Celecoxib proved to be a valuable adjuvant in cutaneous melanoma in preclinical studies. Our in vitro study evaluated for the first time whether celecoxib could prevent phenotype switching in two human melanoma cell lines treated with dabrafenib. Methods: All in vitro experiments were carried out on BRAF-V600E-positive A375 and SK-MEL-28 human melanoma cell lines, and subjected to a celecoxib and dabrafenib drug combination for 72 h. Melanoma cells were already in the MITFlow/AXLhigh end of the spectrum. Of main interest was the evaluation of the key proteins expressed in phenotype switching , as well as cell death mechanisms correlated with oxidative stress production. Results: Celecoxib significantly enhanced the apoptotic effect of dabrafenib in each melanoma cell line compared to the dabrafenib group (p < 0.0001). Even though celecoxib promoted low MITF expression, this was correlated with high receptor tyrosine kinase AXL levels in A375 and SK-MEL-28 cell lines (p < 0.0001), a positive marker for the phenotype switch to an invasive state. Conclusion: This preliminary study highlighted that celecoxib might promote MITFlow/AXLhigh expression in cutaneous melanoma treated with dabrafenib, facilitating phenotype switching in vitro. Our results need further confirmation, as this finding could represent an important limitation of celecoxib as an antineoplastic drug.Background: Cutaneous melanoma is a heterogeneous tumor with a rapidly switching molecular and cellular phenotype. The invasive phenotype switching characterized by MITF BRAF-positive melanomas, 90% of which have VBRAF600E substitutions [Cutaneous melanoma is one of the most heterogenic tumors, accounting for ~75% of skin-cancer-related deaths worldwide. More than half are itutions . Understitutions . Recent BRAF regulates MITF expression via PAX3 and BRN2 transcription factors, which act as a rheostat [low/AXLhigh expression characterized by a slow proliferating state, invasion, and metastasis formation. This phenomenon was described in melanoma tumors treated with cisplatin or BRAF/MEK inhibitors [Several forms of resistance to targeted therapies are described. These include MAPK pathway reactivation or substitutive pathways activation, extracellular matrix remodeling, autophagy upregulation, and the selection of a resistant tumor cell subpopulation. Another strategy that allows melanoma cells to bypass BRAF inhibitors (BRAFi) treatment is not genetic, but implies phenotype switching. This facilitates the rewiring of the transcriptional program with the further accommodation of melanoma cells to stressful conditions. It also includes a differentiation/de-differentiation switch linked to the expression of the microphthalmia transcription factor (MITF) and receptor tyrosine kinase AXL, as well as changes in proliferation rates. MITF is essential for melanocyte development, it regulates the expression of pigment-producing enzymes and also plays a central role in melanoma: it induces the escape from innate immunity, reprograms focal adhesion and the extracellular matrix, and regulates starvation-induced autophagy ,4,5. BRArheostat . In addihibitors ,8,9. A shibitors .Cyclooxygenase-2 (COX-2) overexpression is correlated with melanoma initiation and progression, proving a negative prognostic marker . CelecoxV600EBRAF mutation, A375 and SK-MEL-28, that are already in the MITFlow/AXLhigh end of the spectrum. The effect of celecoxib on phenotype switching has never been tested in melanoma before. The aim of this preliminary study was to evaluate if celecoxib could enhance the treatment response of melanoma cells to dabrafenib. In particular, we evaluated for the first time how celecoxib impacts on melanoma phenotype switching in vitro. The effect was tested on two human melanoma cell lines harboring the BRAF-V600E mutation. Each cell line was seeded and maintained in Dulbecco’s modified Eagle Medium (DMEM), enriched with 10% fetal calf serum (FCS), 5 ng/mL amphotericin, and 50 μg/mL gentamicin, all reagents being from Biochrom AG, Berlin, Germany. The protocol followed standard culture conditions: cells were kept at constant temperature (37 °C) and humidity, under a 5% CO2 atmosphere, in an incubator, and were fed twice a week; all experiments were carried out in triplicate (n = 3); cells were used within a maximum of 4 passages. Dabrafenib and celecoxib were purchased from Cayman Chemical, MI, USA.Two human melanoma cell lines were used: A375  and SK-MEL-28 . Both melanoma cell lines were positive for 4/well and incubated for 48 h, at 37 °C. Then, melanoma cells were exposed for 24–48–72–96 h either to dabrafenib, celecoxib, or the chosen combination. Viability tests were conducted separately with well-established concentrations of dabrafenib  and celecoxib . Stock solutions (1 mM) were prepared for each drug using dimethyl sulfoxide (DMSO) solution . Serial dilutions were further obtained using cell culture medium. In addition, two concentrations were selected for each drug to be tested in combination for 72 h: celecoxib (C1 = 50 nM and C2 = 100 nM) and dabrafenib (D1 = 100 nM and D2 = 200 nM) dissolved in the medium. The final DMSO concentration used to prepare 50 nM celecoxib and 200 nM dabrafenib was 0.02%. Treated cells were compared to the control. Subsequent different exposure regimens, cytotoxicity assays, were performed by adding MTS and PMS solutions from CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay  directly to wells. After 4 h of incubation, an ELISA plate reader  was used to measure the absorbance of formazan produced by living cells (540 nm). The results were presented as IC50.Viability tests were performed for each A375  and SK-MEL-28  cell line, as follows: melanoma cells were seeded in ELISA 96 wells micro titration flat bottom plates, at a density of 1 × 10Cell death mechanisms were analyzed at the end of the protocol (after 72 h). Treated melanoma cells were stained with Annexin V-fluorescein isothiocyanate (FITC)/vital dye propidium iodide (PI) . Using a BD FACS Canto II flow cytometer  for flow cytometry, four groups were separated, as follows: viable cells (Annexin V (−)/PI (−)), early apoptotic cells (Annexin V-FITC (+)), late apoptotic cells (Annexin V (+)/PI (+)), and necrotic cells (PI (+)). The FACS analysis was completed using two lasers as excitation sources: blue  and red , as previously outlined. Forward scatter and side scatter were adjusted so that the majority of events appeared in the middle of the dot plot. A gate was drawn around those cells (P1) and only the events included within this gate were examined for FITC (annexin V) and PI signal. Debris was gated out by not including the events with a very low forward scatter (FSC below 5000) and that appeared at the bottom left corner of the dot plot. Moreover, discrimination of doublets was performed by plotting FSC-A (area) vs. FCS-H (height). Events that showed similar height as singlets but an increased area were gated out.Both melanoma cells were evaluated at the end of the protocol for structural changes using light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). A375 and SK-MEL-28 melanoma cells’ response to treatment was evaluated during the protocol using an inverted microscope . Pictures were taken using a digital camera .The first set of cells from the two lines subjected to the various experimental treatments were grown on glass cover-slips placed at the bottom of the wells. At the end of the treatments, they were prefixed in ice-cold 2.7% glutaraldehyde  in 0.1 M phosphate buffer (at pH 7.4) for 1.5 h at 4 °C. The samples were washed with 0.1 M phosphate buffer (2 × 0.5 h at 4 °C), and postfixed with 1.5% OsO4  in 0.15 M phosphate buffer for 1.5 h at 4 °C. The cells were dehydrated with an ethanol  series of increasing concentrations . After the cells complete drying, the cover-slips were placed on 10 mm/Ø9 mm aluminum stubs , and the cells were sputter-coated with gold in a Polaron E-5100 sputter coater . Examination of samples was performed with a Jeol JSM-25S scanning electron microscope , equipped with a Pixie 3000 system  for image acquisition.g. The obtained pellets were redispersed in ice-cold 2.7% glutaraldehyde (Agar) in 0.1 M phosphate buffer (at pH 7.4), and the cells were prefixed for 1.5 h at 4 °C. The samples were washed with 0.1 M phosphate buffer . Postfixation was performed next with 1.5% OsO4  in 0.15 M phosphate buffer for 1.5 h at 4 °C. The cells were further dehydrated with an acetone  series of increasing concentrations , and infiltrated with EMBED 812  . After resin polymerization for 72 h at 60 °C, ultrathin sections of 60–80 nm were cut with a DiATOME diamond knife  on a LKB Ultrotome III Bromma 8800 ultramicrotome . The sections contrasted for 15 min with 13% uranyl acetate  and for 5 min with 2.8% lead citrate  were examined with a JEOL JEM 1010 transmission electron microscope , equipped with a Mega VIEW G2III camera and an iTEM software  for image acquisition.A second set of cells were detached from the culture plate by trypsinization for 4 min and centrifuged for 5 min at 500× Malondialdehyde (MDA) levels, a marker for the peroxidation of membrane lipids, were measured by spectrophotometry using the fluorimetric method with 2-thiobarbituric acid (TBA), as described by Conti et al. All reagents were purchased from Sigma-Aldrich, St. Louis, MO, USA. Data were expressed as nmol/mg protein .