTPs can be involved in the regulation of a variety of cell processes, including cell proliferation, differentiation, mitosis, and even malignant transformation –15. Fat Here, we studied the role of FAT1 and PTPN14 in the regulation of cell proliferation, adhesion, and invasion during esophageal cancer progression and the possible association with the Hippo pathway. This study might provide a theoretical basis on the roles of FAT1 and PTPN14 in the occurrence and development of esophageal cancer.The specimens of 20 cases of esophageal cancer adjacent tissues and 20 cases of esophageal cancer patients were collected from EC patients who came to the The Second People's Hospital of Lianyungang . All patients received no chemotherapy or radiotherapy before. Tissues were collected immediately after resection and were stored at -80°C. This study was approved by the Ethical Committee of The Second People's Hospital of Lianyungang .ΔΔCt- [Total RNA was extracted using TRIzol  according to the manufacturer's instruction. The mRNA expression levels of Yap1, PTPN14, and FAT1 were analyzed by qRT-PCR. The cDNA was synthesized using the PrimeScript RT Reagent Kit  following the manufacturer's instructions. Real-time PCR was performed on a Roche Light Cycler 480 (Roche) using the SYBR Green PCR Master Mix . Each measurement was performed in triplicate, and the results were normalized by the expression of the GADPH gene. Fold change relative to mean value was determined by 2-ΔΔCt .H&E) staining, immersed with 3% H2O2, and incubated with primary antibodies against related proteins . The second antibody (goat anti-rabbit IgG H&L (FITC) or goat anti-rabbit IgG H&L (Biotin), Abcam) was labeled with FITC or biotin and stained with the ABC method. The negative control group, positive control group, and fluorescent marker control group were set up. For quantification, the Allred scoring system was used [For IHC, 10% neutral formalin was immobilized, dehydrated, paraffin-embedded, and cut into 4-micron-thick tissue sections. Samples were then stained with hematoxylin- and eosin- (μg/ml) was administered at the same time.All cell lines used in the present study was purchased from the Cell Bank of Type Culture Collection . Three human esophageal cancer cell lines TE13 (poorly differentiated squamous cell carcinoma), KYSE140 (moderately differentiated squamous cell carcinoma), and SEG1 (well differentiated adenocarcinoma) were cultured strictly in accordance with aseptic culture. TE13 and normal human esophageal epithelial cell line het-1a were selected for aseptic culture. For cell transfection, 6 bottles of TE13 cell line were inoculated in logarithmic phase and transfected with FAT1 overexpressed plasmid (ov-FAT1) or FAT1 small interfering RNA plasmid (si-FAT1) using the lipo6000 reagent  . The ov-FCM analysis for cell cycle and apoptosis was conducted 72 hours posttransfection, using the nuclear stain DAPI  or Annexin V-FITC/7-AAD Kit. Then, the apoptosis characteristics of each group were analyzed using a FSCAN flow cytometer  .3 were seeded into 6-well dishes, incubating for 14 days, then fixed with glutaraldehyde, and stained with crystal violet. The positive colony formation was manually counted under a light microscope .Cell viability was determined by clone formation assay. Briefly, after 72 hours of transfection, cells with density of 4 × 104/ml cells were seeded onto the upper chamber. The cells were then cultured in serum-free media, were incubated for 24 h, and were then fixed and stained using Crystal Violet. Fibronectin-coated dishes were used to detect the characteristics of cell adhesion to extracellular matrix in each experimental group as reported elsewhere [The Transwell method was used to detect the invasion ability of cells in each experimental group. Briefly, 3 × 10lsewhere .Western blotting was used to evaluate the protein expression of Yap1, PTPN14, FAT1, and Myc in each cell line. Samples were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membranes and then blocked by 5% nonfat milk at room temperature for 1 h. Then, membranes were incubated with primary antibodies of anti-Yap1 ab52771 1/5000, anti-PTPN14 ab204321 1/2000, anti-FAT1 ab190242 1/2000, and anti-Myc ab9106 1/2000 (Abcam) at 4°C overnight, following with incubation with a goat anti-rabbit secondary antibody  at 37°C for 45 min. The bound antibodies were detected using the enhanced chemiluminescence plus Western blotting detection system (Bio-Rad) [t test, and 2-tailed P values less than 0.05 were considered significant.All quantified data represent an average of at least triplicate samples or as indicated. Error bars represent the SD of the mean. Statistical significance was determined by the Student's The specimens of 20 cases of esophageal cancer adjacent tissues and 20 cases of esophageal cancer patients were collected. The mRNA levels of Yap1, PTPN14 and FAT1 were analyzed by qPCR. The results showed that Yap1 mRNA was highly expressed in esophageal carcinoma tissues, but low in the adjacent tissues of esophageal carcinoma . The expBesides, immunohistochemistry was conducted on the same samples of esophageal cancer to analyze the relative protein expression of FAT1 and Yap1. The results showed that FAT1 protein was highly expressed in the adjacent tissues of esophageal carcinoma, but low in esophageal carcinoma tissues. The expression of Yap1 was low in the adjacent tissues of esophageal carcinoma, but high in the tissues of esophageal carcinoma .Three human esophageal cancer cell lines TE13, KYSE140, and SEG1 were cultured strictly in accordance with aseptic culture. qRT-PCR results showed that FAT1 was highly expressed in the SEG1, but low in TE13 , suggestTE13 and the normal human esophageal epithelial cell line het-1a were selected for aseptic culture. At the same time, the TE13 cell line was inoculated in logarithmic phase, which was numbered and transfected with FAT1 and si-FAT1. Additionally, cisplatin was given to inhibit the cell development. Then, the apoptosis characteristics of each group were analyzed by flow cytometry (FCM). The results demonstrated that FAT1 inhibited the proliferation of human esophageal cancer cell lines . Cell clTo evaluate the role of FAT1 in the invasion of human esophageal cancer cell lines, Transwell assay was conducted. The result demonstrated that FAT1 inhibited the invasion of human esophageal cancer cell lines . WesternIn this study, we examined the expression levels of Yap1, PTPN14, and FAT1 in human esophageal cancer cell lines and tissues. We also studied FAT1/PTPN14 on proliferation, adhesion, and invasion characteristics of esophageal cancer cell lines by FACS, clone formation assay, and the Transwell method. Our results showed that FAT1 and PTPN14 are downregulated while Yap1 is upregulated in esophageal cancer tissues. FAT1 promotes the expression of PTPN14 and downregulates the expressions of Yap1 and Myc, to inhibit the proliferation, adhesion, and invasion of human esophageal cancer cell lines.Notably, the invasion and metastasis of tumor cells are two of the important biological characteristics of malignant tumors. Abnormal expression of Yap1 can promote the malignant transformation of cells and induce the occurrence and development of tumors. The Yap gene is located in the long arm of chromosome 11, including Yap1 and Yap2 subtypes. Yap1, as a key effector molecule of the Hippo signaling pathway, can directly participate in cell proliferation, apoptosis, and other biological behaviors after phosphorylation –27. In EIn 1995, when scientists were looking for tyrosine phosphatase expressed in normal breast tissue, PTPN14 was cloned and found for the first time . It is aFAT1 often functions as a tumor-suppressor or oncogene in different types of human cancer –18. Some


On the other hand, hydroxyxanthone 3a exhibited the highest anticancer activity (IC50 = 184 ± 15 µM) and the highest selectivity (SI = 18.42) against MCF-7 cancer cells. From the molecular docking study, it was found that hydroxyxanthone 3a interacted with the active sites of Topoisomerase II protein through Hydrogen bonding with DG13 and π–π stacking interactions with DA12 and DC8. These findings revealed that hydroxyxanthones are potential candidates to be developed as antioxidant and anticancer agents in the future.In the present work, three hydroxyxanthones were synthesized in 11.15–33.42% yield from 2,6-dihydroxybenzoic acid as the starting material. The chemical structures of prepared hydroxyxanthones have been elucidated by using spectroscopic techniques. Afterward, the hydroxyxanthones were evaluated as antioxidant agents through radical scavenging assay; and anticancer agents through in vitro assays against WiDr, MCF-7, and HeLa cancer cell lines. Hydroxyxanthone  Several fatal diseases such as cancer, arthritis, cardiovascular, and neurodegenerative diseases are generated by oxidative stress that damages human tissue and organs2. Free radicals are reported as the main factor in the production of oxidative stress in the human body. Free radicals are very reactive; thus, they react rapidly with biomolecules such as DNA and affect cell metabolism3. Additionally, free radicals are also responsible for cholesterol and atherosclerosis deposition inside the blood vessels4. Therefore, researchers are making extensive efforts to prevent and deactivate the free radicals before damaging the cell function5.Oxidative stress diseases have been receiving great attention due to their harmful, severe, and unfavorable effects on the human body6. Almost all antioxidant agents are composed of phenolic compounds because phenolic compounds are able to generate hydrogen donor and electron delocalization mechanisms to suppress the formation of oxidative stress7. Several natural and synthetic antioxidant agents such as chalcone, indole-3-acetamide, cadmium-bismuth microsphere, indazole, and salicylhydrazidehydrazone derivatives have been evaluated; however, their chemical synthesis is not easy, and their antioxidant activity is still unsatisfactory12.Usage of antioxidant agents is the most straightforward approach to deactivate the free radicals16. Furthermore, xanthones display broad pharmacological activities, which are remarkable to be further developed for the drug research21. Xanthone could be obtained from various plants, such as Symphonia globulifera and Garcinia mangostana23. Nevertheless, the isolation process is very complicated to obtain the desired xanthone derivatives in high purity. Furthermore, the isolation and purification of xanthones require a large volume of organic solvents in a time-consuming process24. Therefore, the synthesis of xanthone is much attractive to be developed to afford xanthone derivatives in a higher yield within a faster research period. Moreover, it is possible to obtain xanthone derivatives with specific functional groups at a certain position by using a synthesis reaction, which may not be found in natural sources18.Almost 80–95% of antioxidant and anticancer agents consist of a heterocyclic structure. Among the heterocyclic compounds, xanthone derivatives