DEFA6, ABCB1 and GAL), whereas analysis of chromosomal copy number aberrations revealed no significant variation over time. These data indicate that although CTC lines derived from sequential blood samples during therapy have common traits, treatment-resistant CTC clones with distinct phenotypic characteristics are selected over time.Circulating tumor cells (CTCs) are important clinical indicators for prognosis and treatment efficacy. However, CTC investigation is hampered by their low number, making the establishment of permanent CTC lines very challenging. We derived and characterized nine CTC lines using blood samples from a patient with metastatic colorectal cancer collected before and after chemotherapy and targeted therapy, and during cancer progression. These cell lines displayed an intermediate epithelial/mesenchymal phenotype, stem-cell like characteristics, angiogenesis potential, an osteomimetic signature and the capacity to escape from the immune system. Moreover, they showed changes in mRNA and protein expression (e.g.,  Several large-scale clinical trials and meta-analyses have shown that CTC number is an important indicator of the risk of cancer progression or death in patients with metastatic solid tumors7. However, CTC investigation is hampered by their very low number, especially in blood of patients with colorectal cancer. Therefore, the establishment of in vitro cultures and permanent lines from CTCs has become the most challenging task over the past years. Indeed, CTC lines could be used to identify proteins and pathways involved in cancer cell stemness and dissemination, and also to test new drugs to inhibit metastasis-competent CTCs. Ex vivo CTC cultures have been established for breast9, prostate10, lung11, and head and neck cancer12. We previously described the first permanent cell line (CTC-MCC-41) from circulating colon cancer cells13. However, its establishment was very difficult (blood samples of 168 patients were tested). This could be partly explained by the much lower CTC number in the peripheral blood of such patients than in patients with breast or prostate cancer, making very difficult their enrichment and culture. In addition to its capacity to expand in vitro for more than 4 years, the CTC-MCC-41 cell line shows specific stem-cell like characteristics and shares some features of the original primary tumor and lymph node metastasis13.Over the past years, liquid biopsy of circulating tumor cells (CTC) has received major attention as a strategy to obtain real time diagnostic and prognostic informationWe then established another eight CTC lines from blood samples collected from the same patient at different time points during his follow-up. This unique biological material represents a chance to study clonal selection and resistance mechanisms during tumor progression and treatment. Here, we report the establishment of these new CTC lines from the same patient with metastatic colon cancer, and their characterization . Comparison of all nine autologous CTC lines (the previously described CTC-MCC-41 line and the eight new lines) highlighted their common features and main differences acquired over time.13 from the patient with the highest CTC number (302 CTCs/7.5 mL of blood) and with 38 CTC clusters (<2 to 5 CTCs/microemboli) (patient 044) . Before the first-line treatment, CTC number was evaluated in 7.5 mL of peripheral blood using the CellSearch system, and then another 10 mL of peripheral blood was used for CTC enrichment and culture. CTC number was ≥1 in 57.5% of patients and ≥3 in 39.4% . Only one colon CTC line (CTC-MCC-41) could be established44) Fig. .Figure 1in vitro. In addition, based on the CellSearch cell images, CTCs were present as single cells and clusters in the first samples, and as single cells in the last sample at the time of the third relapse  and colospheres with a size that could reach 100 µm (50%) Fig. , as obseIn the CTC-MCC-41.5 lines, CTCs started to proliferate in different wells at different time points. Therefore, they were kept separated, giving rise to the different lines . The first three CTC-MCC-41.5 lines  appeared after 18 days of culture. They showed different morphologies and behaved differently. CTC-MCC-41.5D cells grew as isolated cells (50%) and also as big colospheres (up to 100 µm). The CTC-MCC-41.5F and CTC-MCC-41.5G lines were composed essentially of isolated cells (70%) and small colospheres (up to 50 µm) for the remaining 30%. After 24 days of culture, three additional CTC lines appeared: the CTC-MCC-41.5A line, characterized by the presence of isolated tumor cells and colospheres (50%-50%); the CTC-MCC-41.5B line, with 70% of small isolated cells and few small colospheres (30%); and the CTC-MCC-41.5C line, made mostly of large colospheres  and few isolated cells (20%). Finally, after 34 days of culture, the last CTC line (CTC-MCC-41.5E) displayed isolated cells (50%) and big colospheres (50%)  were assessed genome-wide in two single cells and one colosphere for each CTC line by next-generation sequencing (NGS). This analysis revealed a wide spectrum of chromosomal aberrations Fig. . TypicalBRAFV600E mutation with a stable allele frequency of 50%, in agreement with a heterozygote mutation (Supplementary Table KRAS was wild type (wt) in all CTC lines. This mutation profile (BRAFmutKRASwt) mirrors the one detected in the primary tumor, lymph node metastasis, and also tumor xenografts from immunodeficient mice after injection of CTC-MCC-41 cells13.Concerning specific mutations, all samples carried the 14, and included genes encoding molecules involved in many tumor cell features .To characterize these CTC lines, the transcriptome profiles of the eight new colon CTC lines were compared with those of the first line (CTC-MCC-41) and of two commercial colon cancer cell lines . The panel of messenger RNAs (mRNAs) to be analyzed (Supplementary Table EPCAM), cytokeratin 19 (CK19), E-cadherin (ECAD), CMET, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), vascular endothelial growth factor (VEGF), snail family transcription repressor 1 (SNAIL) and aldehyde dehydrogenase 1 (ALDH1), but not CD45. This confirmed the epithelial origin of all these cell lines. A second group included transcripts that were differentially expressed in the colon CTC lines and in HT-29 and SW620 cells: (i) interleukin 33 (IL33), cyclin D2 (CCND2), semaphorin 6A (SEMA6A) and osteoprotegerin (OPG) that were expressed only in the colon CTC lines; and (ii) SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily A, member 1 (SMARCA1) and dickkopf WNT signaling pathway inhibitor 1 (DKK1) that were expressed only in HT-29 and SW-620 cells. The third group included genes that (i) were expressed in the colon CTC lines and in SW620 cells: B-cell lymphoma 11A (BCL11A), tumor necrosis factor receptor superfamilly 1B (TNFRS1B), bone morphogenetic protein 7 (BMP7) and fibronectin 1 (FN1); and (ii) were not expressed in the CTC lines and SW620 cells: adam metallopeptidase with thrombospondin type 1 motif 6 (ADAMTS6), Gata binding protein 2 (GATA2), gap junction protein beta 6 (GJB6), transforming growth factor beta 2 (TGFB2), prostaglandin endoperoxidase synthase 2 (PTGS2), bone marrow stromal cell antigen 2 (BST2), Wnt family member 11 (WNT11) and TWIST. The last group included genes that were downregulated in all colon CTC lines and in HT-29 cells, but expressed in SW620 cells: SRY-related HMG-box gene 2 (SOX2) and vimentin (VIM). Hierarchical clustering analysis based on the mRNA expression profile clearly segregated the CTC lines from the HT-29 and SW-620 cell lines , but none of the tested mesenchymal markers (N-cadherin and vimentin). In addition, the expression of CD133 and CD44v6 and the CD24(low)/CD44(high) ratio indicated stem cell-like characteristics15. All CTC lines weakly expressed VEGF and PanCD66 , also known as CEACAM . The absence of CD45 expression confirmed that these cells were not leucocytes. Moreover, the eight new colon CTC lines did not express endothelial proteins (CD31 and CD105), hematopoietic progenitor markers (CD34) and C-X-C chemokine receptor type 4 (CXCR4), a receptor expressed on breast tumor cells and involved in the chemoattraction to specific organs16  was assessed in the nine colon CTC lines by flow cytometry Fig. . As alrein vitro endothelial cell tube formation assay, and ALDH1 activity assay , and secrete VEGF (95% of cells)  could be established from CTCs isolated from the blood of a patient with metastatic colon cancer13 and present data on the establishment of colon CTC lines suggest that a CTC count of approximately 300 (using the CellSearch system) in blood samples from patients with colon cancer is a prerequisite for successful CTC expansion in culture. However, the number of CTCs is lower in the blood of patients with colorectal cancer that with other cancer types. In patients with colon cancer, viable CTCs can be captured in mesenteric blood samples, indicating that many of them are trapped in the liver22. Furthermore, this specific patient had the BRAF mutation, which is relatively rare (about 10%) and highly aggressive in CRC23. Indeed, only 8 out of 95 patients were positive for this rare colorectal mutation. Interestingly, no correlation between the BRAF mutation and a high number of CTCs has been observed in the COLOSPOT study. All these data clearly show how challenging it is to establish a CTC line from patients with colon cancer even with distant metastases, and may explain why no CTC culture or cell line has been reported in the literature, with the exception by our group.Our previousBRAF mutations that were detected with a stable frequency in all CTC lines.To verify that these cell lines are of cancer origin, we performed genotyping analyses. CNA analysis by NGS demonstrated that these CTC lines were cancer cell lines with cancer–specific genomic aberrations. This analysis highlighted CNA differences between samples (colosphere and single cells) from the same CTC line and also between lines, indicating that these lines have a polyclonal origin. However, we did not observe any CNA associated with disease progression. Indeed, the first CTC line (obtained at diagnosis) and the other lines (obtained at different time points after treatment) were all highly heterogeneous at the inter- and intra-line level. This suggests that therapy did not cause any significant change in the cancer CNA signature. However, we cannot exclude therapy-driven selection of resistance-conferring mutations, other than 24. Recent studies have emphasized that CTC clusters may have an increased malignant capacity25, and microemboli were observed in the blood samples of this patient with metastatic colon cancer during his follow-up.Visual monitoring of the cultures showed again a clear intra- and inter-line heterogeneity: colospheres as well as small and big single cancer cells. This suggests the absence of a preferred morphology for these tumor cells, consistent with the observation that both small and large CTCs are linked to unfavorable prognosis26. All nine CTC lines presented a stable epithelial phenotype, as indicated by the expression of epithelial markers, such as EPCAM and cytokeratins . Concerning markers of mesenchymal transition, all nine CTC lines expressed FN1 and SNAIL, but not vimentin or N-cadherin (mRNA and protein). Moreover, in all CTC cell lines, ECAD was weakly expressed and ADAMTS6, GATA2, GJB6, TGFB2, PTGS2 and BST2, genes encoding cell-cell junctions, focal adhesion or cytoskeletal proteins, were clearly downregulated14. This is in accordance with CTC loss of adherent cell properties to leave the tumor and reach the bloodstream. These data indicate that these colon CTC lines are tumor cells with an intermediate EMT phenotype. The acquisition of mesenchymal features by CTCs could reveal some plasticity required for dissemination to distant organs26. In addition, the expression of TNFRSF1B and BCL11A, linked to apoptosis suppression27, could indicate another CTC hallmark called «anoikis resistance», a mechanism to avoid cell death when tumor cells are not attached, but swim in the bloodstream18.Epithelial-to-mesenchymal transition (EMT) is a complex process that supports the migratory capacity of epithelial tumor cells. EMT also plays a crucial