−3 M nicotineamide adenine dinucleotide (NAD), and 7.75 × 10−2 M lactate  was added to each sample. All reagents were purchased from Abcam, Cambridge, UK. Absorbance was measured at 340 nm and data were expressed as U/mL [Lactate dehydrogenase (LDH), a cell membrane damage marker, was quantified from the melanoma culture medium using the spectrophotometric method. A mixture of 0.05 M sodium pyrophosphate buffer (pH 8.8), 5.25 × 10 as U/mL .Key molecules were assessed via Western blot (WB), focusing on phenotype switching , inflammation (cyclooxygenase-2—COX-2), and melanogenesis (tyrosinase). Following the 72-h protocol, cells were collected for each group and prepared for cell lysates according to the Bradford method , considering bovine serum albumin as the standard. All lysates obtained were adjusted by total protein concentration. Cell lysates (20 μg protein/lane) were subsequently separated via electrophoresis on SDS PAGE gels and then transferred to polyvinylidene difluoride membranes using Bio-Rad Miniprotean system . Protein migration was blocked using Blocking Buffer. The WB membranes were each incubated  with primary antibodies against: MITF (C5) (1:500), AXL (H-3) (1:500), YAP1 (sc-101199) (1:500), TAZ (D-8) (1:500), COX-2 (H-3) (1:500), tyrosinase (H-109) (1:1000), GAPDH (FL-335) (1:1000) , and TGF-β (#3711) (1:1000) . Membranes were washed and further incubated at room temperature, for 4 h, with corresponding secondary antibodies: anti-rabbit secondary antibody for MITF and tyrosinase; anti-mouse secondary antibody for TGF-β, AXL, YAP1, TAZ, COX-2, and GAPDH . Supersignal West Femto Chemiluminescent substrate , as well as Gel Doc Imaging system equipped with a XRS camera and Quantity One analysis software  helped detect the proteins of interest. The protein-loading control was GAPDH from Trevigen Biotechnology Gaithersburg, MD, USA. The volume for each lane was quantified using Image Lab Software for PC Version 6.1, Bio-Rad, Hercules, CA, USA.p value was less than 0.05 after comparing treated groups with the control. The suitable dose for the therapeutic combination was established after calculating the inhibitory concentration 50% (IC50) via Nonlinear regression (curve fit), dose–response inhibition. The panels illustrating the drug combination was generated using Excel for Microsoft 365 .All data were collected using Microsoft Excel for Windows 10 and then statistical analysis was performed using the one-way ANOVA, Tukey Post-tests and Nonlinear regression (curve fit) via GraphPad Prism version 8.00 for Windows, GraphPad Software, San Diego, CA, USA. Statistically significant results were considered when 50 doses of celecoxib (C) and dabrafenib (D) drug combination were chosen after performing successive strategic viability tests on each melanoma cell line, A375 and SK-MEL-28. Viability quantified by colorimetry started using six different preset concentrations for each drug, evaluated for different exposure times . Following our previous experience demonstrating that low doses of celecoxib can have an antineoplastic effect, we chose the same doses for celecoxib, 10, 20, 50, 100, 200, and 250 nM  and celecoxib  to be further tested as a combination (C + D) on each melanoma cell line, in quadruplicate  and 200 nM dabrafenib  (C1 + D2) was chosen for both melanoma cell lines for further assays.The ICd 250 nM a. While d 250 nM b. There uplicate c. As indFlow cytometry (fluorescence-activated cell sorting—FACS) was carried for both melanoma cell lines in order to assess the cell death mechanisms (p < 0.01). When tested in combination with dabrafenib, we observed a significant decrease in the percentage of viable cells in the celecoxib and dabrafenib group compared with the untreated cells  or the dabrafenib group . The main cell death mechanism was early apoptosis.The percentage of viable cells in the celecoxib group was not significantly different from the control, as celecoxib showed a reduced apoptotic effect on A375 cells compared to the control , matching the viability tests and the microscopic evaluation. Regardless of these results, celecoxib effectively increased dabrafenib-induced cell death via apoptosis. SK-MEL-28 melanoma cells treated with the combined regimen underwent apoptosis in a significantly greater percentage compared to the control  and dabrafenib alone , proving superior therapeutic efficacy. Moreover, different from A375 cell lines, there was a significant percent of necrotic cells observed in the dabrafenib and celecoxib + dabrafenib group. Additionally, judging by the total amount of viable cells left in the last group (77.2%), SK-MEL-28 seemed more resistant to treatment than A375.In SK-MEL-28 culture cells, celecoxib showed slight apoptosis induction compared to the control . Results suggest that both celecoxib and dabrafenib increase oxidative stress, resulting in significantly higher MDA levels in the last group compared to the dabrafenib group . Lipid oxidation gives rise to malondialdehyde production (MDA). It is well known that both dabrafenib and celecoxib increase oxidative stress in targeted cells after upregulating reactive oxygen species (ROS) and MDA levels in a dose-dependent manner. Melanoma cells were subjected to a celecoxib and dabrafenib therapeutic combination for 72 h and MDA levels were evaluated as a biological marker of oxidative stress, as shown in p < 0.0001 for A375; Mean Diff. 4.54, 95% CI 4.063 to 5.016, p < 0.0001 for SK-MEL-28), as well as the dabrafenib group . Results correlate with the cell viability and FACS results.LDH is a cytosolic enzyme released into the cell culture medium after damage of the plasma membrane. The LDH level in the spent culture medium is inversely proportional to the viable cell population with intact membrane integrity in the culture. Following the different exposure regimens, the medium was collected and analyzed for LDH levels for both A375 and SK-MEL-28 melanoma cell lines a,b. The Following different treatment regimens, the expression of key proteins involved in the Hippo pathway were further analyzed via Western Blot tests in A375  and SK-MTGF-β. TGF-β release controls extracellular matrix stiffness (ECM), and promotes EDNRA receptor activation and nuclear YAP1 expression with further intracellular AXL production. This cascade is used by melanoma cells when switching to a de-differentiated/invasive state, resistant to RAF/MEK inhibitors. We observed the same pattern in both the BRAF mutant melanoma cells. Surprisingly, the highest levels of TGF-β were obtained in the celecoxib group. Low doses of celecoxib increased the TGF-β release compared to the untreated cells in both A375  and SK-MEL-28  melanoma cells. The celecoxib and dabrafenib treatment combination increased TGF-β expression compared to untreated cells in the A375 cell line , while in SK-MEL-28, we obtained lower levels of TGF-β in the last group compared to the control . Dabrafenib significantly decreased TGF-β expression compared to the control group  and the celecoxib + dabrafenib group  in both melanoma cells. Results suggest that adding celecoxib to dabrafenib reduces the therapeutic response of melanoma cells.MITF. Phenotype switching in melanoma is of crucial importance when talking about resistance to BRAF inhibitors. The most important phenomenon is represented by the MITF expression that correlates inversely with AXL levels. The highest MITF expression was obtained in the dabrafenib group in both A375  and SK-MEL-28  melanoma cells. There was no difference in MITF expression between the celecoxib group and untreated cells (p > 0.05). As a result, the last group expressed increased levels of MITF compared to the control , but lower levels compared to the dabrafenib group .AXL. AXL expression was recently associated with motility and invasion. Melanoma tumors expressing MITFlow/AXLhigh levels adopt a de-differentiated/invasive state and become resistant to targeted therapies. In A375 melanoma cells treated with dabrafenib, we obtained the lowest AXL levels, lower than the control group  and lower than the last group of interest . The highest AXL level was obtained in the celecoxib group compared with the control . As a result, AXL levels were significantly increased in the last group compared to the control . In SK-MEL-28, there was no difference in AXL expression between the first two groups. In this melanoma cell line, celecoxib raised AXL expression in the last group as well compared to the control  or dabrafenib group . Overall, celecoxib decreased therapeutic efficacy, contributing to a tendency of MITFlow/AXLhigh expression in both melanoma cell lines compared to the dabrafenib group.YAP1. When Hippo signaling is reduced in cancer this leads to the accumulation of YAP1/TAZ complexes in the nucleus in order to promote cell proliferation and survival. Increased oncoprotein YAP1 levels facilitate increased AXL expression. In the A375 melanoma cell line, dabrafenib strongly inhibited YAP1 expression , while the celecoxib group registered the highest levels of YAP1  compared with the control group. The therapeutic combination had a higher YAP1 expression than the dabrafenib group , but lower than the control . YAP1 results explain AXL expression. In the SK-MEL-28 melanoma cell line, dabrafenib also showed the highest inhibitory effect on YAP1 compared to the control . The therapeutic combination had the highest YAP1 expression compared to the untreated cells  or the dabrafenib group , correlating with AXL levels as well.TAZ. YAP1/TAZ are not only essential for cancer initiation, but they also function as sensors of the mechanical or structural features of the cell microenvironment. In both cell lines, the highest levels of TAZ were observed in the group of the celecoxib and dabrafenib combination compared to the control . Furthermore, in each case, celecoxib raised TAZ expression compared to the control  or dabrafenib group, suggesting once again that celecoxib strongly promotes YAP1/TAZ activation with AXL production. COX-2. YAP1 transcriptionally regulates COX-2 expression, an important enzyme for the chronic inflammatory state in melanoma. In both melanoma cell lines, we observed the same pattern. The lowest COX-2 expression was obtained in the celecoxib group, followed by the control and the combined regimen. Dabrafenib registered the highest levels of COX-2 expression compared to the group of interest in A375  and SK-MEL-28 . COX-2 expression in the last group was significantly higher than the control in each cell line .TYROSINASE. The overexpression of the tyrosinase enzyme as a marker of melanin production is frequently observed in highly aggressive melanocytic tumors. Tyrosinase activity increased under dabrafenib treatment compared to untreated cells in both A375  and SK-MEL-28  cell lines. Even though the therapeutic combination reduced tyrosinase expression compared to the dabrafenib group, its expression was still higher than the control in both cell lines . These results suggest that celecoxib and dabrafenib therapeutic combination might rather fail to inhibit melanogenesis in these melanoma cell lines.V600EBRAF mutation in more than 50% of cutaneous melanomas has forced the discovery of BRAF and MEK inhibitors, called targeted therapies. Although at first, the combination of targeted inhibitors reduces the tumor burden and prolongs the patients’ overall survival by more than 2 years, eventually, melanomas develop resistance and restart progressing while on treatment [2) inhibition, is that it suppresses cancer cell growth and promotes apoptosis via Wnt/β-catenin and m-TOR (mechanistic target of rapamycin) substitutive pathway inhibition, and reduces the epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and the signal transducer and activator of transcription 3 (STAT3) expression [The presence of the activating reatment ,23. Manyreatment . Among treatment . In a prreatment . Moreovepression ,26,27,28BRAFV600E mutation. The therapeutic association of celecoxib (50 nM) and dabrafenib (200 nM) was tested in vitro for 72 h on SK-MEL-28 and A375 melanoma cell lines. According to the literature and suggested by our own WB results, SK-MEL-28 and A375 melanoma cell lines are already in the MITFlow/AXLhigh end of the spectrum [This study evaluated, for the first time, the chemopreventive effect of celecoxib in overcoming the phenotype switching phenomenon in two metastatic melanoma cell lines encoding the spectrum . We were50 (200–700 nM) [Starting from viability tests we observed that melanoma cells manifested sustained resistance to usual doses of dabrafenib (1–100 nM—data not shown), therefore, we had to increase dabrafenib doses in order to achieve IC–700 nM) . Similar–700 nM) . However–700 nM) . 