with a dibenzo-γ-pyrone skeleton exhibit unique physicochemical propertieso-hydroxybenzoate, and acetic anhydride. Unfortunately, this method generates an unfavorable side reaction; thus, the obtained yield was very low. Eaton’s reagent (a mixture of P2O5 and CH3SO3H) has recently been introduced to give a higher yield for xanthone derivatives25. For example, Zhou et al. successfully synthesized 1,3-dihydroxydinitroxanthone and 7--1,3-dihydroxyxanthone in up to 76% yield; however, their antioxidant activity was still lower compared to the commercial antioxidant agents26. Because of that, synthesis and modification of other xanthone derivatives are important to find the active antioxidant agents to be used for commercial purposes.The synthesis of xanthone was firstly established in 1892 by Michael and Kostanecki by reacting phenol, acetic acid-28. Hydroxyxanthones exhibit good anticancer activity against lung, breast, hepatoma, cervical, colorectal, and other cancer cell lines29. Hydroxyxanthones could lead to cell apoptosis by stimulating the caspase enzyme30. Furthermore, hydroxyxanthones are able to inhibit Topoisomerase II protein by intercalation mechanism on the DNA cleavage sites31. This anticancer mechanism occurred as the hydroxyxanthones were constructed by three fused aromatic systems, which were reported as the essential pharmacophoric feature of Topoisomerase II inhibitors32. Furthermore, the three fused aromatic structure of hydroxyxanthones is similar to doxorubicin and mitoxantrone as standard DNA intercalators28. Therefore, hydroxyxanthones are potential to be further investigated as anticancer agents.Xanthone, benzylhydrazone, and quinoxaline derivatives have been reported to give remarkable anticancer activities33. In continuation of our previous research, we synthesized other hydroxyxanthone derivatives to give a broader map of their antioxidant and anticancer activities through in vitro and molecular docking studies. In this work, three hydroxyxanthone derivatives, i.e., 1,3,8-trihydroxyxanthone (3a), 1,6-dihydroxyxanthone (3b), and 1,5,6-trihydrokxyxanthone (3c) were prepared from 2,6-dihydroxybenzoic acid with phloroglucinol, resorcinol, and pyrogallol, respectively. The chemical structure of these hydroxyxanthones was elucidated using Fourier transform infrared (FTIR) spectrophotometer, mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectrometers. These compounds were evaluated as antioxidant and anticancer agents through in vitro assay using 1,1-diphenylpicryl-2-hydrazyl (DPPH) and 3--2,5-diphenyltetrazolium bromide (MTT) methods, respectively. The inhibition mechanism of the most active anticancer agent against Topoisomerase II protein was further studied through a molecular docking study.In our previous work, several hydroxyxanthone derivatives, i.e., 1,3-dihydroxyxanthone, 3,4-dihydroxyxanthone, 3,6-dihydroxyxanthone, 1,3,6-trihydroxyxanthone, and 3,4,6-trihydroxyxanthone have been synthesized and evaluated as the anticancer agent against WiDr cancer cell line7H6O4), phloroglucinol (C6H6O3), resorcinol (C6H6O2), pyrogallol (C6H6O3), Eaton’s reagent , DPPH (C18H12N5O6), n-hexane (C6H14), ethyl acetate (C4H8O2), and methanol (CH3OH) were purchased from Merck in pro analytical grade. In addition, butylated hydroxytoluene  and MTT (C18H17N5S) were obtained from Aldrich in pro analytical grade.The chemicals used in the synthesis, i.e., 2,6-dihydroxybenzoic acid (Cn-hexane and ethyl acetate 1:1 v/v was used as the mobile phase to afford the target compound. The products were then characterized using FTIR (Shimadzu-Prestige 21), MS (Shimadzu QP-2010S), 1H– and 13C–NMR (JEOL JNMECA 500 MHz) spectrometers.A mixture of 2,4-dihydroxybenzoic acid , the substituted phenol  , and Eaton’s reagent (8.00 mL) was heated at 80–85 °C for 3 h. Then, the mixture was allowed to reach room temperature, and the mixture was poured with cooled water (50 mL). The resulting solid residue was collected by filtration, washed with water until neutral, dried, and then purified by preparative thin layer chromatography. In the preparative thin-layer chromatography, silica was used as the stationary phase, while a mixture of 3a). The compound 3a was obtained as a yellow solid in 16.14% yield. m.p. 239–240 °C. FTIR (KBr) v/cm–1: 3448 (O–H), 1612 (C = O), 1418 (C = C), 1296 (C–O–C). 1H–NMR  δ/ppm = 6.09 , 6.20 , 6.64 , 6.78 , 7.49 . 13C–NMR  δ/ppm = 95.8 (C7), 100.0 (C5), 107.9 (C8a), 111.4 (C8b), 115.9 (C4), 128.4 (C2), 137.6 (C3), 157.4 (C4a), 159.4 (C4b), 162.3 (C1), 164.2 (C6), 169.9 (C8), 185.1 (C9). MS m/z = 244 [M]+.1,3,8-trihydroxyxanthones (3b). This compound was obtained as a yellow solid in 33.42% yield; m.p. 248–249 °C. FTIR (KBr) v/cm–1: 3425 (O–H), 1604 (C = O), 1465 (C = C), 1273 (C–O–C). 1H–NMR  δ/ppm = 6.67 , 6.71 , 6.81 , 6.85 , 7.52 , 7.99 . 13C–NMR  δ/ppm = 102.7 (C5), 107.3 (C4), 108.8 (C8b), 110.5 (C2), 113.2 (C8a), 115.4 (C7), 127.9 (C8), 136.6 (C3), 157.2 (C4a), 159.4 (C4b), 162.4 (C6), 167.5 (C1), 181.9 (C9). MS m/z = 228 [M]+.1,6-dihydroxyxanthones (3c). This compound was obtained as a yellow solid in 11.15% yield. m.p. 251–252 °C. FTIR (KBr) v/cm–1: 3448 (O–H), 1604 (C = O), 1458 (C = C), 1273 (C–O–C). 1H–NMR  δ/ppm = 6.67 , 6.78 , 6.84 , 7.51 , 7.97 . 13C–NMR  δ/ppm = 103.4 (C4), 107.8 (C8b), 109.4 (C2), 110.9 (C7), 113.2 (C8a), 116.6 (C8), 128.3 (C5), 137.0 (C3), 157.7 (C4b), 160.1 (C6), 162.9 (C4a), 169.4 (C1), 182.3 (C9). MS m/z = 228 [M–16]+.1,5,6-trihydroxyxanthones (3a-c) was dissolved in methanol at a concentration range from 10 to 100 µg mL–1. The solution was then mixed with 100 µg mL–1 DPPH in methanol and allowed for 30 min in a dark condition. The absorbance value of the absorption signal at 517 nm was measured using a UV–Vis spectrophotometer (Shimadzu UV 1800). The radical scavenging percentage was calculated using Eq. (blank and Asample are the absorbances at 517 nm for blank (methanol) and sample (hydroxyxanthone), respectively.The antioxidant activity of hydroxyxanthones was evaluated by using a DPPH scavenging assay. Each hydroxyxanthone (50) value was calculated by fixing the radical scavenging percentage equal to 50%. The in vitro antioxidant assay for each sample was performed in three replications.Afterward, the radical scavenging percentage was plotted versus the concentration of the antioxidant agent. The half-maximal inhibitory concentration , MCF-7 (breast cancer), and HeLa  cells were suspended in medium with 10% Fetal Bovine Serum. Concisely, the cell lines were added to 96-well plates and cultured in a medium with various concentrations of each hydroxyxanthone 3a–c. The mixture was stored in a CO2 incubator. After 24 h, the MTT reagent was added, and the mixture was further incubated for an additional 3 h. Afterward, the sodium dodecyl sulfate (SDS) page was added to the mixture, and the mixture was re-incubated for 24 h in a darkroom. The absorbance value of the solution at 495 nm was measured using an Elisa reader (Benchmark) to calculate viability cells’ percentage  cell lines were supplied by the Parasitology Laboratory, Universitas Gadjah Mada, Indonesia. The cells were cultured at 37 °C in an incubator containing 5% CO50 value was calculated by fixing the viability cells’ percentage equals 50%. The in vitro anticancer assay for each sample was performed in three replications.Afterward, the viability cells’ percentage was plotted versus the concentration of the anticancer agent. The IC34. Finally, the formed interactions between hydroxyxanthone and the active site of Topoisomerase II protein were visualized by using Discovery Studio software.The human Topoisomerase II with 4G0V as the protein code was retrieved from the Protein Data Bank. At first, the water molecules were removed from the Topoisomerase II protein structure. Then, the hydrogen atoms were added to build up the structure of the Topoisomerase II protein using AutoDock Tools software. The molecular docking process was performed by using AutoDock Vina software. The validity of the molecular docking study is represented by the root-mean-square deviation (RMSD) value less than 2.0 Å3a–c) were prepared through a cyclo-acylation reaction of 2,6-dihydroxybenzoic acid (1) with phenolic derivatives (2a–c). From the spectroscopic elucidation, the correct chemical structure of hydroxyxanthones 3a–c has been confirmed. The FTIR, MS, 1H–, and 13C–NMR spectra of hydroxyxanthones 3a–c are depicted in Figs. −1 while the C=O functional group appeared as a sharp signal at 1604–1612 cm−1. On the other hand, the C=C aromatic and C–O–C heterocyclic moieties were observed at 1418–1465 and 1273–1296 cm−1, respectively  in their MS spectra  gave higher IC50 values than the dihydroxyxanthone 3b. It shows that a higher amount of hydroxyl groups on the xanthone structure gave a lower antioxidant activity due to a stronger hydrogen bonding among the hydroxyl groups as previously reported36. Dihydroxyxanthone 3b exhibited the strongest antioxidant activity (IC50 = 349 ± 68 µM). Since the hydroxyl groups were located far from each other, the DPPH free radicals could easily attack the hydroxyl groups of dihydroxyxanthone 3b, yielding a strong antioxidant activity. This phenomenon was in agreement with the previous report37. The plausible reaction mechanism of DPPH free radicals with dihydroxyxanthone 3b is shown in Fig. 3b.Antioxidant activity assay of hydroxyxanthones ue Table . A lower50 value  while hydroxyxanthone 3c was the most active anticancer agent against WiDr (IC50 = 209 ± 4 µM) and HeLa (IC50 = 241 ± 13 µM) cell lines. These results demonstrated that trihydroxyxanthones were more active as the anticancer agent than dihydroxyxanthone 3b. However, trihydroxyxanthone 3c was more toxic to normal Vero cell lines (IC50 = 224 ± 14 µM) rather than either MCF-7 (IC50 = 419 ± 27 µM) or HeLa (IC50 = 241 ± 13 µM) cell lines. In contrast, trihydroxyxanthone 3a was more toxic to MCF-7 (IC50 = 184 ± 15 µM), WiDr (IC50 = 254 ± 15 µM), and HeLa (IC50 = 277 ± 9 µM) cancer cell lines rather than normal Vero cell line (IC50 = 3395 ± 435 µM), which was remarkable.Among the prepared hydroxyxanthones, hydroxyxanthone 38. The selectivity index (SI) was a critical parameter to describe selective anticancer activity. The SI value was calculated by dividing the IC50 of Vero cell lines with IC50 of cancer cells of hydroxyxanthone 3a–c. A higher SI value describes a more selective anticancer agent. The SI value of hydroxyxanthones 3a–c is listed in Table 3a was found as the most selective anticancer agent due to its highest SI value against MCF-7, WiDr, and HeLa cancer cell lines among the other hydroxyxanthones in this work.Good anticancer drug candidates must be selective, i.e., toxic only to cancer cells but safe for normal cell lines3a–c and the previously reported hydroxyxanthones against WiDr cancer cell line. Compared to the other hydroxyxanthone derivatives, dihydroxyxanthones (IC50 = 355–1255 µM) gave higher IC50 values than trihydroxyxanthones (IC50 = 38–384 µM). Among the dihydroxyxanthones, the order of the anticancer activity against the WiDr cancer cell line was 