role in promoting cancer metastasis by various mechanisms, including CTC release from the primary tumor and distant metastases15, BMP7, GAL and SEMA6A, and the CD44high/CD24low ratio28, were detected in all nine CTC lines. Moreover, they all showed ALDH activity, a well-known stem cell marker17. The combination of EMT phenotype and stem-cell characteristics should favor their dissemination and ability to form distant metastases29.Stemness features, such as expression of CD133, CD44v6 OPG, BMP7 and TNFRSF1B (involved in osteogenic activity) suggests that these CTCs have the ability to lodge in the bone marrow. However, this patient developed liver metastases, without any evidence of bone metastases. Therefore, we hypothesize that these CTCs could have been released from the bone marrow where they were preserved in special niches as dormant tumor cells with the ability to mimic osteogenic cells.In addition, the expression of 30. However, PD-L1 expression was not detected in any of the nine CTC lines from this patient with colon cancer. Indeed, PD-L1 expression can vary, and it is still debated whether this protein is expressed more at early31 or late, metastatic stages33. Conversely, CEACAM1, DEFA6 and IL33, three genes encoding proteins also involved in immune escape, were expressed by all nine CTC lines. CEACAM1 upregulation at advanced stages of colorectal cancer is associated with invasiveness, metastasis, tumor progression and escape from the immune system34. Upregulation of DEFA614 and IL3335 also is involved in immune mechanisms. Therefore, these three factors could be attractive targets for immunotherapy.Concerning the immune system escape and survival, the PD-1/PD-L1 axis has been extensively studied in the last years after the introduction of cancer immunotherapy. Recently, we demonstrated that CTCs in breast cancer can express PD-L1 as a camouflage against T-lymphocytes13. Moreover, a functional test based on in vitro tube formation using endothelial cells showed that CTC-MCC-41, 41.4, 41.5B, 41.5E and 41.5F cell supernatants induced a denser network of endothelial tubes than those of CTC-MCC-41.5C, D and G cells. These data highlight again the heterogeneity of the nine CTC lines. As the fluoro-EPISPOT assay showed that VEGF secretion was comparable in all nine CTC lines, other factors must be involved in CTC-induced angiogenesis. For instance, semaphorin 6A, a member of the semaphorin family involved in cell motility and proliferation, also contributes to vascular development and particularly to angiogenesis by modulating VEGFR2 signaling in endothelial cells36. The stronger SEMA6A expression in the CTC-MCC-41, 41.4, 41.5B, 41.5E and 41.5F lines could explain the heterogeneous results of the functional assay. Moreover, Loria et al. showed that forced SEMA6A overexpression induces anchorage-independent growth and significantly increases invasiveness. Interestingly, SEMA6A is overexpressed in BRAFV600E melanoma cells36. This BRAF mutation was detected (i) in the primary tumor, (ii) in all the CTC lines, (iii) the lymph node metastasis, and also (iv) in tumor xenografts obtained by injection of CTC-MCC-41 cells13. Thus, SEMA6A might represent a new potential therapeutic target in BRAFV600E cancers.Another important feature of aggressive cancer cells is the capacity to induce neo-angiogenesis that is required to import nutrients and oxygen in the growing tumor. All the new CTC lines expressed and secreted VEGF, a key inducer of angiogenesis, as already shown for the CTC-MCC-41 lineIn conclusion, we established nine permanent colon CTC lines from the same patient with metastatic colon cancer at different time points during disease progression. We then thoroughly characterized these CTC lines, highlighting their heterogeneity, which suggests that they are derived from different clones, and also their common features. Indeed, they all show (i) an intermediate epithelial/mesenchymal phenotype, (ii) stem-cell like characteristics, (iii) the potential to induce angiogenesis, (iv) an osteomimetic signature, and (v) the capacity to escape from the immune system. These features suggest that they could initiate metastases at distant sites. Moreover, in these CTC lines, we observed changes in RNA and protein expression, while the analysis of chromosomal CNA revealed no significant pattern of selected genomic alterations. For instance, the stem-cell like characteristics progressively increased in the CTC lines isolated at later stages of cancer progression. These data indicate that CTCs cultured from sequential liquid biopsies during therapy have common traits, but that treatment-resistant clones with distinct phenotypic characteristics are selected.in vitro CTC culture.After signature of the informed consent, peripheral blood samples from patients with non-resectable metastatic colorectal adenocarcinoma were collected before the start and during first-line chemotherapy (FOLFIRI and bevacizumab) in the framework of the COLOSPOT study . The study was carried out in accordance with the World Medical Association Declaration of Helsinki and the guidelines for experimentation with humans by the Bioethical committee “Sud Méditerranée III”. The experimental protocol was approved  by the Bioethical committee “Sud Méditerranée III”. All included subjects provided written informed consent for participation in this study and the publication of results. The Blood (10 mL) was collected in CellSave tubes  for CTC enumeration and in EDTA tubes (10 mL) for Patient 044 was diagnosed at an advanced stage of colon cancer . He showed good tolerance to the first-line treatment (FOLFIRI + bevacizumab), but, after 5 cycles, had to stop it because of cancer progression (RECIST criteria). A second-line treatment was immediately initiated (FOLFOX + bevacizumab), but again, the treatment was stopped after three cycles due to clinical progression , and no other treatment was planned. The patient was hospitalized for abdominal pain and occlusive syndrome, and a new relapse was diagnosed. He died 6 months after the diagnosis of cancer, 2 months after the last treatment, and 1 week after the last blood sample collection. Additional clinical data about this special patient are available in the Supplementary Table (+) cells using the RosetteSep Human Circulating Epithelial Tumor Cell Enrichment Cocktail , according to the manufacturer’s protocol. Then, CD45(−) CTCs were incubated at 37 °C in 24-well non-adherent plates with CTC culture medium , as previously described13. Colospheres and tumor cells were observed during the first weeks of culture and were then transferred to new 24-well plates for further growth and to T25 flasks for cell expansion. In these conditions, CTCs could be quickly expanded and after a few months, billions of tumor cells were obtained.The blood collected in EDTA tubes was immediately used for CTC culture. First, viable CTCs were enriched through depletion of hematopoietic CD4537. HT-29 and SKBR3 cells were maintained in Dulbecco’s Modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin. A549 cells were maintained in DMEM supplemented with 10 mM HEPES, 10% FBS and 1% penicillin/streptomycin. SW620 cells were maintained in RPMI 1640 with L-glutamine and 10% FBS, and the colon CTC lines and BC-M1 cells in CTC culture medium. Endothelial cells were cultured in M199  containing 0.4 ng/ml endothelial growth factors , 20 µg/ml heparin , 1% penicillin/streptomycin and 20% FBS .The colorectal cell lines HT-29 (ATCC HTB-38) and SW620 (ATCC CCL-227) were used as control for several tests, as well as the lung cancer cell line A549 (ATCC CRM-CCL-185), the mammary cancer cell lines SKBR3 (ATCC HTB-30) and BC-M1 (an established breast cancer cell line from a single disseminated tumor cell in the bone marrow of a patient with non-metastatic breast cancer patient)One colosphere and two single cells were transferred to individual PCR tubes (0.2 mL) for whole genome amplification using the PicoPlex WGA Kit, according to the manufacturer’s protocol . Next, libraries were prepared using the TruSeq DNA Sample Prep Kit according to the manufacturer’s protocol. NGS was performed on a HiSeq. 2500 apparatus  using single reads for 100 cycles. Sequence analyses and CNA identification were performed using Control-FREEC and a custom-made script in MatLab.DNA was extracted from the nine colon CTC lines using the QIAamp DNA Mini Kit , according to the manufacturer’s instructions. DNA yield and purity were assessed with a Qubit spectrophotometer . DNA was stored at −20 °C in 10 mmol/L Tris and 0.5 mmol/L EDTA (pH 7.6).38.Genomic DNA samples were genotyped using MassArray iPlex Pro (AGENA BIOSCIENCE), a Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry assay. A specific colorectal cancer panel  was customized for the detection of mutations in six oncogenes  was used as reference housekeeping gene. One µL of each RT product was added to 9 µL of mix containing the two primers (6 µM each) and SYBR Green . PCR reactions were performed on a QuantStudio 5 real-time PCR instrument  as follows: 95 °C for 3 min, followed by 40 cycles of 95 °C for 5 s, 60 °C for 10 s, and then 1 cycle at 95 °C for 1 min, 55 °C for 5 s, 95 °C for 30 s, 37 °C for 30 s. Results were analyzed using the QuantStudio Design and Analysis Software. Hierarchical clustering was computed with the Cluster and Treeview software packages (PMID: 9843981).Messenger RNA (mRNA) was extracted from each CTC line using the RNeasy Mini Kit , and complementary DNA (cDNA) was obtained by reverse transcription (RT) using the SuperScript III First-Strand Synthesis Super Mix kit , according to the manufacturer’s instructions. Semi-quantitative PCR that targeted a wide range of sequences  and data interpreted with the Kaluza software (BECKMAN COULTER). In parallel, labeled tumor cells were also loaded on glass slides, using a cytospin, and observed under a fluorescent microscope .40.The fluoro-EPISPOT assays for CK19, VEGF and OPG were performed as previously described5 cells/well) were then seeded and cultured for 6 h with different media: RPMI 1640 alone , endothelial cell medium with 20% FBS (positive control), and supernatant from the CTC lines cultured with basal medium for 48 h.24-well plates were coated with 250 µL/well Matrigel  and left at 37 °C for 30 min. Endothelial cells (10ALDH1 activity was measured using the ALDEFLUOR kit (01700 – STEMCELL TECHNOLOGIES) following the manufacturer’s instructions. Briefly, 5 µL of ALDEFLUOR Reagent was added to the cell suspension and half of the cell mixture was immediately transferred to another tube with the ALDEFLUOR DEAB Reagent (an ALDH activity inhibitor) to serve as internal negative control to quantify ALDH activity in each cell line. A549 cells were used as positive control, as recommended by the manufacturer.Only metastasis-competent CTCs lead to cancer relapse, but their specific signature is not known. The nine sequential autologous CTC lines we derived from a patient with metastatic colorectal cancer will help to study CTC changes during cancer progression, a prerequisite to discover new therapeutic targets and to propose personalized treatments.Supplementary information