2 consumption [Oxidative stress was related to melanoma cells’ resistance to BRAFi. On one hand, BRAFi treatment represented by dabrafenib induced ROS production, allowing metabolic processes to rewire and influence melanoma cells’ growth . On the sumption ,34. Oversumption . FACS anlow and AXLhigh expression, the prototype for a de-differentiated/mesenchymal/invasive phenotype. Thus, in aggressive melanoma tumors with acquired resistance, MITF and AXL levels correlate inversely [low (de-differentiated/invasive) and MITFhigh (differentiated/proliferative) cells to coexist. It seems that the MITF modulates the production of an inflammatory secretome and the expression of programmed cell death—ligand 1 (PD-L1). In this manner, the MITF directly controls the infiltration and activation of the immune cells [The Hippo signaling pathway tactfully controls the proliferation vs. apoptosis balance. The pathway is extracellularly regulated by diffusible chemicals, mechanical stimuli, and signals sensed by G-protein coupled receptors . The Hippo signaling pathway also supervises organ size in animals after negatively regulating the expression of YAP/TAZ downstream oncoproteins . The actnversely . Melanomne cells . Müller ne cells . These dThe celecoxib and dabrafenib drug combination tested on two aggressive melanoma cell lines, A375 and SK-MEL-28, followed almost the same pattern. The highest levels of TGF-β, suggestive for ECM stiffness, were obtained in the celecoxib group compared to the control, while TGF-β was unanimously inhibited by dabrafenib in both cell lines. Even though A375 showed a better therapeutic response with significantly increased cell death after the combined regimen (44% viable cells), this activated a TGF-β release in the last group greater than the control and dabrafenib group. The percent of viable SK-MEL-28 cells was not that impaired in the last group (77% viable cells) and TGF-β levels were significantly lower in the fourth group compared to the control. Increased TGF-β makes the switch from the EDNRB G-protein-coupled receptor-mediated MITF and tyrosinase expression to YAP/TAZ activation via EDNRA G-protein-coupled receptors, promoting AXL production [The correlation between COX-2 expression and the tendency to growth and invasion is still partially understood in metastatic melanoma . In thatMelanogenesis, measured by tyrosinase levels, was significantly reduced in the last group compared to dabrafenib used alone, but remained higher than the levels expressed in untreated cells. These results confirm the rule that low pigmentation and reduced proliferation corresponds to invasion and metastasis formation .From the beginning, we preferred a simple experimental design to evaluate the impact of celecoxib on phenotype switching ex vivo, in melanoma. Our results might rather indicate a lack of synergism between celecoxib and dabrafenib regarding phenotype switching in vitro. Even though celecoxib association to dabrafenib inhibited cell viability and promoted oxidative stress-mediated cell death, surprisingly, celecoxib reduced the antineoplastic effect of dabrafenib, promoted ECM stiffness, induced YAP1/TAZ expression, and reduced the MITF/AXL ratio. These findings remind us that melanoma is a versatile tumor and its resistance mechanisms need to be evaluated as a whole. Overall, celecoxib seemed to inhibit the Hippo pathway and promote the switch to a mesenchymal/de-differentiated/invasive phenotype in A375 and SK-MEL-28 melanoma cells.Breast cancer cells are also characterized by these dynamic morphological phenotypes in response to the local social environment . An inteOne also has to be cautious that these are only preliminary results. Our findings require further validation in other melanoma cell lines, in co-cultures and on animal models. Another limitation would be that low doses of celecoxib were used for the protocol. It would be interesting to evaluate if celecoxib is still able to promote the invasive phenotype in higher doses (μM range).high/MITFlow expression in A375 and SK-MEL-28 melanoma cells. These findings clearly need further in vitro and in vivo confirmation and could represent a hypothesis that partially explains the lack of clinical validation of celecoxib in melanoma.There is reticence about the use of celecoxib as a chemopreventive agent in melanoma clinical studies before depicting melanoma biology and its resistance mechanisms. Our preliminary study emphasized there is still much to learn about melanoma microenvironment interactions and how celecoxib exerts its antineoplastic effects in melanoma. Low doses of celecoxib might inhibit the Hippo pathway and promote an invasive phenotype via AXL


CDK1A is one of the most important genes that have different key roles in cell lines. This gene has several transcript variants. Investigating of expression of each one actually can be so important because any one of them may have a separate unknown role in cancer cells so can be used to increase therapeutic efficacy. A549, MDA–MB–231 and Hek–AD cell lines were used in this study. Firstly, three primers for variants of p21 gene were designed by Snapgene and BLAST software. Secondly, the variants expression was checked for each cell lines by RT–qPCR technique, separately. Then the variants that expressed in the cells were selected for more investigation. Finally 2 Gy irradiation was used to evaluate the effect of that on variants expression. The results show that for all cell lines, primer num1 and 3 expressed before any stimuli. After irradiation, for MDA–MB–231 and A549, the expression of primer num3 was decreased, while for Hek–AD no change was observed. The primer num1 expression after the irradiation was different for the cells, V1 expression was decreased in A549 by fold of 0.03 while expression of this for MDA–MB–231 cells was not changed after 2Gy irradiation. It is very necessary to pay attention to the function of each splice variant as well as the response to external stimuli. Understanding the role of each variant in a gene is critical and researchers can use that to improve radiotherapy as well. The generation of transcript variants by alternative splicing is a common process in human gene expression; indeed, >70% of human genes are known to express transcript variants . Various studies have shown that the transcript variants of a gene may have different and even opposing roles, and alternative splicing is known to be a key factor in cancer progression. For example, Bcl-x, which is associated with cell survival/apoptosis, has two isoforms, Bcl-xL and Bcl-xS. The longer Bcl-xL isoform acts as an apoptotic inhibitor, whereas the shorter form acts as an apoptotic activator . In prostate adenocarcinoma, transcripts V4 and V1 were significantly upregulated but only transcript V4 upregulation was associated with poor recurrence-free survival in The Cancer Genome Atlas data . Splice variant (sv) AC333 inhibited MCF7 cell proliferation. Similarly, svAC333 knockdown by RNA interference was shown to have the opposite effect by repressing the cell cycle progression of breast cancer cells . p21. This gene encodes a potent cyclin-dependent kinase inhibitor. The CDK inhibitor p21, also known as p21waf1/cip1 or cyclin-dependent kinase inhibitor1A , belongs to the Cip/Kip family of cdk inhibitors . CDKN1A gene located on chromosome 6 (6p21.2) in humans . One of the most important genes in cells is p21, which stops cells from entering into the S-phase .Using immunohistochemistry, recent studies has revealed that p21 was expressed in a variety of human malignancies, and is correlated with tumor progression and a poor prognosis in various carcinomas . Evidence exists that p21 is an important factor implicated in clinicopathological features and predicated the prognosis of several cancer such as bladder , breast , colorectal , prostate  cancer. There are several treatment modalities including radiotherapy with ionizing radiation (IR) for cancer. In response to IR, p53 protein is activated and it has various downstream targets including genes involved in cell-cycle regulation, apoptosis, and DNA repair. Regulation of these processes by p53 controls the cellular response to IR-induced damage. As soon as DNA is damaged by radiation, binding of p53 protein induces transcription of the downstream gene p21 is encoded by ten transcript variants in humans . The difference of transcript variants is in their 5’ UTR s but are similar in their coding. Vogel et al suggested that translation regulation contributes to the protein variation as several parameters related to translation like 5′ UTR, 3′ UTR, coding sequence length, presence of uORFs and amino acid composition, and so forth showed good correlations with the obtained mRNA/protein ratios .Studies have been done on the role of various p21 variants . However, according to our search in several databases, there is little strong information about the regulation of CDKN1A expression via its transcript variants. While regulation of p21 expression is known, the regulation of the individual transcript variants is Unknown. Furthermore, the effects of transcript variants expression has not been extensively studied. External stimuli such as ionizing radiation have a significant effect on gene expression and will cause noteworthy changes in cell proliferation cycle.Radiotherapy is a cancer treatment that uses high doses of radiation to kill cancer cells and shrink tumors. When used to treat cancer, radiation therapy can cure cancer, prevent it from returning, or stop or slow its growth. But in some cancer cells caused radioresistance. By knockdown or knockout a transcript variant of the responsible gene, we can improve radiotherapy efficiency. In the First step, to access this goal, it is necessary to examine how the expression of each variant changes in response to ionizing radiation and what effect it has on the cells.p21 gene after ionizing radiation. We believe understanding of the function and behavior of transcript variants of p21 gene, with two controversy roles, is so important and effectiveness. These results can help researchers and oncologists for target and irradiation therapy and finally improve therapeutic efficiency. We investigated the changes in the expression of p21 variants in three different cell lines followed by 2Gy irradiation..The purpose of this study was to explore transcript variants expression of Cell lines. MDA–MB–231breast cancer, A549 lung cancer and Hek–AD Human embryonic kidney cell lines were purchased from the Iranian Biological Resource Center  and was cultured in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% FBS  and 5% penicillin/ streptomycin. Cultures were maintained at 37°C with 5% CO2 in a humidified incubator. p21 gene sequence were collected from the https://www.ncbi.nlm.nih.gov. Then BLAST and Snapgene softwares were used for designing each primer individually, and after checking primer design by Oligo