1,6-dihydroxyxanthone > 3,6-dihydroxyxanthone > 1,3-dihydroxyxanthone > 3,4-dihydroxyxanthone. Meanwhile, the order of the anticancer activity of trihydroxyxanthones was 3,4,6-trihydroxyxanthone > 1,5,6-trihydroxyxanthone > 1,3,8-trihydroxyxanthone > 1,3,6-trihydroxyxanthone. From the reported quantitative structure–activity relationship (QSAR) study, we found that the IC50 value of hydroxyxanthone as the anticancer agent against WiDr cancer cell line depends on the net atomic charge on carbon atoms, dipole moment, and octanol/water partition coefficient33. Furthermore, the SI values of trihydroxyxanthones against WiDr cells (SI = 1.072–66.39) are higher than the dihydroxyxantones (SI = 0.870–2.230), demonstrating that trihydroxyxanthones are more potential anticancer drug candidates than dihydroxyxanthones.Table 3a exhibited the strongest and the most selective anticancer activity among the other hydroxyxanthones in this work. Therefore, a further investigation was conducted for hydroxyxanthone 3a through a molecular docking study to reveal the inhibition mechanism on the active site of Topoisomerase II protein. Topoisomerase II protein is selected as the target protein since Topoisomerase II serves a pivotal role in the DNA replication and transcription of cancer cells. Doxorubicin is an anticancer agent with strong inhibitory activity towards Topoisomerase II protein. Doxorubicin consists of two fused aromatics with a carbonyl structure, which is similar to the prepared hydroxyxanthone 3a.The experimental in vitro assay found that hydroxyxanthone 3a with the DNA chains of Topoisomerase II, such as hydrogen bond with DG13, as well as π–π stacking with DA12 and DC8. These kinds of interactions have been reported to be critical for the inhibitory activity against Topoisomerase II protein. Etoposide, a standard Topoisomerase inhibitor, was reported to generate hydrogen bond interactions with Asp463 and DG13 on the active site of Topoisomerase II40. On the other hand, the binding interaction of the anticancer agent with DG13 was reported as pivotal interaction as this interaction stimulates the formation of DNA damage that is toxic to cancer cells41. Similar binding modes with DA12 and DC8 through π–π stacking were also reported for the most active benzoxazole and olivacine derivatives as the anticancer agents42.The formed interactions from the molecular docking study are displayed in Fig. 3a generated no interaction with Ser480 and Gln778. Therefore, the theoretical binding energy and binding constant of hydroxyxanthone 3a were lower than doxorubicin. The binding energy and binding constant of hydroxyxanthone 3a with Topoisomerase II were −8.0 kcal mol−1 and 1.362 µM, respectively, which were weaker than doxorubicin . Nevertheless, it is worthy to note that hydroxyxanthone 3a was able to disturb the function of Topoisomerase II protein, thus increasing the cell apoptosis, which led to the death of cell cancers as previously reported31.On the other side, doxorubicin interacted to the active site of Topoisomerase II through hydrogen bond , carbon-hydrogen bond (DG13), and π–π stacking  interactions. Compared to doxorubicin, hydroxyxanthone 3a–c) have been successfully synthesized from 2,6-dihydroxybenzoic acid and phenolic compounds through an acylation reaction using Eaton’s reagent. The chemical structures of hydroxyxanthones have been clearly elucidated by using FTIR, MS, 1H–, and 13C–NMR. The in vitro antioxidant assay showed that trihydroxyxanthones 3a and 3c gave higher IC50 values than dihydroxyxanthone 3b due to stronger hydrogen bonds. It was found that dihydroxyxanthone 3b exhibited the strongest antioxidant activity (IC50 = 349 ± 68 µM). On the other hand, trihydroxyxanthones gave higher anticancer activity than the dihydroxyxanthone one. Trihydroxyxanthone 3a exhibited higher toxicity against MCF-7 (IC50 = 184 ± 15 µM), WiDr (IC50 = 254 ± 15 µM), and HeLa (IC50 = 277 ± 9 µM) cancer cell lines rather than normal Vero cells (IC50 = 3395 ± 435 µM). Consequently, trihydroxyxanthone 3a yielded the highest selectivity index (12.25–18.42) among the prepared hydroxyxanthones in this work. The molecular docking study reveals that hydrogen bond and π–π stacking interactions were observed between trihydroxyxanthone 3a with the DNA chains of Topoisomerase II. The trihydroxyxanthone 3a was able to disturb the function of Topoisomerase II protein. These findings are important for the future development of anticancer agents based on hydroxyxanthone derivatives.Three hydroxyxanthones (Supplementary Figures.


CRISPR/Cas has emerged as an excelle nt gene-editing technology and is used worldwide for research. The CRISPR library is an ideal tool for identifying essential genes and synthetic lethality targeted for cancer therapies in human cancers. Synthetic lethality is defined as multiple genetic abnormalities that, when present individually, do not affect function or survival, but when present together, are lethal. Recently, many CRISPR libraries are available, and the latest libraries are more accurate and can be applied to few cells. However, it is easier to efficiently search for cancer targets with their own screenings by effectively using databases of CRISPR screenings, such as Depmap portal, PICKLES , iCSDB, Project Score database, and CRISP-view. This review will suggest recent optimal CRISPR libraries and effective databases for Novel Approaches in the Discovery and Design of Targeted Therapies. The proScience by two groups: Shalem et al. and Wang et al. in 2013 [CRISPR libraries for random mutagenesis were published simultaneously in  in 2013 ,17. Shal in 2013 . Various in 2013 .Streptococcus pyogenes Cas9 nuclease is usually used. A lentiviral vector backbone is usually used because of its high transduction rate, easy manipulation, and permanent expression of gRNA after built-in cell line DNA. One plasmid system includes a gRNA library and Cas9 plasmids in one same plasmid and is easy to be handled. However, in the multiple vector system, the vector size is generally small and has a relatively high efficiency of the transduction ratio. For library screening, transduction efficiency is important, and multiplicity of infection (MOI) is an important parameter. MOI can be defined as the ratio of the number of virus particles to the number of target cells. For CRISPR screening, the MOI usually ranges in 0.4–1.0. To maintain the diversity of gRNAs in CRISPR libraries in the screening, scientists should use numerous cells. For example, one hundred times coverage is expected for 64,800 gRNAs, and 6.48 × 106 cells are needed. Indeed, transduction efficiency is not 100% and needs more cells. Therefore, reducing the number of gRNAs has a benefit for precious cells, such as primary culture cells.Before applying gRNA to human cells, the Cas9 nuclease must be introduced to mammalian cell lines. S. pyogenes Cas9 using a gRNA design algorithm method and developed the Brunello library with three to four sgRNAs per target gene but improved on-target and off-target effects [The GeCKO library v2, published in 2014, has been the KO library . Initial effects . The Bru effects .In the KO library, essential genes may be missed, along with cell death, because they play key roles in cell survival. In addition, genes with high copy numbers may mimic essential genes because of the potential for accumulation of dsDNA cleavage, which may cause cell death. In such cases, CRISPRi is useful for loss of function analysis. However, the reliable loss of function was inferior to the KO library. Doench et al. used the FANTOM database to design sgRNAs with an optimal window for the TSS and designed the library . In the CRISPRa is suitable for identifying genetic mutations that favor cell survival and proliferation  and drug resistance factors under anticancer drug administration (synthetic rescue) by analyzing the gRNAs of cells that remain or proliferate under screening conditions. Using CRISPRa , which targets transcription factors, has enabled a comprehensive analysis of the lnc RNA region, which has been difficult to analyze in the past. Bester et al. searched for factors involved in Ara-C resistance, a therapeutic drug for acute myeloid leukemia, by combining a comprehensive cell line database and CRISPRa for non-coding regions  (positive selection) ,34. FurtThe conventional and widely used CRISPRa library—a Synergistic Activation Mediator (SAM)—has a potential problem in the vector of MS2-p65-HSF1, a large vector size, which results in low efficiency of infection and hence low efficiency of activation. To improve the efficiency, two MS2 and two PP7 stem loops were introduced into the Calabrese library on tracrRNAs to facilitate the recruitment of transcription factors by attaching PCP, p65, and HSF complexes . HoweverBRCA1 or BRCA2, which are involved in DNA double-strand breaks, the mutations by themselves are not detrimental to the survival of the cancer cells , but the administration of a DNA single-strand break repair enzyme inhibitor, PARP inhibitor, results in death. By administering PARP inhibitors, the BRCA-mutated cancer cells are prevented from repairing their DNA, and are made to die. Thus, a good synthetic lethal drug lacks lethal effects on normal tissues and cells, but is lethal to cancer cells with certain mutations.Recently, the concept of synthetic lethality has gained ground in cancer treatment, and the development of anticancer drugs regarding synthetic lethality is expected . SynthetSynthetic lethality research requiring complete knockdown of gene expression and CRISPR screening, which introduces genome-wide mutations, is well suited for screening for synthetic lethality and novel cancer therapies. Many synthetic lethality CRISPR screenings have been performed . CRISPR In CRISPR KO and CRISPRi, loss of function (LOF) analysis was performed by subtracting the gRNAs of surviving cells from the original library of dead cells (negative selection) a. Thus, KRAS mutation, such as KRAS (G13D), is the most frequent cancer driver mutation and an intractable anticancer drug. Yau et al. used the GeCKO v2 library (KO library) for KRAS mutant HCT116 colorectal cancer cell lines and transplanted to mice (in vivo xenograft transplantation model) [KRAS mutation. Like the SWI/SNF complex, INO80 is a large multi-subunit complex maintaining genome stability through nucleosome editing, and INO80C is the homolog of the INO80. Thus, INO80C may be a novel target for KRAS mutant tumors.We introduce important reports about synthetic lethality using CRISPR screening. n model) . They foRNF43-mutant) and identified the Wnt pathway, WLS, CTNNB1, TCF7L2, and LRP5, and Fizzled (FZD) receptor genes, FZD5, WNT7B, and WNT10A, as essential factors for HPAF-2 cells [Steinhart et al. introduced the TKO gRNA library to HPAF-2 cells  xenograft mouse models, both GPX4 and FSP1 knock-out cells showed rapid death—synthetic lethality.Ferroptosis is a way of cell death, and glutathione peroxidase 4 (GPX4) plays a pivotal regulator role in ferroptosis. GPX4 is upregulated in various tumors, like malignant lymphoma , and