Ubiquinol cytochrome c reductase binding protein (UQCRB) is known to play crucial roles in the development of various types of diseases. However, the link between UQCRB and microRNAs remains unknown. In the present study, we performed microRNA sequencing of mutant UQCRB-expressing stable cell lines that exhibited pro-oncogenic activities caused by expression of the mutant UQCRB gene. Results showed that hsa-miR-10a-5p was significantly downregulated in the mutant UQCRB-expressing cell lines. Furthermore, mRNA sequencing and gene ontology analysis of differentially expressed genes (DEGs) revealed that the cholesterol biosynthesis pathway might be activation by mutant UQCRB expression. Moreover, inhibition of cholesterol synthesis in mutant UQCRB-expressing cells via treatment with the specific inhibitors suppressed the cell proliferation. Transfection with a hsa-miR-10a-5p mimic validated that lanosterol synthase (LSS) is a target of hsa-miR-10a-5p. In addition, hsa-miR-10a-5p was found to be downregulated in liver cancer cell lines overexpressing UQCRB. Taken together, our findings highlighted the potential use of hsa-miR-10a-5p as a biomarker for UQCRB related diseases. The electron transport complex (ETC) in the mitochondria consists of five complexes that involves in producing an electrochemical proton gradient for energy production by ATP synthesis1. The ubiquinol-cytochrome c reductase binding protein (UQCRB) is one of the subunits of mitochondrial complex III that plays a role in electron transport and maintenance of the mitochondrial complex III6. Identification of a target protein of terpestacin, an anti-angiogenic natural product, revealed a new role of UQCRB in regulation of mitochondrial ROS (mROS) generation and angiogenesis7. Moreover, many reports have implicated UQCRB variants in a number of diseases, including hepatocellular carcinoma8, ovarian cancer9, pancreatic ductal adenocarcinoma10, and colorectal cancer11.Mitochondrial dysfunction has been implicated to play a key role in various diseases, such as metabolic diseases and cancer12. Based on the above findings, our group generated mutant UQCRB-expressing stable cell lines, namely, MT1 and MT2, and investigated their angiogenic properties. The MT1 showed a higher expression level of mutant UQCRB protein than MT2 and both cell lines showed significantly faster cell growth and pro-angiogenic activities than those of control host human embryonic kidney cells 293 (HEK293). In addition, we demonstrated that treatment of these mutant UQCRB-expressing stable cell lines with UQCRB inhibitors significantly suppressed cell proliferation of the cells13.In a recent case study, a Turkish female harboring a deletion in the gene encoding the UQCRB and isolated complex III defect presented hypoglycemia and lactic acidosis during a metabolic crisis in her babyhood; however, these conditions did not continue to her childhood15. Multiple studies have reported the use of miRNAs as biomarkers for specific diseases17. Furthermore, miRNAs have been implicated in mitochondrial function, metabolism, and metabolic disorders, such as cholesterol metabolism20.MicroRNAs (miRNAs) are non-coding, single-stranded RNAs containing approximately 22 nucleotides. MicroRNAs are crucial regulators of numerous physiological and pathological processesHowever, the link between UQCRB and miRNAs remains to be mostly uncovered. In the present study, we performed microRNA and mRNA deep sequencing of mutant UQCRB-expressing stable cell lines with control host cell HEK293 to identify novel microRNA biomarkers for UQCRB related diseases.Mutant UQCRB-expressing stable cell lines were subjected to miRNA sequencing to identify differentially expressed miRNAs that are specific to mutant UQCRB. The mutant UQCRB-expressing cell lines (MT1 and MT2) were previously generated based on a human case report expressing the mutant UQCRB gene. miRNA sequencing was conducted to compare the expression patterns of the generated mutant UQCRB-expressing cell lines with those of normal HEK293.2FC| > 1, log2CPM > 2, and FDR < 0.15. Of these, nine miRNAs were downregulated , whereas three miRNAs were upregulated  compared to the control  and related pathways to identify novel mutant UQCRB-associated phenotypes. mRNA sequencing was performed to compare gene expression patterns between HEK293 wild-type cells and mutant UQCRB-expressing cell lines (MT1 and MT2). For DEG analysis, candidate differentially expressed mRNAs were selected based on the criteria |fold change| > 1.5 and p-value < 0.01.In addition, we performed gene set analysis to identify novel pathway. Results of gene set analysis showed significant upregulation (Benjamini <0.05) of eight different ontologies in both mutant UQCRB-expressing cell lines, which include sterol metabolic process, cholesterol metabolic process, steroid metabolic process, sterol biosynthetic process, steroid biosynthetic process (GO:0006694), terpenoid backbone biosynthesis, isoprenoid biosynthetic process, and cholesterol biosynthetic process. Additionally, the MT1 cell line showed upregulation in lipid biosynthetic process, steroid biosynthesis (hsa00100), MAPK signaling pathway, and biosynthesis of unsaturated fatty acids, whereas the MT2 cell line showed upregulation of mRNAs involved in isoprenoid metabolic process , Tarbase (ver. 5), and mirTarbase (ver. 4.5), which contained information on the validated mRNA targets of miRNAs. We also used miRNA target prediction databases, namely, TargetScanHuman , microRNA.org (August 2010), Microcosm (ver. 5), and miRDB (January 2012) Fig. .Figure 2Results showed that seven mRNA targets, namely, ANKRD1, SCD, CNST, SLC2A3, DUSP3, LSS, and HSPA1B had significantly high target prediction values for hsa-miR-10a-5p Fig. . The resNext, we performed mimic transfection to validate that LSS could be the putative target of hsa-miR-10a-5p. Treatment with 5 nM and 20 nM of hsa-miR-10a-5p mimic miRNA was found to upregulate miRNA levels in a dose-dependent manner Fig. . LSS mRNTo further investigate the relationship between hsa-miR-10a-5p and UQCRB, we analyzed hsa-miR-10a-5p levels in cell lines of UQCRB expressing cancer, namely, HepG2 (human hepatocellular carcinoma), PC3 (human prostate cancer) and HCT116 cells (human colon cancer cell). Results of qRT-PCR analysis showed that hsa-miR-10a-5p levels were downregulated in all three cell lines tested with a variable degree than HEK293. In addition, hsa-miR-10a-5p level was also down-regulated in HeLa , which is also cell line of UQCRB expressing cancer (data is not shown). Notably, hsa-miR-10a-5p level was significantly downregulated in HepG2 was even lower than that in MT1 cells Fig. . FurtherGiven that hsa-miR-10a-5p levels were significantly downregulated in HepG2 cells than HEK293, we evaluated expression levels in a different liver cancer cell line, Huh7. Similar to the results from HepG2 cells, hsa-miR-10a-5p expression levels were significantly downregulated in Huh7 cells relative to those in Chang, a normal liver cell line Fig. . The mRN21; Mevastatin, a HMG-CoA reductase (HMGCR) inhibitor; and YM-53601, a squalene synthase (FDFT1) inhibitor22. These inhibitors act on distinct points in the cholesterol synthesis pathway.To further validate the relationship between mutant UQCRB and cholesterol synthesis, cell proliferation was evaluated after treatment with the following three different cholesterol synthesis inhibitors: Fatostatin, a sterol regulatory element-binding protein (SREBP) inhibitor13. Notably, treatment of the mutant UQCRB-expressing cell lines with these inhibitors suppressed the cell growth in a dose-dependent manner as similar as HEK293. Similar results were obtained using liver cancer cell lines, as well. However, the efficacy of the cholesterol inhibitors varied depending on the liver cancer cell line used. Treatment with Fatostatin significantly inhibited cell proliferation in Huh7 cells. Mevastatin only slightly inhibited cell proliferation in both HepG2 and Huh7 cells. However, YM-53601 significantly inhibited cell proliferation in both HepG2 and Huh7 cells  as the metric to reliable factors which even considered the basal expression level not only the difference of expression level in cell type.de novo cholesterol biosynthesis and for the production of various intermediate metabolites that participate in other essential cellular processes23. For example, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are critical for post-translational modifications of GTPase proteins, including Ras and RhoA24. The mevalonate pathway has been demonstrated to be activated or inhibited in a number of different tumors and has been implicated in tumorigenesis. Furthermore, inhibitor of HMG-CoA reductase (HMGCR), a rate-limiting enzyme of mevalonate pathway, has been demonstrated to exert inhibitory effects on various types of cancers, including breast cancer30. Based on the above-mentioned findings, the oncogenic phenotype in mutant UQCRB-expressing cell lines (MT1 and MT2) could be induced at least in part by promoting cholesterol biosynthesis.Results of mRNA sequencing showed that cholesterol biosynthesis was activated in mutant UQCRB-expressing cell lines. Cholesterol synthesis is initiated by the mevalonate pathway, in which acetyl-CoA is a starting precursor and isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are the end products. The mevalonate pathway is crucial for et al. reported that hsa-10a-5p was down-regulated in CaOV3 and RMUG-S cells under hypoxia as compared to normoxia31. Consistent with this, our study also suggested that mutant UQCRB stable cell lines down-regulated hsa-miR-10a-5p that might lead to higher cell proliferation via promotion of cholesterol biosynthesis. On the contrary, however, Wu C et al. suggested that miR-10a was up regulated in hypoxia condition32 and Zhi F et al., suggested that hypoxia caused up-regulation miR-10a in rat liver33. Accordingly, the cause of down regulation of miR-10a-5p in mutant UQCRB stable cells and exact mechanism underlying this regulation needs to be investigated for addressing the complicated responses of miR-10a-5p upon cell types and physiological conditions in coming studies.In the present study, we performed integrated analysis using established databases that provide validated microRNA-mRNA target pairs, as well as prediction databases that provide predicted microRNA-mRNA target pairs. Hsa-miR-10a-5p has been reported to regulate the metabolism in various cells including hypoxia related mechanism. Kinose Y In the present study, the pro-proliferative activity of mutant UQCRB-expressing cell lines was regulated by cholesterol synthesis inhibitors. Accordingly, we thought that cholesterol synthesis pathway could play a role in pro-proliferative activity of these cell lines. As up-regulation of specific genes could influence to increase sensitivity or resistance to drug, further studies are also needed to explore the exact cause of these effects of inhibitors in respect with their sensitivity or toxicity to the cells.Notably, the regulation of proliferative activity by cholesterol synthesis inhibitors in liver cancer cells was not so much effective compared to mutant UQCRB stable cell lines. This is probably because cholesterol synthesis may partially play a role in proliferative activity of mutant UQCRB cells. Because the other miRNAs also were down regulated in MT cells and these miRNAs could also play roles in proliferation through regulating other pathways of the cells, the effect of cholesterol synthesis inhibitors could be different in liver cancer cell lines.7 and PTD-UQCRB treatment in cells and tissues34 increased mROS, stabilized HIF-1 and increased expression of VEGF. Consistently, mutant UQCRB-expressing cell lines showed similar properties as like these activities of wild UQCRB overexpression13. However, we are not able to exclude the fact that mutant UQCRB-expressing cell lines are not same with wild UQCRB expressing cell lines, exactly. To address this issue, detail comparative studies using wild and mutant UQCRB expressing cells are under investigation.In this study, we used mutant UQCRB stable cell lines to identify miRNAs and novel pathways related UQCRB. We consider that mutant UQCRB stable cell lines are similar with wild UQCRB expressing cells as UQCRB related pathways are similarly activated in the mutant UQCRB-expressing cells. According to previous studies of our group, supplement of UQCRB protein by transfection of wild type UQCRB geneCollectively, our present study revealed that hsa-miR-10a-5p is associated with genes involved in the cholesterol synthesis pathway in mutant UQCRB-expressing cells. Moreover, LSS was validated as a hsa-miR-10a-5p target gene based on mimic transfection of mutant UQCRB-expressing cell lines. These results demonstrated that hsa-miR-10a-5p can be used as a biomarker for UQCRB related diseases.2 in air (pH 7.4). PC3 and HCT116 cells were grown in RPMI1640 (Invitrogen) under the same conditions described above. Mutant UQCRB stable cell lines were constantly provided with 1 mg/mL G418.Control (HEK293) cells, mutant UQCRB-expressing cell lines (MT1 and MT2), HeLa and HepG2 cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and 1% antibiotics (Invitrogen). All cells were incubated at 37 °C in a humidified incubator with 5% COTM RNA isolation kit (Ambion) according to the