and gene Runner programs, ordered for synthesis. Total RNA was extracted using RNeasy Plus-Mini Kit  according to the manufacturer’s protocol. The same amount of RNA quantified via NanoDrop One  was used to synthesize complementary DNA using QuantiTect Reverse Transcription Kit (Qiagen). Finally, the QuantiNova SYBR Green PCR Kit (Qiagen) was used to run the real-time quantitative polymerase chain reaction (RT-qPCR) using LightCycler 96 System .The mRNA expression level was quantified by normalizing over HPRT as the internal control gene. The sequences of the primers used for RT-qPCR were is shown in Reverse transcriptionquantitative polymerase chain reaction (RTqPCR): At first, the−∆∆CT. ∆∆CT = (CT objective gene – CT reference gene) of experimental group − (CT objective gene – CT reference gene) of control group.Fold of difference relative to the reference gene (HPRT) was determined by conversion of 2Irradiation strategy. For more investigation and evaluated the regulation of expressed variants, the cells were exposed to 2Gy X-ray using a linear accelerator (LINAC) which produces X photons of 6 Mev at a dose rate of 200 mGy/min . The SSD was 100cm, and the field size was 10*10 cm.Statistical analysis. All experiments were repeated for at least three times. Data and statistics are presented as means ± SD. The significance was determined by One Way ANOVA using SPSS 24.0. P<0.05 was considered to indicate a statistically significant difference . p21 gene. Secondly, after validation of primers synthesis, expression of these primers was investigated in the cell lines, separately. Finally, we used 2Gy x-ray ionizing irradiation, a routine dose that is used in radiotherapy, as an external stimulus to explore the effect of ionizing irradiation on the expression of p21 transcript variants. Transcript variant 4 of the p21 gene is not expressed.Briefly, in this study, three different cell lines were chosen. MDA–MB–231 breast cancer cell line, A549 lung cancer cell line and Hek–AD human embryonic kidney cell line. Firstly, according to last Refseq update in www.ncbi.nlm.nih.gov, three specific primers were designed. Primer number 1 (num1) transcribes V1 (NM_000389.5) and V5 (NM_001220777.2), primer num2 transcribes V4 (NM_001220778.2), and primer num3 transcribes V2 (NM_078467.3), V3 (NM_001291549.3), and V6 (NM_001374509.1) of theAs mentioned in the Materials and Methods section, RT–qPCR technique was used to examine the expression of the p21 variants. It was found that for all cell lines, primers num 1 and 3 were expressed, but num2 was not expressed in MDA–MB–231, A549, and Hek–AD cell lines. So, V1, 5 (primer num1), V2, 3, 5, and 6 (primer num 3) are expressed in the cell lines. But V4 (primer num2) is not expressed in these.Irradiation has different effects on the expression of p21 variants. The cells were exposed to 2Gy irradiation. After 24h, expression of primers num 1 and 3 was examined.p21 gene has decreased in this cell line after irradiation.RT–qPCR analysis for A549 showed that irradiation significantly decreased the expression of the primer num 1, 3 by a fold of 0.03 and 0.50, respectively . In otheInterestingly, for Hek–AD, primers expression was not changed 24h after 2Gy irradiation (p>0.05) .  p21 gene.The results of RT–qPCR for another cancer cell line, MDA–MB–231 breast cancer cell line, are shown in p21 is known as an inhibitor of cell cycle. This gene includes different variants. Investigating of expression of each one actually can be so important in cancer researches because any one of them may have a separate unknown role in cancer cells. In in vitro researches, primers are designed often expressed all variants altogether.In all of studies checked, the primers was applied to all p21 variants. Furthermore, not enough attention has been paid to the different roles of each variant in a gene. Consequently, the effect of external stimuli especially irradiation, which is used in radiotherapy, on transcript variants expression on the cancer cells has not been strongly investigated.The difference of transcript variants is in their 5’ UTRs and variations in 5’ UTR can function as important switches to regulate gene expression . p21 gene differs in the 5’ UTR, lacks an in-frame portion of the 5’ coding region, and initiates translation at a downstream start codon, compared to variant 3 of this gene. For example, variant (1) of the  p21 gene was investigated. In three different cell lines , three different primers, for variants of p21 gene, were designed. In the first, expression of each one was separately examined. In this study, three different cell lines were chosen. A549 lung cancer cell line, MDA–MB–231 breast cancer cell line, and Hek–AD human embryonic kidney cell line.MDA–MB–231 and A549 are two aggressive cell lines in breast and lung cancer and often radioresistance was observed in these cells. MDA–MB–231 cell line is ER, PR, and E-cadherin negative, triple-negative, invasive, and expresses mutated p53. Also, radiation resistance is often seen in this.Hek–AD human embryonic kidney cell line that is used as a normal cell line. The expression of variants of theThe results showed that for all cell lines, variant primers num 1, 3 were expressed. Indeed we observed that V1, 2, 3, 5, and 6 are expressed in the cell lines. But V4 (primer num2) is not expressed in these. This is a notable result and the question appears why variant 4 is not expressed in these three cell lines although this variant, like the variants 1, 2, and 5, which expressed in these cells, encodes isoform 1 in the CDK1A.In the other word, Is variant 4 not expressed in certain cells, including the cells was used in our study, or is it silenced in all human cells ?P21 is one of the most important gene in cells that has certain and key roles including: regulator of cell cycle progression at G1, cell cycle G1 phase arrest in response to a variety of stress stimuli, and also apoptosis following caspase activation . p21 is a two-faced regulator depending on cell type, cellular localization, p53 status, and the type and level of genotoxic stress. It can acquire either oncosuppressive or oncopromoting properties depending on whether it is in a p53-proficient or p53-deficient environment, respectively . Nozell and Chen (2002) found that a single locus, p21, can express two unique gene products, both of which appear to have a role in cell cycle regulation. p21 gene downregulated in lung cancer cell line but in an aggressive breast cancer cell line no change was observed. . Reports of Collier et al show that just V1 expression was detected in N-TERT cells.We can attribute this observed difference to two different reasons: First, the dissimilarity in the different cancer cell lines has caused this variance. On the other hand, as mentioned above, the p21 gene has different roles in cells . After studying Hek–AD, the results of RT–qPCR showed that no variant expression has changed after 2 Gy irradiation. Therefore, the response to radiotherapy in different cells can be partially justified. X-ray radiotherapy causes cell resistance or cell sensitivity in different cells. Actually, the expression of genes, followed by the expression of proteins in cells, creates these features. However, different functions of the p21 gene can be relate to the different expression of variants in cells. The proteins expression will be necessary to evaluate in others research according to these evidences and can be an interested issue in future studies. High expression of p21 protein in head/neck, esophageal, and oral squamous cell carcinomas has been correlated with poor prognosis, indicating that loss of CDKN1A regulation could promote cancer progression in epithelial cells . Although CDKN1A expression is often dysregulated in a variety of cancers, its expression has been shown to be either increased or decreased depending on the cancer and cell type . Therefore, therapies targeting CDKN1A have proven problematic , and targeting key signaling factors regulating CDKN1A expression may help circumvent these complexities. In second step of this study, the cells received 2Gy irradiation to evaluate the effect of ionizing irradiation on variants expression. 24h after that, the expression of V1, 2, 3, 5, and 6 (expressed primer in step one) was investigated. Interestingly, the expression of these variants was different in the cells after irradiation. In A549 cell line, a decrease in primer num1 expression was observed. While, for MDA–MB–231 there was no difference in this before and after irradiation. It means that V1 of the In our study we observed that stressors such as IR (in this research) and UVB  have different effects on the variants expression and as highlighted above, it is certainly because of difference in the cell lines. All of this conclusions provide necessity of the attention to p21 splice variants as an important gene for cancer therapy.p21 gene, that are responsible for causing cell repair in this, we can increase the radiation sensitivity of cancer cells and did not change the normal cells or even increase their radiation resistance.These features can be used in target therapy to increase the therapeutic efficiency of radiotherapy. By knocking down or knocking out only one or more variants of the In this study, Prime num3 expression  decreased in two cancer cell lines. MDA–MB–231 and A549 are very aggressive and radiation-resistant among their cell lines. We next wished to investigation of the cell cycle distribution in the cells to evaluate that there is any relation between expressions of these variants with regulation of cells cycle (data not published).p21 gene as the most important gene in cells spatially in cancer cells. Our results showed when primers were designed it is eventually necessary to design on different variants of a gene, separately. So researchers can accurately consider the responses of cells and obtain the best treatment for each cancer with target therapy. However, the p21 variants require more exploration in different cells and proteins expression.In conclusion, the importance of gene and target therapy for cancer treatment is clear for all and likewise, it is proven that each cell line has a specific response to stressors that is due to several genes function. Primer designing is one of the most important in biology. It is very necessary to pay attention to the function of each splice variant as well as the response to external stimuli. Understanding the role of each variant in a gene is critical and researchers should be careful when designing it. Because, as the results of this study show, irradiation has caused different responses in different p21 variants in the cell lines. We have chosen the Halime MANSOURY ASL: collected the data, wrote the paper. Amir DANYAEI: wrote the paper, performed the analysis. Ali TEIMOORI: conceived and designed the analysis. Hashem KHANBABAEI: collected the dataAll data of this study is documented and will be provided to the journal if necessary.There is no conflict of interest.