ferRecently, Olivieri et al. focused on the DNA damage response induced by anticancer drugs and used the CRISPR library to identify novel genes that are candidates for synthetic lethality with anticancer drugs . RPE1-hTBrd9&Jmjd6, Kat6a&Jmjd6, and Brpf1&Jmjd6 [To find the effective combination of molecular targets and synthetic lethality in a broad sense for cancer treatments, the combination of dual CRISPR screenings were also invented. Indeed, Han et al. invented a CRISPR-based double knock-out (CDKO) system that can provide the efficiency of combinatorial genetic screening with a robust statistical scoring method for calculating genetic interactions. They used 700 × 700 = 490,000 double-sgRNAs directed against 21,321 pairs of drug targets and found that the combination against both BCL2L1 and MCL1 inhibitors was effective for imatinib-resistant leukemic cells . In addiIn CRISPR screening, many candidate gRNAs are identified, regardless of whether they are functional or not. Many of them may be off-target or deviate from the target phenotype due to unknown incidents during screening. To accurately identify target molecules, it is important to set the MOI below 1.0 and avoid duplication of gRNAs in a single cell. Although, in theory, it is easy to manipulate the MOI, the actual transduction efficiency may change depending on the cell conditions. Therefore, checking the copy numbers of viral integration would be helpful. Moreover, to narrow down the candidate genes, it is imperative to use available databases to facilitate the research. Since the use of the CRISPR library is widespread worldwide, considerable knowledge has been accumulated . Using thttps://depmap.org/portal/ (accessed on 24 Octorber 2021)) integrates the CRISPR knockout screening dataset (Achilles), cell line database (CCLE), and drug susceptibility databases (Profiling Relative Inhibition Simultaneously in Mixtures (PRISM). The Cancer Therapeutics Response Portal (CTRP) provides a comprehensive database of cancer cell vulnerability [The DepMap portal , it is also possible to compare and integrate with the CRISPR library dataset. High essentiality is an essential factor for cell line survival, and KO tends to make the cell line unable to survive and grow. In addition, cell lines can be sorted and ranked in the tabs of cancer type, mutation, copy number, and expression, and they can be browsed by a dot. The ability to examine whether the candidate genes identified in the CRISPR KO library can affect the survival and proliferation of the target cell line in PICKLES can greatly reduce the time and effort required for candidate gene selection experiments. In the recently updated version, it is also possible to refer to the co-essentiality of primary and secondary genes, which is a great help in narrowing down multiple candidate genes.The Hart lab, PICKLES , integrates multiple CRISPR library results to determine the essentiality of each cell line for a specific gene product. The results of multiple CRISPR libraries are merged, and the essentiality of each cell line for a particular gene product is ranked by a score called BF (Bayes Factor), which can be searched . For exaRecently, iCSDB, an integrated database of CRISPR screens, has integrated DepMap with BioGRIS ORCS, a database of CRISPR screening results from PubMed articles, to enable more clinical searches. We integrated DepMap and BioGRIS ORCS—databases of CRISPR screening results from PubMed articles—to enable a more clinical search. In addition, clinical and molecular data were added as annotated data for easier searching than cell lines .BRCA1 deletion and mutation groups. In the mutation group, the PARP1 BF exceeded that of the BRCA1 wild type. These results indicate that PARP1 is highly dependent on BRCA1 deletion mutations in breast cancer cell lines and that PARP1 may be a good target for synthetic lethality in BRCA1 mutant lines.To assess the co-essentiality of interesting genes in cell lines, PICKLES is effective. For example, on the top screen of PICKLES, check “Breast cancer” in the “Cancer type” field, enter “PARP1” in the “Primary gene” field, and “BRCA1” in the “Secondary gene” field (the results of mutation and copy number are reflected in the Secondary gene). In the Co-essentiality tab, PARP1 BF and BRCA1 BF are negatively correlated a. In theIn the DepMap portal, when PARP1 is entered, CRISPR  is 29/978 in the Dependent cell line, indicating that it is not Common essential b. In TarThe iCSDB is useful for searching by cell line, and the Choose by cell line annotation in the Cell line selector section allows you to select a cell line. For example, if you select HCC1187 and MDAMB231 cells, which are triple-negative cell lines in the breast cancer cell line, and click the “Search CRISPR screen,” the essential genes in both cell lines will be displayed in boxplot order c. HoweveIn this way, virtual CRISPR screening can be used to narrow down and validate candidate genes using the database while conducting our screening. However, the data in the database are obtained from cancer-derived cell lines, and information on essentiality in normal tissues will be required for clinical application, so the question is how to apply CRISPR library screening.A method for performing CRISPR screening across numerous cell lines by assigning gene barcodes has also been established, allowing genome-wide analysis of numerous cells at once and linking to drug information. PRISM is a system that can screen more than 10,000 compounds in more than 1000 different cell lines at once by introducing genetic barcodes into the cell lines . Yifeng In this study, we reviewed the principles of the CRISPR-Cas9 system, types of CRISPR libraries, methods, databases, and research results. The database has been renewed and reborn daily. In the future, the database only, without using CRISPR screening for cell lines, will make us discover new candidate genes for synthetic lethal targets of cancer. As databases become enriched, CRISPR screening conditions will be required for the more specific ingenuity mentioned above. In addition, there is limited data on CRISPR inhibition and activation compared to knock-out screenings.In the future, culturable tissues, such as organoids, will be generated from cancer tissues collected from patients, and small-sized but efficient CRISPR library screening will be used to discover individual therapeutic targets, including synthetic lethal targets for cancer, which is expected to be the ultimate precision medicine. By consolidating these data and updating the database, an era in which humanity can truly conquer cancer will arrive.


Usher syndrome (USH) is the leading cause of inherited combined hearing and vision loss. As an autosomal recessive trait, it affects 15,000 people in the United States alone and is responsible for ~21% of inherited blindness and 3 to 6% of early childhood deafness. Approximately 2/3 of the patients with Usher syndrome suffer from USH2, of whom 85% have mutations in the USH2A gene. Patients affected by USH2 suffer from congenital bilateral progressive sensorineural hearing loss and retinitis pigmentosa which leads to progressive loss of vision. To study the molecular mechanisms of this disease and develop a gene therapy strategy, we generated human induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) obtained from a patient carrying compound heterozygous variants of USH2A c.2299delG and c.1256G>T and the patient’s healthy sibling. The pluripotency and stability were confirmed by pluripotency cell specific marker expression and molecular karyotyping. Subsequent CRISPR/Cas9 genome editing using a homology repair template was used to successfully correct the USH2A c.2299delG mutation back to normal c.2299G in the generated patient iPSCs to create an isogenic pair of lines. Importantly, this manuscript describes the first use of the recombinant Cas9 and synthetic gRNA ribonucleoprotein complex approach to correct the USH2A c.2299delG without additional genetic effects in patient-derived iPSCs, an approach that is amenable for therapeutic genome editing. This work lays a solid foundation for future ex vivo and in vivo gene therapy investigations and these patient’s iPSCs also provide an unlimited resource for disease modeling and mechanistic studies. Usher syndrome type II (USH2) is the most prevalent clinical form of USH, accounting for approximately two-thirds of USH patients. USH2 patients display less severe congenital hearing loss than USH1 patients and normal vestibular function. USH2 has three identified genomic loci that correspond to three distinct genes: USH2A (Usherin), USH2C (ADGRV1), and USH2D (Whirlin). Furthermore, USH2A mutations are responsible for up to 85% of USH2 . As. AsUSH2AFirst, we applied reverse transcription (RT) and quantitative real time PCR to examine the specific gene expression profiles of these iPSC lines. We selected blood cell-specific markers CCR7, CD3D, CD8A, and CD4 and stem cell-specific markers POU5F1/OCT4, NANOG, SOX2, and TFCP2L1 to validate the successful reprogramming of these lines. As expected, expression of the blood-cell-specific markers were significantly reduced in the iPSC compared to those in the PBMCs: for JY001, CCR7 (0.0012 fold), CD3D (0.00065 fold), CD8A (0.0054 fold), CD4 (0.011 fold); for JY002, CCR7 (0.00037 fold), CD3D (0.00016 fold), CD8A (0.00073 fold), CD4 (0.0024 fold). On the other hand, expression of the stem cell-specific markers was significantly upregulated in the iPSC lines compared to that of the original PBMCs: for JY 001, POU5F1/OCT4 (25.99 fold), NANOG (59.71 fold), SOX2 (84.45 fold), TFCP2L1 (133.44 fold); for JY002, POU5F1/OCT4 (5.54 fold), NANOG (19.56 fold), SOX2 (21.26 fold), TFCP2L1 (4.20 fold). To corroborate our RT-qPCR results, immunocytochemical (ICC) analysis was performed by staining the iPSC lines using antibodies specific for stem/progenitor cell markers. These iPSC lines stained positively for the nuclear markers OCT4 and SOX2 and for the cell surface markers TRA-1-60 and SSEA4 C,D. The The reprogramming process and subsequent culturing of the iPSC lines carries with them the potential for the introduction of chromosomal abnormalities. Therefore, a molecular karyotyping was performed using the Nanostring Human Karyotyping Panel. Cells known to have a normal karyotype were used as a reference in this experiment. This analysis showed that each iPSC line—JY001 and JY002—had a normal karyotype with no evidence of insertions or deletions within the detectable range of the assay. Sex chromosome profiles were also normal: JY001 is XY and JY002 is XX.Short tandem repeat (STR) profiling was used to verify the identity of the PBMC-derived iPSC lines. This approach is a well-established human cell line authentication method due to its wide application in forensics identification. The iPSC lines JY001  and JY00CRISPR/Cas9 genome editing and HDR were used to correct the c.2299delG mutation in our patient-derived iPSC line. Small guide RNAs (sgRNA) were designed using sequencing information derived from the University of California, Santa Cruz (UCSC) genome browser and are shown in Single CRISP-edited iPSC clones were derived by seeding individual iPSC cells into a 96-well plate following RNP treatment. The cells expanded into distinct colonies, which were manually collected and propagated. Forty-five individual clonal lines were expanded. Genomic DNA was extracted, the region around the deletion PCR was amplified, and the resulting product was Sanger sequenced to identify clonal iPSC lines in which the deletion had been corrected. Among these clonal iPSC lines, 2 clones (clone #16 and #27) were identified to have corrected the deletion and modified the PAM sequence without any additional alterations ) C. In addUSH2A c.2299delG mutation study. When we examined closely the mouse Ush2a gene structure of exon 12, which is a counterpart of the human USH2A gene exon 13 that contains c.2299, the gRNA site remained as shown in USH2A and mouse Ush2a genes. For the human USH2A gene, c.2299delG results in an early protein translation termination as shown in Ush2a gene, c.2299delG does not result in an early termination and the entire exon can be read through as seen in USH2A c.2299delG study needs to be addressed cautiously. Potential solutions could be to prepare a humanized mouse at the c.2299 site by replacing mouse exon 12 with human exon 13 or simply introducing a point mutation to change the codon TGC into TGA for the same early termination as in humans.Finally, we wanted to explore the feasibility of utilizing a mouse model for a human Induced pluripotent stem cell (iPSC) technology is a powerful tool that provides a way to model the impact of genetic variants on disease pathogenesis in disease-relevant cells. This approach was first developed over a decade ago by the work of Yamanaka and colleagues and was quickly adopted by many labs ,30. PatiUSH2A: c.2299delG. Since these variants in USH2A act in an autosomal recessive manner, correction of only one of the alleles would be sufficient to restore the wild-type functionality. This iPSC line offers unlimited material for disease modeling and therapeutic studies of USH2A c.2299delG disorders. In the hearing loss field, patient-derived iPSCs could be differentiated into inner ear organoids and multiple cell types, including hair cell-like cells, spiral ganglion cells, and Schwann cells for therapeutic and mechanism studies [In this study, we generated patient-derived iPSCs from an individual bearing a compound heterozygous mutation in USH2A, including the most common mutation of  studies ,40,41,42 studies ,43. We u studies ,45. Neve studies . The priAlthough USH2A c.2299delG iPSC generation and correction has been previously published, this project has multiple novelties: we generated patient-derived human iPSCs with the novel c.1256G > T (p.Cys419Phe) in addition to the more common c.2299delG (p.Glu767Serfs*21) variant; the derived iPSCs were from patient PBMCs rather than fibroblasts or keratinocytes, making this approach more patient-friendly while reducing the barrier to patient enrollment in studies; and instead of using plasmid-based genome editing tools, we used the ribonucleoprotein (RNP) approach, reducing the possibility of plasmid DNA integration into the cell genome and reducing the potential off-target effects. As for the impact of the correction of c.2299delG on the compound mutation, the literature suggests that one normal USH2A allele is necessary for the normal cochlea and retinal function , and hetUSH2A compound mutation iPSC line and a healthy control line from an unaffected sibling. These lines provide valuable resources and materials for USH2A disease mechanism studies, drug screening, and therapy development. In addition, we established a protocol to correct the c.2299delG mutation in the patient-derived iPSCs. In future, we will test if the genetic correction could rescue the phenotype of the mutation in the auditory and visual systems using series comparison studies between mutated and corrected iPSCs. The corrected iPSCs can be further explored for in vivo cell-based therapies, reducing the obstacles for in vivo delivery and differentiation into auditory and visual system cells. Our work on correcting the USH2A variants supports the use of this approach to test the effect of numerous variants in a variety of deafness genes using a single normal human iPSC cell line. These isogenic iPSC lines could then be differentiated into cells from the inner ear or more complex 3D inner ear organoids to disease phenotypes and therapeutic strategies.In summary, we generated a unique


No relevant improvement in patient outcomes could be achieved in the last decade in metastasized penile cancer due to insufficient identification of molecular hubs crucial for tumor evolution. We investigated the potential of the cellular receptor c-MET and selected other proteins linked to its activity to predict outcomes and for exploitation in targeted treatment. Assessing tumor tissue as well as primary cells both naïve and resistant to systemic drugs, we illustrate the most promising role of c-MET. Indeed, its elevated expression was strongly associated with inferior tumor-related survival. Moreover, its upregulation in treatment-resistant cell lines compared to naïve cells was observed. Treating cell lines with the c-MET inhibitors cabozantinib and tivantinib mediated an effective decrease in cell growth, while the first agent was more efficacious in the naïve cells and the second agent in the resistant cells. Therefore, c-MET blockade warrants further investigation in the setting of metastasized penile cancer.Whereas the lack of biomarkers in penile cancer (PeCa) impedes the development of efficacious treatment protocols, preliminary evidence suggests that c-MET and associated signaling elements may be dysregulated in this disorder. In the following study, we investigated whether c-MET and associated key molecular elements may have prognostic and therapeutic utility in PeCa. Formalin-fixed, paraffin-embedded tumor tissue from therapy-naïve patients with invasive PeCa was used for tissue microarray (TMA) analysis. Immunohistochemical staining was performed to determine the expression of the proteins c-MET, PPARg, β-catenin, snail, survivin, and n-MYC. In total, 94 PeCa patients with available tumor tissue were included. The median age was 64.9 years. High-grade tumors were present in 23.4%, and high-risk HPV was detected in 25.5%. The median follow-up was 32.5 months. High expression of snail was associated with HPV-positive tumors. Expression of β-catenin was inversely associated with grading. In both univariate COX regression analysis and the log-rank test, an increased expression of PPARg and c-MET was predictive of inferior disease-specific survival (DSS). Moreover, in multivariate analysis, a higher expression of c-MET was independently associated with worse DSS. Blocking c-MET with cabozantinib and tivantinib induced a significant decrease in viability in the primary PeCa cell line UKF-PeC3 isolated from the tumor tissue as well as in cisplatin- and osimertinib-resistant sublines. Strikingly, a higher sensitivity to tivantinib could be detected in the latter, pointing to the promising option of utilizing this agent in the second-line treatment setting. Cancer treatment based on the principle of interfering with molecules crucial for tumor cell growth, proliferation, and dissemination capacity has achieved a stunning improvement in clinical outcomes in a plethora of malignancies lately. For instance, targeted therapies have strikingly expanded the treatment armamentarium of advanced thyroid, breast, colorectal, lung, and ovarian cancers as well as leukemia ,2. AmongSeveral obstacles hamper the development of efficacious concepts of management in advanced PeCa. With an incidence of 0.45–1.7 per 100.000 population in European countries and the USA , whereasIn this context, the scientific spotlight is increasingly shone on the receptor tyrosine kinase cellular mesenchymal-epithelial transcription factor (c-MET) and the associated signaling apparatus. Contemporary evidence points to sustained c-MET stimulation, overexpression, or mutation in a variety of malignancies . Since tAfter obtaining ethical board approval, we included PeCa patients with available formalin-fixed, paraffin-embedded tissue samples provided by the tissue bank of the University Medical Center Mainz in accordance with the regulations of the tissue bank. All patients with non-metastatic invasive penile cancer treated from 1980 until 2021 were included. Patient data including age at diagnosis, concomitant diseases, primary tumor surgery, tumor grading, pathologic T (pT) and N (pN) stage, HPV status, disease recurrence, receipt of adjuvant therapy, and survival data were collected. Pathological staging was documented according to the currently valid World Health Organization (WHO) and TNM classifications. Four older cases with a missing T-stage classification were included because the pathology report described invasive cancer extension to or beyond the subepithelial connective tissue.A tissue microarray (TMA) for 94 patients with invasive PeCa was constructed as previously described . The preBiomarker expression was determined by the product of staining intensity and percentage of positively stained cells, ranging from 0 to 9 . The selrCIS2) or 2 μM osimertinib (UKF-PeC3rOSI2) by continuous exposure to increasing concentrations of the respective drugs, as described before [The UKF-PeC3 cell line was generated from a patient with a histopathologically and immunohistochemically confirmed pT3 pN0 L0 G2 R0, HPV DNA Typ 33 (high risk) positive PeCa, as previously described . The celd before . The halrCIS2 and UKF-PeC3rOSI2 were grown at 37 °C in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal calf serum , 1% Anti/Anti , and 1% glutamax , containing 2 μg/mL cisplatin (UKF-PeC3rCIS2) or 2 μM osimertinib (UKF-PeC3rOSI2) in respective resistant cell lines. Cell line authentication and validation were performed by short tandem repeat (STR) profiling . Exome sequencing did not reveal mutations in the c-MET gene .UKF-PeC3 and resistant cell lines UKF-PeC3Cisplatin was purchased from Selleckchem  and osimertinib from MedChemExpress . Cabozantinib and tinvantinib, both obtained from Selleckchem , were used for pharmacological inhibition. Cabozantinib, a receptor tyrosine kinase inhibitor (TKI) that targets multiple tyrosine kinases including VEGFR2, RET, AXL, FLT3, c-KIT, and c-MET, is clinically approved for the treatment of medullary thyroid cancer, renal cell carcinoma, and hepatocellular carcinoma . TivantiCell growth was assessed by using the 3--2,5-diphenyltetrazolium bromide (MTT) dye reduction assay. Tumor cells were seeded into 96-well microtiter plates containing serial dilutions of the indicated substances. After 24, 48, and 72 h incubation, MTT (0.5 mg/mL) was added for 4 h. Cells were lysed in a solubilization buffer containing 10% SDS in 0.01 M HCl. The plates were then incubated overnight at 37 °C. Absorbance for each well was detected at 570 nm using a microplate enzyme-linked immunosorbent