manufacturer’s instructions.Cells were collected using TRIzol reagent . Total RNA was extracted using PureLink35. Data were normalized via the quartile method using the edgeR package. Finally, candidate miRNAs were identified based on fold change (FC), count per million (CPM), and false discovery rate (FDR). The FDR was controlled by adjusting the p-values based on the Benjamini-Hochberg algorithm. The transcriptome analysis data of mutant UQCRB-expressing cells are available at Korean BioInformation Center (KBRS20171018–0000001~KBRS20171018–0000336).Isolated total RNAs were subjected to miRNA sequencing (Illumina HiSeq2000). Raw sequencing reads were provided by Macrogen . Briefly, for miRNA-sequencing, small RNA sample preparation was performed according to the standard the Illumina protocols. The 5′ and 3′ adapters were sequentially ligated to small RNAs (18–30 bp) that were gel-purified from 5–10 µg of total RNA. Adapter-ligated small RNAs were reverse transcribed, amplified, and sequenced on a HiSeq2000 instrument (Illumina) according to the manufacturer’s instructions. Next, adapters were trimmed and mapped to the reference sequence using bowtie, an ultrafast, memory-efficient short read aligner designed to align large sets of short DNA sequences (reads) to large genomeshttp://genome.ucsc.edu). Read counts were analyzed, and differentially expressed mRNAs were identified based on fold change and p-value cutoffs using Cuffdiff.Isolated total RNAs were subjected to mRNA-sequencing (Illummina HiSeq2000). Raw reads were obtained from Macrogen . cDNA library construction was performed using the TruSeq RNA library kit. Briefly, 1 µg of total RNA was subjected to polyA-selected RNA extraction, RNA fragmentation, random hexamer primed reverse transcription, and 100-nt paired-end sequencing on an Illumina HiSeq2000 instrument. The generated libraries were quantified via qPCR according to the qPCR Quantification Protocol Guide and validated using an Agilent Technologies 2100 Bioanalyzer. Next, the adapters were trimmed, and the resulting reads were mapped to the reference sequence using TopHat2. The reference genome sequence  and annotation data were downloaded from the UCSC website  and Taqman primers (Applied Biosystems). Real-time PCR was performed on a HT Fast Real Time PCR system (Applied Biosystems) using Taqman Fast Universal PCR master mix (Applied Biosystems) or on a LightCycler 96 system (Roche) with FastSTtart Essential DNA Probes Master (Roche) and TaqMan primers (Applied Biosystems) according to the manufacturer’s instructions. Data were analyzed based on the 2Isolated RNAs were reverse-transcribed using the following target-specific primers: wild-type UQCRB, forward: 5′-ATGTGAATTCATGGCTGGTAAGCAGGCC-3′ reverse: 5′ ATGCCTCGAGCTTCTTTGCCCATTCTTC-3′; mutant UQCRB, forward: 5′-ATGTGAATTCATGGCTGGTAAGCAGGCC-3′; reverse: 5′-CTC GAGGCCGTCCTCGTAGCAGCTGCAGCCGCACACCTCCACCACGTGGTTGCTGCGCTGGCCGTTCTTTTCTTTTCTTTCCCGAAT-3′; HMG-CoA reductase (HMGCR), forward: 5′-GTC ATT CCA GCC AAG GTT GT-3′; reverse: 5′-GGG ACC ACT TGC TTC CAT TA-3′; Mevalonate (diphospho) decarboxylase (MVD), forward: 5′-CTG CCT GAC TGC CTC AGC-3′; reverse: 5′-ACC TCT CCT GAC ACC TGG G-3′; lanosterol synthase (LSS), forward: 5′-GGC AGA CGT GGA CCT ACC T-3′; reverse: 5′-GAA AAG TGG GCC ACC ATA ATC-3′; GAPDH, forward: 5′-AACAGCGACACCCACTCCTC-3′; reverse: 5′-GGAGGGGAGATTCAGTGTGGT-3′). Expression levels were quantified using ImageJ 1.41o  and Image LabTM software (Bio-rad).TM software (Bio-rad).Cell lysates were analyzed via 10% and 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidenedifluoride membranes  following standard methods. Blots were incubated at 4 °C overnight with the following primary antibodies: anti-UQCRB , anti-HMGCR (Millipore), anti-MVD , anti-LSS , and β-actin . Immunolabeling was performed using Clarity Western ECL substrate . Images were quantified using Image Lab5 cells were seeded onto six-well plates at 24 h prior to transfection. Transfection was performed using Lipofectamine 2000 (Invitrogen) reagent according to the manufacturer’s instructions. Next, cells were transfected with 5 and 20 nmol/L miRNA mimics after 48 h of incubation. Cells were then lysed in TRIzol reagent (Invitrogen).Mutant UQCRB-expressing cells were transfected with a miR-10a-5p mimic purchased from Ambion . Afterwards, 1.5 × 1050 values in cells. Liver cancer cells  were seeded onto 6-well plates and incubated for 36 h with of A1938 (30 µM), a UQCRB inhibitor. A1938 was dissolved in DMSO.Cells were seeded onto 96-well plates and incubated for 24 to 72 h. Cell proliferation was measured using 3--2,5-diphenyltetrazolium bromide  colorimetric assay. Cells were treated with Fatostatin (Sigma), Mevastatin (Sigma) and YM-53601  at or above their reported IChttp://david.abcc.ncifcrf.gov/)36. Data were filtered based on the cutoff value of Benjamini <0.05.Forgene functional analysis of differentially expressed mRNAs (DEGs) was performed using the DAVID tool (www.graphpad.com). Student’s tests were performed to determine significant differences between the control and test groups. Statistically significant differences were considered at p-value < 0.05 .Results were expressed as mean ± standard error (±S.E.M.). All statistical analyses were performed using GraphPad Prism (ver. 5.00 for Windows, GraphPad Software, San Diego, CA, Supplementary Information


The Klotho protein is a recently discovered protein and its overexpression is associated with life extension. Klotho deficiency or silencing of the Klotho gene in mice leads to an accelerated aging and short life, whereas overexpression of Klotho in mice extends lifespan. Klotho participates in many metabolic pathways and is highly expressed in the kidneys, the choroid plexus and neurons. It plays a key role in the calcium-phosphate metabolism, remyelination, and cognitive processes. The present paper is a short review of the literature on the role of Klotho in neurodegenerative disorders, with special attention paid to multiple sclerosis. The neuroprotective function of Klotho is also reported. It is also important to consider potential clinical applications of Klotho that might be useful in the treatment of many diseases. The Klotho protein was discovered in 1997 . The KloBoth in mice and humans, the Klotho gene is located on chromosome 13q12; it encompasses 50 kb and consists of 5 exons. As in the mouse gene, in the human Klotho, two transcripts encoding membrane or secretory protein are formed from one gene by alternative RNA splicing . The Klo2+ absorption by stabilizing the Ca2+ transient receptor potential vanilloid 5 (TRPV5) channel in the plasma membrane. It inhibits renal 1-alpha 25 hydroxylase activity, thus decreasing the levels of circulating calcitriol. Therefore, hyperphosphatemia, hypercalcemia, elevated plasma calcitriol, vascular calcification, and premature aging can be observed in Klotho-deficient mice [Three types of the Klotho protein are distinguished, i.e., cell membrane-related, intracellular, and secretory forms. Klotho associated with the cell membrane (transmembrane Klotho) can be detected in the kidneys, pituitary gland, inner ear, brain, parathyroid glands, pancreas, large intestine, skeletal muscles, bladder, ovaries, testes, and epithelial breast cells . The larent mice . FGF23 lent mice .Klotho has been shown to inhibit 1-α hydroxylase, an enzyme responsible for the production of 1,25-dihydroxyvitamin D3, which is an active form of vitamin D . Lower lOn the molecular level, permanent chronic inflammation, cell proliferation disorders, or cellular aging leads to the formation of a number of age-related chronic diseases, such as obesity, diabetes, atherosclerosis, Alzheimer’s disease (AD), cancer, renal diseases, or degenerative diseases . The secKlotho is produced in large amounts in the brain by ependymal cells of the choroid plexus, Purkinje EC cells, and hippocampal neurons. It is also detected in the cerebral white matter . It was Aging of the brain is related to structural atrophy of the cortex and functional deterioration, especially in the frontal and temporal regions. The process depends on genetics. Inhibition of aging was detected in mice which were carriers of one copy of the KL-VS haplotype , which is associated with longevity . YokoyamM146V, APPswe, tauP301L) and healthy animals [The neural Klotho can protect against the development of age-related neurodegenerative diseases. Masso et al. compared the levels of secreted Klotho (s-KL) in different brain regions between young and older mice and between triple transgenic 3xTg-AD models of AD  . BindingMultiple sclerosis (MS) is a chronic inflammatory disease that is identified as one of the major neurological disorders in young adults. The disease is characterized by the infiltration of autoreactive immune cells into the central nervous system, resulting in degeneration of the myelin and axons . Costa eZeldich et al.  conducte3) for 6 months and 22 patients without MS (control group). In each group, the amount of the secreted form of Klotho and the “redox status” of CSF samples were investigated (using the ELISA kit and a ferric reducing antioxidant power assay—FRAP). The amount of Klotho in the CSF in RRMS was significantly lower (233.62 pg/ml) compared to that in the control group (541.62 pg/ml)—a nearly 2.3-fold decrease in comparison to the controls. The level of the FRAP in MS patients was 1.5-fold lower than in the controls—the mean for the control group was 314.93 μM, and 211.98 μM for MS patients. Additionally, a significant negative correlation was found between Klotho concentration and the Expanded Disability Status Scale (EDSS). However, a significantly positive correlation was observed between Klotho concentration and the FRAP in the CSF. Those results indicated that a decreased level of Klotho in the CSF was related to increased disability in patients with MS. Furthermore, Klotho regulates antioxidant properties of the CSF. The antioxidant power of the CSF was significantly lower in MS patients. Those results were consistent with the hypothesis according to which Klotho exerts neuroprotective effects by modulating oxidative stress.Emami Aleagha et al. conducted another interesting study on the role of Klotho in MS . CerebroAhmadi et al.  tried toCurrently, scientists try to find a potential strategy to increase the level of Klotho in the brain. The administration of exogenous Klotho is limited by the protein weight (130 kDa)—such a large protein cannot cross the blood-brain barrier. Another way is to find molecules that act as Klotho mimetics which could enhance DNA transcription and translation and reduce Klotho degradation. Abraham et al. initiated a program to identify small molecules that could increase Klotho at the transcriptional level . Two comLatanoprost (LA) is an ocular hypotensive agent used in the treatment of glaucoma targeting prostaglandin F2α (FP) receptor. The study of Yamamoto et al. demonstrated that a neuroprotective effect of LA is due to the Klotho-mediated suppression of calpain activation . It was Klotho is highly expressed in the brain. Many studies have demonstrated a neuroprotective role of Klotho. In the human brain, the secreted isoform of the Klotho protein plays a crucial role in neuronal activity, which is the key to aging and neurodegenerative processes. Due to activation of OPCs, soluble Klotho could be used as a therapeutic strategy for brain tissue regeneration. As a cofactor that modulates affinity for various receptors, Klotho regulates calcium homeostasis and oxidative stress. In the CSF, Klotho can participate in the suppression of cytokine/chemokine expression in the central nervous system, supporting the activation of neuronal cells. From a clinical point of view, the Klotho level in the CSF can be used as a biomarker of MS severity. Although it is still not known why Klotho suppresses aging, maintaining Klotho levels by stimulating endogenous Klotho production or administering exogenous Klotho protein may be a potential therapeutic target to slow down the aging process and reduce the incidence of age-related diseases. Finally, as a regulator of remyelination in the white matter, Klotho can become a new therapeutic substance for MS and other demyelinating and neurodegenerative diseases.