Protein tyrosine phosphatases function in dephosphorylating target proteins to regulate signaling pathways that control a broad spectrum of fundamental physiological and pathological processes. Detailed knowledge concerning the roles of classical PTPs in human cancer merits in-depth investigation. We comprehensively analyzed the regulatory mechanisms and clinical relevance of classical PTPs in more than 9000 tumor patients across 33 types of cancer. The independent datasets and functional experiments were employed to validate our findings. We exhibited the extensive dysregulation of classical PTPs and constructed the gene regulatory network in human cancer. Moreover, we characterized the correlation of classical PTPs with both drug-resistant and drug-sensitive responses to anti-cancer drugs. To evaluate the PTP activity in cancer prognosis, we generated a PTPscore based on the expression and hazard ratio of classical PTPs. Our study highlights the notable role of classical PTPs in cancer biology and provides novel intelligence to improve potential therapeutic strategies based on pTyr regulation. Protein tyrosine phosphorylation is a dynamic and reversible post-translational modification regulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). It is a fundamental mechanism for regulating innumerable biological processes , 2. SincThis study comprehensively characterized the molecular regulatory mechanisms and clinical relevance of classical PTPs in 33 types of cancer. We discovered that copy number variations (CNVs) are the predominant factor leading to abnormal expression of classical PTPs across cancer types. We further displayed the architecture and features of gene regulatory networks (GRN) by transcription factors (TFs) and microRNAs (miRNAs). In addition, we assessed the clinical prognosis for classical PTPs and constructed a rigorous model to evaluate the PTP activity in cancer. Finally, we explored the interaction network of classical PTPs with FDA-approved drugs and identified that classical PTPs are promising drug targets for improving therapeutic strategies.All human samples used in this study were collected from patients subjected to clinical surgery in the First affiliated hospital of Bengbu medical college. Before RNA isolation and protein extraction, samples were stored at -80 ℃. Collections of human samples were obtained and approved by the ethics committee of Bengbu medical college. Written informed consent was obtained from individual or guardian participants.https://gdac.broadinstitute.org/). GISTIC2 was employed to define the significant gain or loss in genomic regions  Research Network. We systematically analyzed 38 classical PTPs in 33 TCGA cancer projects. Every project indicates a particular cancer type. RNA-seq data for the transcriptional expression of 33 types of cancer were downloaded using the TCGAbiolinks R package . The CNV regions . The cli regions  or downl regions , the Gen regions , and the regions  were useThe omics data of cell lines, including gene expression profiles, somatic mutations, and CNVs, were acquired from the Broad Institute Cancer Cell Line Encyclopedia (CCLE) and the Genomics of Drug Sensitivity in Cancer databases (GDSC) , 16. TheTo avoid instability between tumor and normal samples in differential analyses caused by batch effect, we downloaded the gene expression profiles from the TCGA project, and the Genotype-Tissue Expression (GTEx) project that is re-computed by the UCSC Xena project depended on a defined pipeline. Thirty-eight differentially classical PTPs were acquired using the limma R package . The BH-Gene Set Variation Analysis (GSVA) was performed to compute the activity of 50 cancer hallmark-related pathways across 33 types of cancer. After that, the spearman correlation between the expression to classical PTPs and pathway activity was estimated. BH-adjusted p-valued< 0.05 and |Rs| > 0.3 were defined as significant.To identify the interactions between miRNA and target genes , we collected miRNAs from four target prediction databases: miRDB, miRTarBase, PITA, and TargetScan –23. The Next, we downloaded the promoter region sequence (from -1000 to +200 bp around the transcription start site) of target genes from the UCSC Table Brower depending on the human wgEncodeGencodeBasic V37 database. TF-binding sites were identified using the TFBSTools R package combined with JASPAR2020 datasets , 25. P-vhttps://www.uth.edu/bioinfo/software.htm). The raw FFLs networks were further refined using TCGA expression profiles to improve the accuracy. The spearman correlation calculated TFs, miRNAs, and target genes for every FFLs in the raw network. For miRNAs-genes pairs, p-value< 0.05 and Rs< 0 was defined as significant. For TFs-genes pairs, p-value< 0.05 and Rs > 0 was defined as significant. The less significant FFLs networks were removed from raw networks to produce the final refined co-regulatory networks in each cancer type. In addition, the refined pan-cancer network was used to perform centrality measure and MCC analysis using the cytoHubba plugin  were extracted from the combination of miRNAs-genes and TFs-genes using MotifPredictor  were retrieved from prior research as FDA-approved therapeutic drugs or biomarker targets . The theWe obtained the drug sensitivity area under the dose-response curve (AUC) and gene expression profiles for cancer cell lines from GDSC to examine drug sensitivity in cancer cell lines . We compThe survival analysis of classical PTPs was analyzed using the Kaplan-Meier method with the log-rank test by the survival R package. The cut-off point in each set was estimated using the survminer R package. P-value< 0.05 was defined as significant. Moreover, the Cox proportional hazard regression model was applied to estimate the hazard ratio (HR) for every classical PTP.The log2 transformed expression profiles were first z-normalized for the classical PTPs with significant HR values across the analyzed samples . PTPscorn is the number of analyzed samples, and e is the z-normalized expression data of involved PTPs with a significant HR.where To identify classical PTPs essential to the proliferation and survival of cancer cells, we applied CERES across 342 cancer cell lines . CERES i2 chamber. Lipofectamine 3000 (Thermo Fisher) was applied to transiently transfection for plasmids and siRNAs following the manufacture`s instruction. After 48 h transfection, cells were lysed on the ice, and western blotting analysis was performed as previously described  supplemented with 10% fetal bovine serum  and 1% Penicillin/Streptomycin. U-87 cell line was cultured in Eagle’s Minimum Essential Medium  supplemented with 20% fetal bovine serum and 1% Penicillin/Streptomycin. All cell lines were maintained at 37℃ in a humidified 5% COescribed .Cells were co-transfected with siRNA or plasmid and luciferase reporter plasmid containing the target promoter for 48 h. The luciferase activity was detected by the Luciferase Assay System  and plate reader .Cell cycle and apoptosis were analyzed using propidium iodide (1 mg/ml) and ribonuclease-A (10 g/ml) , and Annexin V/PI assay by flow cytometry  respectively as we previously described .Cells were seeded in 96-well plates and evaluated with Cell Counting Kit-8  according to the manufacturer`s instruction at the indicated time points. A plate reader  was used at the endpoint to assess the results.Cells were seeded in six-well plates at a density of 1000 cells. After two weeks of growth, colonies were fixed with paraformaldehyde for 30 minutes and marked with 0.1% crystal violet solution for 15 min. Finally, an optical microscope was used to counter the number of colonies.6 cells in serum-free DMEM). Mice were anatomized after two weeks. Western blot was used to detect the protein level of the target gene. The animal study was reviewed and approved by the Animal Care and Use Committee of Northeastern University.Male mice  were acquired from Charles River  and fed in the house in the pathogen-free condition. All procedures were approved by the Institutional Committee on Animal Care of Northeastern University. U-257 cells stably expressing PTPN12 and empty vector were injected subcutaneously into nude mice’s right super lateral tissue . The student’s t-test and the Wilcoxon rank-sum test were utilized to compare normally distributed variables and non-normally distributed variables. The p-values were two-sided and adjusted according to the Benjamini–Hochberg (BH) approach to control the false discovery rate (FDR). A BH-adjusted p-value< 0.05 was considered statistically significant unless otherwise indicated.Statistical analysis and graphical visualization were performed in R, version 4.0.0 (-8), and PTPN6 was negatively correlated with PTPRG .In this study, we reviewed the literature and explored the roles of classical PTPs in human cancer (-32) and PTPN2 in UCS , and some abnormal down-regulations were associated with CNV loss such as PTPN23 in UCS  and PTPRS in OV   into three categories referenced in the published study , includiTo describe the regulatory mechanisms of GRNs in extenso, we constructed the co-regulatory network and integrated TCGA expression data to refine the FFL patterns  and linked to drug resistance to 33 anti-cancer drugs . Dysregulation of the EGFR signaling pathway is an established feature in multiple cancer types, and EGFR signaling can be regulated by classical PTPs  and RTK pathway inhibitor Axitinib , Lestaurtinib , Sorafenib  and PD-173074   as an example, we identified 20 PTPs that are highly associated with the sensitivity of 94 anti-cancer drugs and are targeted by 34 TFs and 42 miRNAs. The expression of these PTPs and paired TFs is negatively associated (drug-sensitive) with the response to anti-cancer drugs. In contrast, their paired miRNAs are positively associated (drug-resistant) with identical drugs , 44, andImmunotherapy has become an efficient clinical approach for cancer treatment. We, therefore, estimated the roles of classical PTPs in anti-PD-L1 treatment  is widely regarded as the gold standard in cancer research, having the most straightforward and clinically meaningful endpoint definition: dead or alive. Furthermore, disease-specific survival (DSS) was superior to OS in patient outcomes . To expl-10; LGG, logFC = 1.50, FDR< 3.13 × 10-10), protein alteration in three cancers , CNV alteration in two cancers  and methylation alteration in nine cancers , logFC = 0.48, FDR< 2.55× 10-11). Surprisingly, 98.3 percent (116/118) of CGAs were connected with at least one kind of PTPscore-associated molecular signature in at least one cancer type. Interestingly, PTPs has an effect on a number of immunotherapeutic targets. PDCD1 (PD-1) was shown to be strongly expressed in PTPscore-high LUSC samples , suggesting that PDCD1 inhibitors, such as nivolumab and pembrolizumab , which is in line with the Phase II multicenter trial in melanoma patients (-24) and Axitinib in LIHC , indicating a possible therapeutic advantage of combining cancer therapy with phosphatase-targeted therapy for individuals with THYM or LIHC. Strikingly, some high-PTPsocre tumors may develop sensitivity to other drugs, including Gefitinib in uveal melanoma (UVM)  and AZD6244 in TGCT , which shows that phosphatase-targeted treatment may not be beneficial for patients with these malignancies.We next assessed the PTPscore on drug response using the imputed drug data from the TCGA samples. We observed that the response to sorafenib was positively associated (drug-resistant) to PTPscore in BRCA , Key Research and Development Plan of Liaoning Province (2020JH2/10300080), National Natural Science Foundation of China , and the Fundamental Research Funds for the Central Universities of China .