assay (ELISA) reader. In order to illustrate the kinetics of dose–response, 24 h data were set to 100%.Protein concentration was determined by the bicinchoninic acid (BCA) assay. Tumor cell lysates were applied to a polyacrylamide gel and were separated for 10 min at 80 V and for 1 h at 120 V. Proteins were transferred to a nitrocellulose membrane which was blocked with non-fat dry milk for 60 min. The membranes were then incubated overnight with the following primary antibodies directed against: snail (clone C15D3), rabbit immunoglobulin IgG1, dilution 1:1000, Cell Signaling; PPARg (C26H12), rabbit immunoglobulin IgG1, dilution 1:1000, Cell Signaling; survivin (clone 71G4B7), rabbit immunoglobulin IgG1, dilution 1:1000; n-myc (clone 51705S), rabbit immunoglobulin IgG1, dilution 1:1000; β-catenin (clone D10A8), rabbit immunoglobulin IgG1, dilution 1:1000; c-MET (clone D1C2), rabbit immunoglobulin IgG1, dilution 1:1000. Horseradish peroxidase-conjugated goat-anti-rabbit IgG  served as the secondary antibody.Antibody complexes were detected using the ECL reagent  and subsequently visualized by Sapphire Imager . Membranes were stained by Coomassie brilliant blue R-250 and measured by Sapphire Imager for an internal loading control. The protein intensity/whole protein intensity ratio was determined and expressed in percentage, related to untreated controls, set to 100%.All statistical analyses were performed within the R environment for statistical computing . The non-parametric Mann–Whitney U test was used to compare differences between two independent groups when dependent variables were either ordinal or continuous. Categorical variables were compared using Fisher’s exact test. Overall survival (OS) was defined as the interval between surgical resection and death, regardless of etiology or the last follow-up. Recurrence-free survival (RFS) was defined as the interval from surgical resection to the detection of progression or death from any cause. Disease-specific survival (DSS) was calculated as the interval from surgical resection to death caused by disease. Last follow-up and death of other causes were considered censored events. Association between patient survival and biomarker expression was calculated by the log-rank test and Kaplan–Meier plots. Univariate and multivariate Cox regression models were used for statistical analysis by calculation of the hazard ratios and 95% confidence intervals. Biomarker correlations were calculated using Spearman’s coefficient analysis.p < 0.05 were considered significant.In vitro experiments were performed at least three times. Statistical significance was calculated with the GraphPad Prism 7.0  two-way ANOVA test and two-sided t-test. Differences with error probabilities of We identified a total of 94 PeCa patients with available tissue of invasive primary PeCa treated from 1980 until 2021 at our institution . The medp ≤ 0.05)  , whereasp ≤ 0.05; Survivin expression was increased in 23 males 24.5%; . MoreoveFurthermore, c-MET expression was augmented in 41 men 43.6%; .In univariate analysis, there were no significant associations between the protein expression level of the biomarker candidates c-MET, n-myc, PPARg, and snail and clinicopathological features. In contrast to β-catenin, there was no association between c-MET or PPARg expression and HPV infection .p = 0.03 and p = 0.01, respectively). Further, a positive association between snail and β-catenin was detected (p = 0.01) (c-MET expression was positively associated with PPARg and survivin expression ( = 0.01) .The median follow-up of the current study cohort was 32.5 months. Overall, 28 patients (29.8%) developed recurrent disease, of which 18 patients (19.2%) had local recurrence and 10 patients (10.2%) had systemic recurrence . A total of 16 patients (17.0%) underwent adjuvant chemotherapy, 1 patient (1.1%) received radiotherapy, and 2 patients (2.6%) were treated with chemoradiotherapy. Overall, 30 patients (31.8%) died during follow-up. Twelve patients succumbed to PeCa (12.8%).p = 0.02). Furthermore, node-positive disease (N1–N3) was associated with decreased DSS (p = 0.001)  was associated with shortened RFS (= 0.001) . There w= 0.001) .p = 0.019 and p = 0.0061, respectively), whereas no difference in RFS or OS could be detected . Furthermore, a trend for unfavorable DSS associated with a higher expression of PPARg was observed (p = 0.06)  .p < 0.001)  was an independent predictor of inferior DSS in multivariate analysis  .rCIS2) or 2 μM osimertinib (UKF-PeC3rOSI2). The IC50  value for cisplatin (5.48 μg/mL vs. 61.76 μg/mL) and osimertinib (5.04 μM vs. 17.02 μM) was distinctly increased in resistant cells compared to treatment-naïve cells, confirming acquired resistance in UKF-PeC3rCIS2 and UKF-PeC3rOSI2  was decreased significantly in resistant cells compared to treatment-naïve cells. In contrast, cabozantinib had a higher impact on tumor cell growth in the parental cell line UKF-PeC3 compared to the resistant cell lines since the IC50 value for cabozantinib (0.67 µM in UKF-PeC3 vs. 2.53 µM in UKF-PeC3rCIS2 and 2.18 µM in UKF-PeC3rOSI2) was decreased in UKF-PeC3 compared to the resistant cell lines. The trypan blue exclusion test did not show signs of toxicity.A significant growth reduction was detected in all PeCa cell lines after 72 h treatment. Thereby, the effect of tivantinib was clearly stronger in both resistant cell lines since the IC50 value for tivantinib  . cMET acValidating our observation in human tissue, c-MET expression was detected in both treatment-naïve and resistant PeCa cells, whereas its level was significantly elevated in both cisplatin- and osimertinib-resistant cell lines. This effect was the most pronounced one among all candidate molecules. While cisplatin is a backbone of chemotherapeutic regimens for the management of metastatic PeCa , no stanCorroborating the findings in our cisplatin-resistant cell line, increased expression of c-MET has been reported to be associated with resistance to chemotherapy as well as EGFR inhibitors. Li and collaborators described an elevated expression of c-MET in cisplatin-resistant cell lines as compared to parental cell lines of ovarian cancer cells . BlockadStrikingly, we observed an inverse trend of response to cell treatment with the multi-kinase inhibitor cabozantinib, approved for the therapy of advanced medullary thyroid cancer and hepatocellular and renal cell carcinoma , and theOf note, resistant cell lines exhibited diminished expression of β-catenin corresponding to its reduced staining in the tumor tissue of a higher grade in our assessment. This is in accordance with an observed decrease in the number of β-catenin-positive cases and the intensity of expression in parallel with a reduction in tumor differentiation in an immunohistochemical study on oral squamous cell carcinoma . NotablySnail, a transcriptional repressor controlling EMT, and survivin, a member of the inhibitor of apoptosis protein family, have both been shown to be activated and correlate with tumor grade, nodal metastasis, unfavorable outcomes, and drug resistance in different metastasized tumors ,47,48. IAmbivalent evidence exists on the role of PPARg, a ligand-dependent transcription factor, in tumorigenesis. While its oncogenic effects on tumor development and progression have been described in prostate cancer, studies on colon, esophageal, lung, and head and neck cancers display its antiproliferative and proapoptotic characteristics ,50. In aOwing to the rarity of PeCa, the main shortcoming of our work is the restricted number of cell lines as well as the sample size curtailing the validity of statistical models and hence the generalization of the findings. Moreover, pathological information on lymphovascular invasion and the resulting discrimination of pT1 in pT1a and pT1b disease was first available from 2010, thus ruling out inclusion of these variables in statistical modeling due to the missing values, as previously described ,51,52. NIn our study, c-MET was considered the most promising biomarker. This protein might offer an intriguing approach as a prognostic or therapeutic target, thus enabling novel therapeutic options for PeCa patients.


The results were similar to those of the A549 and U87 MG cell lines. Without T cell infiltration, PD-L1 did not increase even with upregulated interferon-γ signaling in cancer cells. In conclusions, in the glioblastoma cell line, immune-related signals were significantly upregulated at 24 and 48 h after irradiation. Therefore, the time interval between daily radiotherapy might not be enough to expect full immune responses by combined immune checkpoint inhibitors and newly infiltrating immune cells after irradiation.The time-sequential change in immune-related gene expression of the glioblastoma cell line after irradiation was evaluated to speculate the effect of combined immunotherapy with radiotherapy. The U373 MG glioblastoma cell line was irradiated with 6 Gy single dose. Next-generation sequencing (NGS) transcriptome data was generated before irradiation (control), and at 6, 24, and 48 h post-irradiation. Immune-related pathways were analyzed at each time period. The same analyses were also performed for A549 lung cancer and U87 MG glioblastoma cell lines. Western blotting confirmed the programmed death-ligand 1 (PD-L1) expression levels over time. In the U373 MG cell line, neutrophil-mediated immunity, type I interferon signaling, antigen cross-presentation to T cell, and interferon- Recently, both phase III studies of nivolumab, an immune checkpoint inhibitor of programmed death-1 (PD-1) plus radiotherapy in newly diagnosed glioblastoma (CheckMate-548 (NCT02667587) and -498(NCT02617589)) failed to meet the primary endpoint  expression, allowing T cells to recognize tumor cell targets and infiltrate into tumors   using 6 MV X-ray at 400 MU/min rate via a linear accelerator . For comparison with a wild type cell line, a cell line of human lung cancer, A549, was also cultured under the same conditions (KCLB no.10185) , with inactive mutant p53, was used in this study  via Cesium-137 Gammacell irradiator with a dose-rate of 0.48 Gy/min. We chose data with 8 Gy irradiation, which is similar to our study setting of 6 Gy.We extracted open source data of human U87 MG glioblastoma cell line  from the gene expression omnibus . The original study demonstrates cell culture and irradiation protocols for this cell line  were performed on the U373 MG and A549 cell lines before irradiation , and 6, 24, and 48 h (test groups) post-irradiation. The total RNA concentration was calculated using the Quant-IT RiboGreen (Invitrogen). Samples were run on TapeStation RNA ScreenTape (Agilent) to assess the integrity of the total RNA. Only high-quality RNA preparations (RNA integrity number > 7.0) were used for RNA library construction.A library was prepared using 1 μg of total RNA from each sample using the Illumina TruSeq mRNA Sample Prep kit . First, the poly (A)-containing mRNA molecules were purified using poly-T-attached magnetic beads. Then, the mRNA was fragmented into small pieces