L. coronopifolia against human breast carcinoma cell line (MDA-MB-321). Breast cancer is the most common cause of deaths in women. The search for traditionally used medicinal plants which can serve as non-toxic and affordable anticancer drugs is the need of the hour. This study aimed to investigate the anticancer potential of extracts of L. coronopifolia for 24 h. The cytotoxic response of different extracts was measured by MTT assay, neutral red uptake (NRU) assay and cellular morphological alterations under the microscope.The MDA-MB-231 cells were plated in 96 well plates and exposed to 10-1,000 µg/ml of L. coronopifolia exposed MDA-MB-231 cells. The results also exhibited morphological alterations in MDA-MB-231 cells exposed to various extracts. The cells treated with 250- 1000 µg/ml lost their original morphology and cell linkage as compared to control cells. A concentration-dependent decrease in the cell viability of MDA-MB-231 cells was observed after the exposure of petroleum ether, ethyl acetate, chloroform, and ethanol extracts of L. coronopifolia. The cell viability was found to be 82%, 89% and 98% at 1000, 500 and 250 µg/ml, respectively in petroleum ether, 37%, 75% and 88% at 1,000, 500 and 250 µg/ml, respectively in ethyl acetate extract, 30%, 35% and 64% at 1,000, 500 and 250 µg/ml, respectively in chloroform extract and 44%, 65% and 82% at 1000, 500 and 250 µg/ml, respectively in ethanolic extract of L. coronopifolia against MDA-MB-321 cells. Further studies are required to know the mechanism(s) involved in the cell death.These preliminary results suggest the promising anticancer potential of petroleum ether, ethyl acetate, chloroform, and ethanol extracts of  The cancer has been recognized as second foremost cause of death worldwide after cardiovascular diseases . Among women, breast cancer is the most common type of cancer and the major cause of death . In developing countries, breast cancer is prominent reason of death and in developed countries, breast cancer is second leading cause of death . In spite of new diagnosis techniques such as mammography and needle sampling, no significant change has been observed in the mortality rate since many decades. The increasing tumor heterogeneity, drug resistance and high cost of available therapeutic methodologies are present concern that are connected with effective treatment of breast cancer . Although, excessive progresses have been made to understand the pathophysiology of disease and the development towards conventional chemotherapy even though there is still need to explore effective drug to treat the breast cancer . Thus the novel, non-toxic, target oriented chemotherapeutic agents at affordable cost are yet to be achieved. L. augustifolia exhibited anti-inflammatory and analgesic activities . L. stoechas displayed hepato- and neuroprotective properties . L. pubescens is known for possessing a broad spectrum antimicrobial , anti-inflammatory and hepatoprotective activities . L. coronopifolia Poir, L. dentata and L. pubescens Decne are the three species found in Saudi Arabia. These plants have been used in traditional system of medicine. The infusion of flowers of L. dentata is used for urine retention and removal of stones from kidney and ureter. Similarly L. pubescens is known to relieve headache and cold . The literature survey also reveals L. coronopifolia possess antioxidant , antibacterial  and hepatoprotective potential . However, the anticancer properties of L. coronopifolia have not been reported so far. Hence, this study was designed to explore the anti-cancer effect of various solvent extracts of L. coronopifolia on human breast carcinoma cell line (MDA-MB-321).For centuries now, plants have been used in nutrition, cosmetics and medicine. Owing to the presence of various bioactive metabolites, medicinal plants exhibit various pharmacological activities viz. antifungal, antibacterial, antioxidant, spasmodic, anticancer etc. . Traditional medicinal plants have been proven to play an important role in the treatment of various cancer diseases, since ancient . Therapeutically significant bioactive compounds, such as Taxol, Vinblastine, Vinflunine, Vinorelbine, Camptothecin, Vincristine and Vindesine have been explored from plants and are established to treat the various cancer diseases . The genus Lavandula consists of 39 species of flowering plants in family Lamiaceae . The plants belonging to this genus are mainly are annuals, shrubs and herbaceous plants. Lavandula plants are known for their antioxidant, insecticidal, anti-inflammatory, sedative and spasmodic properties . Chemicals and consumablesCell culture medium (DMEM), Fetal bovine serum, and trypsin-EDTA (0.25%) were procured from Gibco, Invitrogen, USA. Cell culture plates and other plastic wares used in present study were purchased from Nunc. MTT, neutral red dye, other specified chemicals and solvents were obtained from Sigma. Plant material and extractionsLavandula coronopifolia plant were air dried and grounded to a coarse powder. For the extraction, powdered plant material was macerated in petroleum ether, ethyl acetate, chloroform, and ethanol successively. The procedure was repeated several times. At each stage the filtrate was collected and the solvent was then evaporated to obtain the extracts of respective solvents. The plant used in present investigation was collected from Shaza Mountains in Saudi Arabia. The plant was authenticated by a taxonomist and a voucher (# 15799) was submitted at the herbarium, Department of Pharmacognosy, College of Pharmacy, King Saud University. The aerial parts of Cell culture2 incubator at 37oC in high humid atmosphere. MDA-MB-231, a human breast cancer cell line was originally purchased from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. The cell line, MDA-MB-231 was grown in DMEM medium with 10% fetal bovine serum, 0.2% NaHCO3 and 1% antibiotic/antimycotic solution in a COExperimental designL. coronopifolia for 24 h. After the treatment, cytotoxicity assessment of L. coronopifolia extracts was done using MTT assay, neutral red uptake assay, and morphological assessments on MDA-MB-231 cell line. MDA-MB-231 cell line was exposed to 10 - 1,000 µg/ml of petroleum ether, ethyl acetate, chloroform, and ethanol extracts of Cytotoxicity Assessment of L. coronopifolia extracts (MTT assay)L. coronopifolia extracts was performed using MTT assay as described by Mossman et al., 1983. In brief, MDA-MB-231 cells  were seeded in 96 well culture plates and allowed to adhere in CO2 incubator for overnight. Then, MDA-MB-213 cells were treated with 10 - 1,000 µg/ml of petroleum ether, ethyl acetate, chloroform, and ethanol extracts of L. coronopifolia for 24 h. After exposure, 10 μl/well MTT solution (5 mg/ml) was added and incubated further for 4 h. Supernatant was removed from the wells and DMSO (200 µl) was added in each well and mixed. The absorbance of plate was then read at 550 nm wave length.Cytotoxic effects of Cytotoxicity Assessment of L. coronopifolia extracts (NRU assay)L. coronopifolia extracts was also assessed by NRU assay as described by Borenfreund and Puerner, 1984. In brief, MDA-MB-231 cells  were seeded in 96 well culture plates and allowed to adhere in CO2 incubator for overnight. Then, cells were exposed to various concentrations  of petroleum ether, ethyl acetate, chloroform, and ethanol extracts of L. coronopifolia for 24 h. After the treatment, neutral red (50 μg/ml) was added in wells and further incubated for 3 h. Then, solution was rapidly washed with solution  and the dye was extracted in a solution of 1% acetic acid and 50% ethanol. The absorbance of plate was then read at 550 nm wave length.Cytotoxic effects of Morphological analysisLavandula coronopifolia for 24 h. After the treatment with Lavandula coronopifolia, cell images of MDA-MB-231 cells were grabbed under the phase contrast inverted microscope at 20× magnification. For the analysis of morphological assessment, the MDA-MB-231 cell line was exposed to 10 - 1,000 µg/ml of petroleum ether, ethyl acetate, chloroform, and ethanol extracts of Statistical analysisThe p values <0.05 were taken as statistically significant between control and treated groups using One-way ANOVA. The results are expresses as Mean + SD from three independent experiments.Cytotoxicity Assessment of L. coronopifolia extracts (MTT assay)L. coronopifolia against MDA-MB-231 cell line obtained by MTT assay are presented in L. coronopifolia for 24 h exhibited significant decrease in the cell viability of MDA-MB-231 cell line in a concentration dependent manner. The percent cell viability was found as 82%, 89% and 98% at 1,000, 500 and 250 µg/ml, respectively in petroleum ether extract L. coronopifolia obtained by NRU assay. Like MTT assay, similar kind of cytotoxic effects in MDA-MB-231 cells treated with L. coronopifolia extracts were also observed by NRU assay. A concentration dependent cytotoxic effects was observed in MDA-MB-231 cells exposed to 10-1,000 µg/ml for 24 h. The percent cell viability recorded by NRU assay was 83%, 90% and 97% at 1,000, 500 and 250 µg/ml, respectively in petroleum ether extract  cell line. The MDA-MB-231 cell line in this study was chosen, because it has been proven as a good in vitro model system to investigate the cytotoxicity and anticancer potential of various plant extracts against breast cancer . Traditionally medicinal plants have been proven to play an important role in the treatment of various cancer diseases . Numerous researches have been shown beneficial effects of plant extracts against various cancer cell lines . It is well documented that plants and their components could act as cytotoxic/antiproliferative effects and induced apoptosis in various cancer cell lines . These days, the research on natural/plant phytochemicals are increasing actively because of their potential health benefits and less side effects. In this study, we have chosen Lavandula coronopifolia plant because it is reported that L. coronopifolia possess antioxidant , antibacterial  and hepatoprotective potential against ethanol induced oxidative stress and cytotoxicity . But anticancer properties of Lavandula coronopifolia have not been reported against human breast carcinoma cells. The cytotoxic effects of potential of different extracts of L. coronopifolia has been assessed using various parameters, such as MTT assay, NRU assay and morphological alterations in MDA-MB-231 cell line treated with different concentrations for 24 h. The MTT and NRU assays showed that petroleum ether, ethyl acetate, chloroform, and ethanol extracts of L. coronopifolia decreases the cell viability of MDA-MB-231 cell line in a concentration dependent manner. Breast cancer is one of the vigorous problems and major cause of mortality in female globally. Therefore, the aim of this study was to investigate anticancer potential of Lavandula coronopifolia did not show significant cytotoxicity to MDA-MB-231 cells. The concentrations range selected in study was chosen from previously reported literature, since exposure of different natural products have been studied by different investigators under in vitro conditions . The results of the present study are in accordance to previous report on cytotoxic potential of different plant extracts . The ethanolic extract of Nigella sativa plant have also been reported to possess cytotoxic effects against P388, HepG2, Molt4 and Lewis lung carcinoma cell lines . The ethyl acetate extract of Bismillah leaf  induced cytotoxicity and morphological alterations have also been reported against human glioblastoma cell line (U-87) . In other study, the cytotoxic/antiprolifertaive potential of ethyl acetate extract against HT-29 human colon cancer and MCF-7 human breast cancer cells have also been reported . The anticancer potential of chloroform extract and sub-fractions of Nepeta deflersiana against MCF-7 human breast cancer and A549 human lung carcinoma cell lines have been studied . Recently, Usmani et al., (2018) have also reported the antiproliferative and cellular oxidative stress induced by Cordia dichotoma (Linn.) extract and its fractions on human cervix epitheloid (HeLa) and human lung (A549) carcinoma cells. The anticancer/cytotoxic effects of L. coronopifolia extracts in this investigation suggest that the cytotoxic response of different extracts obtained could be due to the presence of active components, which is showing the anticancer activity of L. coronopifolia extracts against human breast cancer cells MDA-MB-231. The chloroform extract was found to be more cytotoxic towards MDA-MB231 followed by ethyl acetate extract and ethanolic extract. Petroleum ether extract was found to be less cytotoxic as compared to other extracts. The cytotoxicity of ethyl ether, chloroform and ethanolic extracts were found at 250 µg/ml and above concentrations of Lavandula coronopifolia, whereas, 100 µg/ml and lower concentrations of L. coronopifolia decreased the cell viability and altered the cell morphology of human breast cancer cell line (MDA-MB-321). The results also showed that chloroform extract was found to be more cytotoxic towards MDA-MB-231 followed by ethyl acetate extract and ethanolic extract. Petroleum ether extract was found to be less cytotoxic as compared to other extracts. Further, more studies are required to know the mechanism(s) of cell death involved in this process. In conclusion, the results from present investigation demonstrated that petroleum ether, ethyl acetate, chloroform, and ethanol extracts of