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This resource will allow exploration of these expression patterns, revealing important insights into cellular physiology and disease, and will aid with cell line selection and the interpretation of results when studying important cellular processes that depend on protein S-palmitoylation.The reversible lipid modification protein S-palmitoylation can dynamically modify the localization, diffusion, function, conformation and physical interactions of substrate proteins. Dysregulated S-palmitoylation is associated with a multitude of human diseases including brain and metabolic disorders, viral infection and cancer. However, the diverse expression patterns of the genes that regulate palmitoylation in the broad range of human cell types are currently unexplored, and their expression in commonly used cell lines that are the workhorse of basic and preclinical research are often overlooked when studying palmitoylation dependent processes. We therefore created CellPalmSeq ( S-palmitoylation is a reversible post-translational modification, in which the 16-carbon fatty acid, palmitate, is attached to a thiol group of specific cysteine residues via a labile thioester bond. As the most common form of S-acylation, this modification has profound influence over protein function and cellular signaling in the brain  and other purpose designed web resources. However, there are often barriers to easy access of this data, particularly when web resources for data viewing are not available.S-palmitoylation in the mouse brain, and demonstrated how these expression patterns can be used to better understand the etiology of diseases that are related to dysregulated S-palmitoylation, and to predict and validate enzyme-substrate interactions  to allow easy interrogation of this multi-study data using simple interactive heatmaps.A recent study from our lab collated and analyzed publicly available RNAseq data revealing considerable heterogeneity in the expression patterns of the genes that regulate ractions . HoweverS-palmitoylation, curated from multi-study RNAseq datasets from non-diseased human tissues, human cancer cell lines and commonly used non-human laboratory cell lines. We processed data for the 24 ZDHHC enzymes , the known ZDHHC accessory proteins . Therefore, we plotted individual heatmaps of ZDHHC expression within each cell-type cluster, with heatmaps scaled separately for each cell type cluster  data are available on CellPalmSeq to plot expression data according to user preferences.We first curated single-cell RNAseq data from the Human Protein Atlas, which analyzed collated data from 26 studies of cells from non-diseased human tissues and organs, identifying 76 broad single-cell types, divided into 15 cell type clusters  from which the cell lines were isolated, and plotted heatmaps that revealed highly heterogeneous expression of the genes that regulate S-palmitoylation across cancer cell lines for both the CCLE  between RNA and protein z-scores , a database repository of high throughput gene expression data, to manually curate available RNAseq data for the several of the most commonly used cell lines including: NIH/3T3, PC12, MDCK, BHK21, CHO, VeroE6, and Calu3 (https://ensembl.org) gene set for each species was used to extract the S-palmitoylation genes but as expected, not all genes we previously analyzed in human are identified in each species, therefore only those identified are included. All of these datasets are available for interactive visualization and download on CellPalmSeq.Numerous non-human cell lines are widely utilized in basic and pre-clinical research and have been used for the study of basic cell biology, drug toxicity, gene therapy and vaccine production . Howevernd Calu3 . We curaPubMed.gov. We found that expression patterns for the genes that regulate S-palmitoylation were very similar across four independent studies, particularly for the ZDHHC enzymes, despite the cell lines originating from three independent sources  are the most reliable resource for assessing relative trends in expression profiles of these genes.S-palmitoylation machinery in the human body and cell line model systems, and therefore will facilitate research into the role of S-palmitoylation in cellular biology and disease.CellPalmSeq will be an invaluable resource that will enable researchers and clinicians to easily interrogate the expression patterns of the www.proteinatlas.org/download/rna_single_cell_type.tsv.zip) were directly downloaded from (www.proteinatlas.org/about/download) and data were extracted (nTPM) for the genes that regulate S-palmitoylation.For Human Protein Atlas, normalized single-cell RNAseq data collated from 26 datasets  and averaged by cell lineage and primary disease. Proteomic data were downloaded from https://gygi.hms.harvard.edu/publications/ccle.html (file For Cancer Cell Line Encylclopedia (CCLE), RNAseq data were downloaded from https://cellmodelpassports.sanger.ac.uk/downloads  and averaged by tissue and cancer type.For Cell Model Passports (CMP), data were downloaded from https://ensembl.org. The RNAseq reads were downloaded from the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/), the series accession numbers and individual sample numbers are listed in For datasets downloaded from the GEO, the reference transcriptome of each species for which cell lines were analyzed from raw sequencing reads was acquired from All plots for the CellPalmSeq database were generated using curated RNA sequencing datasets. Python 3 and Javascript scripts were used with the plotting library Bokeh to generate the interactive heatmaps to display and compare these datasets on the CellPalmSeq website .https://www.displayr.com). Bar charts were plotted in GraphPad Prism 9.2.0 .Heatmaps within the manuscript were plotted in Displayr (


More than 100 human adenovirus (Ad) types were identified, of which species D comprises the largest group. Heparan sulfate proteoglycans (HSPGs) were shown to function as cell surface receptors for cell binding and uptake of some Ads, but a systematic analysis of species D Ads is lacking. Previous research focused on Ad5 and blood coagulation factor X (FX) complexes, which revealed that Ad5 can transduce cells with low expression levels of its main coxsackievirus-adenovirus receptor in the presence of high HSPG expression levels in a FX dependent manner. Based on our reporter gene-tagged Ad-library, we explored for the first time a broad spectrum of species D Ads to study the role of HSPG on their cellular uptake. This study was performed on three Chinese Hamster Ovary (CHO) cell lines with different forms of HSPG (only proteoglycan (745), non-sulfated HSPG (606) or sulfated HSPG (K1)). The effect of Ad:FX complexes on Ad uptake was explored in the presence of physiological levels of FX in blood (6–10 µg/mL). We found that sulfation of HSPG plays an important role in cellular uptake and transduction of FX-bound Ad5 but neither HSPG nor FX influenced uptake of all tested species D Ads. Because FX has no influence on transduction efficiencies of species D Ads and therefore may not bind to them, these Ads may not be protected from attack by neutralizing IgM antibodies or the complement pathway, which may have implications for species D Ads used as vaccine and gene therapy vectors. Human adenoviruses (Ads) are non-enveloped viruses with an icosahedral shaped capsid and which have a linear double-stranded DNA genome of about 36 kbp. The capsid comprises 240 hexon bases, which build the 20 faces of the capsid, and 12 penton bases at each vertice with protruding fiber and knob  of whichBecause of the strong negative charge, HS can interact electrostatically with the viral surface of enveloped viruses or the capsid of non-enveloped viruses . Therefov integrins are needed as secondary receptors for a successful uptake of Ad5:FX-complex via HSPG into the host cell [Physiological concentrations of FX in the blood plasma range from of 6 to 10 µg/mL ,24. It iost cell .Because of the potency of species D Ads as vaccines and for gene therapy, virus–host cell receptor interactions need to be explored in further detail. There is some information about the receptor usage of single species D Ad types in the literature ,26, e.g.2.The Chinese hamster ovarian cell lines CHO-K1 , CHO-pgsAll used Ads, including HAdV-C5 as control and Ad types HAdV-E4, HAdV-B3, -14, -16, -21, -35, -50 and HAdV-D9, -10, -17, -20, -24, -26, -27, -33, -37, -69, -70, -73 and -74 with a GLN-cassette were previously generated [For identifying HSPG on the used CHO cell lines, the cells were detached from the cell culture plate by scraping, to keep the extracellular HSPG intact. Cells were then counted, aliquoted to 300,000 cells, washed twice with DPBS  and resuspended in 100 µL of DPBS. The first antibody, mouse anti-human heparan sulfate IgM . CHO cells were prepared with 100,000 cells/mL, seeded in a 96-well plate for 24 h and infected with different virus particles per cell (vpc). At 24 h after infection, the luciferase assay was performed by adding 1:1 luciferase buffer and substrate mix to each well. After 5 min incubation, the cells were transferred to a non-treated Nunc® MicroWell™ 96-well plate  and luminescence was measured in a plate reader .For testing species D Ad uptake with and without the physiological levels of FX, the same experimental setup was used as described above except adding simultaneously 6, 8 or 10 µg/mL FX  in Opti MEM®  during infection with the Ads. OMEM was used because it is a serum-reduced media to avoid additional serum interference on the effect of FX. As a positive control for the impact of FX, HAdV5 was used.To compare the virus entry into all cell lines by preincubating with or without FX, a duplex qPCR was performed. Previously, all cell lines were seeded in a 24 well plate with 200,000 cells/well for 24 h. The medium was changed to Opti-MEM® (OMEM) and was infected with 1000 vpc with or without FX in a concentration of 10 µg/mL. After 3 h incubation, cells were collected, and the total genomic DNA (gDNA) was isolated with the Monarch Genomic DNA Purification Kit . For duplex qPCR, as the house-keeping gene for the CHO cell lines, glyceraldehyde 3-phosphate dehydrogenase  was used. Ads were detected by targeting the neomycin resistance gene on the GLN-cassette (FAM). As polymerase, the Takyon™ No Rox Probe MasterMix dTTP  was used. The primers were used in an overall concentration of 300 nM and the probe of 200 nM .p ≤ 0.05 (*) was considered as a significant difference to the respective control group, p ≤ 0.01 (**) very significant and p ≤ 0.001 (***) highly significant.All shown data are displayed as mean ± SEM. Statistical comparison was made using the two-tailed t test. A value of To prove the different HSPG depletions of the three used CHO cell lines, flow cytometric analysis was performed. In To determine the transduction efficiency of our GLN-encoding Ads, the three different CHO cell lines were infected with a range of 1000 to 10,000 vpc for each virus. At 24 hours post infection (hpi), luciferase assays were performed to compare the transduction rates of all used Ads among the HSPG-depleted CHO cells. The results displayed in In the non-sulfated HSPG-expressing CHO cell line 606, species C and E showed a significant decrease in transduction compared to the wild type HSPG B. All thFurthermore, for all tested species B Ads, except Ad3, and for the used species C and E Ads, significantly lower or similar transduction efficiency can be observed in the non-HSPG-expressing CHO cell line 745 compared to wild type HSPG-expressing CHO cell line K1 A,B. Ten Compared with the results from the sulfation-ablated CHO-606 cells, the HSPG-ablated CHO-745 cells showed a similar trend in transduction efficiencies of analyzed Ads, supporting the fact that the sulfation status of the heparan chains play a significant role in Ad transduction.According to literature, species D Ads can use the blood coagulation factor X (FX) as an adapter for virus uptake into the cells ,37,38 anTo test the conditions for using FX as an adapter for bridging the Ad to HSPG and as proof of concept, Ad5 was used to show enhanced transduction in CHO-K1 cells, because they express the wild type form of HSPG ,18,19,20In addition, the effect of different FX concentrations was determined in a range from 6, 8 and 10 µg/mL . For this purpose, CHO-K1 cells were infected with Ad5 ranging from 100 to 5000 vpc and 24 hpi, luciferase assays were performed. In The effect of FX on all species D Ads was tested in the HSPG-depleted CHO cell lines. The CHO cells were infected with 5000 vpc  on uptake and transduction of human species D Ads into host cells via HSPGs. For this purpose, three different Chinese Hamster Ovarian cell lines with depleted forms of HSPGs were used. Previous research revealed the function of FX as a bridge between adenovirus 5 (Ad5) and HSPG which functions as an entry receptor into cells with low expression levels of the main Ad5 receptor CAR. Because species D comprises the largest group of human Ads , this spFor proof of concept, we used the well-studied Ad5 as a positive control for enhanced uptake and transduction into host cells via wild type HSPG and FX as an adapter ,20,21,22All tested species D Ads from our reporter gene-tagged library  showed nThe fact that CHO cells display low human integrin expression levels ,40 leadsAs a conclusion, the tested species D Ads in combination with FX showed no significant enhancement in uptake and transduction via HSPG into the used CHO cell lines.