with divalent cations at elevated temperatures. SuperScript II reverse transcriptase (Invitrogen) and random primers were used to copy the cleaved RNA fragments into the first-strand cDNA. Subsequently, second-strand cDNA synthesis using DNA polymerase I and RNase H were performed. These cDNA fragments then underwent an end repair process, the addition of a single ‘A’ base, and the ligation of indexing adapters. The products were purified and enriched via PCR to generate the final cDNA library. The libraries were quantified using qPCR, according to the qPCR Quantification Protocol Guide (KAPA Library Quantification Kits for Illumina sequencing platforms), and validated using TapeStation D1000 ScreenTape (Agilent Technologies). Indexed libraries were then sequenced on the HiSeq2500 platform (Illumina) by Macrogen Incorporated.The study that provided the corresponding data for the U87 MG cell line harvested cells at various time points postirradiation. We chose time points similar to our study .The fragments per kilobase of transcript per million mapped reads (FPKM) of genes for each period  were extracted, and genes with more than five FPKM in at least one period alone were selected for further analysis of the U373 MG and A549 cell lines. For these genes, the log2-fold change (LogFC), comparing the expression level between the test groups  and the control group , was calculated.https://software.broadinstitute.org/morpheus/). After hierarchical analysis according to the LogFC patterns over time, functional enrichment analysis for each clustered group was performed using the Enrichr (https://amp.pharm.mssm.edu/Enrichr/enrich) and Reactome programs (https://reactome.org/).First, a heatmap was generated according to the LogFC value in each period using the Morpheus program -based adjusted P-value  was automatically evaluated. A P- or Q-value < 0.05 was considered statistically significant.Second, in each time group , genes upregulated than before irradiation with a fold change ≥1.2 (LogFC ≥0.263034) were extracted. Using the Enrichr and Reactome programs, functional enrichment analyses for the gene sets were performed based on the gene ontology (GO) terms and the Reactome’s stable identifiers, respectively. We mainly observed the time-sequential change of immune-related signals including the expression level of neutrophil-mediated immunity, type I interferon, antigen-presenting, and interferon-Third, we examined the time-sequential change in LogFC values of PDL1 and TGFB1 which encode PD-L1 and TGF-ß1 in the NGS data.Cell culture, irradiation, RNA isolation, sequencing, and functional enrichment analysis were performed for the U87 MG cell line using the same methods as for the U373 MG cell line. In the GEO datasets, the p53 wild type glioblastoma cell line (U87 MG) dataset was selected (GSE56937). Six samples obtained, three each from the control (no irradiation) and test (8 Gy irradiation) groups after day one were compared because the test scheme was similar to our study. By using the GEO2R program, LogFC was automatically calculated. Genes with a fold change ≥1.2 (LogFC ≥0.26) were extracted and functional enrichment analysis was performed. The result was compared with that of the U373 MG and A549 cell lines.Cell lysates were prepared in cell lysis buffer (iNtRON Biotechnology). The total cellular proteins were separated via SDS-PAGE (10 μg for each period) and transferred to nitrocellulose membranes (Millipore Corp.). The membranes were blocked with blocking solution in 5% nonfat dry milk  for 1 h and probed overnight at 4°C with primary rabbit polyclonal IgG antibody at a dilution of 1:1000 . The antibodies for CD274 and TGFB1 were purchased from Cell Signaling Technology (Beverly). The membranes were incubated with blocking solution containing a dilution of HRP-conjugated goat anti-rabbit IgG as a secondary antibody  at 1:3000 for 2 h. Western blot protein detection was performed using the ECL kit (Intron Biotechnology) according to the manufacturers’ recommendations. As a control, a monoclonal antibody against actin  was used. The expression level of PDL1 and TGFB1 of the U373 MG cell line were examined via western blotting. The cells were treated with media, irradiated with 6 Gy, and then collected at 0 h (before irradiation), and then, 6, 24, and 48 h after radiation, as for the previous NGS data analysis.We examined the time-sequential change of immune-related pathways after irradiation in glioblastoma to estimate the effect of combined immuno-radiotherapy. We hypothesized that the time-series characteristics of regulation after radiation may differ for each immune-related pathway. Since conventional radiotherapy for glioblastoma is a daily schedule, we examined the time-sequential change pattern of radiation-induced expression up to 6, 24, and 48 h after irradiation.After NGS of the U373 MG cell line at each designated time period post-radiation, genes with >5 FPKM in at least one period were selected. A total of 9278 genes were detected. A hierarchical heatmap analysis of LogFC value of each gene at each time period generated four clusters with the gene numbers 2572, 631, 5503, and 572.Functional enrichment analysis was performed for each of these four gene groups . The firP = 1.63E-08; FDR Q = 4.27E-07; Odds ratio [OR], 1.861), while the neutrophil-mediated immunity increased gradually until 48 h .Gene expressions of innate immune response activating cell surface receptor signaling pathway increased at 6 h after irradiation and decreased over time  and the cellular response to type I interferon was gradually upregulated until 48 h .Type I interferon was actively produced at 6 h after irradiation , while the expression levels of folding, assembly and peptide loading of class I MHC molecules  and cross-presentation to stimulate CD8+ T cell immunity  were progressively upregulated until 48 h after irradiation.The expression of antigen processing and presentation related to ubiquitination and proteasome degradation was predominant at 6 h after irradiation and reduced over time  when observed up to 48 h after irradiation.The interferon-These results suggest that immune cells (neutrophil and antigen-induced cytotoxic T cells) would be recruited toward tumor cells at least after 24 h post-irradiation, resulting in a narrow window period for immune-mediated tumor cell death before the next radiotherapy schedule in glioblastoma.The previous clustering analysis was an analysis of time-sequential changes in each immune pathway. Inversely, we further assessed the significantly upregulated immune pathways in each period and verified whether the results are similar to the previous clustering analysis.γ-mediated signaling pathway (GO:0060333) were observed. These all signal pathways were significantly upregulated at 24 h after irradiation and the significances were maintained until 48 h  in the U373 MG cell line was performed. Immune-related signals – neutrophil-mediated immunity (GO:0002446), type I interferon signaling pathway (GO:0060337), antigen processing and presentation of peptide antigen via MHC class I (GO:0002474), and interferon-P = 1.97E-08; FDR Q = 2.01E-05; OR, 4.341 and TP53 Regulates Transcription of Cell Death Genes; R-HSA-5633008; P = 1.43E-05; FDR Q = 0.012). However, the immune-related signals showed a similar result with the U373 MG cell line. All immune-related signals were significantly upregulated at 24 and 48 h after irradiation . Lung cancer cell line was selected considering a recent successful immunotherapy trial in lung cancer to analyze the difference in immunologic pathway after irradiation between glioblastoma and lung cancer  from a study performed at Center of Cancer Systems Biology . Neutrophil-mediated immunity showed borderline significance . These results show that the immunologic signals increased gradually in both cell lines up to at least 48 h post-irradiation, and 24 h post-irradiation may not be the optimal time for a complete immune response.The number of genes upregulated in the U373 and A549 cell lines increased over time . The number of genes commonly upregulated in both cell lines also increased . This corresponds to 2.5%, 22.6%, and 29.1% of the upregulated genes in the U373 cell line. There were no statistically significant immune signals at 6 and 24 h in the enrichment analysis of these genes. However, at 48 h, the type I interferon signaling pathway was detected as a significantly increased signal  and 250  between the U87 and A549 cell lines. No significant immune signals were observed in enrichment analyses for these common genes. Gene lists are provided in Supplementary Tables S1–S5.γ signaling increased in the U373 MG cell line in the NGS data analysis, the expression level of PDL1 gene did not increase over time, suggesting that without T cell infiltration, the 6 Gy single dose irradiation to the U373 MG glioblastoma cell line did not upregulate PD-L1 1 therapy with daily fractionated radiotherapy might not be fully effective in glioblastoma because of the time required for re-infiltration of tumor antigen-specific T cells after the previous infiltrating T cells were killed by radiotherapy.Our study observed the time-sequential change in immune-related gene expressions after irradiation of glioblastoma cells. We mainly focused on speculating the optimal time points for combining radiotherapy and anti-PD-(L)1 treatment. We found that neutrophil-mediated immunity, antigen processing/cross-presentation to activate cytotoxic T cells, and radiation-induced-interferon-γ by infiltrating T cell, and increased interferon-γ provokes tumor cells to generate PD-L1 to make T cells dysfunctional  after irradiation . Therefore, the results of this study suggest that T cell infiltration induced by interferon-γ from tumor cells rarely occurs until at least 1–2 days after irradiation.In our study, although interferon-γ was reduced significantly 1 therapy may not be effective.Second, since there was no T cell in our cell line study, the amount of PD-L1 might be less than when T cells are present. When T cells were inhibited, the amount of interferon-Recent trials showed improved results when radiotherapy and immunotherapy were combined as an adjuvant (after radiotherapy) or neoadjuvant (before radiation) settings. The Pacific trial demonstrated a survival benefit from the adjuvant anti-PD-1 therapy after chemo-radiotherapy in stage III non-small-cell lung cancer -radiotherapy in newly diagnosed glioblastoma after surgery  1 treatment, enough density of infiltrating tumor antigen-specific T cells is indispensable. Radiotherapy affects all cells in the target field. Moreover, lymphocytes are radio-sensitive. Therefore, after irradiation, it would require time to re-infiltrate new T cells in the tumor microenvironment.PD-L1 expression was higher in the p53 mutant tumor than in the p53 wild type cancer, and the p53 mutant tumor was highly sensitive to the anti-PD-L1 therapy 1 therapy against T cell-mediated PD-L1 in tumor cells might not be fully synergistic with daily radiotherapy treatment schedule.Click here for additional data file.