EWSR1-FLI1, PAX3-FOXO1 and SS18-SSX, respectively. Finally, animal experiments confirmed the antitumor activity of ZSTK474 in vivo, with superior efficacy observed in translocation-positive cells. These results suggest that ZSTK474 could be a promising drug candidate for treating sarcomas, especially those harboring chromosomal translocation.Treatment of patients with advanced sarcoma remains challenging due to lack of effective medicine, with the development of novel drugs being of keen interest. A pan-PI3K inhibitor, ZSTK474, has been evaluated in clinical trials against a range of advanced solid tumors, with clinical benefit shown in sarcoma patients. In the present study, we developed a panel of 14 human sarcoma cell lines and investigated the antitumor effect of 24 anticancer agents including ZSTK474, other PI3K inhibitors, and those clinically used for sarcoma treatment. ZSTK474 exhibited a similar antiproliferative profile to other PI3K inhibitors but was clearly different from the other drugs examined. Indeed, ZSTK474 inhibited PI3K-downstream pathways, in parallel to growth inhibition, in all cell lines examined, showing proof-of-concept of PI3K inhibition. In addition, ZSTK474 induced apoptosis selectively in Ewing's sarcoma (RD-ES and A673), alveolar rhabdomyosarcoma (SJCRH30) and synovial sarcoma  cell lines, all of which harbor chromosomal translocation and resulting oncogenic fusion genes,  EWSR1  gene and one of the ETS transcriptional factor genes (FLI1 or ERG) [SS18  gene and one of the SSX  genes  [Sarcomas are rare malignant tumors of mesenchymal origin, such as muscle, fat, bone and other connective tissues, with > 50 different subtypes reported to date . It is e or ERG)  and synoor SSX4) –9. Theseor SSX4) –12. Anotor SSX4) .Although several animal models and patient-derived xenograft models have been established to understand the molecular mechanism underlying sarcoma development, few therapeutics options are available thus far due to its complex character . The priin vitro tool to identify novel antitumor agents and predict modes of action, as well as to identify predictive biomarkers relating to antitumor efficacy. “Fingerprints” are defined as the patterns of differential drug efficacy across the panel of cell lines, and have been found to be reflective of mode of action. Indeed, the mode of action of novel anticancer compounds could be predicted by correlation analysis of its fingerprint to those of reference compounds via a bioinformatic approach called COMPARE [in vitro and in vivo [We previously established a panel of 39 human cancer cell lines (JFCR39) composed of various carcinoma types –24. The  COMPARE . Using t COMPARE . This co in vivo –30. Clin in vivo . Interesvia the use of a cell line panel approach, akin to JFCR39. We collected 14 commercially-available sarcoma cell lines from various origins and established a sarcoma panel. A total of 24 anticancer agents including ZSTK474, other PI3K inhibitors, and those clinically used for sarcoma treatment were examined with respect to their antitumor profiles across the panel of sarcoma cell lines in terms of effects on tumor growth, PI3K-downstream signaling pathway alterations and apoptosis induction in vitro and in vivo. These profiles were then compared to genetic backgrounds of sarcoma cells in an attempt to identify candidate predictive markers for the antitumor efficacy of ZSTK474.In the present study, we characterized the antitumor profile of ZSTK474 in sarcoma cells TP53, which was the most frequently mutated gene among the genes examined. In addition, gain of function mutations, which gave new activity or enhancement of its activity to the proteins, were commonly found in KIT , BRAF  and NRAS  genes. In contrast, none of the cell lines in this panel harbored known gain of function mutations in the PIK3CA gene at the hotspot residues . Missense mutations were not observed in the PTEN gene in these cell lines, while intronic deletions were observed in the HT-1080, RD and RD-ES cell lines.We first examined the genetic background of 14 cell lines within the sarcoma panel by amplicon sequencing. The origin and mutation status of each of the cell lines is shown in Table We next examined the expression and the activation status of signaling proteins involved in PI3K-AKT-mTOR and RAS-MEK-ERK pathways by immunoblot analysis, as shown in Figure NRAS, KRAS or BRAF genes expressed phosphorylated MEK and ERK proteins at a significantly higher level than wild-type cell lines  concentrations also calculated , docetaxel (r = –0.63) or gemcitabine (r = –0.57). In addition, we found a significant positive correlation between phosphorylated IGF1R and the IGF1R-TKI, linsitinib (r = 0.63) or the PI3K-alpha specific inhibitor alpelisib (r = 0.55), and between phosphorylated MEK and sensitivity to selumetinib (r = 0.66) or vemurafenib (r = 0.55).We secondly examined the correlation between drug efficacy and activation status; in vitro, we examined the phosphorylation status of PI3K-downstream signaling molecules after treatment with ZSTK474. As shown in Figure To confirm proof of concept of PI3K inhibition in vitro, we next exploited sarcoma xenograft models to determine antitumor activity of ZSTK474 in vivo. Of the 14 sarcoma cell lines, three cell lines  were subcutaneously transplantable and exhibited reproducible tumor growth in nude mice (data not shown). Therefore, we examined the effect of ZSTK474 on growth of xenografted tumors derived from these cell lines, and compared it with that affected by doxorubicin and pazopanib. As shown in Figure via inhibiting the PI3K signaling pathway in vivo, as well as in vitro.Given that PI3K inhibitors showed antiproliferative activity in sarcoma cell lines via G1 arrest of the cell cycle, but not by a cytotoxic effect via apoptosis, using carcinoma cells from various origins both in vivo and in vitro [50 for an extended period  [The most common chromosomal translocation found in Ewing's sarcoma, t, generates an aberrant transcription factor (BEZ235) , suggestEWSR1-FLI1-negative osteosarcoma cell line, HOS. This result suggested that downregulation of Id2 protein expression observed in Ewing's sarcoma cell lines upon treatment with ZSTK474 could be mediated via transcriptional inactivation of EWSR1-FLI1 from the Id2 promoter.Then, we next examined the effect of ZSTK474 on transcriptional activity of EWSR1-FLI1 by reporter assays. As shown in Figure in vivo. A673 cells were subcutaneously transplanted in nude mice, with the rapid growth of tumors subsequently observed. ZSTK474 administration for two weeks exerted a robust antitumor activity against A673-derived tumors -SSX2 fusion gene, underwent apoptosis upon treatment with ZSTK474, as determined by emergence of cleaved PARP  compared to the MEK inhibitor selumetinib (~1000-fold range). These results suggested that ZSTK474 effectively suppressed tumor growth in sarcoma cell lines irrespective of their subtype. Moreover, ZSTK474 downregulated phosphorylation of PI3K-downstream signaling factors including AKT and ribosomal S6 protein in all of the sarcoma cell line tested, suggesting proof of concept of PI3K inhibition.In the present study, we characterized the antitumor profile of PI3K inhibitors, in particular ZSTK474 which we developed, against sarcomas in preclinical models by exploiting a panel of sarcoma cell lines derived from various origin; in this respect, effects on tumor growth, PI3K-downstream signaling pathway and apoptosis induction KRAS or BRAF genes and overexpression of IGF1R were found to be negative predictors whereas high expression of phosphorylated AKT was observed to be a positive predictor for ZSTK474 efficacy [KRAS/NRAS/BRAF nor overexpression of IGFR/phosphorylated AKT displayed any significant correlation with ZSTK474 efficacy in the sarcoma panel assessed here. HT-1080, one of the less effective cell lines examined, harbored gain of function mutation of NRAS, suggesting that NRAS mutation could be involved ZSTK474 inefficacy; however, RD, another NRAS mutant cell line, responded to ZSTK474 at a moderate level. Further studies are needed to clarify whether NRAS mutation predicts ZSTK474 inefficacy. In addition, we could not determine whether PIK3CA mutation could predict the efficacy of ZSTK474, since none of the 14 sarcoma cell lines examined harbored a gain of function mutation of PIK3CA despite gain of function mutations in this genes being commonly found in in 18–25% of patients with liposarcomas [In our previous study using the JFCR39 carcinoma cell line panel, gain of function mutations of the efficacy ; howeversarcomas , 42. LosEWSR1-FLI1 in Ewing's sarcoma RD-ES cell line and PAX3-FOXO1 in alveolar rhabdomyosarcoma SJCRH30 cell line, respectively) [SS18-SSX2 fusion gene [In contrast to growth inhibitory activity, apoptosis induction was selectively observed following ZSTK474 treatment. As summarized in Table ctively) , 44. We ion gene , and fouvia a reporter assay, and suppressed expression of the Id2 protein in both of the Ewing's sarcoma cell lines examined. Actually, a previous study demonstrated that the EWSR1-FLI1 fusion could transform cells [SS18-SSX fusion genes are capable of transforming cells and knockdown of SS18-SSX expression can greatly reduce viability of synovial sarcoma cells, as determined by colony formation assays [via PI3K/AKT inhibition. However, the causality between these events observed in chromosomal translocation positive sarcoma cells and the induction of apoptosis were unclear in the context of this study. In fact, we detected neither common nor specific changes in the expression levels of Bcl-2 and IAP (inhibitor of apoptosis) family proteins in these cells upon treatment with ZSTK474 (data not shown). Further studies are needed to understand the precise mechanism of apoptosis induction in such types of sarcoma cells.Here, we demonstrated that ZSTK474 partially downregulated transcriptional activity of the EWSR1-FLI1 transcription factor from the Id2 promoter rm cells , and down assays . Interesn assays , 50. Sinn assays , 52, seln assays , and thuin vivo antitumor effect of ZSTK474 using various sarcoma cell lines subcutaneously xenografted in nude mice. ZSTK474 exerted a significant in vivo antitumor effect against all of sarcoma cell lines examined, comparable to the existing antitumor drugs doxorubicin and pazopanib. Of note, in vivo effect was most obvious in the three chromosomal translocation-positive sarcoma cell lines that were characterized with apoptosis induction . Doxorubicin was purchased from Cayman Chemical Company  and dissolved with saline . Pazopanib was purchased from Cellagen Technology , dissolved with