KRAS), Tumor protein p53 (TP53)). MK-2206 and Buparlisib demonstrated pronounced cytotoxic effects and limited cell-line-specific effects in cell death induction. WES revealed two sequence variants within the direct target genes (PIK3CA c.1143C > G in Colo357 and PIK3CD c.2480C > G in Capan-1), but a direct link to the Buparlisib response was not observed. RNA-seq demonstrated that the expression level of the inhibitor target genes did not affect the efficacy of the corresponding inhibitors. Moreover, increased resistance to MK-2206 was observed in the analyzed cell lines carrying a KRAS variant. Further, increased resistance to both inhibitors was observed in SU.86.86 carrying two TP53 missense variants. Additionally, the presence of the PIK3CA c.1143C > G in KRAS-variant-carrying cell lines was observed to correlate with increased sensitivity to Buparlisib. In conclusion, the present study reveals the distinct antitumor effects of PI3K/AKT pathway inhibitors against PDAC cell lines. Aberrations in specific target genes, as well as KRAS and TP53, individually or together, affect the efficacy of the two PI3K/AKT pathway inhibitors.The aberrant activation of the phosphoinositide 3-kinase (PI3K)/ protein kinase B (AKT) pathway is common in pancreatic ductal adenocarcinomas (PDAC). The application of inhibitors against PI3K and AKT has been considered as a therapeutic option. We investigated PDAC cell lines exposed to increasing concentrations of MK-2206 (an AKT1/2/3 inhibitor) and Buparlisib (a pan-PI3K inhibitor). Cell proliferation, metabolic activity, biomass, and apoptosis/necrosis were evaluated. Further, whole-exome sequencing (WES) and RNA sequencing (RNA-seq) were performed to analyze the recurrent aberrations and expression profiles of the inhibitor target genes and the genes frequently mutated in PDAC (Kirsten rat sarcoma virus ( Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive human cancer types and is currently the fourth leading cause of cancer-related deaths in both men and women . Due to The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway is an intracellular signaling pathway important in regulating the cell cycle. PI3Ks have been reported to be involved in several cell functions, such as cell growth, proliferation, differentiation and intracellular trafficking, which in turn contribute to cancer development . AdditioKRAS) wild-type PDAC cell line BxPC-3 in vivo model, while PX-866 showed a slight effect against KRAS mutant PDAC cell lines Panc-1 and MIA Paca-2 [Since the PI3K/AKT pathway plays a critical role in the development and prognosis of PDAC, inhibiting the activation of the PI3K/AKT pathway has become a focus for PDAC therapy. Furthermore, the inhibition of the PI3K/AKT pathway also enhances the chemosensitivity of PDAC cell lines in vitro and in vivo . Key proA Paca-2 . AnotherA Paca-2 . TherefoA Paca-2 ,23,24.KRAS and TP53 are the two most important and most frequently mutated genes among all PDAC hotspot genes, and the mutation rates in PDAC are approximately 92% and 70%, respectively [KRAS and TP53 also interact to increase the malignancy of tumors, including immune evasion, which results in poor patient prognosis [Due to the promising results shown by PI3K/AKT inhibition in PDAC experiments and clinical trials, we investigated the cytostatic/cytotoxic- and apoptosis/necrosis-inducing effects of the AKT1/2/3 inhibitor (MK-2206) and the pan-PI3K inhibitor (Buparlisib) in ten PDAC cell lines . In addition, all cell lines were characterized by whole-exome sequencing (WES) and RNA-seq transcriptome analysis. ectively ,26. Bothectively ,27. Morerognosis . Here, wWhen treating the PDAC cell lines with the AKT1/2/3 inhibitor MK-2206 for 72 h, the cell proliferation and biomass of PDAC were significantly inhibited, starting at a concentration of 1 μM . HoweverThese IC50 values were clustered by unsupervised machine learning into three sensitivity groups: low (Colo357 and SU.86.86), moderate , and high (PaTu8988S and BxPc-3) sensitivity groups .When treating the cell lines with the Pan-PI3K inhibitor Buparlisib for 72 h, it significantly inhibited cell proliferation, metabolic activity, and cell biomass at a concentration of 0.5 μM . In the Based on the same method described above, ten PDAC cell lines were separated into three groups: low (Panc-1 and SU.86.86), moderate , and high  sensitivity groups .MK-2206 induced a significant increase in cell death only in AsPc-1 (10 μM), BxPc-3 (1 μM), and Colo357 (10 μM). In addition, in all cell lines, even in AsPc-1, BxPc-3, and Colo357, the observed percentage of dead cells was less than 20% at all tested concentrations . CompareBuparlisib induced apoptosis/necrosis in all tested PDAC cell lines. Compared with the DMSO control group, a significant induction effect was observed, starting at 1 μM. When Buparlisib concentrations reached 5 μM, more than 50% of AsPc-1, BxPc-3, and T3M4 cells were dead. However, although we observed a significant induction of cell death in Panc-1, SU.86.86, and PaTu8988T, the percentage of apoptotic/necrotic cells was still less than 20% even at the highest tested concentration (10 μM) .AKT1, AKT2, AKT3; for Buparlisib: PIK3CA, PIK3CB, PIK3CG, PIK3CD) was evaluated in all cell lines by RNA-seq. The expression level was displayed as Log2 (TPM + 1) ; AKT3 in AsPC-1 and PaTu8988S (0.00–0.07 vs. 1.52); PIK3CG in AsPc-1, Colo357, Panc-1, PaTu8988T, PaTu8988S, PaTu8902, and T3M4 (0.00–0.10 vs. 0.12); and PIK3CD in AsPc-1, Colo357, and PaTu8988S (0.24–0.89 vs. 1.08). In addition, the expression of these target genes was higher in other PDAC cell lines than in the control. In particular, the expression of AKT1 (6.66–8.96 vs. 5.13), PIK3CA (3.68–4.97 vs. 1.52), and PIK3CB (3.73–6.00 vs. 3.10) was higher than the control in all cell lines.The transcriptional activity of the target genes for each inhibitor (for MK-2206: TPM + 1) . Specifi control . These lAKT1, AKT2, AKT3) and Buparlisib  were selected to analyze transcript variants by WES.The identical target genes for MK-2206  was identified in AsPc-1 : 100), Colo357 (VAF: 23.8), Panc-1 (VAF: 62.1), and SU.86.86 (VAF: 83.7). KRAS c.35G > T  was identified in Capan-1 (VAF: 97.1), PaTu8902 (VAF: 100), PaTu8988S (VAF: 96.9), and PaTu8988T (VAF: 98). KRAS c.183A > C (p.Gln61His) was identified in T3M4 (VAF: 32.6).WES demonstrated ll lines . Three dKRAS in all PDAC cell lines was higher than in the control (4.16–7.09 vs. 2.14)  and SU.86.86 (7.09), respectively. The expression of all KRAS c.35G > T mutations, which were identified in Capan-1, PaTu8988S, PaTu8988T, and PaTu8902, was similar to wild type BxPc-3 , and the expressions of KRAS c183A > C in T3M4 and KRAS c.35G > A in AsPc-1, Panc-1, and SU.86.86 were higher than wild type BxPc-3 (4.79–7.09 vs. 4.53).The expression of s. 2.14) a,b. BothKRAS status of all cell lines revealed that KRAS variants alone have no major influence on the inhibitory effect of Buparlisib, since cell lines harboring a KRAS mutation were classified into all sensitivity groups  and TP53 c.267delC (p.Ser90fs) were identified in AsPc-1 (VAF: 96.4) and Colo357 (VAF: 100), respectively. The missense variants TP53 c.476C > T  and TP53 c.818G > A (p.Arg273His) were identified in Capan-1 (VAF: 100) and Panc-1 (VAF: 98.8), respectively. Double missense mutations, including TP53 c.733G > A (p.Gly245Ser) and TP53 c.1079G > T , were identified in SU.86.86 . TP53 c.659A > G (p.Tyr220Cys) was identified in BxPC-3 (VAF: 99) and T3M4 (VAF: 100). TP53 c.844C > T (p.Arg282Trp) was identified in PaTu8902 (VAF: 100), PaTu8988S (VAF: 100), and PaTu8988T (VAF: 100).Two different types of variants, including frameshift (fs) variants and missense variants of ll lines . The fs TP53 with fs variants were lower than that of missense variants (1.24–2.13 vs. 4.39–5.42) and normal controls (2.83) (The expression levels of s (2.83) a,b.TP53 status of all cell lines revealed no obvious relationship between the status of this tumor suppressor gene and the efficacy of the inhibitors. Interestingly, SU.86.86, which carries two missense variants in TP53, was classified in the low-response group for both inhibitors  of patients with PDAC, in 87.76% (43/49) of them, the overexpression of the genetic modulation of AKT2 was observed [This study also confirmed that MK-2206 inhibited cell proliferation, metabolic activity, and biomass in a dose-dependent manner. However, the effects of apoptosis/necrosis induction were not distinct, and the percentage of dead cells was less than 20% at all tested concentrations in all cell lines. These results indicate that the efficacy of MK-2206 at inhibiting PDAC cell lines is not mainly caused by the induction of apoptosis/necrosis. Moreover, our experiments have also revealed that the anti-proliferative and cytotoxic effects of MK-2206 are similar to, but nevertheless differ from, the observed metabolic effects, especially in Panc-1, PaTu8902, and PaTu8988T. It has been reported that some inhibitors induce cellular stress that alters cellular metabolic activity, and we observed similar properties with MK-2206 ,32. This of PDAC ,33,34. Oobserved . An analKRAS in nine of ten PDAC cell lines, including KRAS p.12Gly > Asp (c.35G > A), KRAS p.12Gly > Val (c.35G > T), and KRAS p.Gln61His (c.183A > C). In addition, it has been reported that KRAS mutations can be found in approximately 92% of pancreatic cancers, and patients with KRAS mutations showed a bad response to first-line gemcitabine-based therapy and presented a poor prognosis [KRAS variants and PDAC cell lines and on patients’ responses to PI3K/AKT pathway inhibitors are currently lacking. A comprehensive analysis of the Buparlisib sensitivity groups and KRAS variants did not demonstrate any relationship. This is obvious, especially in the high sensitivity group, which included not only cell lines carrying KRAS variants but also a wild-type KRAS. These results suggest that the KRAS status alone does not influence the sensitivity to Buparlisib in PDAC cell lines. On the other hand, analysis of MK-2206 demonstrated that carrying the KRAS variant appeared to cause a decrease in the sensitivity of PDAC cell lines to this inhibitor. Consistent with these data, one study demonstrated that, in cell lines of colorectal cancer, lung cancer, breast cancer, and melanoma, KRAS mutations were associated with significant resistance to AKT1/2 inhibition [KRAS status before using MK-2206 to treat patients with PDAC.We identified three different amino acid substitution variants of rognosis ,37. Howehibition . This rehibition . The prePI3K variants (PIK3CA c.1143C > G and PIK3CG c.2480C > G) in PDAC cell lines. We further analyzed the response of cell lines carrying PI3K and KRAS double mutations and a KRAS single mutation to Buparlisib. In four cell lines carrying the KRAS c.35G > A mutation , we identified that Colo357 also carries the PIK3CA c.1143C > G variant. Interestingly, Colo357 was highly sensitive to Buparlisib, while the other three cell lines were less sensitive. This might indicate that there are unknown interactions between the PIK3CA c.1143C > G variant and the KRAS c.35G > A variant. This PIK3CA variant could reduce the negative effects of KRAS on the sensitivity to Buparlisib. However, we did not observe any interaction when analyzing another PI3K mutation (PIK3CG c.2480C > G) in cell lines bearing the KRAS c35G > T variant  when using either inhibitor. However, cBioPortal demonstrated that only 2.5% (31/1228) of patients with PDAC harbor PIK3CA and KRAS double aberrations, and 1.95% (24/1228) of patients harbor PIK3CG and KRAS double aberrations [PIK3CA and KRAS mutation in the cell line. For patients with the same gene aberration, further experiments are still needed to verify the efficacy of the inhibitor.We identified two TP53 variant that can cause amino acid or RNA structure changes, except SU.86.86, which carries two TP53 variants. It has been reported that patients with advanced PDAC who have two TP53 mutations and who were treated with the EGFR-inhibitor Erlotinib demonstrated rapid disease progression, which suggests that multiple TP53 mutations reduce the efficacy of specific inhibitors against PDAC [TP53 variants revealed that SU.86.86 is in the low-sensitivity group when testing both inhibitors, suggesting that two TP53 mutations are related to reducing the efficacy of PI3K/AKT pathway inhibitors  and MK-2206 (AKT1/2/3 inhibitor) were purchased from Selleck Chemicals . According to the manufacturer’s instructions, Buparlisib and MK-2206 were separately dissolved in dimethyl sulfoxide (DMSO)  as a stock solution at a final concentration of 10 mM. The stock solutions were stored at −80 °C and diluted into corresponding working concentrations before each experiment.2 humidified atmosphere incubator at 37 °C.PDAC cell lines AsPc-1, BxPc-3, Capan-1, Colo357, Panc-1, PaTu8902, PaTu8988T, PaTu8988S, SU.86.86, and T3M4 were kindly provided by the University of Greifswald. AsPc-1, BxPc-3, Colo357, Panc-1, SU.86.86, and T3M4 were cultured in RPMI1640 medium  supplemented with 10% heat-inactivated fetal calf serum (FCS)  and 1% Penicillin-Streptomycin (P/S) solution  . PaTu8902, PaTu8988T, and PaTu8988S were cultured in DMEM/F12 medium  supplemented with 10% heated-inactivated FCS and 1% P/S solution. Capan-1 was cultured in RPMI1640 medium supplemented with 15% heat-inactivated FCS and 1% P/S solution. After verifying that all cell lines were not contaminated by mycoplasma, these PDAC cell lines were maintained in a 5% CO4 cells per milliliter in 6-well plates , 24-well plates , and 96-well plates . After 24 h, the supernatant was discarded, and media containing increasing concentrations (range from 1 μM–10 μM for MK-2206 and 0.5 μM–10 μM for Buparlisib) of the inhibitors or vehicle  were added to the corresponding PDAC cell lines. The treated cells were incubated for up to 72 h at 37 °C with 5% CO2. At the indicated time points, cell proliferation, metabolic activities, cell biomass, and apoptosis/necrosis were evaluated in at least three biologically independent replicates.For all assays, the PDAC cell lines were seeded at a density of 3.3 × 10Cell proliferation was evaluated by absolute cell counting and trypan blue  staining. After inhibitor exposure in 24-well plates, the cells were harvested and washed with 1× PBS . In the following step, the cells were stained with trypan blue, and the numbers of viable cells were determined by counting with a hemocytometer. Proliferation was expressed as the percentage of viable cells treated with the inhibitor compared to the 100% DMSO control.®-Multi Microplate Multimode Reader . The metabolic activity was calculated as recommended by the manufacturer. Metabolic activity is expressed as a percentage of the inhibitor-treated group compared to vehicle-treated controls (control = 100%).Metabolic activity was tested by Water Soluble Tetrazolium—1 (WST-1) . After exposure to the corresponding inhibitor, the cells were incubated with 15 μL WST-1 for up to 2 h in 96-well plates. Absorbances at 450 nm and the reference wavelength of 620 nm were measured by Promega GloMax®-Multi Microplate Multimode Reader. For background normalization, the absorbance of each group was subtracted from the absorbance of pure culture media. The amount of CV directly correlates to the cell biomass. The result is expressed as a percentage of the inhibitor-treated group compared to vehicle-treated controls (control = 100%).Biomass quantification was carried out by Crystal Violet (CV)  staining. After exposure to the corresponding inhibitor in 96-well plates, the cells were washed once with PBS and stained with 50 μL of 0.2% CV solution on a shaker at room temperature for 10 min. Thereafter, the plates were washed twice with PBS. To elute bound CV, 100 μL 1% sodium dodecyl sulfate (SDS)  was added to each well and incubated on a shaker at room temperature for 10 min. Finally, absorbances at 570 nm and a reference wavelength at 620 nm were measured by Promega GloMaxIC50 values were calculated independently based on cell proliferation, metabolic activity, or biomass after 72 h of inhibitor exposure. GraphPad Prism Version 8.0.2  was used to evaluate IC50. Briefly, after transforming concentrations and normalizing the results for the three vitality assays, a nonlinear regression model  was used to evaluate the IC50 values. We calculated the IC50 corresponding to the three vitality assays and applied these results to a response-based clustering analysis in order to evaluate the sensitivity of the cell lines to inhibitors.−/PI− cells are considered viable cells, YO-PRO-1+/PI− cells are considered apoptotic cells, and PI+ cells are considered necrotic cells. Flow cytometry measurements were performed on FACSVerse , and data were analyzed by BD FlowJo software .Apoptosis and necrosis were evaluated by YO-PRO-1  and Propidium iodide (PI)  double staining by flow cytometry. After exposure to the corresponding inhibitor, supernatants were collected, and cells were harvested and washed twice with cold PBS. Following that, cells were resuspended in 200 μL YO-PRO-1  solution. After incubating at room temperature for 20 min in the dark, cells were washed twice in cold PBS and resuspended in 400 μL cold PBS. Cells were then stained with PI  immediately before measurement. Unstained and single-stained cells were used as controls and measured in every single experiment. YO-PRO-1® Tissue Kit  according to the manufacturers’ instructions. In brief, 5 × 106 cells were harvested from each continuous cultural cell line and washed twice with cold sterile PBS. Cell pellets were lysed, and then the lysis that contained genomic DNA was extracted and purified by a silica membrane of NucleoSpin column. Lastly, genomic DNA was eluted with 30 μM of nuclease-free water.Genomic DNA was extracted by NucleoSpin6 cells were harvested from each continuous cultural cell line and washed twice with cold sterile PBS. Cell pellets were resuspended in 700 μL QIAzol Lysis Reagent , and then the aqueous phase that contains the total RNA of the lysed cells was extracted and purified by a silica membrane of RNeasy Mini spin columns. Finally, total RNA was eluted in 30 μL of nuclease-free water.Total RNAs were extracted by miRNeasy Mini Kit  according to the manufacturers’ instructions. In brief, 5 × 10After extraction, nucleic acid concentrations, as well as OD260/280 and OD260/230 ratios, were measured with a NanoDrop 1000 Spectrophotometer .Barcoded sequencing libraries were generated after enrichment with the SureSelect Human All Exon kit , pooled and sequenced on a HiSeq4000  instrument using a 150 paired-end protocol to yield at least 20× coverage for >98% of the target region and an overall average depth of coverage above 100×. An in-house bioinformatics pipeline was used, including read alignment to human genome reference hg 19, variant calling , and variant annotation with publicly available databases.After WES, the sequencing data from ten PDAC cell lines were obtained and filtered in order to select variants with the expected highest impact on gene function. Briefly, variants were filtered based on quality , variant allele frequency (VAF), depth of coverage (DP), and variant type. In order to exclude false positive variants, only variants with qual > 100, VAF > 20, and DP > 9 were included in our analysis. Germline mutations were excluded by comparison with COSMIC and dbSNP databases. Then, variant types were excluded if they were unable to cause amino acid substitution, RNA structure change, or base insertions/deletions (indels). These variant types include synonymous variants, intron variants, upstream or downstream variants, and 3 prime or 5 prime untranslated region (UTR) variants. After this filtering procedure, missense variants, splice region variants, inframe indels, frameshift variants, gene fusion, and start/stop gain or lost were chosen for further analysis .Barcoded sequencing libraries were prepared with the TruSeq Stranded mRNA kit , pooled and sequenced on a NextSeq 500 System  using the 75 bp paired-end protocol. At least 30 million reads were obtained for each sample. The reads were aligned to reference genome GRCh37/Release 38 with STAR V.2.7.6a using the two-pass mode . TranscrThe expression data of non-neoplastic pancreatic tissue from The Genotype-Tissue Expression (GTEx) and the Cancer Genome Atlas Program (TCGA) were chosen as controls.The classification of cell lines into distinct sensitivity levels was performed by k-means++ clustering based on an unsupervised machine learning algorithm . Brieflyp < 0.033, **: p < 0.002, ***: p < 0.001 versus the control group.Data have been replicated with at least three biologically independent experiments. GraphPad Prism Version 8.0.2 was used for statistical analysis. The results of proliferation, metabolic activity, biomass quantification, and apoptosis/necrosis analysis were expressed as mean ± standard deviation (SD). Statistical significance was determined by one-way ANOVA (after proving that the data within each group conformed to the Gaussian distribution) or the Kruskal-Willas-Test (for the data within each group that conformed to a non-Gaussian distribution) and displayed as *: PIK3CA nor PIK3CG aberration alone affect the inhibitor response of PDAC cell lines to Buparlisib or MK-2206. Moreover, in the relationship between the observed inhibitor response and aberrations of KRAS and TP53, KRAS point mutations  alone are not able to determine the level of sensitivity to Buparlisib, but they do appear to be related to the level of sensitivity to MK-2206. Cell line carrying a specific PIK3CA variant is associated with enhanced Buparlisib inhibition in KRAS-mutated cell lines. In addition, carrying two TP53 missense variants appears to be associated with reduced sensitivity to PI3K/AKT pathway inhibitors. Thus, our study suggests that blocking the PI3K/AKT pathway is an optional strategy for the treatment of patients with PDAC but that it is still necessary to choose inhibitors based on genetic background.Our present study reveals distinct antitumor effects against PDAC cell lines when inhibiting the PI3K/AKT pathway. Exploring the inhibitor response and the corresponding target gene aberrations shows that neither