Cell cycle regulators are frequently altered in Triple-Negative Breast Cancer (TNBC). Emerging agents targeting these signals offer the possibility to design new combinatorial therapies. However, preclinical models that recapitulate TNBC primary resistance and heterogeneity are essential to evaluate the potency of these combined treatments.Methods: Bioinformatic processing of human breast cancer datasets was used to analyse correlations between expression levels of cell cycle regulators and patient survival outcome. The MetMMTV-R26 mouse model of TNBC resistance and heterogeneity was employed to analyse expression and targeting vulnerability of cell cycle regulators in the presence of BCL-XL blockage. Robustness of outcomes and selectivity was further explored using a panel of human breast cancer cells. Orthotopic studies in nude mice were applied for preclinical evaluation of efficacy and toxicity. Alterations of protein expression, phosphorylation, and/or cellular localisation were analysed by western blots, reverse phase protein array, and immunocytochemistry. Bioinformatics was performed to highlight drug's mechanisms of action.Results: We report that high expression levels of the BCL2L1 gene encoding BCL-XL and of specific cell cycle regulators correlate with poor survival outcomes of TNBC patients. Blockage of BCL-XL confers vulnerability to drugs targeting CDK1/2/4, but not FOXM1, CDK4/6, Aurora A and Aurora B, to all MetMMTV-R26 and human TNBC cell lines tested. Combined blockage of BCL-XL and CDK1/2/4 interfered with tumour growth in vivo. Mechanistically, we show that, co-targeting of BCL-XL and CDK1/2/4 synergistically inhibited cell viability by combinatorial depletion of survival and RTK/AKT signals, and concomitantly restoring FOXO3a tumour suppression actions. This was accompanied by an accumulation of DNA damage and consequently apoptosis.Conclusions: Our studies illustrate the possibility to exploit the vulnerability of TNBC cells to CDK1/2/4 inhibition by targeting BCL-XL. Moreover, they underline that specificity matters in targeting cell cycle regulators for combinatorial anticancer therapies. Targeting of the anti-apoptotic protein BCL-XL together with anti-mitotic agents has been proposed as an efficient therapeutic strategy for different human cancers, including triple-negative breast cancer (TNBC), a particular aggressive subtype of breast cancer MetMMTV-R26 mice) leads to spontaneous tumour formation faithfully recapitulating TNBC features MetMMTV-R26 TNBC model to identify a highly effective therapeutic protocol based on the combined inhibition of the anti-apoptotic molecule BCL-XL and of the cell cycle checkpoint regulator WEE1 INK4 is frequent across TNBC subtypes We have recently reported the generation of a rather unique mouse model in which a subtle increase in the wild-type MET RTK expression levels in the mammary gland MMTV-R2Met mice MetMMTV-R26 model system that recapitulates TNBC heterogeneity and primary resistance to explore the effect of inhibiting specific cell cycle regulators while blocking BCL-XL function. Our results show that targeting specific cell cycle components is important and uncover a detrimental effect on TNBC cells of CDK1/2/4 plus BCL-XL inhibition in vitro and in vivo. We provide evidence that this combinatorial targeting significantly reduces the levels of RTK and AKT signalling besides perturbing cell cycle and DNA repair.In the present study, we employed the MetMMTV-R26 MGT cell lines were derived from independent MetMMTV-R26 tumours and established as previously described 2 atmosphere. PCR-based assays were performed on all cell lines to verify that they were free of Mycoplasma contamination. Mouse embryonic fibroblasts were kindly provided by P. Perrin, either untreated or treated with Mitomycin C (Sigma).All human TNBC  and non-TNBC  cell lines used in this study were obtained from the American Type Culture Collection (ATCC) without further authentication and were tested by PCR-based assay to verify that they were free of Mycoplasma contamination. These human breast cancer cells were grown in DMEM/F12 medium supplemented with P/S, glutamine (2 mM), sodium pyruvate , non-essential amino acids (ThermoFisher Scientific), and insulin (10 µg/mL).6) were injected bilaterally into the fourth mammary fat pad of 6-week-old female nude (Rj:NMRI-Foxn1 nu/nu) mice. Eleven days later, mice were randomized into two groups (n = 8 per group) and treated with either vehicle or the A1155463 + R547 drug combination. A1155463 was administered intraperitoneally  + 5% Tween 80). The R547 compound was injected into the tail vein twice per week ). Mice were treated for 20 days. Mouse body weight and tumour progression were monitored 3 times per week. Tumour volumes were determined using a digital calliper and calculated according to the following formula: V (volume) = L (length) × W (width)2 / 2. At the end of the experiment, mice were sacrificed, tumours were excised. Tumour volumes and weights were then measured.All procedures involving the use of animals were carried out in accordance with the European Community Council Directive of 22 September 2010 on the protection of animals used for experimental purposes (2010/63/EU). The experimental protocols were performed according to the institutional Ethical Committee guidelines for animal research  and in compliance with the French law under the agreement number D13-055-21, delivered by the “Préfecture de la Région Provence-Alpes-Côte-d'Azur et des Bouches-du-Rhône”. The mouse project authorization of the Maina laboratory is: APAFIS #8214-2016121417291352.v5, delivered by the “Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation”. For orthotopic studies, MDA-MB-231 cells , R547 (3 µM), alone or in combination. After trypsinisation, cell suspension was then processed as previously described Protocols used were as described in Protein extracts were prepared and western blot analysis was performed as previously described MetMMTV-R26 tumours and control mammary glands (from MMTV-Cre mice), or MGT cells treated or not with A1155463 (1 µM), R547 (3 µM), or the drug combination A1155463 + R547  for 12 h, were prepared according to the MD Anderson Cancer Center platform instructions. RPPA of cells treated with Adavosertib (3 µM), or A1155463 + Adavosertib  was previously reported Protein lysates of either MetMMTV-R26 tumours (n = 4) and control mammary gland tissues (n = 3) was processed for transcriptome analysis. RNA integrity was assessed using the Agilent RNA 6000 Pico kit and Agilent 2100 Bioanalyzer  according to the manufacturer's instructions. Sequencing was performed as previously described Total RNA from dissected Studies were performed as previously described BCL2L1 gene encoding BCL-XL, and/or specific cell cycle regulator levels. The NCBI dataset used was the GSE31519 Affymetrix Human Genome U133A Array. The database includes 580 TNBC patients phenotyped by immunohistochemistry.Analysis of publicly available microarray data: Kaplan-Meier curve reporting the probability (in percent) of the overall survival of human TNBC patients according to the MetMMTV-R26 tumours versus controls. Ranking of the enriched pathways was then performed using the Enrichr software. RPPA analyses of drug perturbation effects were done using biological duplicates. Expression levels of proteins were Log2 transformed before analysis. GSEA was employed to determine the statistically significant enriched pathways between cells treated with either A1155463 + R547 or A1155463 + Adavosertib. Differential gene expressions were obtained from the RPPA data outcomes. GSEA v4.1.0 was obtained from the Broad Institute (http://software.broadinstitute.org/gsea/index.jsp). The collection of annotated gene sets of REACTOME was obtained from the GSEA website .Raw RNA-seq reads were mapped against the latest release (mm10) of the mouse genome using STAR aligner The probability of overall survival rates was calculated using the Kaplan-Meier method. P values were computed using the Logrank (Mantel Cox) test. P values are indicated in figures. P > 0.05 was considered as non-significant (ns). * P < 0.05; ** P < 0.01; *** P < 0.001. For RPPA studies, analysis of fold-change proteins and P values to determine significantly differentially expressed proteins were done by the Limma package in R. Data are presented as the mean ± standard error of the mean (s.e.m.), according to sample distributions. For two sided comparisons, unpaired Student's t test was used for data showing normal distributions, and Wilcoxon test was used in other situations. For multiple comparisons, we used ANOVA test followed by Tukey test. All statistical analyses were performed using the GraphPad Prism and software.+ and TNBC patients show the highest frequency of alteration in cell cycle regulators  have reported a strong enrichment in cell cycle pathway genes s Figure D. Gene as Figure D. We nexe Figure . Interess Figure . Intrigul Figure . The apps Figure . Furtherpressers A,B. ThesMetMMTV-R26 TNBC model system, employing RNA-seq analysis, proteomic profiling, and cell viability assay as illustrated in Figure MetMMTV-R26 tumours (n = 4) to control mammary gland tissues , we found a striking enrichment in genes related to cell cycle regulation , Aurka and Aurkb (cell cycle regulated kinases involved in microtubule formation and/or stabilization at the spindle pole during chromosome segregation), and Cyclin-dependent kinases Cdk1, Cdk2, and Cdk4 (which control progression through the cell cycle in concert with their cyclin regulatory subunits). Specific cyclin genes such as Ccna2, Ccnb1, Ccnb2, and Ccne1 encoding CYCLIN A2, B1, B2, and E1, respectively, were also upregulated in MetMMTV-R26 tumours  cell lines  derived from the in vivo experiments by injecting orthotopically MDA-MB-231 human TNBC cells into immunecompromised nude mice , the molecular and biological consequences of CDK1/2/4 targeting (by R547) ll lines . We firs5463, thell lines  and S10.ll lines F. CollecWe then analysed the cell cycle effects of R547 alone or in combination with BCL-XL targeting (with A1155463), by following the distribution of cells in the cycle phases through flow cytometry. In the presence of R547, we observed a decrease in the percentage of cells in S phase accompanied by an accumulation in G2 Figure . These r10Histone H3 (pH3)-positive cells and did not trigger any mitotic catastrophe events in comparison to those observed in BCL-XL + WEE1 treated cells , while BCL-XL + WEE1 targeting mainly impacted regulators of cell cycle and DNA repair Figure D. DNA daMetMMTV-R26 MGT cells treated with BCL-XL + CDK1/2/4 inhibitors  and three non-TNBC  cell lines, we observed strong MET expression in all three TNBC cell lines analysed (Figure PUMA and FasL, two FOXO3a target genes involved in apoptosis (Figure The correlation between reduced phosphorylation of AKT and FOXO3a was particularly interesting, considering that AKT phosphorylates FOXO3a leading to its translocation from the nucleus to the cytoplasm, thus preventing its tumour suppressor function s Figure I. TogethMetMMTV-R26 tumours. Such combination appears to be less effective on non-TNBC cells, based on the cell lines used in this study.In this study, we provide evidence that combinatorial targeting of BCL-XL with CDK1/2/4 could be an efficient therapeutic approach for TNBC. This drug combination was potent across different TNBC subtypes, as demonstrated by its high efficacy in different human TNBC cell lines and in the heterogenous cell lines generated from distinct c-Myc activation, p53 mutations, PTEN-loss, and CDKs/Cyclins overactivation/overexpression CDK1, CDK6, and WEE1, are characterised by a worse prognosis compared to those with low expression levels. Interestingly, we have also shown that poor TNBC prognosis is further exacerbated by high expression levels of BCL-XL.The alteration of cell cycle regulators in several types of cancer MetMMTV-R26 model, which recapitulates several features of TNBC including primary therapy resistance and marker intertumoral heterogeneity. This model has previously allowed us to uncover the potency of WEE1 targeting while lowering survival inputs through BCL-XL inhibition The identification of vulnerabilities of cancer cells is particularly challenging for those types of cancer, like TNBC, highly heterogeneous and lacking major drivers to which cells are addicted to CDK1, CDK2, CDK4, FoxM1, Aurora A/B, but not of CDK3, CDK5, CDK6, genes in MetMMTV-R26 tumours versus normal mammary glands, as reported in TNBC patients MetMMTV-R26 TNBC cell lines we examined. This apparent contradiction to previous studies MetMMTV-R26 TNBC cells In this regard, an intriguing aspect emerging from the present studies is that in the presence of BCL-XL, the specific subset of cell cycle regulator targeted determines the effect on TNBC cell viability. Specifically, TNBC cells are sensitive to combined inhibition of CDK1/2/4 by R547, but not of FOXM1, CDK4/6, Aurora A and B. FOXM1 is a transcription factor overexpressed in most solid tumours, such as breast, liver, prostate, colon, and pancreas Besides regulating cell cycle, new functions of CDK1/2/4 have recently emerged. It has been shown that CDK1/2 targets are hyperphosphorylated in basal-like breast cancer, generating genome integrity vulnerability Supplementary figures and tables.Click here for additional data file.