DMSO for in vitro studies, and suspended with 5% hydroxypropylmethyl cellulose (HPMC) for in vivo studies.Buparlisib (BKM-120) and everolimus (RAD-001) were purchased from AdooQ BioScience . Sorafenib, doxorubicin and gemcitabine were purchased from Cayman Chemical Company . Cisplatin, pictilisib (GDC-0941) and pazopanib were purchased from Cellagen Technology . Docetaxel and selumetinib (AZD6244) were purchased from Focus Biomolecules . Carboplatin and ifosfamide were purchased from LKT Laboratories, Inc. . Alpelisib (BYL-719), vemurafenib, imatinib, sunitinib and gefitinib were purchased from Active Biochemicals Co., Ltd. , Axon Medchem BV. , BioVision Inc. , R&D Systems, Inc.  and Synkinase Pty Ltd. , respectively. ZSTK474, ZSTK534, ZSTK778, ZSTK1741 and ZSTK2209 were kindly gifted from Zenyaku Kogyo Co., Ltd. For 2. A673 was also purchased from American Type Culture Collection  and cultured in the same condition mentioned above. SYO-1 cell line provided by Dr. Akira Kawai  and Aska-SS and Yamato-SS cell lines provided by Dr. Norifumi Naka  were cultured in the same condition.Cell lines used in the sarcoma cell line panel assay are listed in Table via the TaKaRa Amplicon sequencing service by using the Ion Ampliseq Cancer Hotspot Panel v2, which includes primer sets to amplify 207 amplicons covering approximately 2,800 COSMIC mutations from 50 oncogenes and tumor suppressor genes using human hg19 reference genome as described in the manufacturer's web site .Genomic DNA was isolated from each cell line by DNeasy Blood & Tissue Kit according to the manufacturer's protocols . Sequencing library construction and Ion torrent amplicon sequencing were performed Inhibitory effects of each agent against the sarcoma cell lines was assessed by using the sulforhodamine B (SRB) assay . PercentPercentage growth (%) = 100 × (T–I)/(C–I)T: absorbance for the test well after incubation with drug for 48 hI: absorbance for the test and control well at starting point (addition of drugs),C: absorbance for the control well (C) after incubation with vehicle for 48 h50 was calculated as described previously . All drugs were tested in duplicate. The GI50 values were log transformed and were used for all analyses. Cluster analysis and visualization of heatmaps was performed using the Cluster 3.0 and TreeView software .The drug concentration of the GIvia fluorescence microscopy. The number of the cells with nuclear condensation and DNA fragmentation was counted and the percentage of apoptotic cells relative to total cells was calculated. At least 100 cells were counted in each group.Apoptosis was detected by Hoechst 33342 staining. Briefly, after incubation with ZSTK474 for 48 h, the cells were trypsinized, collected and fixed in ice cold 1% glutarardehyde-PBS (–) at 4° C for 24 h. After washing with PBS (–), the fixed cells were stained with 167 μM Hoechst 33342 , and observed ad libitium. Animal studies were performed in accordance with the procedures approved by the animal use and care committee of the Japanese Foundation for Cancer Research.Female nude mice with BALB/c genetic backgrounds  were purchased from Charles River Japan, Inc. , maintained under specific pathogen-free conditions in a 12:12 h light-dark cycle, and provided with sterile food and water Ectopic sarcoma xenograft models were previously established elsewhere by subcutaneous injection of SJCRH30 , SK-UT-12)/2,TW =  = 100 × average TW of drug-treated group/average TW of control group.3VO4, 5 mM EDTA, aprotinin at 100 Kal U/mL, 1 mM phenylmethylsulfonyl fluoride, 0.5% Nonidet P-40, and 0.1% sodium dodecyl sulfate (SDS). Tumor samples were resected from mice 4 h after the final drug administration, minced with scissors and homogenized by a glass homogenizer with a Teflon pestle in the same buffer described above. The protein concentration of each sample was measured by a BCA Protein Assay Kit . Then, 10 μg of lysate from each cell line or tissue was separated by SDS-polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane. After blocking with 5% skim milk, the membrane was incubated with a primary antibody. Targeted antigens were detected by incubating with anti-phospho AKT (Ser473) antibody, anti-phospho AKT (Thr308) antibody, anti-pan AKT antibody, anti-phospho S6 antibody, anti-S6 antibody, anti-phospho IGF-1R β (Tyr1135/1136) antibody, anti-phospho MEK 1/2 (Ser217/221) antibody, anti-phospho ERK 1/2 (Thr203/Tyr204) antibody, anti-phospho 4E-BP1 (Thr37/46), anti-AKT antibody, anti-S6 antibody, anti-cleaved PARP at Asp214 antibody, anti-PTEN antibody, anti-Id2 antibody, anti-GAPDH antibody , anti-phospho IGF-1R antibody  or anti-α tubulin antibody , followed by fluorescently tagged secondary antibodies . Visualization of bound antibody was carried out using fluorescently tagged secondary antibodies  and the signals were detected and digitized by an Odyssey CLx Imaging System . An anti-α tubulin antibody  was used to provide data for a loading control.Cells were washed with ice cold PBS (–) twice and lysed with 10 mM Tris-HCl buffer at pH 7.4 containing 50 mM NaCl, 50 mM NaF, 30 mM sodium pyrophosphate, 50 mM Na6 of cells (RD-ES and A673) or 2.5 × 105 cells (HOS) were seeded into 6 well culture plates and cultured for 24 h. Each cell was co-transfected with 1 μg of pGL3-Id2-2755 and 1 μg of Renilla (Renilla reniformis) luciferase internal control plasmid, pGL3-CMV  via the use of Lipofectamine 2000 . The culture medium was changed after 6 h of transfection, and cells were passaged to a 96 well plate and treated with ZSTK474 at final concentrations of 0.3 μM, 1 μM, 3 μM or 10 μM. Luciferase activity was detected using the Dual Glo Luciferase Assay System . The percentage of luciferase activity was calculated by the luciferase activity of firefly  luciferase derived from pGL3-Id2-2755 divided by the Renilla luciferase from pGL3-CMV. pGL3-Id2-2755 was kindly gifted by Dr. Tokino [1 × 10. Tokino .2O2 in PBS (–) and blocked with 10% normal goat serum. Following this, they were incubated with anti-p-S6 antibody, anti-cleaved PARP antibody  or anti-CD31 antibody . The bound antibodies were visualized by using EnVision+System HRP labeled polymer antibody and DAB kit , and the counterstaining was performed with hematoxylin. The number of nucleus of CD31-positive cell was counted as vascular endothelial cells.Tumors were resected from mice, fixed with neutral formalin and embedded in paraffin. Four μm sections were cut and deparaffinized in xylene, followed by rehydration through a graded ethanol series (100% to 50%) to PBS (–). After incubation with Dako Real Target Retrieval Solution, pH 6  in boiling water bath for 40 min, tumor sections were treated with 3% Hin vivo studies, n = 6). Statistical significance of antitumor efficacy was assessed by a Mann–Whitney U test or Student's t test or Welch t test for comparison of two samples on days 10 (MES-SA and MES-SA/Dx5) and days 14 . The statistical significance of observed anti-angiogenic effects was assessed by a Student's t test for comparison of two samples. The statistical significance of aberrant gene status and protein expression levels was determined by either a Mann–Whitney U test or Welch t test. The statistical significance of luciferase activity was assessed by a Student's t test. In all analyses, P < 0.05 was considered statistically significant.Data was expressed as mean ± SEM (


The HER2-enriched (HER2-E) subtype within HER2-positive (HER2+) breast cancer is highly addicted to the HER2 pathway. However, ∼20–60% of HER2+/HER2-E tumors do not achieve a complete response following anti-HER2 therapies. Here we evaluate gene expression data before, during and after neoadjuvant treatment with lapatinib and trastuzumab in HER2+/HER2-E tumors of the PAMELA trial and breast cancer cell lines. Our results reveal that dual HER2 blockade in HER2-E disease induces a low-proliferative Luminal A phenotype both in patient’s tumors and in vitro models. These biological changes are more evident in hormone receptor-positive (HR+) disease compared to HR-negative disease. Interestingly, increasing the luminal phenotype with anti-HER2 therapy increased sensitivity to CDK4/6 inhibition. Finally, discontinuation of HER2-targeted therapy in vitro, or acquired resistance to anti-HER2 therapy, leads to restoration of the original HER2-E phenotype. Our findings support the use of maintenance anti-HER2 therapy and the therapeutic exploitation of subtype switching with CDK4/6 inhibition. HER2-enriched breast cancers within the HER2-positive subtype are addicted to the HER2 pathway. Here, the authors analyse gene expression before, during, and after treatment with anti-HER2-based therapies in the phase II PAMELA clinical trial, finding phenotypic changes induced by treatment. Among them, the HER2-E is characterized by high expression of growth factor receptor-related genes  and cell cycle-related genes, low expression of estrogen-related genes such as ESR1 and PGR, and low expression of basal-related genes6. Although the HER2-E subtype is related to HER2+ disease as defined by immunohistochemistry, important discrepancies exist and HER2-E subtype represents ∼60%, ∼80% and ∼40% of HER2+, HER2+/hormone receptor-negative (HER2+/HR-negative) and HER2+/HR+ tumors, respectively7.Breast cancer consists of four molecular intrinsic subtypes  and a normal-like group20. However, not all HER2-E tumors achieve a complete response following anti-HER2-based therapies. For example, the pathological complete response (pCR) rates in HER2-E subtype following anti-HER2-based neoadjuvant therapy, with or without chemotherapy, are 40–80%21. At the same time, HER2-E tumors that do not achieve a pCR have a poor survival outcome22. Thus, there is a need to better understand the biology associated with incomplete response to HER2-targeted therapy in HER2-E disease. Here, we study patient’s tumor samples and preclinical models before, during and after anti-HER2 therapy to shed light on this clinical observation.The clinical value of the HER2-E subtype in HER2+ breast cancer is starting to be elucidated. From a therapeutic perspective, the HER2-E subtype has shown, across 14 clinical trials and ∼2,000 patients, higher sensitivity to anti-HER2-based therapies than the non-HER2-E subtypes7. The expression of the 50 PAM50 genes and 6 signatures  was explored at both time-points  and HR-negative (n = 61) disease, respectively  < 0.001) . Of note, normal tissue contamination at day 14 did not differ between PAM50 Luminal A tumor samples compared to PAM50 non-Luminal A tumor samples   with HER2+/HR+/HER2-E tumors (n = 38). The analysis revealed a strong correlation  between both gene lists, suggesting that anti-HER2 therapies have a higher impact on molecular tumor changes at day 14 than endocrine therapy  in the PAMELA phase II clinical trialnts Fig. . Among t23, where patients were treated for 2 weeks with either lapatinib, trastuzumab or the combination. Among the 56 genes/signatures, 85.7% were found differentially expressed (Supplementary Table p < 0.001)  or the lack of endocrine therapy.Next, we assessed the expression of the 56 genes/signatures in paired tumor samples from 36 patients with HER2+/HER2-E disease recruited in the LPT109096 phase II clinical trialPIK3CA that might confer resistance to anti-HER2 therapy24, or MCF7, which does not overexpress HER2  and 1 HER2-negative cell line . PAM50 subtyping of the 3 cell lines revealed that BT474, SKBR3 and HCC1954 are HER2-E and MCF7 is Luminal B  and a TKI (given at the IC50 for each cell line). Regarding individual PAM50 genes and signatures, 100% and 92.8% were found differentially expressed in BT474 and SKBR3, respectively (Supplementary Table p < 0.001) and the gene expression profile of each cell line was moderately correlated  with the gene expression profiles obtained from PAMELA and LPT109096  and the proliferation signature . Finally, BT474 cell line was identified by PAM50 as HER2-E as the parental cell line once anti-HER2 therapy was withdrawn  cell line and a BT474-derived tucatinib and trastuzumab resistant (BT474-TuRTR) cell line. Cell viability assays demonstrated that BT474-LRTR and BT474-TuRTR cells were resistant to combinations of lapatinib plus trastuzumab, or tucatinib plus trastuzumab, as opposed to parental BT474 , with FGFR4 being the most upregulated gene .To understand the changes in molecular phenotypes induced by chronic HER2 inhibition, we established a BT474-derived lapatinib and trastuzumab resistant  and leads to anti-HER2 resistance. For example, HER2-E tumors that became Luminal A at day 14 in the PAMELA trial32. Interestingly, a re-bound effect after stopping treatment has also been seen with other therapies such as CDK4/6 inhibitors28. Second, subtype switching from HER2-E to Luminal A might open an opportunity to treat the acquired phenotype with drugs that are known to be active in Luminal A, such as endocrine therapy and/or CDK4/6 inhibitors. For example, the PATRICIA phase II trial29 in advanced HER2+/HR+ disease revealed that the Luminal A tumors had improved progression-free survival when treated with palbociclib and trastuzumab as compared to HER2-E tumors. It is important to note that patients in the PATRICIA trial had been previously treated with a mean of 3 prior lines of anti-HER2-based therapy. Thus, HER2-E tumors at this stage are likely to have high anti-HER2 resistance and switching to a Luminal A phenotype is unlikely. Although this is a hypothesis and needs further validation, our preclinical data using HER2+ cell lines with a complete resistance to anti-HER2 treatment supports this observation.Our study highlights two additional aspects. First, the subtype switch seems reversible upon stopping anti-HER2 therapy. This could explain why maintenance therapy with anti-HER2 therapy both in the adjuvant and metastatic settings are beneficial to patients, especially in high-risk patients33, this provides a rationale for the concomitant HER2 and CDK4/6 inhibition in patients. Indeed, combinations of anti-HER2 therapies with CDK4/6 inhibitor and endocrine therapy are being investigated in 2 phase III trials for advanced HER2+/HR+ disease . Both trials differ in the population being evaluated. PATINA focuses on maintenance therapy in HER2+ tumors that are responding to anti-HER2 therapy, whereas monarcHER35 focuses on tumors that have progressed to at least 2 prior lines of anti-HER2 therapy. Concordant with our hypothesis, the monarcHER main results presented at ESMO 201936 revealed that abemaciclib + fulvestrant + trastuzumab was superior to chemotherapy + trastuzumab  most likely due to the luminal tumors deriving larger benefit form CDK4/6 inhibition and endocrine therapy compared to chemotherapy.The HER2 signaling pathway directly regulates the expression of Cyclin D1, affecting its interaction with CDK437, we have not explored the molecular effects in vitro of dual HER2 blockade with trastuzumab and pertuzumab, as our in vitro models cannot recapitulate antibody-dependent cellular cytotoxicity. Second, although our preclinical data suggest that a switch to Luminal A could be leveraged with CDK4/6 inhibition, we do not have clinical evidence that patients with HER2-E tumors that become Luminal A upon anti-HER2 therapy benefit from CDK4/6 inhibitors. Further clinical studies are needed to address this preclinical hypothesis.Our study has  two limitations worth noting. First, although a switch from HER2-E to Luminal A has been described after combinations of trastuzumab, pertuzumab and chemotherapy in clinical samplesIn conclusion, our findings support the use of maintenance anti-HER2 treatment in HER2+ breast cancer sensitive to anti-HER2 therapies, and warrant further research into exploiting molecular subtypes changes  to improve patient outcomes.7. This study is registered with ClinicalTrials.gov, number NCT01973660, and it is completed. The study protocol was approved by independent ethics committees at each center. In this trial, 151 patients with early HER2+ breast cancer were treated with neoadjuvant lapatinib (1000 mg daily) and trastuzumab (8 mg kg−1 i.v. loading dose followed by 6 mg kg−1) for 18 weeks. Patients with HR+ breast cancer received letrozole or tamoxifen according to menopausal status. Tumor samples were collected at baseline, day 14 and surgery and subsequently formalin-fixed paraffin-embedded (FFPE). As previously reported7, Ki67 was evaluated by IHC on FFPE tissue sections from core biopsies at baseline and at day 14 of treatment. These data are provided as a Source Data file.The main results of the PAMELA neoadjuvant phase II study have been previously reported23. This study is registered with ClinicalTrials.gov, number NCT00524303. In this trial, 100 patients with early HER2+ breast cancer were randomized to lapatinib, trastuzumab or both. Patients received 2 weeks of treatment without chemotherapy, and after a tumor biopsy, they received the same anti-HER2 treatment in combination with chemotherapy consisting of 5FU 500 mg m−2 + epirubicin 75 mg m−2 + cyclophosphamide 500 mg m−2 i.v. every 21 days (FEC75) for four cycles followed by weekly paclitaxel 80 mg m−2 for 12 weeks. Trastuzumab was administered every week (4 mg kg−1 i.v. loading dose followed by 2 mg kg−1). Lapatinib was administered every day . Tumor samples were collected at baseline and day 14 and subsequently FFPE according to protocol.The main results of the LPT109096 neoadjuvant phase II study have been previously reportedRTR) cell line and a BT474-derived tucatinib and trastuzumab resistant (BT474-TuRTR) cell line were established by treating BT474 with the corresponding TKI plus trastuzumab for 6 months, starting at low concentrations and increasing them at each passage. BT474-LRTR are maintained with 2 nM lapatinib + 2 µg ml−1 trastuzumab, while BT474-TuRTR are maintained with 2 nM tucatinib + 2 µg−1 ml trastuzumab.The breast cancer cell lines BT474, SKBR3 and MCF7 were purchased from the American Type Culture Collection . All cell lines were maintained as recommended by the suppliers. All cell lines were authenticated using Human 9-Marker STR Profile and Interspecies Contamination Test by IDEXX BioAnalytics. Lapatinib, neratinib, tucatinib and palbociclib were purchased from Selleckchem . Trastuzumab was obtained from Roche Farma . A BT474-derived lapatinib and trastuzumab resistant  from the PAMELA trial and 36 pairs of FFPE tumor samples (baseline and day 14) from the LPT109096 study using the High Pure FFPET RNA isolation kit  following manufacturer’s protocol. At least 1–5 10 μm FFPE slides were used for each tumor specimen, and macrodissection was performed to avoid contamination with normal breast tissue if needed. The RNeasy Mini Kit  was used to extract RNA from cell lines plated in triplicates in 6-well plates at a density of 150,000 cells per well. RNA was quantified at the NanoDrop spectrophotometer .RNA samples of the PAMELA trial from baseline and day 14 of treatment were previously extracted as reportedn = 101) and day 14 of treatment (n = 96) were previously analyzed7. Here, the nCounter platform  analyzed RNA samples from 57 residual tumors from the PAMELA trial, RNA from 36 paired tumors from the LPT109096 study, and RNA from cell lines treated with anti-HER2 and anti-CDK4/6 therapies. A minimum of 100 ng of total RNA was used to measure the expression of 50 genes of the PAM50 intrinsic subtype predictor assay and 5 housekeeping genes . Expression counts were then normalized using the nSolver 4.0 software and custom scripts in R 3.4.3. For each sample we calculated the PAM50 signature scores  and the proliferation signature score38. These data are provided as a Source Data file.RNA samples of the PAMELA trial from HER2-E tumors at baseline (−1 trastuzumab. Cell viability was determined 72 h after treatment using CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS)  following the manufacturer’s instructions, and quantified using the Gen5 Microplate Reader and Imager Software . For anti-CDK4/6 treatments, cells were plated in triplicate at 4000 cells per well in 96-well plates and treated at low doses of TKI or DMSO for 24 h. Cells were then treated with increasing concentrations palbociclib in combination with low doses of TKI or DMSO. At 72 h, cells were labeled with the DNA stain Hoechst 33342  and fluorescence was subsequently determined at the Gen5 Microplate Reader and Imager Software. GraphPad Prism was used for statistics. These data are provided as a Source Data file.Cells were plated in triplicate at 4000 cells per well in 96-well plates. The day after seeding, cells were treated with increasing concentrations of TKI  and 10 µg ml3O4V, 1 µM benzamidine, 1 µg ml−1 aprotinin, 1 µM leupeptin, 1 µM DTT. Total protein extracts were quantified using the DC Protein Assay . 25 μg of proteins were separated in reducing conditions (2.5% β-mercaptoethanol) by SDS–PAGE and transferred to nitrocellulose membranes  for further processing, following standard Western blotting procedures. Primary antibodies used in this study were: HER2 (D8F12), Phospho-HER2 (Tyr1221/1222) (6B12), AKT, Phospho-Akt (Ser473) (D9E), Phospho-RB (Ser807/811) (D20B12), Cyclin D1 (92G2) and anti-GAPDH (14C10) rabbit antibodies from Cell Signaling Technologies . The secondary fluorescent antibody used was the IRDye 800CW Donkey anti-Rabbit IgG . Fluorescent signal was acquired by the Odyssey Imaging System . Uncropped blots are provided as a Source Data file.Cells were plated in triplicate at 150,000 cells per well in 6-well plates and treated with TKI and trastuzumab. Cell lysates were collected after 24 h of treatment with Pierce RIPA buffer  supplemented with protease inhibitors: 5 µM NaF, 1 µM PMSF, 1 µM Na−1 trastuzumab and TKI  or with DMSO control. Propidium Iodide (PI) was used to stain total DNA. Data acquisition was performed at the Cytometry and cell sorting core facilities of the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) using a BD FacsDiva analyzer. GraphPad Prism was used for statistics. These data are provided as a Source Data file.BC cell lines were fixed in 70% cold ethanol after 72 h of treatment with combinations of 10 µg ml−1 trastuzumab and TKI  or with DMSO control. After 10 days, cells were fixed with cold methanol and stained with 0.5% crystal violet solution.Breast cancer cell lines were plated at low density in 6-well plates and treated with combinations of 10 µg mlTo identify genes and signatures whose expression was significantly different between paired samples  or unpaired samples  we used two-class paired Significance Analysis of Microarrays (SAM) with a false discovery rate (FDR) <5%. All statistical tests were two sided, and the statistical significance level was set to <0.05.Further information on research design is available in the Peer Review FileSupplementary InformationReporting Summary


N-glycosylation is essential in biological and biopharmaceutical research as N-glycans have been reported to regulate a wide range of physiological and pathological processes. Monitoring glycosylation in diagnosis, prognosis, as well as biopharmaceutical development and quality control are important research areas. A number of techniques for the analysis of protein N-glycosylation are currently available. Here we examine three methodologies routinely used for the release of N-glycans, in the effort to establish and standardize glycoproteomics technologies for quantitative glycan analysis from cultured cell lines. N-glycans from human gamma immunoglobulins (IgG), plasma and a pool of four cancer cell lines were released following three approaches and the performance of each method was evaluated.The study of protein  N-linked glycosylation is one of the most common post-translational modifications of proteins.2+ from HT29 human colorectal cancer cell line N-glycans released by in-solution PNGaseF.Fig Z. Fragment ion spectra of precursor m/z 1276.57, [M + PROC]+ from HCT15 human colorectal cancer cell line N-glycans released by in-solution PNGaseF.Fig AA. Fragment ion spectra of precursor m/z 814.99, [M + PROC]+ from HCT116 human colorectal cancer cell line N-glycans released by in-solution PNGaseF.(DOCX)Click here for additional data file.